Yongseong Kim

Separation of DNA sequencing fragments up to 1000 bases by using poly(ethylene oxide)-filled capillary electrophoresis.

We have demonstrated that DNA bases up to 1000 base pairs (bp) in a sequencing ladder can be separated using poly(ethylene oxide)-filled capillary electrophoresis (resolution of raw data = 0.5 at 966 bp). Separation performance of this sieving matrix has been tested under different experimental conditions. It was found that the electric field strength played a critical role in the onset of reptation and thus the separation efficiency. Optimized gel composition and concentration is required for good separation, but the total gel concentration should lie between 2.5 and 3.0%. We observed that the capillary length influences the number of theoretical plates and the maximum readable length of DNA. For sequencing up to 500 bp, relatively nonviscous solutions can be used, greatly facilitating the replacement of the sieving matrix in between runs.

Protein analysis with large volume sample stacking with an electrosmotic flow pump: a potential approach for proteomics

Large volume sample stacking using electrosmotic flow pump (LVSEP) in capillary electrophoresis is a useful tool for on-line concentration of low-abundant protein samples for proteomics research. While the stacking of only small anions with high pKa values has been reported at low pH buffers (pH 4) were stacked successfully in our system using LVSEP. Initially, the whole column of the SIL, or LPA-coated capillary was filled with protein samples (∼1 μg/ml), then a high voltage was applied without polarity switching with high pH buffer (pH 8.5). An enhancement factor of more than 100 times was achieved compared to the conventional pressure injection method.

Capillary and microchip gel electrophoresis for simultaneous detection of Salmonella pullorum and Salmonella gallinarum by rfbS allele-specific PCR.

We report the use of capillary gel electrophoresis (CGE) based on a rfbS allele-specific polymerase chain reaction (PCR) for the analysis and simultaneous detection of Salmonella pullorum and Salmonella gallinarum, which are the major bacterial pathogens in poultry. rfbS allele-specific PCR was used to concurrently amplify two specific 147- and 187-bp DNA fragments for the simultaneous detection of S. pullorum and S. gallinarum at an annealing temperature of 54+/-1 degrees C and an MgCl(2) concentration of 2.8-5.6mM. Under an electric field of 333.3V/cm and a sieving matrix of 1.0% poly(ethyleneoxide) (M(r) 600000), the amplified PCR products were analyzed within 6min by CGE separation. This CGE assay could be translated to microchip format using programmed field strength gradients (PFSG). In the microchip gel electrophoresis with PFSG, both of the Salmonella analyses were completed within 30s, without decreasing the resolution efficiency. rfbS allele-specific PCR-microchip gel electrophoresis with the PFSG technique might be a new tool for the simultaneous detection of both S. pullorum and S. gallinarum, due to its ultra-speed and high efficiency.

Patterning Si by using surface functionalization and microcontact printing with a polymeric ink

This paper describes a simple procedure for patterning Si substrate using a combination of surface functionalization and microcontact printing(ΜCP). The Si/SiO2 surfaces were chemically modified to present self-assembled monolayers (SAMs) of siloxanes terminating in reactive carboxylic anhydride groups and then patterned with poly(ethylene imine) (PEI) by, ΜCP We used the patterned thin films of PEI as etch resists on Si surfaces.

Direct Determination of Strontium in Marine Algae Samples by Laser-Induced Breakdown Spectrometry

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Novel chemical scaffolds of the tumor marker AKR1B10 inhibitors discovered by 3D QSAR pharmacophore modeling

Aim:Recent evidence suggests that aldo-keto reductase family 1 B10 (AKR1B10) may be a potential diagnostic or prognostic marker of human tumors, and that AKR1B10 inhibitors offer a promising choice for treatment of many types of human cancers. The aim of this study was to identify novel chemical scaffolds of AKR1B10 inhibitors using in silico approaches.Methods:The 3D QSAR pharmacophore models were generated using HypoGen. A validated pharmacophore model was selected for virtual screening of 4 chemical databases. The best mapped compounds were assessed for their drug-like properties. The binding orientations of the resulting compounds were predicted by molecular docking. Density functional theory calculations were carried out using B3LYP. The stability of the protein-ligand complexes and the final binding modes of the hit compounds were analyzed using 10 ns molecular dynamics (MD) simulations.Results:The best pharmacophore model (Hypo 1) showed the highest correlation coefficient (0.979), lowest total cost (102.89) and least RMSD value (0.59). Hypo 1 consisted of one hydrogen-bond acceptor, one hydrogen-bond donor, one ring aromatic and one hydrophobic feature. This model was validated by Fischers randomization and 40 test set compounds. Virtual screening of chemical databases and the docking studies resulted in 30 representative compounds. Frontier orbital analysis confirmed that only 3 compounds had sufficiently low energy band gaps. MD simulations revealed the binding modes of the 3 hit compounds: all of them showed a large number of hydrogen bonds and hydrophobic interactions with the active site and specificity pocket residues of AKR1B10.Conclusion:Three compounds with new structural scaffolds have been identified, which have stronger binding affinities for AKR1B10 than known inhibitors.

