This ''Applications'' article focuses on superquenching of the fluorescence of conjugated polyelectrolytes and related fluorescent polyelectrolytes and its applications to biosensing. Superquenching can occur for both aqueous solutions of these polymers as well as for various supported formats. For biosensing the supported formats are generally most practical. Three practical sensing applications are reviewed: nucleic acids, protease enzyme activity assays and kinase/phosphatase assays based on metal-ion-mediated superquenching.

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An assay technology for high-throughput screening of kinase and phosphatase activities is introduced. The format is based upon superquenching of fluorescent-conjugated polymers by dyelabeled kinase͞phosphatase peptide substrates. The sensor platform is composed of highly fluorescent-conjugated polyelectrolytes colocated with the phosphate coordinating metal ion gallium on microspheres. Phosphorylated peptide substrates containing a quencher bind specifically to the metal ions by means of phosphate groups, resulting in quench of polymer fluorescence. The modulation of fluorescence signal is proportional to kinase or phosphatase activity and is monitored as a turn-off or turn-on signal, respectively. The assay is homogeneous and simple and can be run either as an endpoint measurement or in a kinetic mode. The assay meets the sensitivity required for high-throughput screening of kinase or phosphatase inhibitors and is a valuable tool for drug discovery. A modified version of the assay allows for the detection of protein phosphorylation. P hosphorylation and dephosphorylation of proteins by kinase and phosphatase enzymes mediate the regulation of cellular metabolism, growth, differentiation, and proliferation (1-3). Aberrations in kinase and phosphatase activities can lead to inflammation and diseases such as cancer (4, 5). More than 500 kinases and phosphatases are thought to be involved in the regulation of cellular activity and are possible targets for drug therapy (6). Of the kinases, Ϸ90% phosphorylate serine residues, 10% threonine, and 0.1% tyrosine residues (7). Although it has become possible to develop anti-phospho-tyrosine antibodies (8), those against phospho-serine and threonine residues are of low affinity and are often specific to only one kinase (9). Currently, non-antibody-based high-throughput screening (HTS) assays are based on methods such as time-resolved fluorescence (TRF) (10), fluorescence polarization (FP) (11-13), or fluorescence resonance energy transfer (FRET) (14). These assays require specialized equipment and͞or suffer from low fluorescence intensity change as a function of enzyme activity and generally cannot be used to detect phosphorylation of natural, chemically unmodified protein substrates. The use of native substrates is attractive because inhibitor screens may yield novel inhibitors that affect the enzyme docking site, which can be at a site distant from the active site.We sought to enhance sensitivity in the measurement of enzymatic activity by amplifying the fluorescence signal using superquenching (15)(16)(17)(18)(19)(20)(21)(22)(23)(24). This phenomenon has been described in several reports and is based on the finding that photoluminescence of conjugated polymers and related polymeric ensembles can be quenched by means of energy and͞or electron transfer to small molecule quenchers (15)(16)(17)22). In previous studies, it was found that one quencher molecule can quench the photoluminescence of up to several hundred polymer repeat units (25)(26)(27).Our sensor platform compri...

Breast cancer (BC) is the most frequently diagnosed cancer and the leading cause of cancer-related death among women worldwide. Evidence indicates that posttranscriptional N6-methyladenosine (m6A) modification modulates BC development. In the present study, we assessed BC and normal tissues to investigate this connection. RNA m6A levels were determined by methylation quantification assay. The effects of methyltransferase-like 14 (METTL14) gain-of-expression or co-transfection with an m6A inhibitor on cell migration and invasion abilities were determined by Transwell assays. The levels of differentially expressed (DE) miRNAs were verified by real-time quantitative PCR (RT-qPCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses (KEGG) were performed to analyze potential function of target genes of the DE miRNAs. The effects of candidate miRNAs modulated by METTL14 on cell migration and invasion abilities were confirmed by Transwell assays. We demonstrated that m6A methyltransferase METTL14 was significantly upregulated in BC tissues compared with normal tissues. METTL14 gain-and loss-of-expression regulated m6A levels in MCF-7 and MDA-MB-231 cells. The cell function assays revealed that METTL14 overexpression enhanced the migration and invasion capacities of BC cells. Moreover, treatment with the m6A inhibitor suppressed this enhanced cell migration and invasion. Additionally, aberrant expression of METTL14 reshaped the miRNA profile in BC cell lines. The remodeled DE miRNA/mRNA network was found to be most enriched in cancer pathways, and DE miRNAs were enriched in cell adhesion terms. hsa-miR-146a-5p modulated by METTL14 promoted cell migration and invasion. METTL14 modulates m6A modification and hsa-miR-146a-5p expression, thereby affecting the migration and invasion of breast cancer cells.

The lack of sensitive and specific biomarkers hinders pathological diagnosis and prognosis for hepatocellular carcinoma (HCC). Since glutaminolysis plays a crucial role in carcinogenesis and progression, we sought to determine if the expression of kidney-type and liver-type glutaminases (GLS1 and GLS2) were informative for pathological diagnosis and prognosis of HCC. We compared the expression of GLS1 and GLS2 in a large set of clinical samples including HCC, normal liver, and other liver diseases. We found that GLS1 was highly expressed in HCC; whereas, expression of GLS2 was mainly confined to non-tumor hepatocytes. The sensitivity and specificity of GLS1 for HCC were 96.51% and 75.21%, respectively. A metabolic switch from GLS2 to GLS1 was observed in a series of tissues representing progressive pathologic states mimicking HCC oncogenic transformation, including normal liver, fibrotic liver, dysplasia nodule, and HCC. We found that high expression of GLS1 and low expression of GLS2 in HCC correlated with survival time of HCC patients. Expression of GLS1 and GLS2 were independent indexes for survival time; however, prognosis was predominantly determined by the level of GLS1 expression. These findings indicate that GLS1 expression is a sensitive and specific biomarker for pathological diagnosis and prognosis of HCC.