The 49th annual SOT meeting was held from March 7 to March 11 at the Salt Palace Convention Center in Salt Lake City Utah. The conference had 210 sessions that accounted for 2,405 presentations. While the exact number of attendees had not yet been announced several vendors suggested the number of attendees (not counting vendors) was around 4,000, which was less than the year before. The poor economy of the previous year was blamed for the drop off in attendance.
While I was not able to attend many of the talks, there were numerous poster presentations that brought large numbers into the exhibition show floor. Unlike many meeting that I have attended, posters were only available for viewing 3 hours, with a new group of posters being put up morning in afternoon each day.
In regards to the exhibition, BioTek was the only microplate instrumentation company exhibiting at the show. Traditional reagent companies were also limited, with Enzo Life Sciences and Promega being the most notable. The exhibition boasted a large number of assay service companies offering assay development and ADME/Tox testing. These contract lab service companies were the vast majority of the exhibitors present; with some have immense 40 x 60 booths. There were numerous animal testing equipment companies present. This equipment was primarily for rodents, but animal models also included dogs, rabbits and primates.
This was the first year that BioTek attended the meeting as a vendor and as such had a low priority for booth location. As a result the booth was located closer to Siberia than the front door of the exhibition hall. Despite being located at the far end of the hall and not having BioTek’s name in the published meeting vendor program listing a number of very satisfied customers stopped by the booth.
As with any scientific meeting the presentations and exhibits is only part of the charm. The location is almost as important as the science and this meeting was no different. The city itself is a haven for skiers with several ski resorts located nearby. The locals claim that Utah has the best snow in the country. I found it hard to argue. Many attendees skipped out of talks, came early or planned on staying after the conference to get in some skiing. The weather was pleasant with temps in the 50s each day despite a large amount of snow in the surrounding mountains. Numerous restaurants, including a couple of brewpubs were within walking distance of the convention center. Next year the 50th annual meeting is being held in Washington DC. It should be a good one as well.
Thursday, March 11, 2010
Thursday, March 4, 2010
Pittsburg Conference Recap
The annual 2010 Pittsburg Conference was held at the Orange County Conference Center in Orlando, FL and boasted nearly 2000 booths with 6700 exhibitors serving the 8000 delegates registered, with more arriving in a continuous stream. As the focus of the conference is on analytical chemistry and applied spectroscopy the conference sponsors included major industry giants such as Agilent, Thermo, Jasco, PerkinElmer, Dionex and myriad others offering a large range of analytical equipment and OEM solutions. The large analytical instrumentation companies made their presence known with expansive booths launching new products with the latest advances in separation technologies feeding samples into high-end MS, AA, ICP-AES and every version of light spectroscopy instrumentation ranging from UV-Vis to NIR.
Waters introduced its latest separations technology launching the Acquity Ultra-high Performance LC system capable of performing separations via up to 8 column/matrices and 24 solvents! Agilent introduced the latest in triple quadrupole mass spectrometers while Shimadzu is in the process of launching a new product for purification and concentration via trapping in a “tunable” particle structure for analysis of dilute analyte as well as the latest in LC-MS instruments. Many of the technologies are focused on analysis with micro- to nanoliter volume samples. In many instances these volumes are pushing the envelope of detection limits forcing instrumentation to become ever more sensitive.
In addition to the exhibition floor, the conference consisted of invited symposia from founders and leaders in the fields of analytical chemistry and spectroscopy, contributed sessions, poster sessions, awards presentations, short courses, networking and technical programs. The technical programs covered a diverse range of focus areas such as Art/Archaeology, Bioanalytical, Drug Discovery, Fuels, Energy and Petrochemicals, Homeland Security/Forensics, Sensors/Integrated Sensor systems, and Spectroscopy with hundreds of presentations each day. BioTek presented a poster at the Sunday evening session titled: Correlation of Sample OD Measurements on a Multi-Volume (Micro) Plate.
