6 research outputs found
Bioelectronic Nose Using Odorant Binding Protein-Derived Peptide and Carbon Nanotube Field-Effect Transistor for the Assessment of <i>Salmonella</i> Contamination in Food
<i>Salmonella</i> infection is the one of the major causes
of food borne illnesses including fever, abdominal pain, diarrhea,
and nausea. Thus, early detection of <i>Salmonella</i> contamination
is important for our healthy life. Conventional detection methods
for the food contamination have limitations in sensitivity and rapidity;
thus, the early detection has been difficult. Herein, we developed
a bioelectronic nose using a carbon nanotube (CNT) field-effect transistor
(FET) functionalized with <i>Drosophila</i> odorant binding
protein (OBP)-derived peptide for easy and rapid detection of <i>Salmonella</i> contamination in ham. 3-Methyl-1-butanol is known
as a specific volatile organic compound, generated from the ham contaminated
with <i>Salmonella</i>. We designed and synthesized the
peptide based on the sequence of the <i>Drosophila</i> OBP,
LUSH, which specifically binds to alcohols. The C-terminus of the
synthetic peptide was modified with three phenylalanine residues and
directly immobilized onto CNT channels using the π–π
interaction. The p-type properties of FET were clearly maintained
after the functionalization using the peptide. The biosensor detected
1 fM of 3-methyl-1-butanol with high selectivity and successfully
assessed <i>Salmonella</i> contamination in ham. These results
indicate that the bioelectronic nose can be used for the rapid detection
of <i>Salmonella</i> contamination in food
Dramatic and widespread changes in insulin signaling molecules in CPEB knockout mice.
<p>(A) Western blot analysis and quantification of IRS1, IRS2, PTEN, PDK1, phospho-Stat3 (S727), total Stat3, Socs3, and tubulin as a loading control from WT and CPEB knockout liver, fat, and muscle. (B) Quantitative RT-PCR analysis of mRNAs encoding IRS1, IRS2, PTEN, PDK1, Stat3, and Socs3 mRNAs from WT and <i>Cpeb1</i> KO liver. Data are represented as mean +/− SEM. At least 3 animals per group were used for the experiment. Asterisks refer to statistical significance at the p<0.05 (*) or p<0.01 (**) levels (Student's t test).</p
CPEB controls the synthesis of Stat3 and PTEN.
<p>(A and B) 3′ UTR sequences of Stat3 and PTEN from human, mouse and cow. The nucleotides in bold represent putative CPEs. (C and D) Western blots of Stat3 and PTEN following CPEB depletion in HepG2 cells. In panels C-H, tubulin served as a negative or input control. (E and F) Quasi-quantitative RT-PCR for Stat3 and PTEN RNAs following CPEB depletion. (G and H) HepG2 cells depleted of CPEB were pulse labeled with <sup>35</sup>S-methionine for 15 min followed by Stat3, PTEN and tubulin (as a control) immunoprecipitation and SDS-PAGE analysis. These same proteins were also analyzed by western blots. (I) Representation of Renilla and firefly luciferase RNAs that were electroporated into HepG2 cells. Renilla luciferase RNA, which contained an irrelevant 3′ UTR, served as a normalization control. Firefly luciferase contained the Stat3 or PTEN 3′ UTRs as noted in panels A and B; in some cases, the CPEs in these 3′ UTRs were mutated. (J and K) The firefly and Renilla RNAs noted above were electroporated into HepG2 cells, some of which were depleted of CPEB. Firefly luciferase was normalized to the Renilla luciferase transfection control; luciferase activity derived from all RNAs was then made relative to the control shRNA. The Stat3 and PTEN data were analyzed with ANOVA; p values were 0.009 and 0.005, respectively. The asterisk refers to statistical significance (p<0.05). Data are represented as mean +/− SEM. The firefly and Renilla luciferase RNAs were also analyzed for relative stability by quasi-quantitative RT-PCR; all the RNAs had similar stabilities. At least 3 animals were used for each experiment.</p
CPEB mediates insulin signaling in the liver.
<p>(A) Western blot and quantification of total and phospho-Akt (serine 473 and threonine 308) from liver of WT and <i>Cpeb1</i> KO mice, some of which were injected with insulin. The pAkt (Ser473) and pAkt (Thr308) data were analyzed with ANOVA with (p<0.05, *; p<0.01, **). Data are represented as mean +/− SEM. In this and all panels, at least 3 animals per group were used for the experiment. (B,C) Phospho-Akt (threonine 308) in CPEB KO fat and muscle, respectively. Analysis as in panel A. (D) Examination of insulin signaling molecules in WT and CPEB KO liver. Analysis as in panel A.</p
Proposed model for CPEB-dependent regulation of the insulin-signaling ribonome regulation.
<p>The dark gray boxes refer to mRNAs that contain conserved CPEs and can potentially be regulated by CPEB. The light gray boxes refer to mRNAs that contain conserved CPEs but because they are not co-immunoprecipitated with CPEB, are probably not regulated by this protein in the current settings. The striped boxes refer to mRNAs that contain conserved CPEs and are regulated by CPEB. Insulin mRNA does not contain a CPE.</p
<i>Cpeb1</i> KO mice are insulin-resistant.
<p>(A) WT and KO mice were fed a normal chow diet and then examined for glucose tolerance test, serum insulin levels, and insulin tolerance. ANOVA values are as indicated in the figure. (B) Measurements for lean mass, fat mass, and total body mass of WT and <i>Cpeb1</i> KO mice fed a high fat diet. (C) Animals fed a high fat diet were subjected to euglycemic clamp analysis that determined glucose infusion rate (GIR), glucose turnover, whole body glycolysis, glycogen synthesis, hepatic glucose production (HGP), and liver insulin action (the ratio of basal to clamped HGP). The HGP data were analyzed with ANOVA with a value of 0.006. The asterisks in this panel as well as panel D refer to statistical significance (p<0.05). (D) Following the euglycemic clamp, liver proteins from WT and KO animals were probed on western blots for total and phospho-Akt (S473 and T308). The pAkt (Ser473) and pAkt (Thr308) data were analyzed with ANOVA with suggestive values of 0.01999 and 0.08335 values respectively. Data are represented as mean +/− SEM. At least 3 animals per group were used for the Western blots and at least 6 animals per group were used to measure the physiological parameters.</p