47 research outputs found
Mouse serum anti-adhesin and antitoxin IgG antibody titers.
<p>Anti-CFA/I, anti-CS1, -CS2, -CS3, -CS4/CS6 and anti-CS5/CS6, and anti-STa and anti-LT IgG antibodies in the serum of each mouse immunized with CFA/I/II/IV-STa<sub>A14Q</sub>-dmLT MEFA protein (ā) and the serum of each control mouse (ā) were titrated in ELISAs. CFA/I, CS1, CS2, CS3, CS4/CS6, CS5/CS6 heat-extracted from <i>E</i>. <i>coli</i> or ETEC strains in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121623#pone.0121623.t001" target="_blank">Table 1</a> (500 ng per well of a 2HB plate), STa-ovalbumin (10 ng per well of a Costar plate), or LT (List Biological Laboratories, Inc.; 100 ng per well of a 2HB plate) and HRP-conjugated goat-anti-mouse IgG (1:3300; the secondary antibodies) were used to titrate IgG antibodies specific to CFA/I, CS1, CS2, CS3, CS4/6, CS5/6 and to STa and LT toxins, respectively. The antibody titer was calculated from the highest dilution of a serum sample that produced an ELISA optical density of greater than 0.3 (above the background) and presented in a log<sub>10</sub> scale. Each dot represented a mouse IgG titer, and the bars indicated the mean titer of the group.</p
Results of <i>in vitro</i> antibody adherence inhibition assays<sup>a</sup>, using serum samples of mice immunized with CFA/I/II/IV-STa<sub>N12S</sub>-dmLT, co-administrated with CFA/I/II/IV MEFA and toxoid fusion 3xSTa<sub>N12S</sub>-dmLT, or the negative control mice.
<p><sup>a</sup>: ETEC field isolates and <i>E</i>. <i>coli</i> recombinant strains expressing CFA/I, CS1, CS2, CS3, CS4/CS6, CS5/CS6 and CS6 (3.5x10<sup>6</sup> CFUs) were individually incubated with serum samples (20 Ī¼l) pooled from mice in the group immunized with CFA/I/II/IV-STa<sub>N12S</sub>-dmLT, co-immunized with the CFA/I/II/IV MEFA and 3xSTa<sub>N12S</sub>-dmLT, or the control mice on a shaker (50 rpm) for 1 hour at room temperature. The serum-bacteria mixture was added to Caco-2 cells (7x10<sup>5</sup> cells; 1 ml final volume) and incubated in a CO<sub>2</sub> incubator for 1 h. After washing off non-adherent bacteria, ETEC or <i>E</i>. <i>coli</i> bacteria adhered to Caco-2 cells (in 1 ml PBS) were serial diluted, plated, cultured overnight, and counted (CFUs).</p><p><sup>b</sup>: serum samples pooled from mice of the group immunized the CFA/I/II/IV-STa<sub>N12S</sub>-dmLT, the group co-immunized with CFA/I/II/IV and 3xSTa<sub>N12S</sub>-dmLT, or the control group. These serum samples were used in the antibody adherence inhibition assay.</p><p><sup>c</sup>: p values were calculated by using a Student <i>t</i> test comparing numbers of ETEC or <i>E</i>. <i>coli</i> bacteria adhered to the Caco-2 cells incubated with mouse serum of each immunization group vs. bacteria adherent to the cells treated with serum of the control group.</p><p>The number of ETEC or <i>E</i>. <i>coli</i> bacteria adhered to Caco-2 cells was used to indicate activity of anti-CFA antibodies against bacteria adherence.</p
Mouse serum <i>in vitro</i> antibody neutralization activity against STa toxin.
<p>Intracellular cyclic GMP concentration (pmol/ml) in T-84 cells incubated with STa toxin and mouse serum was measured with an EIA cGMP ELISA kit (Assay Design) and was used to indicate anti-STa antibody neutralizing activity. STa toxin elevates intracellular cGMP in T-84 cells, whereas neutralizing anti-STa antibodies neutralize the toxin and prevent STa from stimulating cGMP, thus a lower cGMP concentration indicates a stronger neutralization activity of anti-STa antibodies. The serum sample (30 Ī¼l; in a final dilution of 1:33.3) pooled from each group of mice immunized with CFA/I/II/IV-STa<sub>A14Q</sub>-dmLT or CFA/I/II/IV-STa<sub>N12S</sub>-dmLT, co-immunized with CFA/I/II/IV and 3xSTa<sub>N12S</sub>-dmLT, the control group, or the serum sample collected prior to immunization was incubated with STa toxin (2 ng, in 150 Ī¼l cell culture medium) for 30 min at room temperature, and the serum-toxin mixture was added to T-84 cells (1 ml of final volume with cell culture medium). Intracellular cGMP concentration in T-84 cells was measured after 1 hour incubation at a CO<sub>2</sub> incubator, with the mean cGMP and standard deviation (from four to six replicates) of each group indicated as columns and bars. The cGMP levels in T-84 cells cultured with cell culture medium alone (without STa toxin or serum; no STa toxicity) or with STa toxin in culture medium (without serum; STa toxicity) were used as controls.</p
Construction and detection of the CFA/I/II/IV-STa<sub>-toxoid</sub>-dmLT MEFA.
