treating-sca1-mice-with-water-soluble-compounds-to-non-specifically

Treating SCA1 Mice with Water-Soluble Compounds to Non-Specifically Boost Mitochondrial Function

Mitochondrial dysfunction plays a significant role in the aging process and in neurodegenerative diseases including several hereditary spinocerebellar ...

Skidmore College

Electroosmotic flow dynamics during a field-amplified sample stacking experiment have been studied experimentally using the periodic photobleaching of a dilute, neutral fluorophore added to the separation buffer. The effects of hydrodynamically injecting different sample plug lengths containing a mixture of arsenic compounds dissolved in 0.125 mM (120, 240, and 600 s) and 41.7 microM (27, 45, and 74 s) phosphate buffer with a separation buffer concentration of 12.5 mM phosphate buffer were examined. Changes in electroosmotic flow during sample stacking and separation were monitored at a rate of 1 Hz. The observed effects of increasing the sample plug length on electroosmotic flow and electrophoretic current agreed qualitatively with predictions by theoretical models presented in the literature. Electroosmotic flow changes on the order of 100% (1.6-3.3 mm/s) were observed. Broadening of the flow monitoring peaks has been used to examine parabolic flow due to the discontinuous buffer systems used for sample stacking.

Experimental studies of electroosmotic flow dynamics during sample stacking for capillary electrophoresis.

Measurement of illicit drugs on paper currency is of interest for evidentiary purposes in legal cases involving the drug trade. Current methods, primarily based on mass spectrometry, are destructive and prevent re-analysis of the evidence. This article details a method based on Raman microspectroscopy that is able to collect spectra from individual crystals on the surface of paper currency. Mixtures of isoxsuprine and norephedrine, which are non-pharmacologically active drug surrogates, as well as lidocaine and benzocaine, common excipients in street drugs, were doped in small quantities onto US currency. Significant fluorescence interference resulted from the underlying dollar bill. This work presents two methods for reducing the fluorescence background, photobleaching and background subtraction, which both worked well. Finally, a method for determining the percent composition of individual components in heterogeneous mixtures was developed by systematically sampling the surface of the dollar bill. Results were accurate within a few percent, although the method was quite time consuming.

Qualitative and quantitative analysis of illicit drug mixtures on paper currency using Raman microspectroscopy.

Insulin capture by a G-quadruplex DNA oligonucleotide containing a two-repeat sequence of the insulin-linked polymorphic region (ILPR) of the human insulin gene promoter region is reported. The immobilized oligonucleotide was demonstrated to capture human insulin from standard solutions and from nuclear extracts of pancreatic cells with high selectivity, using affinity MALDI mass spectrometry and affinity capillary chromatography. Insulin was preferentially captured by the two-repeat ILPR oligonucleotide over another G-quadruplex-forming oligonucleotide, the thrombin-binding aptamer, as well as over a single repeat of the ILPR sequence that is not capable of forming the G-quadruplex architecture. Binding was shown to involve the beta chain of insulin. The discovery raises the possibility that insulin may bind to G-quadruplex DNA formed in the ILPR in vivo and thereby play a role in modulation of insulin gene expression, and it provides a basis for design of insulin analogues to probe this hypothesis. The availability of a DNA ligand to human insulin has analytical importance as well, offering an alternative to antibodies for in vitro or in vivo detection and sensing of insulin as well as its isolation and purification from biological samples.

Insulin capture by an insulin-linked polymorphic region G-quadruplex DNA oligonucleotide.

CE separations are known for their high separation efficiencies. In systems with EOF, the high efficiencies benefit from the flat, plug profile that is characteristic of EOF. When a velocity gradient is present, such as in separations which have nonuniform buffer ionic strength, surface adsorption or differences in the height of the ends of the capillary, a parabolic flow component is introduced. This deviation from purely EOF yields increased peak dispersion and a subsequent decrease in separation performance. This work details a rapid method for detecting deviations from ideal plug flow during the course of a separation using the radially averaged flow profile of a photobleached fluorophore added to the BGE. By comparing the ratio of two different data analysis procedures, deviations from ideal plug flow can be detected. This method allows rapid measurement of flow character and does not interfere with the concurrent separation. We demonstrate easy detection of the onset of hydrodynamic flow induced by both gravity siphoning and an ionic strength buffer discontinuity. A brief analysis of the radially averaged peak shapes is also presented.

Detecting deviations from pure EOF during CE separations.

