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ObjectiveTo investigate the effect of different oxygen concentration on the proliferation and autophagy of colon cancer cells and to explore the effect of Yangyin Huayu Jiedu Preseription （YHJP） on autophagy and apoptosis of colon cancer cells under hypoxia based on phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） signaling pathway. MethodHCT-116 cells were divided into normoxia group， 1% O2 group， and 5% O2 group. Cell viability was detected by cell proliferation assay （MTS）， and autophagy was observed based on monodansylcadaverine （MDC） staining. HCT-116 cells were treated with YHJP in 5% O2 microenvironment. The cells were divided into normal group， blank serum group， and low-， medium-， high-dose YHJP groups （5%， 15%， 25% serum containing YHJP）. Cell inhibition rate in each group was calculated by MTS， and changes in the rate of autophagy were detected based on MDC staining. Annexin V-fluorescein isothiocyanate （FITC）/propidium iodide （PI） was employed to detect the apoptosis rate of each group. Western blotting was applied to measure the expression of autophagy proteins microtubule-associated protein 1 light chain 3 （LC3Ⅱ/Ⅰ）， yeast Atg6 homolog （Beclin-1）， ubiquitin-binding scaffold protein p62 （p62）， apoptosis-related proteins B-cell lymphoma-2 （Bcl-2）， Bcl-2/adenovirus E1B interacting protein 3 （BNIP-3）， and Bcl-2 associated X protein （Bax）， cleaved cysteine-aspartic acid protease-3 （Caspase-3）， hypoxia-inducible factor-1α （HIF-1α） and pathway proteins PI3K， phosphorylated （p）-PI3K， Akt， and p-Akt. ResultCell survival rates of the 1% O2 and 5% O2 groups were increased compared with that in the normoxia group， particularly the 5% O2 group （P<0.01）. The fluorescence intensity for autophagy in 1% O2 and 5% O2 groups was significantly increased compared with that in the normoxia group， especially the 5% O2 group. In the presence of 5% O2， compared with the blank serum group， medium-dose and high-dose YHJP groups showed high cell inhibition rate， low autophagy rate， high apoptosis rate （P<0.01）， and low expression of Beclin-1 protein （P<0.05）. Compared with low-dose YHJP group， high-dose YHJP group demonstrated low expression of Beclin-1 protein （P<0.05）. Compared with the blank serum group， the three YHJP groups had low expression of LC3Ⅱ/Ⅰ protein （P<0.05， P<0.01）. Compared with the blank serum group， medium-dose and high-dose YHJP groups showed high expression of p62 protein （P<0.01）. Compared with low-dose YHJP group， high-dose YHJP group showed high expression of p62 protein （P<0.05）. Compared with the blank serum group， high-dose YHJP increased the expression of BNIP-3 and Bax and decreased the expression of Bcl-2 （P<0.01）. The expression of Bax protein in the high-dose YHJP group was increased compared with that in the low-dose YHJP group （P<0.05）. The expression of HIF-1α in the medium-dose and high-dose YHJP groups was decreased （P<0.01） and the expression of p-PI3K/PI3K and p-Akt/Akt in the high-dose YHJP group was increased （P<0.05， P<0.01） compared with that in the blank serum group. The expression of p-Akt/Akt was higher in the high-dose YHJP group than in the medium-dose YHJP （P<0.05）. ConclusionHypoxic microenvironment can significantly promote autophagy and proliferation of colon cancer cells. YHJP can significantly inhibit autophagy and proliferation and promote apoptosis of colon cancer cells in 5% O2 environment by up-regulating the PI3K/Akt signaling pathway.
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Yin deficiency is one of the main pathogenesis of traditional Chinese medicine (TCM), and it is also the basicpathogenesis of many clinical syndromes. Discrimination of pathogenesis is the key to effective treatment based onsyndrome differentiation. This paper will discuss the basic research and clinical research on the establishment of theanimal model of yin deficiency, the detection of physiological and biochemical indexes, yin deficiency constitution andTCM in the prevention and treatment of yin deficiency to provide data to clarify the material basis of yin deficiency andlay the foundation for the standardization and unification of the syndrome differentiation and treatment formulation of clinical TCM.
