Platinum Nanoparticle-based Collision Electrochemistry for Rapid Detection of Breast Cancer MCF-7 Cells

Cancer metastasis is the leading cause of death in cancer patients worldwide and one of the major challenges in treating cancer.Circulating tumor cells(CTCs)play a pivotal role in cancer metastasis.However,the content of CTCs in peripheral blood is minimal,so the detection of CTCs in real samples is extremely challenging.Therefore,efficient enrichment and early detection of CTCs are essential to achieve timely diagnosis of diseases.In this work,we constructed an innovative and sensitive single-nanoparticle collision electrochemistry(SNCE)biosensor for the detection of MCF-7 cells(human breast cancer cells)by immunomagnetic separation technique and liposome signal amplification strategy.Liposomes embedded with platinum nanoparticles(Pt NPs)were used as signal probes,and homemade gold ultramicroelectrodes(Au UME)were used as the working electrodes.The effective collision between Pt NPs and UME would produce distinguishable step-type current.MCF-7 cells were accurately quantified according to the relationship between cell concentration and collision frequency(the number of step-type currents generated per unit time),realizing highly sensitive and specific detection of MCF-7 cells.The SNCE biosensor has a linear range of 10 cells·mL^(-1)to 10^(5) cells·mL^(-1)with a detection limit as low as 5 cells·mL^(-1).In addition,the successful detection of MCF-7 cells in complex samples showed that the SNCE biosensors have great potential for patient sample detection.

Immune algorithm for discretization of decision systems in rough set theory

Rough set theory plays an important role in knowledge discovery, but cannot deal with continuous attributes, thus discretization is a problem which we cannot neglect. And discretization of decision systems in rough set theory has some particular characteristics. Consistency must be satisfied and cuts for discretization is expected to be as small as possible. Consistent and minimal discretization problem is NP-complete. In this paper, an immune algorithm for the problem is proposed. The correctness and effectiveness were shown in experiments. The discretization method presented in this paper can also be used as a data pre- treating step for other symbolic knowledge discovery or machine learning methods other than rough set theory.

Cloning and Sequence Analysis of HN and F Protein Genes from a Strain of Goose Paramyxovirus

[ Objective] The study was to clone HN and F genes from GX1 strain of goose paramyxovirus and analyze their sequences. [ Method] According to the full nucleotide sequence of GPV-SF02 strain of goose paramyxovirus, two pairs of pdmers were designed to amplify the HN and F genes from GX1 strain of goose paramyxovirus isolated from diseased goose in Guangxi Zhuang Autonomous Region; the amplified products were ligated into pMD18-T vector and sequenced. [ Result ] HN and F genes of this strain tested were 1 716 and 1 662 bp in full nucleotide length, respectively; both showed the homologues of about 97.3% with GPV- SF02 strain, of 80.3% -97.5% with strains LaSota, F48E9 and JS, of just 84.8% with Miyadera strain. [ Conclusion] The results show that isolated strain BX1 matches to virulent APMV-1 strain, belonging to genotype Ⅶ of APMV-1 strain.

Effects of social isolation stress on immune response and survival time of mouse with liver cancer

AIM： To investigate the effects of isolation stress on mouse with liver cancer and possible associated mechanisms. METHODS： Transplantable murine hepatoma22 （H22） model was used to evaluate the effects of social isolation stress on murine liver cancer. Mice were immunized with sheep red blood cell （SRBC） and intraperitoneally inoculated with H22 cell, then divided into two groups, one reared individually as group （I） and the other reared in groups as group （G）. Titer of antibody to SRBC and interleukin 2 （IL-2） in serum was monitored. The survival time of mouse with liver cancer was observed. RESULTS： The titer of antibody to SRBC in group （G） was 1：24.5 and that in group （I） was 1：11.2. There was a significant difference between these two groups （t = 2.60, P = 0.02）. A significant difference in IL-2 concentration was observed between group （G） （39.6 ng/L） and group （I） （47.1 ng/L, t = 2.14, P = 0.046）. The survival time in group （G） （16.5 d） was markedly longer than that in group （I） （13.2 d, t = 3.46, P = 0.002）. CONCLUSION： Our study suggests that survival time of the mouse bearing H22 tumor is affected by the social isolation stress and the associated mechanism may be the immunological changes under the social isolation stress.