The insulin-linked polymorphic region (ILPR) of the human insulin gene contains tandem repeats of similar G-rich sequences, some of which form intramolecular G-quadruplex structures in vitro. Previous work showed affinity binding of insulin to an intramolecular G-quadruplex formed by ILPR variant a. Here, we report on interactions of insulin and the highly homologous insulin-like growth factor-2 (IGF-2) with ILPR variants a, h, and i. Circular dichroism indicated intramolecular G-quadruplex formation for variants a and h. Affinity MALDI MS and surface plasmon resonance were used to compare protein capture and binding strengths. Insulin and IGF-2 exhibited high binding affinity for variants a and h but not i, indicating the involvement of intramolecular G-quadruplexes. Interaction between insulin and variant a was unique in the appearance of two binding interactions with K(D) approximately 10(-13) M and K(D) approximately 10(-7) M, which was not observed for insulin with variant h (K(D) approximately 10(-8) M) or IGF-2 with either variant (K(D)s approximately 10(-9) M). The results provide a basis for the design of DNA binding ligands for insulin and IGF-2 and support a new approach to discovery of DNA affinity binding ligands based on genome-inspired sequences rather than the traditional combinatorial selection route to aptamer discovery.

A genome-inspired DNA ligand for the affinity capture of insulin and insulin-like growth factor-2.

The ability to accurately and noninvasively analyze illicit drugs is important for criminal investigations and prosecution. Current methods involve significant sample pretreatment and most are destructive. The goal of this work is to develop a method based on Raman spectroscopy to classify simulated street drug mixtures composed of one drug component and up to three cutting agents including those routinely found in confiscated illicit street drug mixtures. Spectra were collected on both a homebuilt instrument using a HeNe laser and on a handheld commercial instrument with a 785 nm light source. Mixtures were prepared with drug concentrations ranging from 10 to 100 percent. Optimal preprocessing for the data set included truncating, Savitzky-Golay smoothing, normalization, differentiating, and mean centering. Using principal component analysis (PCA), it was possible to resolve the spectral differences between benzocaine, lidocaine, isoxsuprine, and norephedrine and correctly classify them 100 percent of the time.

Rapid classification of simulated street drug mixtures using Raman spectroscopy and principal component analysis.

Dynamic buffer conditions are present in many electrophoretically driven separations. Polyelectrolyte multilayer coatings have been employed in CE because of their chemical and physical stability as well as their ease of application. The goal of this study is to measure the effect of dynamic changes in buffer pH on flow using a real-time method for measuring EOF. Polyelectrolyte multilayers (PEMs) were composed of pairs of strong or completely ionized polyelectrolytes including poly(diallyldimethylammonium) chloride and poly(styrene sulfonate) and weak or ionizable polyelectrolytes including poly(allylamine) and poly(methacrylic acid). Polyelectrolyte multilayers of varying thicknesses (3, 4, 7, 8, 15, or 16 layers) were also studied. While the magnitude of the EOF was monitored every 2 s, the buffer pH was exchanged from a relatively basic pH (7.1) to increasingly acidic pHs (6.6, 6.1, 5.5, and 5.1). Strong polyelectrolytes responded minimally to changes in buffer pH (<1%), whereas substantial (>10%) and sometimes irreversible changes were measured with weak polyelectrolytes. Thicker coatings resulted in a similar magnitude of response but were more likely to degrade in response to buffer pH changes. The most stable coatings were formed from thinner layers of strong polyelectrolytes.

Article	Year
Experimental studies of electroosmotic flow dynamics during sample stacking for capillary electrophoresis. Analytical chemistry\| PubMed ID: 14570207	2003
Qualitative and quantitative analysis of illicit drug mixtures on paper currency using Raman microspectroscopy. Applied spectroscopy\| PubMed ID: 16390588	2005
Insulin capture by an insulin-linked polymorphic region G-quadruplex DNA oligonucleotide. Journal of the American Chemical Society\| PubMed ID: 16608332	2006
Detecting deviations from pure EOF during CE separations. Electrophoresis\| PubMed ID: 17632806	2007
A genome-inspired DNA ligand for the affinity capture of insulin and insulin-like growth factor-2. Journal of separation science\| PubMed ID: 19391177	2009
Rapid classification of simulated street drug mixtures using Raman spectroscopy and principal component analysis. Applied spectroscopy\| PubMed ID: 19589210	2009
Assessment of polyelectrolyte coating stability under dynamic buffer conditions in CE. Journal of separation science\| PubMed ID: 21766482	2011
Using forensic science to teach method development in the undergraduate analytical laboratory. Analytical and bioanalytical chemistry\| PubMed ID: 23657453	2013

Kimberley Frederick

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