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The emission of hydrogen sulfide in the waste gas from slaughter plant, fishmeal feed processing and some other food industrial processing could cause serious air pollution to the surrounding environment. The purpose of this study was to screen heterotrophic bacterium strains for the removal of hydrogen sulfide odor. One heterotrophic bacterial mutant ZJNB-B3 was derived from the sulfide degrader Bacillus cereus XJ-2 and its sulfide removal efficiency was 97%. Based on the morphology studies, biochemical tests and 16S rRNA gene analysis, the strain was identified as Bacillus cereus ZJNB-B3. The NCBI GenBank accession number is MF679650. Batch tests showed that the strain tolerated up to 300 mg/L of toxic S²⁻ concentration. Response surface methodology was applied to optimize the conditions of degradation of sulfide. The optimal parameters were as follows: initial sulfide concentration 211.8 mg/L, initial pH 6.72, inoculum volume 5.04%, and incubation temperature 30 ℃. The accumulated sulfate concentration was 63.8 mg/L and the sulfide removal efficiency was 97.3% after 48 h incubation. No sulfuric acid was generated during sulfide oxidation by the strain. Sulfide could be removed effectively by this strain under mild pH conditions. The results suggested that the strain may have great industrial application potential. This study provides the fundamentals for the removal of hydrogen sulfide gas.
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The essence of thinking was the procedure for processing information by knowledge in the frame of world view,which depending on the world view and the existing knowledge and information.The characteristic of traditional Chinese medicine (TCM) thinking was reflected by its unique world view.The world view of TCM thinking was natural view,holism concept and dialectic view,compared with modern scientific thinking mostly in reduction analysis.To develop TCM,the modern TCM thinking system was established through the reforming and innovating of TCM thinking characteristic based on preservation and promotion of them.
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This study aimed at investigating the dose-effect relationship of SFI between the blood viscosity and the early-and mid-stage cardiogenic shock and the mediatory effect on rats.The end or root of left anterior descending coronary arteries (LADCA) was ligatured to establish the rat model of the early-and mid-stage cardiogenic shock.The blood viscosity indexes included low shear rate (LSR,10/s),middle shear rate (MSR,60/s),high shear rate (HSR,150/s) of the whole blood viscosity and plasma viscosity (PV),being observed 60 mins after the venous administration of 0.10,0.33,1.00,3.30,10.00 and 20.00 mL·kg-1 SFI (low dosage range:0.1-1.0 mL·kg-1,middle dosage range:1.0-10 mL·kg-1,high dosage range:10-20 mL·kg-1) with a blood rheometer.Dose-response curves were fitted by GraphPad Prism 6.0 software,the dose-response relationship of SFI between the blood viscosity and the early-and mid-stage cardiogenic shock in rats was evaluated to calculate the dose threshold parameters of the indexes.It was found that the blood viscosity indexes were improved with the dosage of 10 mL·kg-1 SFI in rats with the early-stage cardiogenic shock,while the dose-response curves of LSR,MSR and HSR at the early stage all presented favorable s shapes.Most of the effective dose range [D]2o-[D]80 and the threshold dose [D]20 were between 3.3 and 6.3 mL· kg-1.The four indexes of blood viscosity were improved with the administration of 10 and 20 mL·kg-1 SFI in mid-stage model rats with favorable s shapes in the dose-response curves.Most of the effective dose range and the threshold dose were in the range of 3.3 to 10.0 mL·kg-1.In conclusion,most of the dose-response curves of blood viscosity indexes in early-and mid-stage cardiogenic shock rats presented favorable s shapes with the threshold dose between 3.3 and 10.0 mL·kg-1,indicating an effective middle dosage range,which was converted into clinical dosage about 37.1 to 112 mL each day.The research provided an experimental basis for clinical medication.
