Based on microfabrication technology and electrochemical modification method, a micro electrochemical sensor for nitrate ( NO-3 ) determination was developed. A micro sensor chip with working electrode and counter electrode was used as the signal convertor of the sensor. The area of the micro working_electrode was only 1 mm2 . As an electrocatalysis sensitive material, copper was electrodeposited onto the working electrode by square_wave pulse current electrodeposition method. The morphologies and components of freshly deposited materials were examined by scanning electron microscopy ( SEM ) and X_ray diffraction ( XRD) to explore key factors that affected the electrocatalytic ability of the deposited copper layer for reducing nitrate ions. The experimental results revealed that under the optimal conditions, the deposited copper layer was macroporous and had a larger effective surface area that could serve as a more effective electrocatalyst in facilitating nitrate reduction. Electrochemical response of the macroporous copper layer was characterized by linear sweep voltammetry in acidic supporting electrolytes ( pH=2 ) . The electroanalytical results showed that the modified microsensor had marked sensitivity for standard nitrate samples within the concentration range from 12. 5 to 3000 μmol/L (in the range of 12. 5-200 μmol/L yielded straight line:y1=-0. 1422x-10. 326, R12=0. 9976, while in the range of 200-3000 μmol/L yielded straight line: y2=-0. 0984x-22. 144, R22=0. 9927) with a detection limit of 2 μmol/L (S/N=3). The developed electrochemical microsensor was also employed for nitrate determination in water samples collected from lakes and rivers near the city of Beijing. The results were in good agreement with the data given by qualified water quality detection institute, with the deviations from 3 . 9% to 15 . 4%.

Objective To investigate surgical treatment and effect of secondary endocardial fibroelastosis,based on respective analysis of clinical data and follow-up data of patients with secondary endocardial fibroelastosis (SEF) between 2010 and 2012.Methods A retrospective analysis was performed including 10 patients with secondary endocardial fibroelastosis from January 2010 to December 2012 in Wuhan Union Hospital.All patients were diagnosed by Untrasonic Cardiogram and/or CT angiography of heart and great vessel,and had cardiac insufficiency in different degree [EF 0.37 ± 0.08 (0.26 ～ 0.48)].All patients except 2 patients with anomalous origin of the coronary artery received treatment of digitaloid drugs before operation,which promoted preoperative cardiac function.5 patients with SEF complicated with Congenital Coarctation of the Aorta (CoA),2 patients underwent correction of CoA,2 patients underwent correction of CoA and partial resection of endocardium,1 patient underwent correction of CoA,partial resection of endocardium and mitral vavuloplasty.2 patients with SEF complicated with anomalous origin of the left coronary artery from the pulmonary artery,who were underwent correction of anomalous origin of coronary artery.2 patients with SEF complicated with aortic stenosis,who were underwent aortic commissurotomy and partial resection of endocardium.1 patient with SEF complicated with mitral stenosis and insufficiency,who underwent mitral valve replacement.The intraopertive gross appearance of endocardium was opaque greyish-white not transparent pink.The postoperative pathological examination showed obviously positive dyeing of elastic fibers.In 3,6,12 and 24 months after operation,Untrasonic Cardiogram evaluated cardiac function and endocardium.Results one 6 months patients with origin of left coronary from pulmonary artery died of severe post-operative low cardiac output syndrome,while another 1 months patients with origin of left coronary from pulmonary artery obtained post-operative good recovery,and the Untrasonic Cardiogram show disappearance of endocardial fibroelastosis.The post-operative mean time of using respirator(4.0 ± 1.5) days (2-7 days).Compared with the preoperative data,the cardiac function index (EF) was not significantly better at 2 weeks and 3-6 months[0.38 ± 0.07 (0.28 ～ 0.48),P ＞ 0.05 ; 0.39 ± 0.08 (0.30 ～ 0.50),P ＞ 0.05],and the non-resected fibroelatic endocardium still existed and were not attenuated.But the cardiac function index (EF) significantly increased [0.44 ± 0.08 (0.38 ～ 0.55),P ＜ 0.05] than the pre-operative EF,and the 3 of 5 cases the fibroelatic endocardium were attenuated or disappeared,while 2 of 5 cases the fibroelatic endocardium still existed.Conclusion SEF is the important causes of the infant intractable heart failure,which has the characteristic of high mortality and limited therapy.For SEF patients with anomalous origin of the coronary artery,the SEF is completely reversed by early diagnosis and early correction of the malformation.For SEF patients with CoA or aortic stenosis,the surgical treatment could promote recovery of cardiac function,but whether the SEF were reversed is still subject to further follow-up.The heart transplantation is the best therapy for SEF with severse heart failure.