Radioprotective Effect of Lyophyllum Decastes and the Effect on Immunological Functions in Irradiated Mice

In this study, to explore the radiation protection effects of Lyophyllum Decastes Sing （LDS）, a hot distilled-water extract of LDS was orally administered at a dosage of 250mg/kg every other day for a period of 2 weeks in irradiated mice. An automatic blood cell counter was used to measure white blood cells （lymphocytes, monocyte, and granulocytes） one day before X-ray irradiation, and 3 hours, 12 hours, 24 hours, 3 days, 7 days, 15 days and 30 days after irradiation. The Dunnett test was used to examine statistical significance of differences. The peripheral blood cell counts in the Lyophyllum-administered non-irradiation group revealed an increase in the numbers of ieukocytes, lymphocytes and monocytes. For 2 Gy whole body radiation, a significant statistical difference was found between the X-ray group and the Lyophyllum plus X-ray group in the numbers of leukocytes, lymphocytes and monocytes. The results suggest that Lyophyllum restrains blood cell-count falling after irradiation, which is probably mediated at least in part by hemopoietic function, and NK and LAK activities seems to play a role in preventing secondary irffections associated with irradiation.

Distance Concentration-Based Artificial Immune Algorithm

The diversity, adaptation and memory of biological immune system attract much attention of researchers. Several optimal algorithms based on immune system have also been proposed up to now. The distance concentra- tion-based artificial immune algorithm (DCAIA) is proposed to overcome defects of the classical artificial immune al- gorithm (CAIA) in this paper. Compared with genetic algorithm (GA) and CAIA, DCAIA is good for solving the prob- lem of precocity,holding the diversity of antibody, and enhancing convergence rate.

Initial steroid-free immunosuppression after liver transplantation in recipients with hepatitis c virus related cirrhosis

AIM:Steroids can increase hepatitis C virus(HCV) replication.After liver transplantation(LTx),steroids are commonly used for immunosuppression and acute rejection is usually treated by high steroid dosages.Steroids can worsen the outcome of recurrent HCV infection.Therefore, we evaluated the outcome of HCV infected liver recipients receiving initial steroid-free immunosuppression. METHODS:Thirty patients undergoing LTx received initial steroid-free immunosuppression.Indication for LTx included 7 patients with HCV related cirrhosis.Initial immunosuppression consisted of tacrolimus 2×0.05mg/kg.d po and mycophenolate mofetil(MMF)2×15mg/kg.d po.The tacrolimus dosage was adjusted to trough levels in the target range of 10-15μg/L during the first 3 mo and 5-10μg/L thereafter.Manifestations of acute rejection were verified histologically. RESULTS:Patient and graft survival of 30 patients receiving initial steroid-free immunosuppression was 86% and 83% at 1 and 2 years.Acute rejection occurred in 8/30 patients, including 1 HCV infected recipient.All HCV-infected patients had HCV genotype Ⅱ(lb).HCV seropositivity occurred within the first 4 mo after LTx.The virus load was not remarkably increased during the first year after LTx.Histologically,grafts had no severe recurrent hepatitis. CONCLUSION:From our experience,initial steroid-free immunosuppression does not increase the risk of acute rejection in HCV infected liver recipients.Furthermore,none of the HCV infected patients developed serious chronic liver diseases.It suggests that it may be beneficial to avoid steroids in this particular group of patients after LTx.

Prevalence of occult hepatitis B virus infection

Occult hepatitis B virus(HBV) infection(OBI) is characterized by the persistence of HBV DNA in the liver tissue in individuals negative for the HBV surface antigen.The prevalence of OBI is quite variable depending on the level of endemic disease in different parts of the world,the different assays utilized in the studies,and the different populations studied.Many studies have been carried out on OBI prevalence in different areas of the world and categories of individuals.The studies show that OBI prevalence seems to be higher among subjects at high risk for HBV infection and with liver disease than among individuals at low risk of infection and without liver disease.

Identification of TRPM7 channels in human intestinal interstitial cells of Cajal

AIM： To investigate the characteristics of slow electrical waves and the presence of transient receptor potential melastatin-type 7 （TRPM7） in the human gastrointestinal （GI） tract. METHODS： Conventional microelectrode techniques were used to record intracellular electrical responses from human GI smooth muscle tissue. Immunohistochemistry was used to identify TRPM7 channels in interstitial cells of Cajat （ICCs）. RESULTS： The human GI tract generated slow electrical waves and had ICCs which functioned as pacemak er cells. Flufenamic acid, a nonselective cation channel blocker, and 2-APB （2-aminoethoxydiphenyl borate） and La3＋, TRPM7 channel blockers, inhibited the slowwaves. Also, TRPM7 channels were expressed in ICCs in human tissue. CONCLUSION： These results suggest that the human GI tract generates slow waves and that TRPM7 channels expressed in the ICCs may be involved in the gen- eration of the slow waves.

