Objective: To investigate the effect of RNA interference (RNAi) targeting AKT1 and PI3K P85 on the proliferation and invasion of breast carcinoma MCF-7 cells. Methods: The recombinant adenovirus expression vector, which contained short hairpin RNA (shRNA) targeting open reading frames of AKT1 and PI3K P85 (rAd5-siAKT1-siPI3K), was transfected into human breast carcinoma MCF-7 cells. AKT1 and PI3K P85 mRNA and protein expressions were detected by real-time PCR and Western blotting analysis. The expressions of PCNA, cyclinD1, and P53 were also detected by Western blotting analysis. The proliferation and apoptosis of MCF-7 cells were measured by MTT, flow cytometry and 2-dementinal and 3-dementional matrigel assay. Results: Recombinant adenovirus vector rAd5-siAKT1-siPI3K dramatically down-regulated AKT1 and PI3K P85 mRNA and protein expressions in MCF-7 cells; the downstream factors PCNA and cyclin D1 were also down-regulated, while P53 was up-regulated. Growth of MCF-7 cells was inhibited by over 50% in rAd5-siAKT1-siPI3K group as measured by MTT assay, and cell cycle was arrested in G_1/G_0 phase compared with untransfected and rAd5-siCtrl transfected groups. Cell growth on matrigel matrix showed normal cell shapes, while the cells in rAd5-siAKT1-siPI3K transfected group were detached from the matrix or grew in scattered clustering patterns, forming only small aggregates. Conclusion: shRNA targeting AKT1 and PI3K P85 can significantly down-regulate the expression of AKT1 and PI3K P85 in breast carcinoma MCF-7 cells, and inhibit the growth of MCF-7 cells in vitro.

Objective: To study the effect of and possible mechanism of knockinng down PI3Kp85α using siRNA in MCF-7 human breast cancer cell line. Methods: Oligofectamine was used to transfect PI3Kp85α siRNA to knock down the PI3Kp85α expression level in MCF-7 human breast cancer cell line in vitro. Real-time PCR was conducted to detect the expression of PI3Kp85α. The effect of PI3Kp85αsiRNA on the growth of MCF-7 cells was measured by MTT. The cell cycle distribution and cell apoptosis were detected by cell flow cytometry. Protein expression was evaluated by immunofluorescence staining and Western blot. Results: The expression of PI3Kp85 α was knocked down with PI3Kp85α siRNA in MCF-7 cells. Cell growth was delayed in PI3Kp85αsiRNA-treated group. Conclusion: The suppressive effect of PI3Kp85αsiRNA on the growth of MCF-7 human breast cancer cell line is significant and PI3Kp85α could be a candidate for gene therapy for breast cancer.

It has gradually become a consensus in the industry that the traditional Chinese medicine gypsum should be decocted first, but the understanding of decocting method is not completely unified in the works of doctors since ancient times, and there are occasional disputes about whether it is necessary to decocting first. In this study, the phase determination, physical and chemical characterization, qualitative and quantitative analysis of inorganic and organic components of the decoctions of herbal pairs and the whole prescription Maxingshigan decoction with gypsum as the center, and the pre-decoctions and co-decoctions of them were carried out to explore the scientific connotation of the pre-decoctions of gypsum. Results show that decoction phases were different between the co-decoctions and pre-decoctions of licorice-gypsum (Gancao-Shigao, GC-SG), ephedra-gypsum (Mahuang-Shigao, MH-SG) and almond-gypsum (Xingren-Shigao, XR-SG). The results of the micromorphology, particle size and zeta potential of herbal pairs and prescription (Quanfang, QF) showed that the supramolecular particles in pre-decoctions were smaller, more uniform and more stable than the co-decoctions. The results of organic components analysis showed that different cooking methods did not change the organic composition and content. ICP-OES results showed that the content of inorganic components in pre-decoctions was higher than in co-decoctions for the same boiling time of gypsum. The IR results showed that the pre-decoctions had stronger chemical functional group effect than the co-decoctions. To sum up, compared with the co-decoction, the pre-decoction of gypsum has different phase state and chemical composition interaction, and the difference of inorganic composition is an important material basis affecting the change of phase state compared with the co-decoction. It indicates that the material basis of traditional Chinese medicine decoction is indeed different whether gypsum is decocted first or not, which can provide a basis for the clinical application of decocted gypsum.

OBJECTIVETo investigate the mechanism of chrysin in regulating lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells.

METHODSRAW264.7 cells were treated with different concentrations (0, 5, 10, 20, 40, 60, 80, 100, 150, and 200 µg/mL) of chrysin for 24 h, and the cell viability was measured using CCK-8. RAW264.7 cells were pre-treated with 10, 30, or 60 µg/mL chrysin for 2 h before stimulation with LPS for different times. The levels of TNF-α, IL-6 and MCP-1 were detected by ELISA, and Western blotting was used to detect the phosphorylation of JAK- 1, JAK-2, STAT-1 and STAT-3. The level of reactive oxygen species in RAW264.7 cells was detected by CM-H2DCFDA fluorescence probe. The effect of ROS on LPS-induced JAK-STATs signal and the inflammatory response of RAW264.7 cells was detected by ROS scavenger NAC. The transcription factors STAT-1 and STAT-3 nuclear translocation were observed by laser confocal microscopy.

