New cytotoxic biflavones from Selaginella doederleinii.

Three new biflavones, apigenin-(3',8″)-chrysin (1), (2S)-2,3-Dihydroametoflavone 5,4'-dimethyl ether (2), and (2S)-5″,7″-Dihydroxy-2″-phenoxychromonyl-(4'″,3')-naringenin (3), together with seven known biflavones (4-10) were isolated from the 75% EtOH extract of Selaginella doederleinii. The structures of new compounds were established by application of spectroscopic methods, including 1D and 2D NMR, HRMS, and CD measurements. In addition, all new compounds were evaluated for their cytotoxic potential against three human cancer cell lines A549, MCF-7, and SMMC-7721 in vitro. Compound 2 exhibited potent cytotoxic activity with IC50 values ranging from 6.35 to 10.18 µM.

Palhinosides A-H: Flavone Glucosidic Truxinate Esters with Neuroprotective Activities from Palhinhaea cernua.

Palhinosides A-H (1-8), new flavone glucosidic truxinate esters, including ß-truxinate and µ-truxinate forms, were isolated from Palhinhaea cernua. Their structures were elucidated by extensive spectroscopic methods and chemical analyses. The flavone glucoside cyclodimers possess a unique cyclobutane ring in their carbon scaffolds. Compounds 2-7 represent three pairs of stereoisomers (2/3, 4/5, 6/7). The protective effects of 1-8 against the damage of HT-22 cells induced by l-glutamate were evaluated, and compounds 4 and 5 showed better neuroprotective effects than the positive control, Trolox.

Scavenger receptor class a, member 3 is associated with severity of hand, foot, and mouth disease in a case-control study.

Hand, foot, and mouth disease (HFMD) spreads rapidly and has been recognized as a public health problem in recent years in China. Unfortunately, there is no effective vaccine or antiviral drug currently for EV71 infection. In this study, we aim to identify biomarker which are associated with for severity of EV71 infection cases using high-throughput RNA sequencing approach.RNA sequencing of samples from severe HFMD (S) patients group (n = 10) and control HFMD (C) patients group (n = 10) were performed and the results were verified by qPCR. mRNA with the highest expression level was selected to be validated in an independent cohort comprising of 45 severe EV71 infected patients and 45 control by qPCR assay.Seventeen significant differentially expressed genes were identified. Scavenger receptor class A, member 3 (SCARA3) was one of the significantly upregulated genes with the highest expression level and was selected for validation. The mean relative expression levels in severe HFMD and control HFMD patients were 10.1-fold and 5.0-fold, respectively, P value <.001.We found that SCARA3 is associated with severity of HFMD, and it may be a potential prognostic marker to predict the HFMD progression in EV71 infected patients.

[Association between family environment and developmental coordination disorder in preschool children].

OBJECTIVE: To investigate the influence of family environment on developmental coordination disorder (DCD) in preschool children. METHODS: Stratified random cluster sampling was used to select 1 727 children (4-6 years old). The Movement Assessment Battery for Children was used to screen out the children with DCD. The Family Environment Scale on Motor Development for Preschool Urban Children and a self-designed questionnaire were used to assess family environment. RESULTS: A total of 117 children were confirmed with DCD. There were significant differences in mother's education level and family structure between the DCD and normal control groups. There were also significant differences in the scores of "Let children manage their daily items" and "Arrange all affairs" between the DCD and normal control groups. The multivariate logistic regression analysis indicated that when children's age and gender were controlled, mother's education level, family structure, "Let children manage their daily items", and "Arrange all affairs" were main factors influencing the development of DCD in children (P<0.05). CONCLUSIONS: Family environment may affect the development of DCD in preschool children. Therefore, parents should not arrange all affairs for children and should provide more opportunities for children to manage their daily life, in order to promote the development of early motor coordination and prevent the development of DCD.

A new androstanoid metabolite from a soil fungus Curvularia borreriae strain HS-FG-237.

A new androstanoid metabolite, 4α-methyl-9α-methoxyandrosta-8,15-diene-3,17-dione (1), was isolated from a soil fungus Curvularia borreriae (Pleosporaceae) strain HS-FG-237. Its structure was determined by extensive spectroscopic and X-ray diffraction analyses. Compound 1 exhibited poor cytotoxicity toward HCT-116 cells by CCK-8 assay and weak anti-inflammatory activity in an ANA-1 murine macrophages model.

