Esler B.,University of Oregon |
Lyons T.,Marker Gene Technologies |
Turovets S.,University of Oregon |
Turovets S.,Electrical Geodesics, Inc. |
Tucker D.,Electrical Geodesics, Inc.
Journal of Physics: Conference Series | Year: 2010
We describe instrumentation for low frequency (< 500 Hz) EIT studies of the human head and its calibration, testing and validation in the phantom experiments. Our EIT system prototype is based on a 256 channel commercial EEG system complimented by the current injection module and lock-in detection software. We have designed and built two types of head phantoms: i) a resistor network and ii) a cylinder tank filled with saline and gel insertions with chemically targeted conductivity values. We have developed a technology for fabricating, handling and storage of agar TX151 gel insertions. Independent and direct conductivity measurements of gel samples have been performed using a HP LCR meter in a four electrode conductivity cell specifically designed and built for this purpose. Measurements of saline conductivity were done with commercially available salinity / conductivity meters. Our inverse conductivity estimates in the phantom experiments with EIDORS and in-house software cross-validate the viability of the EIT-EEG system. © 2010 IOP Publishing Ltd.
Carroll C.N.,University of Oregon |
Carroll C.N.,Oregon Nanoscience and Microtechnologies Institute |
Naleway J.J.,Oregon Nanoscience and Microtechnologies Institute |
Naleway J.J.,Marker Gene Technologies |
And 4 more authors.
Chemical Society Reviews | Year: 2010
This critical review will focus on the application of shape-persistent receptors for anions that derive their rigidity and optoelectronic properties from the inclusion of arylethynyl linkages. It will highlight a few of the design strategies involved in engineering selective and sensitive fluorescent probes and how arylacetylenes can offer a design pathway to some of the more desirable properties of a selective sensor. Additionally, knowledge gained in the study of these receptors in organic media often leads to improved receptor design and the production of chromogenic and fluorogenic probes capable of detecting specific substrates among the multitude of ions present in biological systems. In this ocean of potential targets exists a large number of geometrically distinct anions, which present their own problems to the design of receptors with complementary binding for each preferred coordination geometry. Our interest in targeting charged substrates, specifically how previous work on receptors for cations or neutral guests can be adapted to anions, will be addressed. Additionally, we will focus on the design and development of supramolecular arylethynyl systems, their shape-persistence and fluorogenic or chromogenic optoelectronic responses to complexation. We will also examine briefly how the "chemistry in the cuvet" translates into biological media (125 references). © 2010 The Royal Society of Chemistry.
Harlan F.K.,Marker Gene Technologies |
Lusk J.S.,Marker Gene Technologies |
Mohr B.M.,Sonas BioPharma Inc. |
Guzikowski A.P.,Marker Gene Technologies |
And 4 more authors.
PLoS ONE | Year: 2016
Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson's Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker1 Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research, diagnostics and monitoring the effect of secondary therapeutic agents on lysosomal enzyme activity in drug development for the lysosomal storage disorders and allied diseases. © 2016 Harlan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Coleman D.J.,Marker Gene Technologies |
Kuntz D.A.,Ontario Cancer Institute |
Venkatesan M.,Ontario Cancer Institute |
Cook G.M.,Marker Gene Technologies |
And 4 more authors.
Analytical Biochemistry | Year: 2010
A simple and reliable continuous assay for measurement of α-mannosidase activity is described and demonstrated for analysis with two recombinant human enzymes using the new substrate resorufin α-d-mannopyranoside (Res-Man). The product of enzyme reaction, resorufin, exhibits fluorescence emission at 585nm with excitation at 571nm and has a pKa of 5.8, allowing continuous measurement of fluorescence turnover at or near physiological pH values for human lysosomal and Drosophila Golgi α-mannosidases. The assay performed using recombinant Drosophila Golgi α-mannosidase (dGMII) has been shown to give the kinetic parameters Km of 200μM and Vmax of 11nmol/min per nmol dGMII. Methods for performing the assay using several concentrations of the known α-mannosidase inhibitor swainsonine are also presented, demonstrating a potential for use of the assay as a simple method for high-throughput screening of inhibitors potentially useful in cancer treatment. © 2009 Elsevier Inc.