ISOLATION OF HEPATIC OVAL CELLS FROM DIFFERENT MODEL RATS INCLUDING DIABETIC RATS

Objective To acquire oval cells （progenitor stem cells ） from adult rat liver of different models including diabetic rats. Methods Thirty Sprague-Dawley （ SD ） rats were divided into 5 groups randomly： control, 2-acetylaminofluorene （ 2-AAF ）, 2-AAF ＋ partial hepatectomy （ PH ）, 2-AAF ＋ carbon tetrachloride （ CCl4 ）, and diabetic groups. As two-step collagenase perfusion protocol of Seglen, oval cells were isolated by Percoll density gradient centrifugation. Thy1. 1 positive cells were sorted by flow cytometry, and then cultured in Dulbecco＇s minimum Eagle＇s medium （DMEM）. Immunofluorescence staining was applied to labelling Thyl. 1. Results Different rates of Thy1.1 positive oval cells were found in different rat model groups ： 0. 5 % in 2-AAF, 0. 3% in 2-AAF ＋ PH, 0. 2% in 2-AAF ＋ CCl4, 0. 1% in diabetic, and 0. 0% in control. Isolated cells adhered to plate with fusiform or polygon as epithelial cells. Conclusion Progenitor stem cells exist in injured liver tissue including those from diabetic rats.

Linoleic Acid Activates GPR40/FFA1 and Phospholipase C to Increase [Ca^(2+)]_i Release and Insulin Secretion in Islet Beta-Cells

Objective To elucidate GPR40/FFA1 and its downstream signaling pathways in regulating insulin secretion. Methods GPR40/FFA1 expression was detected by immunofluorescence imaging. We employed linoleic acid (LA), a free fatty acid that has a high affinity to the rat GPR40, and examined its effect on cytosolic free calcium concentration ([Ca2+]i) in primary rat β-cells by Fluo-3 intensity under confocal microscopy recording. Downregulation of GPR40/FFA1 expression by antisense oligonucleotides was performed in pancreatic β-cells, and insulin secretion was assessed by enzyme-linked immunosorbent assay. Results LA acutely stimulated insulin secretion from primary cultured rat pancreatic islets. LA induced significant increase of [Ca2+]i in the presence of 5.6 mmol/L and 11.1 mmol/L glucose, which was reflected by increased Fluo-3 intensity under confocal microscopy recording. LA-stimulated increase in [Ca2+]i and insulin secretion were blocked by inhibition of GPR40/FFA1 expression in β-cells after GPR40/FFA1-specific antisense treatment. In addition, the inhibition of phospholipase C (PLC) activity by U73122, PLC inhibitor, also markedly inhibited the LA-induced [Ca2+]i increase. Conclusion LA activates GPR40/FFA1 and PLC to stimulate Ca2+ release, resulting in an increase in [Ca2+]i and insulin secretion in rat islet β-cells.

EFFECT OF HERB-MEDICINE-CAKE-SEPARATED MOXIBUSTION ON SERUM LIPOPROTEIN CONTENTS IN HYPERLIPEMIA RABBITS

Objective: To study the effect of medicinal herb-cake-separated moxibustion on serum lipoprotein levels in hyperlipemia rabbits. Methods: A total of 55 New-Zealand rabbits were randomly divided into control group (n=13), model group (n=14), direct moxibustion group (n=14) and medicinal herb-cake-separated moxibustion (indirect moxibustion) group (n=14). Hyperlipemia model was established by feeding the animals with specialized forage (15% vitellus powder, 5% lard, 0.5% cholesterol and common forage) for 6 weeks. Moxibustion was applied to “Juque”(CV 14), “Tianshu”(ST 25), “Fenglong”(ST 40), etc., 4 moxa-cones for every acupoint, once daily and continuously for 40 days. Serum triglyceride (TG) and total cholesterol (TCh) contents were assayed with colorimentric method. Results: Compared with control group, TCh and TG levels of model group increased significantly (P< 0.01). TCh and TG contents of direct moxibustion and indirect moxibustion groups were significantly lower than those of model group (P<0.01). Comparison between two moxibustion groups showed that serum TCh level of indirect moxibustion group was strikingly lower than that of direct moxibustion group (P<0.01). It indicated that both direct and indirect moxibustion could effectively lower hyperlipemia and the therapeutic effect of indirect moxibustion was significantly superior to that of direct moxibustion in lowering serum TCh level. Conclusion: Both direct and indirect moxibustion can regulate lipid metabolism and the therapeutic effect of medicinal herb-cake-separated moxibustion is superior to that of direction moxibustion in hyperlipemia rabbits.