Objective To study the micro-pore architecture and mechanical properties of porous titanium scaffolds with diamond molecule structure produced by 3D print technology, so as to guide the development of 3D-prinited porous titanium orthopedic implants. Methods Selective laser melting (SLM) and electron beam melting (EBM) were used to fabricate porous Ti6Al4V scaffolds with diamond molecule structure. The micro-pore architectures of those scaffolds were observed using optical microscope and scanning electron microscope (SEM), and universal material testing machine was used to conduct compressive test on the scaffolds. Results Both SLM and EBM techniques had machining error and half-melted metal particles were found on the strut surface. The relative error of strut size produced by SLM and EMB was 20.9%-35.8% and －9.1%-46.8%, respectively. The scaffold with strut width of 0.2 mm could not be produced by EBM. The compressive strength and elastic modulus of the scaffold fabricated by SLM was 99.7-192.6 MPa and 2.43-4.23 GPa, respectively. The compressive strength and elastic modulus of the scaffold fabricated by SLM was 39.5-96.9 MPa and 1.44-2.83 GPa, respectively. Conclusions The manufacturing precision of SLM is higher than that of EBM. Porosity is the main factor that affects the compressive strength and elastic modulus of the scaffolds. In the same process, with the increase of porosity, both the compressive strength and elastic modulus decrease. When the porosities are similar, the scaffolds fabricated by SLM possess higher compressive strength and elastic modulus than those by SLM.

Objective To explore the impacts of different excipients on physical and chemical properties of Acteoside solid lipid nanoparticles (Acteoside-SLN),and make experimental evidence for the study of prescription SLN.Methods Emulsification-evaporation was appropriate for the preparation of Acteoside-SLN.Single variable method was used for fumbling the effects of Compritol 888 ATO,glyceryl monostearate,soy lecithin,Myrj52 and other accessories on the physicochemical properties including nanoparticles particle size,encapsulation efficiency and characterization dispersity (PDI) of Acteoside-SLN.Transmission electron microscope was used to observe the morphology of Acteoside-SLN and the structure of Acteoside in SLN was measured by XRD.Results With the increasing of the amount of Compritol 888 ATO,the particle size of nanoparticles decreased significantly,encapsulation efficiency decreased slightly,PDI increased;With the increasing of amount of glycerol monostearate,particle size increased obviously,encapsulation efficiency decreased slightly,and PDI decreased;With the increasing of the amount of lecithin,particles size increased significantly,encapsulation efficiency decreased,and PDI decreased;With the increasing of Myrj52 amount,the nanoparticles particle size decreased,encapsulation rate and PDI increased slightly.The appearance of Acteoside-SLN was presented uniform spherically.Acteoside was wrapped in SLN in a molecular dispersion state.Conclusion Various additives have a greater impact on physicochemical properties of Acteoside-SLN,and inspiration for prescription screening of SLN is supplied by this study.

Objective:To investigate the inhibitory effects of melanoma differentiation associated gene-7(mda-7/IL- 24)on lymphoma cell line Namalwa in vitro and in vivo.Methods:Using RT-PCR,the expression of mda-7/IL-24 was examined in 10 malignant hematopoietic cell lines,including Namalwa,Raji,K562,NB4,U937,Ramous,CEM,KG1a, HL60,J6-1,etc.The coding region of mda-7/IL-24 was cloned from LPS-treated human peripheral blood mononuclear cells(PBMC)by RT-PCR,and the eukaryotic expression vector pTarget-IL-24 was constructed.The recombinant vector, after sequenced,was transfected into Namalwa cell line via lipofectamine reagent.The stable expression transfectants were selected by G418.The expression of mda-7/IL-24 mRNA and protein was verified by RT-PCR and Western blotting.MTT assay,colony forming assay,apoptosis detection,and tumorigenesis in nude mice were used to assess the effects of mda- 7/IL-24 on tumor proliferation,growth characteristics,colony forming,apoptosis,and tumorigenesis.Results:Expression of mda-7/IL-24 mRNA was not found in any of the 10 malignant hematopoietic cell lines and the expression of mda-7/IL- 24 mRNA and protein was found in Namalwa cells transfected with recombinant plasmid pTarget-IL-24.Significant de- crease in tumor cell viability was observed in Namalwa cells stably transfected with mda-7/IL-24,compared with control cells transfected with empty plasmid pTarget(P

