Hybridization kinetics on microarray surfaces

Kinetic hybridization data are compared to a number of different models. A first-order Langmuir model provides the best fit to the data.     

A cancer protein microarray platform using antibody fragments and its clinical applications

Antibody microarrays have shown great potential for measurement of either a spectrum of target proteins in proteomics or disease-associated antigens in molecular diagnostics. Despite its importance, the applications of antibody microarrays are still limited by a variety of fundamental problems. Among them, cross-reactivity significantly limits the multiplexing ability in parallel sandwich immunoassays. As a result, it is very important to design new capture probes in order to incorporate a universal label into the assay configuration. In this report, an antibody fragments (F(ab')2) microarray platform for serum tumor markers was developed. Each antigen was detected at different concentrations to assemble its calibration curve, and combinations of different markers were tested to examine the specificity of simultaneous detection based on the F(ab')2 microarrays. Diagnostics of serum samples with this cancer antibody microarray platform and immunoradiometric assays (IRMA) were also performed. Wide range calibration curves (0-1280 U mL(-1)) were obtained for each tumor marker. Comparative studies demonstrated that such F(ab')2 microarrays exhibited both moderately improved sensitivity and better specificity than full-sized monoclonal antibody microarrays. It is also demonstrated that this microarray platform is quantitative, highly specific and reasonably sensitive. More importantly, clinical applications of our F(ab')2 microarray platform for upwards of 100 patient serum samples clearly show its potential in cancer diagnostics.     

Development and validation of a liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of D-amphetamine and diphenhydramine in beagle dog plasma and its application to a pharmacokinetic study

A new drug, quick-acting anti-motion capsule (QAAMC) composed of d-amphetamine sulfate, dimenhydrinate and ginger extraction has been studied for anti-motion-sickness use. We have developed a sensitive, specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantitative determination of d-amphetamine and diphenhydramine, the main effective components of the QAAMC, using pseudoephedrine as the internal standard. The analytes and internal standard were isolated from 200 microL plasma samples by a simple liquid-liquid extraction (LLE). Reverse-phase HPLC separation was accomplished on a Zorbax SB-C18 column (100 mm x 3.0 mm, 3.5 microm) with a mobile phase composed of methanol-water-formic acid (65:35:0.5, v/v/v) at a flow rate of 0.2 mL/min. The method had a chromatographic total run time of 5 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 136.0-->91.0 (D-amphetamine), 256.0-->167.0 (diphenhydramine) and 166.1-->148.0 (IS) used for quantitation. The method was sensitive with a lower limit of quantitation (LLOQ) of 0.5 ng/mL for d-amphetamine and 1 ng/mL for diphenhydramine, with good linearity in the range 0.5-200 ng/mL for D-amphetamine and 1-500 ng/mL for diphenhydramine (r(2)> or =0.9990). All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic study of the QAAMC in beagle dogs.     

MDM2 splice variants predominantly localize to the nucleoplasm mediated by a COOH-terminal nuclear localization signal

Of the >40 alternative and aberrant splice variants of MDM2 that have been described to date, the majority has lost both the well-characterized nuclear localization signal (NLS1) and the nuclear export signal (NES) sequence. Because cellular localization of proteins provides insight regarding their potential function, we determined the localization of three different MDM2 splice variants. The splice variants chosen were the common variants MDM2-A and MDM2-B. In addition, MDM2-FB26 was chosen because it is one of the few variants described that contains the complete p53-binding site. All three splice variants predominantly localized to the nucleus. Nuclear localization of MDM2-A and MDM2-B was controlled by a previously uncharacterized nuclear localization signal (NLS2), whereas nucleoplasmic localization of MDM2-FB26 was mediated by NLS1. p53 and full-length MDM2 colocalized with the splice variants in the nucleus. MDM2-A and MDM2-B both contain a COOH-terminal RING finger domain, and interaction with full-length MDM2 through this domain was confirmed. MDM2-FB26 was the only splice variant evaluated that contained a p53-binding domain; however, interaction between MDM2-FB26 and p53 could not be shown. p14(ARF) did not colocalize with the splice variants and was predominantly expressed within the nucleoli. In summary, nuclear localization signals responsible for the nucleoplasmic distribution of MDM2 splice variants have been characterized. Colocalization and interaction of MDM2-A and MDM2-B with full-length MDM2 in the nucleus have important physiologic consequences, for example, deregulation of p53 activity.     