Variation in the immunoglobulin levels in turbot(Scophthalmus maximus) after vaccination with Streptococcus iniae

A pathogenic bacterium （S636）, identified as Streptococcus iniae, was isolated from turbot （Scophthalrnus maximus） in 2005. We immunized turbot with formalin-killed S. iniae four times （on days 1, 14, 21, and 28） by intraperitoneal inoculation. After each vaccination, we obtained serum samples and isolated the lymphocytes from the peripheral blood, spleen, pronephros, and mesonephros. We measured surface Ig-positive （sIg＋） lymphocytes and serum antibody levels from these organs using flow cytometry and enzyme-linked immunosorbent assay （ELISA）, respectively, using monoclonal antibodies against turbot immunoglohulin. We confirmed that the antibody reacted with both the surface and plasma Ig by confocal laser scanning microscopy and electron microscopy. The percentage of sIg＋ in the lymphocytes increased following each successive vaccination. The mean percentage increased from 31.96% （control） to 37.49%, 38.36%, 42.9%, and 51.63% in the peripheral blood; from 27.09% to 36.63%, 36.81%, 39.28%, and 46.0% in the spleen; from 22.2% to 28.99%, 29.21%, 32.83%, and 41.58% in pronephros; and from 18.12% to 22.17%, 22.45%, 25.69%, and 31.68% in the mesonephros. The ELISA results were consistent with these results. Both the total and specific antibody levels increased with each vaccination. The mean OD value of the specific antibody assay increased from 0.094, to 0.269, 0.283, 0.333, and 0.421; for total antibody the mean OD value increased from 0.133, to 0.292, 0.323, 0.413, and 0.527.

Simultaneous identification and quantification of tetrodotoxin in fresh pufferfish and pufferfish-based products using immunoaffinity columns and liquid chromatography/quadrupole-linear ion trap mass spectrometry

In this study, we established a comprehensive method for simultaneous identification and quantification of tetrodotoxin （TTX） in fresh pufferfish tissues and pufferfish-based products using liquid chromatography/quadrupole-linear ion trap mass spectrometry （LC-QqLIT-MS）. TTX was extracted by 1% acetic acid-methanol, and most of the lipids were then removed by freezing lipid precipitation, followed by purification and concentration using immunoaffinity columns （IACs）. Matrix effects were substantially reduced due to the high specificity of the IACs, and thus, background interference was avoided. Quantitation analysis was therefore performed using an external calibration curve with standards prepared in mobile phase. The method was evaluated by fortifying samples at 1, 10, and 100 ng/g, respectively, and the recoveries ranged from 75.8%--107%, with a relative standard deviation of less than 15%. The TTX calibration curves were linear over the range of 1-1 000 ~tg/L, with a detection limit of 0.3 ng/g and a quantification limit of 1 ng/g. Using this method, samples can be further analyzed using an information- dependent acquisition （IDA） experiment, in the positive mode, from a single liquid chromatography-tandem mass spectrometry injection, which can provide an extra level of confirmation by matching the full product ion spectra acquired for a standard sample with those from an enhanced product ion （EPI） library. The scheduled multiple reaction monitoring method enabled TTX to be screened for, and TTX was positively identified using the IDA and EPI spectra. This method was successfully applied to analyze a total of 206 samples of fresh pufferfish tissues and pufferfish-based products. The results from this study show that the proposed method can be used to quantify and identify TTX in a single run with excellent sensitivity and reproducibility, and is suitable for the analysis of complex matrix pufferfish samples.

Aluminum（Ⅲ）ion sensor based on the sensitive membrane containing nano.sizedAL2O3 powders

An AI^3＋ sensor based on the membrane of acetyl cellulose containing nano γ-Al2O3 crystals was studied. In the buffer solution of 0.5 mol/L CH3COOH-CH3COONa （pH=5.0）, the sensor responds to AI^3＋ in a linear range from 1.00×10^-5 to 1.00x10-^2 mol/L. A near-Nernstian response was obtained and the regression equation was E （mv） = -161.4-26.54 Ig [AI^3＋] with a detection limit of 7.90x10^-6 mol/L. More than 14 different ions as the considered interferences were tested and the relevant selectivity coefficients were determined using the separate solution method （SSM）. The sensor possesses many advantages including short conditioning time, fast response, and, especially, very good selectivity over a wide variety of other co-existing ions. The sample analysis on the aluminium migration amount from aluminium utensils to the solution was determined by this sensor. The analytical results were agreed with that of inductively coupled plasma-atomic emission spectroscopy（ICP-RES）.

