Modulus Microplate Multimode Reader
Dr. Martin Smilkstein
Portland VA Medical Center

The Modulus is in place at the Shoklo Malaria Research Unit. We tested more than 70 clinical isolates (blood samples from patients with falciparum malaria) from the clinics served by SMRU. These clinics treat almost exclusively refugees and migrant workers from Burma, primarily members of the Karen hill tribe. The isolates were grown in vitro in the presence of serial dilutions of 8 different antimalarial drugs (chloroquine, quinine, piperaquine, mefloquine, artesunate, dihydroartemisinin, lumefantrine, and doxycycline) to determine susceptibility or drug resistance. Matching plates were set up for each isolate, one assayed using a pLDH DELI absorbance-based plate reader method, and the other assayed using our SYBR green fluorescence-based method. All plates were read on the Modulus, and a randomly selected subset of these were also read using transfer to mini-tubes and the TBS-380. In short, the assay and the Modulus out-performed the DELI (less failed assays, more valid results so better capture of epidemiologic data) at far lest cost and will be their standard from now on. This also validates that top-reading can work (although I still think it is not optimal) for this purpose in a challenging setting and should make the Modulus an immediate candidate for this role. Basically, I think there will be no further debate as to the method, and attention will shift back to the choice of reader method, making it very timely.

Modulus Single Tube Fluorometer
Richard Lasker, Director
Brabant Research, Inc.
Bent Mountain, Virginia
www.brabantresearch.com

 
Our work centers about analyzing the nutrient content of raw fruits & vegetables. This helps us determine what is, and is not, in the food we all consume for our nutritional needs. The accumulation of this data, and the comparison of the USDA "label" nutritional levels that are used throughout the food industry, blanket without any confirmation, will help us design better growing programs coupled with better varieties to improve human nutrition and therefore decrease human illness issues. This research will be done through Extraction of vitamins, amino acids and other nutritional compounds via wet chemistry to quantify the levels of nutritional compounds in raw fruits & vegetables. We entirely self-fund the work we do in human nutrition. We have, to this date, received no monies from any source. Every dollar we make on contracts, royalties, co-operative projects or consulting we spend doing the nutritional research. This includes growing varieties in order to be able to determine how much of an increase we can attain in the selected fruits & vegetables in order to qualify selections and delineation. Every dollar we do not have to spend on necessary equipment would help speed the end-result: identifying those foods that can help prevent/cure diseases. Our work is unique in that no one, anywhere, has absolutely no agenda other than the identification and improvement of the foods consumed by us. All groups we have met all have their own agenda which often conflicts with the data and results of their work. No one is doing complete nutritional analysis of commercially grown produce. Even cancer and medical research trials utilize the USDA/FDA "label" nutritional values in their calculations. Hence why
the whole foods trials are always unsuccessful. Our preliminary work, in bone cancer and lymphoma has a 100% success rate preventingrelapse after initial chemo/radiation therapy.

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Modulus Single Tube Fluorometer
Sujey  Carro, Thesis Student (graduate-research)
UMET School of Environmental Affairs
San Juan, Puerto Rico
www.umet.edu

My project is to determine if:
1) Apoptosis is the mode of cell death using the mitochondrial transmembrane potential as an indicator.
2) ROS generation, ATP determination to see if there is mitochondrial dysfunction, impairment of cellular respiration and if the generated ROS are contributors of cellular oxidative stress.
3) Adduct formation in mitochondrial DNA. Since we already know there is adduct formation with Calf thymus DNA and nuclear DNA we would expect to see adducts in the mitochondrial DNA.

All of this will be assessed after exposing cancer cell line A431 to three different concentrations at three different times of exposure to potential anticancer drug NBQ.

I plan to publish my results to gain experience in the professional scientific community.

1)  For apoptosis we would we are currently using mitochondrial membrane permeabilization as an indicator of apoptosis.
2)   Our NBQ drug agents are autoflourescent and we propose to measure how much the adherent A431 cells uptake the drug. We can estimate the amount of drug uptake by cells measuring the absorbance change in the RPMI residual medium that will be removed every 24 hours over a 72 hour exposure period. Subtracting the amount of absorbance of the initial drug dose with RPMI from the RPMI residual will result in the change in absorbance of drug and RPMI over the 72 hours. The absorbance readings can be made with a UV-Spectrophotometer.
3)   For ROS we would like to measure hydrogen peroxide production measuring fluorescence with horseradish peroxidase.
4)   We will determine ATP using luciferase luciferin bioluminescent kit.
5)   Adduct detection would be performed by isolating mitochondria, isolating mtDNA, digest mtDNA and then use the HPLC-ESI MS/MS for detection.