Objective To reversing methicillin-resistant Staphylococcus(MRS) to methicillin-susceptible Staphylococcus(MSS) by changing nutritional conditions and continuous transfer of culture .Methods MRS trains separating from clinical specimens were cultured in different conditions ,continuous cultural transfer ,and drug sensitive test were proceeded periodically to observe the phe-notypic and chemical reaction change of MRS .The mecA gene were detected of the original and mutant strains by polymerase chain reaction(PCR) ,then the gene sequenced and compared .Results 53 MRS strains were studied .6 strains were phenotype successful-ly converted to MSS in different cultural conditions ,among them mecA gene was undetected in 2 strains ,and down expressed in 4 strains .Conclusion The MRS strains separated from clinical specimens may revert to MSS by culture under different nutritional conditions .The mecA gene of MRS may be lost or lower expressed and the MRS and mutant strains may be different in genomics .

Application of HPLC-ESI-ITMS in the quality control of carboxyterminal sequence confirmation for insulin and insulin chain B was studied. The solution of intact insulin or insulin chain B was added to the solution of carboxypeptidase P (CPP) and carboxypeptidase Y (CPY). Fractions of appropriate volume were removed at some appointed time points, acidified with the same amount of 1% formic acid to stop the digestion, and then briefly vortexed for HPLC-ESI-ITMS analysis. Mobile phase A consisted of 0.02% TFA in 98% ultra-pure water and 2% acetonitrile. Mobile phase B consisted of 0.02% TFA in 98% acetonitrile and 2% ultra-pure water. The solution used for post-column fix consisted of propionic acid and isopropyl alcohol (20 : 80, v/v). Chromatographic separation was carried out on a reversed-phase column (Zorbax Prosphere C18, 300A, 5 microm, 2.1 mm ID x 150 mm length). The molecular weights of the multiply charged ions representing consecutive truncated losses of carboxyterminal amino acids were determined by the use of HPLC-ESI-ITMS. The differences between the consecutive truncated peptides are the experimental weights of the carboxyterminal amino acid residues. The carboxyterminal amino acid residue Ala, which released from chain B of intact insulin, was confirmed in the nanomolar concentration range by analyzing the molecular weight of the truncated peptides. Another one carboxyterminal amino acid Ala was confirmed in the nanomolar concentration range of insulin chain B. In the quality control for recombinant DNA product or natural protein, the confirmation of 1 - 3 carboxyterminal amino acid residues is regarded to be up to standard. One amino acid residue of insulin or insulin chain B could be confirmed accurately in the nanomolar concentration range. The results showed that intact insulin could be directly sequenced in the quality control without separating chain B from chain A. There would be no need to separate chain A from chain B to identify carboxyterminal of intact insulin. Furthermore, the method saved us a lot of trouble from the preparation and purification of insulin chain A and chain B.
Amino Acid Sequence ; Carboxypeptidases ; chemistry ; Cathepsin A ; chemistry ; Chromatography, High Pressure Liquid ; methods ; Insulin ; chemistry ; standards ; Molecular Sequence Data ; Molecular Weight ; Peptide Fragments ; chemistry ; Quality Control ; Spectrometry, Mass, Electrospray Ionization ; methods

OBJECTIVETo select the effective siRNA which could inhibit the expression of DNA methyltransferase 1 (DNMT-1) in adenoid cystic carcinoma (ACC) and discuss the time-, and dose-dependent effect of RNA interference (RNAi).

METHODSFour pairs of siRNA were designed, synthesized and transfected through oligofectamine reagent into ACC cell lines ACC-2, ACC-3 and ACC-M. 24, 48 and 72 h after transfection, total RNA and protein were harvested respectively. mRNA and protein expression level of DNMT-1 were detected by real time PCR, RT-PCR and Western blot, and then the effective siRNA was subsequently selected. ACC-3 as also transfected by different concentration of siRNA and the dose-dependent effect of RNAi was discussed. Cy(3) labelled GAPDH siRNA was used as a positive control.

