SOX9 regulated proliferation and apoptosis of human lung carcinoma cells by the Wnt/ß-catenin signaling pathway.

OBJECTIVE: Sex-determining region Y-box 9 (SOX9) is a transcription factor linked to stem cell maintenance and commonly over-expressed in solid cancers. In the present study, the effects of SOX9 on proliferation and apoptosis of human lung carcinoma cells and its mechanisms were investigated. MATERIALS AND METHODS: Following over-expression or knock-down of SOX9 in human lung carcinoma cell line A549, cell viability was evaluated using XTT method, and cell apoptosis was measured by Flow cytometry. Caspase-3, Caspase-8, Caspase-9 and SOX9 expression was measured by RT-PCR, and Wnt, phosphorylated Wnt (p-Wnt) and ß-catenin expression was detected by Western Blot. RESULTS: Results showed that SOX9 expression was elevated in human lung carcinoma cells. Knocking down cellular SOX9 by short hairpin RNA (shRNA) decreased cell proliferation while promoted apoptosis of A549 cells. Furthermore, down-regulation of p-Wnt and ß-catenin expression levels was detected in A549 cells lack of SOX9. However, over-expression of SOX9 played the opposite roles in proliferation and apoptosis of human lung carcinoma cells. To further demonstrate the functions of the Wnt/ß-catenin signaling pathway in SOX9 regulated-cell functions, the inhibitor IWP-2 was used to block the activation of Wnt/ß-catenin signal. No significant differences between IWP-2-treated cells and SOX9 plus IWP-2-treated cells suggested the existence of a regulatory role for SOX9 through targeting the Wnt/ß-catenin pathway. CONCLUSIONS: These findings establish the significance of SOX9 in lung cancer pathobiology and heterogeneity, with implications for targeting the Wnt/ß-catenin-SOX9 signaling pathway as a rational therapeutic strategy.

Elevated non-esterified fatty acids impair survival and promote lipid accumulation and pro-inflammatory cytokine production in bovine endometrial epithelial cells.

Elevated non-esterified fatty acids (NEFAs) are associated with negative effects on bovine theca, granulosa and oviductal cells but the effects of NEFAs on bovine endometrial epithelial cells (bEECs) are not as well documented. The objective of this study was to define the effects of NEFAs on bEECs. Postprimary bEECs were treated with 150, 300 or 500µM of either palmitic acid (PA), stearic acid (SA) or oleic acid (OA) or a mixture of NEFAs (150µM of each FA) or 0.5% final concentration of vehicle ethanol (control). Viability and proliferation of bEECs exposed to 150µM of each NEFA or a mixture of NEFAs were unaffected. Increased lipid accumulation was found in all treated groups (P<0.01). In cells exposed to 500µM of each NEFA and 300µM PA decreased cell viability (P<0.001), proliferation (P<0.05) and increased apoptosis (P<0.05) were observed. Treatment with 500µM OA, PA and SA had the strongest effects on cell viability, proliferation and apoptosis (P<0.05). Treatment with PA and OA increased interleukin-6 (IL-6) concentrations (P<0.05), whereas only the highest concentration of PA, OA and SA stimulated IL-8 production (P<0.05). These results suggest that high concentrations of NEFAs may impair endometrial function with more or less pronounced effects depending on the type of NEFA and time of exposure.

High non-esterified fatty acid concentrations promote expression and secretion of fibroblast growth factor 21 in calf hepatocytes cultured in vitro.

Negative energy balance is considered as the pathological basis of energy metabolic disorders in periparturient dairy cows. Serum non-esterified fatty acids (NEFA) are one of the most important indicators of energy balance status. Fibroblast growth factor 21 (FGF21) has been identified as a hepatokine involved in regulation of metabolic adaptations, such as promoting hepatic lipid oxidation and ketogenesis, during energy deprivation. However, the direct effects of NEFA on FGF21 expression and secretion in bovine hepatocytes are not entirely clear. The objective of this study was to investigate the effects of different NEFA concentrations on FGF21 expression and secretion in calf hepatocytes cultured in vitro. NEFA were added to the culture solution at final concentrations of 0.6, 1.2, 1.8 and 2.4 mmol/L. After 24 hr of continuous culture, FGF21 mRNA and protein expression levels in the hepatocytes were determined by real-time PCR and Western blot respectively. FGF21 secretion in the supernatant was determined by enzyme-linked immunosorbent assay (ELISA). The results showed that expression and secretion of FGF21 at 0.6 mmol/L NEFA-treated hepatocytes was higher than that of the control group (p < .05). The FGF21 expression and secretion were similar at 1.2, 1.8 and 2.4 mmol/L NEFA-treated hepatocytes and significantly higher than those observed for controls (p < .01). These data suggest that high concentrations of NEFA significantly promote FGF21 expression and secretion in bovine hepatocytes. In particular, this promotion occurs in a dose-dependent manner and may be involved in the pathological processes of energy metabolism disorders of dairy cows in the peripartum period.