This conference appeals to the R&D engineer and systems manufacturers as a large portion of the smaller exhibits showcased the latest in gratings, mirrors, fiber optics, power supplies, heating elements, ceramics, valves, electron generators, controllers, etc., etc. Additional exhibits included labware and analyzers of all fashions as well as pharmaceutical equipment spanning from R&D to production/QC to packaging. Certainly the conference is a spectacle to be seen and excellent resource for information for anyone working in a scientific discipline.
Tuesday, February 23, 2010
Detection of Drug-Induced Lysosomal Cytoxicity
The identification of cytotoxic effects is a critical element in the pre-clinical small molecule drug discovery process. In-vitro cell-based assays are typically used early in the process following screening which requires the ability to automate workflows to handle the number of compounds tested. Cytotoxic effects of drug molecules are often first observed as perturbations of normal cellular organelle functionality. For example, Lysosome perturbation as a result of ion trapping of amine containing compounds has been demonstrated to cause the formation of autophagosomes and autophagic cytopathology. Cationic amphiphilic compounds (i.e. small molecule drug compounds) can be absorbed by cells by simple diffusion and accumulate inside the acidic cellular organelles, a process referred to as lysosomotropism. While many drugs require the presence of a cationic moiety for bioactivity, their accumulation into subcellular organelles can lead to many undesirable effects [1].
Figure 1. Image of lysosomal staining by Lyso-ID® Red dye. Increase in lysosome volume and number resulting from 200 µM verapamil.
Many drugs cause an accumulation of phospholipids and lysosomes in the cytoplasm. For example amioderone induces an abnormal accumulation of phospholipids that appear as vacuoles with multilamellar inclusions often referred to as autophagosomes [2]. Other organic amines cause vacuolar-ATPase driven ion trapping, which has been associated with vacuolar and autophagic cytopathology.
Here we describe the use of the EL406™ Combination Washer Dispenser to automatically aspirate media, wash cells and dispense reagents for the Lyso-ID® Red Detection kit, part of the CELLestial® Live Cell Analysis platform from Enzo Life Sciences.
Lyso-ID® Red dye is a fluorescent reagent that accumulates in lysosomes. An increase in signal is indicative of an increase in the number or size of cellular lysosomes and lysosome-derived vacuoles. In addition to the lysosomal specific dye, the assay also uses Hoechst 33342 nuclear stain. A decrease of 30% or greater of the Hoechst signal is indicative of generalized cytotoxicity.
H-mesothelioma (H-Meso) cells were seeded at 20,000 cells per well and allowed to attach overnight. The following morning the cells were treated with increasing doses of chloroquine, verapamil or thiostrepton. After an 18-hour exposure to the drugs the cells were washed and Lyso-ID® Red and Hoechst 33342 dyes were added using the EL406 Washer Dispenser. After 30 minute incubation, excess dye was removed by washing 3 times with wash solution, followed by a final addition of 80 µL of wash solution. The fluorescence was then determined using a Synergy Mx reader. Lyso-ID® Red dye (red fluorescence) was measured using an excitation of 540 nm and an emission of 680 nm, while Hoechst dye (blue fluorescence) was determined with an excitation of 340 nm and an emission of 480 (Figure 2).
Figure 2. Schematic of the Automated Lyso-ID® Red Process Carried out by the EL406 Washer Dispenser and Synergy™ Mx Reader.
Initial experiments focused on the response of human mesothelioma cells to agents known to cause drug-induced phospholipidosis. When H-meso cells were treated with various concentrations of verapamil a dose dependent increase in Lyso-ID® Red signal is observed (Figure 3). The presence of 100 µM verapamil resulted in a 2-fold increase in fluorescence. This indicates that this drug causes an increase in the number of cellular lysosomes and/or an increase in lysosomal volume in H-Meso cells. Staining with Hoechst 33342, a monitor for cytotoxic cell loss, did not change over the tested concentrations.