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The effects of fucoidan on the lipidomics profiling of juvenile yellow catfish (pelteobagrus fulvidraco) during different feeding time (week 1, week 2, week 3 and week 8) were investigated by ultra-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry (UPLC-Q-TOF-MS) combined with multivariate data analysis, e.g.principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA).Based on VIP and p values, 11 lipid biomarkers were screened including lysophosphatidylcholine (Lyso-PC) 16∶0, phosphatidylcholine (PC) 22∶6/16∶0, phosphatidylethanolamine (PE) 22∶6/16∶0, phosphatidylinositol (PI) 18∶0/22∶6, diacylglycerol (DAG) 16∶0/16∶0,DAG 16∶0/18∶1, DAG 18∶1/18∶1, triglycerides (TAG) 20∶5/16∶0/18∶3, TAG 20∶4/16∶1/18∶2, TAG 16∶0/18∶2/20∶5 and TAG 18∶2/14∶0/18∶1.It was found that Lyso-PC, PC, PE and PI had the largest levels at the eighth week;the levels of DAG were decreased at the second and fourth weeks, and increased at the eighth week.While TAG was different: the content of TAG 18∶2/14∶0/18∶1, TAG 20∶5/16∶0/18∶3 and TAG 20∶4/16∶1/18∶2 increased in response to fucoidan, but TAG 16∶0/18∶2/20∶5 decreased little.Therefore, for juvenile yellow catfish, fucoidan can affect its lipid metabolism, which provides a theoretical basis for investigation of the influence of fucoidan on the response mechanism of lipid metabolism of juvenile yellow catfish.
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The free radical scavenging effect of flaxseed was screened by HPLC-DPPH ( 2 , 2-diphenyl-1-picrylhydrazyl-high performance liquid chromatography assay ) and colorimetric DPPH methods. To test the effectiveness of the approach, three Lignans ( secoisolariciresinol diglucoside ( SDG ) , secoisolariciresinol ( SECO) and enterodiol( ED) ) with antioxidative properties were investigated both in monomer and mixture. HPLC conditions were optimized using following methods: Waters XBridge C18 was used as stationary phase, acetonil/H2 O was used as mobile phase and detective wavelength was set at 280 nm. Antioxidant activity of standards was investigated by reaction with or without DPPH radical for 20 min as sample and control, respectively. Both of them were analyzed by high performance liquid chromatography. According to the changes of amount of sample and control, the antioxidant activities of standards were calculated as following order:SDG>SECO>ED. Based on above DPPH-HPLC assay and UPLC-Q-TOF-MS, antioxidants extracted from flaxseed were separated, identified and screened. The radical scavenging activities were in the following order:SDG isomer (5)>SDG (4)>7α-[(β-D-glupyranosyl) oxy]-1-methoxyisolariciresinol (1)>(6R,7R, 8S)-1-methoxyisolariciresinol (2)>herbacetindiglucoside (3). It indicated that the HPLC-DPPH assay could be successfully used for the antioxidant activity screening of complex flaxseed extract.
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Cell therapy has achieved tremendous success in regenerative medicine in the past several decades. However, challenges such as cell loss, death and immune-rejection after transplantation still persist. Biomaterials have been designed as carriers to deliver cells to desirable region for local tissue regeneration; as barriers to protect transplanted cells from host immune attack; or as reactors to stimulate host cell recruitment, homing and differentiation. With the assistance of biomaterials, improvement in treatment efficiency has been demonstrated in numerous animal models of degenerative diseases compared with routine free cell-based therapy. Emerging clinical applications of biomaterial assisted cell therapies further highlight their great promise in regenerative therapy and even cure for complex diseases, which have been failed to realize by conventional therapeutic approaches.