Effects of Fungicides and Spray Application Interval on Controlling Marssonina Blotch of Apple in the Loess Plateau Region of China.

Marssonina blotch, caused by the fungus Marssonina coronariae, is a serious foliar disease on apple in East Asia as well as in other moist temperate regions in Asia, Europe, and South America. Several fungicides were investigated for their toxicity to mycelial growth and conidial germination of the pathogen in vitro. Tebuconazole, kresoxim-methyl, hexaconazole, propiconazole, and a mixture of tebuconazole and benziothiazolinone sharply inhibited mycelial growth but had less effect on conidial germination. Field tests were conducted in a commercial orchard in Baishui County, Shaanxi Province, China, during 2012, 2013, 2014, and 2015 in order to develop recommendations for apple growers. Three applications of tebuconazole, hexaconazole, propiconazole, or a mixture of tebuconazole and benziothiazolinone at 20-day intervals from early July to late August resulted in defoliation incidence of <5%. When sprays of Bordeaux mixture + tebuconazole, Bordeaux mixture + propiconazole, and Bordeaux mixture + tebuconazole and benziothiazolinone were alternated, the spray interval was extended to 25 days and defoliation incidence remained <5%. Based on historical records and our results, scouting for symptoms should begin in mid-June. We recommend commencing the spray period in early July in years with normal rainfall patterns, and spraying in mid- to late June in years with much rainfall. The findings of this study create a foundation for implementation of an efficient spray program against Marssonina leaf blotch in apple orchards in the Loess Plateau Region of China.

Management of Valsa Canker on Apple with Adjustments to Potassium Nutrition.

Valsa canker, caused by the fungus Valsa mali, is one of the most destructive diseases of apple in the primary production areas of China and other East Asian countries. Currently, there are no effective control methods for this disease. We investigated the occurrence of Valsa canker in 24 apple orchards in Shaanxi Province in concert with foliar nutrient analysis, and found that there was a significant negative correlation of leaf potassium (K) content with incidence and severity of Valsa canker. Fertilization experiments showed that increasing tree K content enhanced resistance to pathogen colonization and establishment. Apple trees with leaf K content greater than 1.30% exhibited almost complete resistance to Valsa mali. Field trials demonstrated that increasing K fertilization could significantly reduce disease incidence. Improved management of tree nutrition, especially K content, could effectively control the occurrence and development of Valsa canker.

Association of the IGF-1 rs35767 and rs972936 polymorphisms with the risk of osteoporosis in a Chinese postmenopausal female population.

The aim of our study was to conduct a case-control study in a Chinese postmenopausal population to evaluate the roles of the IGF-1 rs35767 and rs972936 polymorphisms on bone mineral density (BMD) levels and osteoporosis risk. A total of 272 consecutive postmenopausal women with a primary diagnosis of osteoporosis and 272 controls were enrolled in the study between 2012 and 2014. The polymerase chain reaction-restriction fragment length polymorphism method was used to genotype the rs35767 and rs972936 IGF-1 polymorphisms. By comparing the demographic characteristics between patients and controls, patients with osteoporosis were found to be more likely to have a habit of alcohol drinking (P = 0.023). Furthermore, the BMD levels of the L1-L4 vertebrae, femoral necks, total hips, and trochanters in patients with osteoporosis were significantly lower than those in controls. By conditional regression analysis, we found that the IGF-1 rs2288377 and rs972936 gene polymorphisms were not associated with the risk of osteoporosis (P < 0.05). However, the CT+TT genotype of rs35767 and the AG+GG genotype of rs972936 were significantly associated with lower BMD levels in the femoral neck. Overall, our study suggests that IGF-1 rs2288377 and rs972936 gene polymorphisms do not influence the risk osteoporosis.

8-Chloro-methyl-5-(2,5-dioxooxolan-3-yl)-3,3a,4,5-tetra-hydro-1H-naphtho-[1,2-c]furan-1,3-dione.

