Detection of Hepatitis B Virus (HBV) DNA at femtomolar concentrations using a silica nanoparticle-enhanced microcantilever sensor

We report Hepatitis B Virus (HBV) DNA detection using a silica nanoparticle-enhanced dynamic microcantilever biosensor. A 243-mer nucleotide of HBV DNA precore/core region was used as the target DNA. For this assay, the capture probe on the microcantilever surface and the detection probe conjugated with silica nanoparticles were designed specifically for the target DNA. For efficient detection of the HBV target DNA using silica nanoparticle-enhanced DNA assay, the size of silica nanoparticles and the dimension of microcantilever were optimized by directly binding the silica nanoparticles through DNA hybridization. In addition, the correlation between the applied nanoparticle concentrations and the resonant frequency shifts of the microcantilever was discussed clearly to validate the quantitative relationship between mass loading and resonant frequency shift.HBV target DNAs of 23.1 fM to 2.31 nM which were obtained from the PCR product were detected using a silica nanoparticle-enhanced microcantilever. The HBV target DNA of 243-mer was detected up to the picomolar (pM) level without nanoparticle enhancement and up to the femtomolar (fM) level using a nanoparticle-based signal amplification process. In the above two cases, the resonant frequency shifts were found to be linearly correlated with the concentrations of HBV target DNAs. We believe that this linearity originated mainly from an increase in mass that resulted from binding between the probe DNA and HBV PCR product, and between HBV PCR product and silica nanoparticles for the signal enhancement, even though there is another potential factor such as the spring constant change that may have influenced on the resonant frequency of the microcantilever.     

Sudden failure of biological nitrogen and carbon removal in the full-scale pre-denitrification process treating cokes wastewater

A full-scale pre-denitrification process treating cokes wastewater containing toxic compounds such as phenols, cyanides and thiocyanate has shown good performance in carbon and nitrogen removal. However, field operators have been having trouble with its instability without being able to identify the causes. To clarify the main cause of these sudden failures of the process, comprehensive studies were conducted on the pre-denitrification process using a lab-scale reactor system with real cokes wastewater. First, the shock loading effects of three major pollutants were investigated individually. As the loading amount of phenol increased to 600 mg/L, more COD, TOC and phenol itself were flowed into the aerobic reactor, but phenol itself did not inhibit nitrification and denitrification, owing to the effect of dilution and its rapid biodegradation. Higher loading of ammonia or thiocyanate slightly enhanced the removal efficiency of organic matter, but caused the final discharge concentration of total nitrogen to be above its legal limit of 60 mg-N/L. Meanwhile, continuous inflow of abnormal wastewater collected during unstable operation of the full-scale pre-denitrification process, caused a sudden failure of nitrogen removal in the lab-scale process, like the removal pattern of the full-scale one. This was discovered to be due to the lack of inorganic carbon in the aerobic reactor where autotrophic nitrification occurs.     

Design,synthesis, screening,and molecular modeling study of a new series of ROS1 receptor tyrosine kinase inhibitors

A series of rationally designed ROS1 tyrosine kinase inhibitors was synthesized and screened. Compound 12b has showed good potency with IC₅₀ value of 209 nM, which is comparable with that of the reference lead compound 1. Molecular modeling studies have been performed, that is, a homology model for ROS1 was built, and the screened inhibitors were docked into its major identified binding site. The docked poses along with the activity data have revealed a group of the essential features for activity. Overall, simplification of the lead compound 1 into compound 12b has maintained the activity, while facilitated the synthetic advantages. A molecular interaction model for ROS1 kinase and inhibitors has been proposed.     

Spiropiperidine CCR5 antagonists

A novel series of CCR5 antagonists has been identified, utilizing leads from high-throughput screening which were further modified based on insights from competitor molecules. Lead optimization was pursued by balancing opposing trends of metabolic stability and potency. Selective and potent analogs with good pharmacokinetic properties were successfully developed.     

Identification of compounds exhibiting inhibitory activity toward the Pseudomonas tolaasii toxin tolaasin I using in silico docking calculations,NMR binding assays,and in vitro hemolytic activity assays

Using in silico docking calculations, NMR analysis of target–ligand binding, and hemolytic activity assays, we searched a 30,000-compound library for an effective inhibitor of tolaasin I, a Pseudomonas tolaasii toxin that causes virulent infection in mushrooms. Of more than 30,000 compounds screened in silico, two compounds were selected. One of these compounds, sorbitololeic acid, bound to tolaasin I and inhibited its hemolytic activity in vitro. Therefore, sorbitololeic acid can be a potential inhibitor of tolaasin I.     

Genetic selection system for improving recombinant membrane protein expression in E. coli

A major barrier to the physical characterization and structure determination of membrane proteins is low yield in recombinant expression. To address this problem, we have designed a selection strategy to isolate mutant strains of Escherichia coli that improve the expression of a targeted membrane protein. In this method, the coding sequence of the membrane protein of interest is fused to a C‐terminal selectable marker, so that the production of the selectable marker and survival on selective media is linked to expression of the targeted membrane protein. Thus, mutant strains with improved expression properties can be directly selected. We also introduce a rapid method for curing isolated strains of the plasmids used during the selection process, in which the plasmids are removed by in vivo digestion with the homing endonuclease I‐CreI. We tested this selection system on a rhomboid family protein from Mycobacterium tuberculosis (Rv1337) and were able to isolate mutants, which we call EXP strains, with up to 75‐fold increased expression. The EXP strains also improve the expression of other membrane proteins that were not the target of selection, in one case roughly 90‐fold.     

Promoter hypermethylation and Up-regulation of thyroid-stimulating-hormone-alpha (TSH-α) in thyroid cancer

Thyroid-stimulating-hormone-alpha (TSH-α) is the common subunit of the heterodimeric hormone TSH and also of other glycoprotein hormones. Although both expression and promoter-methylation profiles of the gene have been observed in the pituitary gland and placenta, no observation has been reported in the thyroid gland. We examined TSH-α expression in normal and cancer thyroid tissues. Real-time RT-PCR and immunohistochemistry indicated that TSH-a was repressed in normal tissues while activated in cancer tissues. To identify the epigenetic mechanism of upregulation of TSH-α, the methylation status of the seven CpG sites in the TSH-a promoter was examined in sixty thyroid cancer tissues. Two CpG sites showed remarkably higher levels of methylation in cancer (46 and 45%) than in normal tissues (24 and 23%) (p=0.010 and 0.003). These findings indicate that TSH-α is expressed in the thyroid cancer tissue per se and that its expression can be affected by promoter methylation.