Self-Assembled Monolayer of Lipoic Acid on Gold and Its Application to Rapid Determination of 2, 3, 7, 8-Tetrachlorodibenzo-p-Dioxin

Lipoic acid (LA) has received great attention in the area of gold surface functionalization. In this study, LA was employed as a linker for the attachment of antibody to the gold surface of surface plasmon resonance (SPR) chip. By using this chip in a homemade SPR immunosensor, low molecular weight compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) can be detected at a low level of 0.01 ng/mL. There is a good linear relationship(R2 =0.943 1) between the results of SPR biosensor and enzyme-linked immunosorbent assay(ELISA).

Metal sulfide quantum dots-aggregated PAMAM dendrimer for cadmium ion-selective electrode-based immunoassay of alpha-fetoprotein

An ion-selective electrode(ISE)-based immunoassay has been innovatively designed for the sensitive detection of liver cancer biomarker(alpha-fetoprotein,AFP),using metal sulfide quantum dot(QD)-based nano labels.Cd S QDs-aggregated PAMAM dendrimer(QD-DE)was first synthesized and functionalized with polyclonal rabbit anti-human AFP antibodies.Thereafter,a sandwich immunoreaction was implemented on monoclonal mouse anti-human AFP antibody-coated microplate by using antibody-functionalized QD-DE as the secondary antibody.Accompanying the immunocomplex,subsequent potentiometric detection of cadmium ion dissolved from the QD-DE under acidic condition was conducted on a portable cadmium ion-selective electrode(Cd-ISE).Results revealed that the electrode potential of the Cd-ISE increased with the increment of AFP concentration from 0.1 to 100 ng m L^(-1)at a detection limit(LOD)of 68 pg m L^(-1).The relative standard deviations(RSD)were below9.09%and 10.54%for the intra-and inter-assay,respectively.Additionally,six human serum specimens were determined on CdISE-based immunosensor by using commercial human AFP ELISA kit as the reference,and gave good relationship between two methods.Importantly,Cd-ISE-based immunoassay offers the promise for simple and cost-effective screening of disease-related biomarkers.

Global stability of a delayed virus dynamics model with multi-staged infected progression and humoral immunity

In this paper, we propose a nonlinear virus dynamics model that describes the interac- tions of the virus, uninfected target cells, multiple stages of infected cells and B cells and includes multiple discrete delays. We assume that the incidence rate of infection and removal rate of infected cells are given by general nonlinear functions. The model can be seen as a generalization of several humoral immunity viral infection model presented in the literature. We derive two threshold parameters and establish a set of conditions on the general functions which are sufficient to establish the existence and global stability of the three equilibria of the model. We study the globa! asymptotic stability of the equilibria by using Lyapunov method. We perform some numerical simulations for the model with specific forms of the general functions and show that the numerical results are consistent with the theoretical results.

Proteome analysis of hepatic non-parenchymal cells of immune liver fibrosis rats

Elucidation of the mechanisms of liver fibrogenesis is important to treat liver fibrosis.In this study,we established rat models of liver fibrosis with stages from 0–1,2,and 3–4 to 4 at 2,4,6,and 8 weeks,respectively,by injection of pig serum.Liver fibrogenesis was detected by Masson’s trichrome staining.Rat non-parenchymal cells(NPCs)were enriched 4-fold by Percoll density gradient centrifugation.Protein extracts from NPCs were prepared at 4 and 8 weeks,separated by two-dimensional electrophoresis,and then stained with Coomassie Blue G-250.At 4 weeks,we identified 18 non-redundant differentially expressed proteins of which protein disulfide-isomerase associated protein 3(PDIA3)and NDUV showed consistent expression at protein and mRNA levels from 4 to 8 weeks.PDIA3 was found to be down-regulated by Western blotting in the rat model and immunohistochemically in human liver.Our results revealed important aspects of the pathogenesis/progression of liver fibrosis and demonstrated important changes in protein expression levels of NPCs at various stages of liver fibrosis.