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Animals , Humans , Biocompatible Materials , Chemistry , Pharmacology , Bioreactors , Cell- and Tissue-Based Therapy , Methods , Drug Carriers , Chemistry , Pharmacology , Regenerative Medicine , MethodsABSTRACT
Objective To explore the relationship among the left ventricular rotation and twist, Tei index and left ventricular function in chronic heart failure patients and provide reliable indicator of chronic heart left ventricular myocardial function and mechanical movement. Methods Seventy-nine chronic heart failure patients with cardiac function classⅡ(32 cases), cardiac function classⅢ(26 cases) and cardiac function classⅣ(21 cases) and 28 healthy volunteer were included. All subjects underwent conventional echocardiography and two dimensional-speckle tracking imaging. The mitral annulus tissue Doppler spectrums at apical four chambers view, two-dimensional images of left ventricle at basal and apical level were acquired. The left ventricular twist, segmental rotation at short axis view and Tei index were measured. The differences were compared and the correlations were analyzed. Results (1) There were signiifcant differences for Tei index among the four group (χ2=88.31, P<0.01). The Tei index of healthy control group and chronic heart failure patients with cardiac function class Ⅱ , class Ⅲand class Ⅳwere 0.33±0.19, 0.37±0.18, 0.46±0.31 and 0.49±0.33. The Tei index gradually increased when the heart function decreased. (2) The left ventricular twist were also signiifcantly different among the four groups (F=25.99, P<0.01). The left ventricular twist of healthy control group and chronic heart failure patients with cardiac function classⅡ, classⅢ and classⅣwere (12.89±2.65)°, (12.29±1.94)°, (12.38±2.13)° and (8.46±2.90)°. The left ventricular twist gradually decreased when the heart function decreased. The differences between chronic heart failure patients with cardiac function classⅣand healthy control group, chronic heart failure patients with cardiac function classⅡand classⅢwere signiifcant (q=6.43, 5.71 and 4.17, all P<0.05). (3) The left ventricular segmental rotations at basal level were significantly different among healthy control group and chronic heart failure patients with cardiac function classⅡ,ⅢandⅣ(F=9.51, 9.47, 7.41, 10.27, 9.42 and 11.34, all P<0.01 ). The left ventricular segmental rotations were positively correlated with Tei index and the segmental rotation of anterior wall was most correlated with Tei index (r=0.327, 0.277, 0.266, 0.321, 0.306 and 0.325, all P<0.01). (4) The left ventricular segmental rotations at apical level were signiifcantly different among four groups (F=6.17, 3.49, 3.46, 3.50, 3.48 and 2.81, all P<0.01). The left ventricular segmental rotations were negatively correlated with Tei index and the segmental rotation of anterior wall was most correlated with Tei index (r=-0.362,-0.278,-0.251,-0.279,-0.290 and-0.288, all P<0.01). (5) The left ventricular twist and Tei index were negatively correlated (r=-0.443, P<0.01). Conclusions The left ventricular twist and segmental rotation at short axis view in patients with chronic heart failure decreased and Tei index gradually increased. The left ventricular twist and Tei index were negatively correlated. The combination of two-dimensional speckle tracking technology and Tei index could achieve noninvasive assessment of left ventricular mechanical movement and left heart function in chronic heart failure patients.
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[Objective] To investigate combined application of multiplex reverse transcription-polymerase chain reaction(mRT-PCR)and karyotype analysis detect of clonal chromosomal aberrations in acute lymphoblasfic leukemia (ALL),and explore the expression of common fusion genes.Methods 189 ALL patients were examined by multiplex RT-PCR and R or G banding techniques.[Results]10 fusion genes were detected in 69 out of 189 ALL patients(36.5％),including E2A/PBX1,TEL/AML1,BCR/ABL,MLL/AF4,MLL/AF6、MLL/AF9,MLL/AF10,MLL/ELL,SIL/TAL1,TLS/ERG.R or G banding techniques could find chromosome structural and numeracy abnormalities in 86 out of 152 patients (56.6％) available for analysis.Combination of mRT-PCR and R or G banding could raise the rate of detecting clonal chromosomal abnormalities to 69.3％.Fusion genes were detected in 33 out of 90 (36.7％) patients with adult ALL and 36out of 99 (36.4 ％) patients with children ALL,there were 22 patients with positive BCR/ABL but no TEL/AML1 in adult ALL group,while there were 24 patients with positive TEL/AML.1 and 2 with positive BCR/ABL in children ALL group.There was significant statistical difference for the expression of RCR/ABL and TEL/AML1 between adult ALL and children ALL (P＜0.01),but no difference for MLL related fusion gene,E2A/PBX1,SIL/TAL1 and TLS/ERG(P＞0.05).BCR/ABL and TEL/AML1 fusion gene could be detected in 66 ALL patients with normal karyotype (36.3％).[Conclusion]There were different biological characteristics between adults and children with ALL.mRT-PCR technique can quickly screen chromosome structural aberrations in patients with newly diagnosed leukemia.It is useful in detection of fusion genes in ALL with normal karyotypes and it would refine the karyotype analysis and provide imrootramt prognosis-relevant information.
