Identifying Contaminants of Emerging Concern in Surface Waters

Contaminants of emerging concern (CECs) are environmental pollutants being detected at an increasing rate in surface waters over the last 20 years. These contaminants are ubiquitous and include anthropogenic and naturally occurring chemicals, pharmaceuticals and personal care product metabolites, engineered nano-materials, and antibiotic resistance genes. Contaminants of emerging concern are not yet regulated in drinking water supplies, and therefore are not commonly monitored.

We are working with the Fond du Lac Resource Management Division (Kari Hedin and Nancy Schuldt) to complete a preliminary study of CECs present at Big Lake compared to an anthropogenically unaltered watershed. We will use classic methodologies including gas chromatography – mass spectrometry for small molecular weight, non-polar compounds, as well as emerging techniques for quantification of polar, high molecular weight CECs, such as high-pressure liquid chromatography.

Anthropogenically sourced contaminants do not always pose an environmental threat. However, many of these contaminants have the potential to cause adverse ecological and/or human health effects. Thus, the effect of CECs on both aquatic and human life is an area of concern for environmental regulatory bodies (i.e. FDL – RMD, EPA, MPCA, etc.). In addition, CECs often undergo transformations in their environment that include bio-degradation, chemical oxidation and reduction, hydrolysis and photolysis. In some cases, the transformation product has been found to be more toxic than the parent compound. To increase the usefulness of our findings, we plan to test the toxicity of select detected CECs and transformation products via a thiazolyl blue tetrazolium bromide cellular viability test on a mouse embryonic fibroblast mammalian cell line. In addition, we will test the potential for a select subset of detected CECs to interrupt endocrine function via an in vitro estrogen receptor and androgen receptor binding assay.

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siRNA-Aptamer Chimeras for Biotherapeutic Applications

It was estimated in 2015 that 17% of the American population above age 45 has diabetes. The disease was the seventh leading cause of death in the United States in 2015, with related healthcare costs of $245 billion dollars.¹ Type 2 diabetes (T2D), which accounts for ~90% of all cases¹, is characterized by decreased insulin production in pancreatic islet cells and insulin resistance in target tissues.² Recent work to elucidate the cause of insufficient insulin production has shown that insulin producing β-cell mass is reduced up to 50% in patients with T2D, contributing to the observed clinical phenotype of hypoinsulinemia.³ Methods to replace β-cell mass have been explored with mixed results; mass transplantation of islets is not currently feasible due to low donor islet availability and the need for subsequent, long-term immunosuppressive therapy, and β-cell directed stem cell differentiation is costly and controversial.⁴ Perhaps the most obvious approach is to pharmacologically induce β-cell proliferation in vivo. Recent findings suggest a kinase inhibitor effective against dual-specificity tyrosine phosphorylation-regulated kinase (DYRK1A), among other tyrosine kinases, induces β-cell proliferation through activation of the transcription factor, nuclear factor of activated T-cells, (NFATc).^5-6 It is well known that NFATc promotes transcription of genes controlling cell cycle progression⁷. Unfortunately, DYRK1A is not exclusively expressed in β-cells, and off-target effects have not been ruled out . An unexplored approach to side-step in vivo cytotoxicity is to specifically deliver a DYRK1A silencing molecule to β-cells. To that end, I propose to develop a ribonucleotide-based aptamer-siRNA chimera that will specifically internalize into β-cells to knock-down DYRK1A expression.

Aptamers are short oligo-nucleotides whose specific sequence and secondary structure specifically recognize epitopes on a cell or organelle of interest. They are “selected” through a process termed systematic evolution of ligands through exponential enrichment (SELEX), which is an in vitro methodology described above. Cell-internalization SELEX has been described for the development of aptamer–siRNA chimeras, capable of delivering bio-active siRNA molecules to specific cell types^12-13. This can be accomplished through an iterative process of cell incubation with a library of single stranded RNA oligonucleotides, removal of unbound or un-internalized oligonucleotides through washing steps, followed by PCR amplification of the internalized RNA oligonucleotides. To this end, we will develop a ribonucleotide-based aptamer-siRNA chimera that will specifically internalize into β-cells to knock-down DYRK1A expression to modulate β-cell proliferation. Internalizing sequences will be selected and manipulated using newly reported¹³, and traditional molecular biology methods¹⁴, but sequenced using next generation sequencing.¹⁵ Selected aptamers will be used to construct siRNA chimeras.¹⁶

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Design and Construction of a Fluorescence-Activated Organelle Sorting Instrument

In the case that β-cell mass cannot be restored in T2D patients, a separate therapeutic approach would be to maintain or increase insulin secretion from β-cells relative to normal levels. Unfortunately, it has recently been that cells from afflicted patients secrete ~3.5 fold less insulin compared to cells from healthy patients after correction for total insulin content.^8-9 After controlling for total cellular insulin content, decreased insulin peptide production is ruled out, leaving dysregulated intracellular trafficking as a potential cause for the observed insulin secretion decrease. Intracellular insulin trafficking is a multi-step process compromising distinct organelles who differ in phenotype and function.²

A detailed analysis of the proteomic and phenotypic sub-populations of these insulin secretory granules (organelles) across secretion-competent and incompetent cells could illuminate functional changes, providing potential therapeutic targets. With extraordinary advances in mass spectrometer mass resolution, range, accuracy and abundance sensitivity, there is an inherent need for extremely pure sub-cellular fractions. Current methods such as differential or density-gradient centrifugation produce relatively crude fractions¹⁰, and while emerging methods such as fluorescence-activated organelle sorting (FAOS) offer improved methodologies for sub-cellular fractionation, they suffer from a lack of sensitivity for sorting very small, weakly-fluorescent particles.¹¹ As a second, longer-term project, we will build a capillary FAOS instrument with post-column laser induced fluorescence detection to combine the low-limits of detection required for organelle analysis, with the ability to selectively sort and collect sub-cellular organelles for downstream proteomic and phenotypic analysis.

While methods such as fluorescence activated cell sorting (FACS) exist to physically separate phenotypically or functionally distinct whole cell populations, very few methods exist to separate individual organelles on the same parameters. We will work to solve this analytically challenging problem through the development and characterization of a capillary-FAOS instrument, which will build on the advances in individual organelle analysis reported by the Arriaga lab at UMN.¹⁹ It will couple the low limits of detection necessary for individual organelle analysis with the possibility for an integrated application of an electric field to separate organelles with unique electrophoretic mobilities and fluorescence signatures in an applied electric field for downstream fraction collection. Students will assist in building a capillary-FAOS instrument based on the capillary electrophoresis instrument first reported by Duffy.²⁰ After successful construction and LabView code integration, we will characterize the instrument for analytical figures of merit (i.e. limit of detection, sensitivity, selectivity, etc.) using fluorescent beads of known composition.

Following characterization, we will collect organelle fractions based on size (forward scatter), granularity (side scatter), and insulin content (fluorescent immunolabel) from DYRK1A knock-down and control β-cells. These fractions will be subjected to an established quantitative 2D-DIGE and LC-MS/MS proteomics strategy^27-28 to identify significant proteomic differences²⁹. The successful completion of this project will yield an operational capillary-FAOS instrument and a list of differentially abundant proteins found on mature (highly granular, small, low-insulin content) and immature (low granularity, large, high-insulin content) insulin granules providing insight into trafficking dynamics in proliferative vs. non-proliferative β-cells.

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