<p>(A) Construction of the CFA/I/II/IV MEFA. The most antigenic epitopes of the CS1, CS2, CS3, CS4, CS5 and CS6 major structural subunits were embedded into CFA/I major subunit by replacing the CfaB surface-exposed but less antigenic epitopes. (B) Construction of the 3xSTa<sub>N12S</sub>-dmLT toxoid fusion. Three copies of the STa toxoid STa<sub>N12S</sub> gene were genetically fused to the monomeric dmLT (LT<sub>R192G/L211A</sub>) gene using SOE (splicing overlap extension) PCRs. (C) Construction of CFA/I/II/IV-STa<sub>N12S</sub>-dmLT MEFA. A substitution of the first 150 amino acids of the 3xSTa<sub>N12S</sub>-dmLT (the N-terminal STa<sub>N12S</sub> and the first 131 amino acids of LT-A subunit) with the CFA/I/II/IV MEFA created the CFA/I/II/IV-STa<sub>N12S</sub>-dmLT MEFA. Four linkers: LGA, GPVD, Gly-Pro linker GPGP, and L-linker were used for the construction. (D) Western blot to detect the CFA/I/II/IV-STa<sub>N12S</sub>-dmLT MEFA protein with anti-CFA/I, anti-CS1, -CS2, -CS3, -CS4, -CS5, and anti-CS6 MAb hybridoma supernatant (1:100; provided by Dr. AM Svennerholm), and rabbit anti-CT (1:3300; Sigma) and anti-STa antiserum (1:3300; provided by Dr. DC Robertson). Extracted MEFA proteins separated in 12% PAGE gel were detected with each anti-adhesin MAb, anti-CT and anti-STa antiserum and IRDye-labeled goat anti-mouse IgG or anti-rabbit IgG (1:5000; LI-COR). Lane (+) indicated the CFA/I/II/IV-STa<sub>N12S</sub>-dmLT MEFA proteins, whereas lane (-) of extracted total proteins of <i>E</i>. <i>coli</i> BL21 host strain as the negative control. Lane M is the protein marker (in kilo Daltons; Precision Plus Protein pre-stained standards; Bio-Rad).</p
<i>Escherichia coli</i> strains and plasmids used in the study.
<p><i>Escherichia coli</i> strains and plasmids used in the study.</p
Joint Network Reconstruction and Community Detection from Rich but Noisy Data
Most empirical studies of complex networks return rich but noisy data, as they measure the network structure repeatedly but with substantial errors due to indirect measurements. In this article, we propose a novel framework, called the group-based binary mixture (GBM) modeling approach, to simultaneously conduct network reconstruction and community detection from such rich but noisy data. A generalized expectation-maximization (EM) algorithm is developed for computing the maximum likelihood estimates, and an information criterion is introduced to consistently select the number of communities. The strong consistency properties of the network reconstruction and community detection are established under some assumption on the Kullback-Leibler (KL) divergence, and in particular, we do not impose assumptions on the true network structure. It is shown that joint reconstruction with community detection has a synergistic effect, whereby actually detecting communities can improve the accuracy of the reconstruction. Finally, we illustrate the performance of the approach with numerical simulations and two real examples. Supplementary materials for this article are available online.</p
Insights into the Correlation of Aluminum Distribution and BroĢnsted Acidity in HāBeta Zeolites from Solid-State NMR Spectroscopy and DFT Calculations
Here we utilized <sup>27</sup>Al
MAS/MQMAS and <sup>31</sup>P MAS
NMR of quantitative adsorption of trimethylphosphine oxide (TMPO)
and DFT calculations to elucidate the relationship between Al distribution
and BroĢnsted acidity of series H-Beta zeolites derived from
dealumination of Al-rich H-Beta zeolite. Three types of BroĢnsted
acid strengths corresponding to different specific Al T-sites were
demonstrated. The removal of one framework Al in 5MR2ā-2Al
and 6MR-2Al sites led to increasing the BroĢnsted acid strength
of dealuminated H-Beta. Our findings on such exact correlation between
specific Al distributions and corresponding BroĢnsted acid sites
may guide the controlling Al distribution to get desired acid properties
through zeolite synthesis or finely tuned dealumination, which has
a great impact on the catalytic activity and selectivity of zeolite
catalysts
GPR26 deficiency causes hyperphagia.
<p>Cummulative food intake was monitored during 12-weeks of high-fat diet from female (<b>A</b>) and male (<b>B</b>) GPR26 knockout mice (KO) and their wild type littermates (WT). Nā=ā8, <i>*p<0.05</i>, **<i>p</i><0.01 when compared with wild type controls.</p
GPR26 deficiency exacerbates hyperinsulinemia and dyslipidemia associated with obesity.
<p>After 12-weeks of high-fat diet, GPR26 knockout mice (KO) and wild type littermates (WT) were fasted overnight and blood samples were collected into a tube that contained heparin and EDTA on ice. (<b>A</b>ā<b>B</b>), circulating levels of insulin, ghrelin, leptin, and adiponectin were analyzed by Linco Research service. (<b>C</b>), the concentrations of plasma triglyceride and cholesterol were determined using the Roche/Hitachi 912 automatic analyzer system. Nā=ā8, <i>*p<0.05</i>, **<i>p</i><0.01 when compared with wild type controls.</p
GPR26-deficicent mice exhibit glucose intolerance.
<p>Oral glucose tolerance tests were carried out in GPR26<sup>ā/ā</sup> mice (KO) and the wild type controls (WT) after 12 weeks of high-fat diet (HFD) or regular chow. Mice were orally gavaged with 2.0g of glucose per kg of body weight. Mouse tail blood samples were collected at indicated time points and analyzed for blood glucose levels by the ACCU-CHEK Blood Glucose Meter. (<b>AāB</b>): blood glucose levels of female (<b>A</b>) and male (<b>B</b>) mice on high-fat diet; (<b>BāD</b>): blood glucose levels of female (<b>C</b>) and male (<b>D</b>) mice on regular diet. Nā=ā8, <i>*p<0.05</i>, **<i>p</i><0.01 when compared with wild type controls.</p