Use of a novel DNA melting profile assay for the identification of PCR-amplified genomic sequences encoding for variant-specific surface proteins from the clonal GS/M-83-H7 line of Giardia lamblia.

During infections, Giardia lamblia undergoes a continuous change of its major surface antigens, the variant-specific surface proteins (VSPs). Many studies on antigenic variation have been performed using G. lamblia clone GS/M-83-H7, which expresses surface antigen VSP H7. The present study was focused on the identification and characterization of vsp gene sequences within the genome of the clonal G. lamblia GS/M-83-H7 line. For this purpose, we applied a PCR which specifically amplified truncated sequences from the 3'-terminal region of the vsp genes. Upon cloning, most of the vsp gene amplification products were shown to be approximately identical in size and thus could not be distinguished from each other by conventional gel electrophoresis. In order to pre-estimate the sequence complexity within the large panel of vsp clones isolated, we elaborated a novel concept which facilitated our large-scale genetic screening approach: PCR products from cloned DNA molecules were generated and then subjected to a DNA melting profile assay based on the use of the LightCycler Instrument. This high-throughput assay system proved to be well suited to monitor sequence differences between the amplification products from closely related vsp genes and thus could be used for the primary, sequence-related discrimination of the corresponding clones. After testing 50 candidates, vsp clones could be divided into five groups, each characterized by an individual DNA melting profile of the corresponding amplification products. Sequence analysis of some of these 50 candidates confirmed data from the aforementioned assay in that clones were demonstrated to be identical within, but different between, the distinct groups. The nucleotide and deduced amino acid sequences of five representative vsp clones showed high similarities both among each other and also with the corresponding gene segment of the variant-specific surface antigen (VSP H7) expressed by the original GS/M-83-H7 variant type. Furthermore, three of the genomic vsp sequences turned out to be identical to vsp sequences that represented previously characterized transcription products from in vivo- or in vitro-switched GS/M-83-H7 trophozoites. In conclusion, the DNA melting profile assay seems to be a versatile tool for the PCR-based genotyping of moderately or highly diversified sequence orthologues.

Maize Stress DIESI-MS Data and R Script

Climatic change is an increasing challenge for agriculture that is driving the development of suitable crops in order to ensure supply for both human nutrition and animal feed. In this context, it is increasingly important to understand the biochemical responses of cells to environmental cues at the whole system level, an aim that is being brought closer by advances in high throughput, cost-efficient plant metabolomics. To support molecular breeding activities, we have assessed the economic, technical and statistical feasibility of using direct mass spectrometry methods to evaluate the physiological state of maize (Zea mays L.) plants grown under different stress conditions.

Learning Models of Gene Expression from Synthetic DNA Sequences

Thesis (Ph.D.)--University of Washington, 2016-06

Analysis of RNA-protein interactions by flow cytometry

Flow cytometry, in combination with advances in bead coding technologies, is maturing as a powerful high-throughput approach for analyzing molecular interactions. Applications of this technology include antibody assays and single nucleotide polymorphism mapping. This review describes the recent development of a microbead flow cytometric approach to analyze RNA-protein interactions and discusses emerging bead coding strategies that together will allow genome-wide identification of RNA-protein complexes. The microbead flow cytometric approach is flexible and provides new opportunities for functional genomic studies and small-molecule screening.

Systematic proteomic strategies to map the human signaling landscape

Thesis (Ph.D.)--University of Washington, 2016-05

Centromere identification and inactivation on a neo-Y chromosome fusion in threespine stickleback fish (Gasterosteus aculeatus)

Thesis (Ph.D.)--University of Washington, 2016-05

Modulation of Translation Efficiency in S. cerevisiae by Codon Pairs and mRNA Structure

Thesis (Ph.D.)--University of Washington, 2016-06

Using the R package Shiny to create web applications that facilitate quantitative gene expression analysis

Thesis (Master's)--University of Washington, 2016-06

Psychiatric genetics in Australia

Australian research in psychiatric genetics covers molecular genetic studies of depression, anxiety, alcohol dependence, Alzheimer's disease, bipolar disorder, schizophrenia, autism, and attention deficit hyperactivity disorder. For each disorder, a variety of clinical cohorts have been recruited including affected sib pair families, trios, case/controls, and twins from a large population-based twin registry. These studies are taking place both independently and in collaboration with international groups. Microarray studies now complement DNA investigations, while animal models are in development An Australian government genome facility provides a high throughput genotyping and mutation detection service to the Australian scientific community, enhancing the contribution of Australian psychiatric genetics groups to gene discovery. (C) 2003 Lippincott Williams Wilkins.

