A Magnetic Bead-Based Sensor for the Quantification of Multiple Prostate Cancer Biomarkers.

Novel biomarker assays and upgraded analytical tools are urgently needed to accurately discriminate benign prostatic hypertrophy (BPH) from prostate cancer (CaP). To address this unmet clinical need, we report a piezeoelectric/magnetic bead-based assay to quantitate prostate specific antigen (PSA; free and total), prostatic acid phosphatase, carbonic anhydrase 1 (CA1), osteonectin, IL-6 soluble receptor (IL-6sr), and spondin-2. We used the sensor to measure these seven proteins in serum samples from 120 benign prostate hypertrophy patients and 100 Gleason score 6 and 7 CaP using serum samples previously collected and banked. The results were analyzed with receiver operator characteristic curve analysis. There were significant differences between BPH and CaP patients in the PSA, CA1, and spondin-2 assays. The highest AUC discrimination was achieved with a spondin-2 OR free/total PSA operation--the area under the curve was 0.84 with a p value below 10(-6). Some of these data seem to contradict previous reports and highlight the importance of sample selection and proper assay building in the development of biomarker measurement schemes. This bead-based system offers important advantages in assay building including low cost, high throughput, and rapid identification of an optimal matched antibody pair

Mechanism underlying synergic activation of Tyrosinase promoter by MITF and IRF4

Background: The transcription factor interferon regulatory factor 4 (IRF4) was identified to be involved in human pigmentation by genome-wide association studies (GWASs). The rs12203592-[T/C], which is located in intron 4 of IRF4, shows the strongest link to these pigmentation phenotypes including freckling, sun sensitivity, eye and hair color. Previous studies indicated a functional cooperation of IRF4 with Microphthalmia-associated transcription factor (MITF), a causing gene of Waardenburg syndrome (WS), to synergistically trans-activate Tyrosinase (TYR). However, the underlying mechanism is still unknown. Methods: To investigate the importance of DNA binding in the synergic effect of IRF4. Reporter plasmids with mutant TYR promoters was generated to locate the IRF4 DNA binding sites in the Tyrosinase minimal promoter. By building MITF and IRF4 truncated mutations plasmids, the necessary regions of the synergy functions of these two proteins were also located. Results: The cooperative effect between MITF and IRF4 was specific for TYR promoter. The DNA-binding of IRF4 was critical for the synergic function. IRF4 DNA binding sites in TYR promoter were identified. The Trans-activation domains in IRF4 (aa134-207, aa300-420) were both important for the synergic function, whereas the auto-mask domain (aa207-300) appeared to mask the synergic effect. Mutational analysis in MITF indicated that both DNA-binding and transcriptional activation domains were both required for this synergic effect. Conclusions: Here we showed that IRF4 potently synergized with MITF to activate the TYR promoter, which was dependent on DNA binding of IRF4. The synergic domains in both IRF4 and MITF were identified by mutational analysis. This identification of IRF4 as a partner for MITF in regulation of TYR may provide an important molecular function for IRF4 in the genesis of melanocytes and the pathogenic mechanism in WS

Intracellular Detection of ATP Using an Aptamer Beacon Covalently Linked to Graphene Oxide Resisting Nonspecific Probe Displacement

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see Liu, Z., Chen, S., Liu, B., Wu, J., Zhou, Y., He, L., … Liu, J. (2014). Intracellular Detection of ATP Using an Aptamer Beacon Covalently Linked to Graphene Oxide Resisting Nonspecific Probe Displacement. Analytical Chemistry, 86(24), 12229–12235. https://doi.org/10.1021/ac503358mFluorescent aptamer probes physisorbed on graphene oxide (GO) have recently emerged as a useful sensing platform. A signal is generated by analyte-induced probe desorption. To address nonspecific probe displacement and the false positive signal, we herein report a covalently linked aptamer probe for adenosine triphosphate (ATP) detection. A fluorophore and amino dual modified aptamer was linked to the carboxyl group on GO with a coupling efficiency of ∼50%. The linearity, specificity, stability, and regeneration of the covalent sensor were systematically studied and compared to the physisorbed probe. Both sensors have similar sensitivity, but the covalent one is more resistant to nonspecific probe displacement by proteins. The covalent sensor has a dynamic range from 0.125 to 2 mM ATP in buffer at room temperature and is resistance to DNase I. Intracellular ATP imaging was demonstrated using the covalent sensor, which generated a higher fluorescence signal than the physisorbed sensor. After the cells were stimulated with 5 mM Ca2+ for ATP production, the intracellular signal enhanced by 31.8%. This work highlights the advantages of covalent aptamer sensors using GO as both a quencher and a delivery vehicle for intracellular metabolite detection.National Natural Science Foundation of China || Grant No. 81301258, 21301195 Hunan Provincial Natural Science Foundation of China || Grant No. 13JJ4029 Specialized Research Fund for the Doctoral Program of Higher Education of China || Grant No. 20130162120078 Postdoctoral Science Foundation of Central South University and China || Grant No. 124896 China Postdoctoral Science Foundation || Grant No. 2013M540644 International Postdoctoral Exchange Fellowship Program ||Grant No. 20140014 Shenghua Scholar Foundation || Natural Sciences and Engineering Research Council |