An Aqueous Two-Phase System for the Concentration and Extraction of Proteins from the Interface for Detection Using the Lateral-Flow Immunoassay

The paper-based immunoassay for point-of-care diagnostics is widely used due to its low cost and portability over traditional lab-based assays. Lateral-flow immunoassay (LFA) is the most well-established paper-based assay since it is rapid and easy to use. However, the disadvantage of LFA is its lack of sensitivity in some cases where a large sample volume is required, limiting its use as a diagnostic tool. To improve the sensitivity of LFA, we previously reported on the concentration of analytes into one of the two bulk phases of an aqueous two-phase system (ATPS) prior to detection. In this study, we preserved the advantages of LFA while significantly improving upon our previous proof-of-concept studies by employing a novel approach of concentrating gold nanoparticles, a common LFA colorimetric indicator. By conjugating specific antibodies and polymers to the surfaces of the particles, these gold nanoprobes (GNPs) were able to capture target proteins in the sample and subsequently be concentrated within 10 min at the interface of an ATPS solution comprised of polyethylene glycol, potassium phosphate, and phosphate-buffered saline. These GNPs were then extracted and applied directly to LFA. By combining this prior ATPS interface extraction with LFA, the detection limit of LFA for a model protein was improved by 100-fold from 1 ng/μL to 0.01 ng/μL. Additionally, we examined the behavior of the ATPS system in fetal bovine serum and synthetic urine to more closely approach real-world applications. Despite using more complex matrices, ATPS interface extraction still improved the detection limit by 100-fold within 15 to 25 min, demonstrating the system’s potential to be applied to patient samples.     

A conserved Pol? binding module in Ctf18-RFC is required for S-phase checkpoint activation downstream of Mec1

Defects during chromosome replication in eukaryotes activate a signaling pathway called the S-phase checkpoint, which produces a multifaceted response that preserves genome integrity at stalled DNA replication forks. Work with budding yeast showed that the ‘alternative clamp loader’ known as Ctf18-RFC acts by an unknown mechanism to activate the checkpoint kinase Rad53, which then mediates much of the checkpoint response. Here we show that budding yeast Ctf18-RFC associates with DNA polymerase epsilon, via an evolutionarily conserved ‘Pol ϵ binding module’ in Ctf18-RFC that is produced by interaction of the carboxyl terminus of Ctf18 with the Ctf8 and Dcc1 subunits. Mutations at the end of Ctf18 disrupt the integrity of the Pol ϵ binding module and block the S-phase checkpoint pathway, downstream of the Mec1 kinase that is the budding yeast orthologue of mammalian ATR. Similar defects in checkpoint activation are produced by mutations that displace Pol ϵ from the replisome. These findings indicate that the association of Ctf18-RFC with Pol ϵ at defective replication forks is a key step in activation of the S-phase checkpoint.     

Targeted inhibition of the EGFR pathways enhances Zn‐BC‐AM PDT‐induced apoptosis in well‐differentiated nasopharyngeal carcinoma cells

Epidermal growth factor receptor (EGFR), a receptor often expressed in nasopharyngeal carcinoma (NPC) cells, is one of the recently identified molecular targets in cancer treatment. In the present study, the effects of combined treatment of Zn‐BC‐AM PDT with an EGFR inhibitor AG1478 were investigated. Well‐differentiated NPC HK‐1 cells were subjected to PDT with 1 µM of Zn‐BC‐AM and were irradiated at a light dose of 1 J/cm² in the presence or absence of EGFR inhibitor AG1478. Specific protein kinase inhibitors of downstream EGFR targets were also used in the investigation. EGFR, Akt, and ERK were found constitutively activated in HK‐1 cells and the activities could be inhibited by the EGFR inhibitor AG1478. A sub‐lethal concentration of AG1478 was found to further enhance the irreversible cell damage induced by Zn‐BC‐AM PDT in HK‐1 cells. Pre‐incubation of the cells with specific inhibitors of EGFR (AG1478), PI3k/Akt (LY294002), or MEK/ERK (PD98059) before light irradiation were found to enhance Zn‐BC‐AM PDT‐induced formation of apoptotic cells. The efficacy of Zn‐BC‐AM PDT can be increased through the inhibition of EGFR/PI3K/Akt and EGFR/MEK/ERK signaling pathways in NPC cells. Combination therapy with Zn‐BC‐AM PDT and EGFR inhibitors may further be developed for the treatment of advanced NPC. J. Cell. Biochem. 108: 1356–1363, 2009. © 2009 Wiley‐Liss, Inc.     

The Melanogenesis-Inhibitory Effect and the Percutaneous Formulation of Ginsenoside Rb1

Ginsenoside Rb1 (Rb1) is the most predominant ginsenoside isolated from the roots of ginseng (Panax ginseng C. A. Meyer). This compound is active in various human biological pathways that are involved in human collagen synthesis and inhibition of cell apoptosis. In this study, the skin-whitening effects of Rb1 were investigated in B16 melanoma cells. Our results showed that Rb1 inhibited melanogenesis in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16 cells in a dose-dependent manner, which collectively indicated that Rb1 may have skin-whitening effects and may be formulated into skin-whitening products for skin care. Accordingly, a ginsenoside collagen transdermal patch was developed as a vehicle to topically deliver Rb1 into pig skin. The percutaneous permeation, retention within skin, and release in vitro of Rb1 from seven transdermal patch formulas were studied. It was determined that the best formula for ginsenoside collagen transdermal patch is made of protein collagen hydrolysate powder (PCHP) 2.0% (w/w), methyl cellulose (MC) 0.5% (w/w), polyethyleneglycol 6000 (PEG6000) 0.5% (w/w), ginsenoside 0.036% (w/w), azone 0.4% (v/w), menthol 0.20% (w/w), and water.     

The Effect of pH and Heat Pre-Treatments on the Physicochemical and Emulsifying Properties of β-lactoglobulin

The overall goal of this research was to investigate structure-function mechanisms associated the emulsifying properties β-lactoglobulin (β-LG). Specifically the physicochemical (i.e., surface charge and hydrophobicity, size and interfacial tension) and emulsifying (i.e., emulsification activity (EAI) and stability indices (ESI)) properties of β-LG were investigated in response to changes in pH (3.0, 5.0 and 7.0) and heat pre-treatment conditions (25, 55 and 85 °C). Hydrophobicity was found to be greatest at pH 5.0/85 °C, whereas at all conditions it was significantly lower. Surface charge on β-LG was found to be neutral at?~?pH 3.9, regardless of conditions. Aggregate size was also found to be highest at pH 5.0/85 °C (avg. hydrodynamic radii of ~714 nm), corresponding to a reduced net surface charge and high hydrophobicity. Little size dependence of aggregates was observed at pH 3.0 regardless to the temperature pre-treatments (radii ~120 nm). In contrast, at pH 7.0 slight temperature dependence was apparent, where treatments at 25, 55 and 85 °C led to radii of 412.8, 307.2 and 232.3 nm, respectively. Overall, the addition of β-LG to a canola oil–water system resulted in a decline in interfacial tension from ~28 mN/m to ~18 mN/m, however the effect of pH/temperature conditions was minimal. EAI was found to be highest when β-LG solutions displayed high surface charge combined with moderate hydrophobicity. In contrast, ESI was higher under conditions where β-LG solutions remained in a native (25 °C) or fully denatured state (85 °C) versus one in where partially unravelling may be occurring (55 °C).