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Objective To explore the value of fluorescence in situ hybridization (FISH) and multiplex RT-PCR in the detection of mixed lineage leukemia (MLL) gene rearrangement in acute leukemia (AL) patients. Methods Dual-color MLL probe, multiplex RT-PCR and R or G banding techniques were used to detect the MLL gene rearrangement in 189 cases of AL.Results MLL gene rearrangements were detected in 9 cases (5.03 ％) by FISH,and 16 cases (8.47 ％) by multiplex RT-PCR,including MLL/AF9,MLL/AF10,MLL/AF6, MLL/AF7, MLL/ELL, MLL/PTD. R or G banding techniques could find 11q23 in 5 out of 189 patients (2.65 ％). There was no statistic difference in the incidence of 6 common MLL gene rearrangements between ALL (73 cases) and AML patients (116 cases) (P ＞ 0.05).Conclusion Multiplex RT-PCR is a powerful technique in the detection of MLL gene rearrangement for tentatively diagnosed AL.It could not only confirm translocation detected by conventional cytogenetic method, but also detect MLL partial tandem duplication which could not been detected by cytogenetic examination or FISH. It plays an important role in guiding therapy and predicting prognosis for AL.
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This study is to investigate the protective effect of astaxanthin against injured hepatocyte L-02 cells induced by sodium azide (NaN3) and reveal the possible mechanisms. Hepatocyte L-02 cells were exposed to 100 mmol.L-1 NaN3 with various concentrations of astaxanthin pre-incubated, then the cell viability was measured by MTT method; The level of reactive oxygen species (ROS) was determined by DCFH-DA method; The changes of mitochondrial membrane potential (MMP) and apoptosis ratio were detected by JC-1 method and Annexin V-FITC/PI double stain method, respectively. Results showed that after cells were exposed to 100 mmol.L-1 NaN3 for 3 hours, the cell viability significantly decreased; ROS level and the percentage of late phase apoptosis increased obviously; MMP was also declined. When cells were pretreated with astaxanthin, the cell damage and late phase apoptosis ratio reduced and MMP was maintained. However, the level of ROS showed insignificant decrease (P>0.05). The beneficial concentration of astaxanthin in improving cell viability and MMP was not in a dose dependent manner and the most effective of which was 0.10 nmol.L-1 (P<0.01). In order to reveal its possible non-antioxidant mechanism, mitochondrial membrane was imitated and H+ transferring function of astaxanthin was also detected by bilayer lipid membrane (BLM) method. Results showed that 2.0% astaxanthin could transfer H+ efficiently. These suggested the mechanisms of astaxanthin in protection of hepatocyte L-02 cells not via its ROS quenching capability but via its H+ transferring function, which improved the mitochondrial function and had the sequence biology effects.
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This study is designed to investigate the anti-tumor and anti-angiogenesis mechanism of carrageenan oligosaccharides. The effects of carrageenan oligosaccharides on basic fibroblast growth factor (bFGF) induced cell proliferation, heparanase activity and bFGF binding ability were evaluated in human cervical cancer cells (HeLa) and human umbilical vein endothelial cells (HUVEC). Results indicate that, at rational concentrations, carrageenan oligosaccharides showed low cytotoxic effect. At relatively low concentrations (0.2-200 microg x mL(-1)), these oligosaccharides could competitively bind bFGF and inhibit bFGF induced cell proliferation. In these samples, oligo-lambda-carrageenans (dp2-8) were the most potent bFGF antagonists. At concentration of 20 microg x mL(-1), their inhibitory ratio reached to 30%. The heparanase enzyme assay revealed that three kinds of carrageenan oligosaccharides showed different inhibitory activities to two cell lines. For HeLa cell, oligo-lambda-carrageenans showed highest inhibitory effect, but for HUVEC, oligo-kappa-carrageenans (dp9-17) were the best inhibitors. Current observations demonstrated that the biological activities of carrageenan oligosaccharides are closely related to the molecular weight, carbohydrate structure and the content and linking position of sulfur groups. Carrageenan oligosaccharides with high sulfate fraction, 2-8 units saccharide size and suitable molecular structure are able to achieve potent heparin sulfate-like compounds.