A lazy learning-based QSAR classification study for screening potential histone deacetylase 8 (HDAC8) inhibitors

Histone deacetylases 8 (HDAC8) is an enzyme repressing the transcription of various genes including tumour suppressor gene and has already become a target of human cancer treatment. In an effort to facilitate the discovery of HDAC8 inhibitors, two quantitative structure–activity relationship (QSAR) classification models were developed using K nearest neighbours (KNN) and neighbourhood classifier (NEC). Molecular descriptors were calculated for the data set and database compounds using ADRIANA.Code of Molecular Networks. Principal components analysis (PCA) was used to select the descriptors. The developed models were validated by leave-one-out cross validation (LOO CV). The performances of the developed models were evaluated with an external test set. Highly predictive models were used for database virtual screening. Furthermore, hit compounds were subsequently subject to molecular docking. Five hits were obtained based on consensus scoring function and binding affinity as potential HDAC8 inhibitors. Finally, HDAC8 structures in complex with five hits were also subjected to 5 ns molecular dynamics (MD) simulations to evaluate the complex structure stability. To the best of our knowledge, the NEC classification model used in this study is the first application of NEC to virtual screening for drug discovery.

Study of matrix and polymer substrate in MALDI-TOF mass spectrometry of DNA

Protein matrices such as 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, SA) and a-cyano-4-hydroxycinnamic acid (CHCA) tend to yield homogeneous dried spots. However, well known MALDI matrices for single- and double-stranded DNA such as 3-hydroxy picolinic acid (HPA) and picolinic acid (PA) forms the crystals at the rim of their spots with uneven distribution of matrix and DNA. This inhomogeneous deposition of DNA-doped matrix crystals at the MALDI spot requires a search for sweet spots. It is important to obtain homogeneous MALDI spots that yield signals not only from the periphery but the entire spot for automated, high throughput MALDI-TOF analysis of short DNA fragments. We have investigated the characteristics of MALDI matrices for DNA and presented a method for improving the homogeneity of MALDI samples by using polymer substrates such as linear polyacrylamide (LPA), poly(ethylene oxide) (PEO), methyl cellulose (MC) and Nafion.

Characterization of single-stranded DNA separation by capillary gel electrophoresis

We have investigated the effect of polymer gel reconditioning, the shape of the capillary, the applied electric field, and the capillary length for single-stranded DNA. The polyethylene oxide gel had deformed under the high electric field causing the degradation of the separation power. By the reintroduction of the fresh polyethylene oxide gel for the next run, one-base resolution was recovered. It turned out that the tip of the capillary at the injection side needed to be clean and symmetric for much improved resolution. Changing DNA motion by the pulsed electric field resulted in the separation of DNA far more than 500 bases.

Crystallization and preliminary X-ray crystallographic analysis of human quinolinate phosphoribosyltransferase

Quinolinate phosphoribosyltransferase (QPRTase) is a key NAD-biosynthetic enzyme which catalyzes the transfer of quinolinic acid to 5-phosphoribosyl-1-pyrophosphate, yielding nicotinic acid mononucleotide. Homo sapiens QPRTase (Hs-QPRTase) appeared as a hexamer during purification and the protein was crystallized. Diffraction data were collected and processed at 2.8 Å resolution. Native Hs-QPRTase crystals belonged to space group P2(1), with unit-cell parameters a=76.2, b=137.1, c=92.7 Å, β=103.8°. Assuming the presence of six molecules in the asymmetric unit, the calculated Matthews coefficient is 2.46 Å3 Da(-1), which corresponds to a solvent content of 49.9%.