The title compound, C17H13ClO6, is an asymmetric alicyclic dianhydride containing a chloro-methyl-substituted tetra-hydro-naphthalene moiety. The cyclo-hexene ring in the tetra-hydro-naphthalene moiety exhibits an envelope conformation with the tertiary C atom as the flap The dihedral angle between the two anhydride rings is 79.96 (6)°, while those between the benzene ring and the non-fused and fused anhydride rings are 71.03 (5) and 42.57 (7)°, respectively. In the crystal, mol-ecules are connected by weak C-H⋯O inter-actions, forming a three-dimensional supramolecular structure.

5-(2,5-Dioxooxolan-3-yl)-8-methyl-3,3a,4,5-tetra-hydro-1H-naphtho-[1,2-c]furan-1,3-dione.

In the title compound, C(17)H(14)O(6), the dihedral angle between the two anhydride rings is 76.01 (8)°while the dihedral angles between the benzene and anhydride rings are 42.60 (7) and 68.94 (7)°. The cyclo-hexene ring of the tetra-hydro-naphthalene unit exhibits an envelope conformation.

Evaluation of normal fetal pulmonary veins from the early second trimester by enhanced-flow (e-flow) echocardiography.

OBJECTIVES: To explore the feasibility of using enhanced-flow (e-flow) imaging technology to identify fetal pulmonary veins and establish gestational age-specific reference values at 12-40 weeks' gestation. METHODS: The pulmonary venous internal diameter, peak systolic and diastolic flow velocities and visualization rate were analyzed in 332 normal fetuses at 12-40 weeks of gestation. Two-dimensional gray-scale (2D) ultrasound, color Doppler and e-flow imaging were used to detect the pulmonary veins in the four-chamber view by taking the lung as the penetration window. RESULTS: The pulmonary veins could be visualized as early as 12 weeks' gestational age by e-flow imaging. The right and left pulmonary venous internal diameters and peak systolic and diastolic flow velocities increased with increasing gestational age. Between 12 and 40 gestational weeks, the internal diameter and both the systolic and diastolic flow velocities of the fetal right pulmonary vein were significantly larger than were those of the left vein (P < 0.05). Of 118 fetuses at 12-22 gestational weeks, the visualization rate of the four pulmonary veins (left superior and inferior, right superior and inferior) was 5.9% (7/118) by 2D ultrasound, 41.5% (49/118) by color Doppler and 61.9% (73/118) by e-flow imaging. The visualization rate by e-flow imaging was significantly higher than that using the other two techniques (P < 0.001). CONCLUSION: e-flow imaging is apparently a feasible and promising technology with which to identify the fetal pulmonary veins in the early stages of the second trimester.

Classifying G protein-coupled receptors and nuclear receptors on the basis of protein power spectrum from fast Fourier transform.

As the potential drug targets, G-protein coupled receptors (GPCRs) and nuclear receptors (NRs) are the focuses in pharmaceutical research. It is of great practical significance to develop an automated and reliable method to facilitate the identification of novel receptors. In this study, a method of fast Fourier transform-based support vector machine was proposed to classify GPCRs and NRs from the hydrophobicity of proteins. The models for all the GPCR families and NR subfamilies were trained and validated using jackknife test and the results thus obtained are quite promising. Meanwhile, the performance of the method was evaluated on GPCR and NR independent datasets with good performance. The good results indicate the applicability of the method. Two web servers implementing the prediction are available at http://chem.scu.edu.cn/blast/Pred-GPCR and http://chem.scu.edu.cn/blast/Pred-NR.

Comparative study of Scutellaria planipes and Scutellaria baicalensis.

Scutellaria planipes, a species of Scutellaria, was explored by comparing to Scutellaria baicalensis, a pharmacopoeia species. Four principle flavonoids in both the plant roots were analyzed by using a reversed-phase chromatographic system with a chemically bonded ODS silica gel column and phosphate buffer:methanol (68:32 and 1:1) as mobile phase. Their contents were similar in both plant roots. Antiallergic and antibacterial activities in vitro and acute toxicity were compared. The results provided valuable data for S. planipes as a potential medicinal resource.

Studies on the constituents of roots of Scutellaria planipes.