[Regulatory Effect of Chinese Drugs for Stasis Removing and Collaterals Dredging on the Expres- sions of podocin and CD2AP in Podocyte Slit Diaphragm of Diabetic Nephropathy Rats].

OBJECTIVE: To observe the regulatory effect of Chinese drugs for stasis removing and collaterals dredging (CDSRCD) on the expressions of podocin and CD2AP in podocyte slit diaphragm (SD) of diabetic nephropathy (DN) rats. METHODS: DN rat model was duplicated in 40 male Sprague- Dawley rats by feeding high fat high glucose diet combined with intraperitoneally injecting 1 % streptozoto- cin (STZ, 35 mg/kg). Totally 36 successfully modeled rats were divided into the model group, the CD- SRCD group,- and the irbesartan group according to random digit table, 12 in each group. Besides, anoth- er 10 normal rats were recruited as a normal group. Rats in the CDSRCD group and the irbesartan group were intragastrically fed with CDSRCD and irbesartan respectively. Rats in the normal group and the mod- el group were fed with equal volume of distilled water at the same time. 24 h urine protein quantitation was detected using ELISA at various time points. Body weight (BW) , kidney weight ( KW), kidney index (KI) , fasting blood glucose (FBG) , serum creatinine (SCr), blood urea nitrogen (BUN), and uric acid (UA) in each group were detected after 16 weeks of intervention. The pathomorphological changes of re- nal tissue were observed under light microscope and electron microscope respectively. The protein and mRNA expressions of podocin and CD2AP were detected by Western blot and Real-time PCR respectively. RESULTS: (1) Compared with the normal group, 24 h urine protein quantitation significantly increased at week 4, 8, 12, and 16, respectively (P <0. 01). BW was decreased; KI and levels of FBG, SCr, BUN, and UA all increased after modeling (P <0. 01). Compared with the model group, 24 h urine protein quan- titation significantly decreased in the CDSRCD group and the irbesartan group at week 4, 8, 12, and 16, respectively (P <0. 01). Besides, it was more obviously reduced in the CDSRCD group than in the irbe- sartan group (P <0. 05, P <0.01). BUN level obviously decreased both in the CDSRCD group and the irbesartan group after modeling (P <0. 05, P <0. 01). (2) Results of renal pathology showed that disar- ranged renal structure, obviously thickened basement membrane, severely proliferated mesenteria, widely fused foot processes in the model group. All these pathological changes were attenuated in the CD- SRCD group and the irbesartan group to some degree. (3) Results of Western blot and Real-time PCR showed, compared with the normal group, protein and mRNA expressions of podocin and CD2AP decreased in the model group (P <0. 01). Compared with the model group, protein and mRNA expressions of podocin and CD2AP increased in the CDSRCD group and the irbesartan group (P <0. 01). Protein and mRNA expressions of podocin and CD2AP increased more in the CDSRCD group than in the irbesartan group (P <0. 05). CONCLUSIONS: CDSRCD could protect renal function by lowering urinary protein in DN rats, improve renal pathological changes. Its mechanism might be related to up-regulating mRNA and protein expressions of podocin and CD2AP.

[Design, synthesis and biological evaluation of novel [1,3] dioxolo [4,5-f]isoindolone derivatives].

A series of [1,3]dioxolo[4,5-f]isoindolone derivatives were designed, synthesized and evaluated as inhibitors of acetylcholinesterases (AChE). Furthermore, their effects on memory impairment of mice induced by scopolamine were investigated with step-through test. The results suggested that most of the target compounds exhibited potential inhibition on AChE with IC50 values at micromolar range. Compounds I1 (IC50 value of 0.086 µmol · L(-1)) and I2 (IC50 value of 0.080 µmol · L(-1)) showed the strongest AChE inhibitory activity, which are equipotent to donepezil (IC50 value of 0.094 µmol · L(-1)). Moreover, compounds I1-I4 could improve the memory impairment induced by scopolamine in mice.

Macrocyclic compounds as anti-cancer agents: design and synthesis of multi-acting inhibitors against HDAC, FLT3 and JAK2.

A novel series of macrocyclic compounds were designed and synthesized as multi-target inhibitors targeting HDAC, FLT3 and JAK2. Some of these compounds exhibited potent HDAC inhibition as well as FLT3 and JAK2 inhibition under both cell-free and cellular conditions. In vitro antiproliferative assay indicated that these compounds were interestingly more cytotoxic to MV4-11 cells bearing FLT3-ITD mutation and HEL cells bearing JAK2(V617F) mutation.

