Category G. Enabling Sciences
G1. The University of Kansas Protein Structure Laboratory
Protein Structure Laboratory, Del Shankel Structural Biology Center, University of Kansas, Lawrence, KS
The COBRE Center in Protein Structure and Function (COBRE-PSF) at The University of Kansas conducts health-related basic research in the area of protein structure and function. One of the three core laboratories at the COBRE-PSF, the Protein Structure Laboratory (PSL), collaborates with investigators from various institutions in an effort to obtain the 3-dimensional structures of proteins using X-ray crystallography. The capabilities and infrastructure of the PSL are presented. In addition, examples of collaborative projects recently completed by PSL are highlighted. These include the inhibitor bound structures of viral proteases from norovirus, poliovirus and transmissible gastroenteritis virus as well as the structure of Pseudomonas Bacterioferritin (BfrB) in complex with its bacterioferritin-associated ferredoxin (Bfd). These projects highlight the significance of obtaining the crystal structures of proteins to facilitate drug discovery efforts.
G2. Microchip Electrophoresis with Dual-Electrode Electrochemical Detection for Detection of Reactive Nitrogen Species
Diogenes Meneses dos Santos1,2, Dulan B. Gunasekara1,3, Pann Pichetsurnthorn1, Ryan J. Grigsby1, Anne R. Regel1, 3, Jose Alberto Fracassi da Silva4, Fabiane Caxico de Abreu2, Susan M. Lunte1, 3
1Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas, Lawrence, KS
2Federal University of Alagoas, Maceio, Brazil
3Department of Chemistry, University of Kansas, Lawrence, KS
4State University of the Campinas, Campinas, Brazil.
Microchip Electrophoresis (ME) becomes a powerful tool in micro-total analysis systems because it is a fast and efficient separation technique, and requires low microliter sample volumes. Electrochemical detection (EC) can be easily coupled with ME and is therefore an ideal detection method for ME. One of the crucial challenges in ME is the isolation of the detector circuits from the separation voltage. We have previously reported an in-channel amperometric method for ME to separate and detect reactive nitrogen species (RNS). However, detection limits were relatively high due to the noise from high voltage power supply used for separation. Thus a dual channel method reported by the Hahn group was adapted to evaluate the influence of separation voltage on noise1, and subsequently detection limits for RNS. This microchip contains two separate channels each containing a platinum electrode. One electrode serves as the working electrode and second electrode serves as the reference electrode. To further improve the signal and signal to noise ratio one of Pt electrodes were converted into a stable Ag/AgCl reference electrode and the platinum black was electrodeposited in the other electrode. The two electrodes are positioned at the exact exit of the channel and amperometric signals could be measured without any potential shift or interference from the applied separation voltage. Then a sample of nitrite, tyrosine and hydrogen peroxide was injected only into the channel where working electrode is contained while buffer is injected into the reference channel with the Pt or Ag/AgCl reference electrode to evaluate the microchip. With this configuration, low noise and high resolution is maintained compared to the end channel configuration. This configuration can also be used for peak identification. Each platinum electrode is set at a different potential with respect to the Ag/AgCl electrode. In this case, sample is injected into both channels and the current ratio is obtained by measuring the response at the two electrodes. The current ratio will be different for analytes that have a different voltammetric profiles. These methods will be employed to detect and identify RNS from stimulated RAW macrophage cells.
G3. Characterization of water-compatible dentin adhesive polymer formed under wet conditions
Ranganathan Parthasarathy1, 4 Anil Misra1,3, Jonggu Park1, Qiang Ye1, Viraj Singh1,2 and Paulette Spencer1,2
1Bioengineering Research Center; 2Mechanical Engineering; 3Civil Engineering; 4Bioengineering Graduate Program , University of Kansas, Lawrence, KS, U.S.A.
In current clinical practice, a dental restoration is achieved by bonding a composite resin to demineralized collagen on acid-etched tooth substrate using an adhesive polymer. The bond is formed when the adhesive monomer fills gaps in the collagen matrix left behind by the mineral to form a hybrid layer. However, the presence of dentinal water in the tooth substrate separates the adhesive monomer into hydrophobic and hydrophilic-rich phases. Therefore, the adhesive polymer inside the hybrid layer increases in hydrophilicity towards the dentin substrate. Such a structure typically presents poor cross-linking, which decreases the mechanical properties and the degradation resistance and leads to restoration failure by a combination of inter-related chemical, mechanical and biological phenomena. We have developed a protocol for testing adhesive formulations for water-compatibility. We use this protocol to evaluate our newly synthesized monomer, BMPB, in comparison to a control adhesive formulation. The protocol consists of the following steps: 1) miscibility tests using visual observation to measure solubility 2) ternary phase diagrams from composition analysis using chemometrics on FTIR spectra 3) degree of polymerization along the phase boundary curve 4) polymerization kinetics along phase boundary curve 5) selection of initiator system for adhesive polymer 6) chemical mapping of a water-oversaturated resin polymer and 7) mechanical modeling of the adhesive formulation using a poromechanics approach. Selected results that provide insight into critical relationships between chemical structures, phase partitioning and mechanical properties of these adhesives will be presented.