RESULTSChrysin below 60 µg/mL did not significantly affect the viability of RAW264.7 cells. At 10, 30, and 60 µg/mL, chrysin dose-dependently inhibited the expression of iNOS induced by LPS. Chrysin treatment also inhibited LPS-induced phosphorylation of JAK-STATs, nuclear translocation of STAT1 and STAT3, release of TNF-α, IL-6 and MCP-1, and the production of ROS in RAW264.7 cells; ROS acted as an upstream signal to mediate the activation of JAK-STATs signaling pathway.

CONCLUSIONChrysin blocks the activity of JAK-STATs mediated by ROS to inhibit LPS-induced inflammatory response in RAW264.7 cells.

OBJECTIVETo explore the effects of slenderstyle acanthopanax root-bark on cyclooxygenase in vivo and in vitro.

METHODContents of 6-keto-PGF1alpha in bovine aorta endothelial cells, PGE2 in mouse abdominal macrophages, and 6-keto-PGF1alpha in rat stomach tissue were determined. The ulcer index in rat gastric mucosa was measured.

RESULTCOX-1 and COX-2 were inhibited by slenderstyle acanthopanax root-bark, and the inhibitive rate of COX-2 was higher than that of COX-1 at the same concentration. Necrotic injuries in health gastric mucosa were not produced, but the injuries previously induced by the ethanol worsened.

CONCLUSIONOne of the antirheumatic mechanisms of slender-root-bark might probably be mediating through inhibition of cyclooxygenase. style acanthopanax

6-Ketoprostaglandin F1 alpha ; metabolism ; Animals ; Aorta ; enzymology ; Cattle ; Cyclooxygenase 1 ; metabolism ; Cyclooxygenase 2 ; metabolism ; Dinoprostone ; metabolism ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Eleutherococcus ; chemistry ; Endothelial Cells ; enzymology ; Female ; Gastric Mucosa ; enzymology ; Macrophages, Peritoneal ; enzymology ; Male ; Mice ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Rats ; Rats, Sprague-Dawley

Aim To explore the novel anti-fatigue a-gents targeting AMPA receptor. Methods Three ben-zoic acids of different methoxy substitutions were em-ployed, including piperonylic acid, 3,4,5-trimethoxy benzoic acid and 3,4-dimethoxy benzoic acid, which as mother nucleus and cyclic aliphatic amine or pheny-lpiperazine derivatives were introduced to modify the nitrogen atoms connecting to amino bonds. Forty-three compounds were synthesized and identified by 1 H-NMR. MTT assay, then pentobarbital induced hypno-sis experiment and mice burden swimming experiment were applied respectively to evaluate the new synthesized compounds’ cytotoxicity, CNS excitability and anti-fatigue activity. Results Compound 2j had low cytotoxicity,presenting certain central excitability and significant advantages on anti-fatigue. Conclusion The further development of compounds 2j with good anti-fatigue activities could be cultivated in further study.

Objective To observe the effect of intracoronary transfer of autologous HO-1 overexpressed MSCs in porcine model of myocardial ischemia ( 1 h)/reperfusion. Methods Apoptosis was assayed and cytokine concentrations in supernatant were measured in cells exposed to hypoxia-reoxygen in vitro. In vivo, Chinese male mini-pigs were allocated to the following treatment groups; control group (saline), MSCs group (MSCs), MSCs transfected with pcDNA3. 1-nHO-l (HO-1-MSCs). 1 x 107 of autologous stem cells or identical volume of saline was injected intracoronary into porcine hearts 1 h after ischemia. MRI assay and postmortem analysis were assessed 3 months after stem cell transplantation. Results In vitro, cell apoptosis rate post hypoxia-reoxygen was significantly reduced in HO-1- MSCs group ( 30. 30% ±7. 64% ) compared with that in MSCs group (56. 93% ±4. 68% , P <0. 001) and LacZ- MSCs group (55. 88% ± 4. 38% , P < 0. 001) , VEGF was also significantly upregulated in HO-1-MSCs group [(768.44±78.38)pg/ml] compared with that in MSCs group[ (555. 27 ±67. 67)pg/ml, P<0.001] and LacZ-MSCs group [ (522. 97 ± 71. 45 ) pg/ml, P < 0. 001 ] . In vivo, cardiac function was significantly improved in both MSCs transplantation groups compared to saline group (all P<0. 05 vs. saline) and the left ventricular ejection fraction was significantly higher in HO-1-MSCs group compared with that in MSCs group at 3 months after transplantation ( 53. 50% ± 2. 09% vs. 49. 54% ± 2. 74% , P = 0. 017 ), capillary density in the peri-infarct area was also significantly higher in HO-1-MSC group than that in MSCs group [ (14.59 ± 2. 39 )/HPF vs. (11.78 ± 2.48 )/HPF, P = 0.033 ] . Conclusions Efficacy of HO-1 overexpressed MSCs on improving cardiac function and promoting angiogenesis was greater than those by MSCs in this porcine ischemia/reperfusion model.