[Design, synthesis and biological evaluation of novel 4-substituted-3-nitrobenzamide derivatives].

A series of novel 4-substituted-3-nitrobenzamide derivatives were designed and synthesized. The structures of the target compounds were confirmed with 1H NMR, 13C NMR, MS and element analysis. Anti-tumor activities against HCT-116, MDA-MB435 and HL-60 cell lines in vitro were evaluated by SRB assay. The results indicated most of the target compounds exhibited potent anti-tumor activity. Compound 4a showed the most potent inhibitory activities against three cancer cell lines with the GI50 values of 1.904-2.111 micromol x L(-1). Compounds 4g, 41-4n exhibited more potent inhibitory activities against MDA-MB435 and HL-60 cell lines with the GI50 values of 1.008-3.586 micromol x L(-1) and 1.993-3.778 micromol x L(-1), respectively. The structure-activity relationship of these compounds is discussed preliminarily.

Mechanisms elevating ORMDL3 expression in recurrent wheeze patients: role of Ets-1, p300 and CREB.

The first genetic factor identified for childhood asthma by genome-wide association study (GWAS) is the locus on chromosome 17q21, harboring the Orosomucoid 1-like 3 (ORMDL3) gene. ORMDL3 is implicated in facilitation of endoplasmic reticulum-mediated inflammatory responses, believed to underlie its association with asthma. In the present study, we demonstrated that mRNA expression of ORMDL3 is significantly increased in the peripheral blood of recurrent wheeze patients compared with normal control subjects by real-time RT-PCR. To elucidate the molecular mechanisms involved in human ORMDL3 regulation, we cloned and characterized the promoter region of ORMDL3. Applying 5'-rapid amplification of cDNA end analysis (RACE), we revealed that ORMDL3 gene used multiple transcriptional start sites (TSSs). Using a series of 5' deletion promoter plasmids in luciferase reporter assays, we identified that the proximal minimal promoter of ORMDL3 was located within the region -84/+58 relative to the TSS. Mutational analysis, RNA interference experiments and sequential chromatin immunoprecipitation (ChIP) assay demonstrated that transcriptional activity of the ORMDL3 gene was cooperatively regulated by multiple transcription factors, including Ets-1, p300 and CREB. The expression levels of Ets-1, p300 and CREB were increased in the peripheral blood of recurrent wheeze patients compared with normal control subjects and showed a strong linear correlation with the expression of ORMDL3. Our findings indicate that Ets-1, p300 and CREB binding to the promoter region drive the ORMDL3 transcription.

Simultaneous detection of eight genetically modified maize lines using a combination of event- and construct-specific multiplex-PCR technique.

To fulfill labeling and traceability requirement of genetically modified (GM) maize for trade and regulation, it is essential to develop an event-specific detection method for monitoring the presence of transgenes. In pursuit of this purpose, we systematically optimized and established a combined event- and construct-specific multiplex polymerase chain reaction (mPCR) technique for simultaneous detection of 8 GM maize lines. Altogether 9 sets of primers were designed, including six that were event-specific for Event176, Bt11, TC1507, NK603, MON863, and Mon810; two that were construct-specific for T25 and GA21, and one for an endogenous zein gene. The transgene in each GM maize line and the endogenous zein gene could be clearly detected and distinguished according to the different sizes of PCR amplicons. The limit of detection (LOD) was approximately 0.25% (v/v), although the detection can be as sensitive as 0.1% as demonstrated by the International Seed Testing Association (ISTA) proficiency test. This study further improves the current PCR-based detection method for GM maize. The method can be used in an easy, sensitive, and cost and time effective way for the identification and quality screening of a specific GM maize line.

Functional characterization of a gene family encoding Polygalacturonases in Phytophthora parasitica.