From the roots of Scutellaria planipes (L.), three new flavonoids, 5,7,3',6'-tetrahydroxy-6,8,2'-trimethoxyflavone, (2 R,3 R)-3,5,7,2',5'-pentahydroxyflavanone and 7,2'-dihydroxy-5-methoxyflavone were isolated, together with ten known flavonoids, skullcapflavone I, wogonin, baicalein, norwogonin, viscidulin III, 5,7,2',6'-tetrahydroxy-flavone, 3,5,7,2',6'-pentahydroxyflavone, wogonin 7- O-beta- D-glucuronide, baicalin, baicalein 7- O-beta- D-glucopyranoside, and three phenethyl alcohol glycosides, acteoside, martynoside, and jionoside D. The structures were elucidated by spectroscopic methods.

Technetium-99m N,N'-bis(2-mercapto-2-methylpropyl)-2-aminobenzylamine: technetium-99m complexes of a novel bis(aminoethanethiol) ligand.

A new N2S2 ligand system, N,N'-bis(2-mercapto-2-methylpropyl)-2- aminobenzylamine, U-BAT, 1, containing uneven amine groups (two amine groups with different pKa values) for complexing [TcVO]3+, was prepared. The reaction of this novel ligand with [99mTc]pertechnetate, in the presence of stannous tartrate as the reducing agent, produces the neutral and lipid-soluble [99mTc]TcVO(U-BAT), TcS2C15H23N2O, 2. However, when the same reaction was carried out at a higher pH, 9-10, and with 30 min of heating (100 degrees C), a second neutral but more lipid-soluble complex, [99mTc]TcVO(OU-BAT), TcS2C15H21N2O, 3, was isolated. The X-ray crystallography data of the 99Tc complexes show square pyramidal coordination with N2S2 as the base and the Tc = O in the apical position. Compound 3 can be derived from 2 by an oxidation of the ligand to form an imine. After iv injection into rats, the neutral and lipid-soluble technetium-99m complexes showed significant brain uptake, 1.54 and 1.07% dose/organ at 2 min for [99mTc]TcVO(U-BAT) and TcVO(OU-BAT), respectively. The novel Tc chemistry of this new ligand system may provide a useful foundation for designing Tc complexes with a built-in redox mechanism.

Localization of IBF as a D-2 dopamine receptor imaging agent in nonhuman primates.

Preliminary study of iodine-123 labeled IBF, (S)-5-iodo-7-N-[(1-ethyl-2-pyrrolidinyl)methyl] carboxamido-2, 3-dihydrobenzofuran, has demonstrated the potential of using this agent to evaluate the status of the CNS D-2 dopamine receptor in humans. To further characterize this ligand and evaluate single-photon emission tomography (SPET) quantitation, a detailed biodistribution study in monkeys (Macaca fascicularis) with 123I- and 125I-IBF was performed. The dual tracer was simultaneously injected for in vivo imaging, bio-distribution, and ex vivo autoradiography in the same monkey. After the injection, SPET data (10 min/frame x 15) were collected with a triple-head gamma camera. Dynamic imaging data indicated that IBF localized in basal ganglia (BG) with a half life of 90-120 min. Other regions, i.e., cerebellum (CB) and cortex (CX), showed very low uptake. At 2.2 h after the injection, the monkey was sacrificed. Organ distribution data indicated that, as expected, there was a significant uptake in basal ganglia (0.029% ID/g), and the BG/CB and BG/CX ratios were 17.8 and 14.2 respectively. Lower ratios were obtained from SPET image analysis (BG/CB = 3.5 at 2.5 h). The eye uptake was observed with SPET, but was only quantified on autoradiograms with significant uptake (0.017% ID/g). Autoradiography of the eye demonstrated that predominant uptake was localized in the ciliary body and the choroid. The selective retention and high BG/CB ratio of 123I-IBF make it a useful agent for in vivo D-2 dopamine receptor imaging with SPET.

A simplified method for quantitation of iodine-123 iodobenzamide in human plasma: a technical note.

To establish a quantitative single-photon emission tomography (SPET) procedure for imaging CNS D2 dopamine receptors, measurement of unchanged iodine-123 iodobenzamide (123I-IBZM) (a selective D2 ligand) in human plasma was investigated. There are three possible radioactive components in human plasma: hydrophilic compounds (iodide ion, etc.), lipophilic metabolites, and unchanged IBZM. Based on the difference in lipophilicity of IBZM and its lipophilic metabolites (LM), a new quantitative method of analysis of 123I-IBZM using a simple solvent extraction was developed. The selective extraction was achieved with n-octane/phosphate buffer (0.1 M, pH 7.4). Extraction efficiency was 93.2% +/- 0.3% (n = 15) for IBZM from plasma, while 99.3% +/- 0.2% (n = 12) of LM remained in the aqueous plasma fraction. Twenty-one confirmation tests with plasma containing known ratios of IBZM/LM, ranging between 0.39 and 7.60, were performed. The experimental results were very close to the values of the true ratios over the wide range (accuracy approximately 99%, relative standard error 6.6%). The data from this simplified method are comparable to those obtained by the high-performance liquid chromatography method. This improved method provides an easy and accurate way to quantify unchanged IBZM in human plasma. With appropriate kinetic modeling and in conjunction with a dedicated SPET imaging device for measuring quantitative information, it may be possible to develop a practical method for measuring D2 receptor density in vivo.

