The binding of estrogen receptor (ER) to estrogen response element (ERE) is essential for genomic pathways of estrogens and gel-based electrophoretic mobility shift assay (EMSA) is commonly used for analyzing ERE binding. Gel-based EMSA, however, requires the use of hazard radio isotopes and they are slow, labor-intensive and difficult to quantify. Here, we present quantitative affinity assays based on microchip electrophoresis using PEG-modified glass microchannels, which bear neutral surfaces against the adsorption of acidic DNA molecules and basic ER proteins. We first demonstrated the feasibility of the method by measuring binding constants of recombinant ERalpha and ERbeta with a consensus ERE sequence (cERE, 5'-GGTCAGAGTGACC-3') as well as with an ERE-like sequence (ERE 1576, 5'-GACCGGTCAGCGGACTCAC-3'). Changes in mobility as a function of protein-DNA molar ratios were plotted and the dissociation constants were determined based on non-linear curve fitting. The minimum amount of ER proteins required for one assay was around 0.2 ng and the run time for one chip analysis was less than 2 min. We further measured the estrogenic compound-mediated dissociation constants with recombinant ER proteins as well as with the extracted ERbeta from treated and untreated A549 bronchioloalveolar carcinoma cells. Dissociation constants determined by this method agree with the fact that agonist compounds such as 17beta-estradiol (1.70 nM), diethylstilbestrol (0.14 nM), and genistein (0.80 nM) assist ERE binding by decreasing the constants; while antagonist compounds such as testosterone (140.4 nM) and 4-hydroxytamoxifen (10.5 nM) suppress the binding by increasing the dissociation constant.
We report on the photopatterning of single carbon nanotube composites with soft hydrogel polymers in glass microchannels. Since the hydrogels by themselves are able to withstand liquid flow within the microchannels, we covalently combined them with single-walled carbon nanotubes to impart mechanical strength. We attempted this approach by patterning the gels within the microchannels without prior surface modifications. Our results show that the 1-cm nanocomposite hydrogels are far stronger than the free hydrogels. Moreover, the nanocomposites were able to concentrate and separate proteins within a 1.5-cm distance using gel-free buffers. The separation cannot only be tuned by changing the running buffer; the lack of gels in the running buffer reduces the chance of channel blockage and thus the lifetime of the device is prolonged. The usefulness of the patterned nanocomposites may be extended by a wide selection of nanocomposite properties and monomers to find a broad range of applications in lab-on-chip technology.
Based on the fatigue test of 6 stainless steel reinforced concrete beams, number of stress cycles, dropping-coefficient of stiffness and crack width are studied. This paper mainly discussed, under different stress states and reinforcement ratio, the influence of the reinforced concrete beam with stainless steel rebar on crack development, deflection and fatigue life. It can be concluded that, the fatigue life of the stainless steel reinforced concrete beam will go up as the stress decreases. Based on the analysis of the test results, the S-N curve can be deduced.
In order to study the elastic-plastic mechanics properties of the stainless steel reinforced concrete columns under low-cyclic load, the engineering open-source earthquake simulation system OpenSees is used to carry out the numerical simulation. The comparison between the computed results and the pseudo-static test results shows that the OpenSees may stimulate the mechanical properties of the stainless steel reinforced concrete columns by using the fiber element model, both of the skeleton curves and hysteretic curves are well agreement with the tests results.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.