As part of a Risk Management we are trying to discover the mechanism of action of these synthesized potential anticancer drugs. We are using the cancer cell line A431 in order to complete the pre-clinical phase of this study (in vitro). This equipment is an essential tool that will help us to discover the mechanism of action of the drug using the mitochondrion as a target organelle. My budget is small but I am much exited about completing it to acquire my Masters Degree.

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Modulus Single Tube Fluorometer
Chin-Chuan Wei, Assistant Professor
Southern Illinois University Edwardsville
Chemistry Department
Edwardsville, Illinois
http://www.siue.edu/~cwei

The main focus of Dr. Wei's laboratory is to study the mechanism of reactive oxygen species (ROS) generated from non-phagocytic NADPH oxidases (NOXs) that play important roles in cancer and disease development. Using the recombinant DNA/protein technology, we express and/or purify the desired proteins fused with fluorescent proteins or purification tags for structural and functional studies.
List of Assays = nucleic acid and protein quantitation , gene expression.
We request a Modulus Fluorometer that allows us to perform routine molecular biology techniques, such as nucleic acid and protein quantitation as well as gene expression. We intend to use this instrument for the measurement of ROS levels that are related to the function of NOXs both in vitro and in vivo in order to understand their molecular mechanism.

The instrument will facilitate our research on the mechanism of ROS generated from NOXs at Southern Illinois University Edwardsville (SIUE), a primarily undergraduate institute, and it will be also used in biochemistry laboratory and advance biochemistry course for gene expression experiments using green and red fluorescent proteins.

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Modulus Single Tube Fluorometer
Betsy Martinez-Vaz, Assistant Professor of Biology
Hamline University
Biology Department
Saint Paul, Minnesota
http://www.hamline.edu/cla/acad/

The primary focus of my research is to investigate the effects of sub-inhibitory concentration of antibiotics in bacterial gene expression and metabolism. My laboratory uses Escherichia coli K12 strain MG1655, a model laboratory organism, to study how gene regulation is affected by sub-inhibitory concentrations of antibiotics. Most research on antimicrobial drugs has focused on microorganisms isolated due to their high level of resistance to one or to multiple antibiotics. Bacteria are frequently exposed to low concentrations of antibiotics in laboratory environments and in their natural surroundings; the cellular effects of sub-inhibitory concentrations of antibiotics in bacteria have not been studied in detail. The significance of this research is twofold. First, elucidating how bacteria respond to sub-inhibitory concentrations of antibiotics is essential to understand the ways in which microbes overcome the effects of antimicrobial drugs. Secondly, understanding the transcriptional responses triggered by antibiotics is crucial to elucidate the natural function of antibiotics. Antibiotics were originally discovered because of their ability to adversely affect microbial species. However, many antibiotics are naturally occurring molecules (produced by microbes) which cellular function is not fully understood. My research laboratory is located at Hamline University, a primarily undergraduate institution in Saint Paul, Minnesota. Accordingly, my research program focuses on using the effects and the modes of action of antibiotics as a framework to introduce undergraduate students to scientific research while teaching the principles of molecular biology and microbiology. In order to study how antibiotics affect gene expression and trigger cell-signaling responses, my students are in the process of constructing a collection of GFP transcriptional fusions, which includes the promoters for the main transcriptional regulators present in Escherichia coli. The next step of their research will consist of measuring the activity of the GFP fusions in the presence of different classes of antibiotics at several concentrations.
By identifying E.coli promoters that are responsive to antibiotics, we will get insights into the following questions:
1) Does exposure to low concentration of antibiotics triggers specific gene expression patterns in the cell?
2) Can antibiotics act as signaling molecules?
This will allow students to learn about the role of antibiotics and the regulatory networks that control antimicrobial resistance in microbial cells.
List of Assays = 1) Measurement and quatification of fluorescence from GFP-transcriptional fusions.
2) Measurement and quantification of nucleic acids
3) measurement of microbial growth by monitoring absorbance at 600 nm.
The main application of the Modulus laboratory Fluorometer will be to quantify gene expression by measuring the activity of GFP-promoter fusions. In addition, the fluorometer will be useful to quantify nucleic acids prior to PCR and other molecular biology experiments.