G5. Electrochemical Detection using an all PMMA Microfluidic Flow-cell with an Integrated Carbon Microelectrode
Anne Regel A,C and Susan Lunte A,B,C
ADepartment of Chemistry, University of Kansas, BDepartment of Pharmaceutical Chemistry, University of Kansas, CRalph N. Adams Institute for Bioanalytical Chemistry
The interest in developing a miniaturized separation based sensor has increased significantly over the last 20 years. This is especially true when used in conjunction with electrochemical detection, as the supporting electronics are also miniaturized, leading to a truly portable analytical system. Miniaturized analytical systems have many advantages, sample and reagent volumes are drastically reduced (mL – mL) and analysis time can be much faster (s-min), compared to their standard size analytical systems. However, the fabrication of miniaturized devices with integrated electrodes can be difficult and prohibitively expensive, in both time and money. We have developed a method to embed a graphite PMMA composite electrode into a thermoplastic microfluidic device for electrochemical detection. These electrodes are inexpensive (< $0.10 for a 2x2 in substrate), can be fabricated quickly and in mass (> 50 electrodes fabricated per day, start to finish).
G6. TIRF: A Novel Non-PCR Based Approach to Genotyping Low Copy Number DNA
Tanya M. Simms, Sarah E. LeGresley and Matthew D. Antonik
Department of Physics and Astronomy, University of Kansas, Lawrence, KS 66047
Short tandem repeats (STRs), both autosomal and Y-chromosome specific, are routinely employed in the forensic laboratory for human identification purposes. However, in some instances, the evidence collected from the crime scene contains too few copies of DNA for amplification via traditional and/or quantitative polymerase chain reaction (PCR) techniques, which require DNA concentrations of 0.5 nanograms and 30 picograms, respectively. This type of sample, commonly referred to as low copy number (LCN) DNA, typically generates stochastic artifacts (e.g., stutter peaks, allele dropout, allele drop-in, pull-up and non-template addition) during the PCR amplification process, ultimately resulting in an STR profile that is difficult to interpret. As such, we are developing a novel mechanism of genotyping LCN DNA using total internal reflection fluorescence (TIRF) microscopy, a well-established single-molecule technique that allows repeated interrogation of a single fluorescently labeled DNA molecule that is anchored to the surface of a slide.
G7. Nonlinear Viscoelastic-Damage Model Using Granular Micromechanics forDentin Adhesive
Bioengineering Research Center, Mechanical Engineering, KU
In the mouth, the interplay of chemical and mechanical stresses can lead to a change in the mechanical properties of the adhesive.
The aim of the current study is to investigate the creep behavior of a model dentin adhesive under conditions that simulate the wet, oral environment and further develop mathematical model to explain the creep behavior in dentin adhesives.
The model dentin adhesive used in this study consisted of following co-monomers, (a) bisphenol-A diglycidyl ether dimethacrylate (bisGMA) and (b) 2-hydroxyethyl methacrylate (HEMA) [HEMA /bisGMA, 45/55 w/w], with 3-component photoinitiator system: camphorquinone (0.5wt%), 2-ethyl-4-aminobenzoate (0.5wt%) and diphenyliodonium hexafluorophosphate (0.5wt%). These co-monomers were mixed and continuously shaken for 48 hours to yield well-mixed resin solutions. Further, rectangular beam specimens, of 1mm×1mm cross-section and 15mm length, were casted using Borosilicate glass tubing. These beam specimens were then subjected to a load of 4.5 MPa under four different environmental conditions for 24hr-48hrs. Series 1: samples stored dry and tested dry, series 2: samples submerged in water for 5 days and tested submerged in water, series 3: specimens stored dry and tested submerged in water, series 4: specimens stored dry and tested in the dry condition for the first 200 minutes and then water was added and the test continued with the samples submerged in water.
Further we have used micromechanical approach to model the rate-dependent behavior of dentin adhesives. In this approach, the cross-links are modeled as rheological elements that undergo water-induced damage. The derived model is used to predict the creep response of model dentin adhesive samples that have been tested under different moisture conditions.
Supported NIH/NIDCR R01 DE014392, DE014392-08S109
G8. Genome Sequencing Core Lab at KU-Lawrence
Wang, Xinkun1,2, Erik Lundquist1,3, Sue Lunte1,4,5,
1Center for Molecular Analysis of Disease Pathways,
2Higuchi Biosciences Center,
3Department of Molecular Biosciences,
4Department of Chemistry,
5Department of Pharmaceutical Chemistry, University of Kansas
The Genome Sequencing Core (GSC) is one of three research core labs in the newly established NIH COBRE Center for Molecular Analysis of Disease Pathways (CMADP) at KU. The major mission of the GSC is to provide researchers with next-generation sequencing (NGS) technologies as well as expertise in experimental design and analysis of sequence data. NGS, carried out in a massively parallel fashion, has been revolutionizing bio-medical research and used in a growing list of applications. Projects supported by the GSC include de novo genome assembly, genome re-sequencing for identification of mutations and polymorphisms, transcriptome analysis (RNA-Seq), epigenomic and gene regulation studies such as ChIP-Seq, Methyl-Seq, small RNaA discovery and analysis. The Genome Sequencing Core enhances the genomics infrastructure already at KU, in the KU Genomics Facility and the Natural History Museum first generation sequencing facility, by bringing the astronomically high-throughput Illumina HiSeq 2500 sequencing capabilities to researchers at KU-Lawrence and across Kansas and the region. This next-generation sequencer has the capacity to generate 3-6 billion reads of 100bp per run of two eight-lane flow cells (600Gb data). In its rapid mode, it can generate 1.2 billion reads of 150 bp per run on two two-lane flow cells (120Gb data) in 27 hours. To capture the full power of NGS, we provide a whole range of project support, from project consultation, sample QC, library construction, cluster generation, data generation, to data analysis.