Adolescent ; Adult ; Aged ; Emergency Treatment ; Female ; Humans ; Injury Severity Score ; Male ; Middle Aged ; Multiple Trauma ; diagnosis ; therapy

OBJECTIVETo investigate the epidemiological features, genetic drift in the epitopes of hemagglutinin (HA) of the novel influenza A (H1N1) virus and oseltamivir-resistant variants characterized by H275Y and N295S mutations in children in Shanghai since the outbreak.

METHODBetween June 2009 and May 2012, a prospective surveillance study was carried out in Shanghainese children who attended the outpatient clinic of Children's Hospital of Fudan University for influenza-like illness. One-step real-time fluorescence quantitative RT-PCR was performed to detect seasonal influenza A and influenza B virus and the novel influenza A (H1N1) virus in the respiratory samples. Genetic drift from the vaccine strain in HA epitopes of the novel influenza H1N1 virus and the molecular markers associated with oseltamivir resistance in neuraminidase (NA) were analyzed.

RESULTOut of 3475 enrolled cases, the novel influenza A (H1N1) virus was confirmed virologically in 222 (6.4%) otherwise healthy children with 133 (59.9%) being boys and 89 (40.1%) girls. The median ages of children with the novel influenza A (H1N1) virus infection during the first wave from August 2009 to February 2010 and the second wave from December 2010 to February 2011 were 53.5 months and 32.0 months, respectively (Z = -4.601, P = 0.000); 119 (46.9%) had the close contact with persons suffering from fever or respiratory infection, of whom, 68 (57.1%) contacts were family members and 47 (39.5%) contacts were classmates. During the outbreak in 2009-2010 season, 66 (40.9%) were exposed to primary index cases, school students were the major exposure subjects, accounting for 50.0%. The nucleotide sequences of HA1 gene were highly homologous between the vaccine strain A/California/07/2009 and Shanghai circulating novel influenza A (H1N1) strains and only S83P mutation in epitope E of HA was detected inclusively in the circulating strains. The H275Y and N295S amino acid mutations associated with oseltamivir resistance were not found in the circulating novel influenza (H1N1) strains.

CONCLUSIONTwo major waves of the novel influenza A (H1N1) outbreaks occurred in Shanghainese children during 2009-2011. Institutional children were the major affected individuals during the 2009 pandemic wave. Households and schools were the main sites of transmission among children during influenza pandemic. Influenza vaccination should be enhanced in children and their close family contacts. The novel influenza A (H1N1) virus in Shanghai has not undergone significant genetic changes. Oseltamivir is effective for the treatment of the novel influenza A (H1N1) virus.

Adolescent ; Amino Acid Sequence ; Antiviral Agents ; pharmacology ; Child ; Child, Preschool ; China ; epidemiology ; Drug Resistance, Viral ; Female ; Hemagglutinins, Viral ; genetics ; Humans ; Infant ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; drug therapy ; epidemiology ; pathology ; virology ; Male ; Molecular Epidemiology ; Molecular Sequence Data ; Neuraminidase ; genetics ; Oseltamivir ; pharmacology ; Pandemics ; Viral Vaccines ; genetics ; immunology

OBJECTIVETo identify potential mutation responsible for synpolydactyly (SPD) in a large Chinese kindred and to offer genetic counseling and prenatal diagnosis for the members of the family.

METHODSAll family members were examined clinically, and blood samples were obtained for linkage analysis and mutation screening. Ultrasound examinations were conducted at 16-21 weeks. Amniotic fluid sample was obtained by ultrasound-guided amniocentesis at 18 weeks of gestation.

RESULTSA large kindred affected with SPD was identified and characterized. With two short tandem repeat (STR) markers (D2S1238 and D2S1245) flanking the HOXD13 gene, the disease was mapped to 2q31. A heterozygous 27 bp expansion within the imperfect GCN triplet-repeat of exon 1, c. 184_210dup, was identified. The mutation resulted in a gain of 9 alanine residues between the 14th and 15th alanine of the normal 15-amino-acid-long polyalanine tract. On ultrasound examination, all fingers and toes of the fetus appeared to be normal. Linkage analysis and mutation detection confirmed that the fetus did not inherit the mutant allele from his affected mother.

CONCLUSIONHOXD13 gene mutation was responsible for the SPD phenotype in this family. Accurate prenatal diagnosis of SPD was achieved with combined ultrasound and molecular analysis.

Adolescent ; Adult ; Base Sequence ; China ; DNA Mutational Analysis ; Female ; Fingers ; abnormalities ; Genetic Linkage ; Homeodomain Proteins ; genetics ; Humans ; Male ; Middle Aged ; Pedigree ; Pregnancy ; Syndactyly ; diagnosis ; genetics ; Toes ; abnormalities ; Transcription Factors ; genetics ; Ultrasonography, Prenatal ; Young Adult