Phytophthora parasitica is an oomycete plant pathogen that causes severe disease in a wide variety of plant species. In our previous study, we discovered a multigene family encoding endopolygalacturonases (endoPG) in Phytophthora parasitica. Here, we screened the genomic library of Phytophthora parasitica for the genes encoding endoPG named pppg2 through pppg10, and analyzed their functions. Results obtained by real-time quantitative reverse transcriptase-polymerase chain reaction demonstrated that some of these genes are highly induced during plant infection, which suggests their important roles in the pathogenesis of Phytophthora parasitica. Analysis by in-gel activity assay of recombinant proteins obtained from Pichia pastoris indicated that each of these genes encodes a functional endoPG. Investigation of the function of pppg genes in planta by a Potato virus X agroinfection system in tobacco revealed that each pppg caused specific effects, varying from no symptoms to dwarfism, necrosis, leaf curl, silvery leaf, and cracks in leaf stalks. Appearance of these effects depends on the expression of a pppg protein with a normal active site in the apoplast. These results indicated that each pppg plays a distinct role in the decomposition of plant cell wall.

[Experimental study and clinical application of early pedicle division of skin flap by ligation].

OBJECTIVE: To establish a new method of early cut-off skin flap by ligating to stegnosis pedicle and assess its feasibility and clinical application. METHODS: Twenty New Zealand rabbits were used to make skin flaps with the size of 8.0 cm x 4.0 cm on both sides of the back respectively. And one side was experimental group with the pedicles of skin flaps horizontally oversewn by several pairs of silk thread, the other side was control group. Two pairs of silk thread in the two sides of the pedicle of skin flap of experimental group were ligated on the 3rd day. On the 5th day the pedicle was wholly ligated. All pedicles were divided on the 6th day and the survival area of skin flaps were measured after 3 days. The tissue samples from the skin flaps were collected for histology test on the 4th and 6th day respectively. The pedicles of 78 random flaps from 48 patients were cut off after narrowing them by ligating. RESULTS: The mean flap survival rate of the experimental group was statistically higher than the control group. Histological examination results showed the density and diameter of blood vessel were increased in the skin flaps of the experimental group. The mean time for removal pedicles was shortened to 10 days, and no necrosis was found after resecting. CONCLUSIONS: This method is secure and convenient and the time of pedicle division can be shortened.

Expression of ascorbate peroxidase and glutathione reductase in roots of rice seedlings in response to NaCl and H2O2.

The accumulation of H2O2 by NaCl was observed in the roots of rice seedlings. Treatment with NaCl caused an increase in the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) and the expression of OsAPX and OsGR in rice roots. Exogenously applied H2O2 also enhanced the activities of APX and GR and the expression of OsAPX and OsGR in rice roots. The accumulation of H2O2 in rice roots in response to NaCl was inhibited by the NADPH oxidase inhibitors, diphenyleneiodonium chloride (DPI) and imidazole (IMD). However, DPI, IMD, and dimethylthiourea, a H2O2 trap, did not reduce NaCl-enhanced activities of APX and GR and expression of OsAPX and OsGR. It appears that H2O2 is not involved in the regulation of NaCl-induced APX and GR activities and OsAPX and OsGR expression in rice roots.

Production of two highly active bacterial phytases with broad pH optima in germinated transgenic rice seeds.

Phytate is the main storage form of phosphorus in many plant seeds, but phosphate bound in this form is not available to monogastric animals. Phytase, an enzyme that hydrolyzes phosphate from phytate, has the potential to enhance phosphorus availability in animal diets when engineered in rice seeds as a feed additive. Two genes, derived from a ruminal bacterium Selenomonas ruminantium (SrPf6) and Escherichia coli (appA), encoding highly active phytases were expressed in germinated transgenic rice seeds. Phytase expression was controlled by a germination inducible alpha-amylase gene (alphaAmy8) promoter, and extracellular phytase secretion directed by an betaAmy8 signal peptide sequence. The two phytases were expressed in germinated transgenic rice seeds transiently and in a temporally controlled and tissue-specific manner. No adverse effect on plant development or seed formation was observed. Up to 0.6 and 1.4 U of phytase activity per mg of total extracted cellular proteins were obtained in germinated transgenic rice seeds expressing appA and SrPf6 phytases, respectively, which represent 46-60 times of phytase activities compared to the non-transformant. The appA and SrPf6 phytases produced in germinated transgenic rice seeds had high activity over broad pH ranges of 3.0-5.5 and 2.0-6.0, respectively. Phytase levels and inheritance of transgenes in one highly expressing plant were stable over four generations. Germinated transgenic rice seeds, which produce a highly active recombinant phytase and are rich in hydrolytic enzymes, nutrients and minerals, could potentially be an ideal feed additive for improving the phytate-phosphorus digestibility in monogastric animals.