Effect of dietary patterns on the blood/urine concentration of the selected toxic metals (Cd,Hg, Pb) in Korean children

Food Science and Biotechnology - This study was aimed to examine the association the blood/urinary concentration of toxic metal (Hg, Pb, and Cd) with children’s dietary patterns. This...     

On-chip, cell-based microarray immunofluorescence assay for high-throughput analysis of target proteins

We have developed an immunofluorescence-based assay for high-throughput analysis of target proteins on a three-dimensional cellular microarray platform. This process integrates the use of three-dimensional cellular microarrays, which should better mimic the cellular microenvironment, with sensitive immunofluorescence detection and provides quantitative information on cell function. To demonstrate this assay platform, we examined the accumulation of the alpha subunit of the hypoxia-inducible factor (HIF-1alpha) after chemical stimulation of human pancreatic tumor cells encapsulated in 3D alginate spots in volumes as low as 60 nL. We also tested the effect of the known dysregulator of HIF-1alpha, 2-methoxyestradiol (2ME2), on the levels of HIF-1alpha using a dual microarray stamping technique. This chip-based in situ Western immunoassay protocol was able to provide quantitative information on cell function, namely, the cellular response to hypoxia mimicking conditions and the reduction of HIF-1alpha levels after cell treatment with 2ME2. This system is the first to enable high-content screening of cellular protein levels on a 3D human cell microarray platform.     

Bioinformatics-driven, rational engineering of protein thermostability

A longstanding goal in protein engineering is to identify specific sequence changes that endow proteins with desired functional properties. As opposed to traditional rational and random protein engineering techniques, we have employed a bioinformatic approach to identify specific sequence changes that influence key functional properties of a protein within a defined superfamily. Specifically, we have used the Bayesian sequence-based algorithms PROBE and Classifier to identify a strand-turn-strand motif that contributes to thermophilicity among members of the serine protease subtilase superfamily. By replacing a 16 amino acid sequence in the mesophilic subtilisin E (from Bacillus subtilis) with a bioinformatics-generated thermophilic model sequence, the melting temperature of subtilisin E was increased by 13 degrees C. While wild-type subtilisin E was inactive at 90 degrees C, the mutant retained a substantial fraction of its function, with ca. one-third of the activity that it has at 45 degrees C.