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OBJECTIVE@#To investigate the distribution patterns of microvascular density(MVD) and lymph vessel density (LVD) in nasopharyngeal carcinoma, and to discuss their relationships with tumor recurrence-metastasis and prognosis.@*METHOD@#One hundred and six patients with pathologically proved nasopharyngeal carcinoma were included in this study. Immunostainings for the vascular endothelial marker CD34 and the lymph vessel marker D2-40 were performed. The microvascular and lymph vessels within the tumor and the peritumoral area were respectively quantified with computer assisted morphometric analysis.@*RESULT@#Both microvascular and lymph vessels were positive in the specimen of 106 nasopharyngeal carcinoma patients, with MVD and LVD higher in the peritumoral area than in the tumor. Univariate analysis showed that intra-tumor MVD,intra-tumor and peritumor LVD were correlated with nasopharyngeal carcinoma recurrence-metastasis (P 0.05). Multivariate analysis demonstrated that intra-tumor MVD and peri-tumor LVD were two independent risk factors of nasopharyngeal carcinoma recurrence-metastasis. Higher intra-tumor MVD was associated with adverse prognosis of nasopharyngeal carcinoma (P < 0.05).@*CONCLUSION@#High MVD and LVD may be of significance in predicting poor prognosis in patients with nasopharyngeal carcinoma. Intra-tumor and peri-tumor microvessels may play different roles in the tumor recurrence and metastasis. Intra-tumor MVD and peri-tumor LVD should be the therapeutic targets in the nasopharyngeal carcinoma.
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Adolescent , Adult , Aged , Child , Female , Humans , Male , Middle Aged , Young Adult , Carcinoma, Squamous Cell , Pathology , Lymphangiogenesis , Lymphatic Metastasis , Lymphatic Vessels , Pathology , Microvessels , Pathology , Nasopharyngeal Neoplasms , Pathology , Neoplasm Staging , Neovascularization, Pathologic , PrognosisABSTRACT
<p><b>OBJECTIVE</b>To investigate the antitumor effects of two kinds of bromophenols isolated from marine algae Rhodomela confervoides on three tumor cells of Hela, MGC and BGC-823 and their antitumor mechanism in vitro.</p><p><b>METHOD</b>MTT method was employed to assay the inhibitory effects of marine bromphenols with various concentrations. Flow cytometry (FCM) was used to study the cell cycle and aneuploid induction.</p><p><b>RESULT</b>Both of two bromophenols showed cytotoxic activities on the tested tumor cells. Hela cells were proved to be the most sensitive to the marine bromophenols. Although they couldn't cause apoptosis of the tumor cells, the aneuploid and cell cycle inhibition were detected. For Hela and MGC cells, hypoploid was observed under low drug concentrations, while G1 phase block was caused by higher drug concentrations. For BGC-823 cells, G1 phase inhibition was observed for different drug concentrations, and the inhibitiory effect showed dose-dependent.</p><p><b>CONCLUSION</b>Marine bromophenol can inhibit the proliferation of three tumor cells, and the mechanism was probably aneuploid induction and cell cycle inhibition.</p>
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Humans , Antineoplastic Agents , Pharmacology , Apoptosis , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Phenols , Pharmacology , Rhodophyta , ChemistryABSTRACT
A method was developed to elucidate the structures of sulfated oligosaccharides through establishment of an effective reversed phase ion pair ultra-performance liquid chromatography coupled with electrospray ionization time of flight mass spectrometry( RPIP-UPLC-ESI-TOF-MS). Heptylamine (20 mmol/L,pH4) has been selected as the ion-pairing agent.κ-Carrageenan oligosaccharides have been separated on BEH C_(18) column using MeOH/H_2O with 25% heptylammonium formate as eluent in linear gradient mode. Mass spectra were obtained by ESI-Q-TOF-MS in both positive and negative modes. κ-Carrageenan oligosaccharides were well separated up to pentatetrasaccharide,and ESIMS analysis for κ-carrageenan oligosaccharides up to hepta-cosasccharide. The results showed that all acid hydrolyzed κ-carrageenan oligosaccharides were odd sugars,which was further confirmed by polyacrylamide gel electrophoresis ( PAGE). The characteristic fragmentation pattern of ion-pair oligosaccharides in mass spectra can be applied for rapid structure identification.
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To overcome the present limitations of passive biochip, based on the basic principle of antigen-antibody reaction, we develop an antigen-antibody reaction model of solid-phase surface and design a novel active biochip system according to this model, which introduces the negative pressure and controlling devices to control the immunoreactions on the nitrocellulose (NC) membrane. From the computer simulation results, this is a rapid, stable, robust and practicable system, which can be used to increase the efficiency of immunoreactions and improve the reproducibility and accuracy of biochip analysis.