RESULTSTwo of 4 pairs of siRNA inhibited the mRNA expression of DNMT-1 in three ACC cell lines and the expression of DNMT-1 was downregulated by 67%, 86%, 92% and 76%, 76%, 86% respectively. The gene inhibition was detected at 24 h or 48 h after transfection, maintained only 1 - 2 days and showed direct relationship to the concentration of siRNA. The change of protein expression level of DNMT-1 was consistent to the changes of mRNA.

CONCLUSIONSThe effective siRNA which could inhibited the expression of DNMT-1 of ACC were achieved. The inhibition effect of RNAi was time-dependent and dose-dependent.

Carcinoma, Adenoid Cystic ; genetics ; metabolism ; Cell Line, Tumor ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; metabolism ; DNA Methylation ; Genetic Vectors ; Humans ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Salivary Gland Neoplasms ; genetics ; metabolism ; Transfection

The full-length cDNA of hTIMP-1 was obtained from a surgical patient with HCC by the method of RT-PCR. Then it was cloned into the adenoviral shuttle plasmid pAdTrack-CMV, and subsequently cotransformed into competent BJ5183 cells with the adenoviral backbone plasmid pAdEasy-1. Thereupon, a recombinant adenoviral plasmid containing full-length cDNA of hTIMP-1 was generated by homologous recombination in E. coli. The adenoviruses (AdhTIMP-1) were packaged and amplified in adenoviral packaging cells HEK 293. Then the viral titer was checked by green fluorescent protein (GFP), and the expression of hTIMP-1 was detected by the techniques of Western blot and RT-PCR. The recombinant adenovirus vector carrying human TIMP-1 was successfully constructed and expressed in vitro and may pave the way for further application in liver gene therapy.
Adenoviridae ; genetics ; metabolism ; Carcinoma, Hepatocellular ; metabolism ; Cell Line ; Cloning, Molecular ; DNA, Complementary ; genetics ; Extracellular Matrix ; metabolism ; Genetic Vectors ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Tissue Inhibitor of Metalloproteinase-1 ; biosynthesis ; genetics

OBJECTIVETo study the inhibition of the expression of bcl-xL gene induced by RNA interference in CNE-2Z cell line in addition to the inhibition of its proliferation and apoptotic induction.

METHODSSmall interfering RNAs targeting bcl-xL gene were synthesized by using web design software provided by Amnion and the silencer short interfering RNA (siRNA) construction kit; fluorescein-labeled siRNAs were done by FAM-silencer siRNA labeling kit; siRNAs were transfected into CNE-2Z cells by using lipofectamine 2000 reagent; siRNA transfection efficiencies were analyzed by fluorescent microscopy; down-regulation of bcl-xL was detected by RT-PCR; thiazolyl blue (MTT) assay was used to assess the cell growth; apoptosis of CNE-2Z cells was analyzed by flow cytometry.

RESULTSGreen fluorescence in the cells was seen clearly in FAM-labeled siRNA transfected group under the fluorescent microscope while none in the untransfected group. Different down-regulations of bcl-xL mRNA expression were found in the transfected groups. The expression of bcl-xL mRNA decreased by 10% - 70% in the siRNAs transfected CNE-2Z by RT-PCR scan analysis. The inhibitory rate of cell proliferation depended on time and concentrations to some extent. Different cell apoptosis could be induced by different concentrations of siRNA4.

CONCLUSIONSThe synthesized siRNAs in vitro were able to down-regulate the expression of bcl-xL There were different capabilities of the specific siRNAs down-regulation. The transient transfected bcl-xL siRNA4 could effectively inhibit the growth of the cancer cells and induce theirs apoptosis. It was suggested that the siRNA technique provide not only an extremely powerful tool for the functional analysis of genome but also a new method for anti-nasopharyngeal carcinoma gene therapy.

Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; Nasopharyngeal Neoplasms ; genetics ; pathology ; RNA Interference ; RNA, Messenger ; genetics ; Transfection ; bcl-X Protein ; genetics