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 224.59K | Year: 2015
DESCRIPTION provided by applicant This Small Business Innovation Research Phase I project aims to develop new targeted pharmacological chaperones capable of modulating enzyme degradation in the Golgi apparatus and Endoplasmic Reticulum ER of living cells and tissues If successful the proposed research will provide breakthroughs needed to advance the discovery of promising new therapies and modulating drugs for neurodegenerative disorders including lysosomal storage diseases Alzheimerandapos s disease AD amyotrophic lateral sclerosis ALS Parkinsonandapos s disease PD myeloid leukemia glioblastoma Type diabetes Lowe syndrome and allied degenerative diseases and medical conditions involving protein misfolding In Phase I of this project Marker Gene Technologies Inc will establish the feasibility of the technology by preparing new targeted pharmacological chaperones demonstrating improved loading and localized accumulation in the Golgi and ER and demonstrating efficacy for increasing lysosomal enzyme activity in living cells that are of disease origin in comparison to those from normal controls In Phase II these and additional new targeted drug conjugates will be evaluated in vitro and in vivo for their ability to affect specific and localized induction of tese enzymes in living cells as well as alleviate unwanted protein degradation or improve protein trafficking in a cell or tissue specific manner using a variety of delivery methods These new pharmacological chaperones and the resulting targeting systems will provide innovative methods to modulate Golgi and ER organelle function and thereby screen for the influence of secondary drug or protein administration affect intracellular trafficking of proteins or improve transport or secretion of proteins making them useful analytical tools for drug discovery and basic research in a variety of significant medical applications Our very preliminary results indicate the proposed methods have the potential to increase intracellular loading and targeting of pharmacological chaperones in human cell lines from patients with Gaucher disease thereby providing a new tool to the arsenal of available therapeutics for clinical treatment of neurodegenerative disorders PUBLIC HEALTH RELEVANCE The success of this project opens up enormous commercial possibilities in the fields of medical intervention in a variety of neurodegenerative diseases such lysosomal storage diseases Alzheimerandapos s disease AD amyotrophic lateral sclerosis ALS Parkinsonandapos s disease PD myeloid leukemia glioblastoma Type diabetes Lowe syndrome and allied medical conditions as well as in the screening of new proteins and drugs in cell culture systems for efficacy in modulating intracellular enzyme activity in these diseases The development of new general and organelle specific targeting strategies will find use in potential treatment of organelle specific viral and bacterial diseases such as Cholera Chlamydia or malarial infection The developed products will also be useful in non mammalian cells systems that exhibit defective protein folding and ER Golgi accumulation or degradation including plant yeast and bacterial species and will lead to commercial and licensable products in these areas
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 1.53M | Year: 2012
DESCRIPTION (provided by applicant): This Small Business Innovation Research Phase II project aims to develop new targeted fluorogenic substrates capable of measuring lysosomal enzyme activity in living cells and tissues. If successful, the proposed research will provide breakthroughs needed to advance the discovery of promising new therapies and modulating drugs for lysosomal storage diseases and allied medical applications. In Phase I of this project, Marker Gene Technologies, Inc. established the feasibility of the technology by preparing new fluorogenic glycosidase, esterase, phosphatase, lipase and sulfatase substrates for lysosomal enzymes and demonstrated differential staining in living cells that were from normal or were of disease origin or upon induction of inhibition of lysosomal enzyme activities. In Phase II, these and additional new substrates will be assayed in vitro for their ability to measure specific and localized inhibition or induction of lysosomal enzymes in living cells as well as differentiate individual enzyme activities in a cell- or tissue-specific manner. These new systems will be validated for use in high-throughput screening for drug discovery, for use in clinical diagnostics to evaluate the occurrence and progression of disease,and for use in monitoring the effectiveness of existing or emerging therapeutic interventions for these disorders. The new substrates and the resulting detection systems will also provide innovative methods to quantitate lysosomal enzyme function and to screen for the influence of secondary drug or protein administration, making them useful medical research tools for a variety of significant biochemical and medical applications. The company has engaged the collaboration of several noted research laboratories and institutions as well as major pharmaceutical companies in this arena, who are eager to test the methods and systems in their existing clinical applications. The resulting assays and products will be marketed to the research, pharmaceutical, biotechnology and diagnostic industries. PUBLIC HEALTH RELEVANCE: The success of this project opens up enormous commercial possibilities in the fields of medical intervention in lysosomal storage disorders such as Sandhoff's disease, Tay-Sachs syndrome, Krabbi's disease and Gaucher's disease through discovery of new proteins and drugs for use in modulating lysosomal enzyme activity in these and other related diseases. These assays will also enable clinicians to determine the efficacy of current and emergingtherapies as well as add a new set of clinical diagnostic assays for patient assessment. In addition, it will lead to commercial and licensable products in these areas.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 149.76K | Year: 2013