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Modulus Single Tube Fluorometer
Timothy Mattes, Assistant Professor
The University of Iowa
Civil and Environmental Engineering Department
Iowa City, Iowa
www.cee.engineering.uiowa.edu

In general, I am interested in the mechanisms that govern evolution of xenobiotic biodegradation pathways.  I am also interested developing molecular tools for the detection and quantification of aerobic bacteria that degrade vinyl chloride, a known
human carcinogen, in the environment.
List of Assays = Molecular biology techniques such as cloning, sequencing, real-time PCR, real-time reverse transcription PCR, in vitro transcription
We are starting to develop techniques for real-time PCR and real-time RT-PCR of specific catabolic genes using an internal mRNA reference technique for quantification.  This requires that we accurately quantify DNA and RNA using fluorescence techniques (e.g. Picogreen, ribogreen)

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Modulus Single Tube Luminometer
Radhika Andavolu, Director
Genetics Research Institute of the Desert
Rancho Mirage, California
www.gridonline.org
Microarray technology to define molecular signatures of prostate tumors that will provide additional predictive value over conventional markers of outcome and be useful in helping to guide clinical management. The assays I am running are Panomics Quantigene assays for Gene Expression in Tumor and normal tissues.

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Modulus Single Tube Luminometer
Dr. Raju C. Reddy is an Assistant Professor of Internal Medicine in the Division of Pulmonary and Critical Care Medicine at the University of Michigan School of Medicine. He was granted a Modulus Luminometer to study the role of the nuclear hormone receptor, peroxisome proliferator-activated receptor-gamma (PPAR-gamma), in lung tumorigenesis, pulmonary fibroproliferative responses, and alveolar macrophage function. Further characterizing the functional significance of PPAR-gamma in pulmonary (patho)biology will potentially result in the identification of novel therapeutic targets.

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Modulus Microplate Multimode Reader set up as a Luminometer (Granted once per year in June – This is our first grant recipient for the year 2007)
Tanja Stoyan, Ph.D. Coordinator Undergraduate Teaching Laboratory
University of California Santa Barbara
Santa Barbara, California
I coordinate and teach laboratory classes for undergraduates in pharmacology and immunology. Approximately 60 students graduate from these classes each year with a B.S. in Pharmacology or  a B.S. in Microbiology/Cell Biology. These are classes for seniors and many of our students go on to work in the Biotech and Pharmaceutical Industries after they graduate. We are intending to expose our students to a wide variey of biochemical techniques that apply to molecular pharmacology. Recently we have developed new exercises for our classes using Flow Cytometry. My goal is to introduce our Pharmacology and Immunology students to the field of Luminometry and to develop several exercises that use this powerful technique. I will give you a brief overview about the Pharmacology program. The lab courses require pharmacology majors to spend one day each week for two quarters performing experiments to demonstrate the site and mechanism of action of a wide variety of drugs and chemical agents using different in vitro isolated tissue preparations, as well as in vivo, using anesthetized mice and rabbits. The winter quarter lab is designed to teach common biochemical techniques that are routinely used to investigate site and mechanism of action of drugs. The series of laboratory exercises include an ELISA assay to determine the concentration of protein in a sample, a radioactive drug-receptor-binding filter assay, a pharmacokinetic study to identify metabolites following different routes of administration of a therapeutic agent, isolation of liver microsomes for drug metabolism study, including identification of p450 enzymes by gel electrophoresis and immuno-blot, the amplification of G protein cDNA using PCR and a new Flow Cytometry exercise to determine the EC50 of drugs on apoptotic Jurkat cells. In addition to the experiments described above the students are required to complete a 3-dimensional molecular modeling computer exercise teaching them how to interpret structure-activity relationship of drugs and molecular binding at drug-receptor sites. The students use various software packages to analyze their data (Excel, Sigmaplot, Flow Cytometry software). Our goal is to keep the class up to date with new technologies and instrumentation. We feel that luminometry is a very important research tool.
We will be using Cell Viability and Apoptosis assays, Cell titer glo, Caspase-Glo, High Through put compound screening: p450 glo, kinase-glo plus assay, pGL4, luciferase reporter vectors, Dual-glo luciferase assay system. One assay that we are interested in developing is an assay to screen for GPRC modulators which is described in Cell Notes, Issue 16, 2006 from Promega. We would introduce a luciferase reporter plasmid into various cell lines and have the students add drugs from an 80 compound library to the cells (alternatively a series of dilutions of one single drug). We would then assay for firefly and Renilla luciferase activity using the Dual-glo assay system. The results would be measured in a 96 microplate luminometer. Another application would be to assay apoptotic activity of drugs in our compound library on Jurkat cells with a luminescent assay. A high throughput screen using Flow cytometry has been difficult to do in the class format since the time for reading the 96 well plate is too long.

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