Antisense RNA-mediated suppression of Bmi-1 gene expression inhibits the proliferation of lung cancer cell line A549

The oncogene Bmi-1 regulates cell proliferation and senescence. It is reported that it controlled the self-renewal of leukemic and breast cancer stem cell and was overexpressed in some solid tumors and hematologic malignancies. In this study, the effects of inactivation of Bmi-1 mediated by a plasmid-expressing antisense Bmi-1 RNA on the proliferation of lung cancer cell line A549 were investigated. As a result, when the plasmid was stably introduced into the cell line, the Bmi-1 protein level was specifically downregulated, and the cell proliferation was significantly inhibited as shown by the cell growth curve and colony forming assay. The cells were found mostly in the phase of G(0)/G(1) and cells in S phase were significantly decreased. Our results suggest that targeting Bmi-1 might be a therapeutic potential for the treatment of non-small-cell lung cancer.     

Dissecting multiple steps of GLUT4 trafficking and identifying the sites of insulin action

Insulin-stimulated GLUT4 translocation is central to glucose homeostasis. Functional assays to distinguish individual steps in the GLUT4 translocation process are lacking, thus limiting progress toward elucidation of the underlying molecular mechanism. Here we have developed a robust method, which relies on dynamic tracking of single GLUT4 storage vesicles (GSVs) in real time, for dissecting and systematically analyzing the docking, priming, and fusion steps of GSVs with the cell surface in vivo. Using this method, we have shown that the preparation of GSVs for fusion competence after docking at the surface is a key step regulated by insulin, whereas the docking step is regulated by PI3K and its downstream effector, the Rab GAP AS160. These data show that Akt-dependent phosphorylation of AS160 is not the major regulated step in GLUT4 trafficking, implicating alternative Akt substrates or alternative signaling pathways downstream of GSV docking at the cell surface as the major regulatory node.     

Generation of pigs with humanized type Ⅱ collagen by precise human COL2A1 gene knock-in

<正>Osteoarthritis (OA) is the most common chronic disease, characterized by progressive cartilage breakdown, subchondral bone sclerosis, and aberrant bone outgrowth (Yucesoy et al., 2015; Hussain et al., 2016). OA is one of the leading causes of cartilage damage. Patients with severe cartilage damage require transplantation of articular cartilage to improve their quality of life. Type Ⅱ collagen is a major component of articular cartilage and intervertebral discs and plays an important rol...     

Mettl13 protects against cardiac contractile dysfunction by negatively regulating C-Cbl-mediated ubiquitination of SERCA2a in ischemic heart failure

Ischemic heart failure(HF) remains a leading cause of morbidity and mortality. Maintaining homeostasis of cardiac function and preventing cardiac remodeling deterioration are critical to halting HF progression. Methyltransferase-like protein 13(Mettl13) has been shown to regulate protein translation efficiency by acting as a protein lysine methyltransferase, but its role in cardiac pathology remains unexplored. This study aims to characterize the roles and mechanisms of Mettl13 in cardiac contra...     

Single-cell-resolution transcriptome map revealed novel genes involved in testicular germ cell progression and somatic cells specification in Chinese tongue sole with sex reversal

Female-to-male sex reversals(pseudomales) are common in lower vertebrates and have been found in natural populations, which is a concern under rapid changes in environmental conditions. Pseudomales can exhibit altered spermatogenesis. However, the regulatory mechanisms underlying pseudomale spermatogenesis remain unclear. Here, we characterized spermatogenesis in Chinese tongue sole(Cynoglossus semilaevis), a species with genetic and environmental sex determination, based on a highresolution sin...