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Antigen-Antibody Reactions , Biosensing Techniques , Equipment Design , Models, BiologicalABSTRACT
Template direct dye-terminator incorporation with fluorescence-polarization (TDI-FP assay) is a technology for genotyping single nucleotide polymorphisms (SNPs). To apply this method in analyses of A647G variation in human papillomavirus (HPV) 16 E7 gene from HPV 16-positive cervical tissues, a total of 91 and 49 HPV 16-positive DNA samples obtained from women with cervical cancer and normal/inflamed cervices living in Shaanxi in northwest China were subjected to the partial E7 gene PCR with nucleotide (nt) 647 in the products. Then, the oligonucleotide probe designed to anneal immediately to nt 647 was hybridized to the template within the PCR amplicons, and extended specifically by TAMRA-ddTTP or R110-ddCTP directed by the base at nt 647. The increasing FP values were read and the base at nt 647 was identified. The prevalence of nt 647 A→G was 35.71% (50/140). The variation 647G detected in 42.86% (39/91) of women with cervical cancer was significantly higher than 22.45% (11/49) detected in those with normal/inflamed cervices (x2 = 5.778, P = 0.016). The odds ratio (OR) between these two groups was 2.59 (95% confidence interval=l.17～5.71). The results demonstrate that TDI-FP method can be potentially applied in analysis of interest point mutations in HPVs. The incidence and risk implication of HPV 16 A647G variant infection in Shaanxi, China, displays significant geographic difference from other areas. The HPV 16 with E7 gene A647G point mutation appears to have a higher risk for invasive cervical cancer in women living in Shaanxi.
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Objective To study the effect of diet containing different proportions of porphyra powder on relieving constipation, volatile organic compounds and inhibiting intestinal E. coli. Method Mice were divided into 3 groups: blank group, 2 test groups. After the mice were allotted for different concentrations of porphyra powder diet for 30 d, the body weight of mice, water-holding capacity, the number and weight of feces within 24 h, the feces pH, E. coli counts and the first black defecating time were detected. The odorous compounds in feces were also measured by headspace solid phase microextraction coupled with GC-MS. Results Compared with the blank group, the water absorbed and number and weight of feces were significantly increased(P
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<p><b>OBJECTIVE</b>To develop a simple, cheap, quick, accurate and practical method for a high throughout genotypes assay of human papillomavirus (HPV) DNA.</p><p><b>METHODS</b>Crude DNA was extracted by a simplified proteinase K digesting method. HPV common conservative primers: GP5+/6+ system was used to amplify HPV DNA in 127 samples of condylomata acuminatum (CA) and cervical scrapes by PCR, then the PCR product was assayed using a template directing terminator incorporation (TDI) and genotypes were detected with fluorescence polarization (FP). Major HPVs type-specific probes (HPV6, 11, 16, 18, 31, 33, 35 and 58) designed by us were hybridized with the specific PCR products and a special fluorescent ddNTP terminator was directly added to the end of the probe under direction of specific PCR products. The results were measured with FP and compared with the results of the DNA sequence.</p><p><b>RESULTS</b>Compared with the results of DNA sequencing, the results detected with fluorescence polarization were all correct. The proposed method could detect more than one type of HPV infection, but DNA sequencing method could not. The positive rate of HPV was 100% in 78 CA biopsies. Among them, there were 14 HPV double infections [HPV6B and 11 (9 cases), HPV11 and 16 (4), HPV11 and 18 (1)], 5 HPV triple infections [HPV6B, 11 and 16 (4), HPV11, 16 and 18 (1)], and one HPV quadruple infection (HPV6B, 11, 16 and 18). The positive rate of HPV was 77% in the 49 cervical scrapes. Six HPV double infections [HPV6B and 11 (2), HPV11 and 16 (1), HPV6B and 16 (1), HPV16 and 18 (1), HPV18 and 58 (1)], 3 HPV triple infections [HPV6B, 11 and 16 (2), HPV11, 16 and 18 (1)] and one HPV quadruple infection (HPV6B, 11, 16 and 18) were detected in cervical cancer scrapes.</p><p><b>CONCLUSIONS</b>The proposed method allowed a high throughout, special, simple, rapid, automatic and economical detection of HPV-DNA genotyping without a use of labeling probes. It can detect multiple HPV genotype infection and will be and useful tool in HPV genotype screening.</p>