119 research outputs found

    Simultaneous Detection of Human C‑Terminal p53 Isoforms by Single Template Molecularly Imprinted Polymers (MIPs) Coupled with Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)-Based Targeted Proteomics

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    Abnormal expression of C-terminal p53 isoforms α, β, and γ can cause the development of cancers including breast cancer. To date, much evidence has demonstrated that these isoforms can differentially regulate target genes and modulate their expression. Thus, quantification of individual isoforms may help to link clinical outcome to p53 status and to improve cancer patient treatment. However, there are few studies on accurate determination of p53 isoforms, probably due to sequence homology of these isoforms and also their low abundance. In this study, a targeted proteomics assay combining molecularly imprinted polymers (MIPs) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for simultaneous quantification of C-terminal p53 isoforms. Isoform-specific surrogate peptides (i.e., KPLD­GEYF­TLQIR (peptide-α) for isoform α, KPLD­GEYF­TLQD­QTSF­QK (peptide-β) for isoform β, and KPLD­GEYF­TLQM­LLDLR (peptide-γ) for isoform γ) were first selected and used in both MIPs enrichment and mass spectrometric detection. The common sequence KPLD­GEYF­TLQ of these three surrogate peptides was used as single template in MIPs. In addition to optimization of imprinting conditions and characterization of the prepared MIPs, binding affinity and cross-reactivity of the MIPs for each surrogate peptide were also evaluated. As a result, a LOQ of 5 nM was achieved, which was >15-fold more sensitive than that without MIPs. Finally, the assay was validated and applied to simultaneous quantitative analysis of C-terminal p53 isoforms α, β, and γ in several human breast cell lines (i.e., MCF-10A normal cells, MCF-7 and MDA-MB-231 cancer cells, and drug-resistant MCF-7/ADR cancer cells). This study is among the first to employ single template MIPs and cross-reactivity phenomenon to select isoform-specific surrogate peptides and enable simultaneous quantification of protein isoforms in LC-MS/MS-based targeted proteomics

    Three-Dimensional Graphene-Carbon Nanotube Hybrid for High-Performance Enzymatic Biofuel Cells

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    Enzymatic biofuel cells (EBFCs) are promising renewable and implantable power sources. However, their power output is often limited by inefficient electron transfer between the enzyme molecules and the electrodes, hindered mass transport, low conductivity, and small active surface area of the electrodes. To tackle these issues, we herein demonstrated a novel EBFC equipped with enzyme-functionalized 3D graphene-single walled carbon nanotubes (SWCNTs) hybrid electrodes using the naturally abundant glucose as the fuel and oxygen as the oxidizer. Such EBFCs, with high stability, can nearly attain the theoretical limit of open circuit voltage (∼1.2 V) and a high power density ever reported (2.27 ± 0.11 mW cm<sup>‑2</sup>)

    Actual pressure-time histories for both the front sensor and rear sensor during the first 0.4 msec after blast.

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    <p>The peak pressure of blast wave significantly decreases from 10.14 psi (pressure before plastic tubings) measured by the front sensor to 0.27 psi (pressure behind plastic tubings) measured by the rear sensor.</p

    Schematic representing cytosolic and peroxisomal acetyl-CoA metabolism in <i>S. cerevisiae</i> under growth on C<sub>2</sub> compounds.

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    <p>(A) reference strain; Acs1p is suggested to be distributed between the cytosol and the peroxisomes, Cit2p is strongly suggested to be located in the peroxisomes, Mls1p stays in the cytosol; pathway I (in green) and II (in blue) indicate the possible shunt in the absence of <i>CIT2</i> during growth on C<sub>2</sub> compounds; (B) <i>acs1</i>Δ deletion strain; Cit2p and Mls1p are compartmentalized in the peroxisomes; (C) <i>cit2</i>Δ deletion strain; Mls1p remains in the cytosol; (D) <i>acs1</i>Δ and <i>cit2</i>Δ double deletion strain; Mls1p is targeted to the cytosol. Location of reactions marked by dashed lines was not identified in this study. Oac, oxaloacetate; Acs1p, acetyl-CoA synthetase 1; Acs2p acetyl-CoA synthetase 2; Cit2p citrate synthase 2; Mls1p malate synthase 1; Icl1p, isocitrate lyase 1; Mae1p, malic enzyme; Pck1p, phosphoenolpyruvate carboxykinase 1; Mdh2p, malate dehydrogenase 2.</p

    Physiological features of reference and single deletion strains.

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    <p>Cell growth of reference strain (A) and mutant strains SCIYC05 (<i>acs1</i>Δ) (B), SCIYC06 (<i>cit2</i>Δ) (C) and SCIYC07 (<i>mls1</i>Δ) (D), cultured in shake flask with minimal medium containing 20 g L<sup>−1</sup> glucose as carbon source. All measurements are mean +/− standard error of three biological replicates.</p

    Growth phenotypes of double deletion and the triple deletion strains.

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    <p>Plate assay for SCIYC14 (<i>acs1</i>Δ <i>cit2</i>Δ), SCIYC15 (<i>cit2</i>Δ <i>mls1</i>Δ) and SCIYC16 (<i>mls1</i>Δ <i>acs1</i>Δ), and the triple deletion strain SCIYC21 (<i>acs1</i>Δ <i>cit2</i>Δ <i>mls1</i>Δ) on minimal medium with glucose, glycerol, ethanol or acetate as carbon source. The plates were recorded photographically after 10 days incubation at 30°C.</p

    Experimental setup and the blast-testing equipments.

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    <p>(a) A photograph of the blast-testing equipment including shock tube, test frame, and front and sensors for assessment of the protective effects of the specially designed blast-resistant body armor against blast overpressure waves; (b) Schematic diagram of the blast-testing equipments. The sample of the body armor is fixed on a test frame that was approximately 40 cm away from the shock tube opening. The front sensor is placed 2 cm before the sample and the rear sensor is placed 4 cm behind the sample.</p

    Growth phenotypes of <i>mls1</i>Δ and <i>acs1</i>Δ strains expressing an SKL-less or intact Mls1p variant.

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    <p>Cells were grown on minimal medium supplemented with glucose, glycerol, ethanol or acetate as carbon source. Uracil (20 µg ml<sup>−1</sup>) was added as required (reference, SCIYC05 (<i>acs1</i>Δ) and SCIYC07 (<i>mls1</i>Δ)). The plates were recorded photographically after 10 days of incubation at 30°C.</p

    Growth phenotypes of reference and single deletion strains on minimal medium with various carbon sources.

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    <p>The plates were incubated at 30°C and recorded photographically 10 days after inoculation. The strains used were reference strain CEN.PK 113-5D, mutant strains SCIYC05 (<i>acs1</i>Δ), SCIYC23 (<i>acs2</i>Δ), SCIYC06 (<i>cit2</i>Δ) and SCIYC07 (<i>mls1</i>Δ).</p
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