DESCRIPTION: This Small Business Innovation Research Phase I project aims to develop new targeted fluorogenic substrates capable of measuring enzyme activities in the Golgi apparatus and Endoplasmic Reticulum (ER) of living cells and tissues. If successful, the proposed research will provide breakthroughs needed to advance the discovery of promising new therapies and modulating drugs for neurodegenerative disorders including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease(PD), Type 2 diabetes, Lowe syndrome, Huntington's disease and allied medical conditions. In Phase I of this project, Marker Gene Technologies, Inc. will establish the feasibility of the technology by preparing new fluorogenic glycosidase, phosphatase andpeptidase substrates for enzymes with localized activity in the Golgi and ER and demonstrating differential staining in living cells that are from normal or are of disease origin. In Phase II, these and additional new substrates will be assayed in vitro and in vivo for their ability to measure specific and localized inhibition or induction of these enzymes in living cells as well as differentiate individual enzyme activities in a cell- or tissue-specific manner. The new substrates and the resulting detection systems will provide innovative methods to quantitate Golgi and ER organelle enzyme function and to screen for the influence of secondary drug or protein administration, making them useful analytical tools for drug discovery and diagnosis in a variety of significant medical applications. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: The success of this project opens up enormous commercial possibilities in the fields of medical intervention in a variety of neurodegenerative diseases such asAlzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Creutzfeldt-Jacob disease (CJD), multiple system atrophy (MSA), Parkinson's disease (PD), spinocerebelar ataxia type 2 (SCA2), myeloid leukemia, glioblastoma, Type 2 diabetes, Lowe syndrome, Niemann-Pick type C (NPC), Huntington's disease (HD), as well as in the screening of new proteins and drugs in cell-culture systems for efficacy in modulating Golgi and ER enzyme activity in these diseases, and development of new, general and specific high-throughput organelle specific enzyme detection strategies. In addition, it will lead to commercial and licensable products in these areas.
Marker Gene Technologies | Date: 2012-12-04
A codon optimized and stabilized luciferase gene and a novel recombinant DNA characterized by incorporating this new gene coding for a novel luciferase into a vector DNA for improved activities in mammalian cells, are disclosed. This new luciferase exhibits long-wavelength light emission, as well as improved thermostability and higher expression levels in mammalian cell systems, compared to native luciferase. Assays using this new enzyme for measuring various biological metabolic functions are described.
Agency: National Science Foundation | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 499.89K | Year: 2009
This Small Business Innovation Research Phase II project aims to develop new, sensitive, accurate, and reliable detection methods for measuring genomic DNA or RNA samples isolated from living cells. The intellectual merit of this project lies in the development of new detection methods that are essential for improving high-throughput genomic microarray analyses of gene activity. Problems with current microarray and genomic analysis techniques, including hybridization perturbation, slow enzymatic labeling methods using expensive labeled nucleotides and sequence dependence, are solved using a direct labeling approach. These new systems will provide the detection tools needed to advance the promising pharmaceutical, research and diagnostic uses of genomic analysis to determine the pattern of gene expression in disease or upon therapeutic treatment. Marker Gene Technologies, Inc. has established the feasibility of these detection methods by preparing new ultrasensitive fluorescent labeling reagents and developing protocols for directly labeling DNA or RNA samples isolated from live cells. These reagents are able to efficiently and sensitively label oligonucleotides for high-throughput microarray analysis. In Phase II these systems will be validated by further analysis of the fluorescent labeling methods and characterization of their ability to monitor changes in gene expression upon application of drugs or other bioactive compounds or in response to biological changes in cell function or disease, in a cell-specific manner. The broader impacts of this project include development and commercialization of new methods for rapid screening of genomic expression patterns in response to specific drug application in normal cells and tissues as well as in disease, bacterial or viral infections. These methods are a significant improvement over existing technologies by using a direct labeling approach that is quicker, more accurate and more cost-effective. These systems will be marketed to the pharmaceutical and diagnostics industries for high-throughput pre-clinical screening of drug efficacy by comparative cellular genomic analysis. In addition, existing collaborations with industrial and research partners assure quick commercial development of the technology. The combined techniques will improve U.S. competitiveness in the burgeoning genomic analysis field as well as in pharmaceutical therapeutic drug development and lead to further job creation based on both the products and systems developed.
Marker Gene Technologies | Date: 2013-05-14
The present invention relates to the visualization of acidic organelles based upon organelle enzyme activity. The organelle substrates of the invention are specific for enzyme activity of the organelle and label these organelles, such as lysosomes, rendering them visible and easily observed. Substrates of the present invention include substrates that produce a fluorescent signal. The fluorogenic acidic organelle enzyme substrates of this invention are designed to provide high fluorescence at low pH values and are derivatized to permit membrane permeation through both outer and organelle membranes of intact cells and can be used for staining cells at very low concentrations. They can be used for monitoring enzyme activity in cells at very low concentrations and are not toxic to living cells or tissues.