Figure 3. Dose Dependent Drug Induced Increase in Lysosomal Content as Measured by Increase in Lyso-ID® Red Fluorescence
Thiostrepton is an antibiotic related to Siomycin A, first described by Donivick et. al. in 1955 [3]. This thiazole compound, while typically used in topical antibiotic mixtures due to its poor water solubility, has been found to target breast cancer cells through inhibition of forkhead box M1 expression [4]. Because tumor cells are selectively targeted as compared to normal cells and the chemical structure of thiostrepton contains several amino groups we sought to examine whether or not this compound is differentially partitioned into lysosomes depending on cellular transformation.
Figure 4. Response change in Lyso-ID® Red dye Fluorescence caused by thiostrepton and verapamil in two mesothelial cell lines
The effect of thiostrepton on lysosomal content was tested with a H-meso malignant mesothelioma cell line, as well as control LP9 mesothelial cells. The verapamil dose response curves were run in parallel as a positive control. As seen in figure 4, verapamil elicited an approximately 2-fold increase in Lyso-ID® staining in both cell lines at higher drug concentrations with no increase in Hoechst signal. This indicates that these cell lines are capable of responding to agents known to cause phospholipidosis, resulting in increased lysosome content. When the effect of thiostrepton was examined, only the non-transformed cell line LP9 was found to respond with increased lysosome content as a result of exposure to the compound. Lyso-ID® Red dye fluorescence increased almost 3-fold with the addition of 5 µM thiostrepton in LP9 cells, while the H-Meso cells displayed no increase as a result to exposure to thiostrepton (Figure 4).
The Lyso-ID® Red kit in conjunction with the EL406 Combination Washer Dispenser provides an optimized workflow to investigate and screen compounds for phospholipidosis. The Lyso-ID® Red fluorescent assay kits provide a much easier way to screen for this potential problem than the gold standard method of electron microscopy. The ELx406 Washer Dispenser provides the liquid handling capabilities to automate many of the tedious fluid handling steps required of this assay. The EL406 instrument has the ability to quickly and reliably add and remove fluid from 96 or 384-well microplates containing live cells, replacing numerous manual pipetting tasks. In addition the device can dispense accurate amounts of Lyso-ID® Red reagent as needed.
References
1.Ikeda , K., M. Hirayama, Y. Hirota, E. Asa, J. Seki and Y. Tanaka (2008) Drug-induced phospholipidosis is caused by blockade of mannose 6-phosphate receptor-mediated targeting of lysosomal enzymes, BBRC, 377:268-274.
2.Lewis,J.H., F. Mullick, , K.G. Ishak, R.C. Ranard, B. Ragsdale, R.M. Perse, E.J. Rusnock, A. Wolke, S.B. Benjamin, L.B. Seeff, H.J. Zimmerman. (1990) Histopatholic Analysis of Suspected Amiodarone hepatoxicity. (Hum. Path. 21:59-67.
3.Donovick R, Pagano JF, Stout HA, Weinstein MJ (1955). Thiostrepton, a New Antibiotic I. In. vitro Studies Antibiotics Annual 3: 554–9.
4.Kwok, J. M-M., S.S. Myatt, C.M. Marson, R.C. Coombies, D. Constantinidou, and E. W-F. Lam (2008) Thiostrepton Selectively Targets Breast Cancer Cells through Inhibition of Forkhead box M1 Expression. Mol. Cancer Ther. 7(7):2022-2032
Figure 1. Image of lysosomal staining by Lyso-ID® Red dye. Increase in lysosome volume and number resulting from 200 µM verapamil.
Many drugs cause an accumulation of phospholipids and lysosomes in the cytoplasm. For example amioderone induces an abnormal accumulation of phospholipids that appear as vacuoles with multilamellar inclusions often referred to as autophagosomes [2]. Other organic amines cause vacuolar-ATPase driven ion trapping, which has been associated with vacuolar and autophagic cytopathology.
Here we describe the use of the EL406™ Combination Washer Dispenser to automatically aspirate media, wash cells and dispense reagents for the Lyso-ID® Red Detection kit, part of the CELLestial® Live Cell Analysis platform from Enzo Life Sciences.
Lyso-ID® Red dye is a fluorescent reagent that accumulates in lysosomes. An increase in signal is indicative of an increase in the number or size of cellular lysosomes and lysosome-derived vacuoles. In addition to the lysosomal specific dye, the assay also uses Hoechst 33342 nuclear stain. A decrease of 30% or greater of the Hoechst signal is indicative of generalized cytotoxicity.