Dicarboxylate diamide dimercaptide (N2S2) technetium-99m complexes: synthesis and biological evaluation as potential renal radiopharmaceuticals.

Novel diamide dimercaptide (N2S2) ligands 4, 5, and 8 have been synthesized and evaluated as potential renal radiopharmaceuticals. The target compounds were prepared in modest overall yields of 22%, 19%, and 20%, respectively, using readily available starting materials. Following in situ deprotection, 99mTc complexes of high radiochemical purity were obtained in excellent yield and were found to be stable for up to 6 h. The 99Tc complex of ligand 8 was isolated as the AsPh4 salt. The X-ray crystallographic data for [99TcO(8)]AsPh4 (space group P2(1)/n: Z = 4, a = 9.342(3) A; b = 18.594(5) A; c = 18.417(7) A; beta, deg = 90.61(3); V, A3 = 3199.1(20)) show that the Tc is bound to both thiolate sulfur atoms and to two deprotonated amide nitrogen atoms. The coordination geometry about the Tc is square-pyramidal with an -yl oxygen atom in the apical position. The Tc-N bond distances (2.002(12) and 1.984(12) A), the Tc-S bond distances (2.300(5) and 2.286(5) A), and the Tc-O bond distance (1.667(11) A) are in good agreement with bond lengths reported for similar complexes. The carboxylate groups are not bonded to the Tc atom in the solid state, nor in CDCl3 solution, as evidenced by X-ray crystal data and solution NMR data, respectively. In the solid state, [99TcO(8)]AsPh4 is monoanionic, therefore, at physiological pH, [99mTcO(8)] is presumably trianionic. Biodistribution studies performed in rats with the 99mTc complexes revealed slow blood clearance and high muscle uptake for these agents. Modest hepatobiliary excretion was observed, and low quantities of the complexes were found in the heart, lungs, and spleen after 1 h. The urinary excretion of the 99mTc complexes of ligands 4, 5, and 8 was found to be slow when compared to the excretion of [131I]OIH in rats (22%, 22%, and 32% vs 85-86%, respectively). Protein binding of 99mTc complexes of ligands 4, 5, and 8 in both rat and monkey plasma was found to be similar to MAG3. While the synthetic schemes reported here supply facile routes to novel N2S2 ligands, biodistribution studies of the 99mTc complexes performed on rats revealed slow renal excretion rates, accompanied by slow blood clearance and high uptake in muscle tissue. Preliminary planar imaging studies in monkeys also revealed slow renal excretion for these agents. The 99mTc complexes evaluated here are poor candidates as renal radiopharmaceuticals.

[Identification and determination of marker substances in niuhuang jiedu pian by three dimensional HPLC].

The Chinese traditional patent medicine Niuhuang Jiedu Pian was analyzed by three dimensional HPLC. The results show that some of the Chinese herbal drugs contained in Niuhuang Jiedu Pian can be identified and the content of the marker substances can be determined. According to the results of determination, the content of baicalin, sennoside A and glycyrrhizin in Niuhuang Jiedu Pian was 4.21, 0.59 and 1.03 (mg/tablet), respectively. The RSD was 1.6, 2.8 and 1.4%, respectively.

[The identification and determination of marker substances in xiaoyao wan by three dimensional HPLC].

A Chinese traditional patent medicine Xiaoyao Wan was analysed by three dimensional HPLC. The result shows that the Chinese traditional medicines involved in Xiaoyao Wan were identified excellently and the content of marker substances can be determined accurately. According to the results of determination, the content of saikosaponin b1, b2, glycyrrhizin, paeoniflorin and atractylenolide III in Xiaoyao Wan was 0.43, 0.53, 5.22, 3.95 and 0.16 (mg/g), respectively; The RSD was 1.2, 0.8, 0.4, 0.3 and 1.0 (%) respectively.