A rapid microtitre plate screening method for in vitro assessment of fibrinolysis: a preliminary report

A novel and precise assay that facilitates high-throughput screening of fibrinolytic agents was developed based on the automated assessment of the euglobulin clot lysis time in microtitre plates. Euglobulin fractions from fresh plasma samples were assessed over 28 days to determine the inter-assay and intra-assay precision. The intra-assay (coefficient of variation range, 0.7-2.6%) and inter-assay precision (coefficient of variation range, 6.8-12.1%) was found to be well within limits required by the Food and Drug Administration. On day 1 and day 28, the results of the microtitre plate euglobulin clot lysis time method were compared with tissue plasminogen activator activity, plasminogen activator inhibitor activity and results produced on fibrin plates. All comparisons were found to correlate significantly. The validity of this method for assaying fibrinolytic agents was assessed by comparing dose-response curves for streptokinase produced using fibrin plates and this method. The critical influence of ambient temperature on the inter-assay reproducibility of this method was established by testing samples over a range of temperatures between 20degreesC and 40degreesC. (C) 2004 Lippincott Williams Wilkins.

A rapid HPLC-mass spectrometry cyclosporin method suitable for current monitoring practices

Objectives: Cyclosporin is an immunosuppressant drug with a narrow therapeutic window. Trough and 2-h post-dose blood samples are currently used for therapeutic drug monitoring in solid organ transplant recipients. The aim of the current study was to develop a rapid HPLC-tandem mass spectrometry (HPLC-MS) method for the measurement of cyclosporin in whole blood that was not only suitable for the clinical setting but also considered a reference method. Methods: Blood samples (50 mu L) were prepared by protein precipitation followed by C-18 solid-phase extraction while using d(12) cyclosporin as the internal standard. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface in positive ionization mode. Results: The assay was linear from 10 to 2000 mu g/L (r(2) > 0.996, n = 9). Inter-day,analytical recovery and imprecision using whole blood quality control samples at 10, 30, 400, 1500, and 2000 mu g/L were 94.9-103.5% and

Biomolecular screening with novel organosilica microspheres

Organosilica microspheres synthesised via a novel surfactant-free emulsion-based method show applicability towards optical encoding, solid-phase synthesis and high-throughput screening of bound oligonucleotide and peptide sequences.

An automatable screen for the rapid identification of proteins amenable to refolding

Insoluble expression of heterologous proteins in Escherichia coli is a major bottleneck of many structural genomics and high-throughput protein biochemistry projects. Many of these proteins may be amenable to refolding, but their identification is hampered by a lack of high-throughput methods. We have developed a matrix-assisted refolding approach in which correctly folded proteins are distinguished from misfolded proteins by their elution from affinity resin under nondenaturing conditions. Misfolded proteins remain adhered to the resin, presumably via hydrophobic interactions. The assay can be applied to insoluble proteins on an individual basis but is particularly well suited for high-throughput applications because it is rapid, automatable and has no rigorous sample preparation requirements. The efficacy of the screen is demonstrated on small-scale expression samples for 15 proteins. Refolding is then validated by large-scale expressions using SEC and circular dichroism.

Comparative gene expression in brain regions of human alcoholics

The mesocorticolimbic system is the reward centre of the brain and the major target for drugs of abuse including alcohol. Neuroadaptive changes in this region are thought to underlie the process of tolerance and dependence. Recently, several research groups have searched for alcohol-responsive genes using high-throughput microarrays and well-characterized human post-mortem material. Comparison of data from these studies of cortical regions highlights the differences in experimental approach and selection of cases. However, alcohol-responsive gene sets associated with transcription, oxidative stress and energy production were common to these studies. In marked contrast, alcohol-responsive genes in the nucleus accumbens and the ventral tegmental area are primarily associated with changes in neurotransmission and signal transduction. These data support the concept that, within cortical regions, changes in gene expression are associated with alcoholism-related pathology. In the dopaminergic tract of the mesocorticolimbic system, alcohol-responsive gene sets suggest long-term neuroplastic changes in synaptic transmission.