H-mesothelioma (H-Meso) cells were seeded at 20,000 cells per well and allowed to attach overnight. The following morning the cells were treated with increasing doses of chloroquine, verapamil or thiostrepton. After an 18-hour exposure to the drugs the cells were washed and Lyso-ID® Red and Hoechst 33342 dyes were added using the EL406 Washer Dispenser. After 30 minute incubation, excess dye was removed by washing 3 times with wash solution, followed by a final addition of 80 µL of wash solution. The fluorescence was then determined using a Synergy Mx reader. Lyso-ID® Red dye (red fluorescence) was measured using an excitation of 540 nm and an emission of 680 nm, while Hoechst dye (blue fluorescence) was determined with an excitation of 340 nm and an emission of 480 (Figure 2).
Figure 2. Schematic of the Automated Lyso-ID® Red Process Carried out by the EL406 Washer Dispenser and Synergy™ Mx Reader.
Initial experiments focused on the response of human mesothelioma cells to agents known to cause drug-induced phospholipidosis. When H-meso cells were treated with various concentrations of verapamil a dose dependent increase in Lyso-ID® Red signal is observed (Figure 3). The presence of 100 µM verapamil resulted in a 2-fold increase in fluorescence. This indicates that this drug causes an increase in the number of cellular lysosomes and/or an increase in lysosomal volume in H-Meso cells. Staining with Hoechst 33342, a monitor for cytotoxic cell loss, did not change over the tested concentrations.
Figure 3. Dose Dependent Drug Induced Increase in Lysosomal Content as Measured by Increase in Lyso-ID® Red Fluorescence
Thiostrepton is an antibiotic related to Siomycin A, first described by Donivick et. al. in 1955 [3]. This thiazole compound, while typically used in topical antibiotic mixtures due to its poor water solubility, has been found to target breast cancer cells through inhibition of forkhead box M1 expression [4]. Because tumor cells are selectively targeted as compared to normal cells and the chemical structure of thiostrepton contains several amino groups we sought to examine whether or not this compound is differentially partitioned into lysosomes depending on cellular transformation.
Figure 4. Response change in Lyso-ID® Red dye Fluorescence caused by thiostrepton and verapamil in two mesothelial cell lines
The effect of thiostrepton on lysosomal content was tested with a H-meso malignant mesothelioma cell line, as well as control LP9 mesothelial cells. The verapamil dose response curves were run in parallel as a positive control. As seen in figure 4, verapamil elicited an approximately 2-fold increase in Lyso-ID® staining in both cell lines at higher drug concentrations with no increase in Hoechst signal. This indicates that these cell lines are capable of responding to agents known to cause phospholipidosis, resulting in increased lysosome content. When the effect of thiostrepton was examined, only the non-transformed cell line LP9 was found to respond with increased lysosome content as a result of exposure to the compound. Lyso-ID® Red dye fluorescence increased almost 3-fold with the addition of 5 µM thiostrepton in LP9 cells, while the H-Meso cells displayed no increase as a result to exposure to thiostrepton (Figure 4).
The Lyso-ID® Red kit in conjunction with the EL406 Combination Washer Dispenser provides an optimized workflow to investigate and screen compounds for phospholipidosis. The Lyso-ID® Red fluorescent assay kits provide a much easier way to screen for this potential problem than the gold standard method of electron microscopy. The ELx406 Washer Dispenser provides the liquid handling capabilities to automate many of the tedious fluid handling steps required of this assay. The EL406 instrument has the ability to quickly and reliably add and remove fluid from 96 or 384-well microplates containing live cells, replacing numerous manual pipetting tasks. In addition the device can dispense accurate amounts of Lyso-ID® Red reagent as needed.
References
1.Ikeda , K., M. Hirayama, Y. Hirota, E. Asa, J. Seki and Y. Tanaka (2008) Drug-induced phospholipidosis is caused by blockade of mannose 6-phosphate receptor-mediated targeting of lysosomal enzymes, BBRC, 377:268-274.
2.Lewis,J.H., F. Mullick, , K.G. Ishak, R.C. Ranard, B. Ragsdale, R.M. Perse, E.J. Rusnock, A. Wolke, S.B. Benjamin, L.B. Seeff, H.J. Zimmerman. (1990) Histopatholic Analysis of Suspected Amiodarone hepatoxicity. (Hum. Path. 21:59-67.
3.Donovick R, Pagano JF, Stout HA, Weinstein MJ (1955). Thiostrepton, a New Antibiotic I. In. vitro Studies Antibiotics Annual 3: 554–9.
4.Kwok, J. M-M., S.S. Myatt, C.M. Marson, R.C. Coombies, D. Constantinidou, and E. W-F. Lam (2008) Thiostrepton Selectively Targets Breast Cancer Cells through Inhibition of Forkhead box M1 Expression. Mol. Cancer Ther. 7(7):2022-2032
Monday, February 22, 2010
Micro-Volume Spectrophotometric Analysis
Micro-volume nucleic acid quantification using short pathlength spectrophotometric devices, such as NanoDrop, simply workflows through omission of dilution steps and conserve samples by using only 1-2 µL of sample, which may even be retained after analysis. The molar absorptivity of dsDNA and RNA at 260 nm is consistent whatever the amount or proportions of purine or pyrimidine bases and sufficient for accurate quantification of samples ranging from a few decades of ng/µL to thousands of ng/µL. This effectively covers the expected dsDNA or RNA concentration isolated from genomic DNA or RNA kits from a wide range of samples and also the isolation of plasmids on the concentration end.
Protein micro-volume spectrophotometric analysis is more problematic. The molar absorptivity of proteins at 280 nm is dependent on the relative amounts and proportions of amino acids with aromatic side chains, such as phenylalanine, tyrosine and tryptophan. The absorptivity of these side chains can also be affected by the 3° structure of the protein. Furthermore, sensitivity at 280 nm is typically limited to protein concentrations ranging from a few tenths of mg/mL to a few decades of mg/mL – a concentration range that is an order of magnitude higher that for nucleic acids. Fortunately, for most samples (i.e. cells, tissues), total protein is about an order of magnitude higher than total RNA.
Do you perform spectrophotometric micro-volume analysis? If so, which device do you use: NanoDrop, NanoVue, Epoch/Take3 or another? Do you use the device for nucleic acid or protein determinations, or both?
Protein micro-volume spectrophotometric analysis is more problematic. The molar absorptivity of proteins at 280 nm is dependent on the relative amounts and proportions of amino acids with aromatic side chains, such as phenylalanine, tyrosine and tryptophan. The absorptivity of these side chains can also be affected by the 3° structure of the protein. Furthermore, sensitivity at 280 nm is typically limited to protein concentrations ranging from a few tenths of mg/mL to a few decades of mg/mL – a concentration range that is an order of magnitude higher that for nucleic acids. Fortunately, for most samples (i.e. cells, tissues), total protein is about an order of magnitude higher than total RNA.
Do you perform spectrophotometric micro-volume analysis? If so, which device do you use: NanoDrop, NanoVue, Epoch/Take3 or another? Do you use the device for nucleic acid or protein determinations, or both?
Monday, February 8, 2010
LabAutomation 2010 Recap
LabAutomation 2010 was another engaging conference and exhibition held in the Californian desert at Palm Springs. Over 4100 delegates and 226 exhibitors participated over five days at the end of January. The conference had something for everyone as their were five concurrent tracks held throughout the scientific program offering symposia topics such as Multiplexed Microfluidic Systems, Biofuels, Process Analytical Technology and Point of Service Assays, among others.
R. Graham Cooks gave a stimulating opening plenary session tracking the evolution of mass spectrometry focusing on the 100 years of development of ion sources and mass analyzers culminating in his vision of portable mass spectrometers. Kei Koizumi gave an overview of OSTP and hinted at levels of Federal R&D investment in the years to come. Hod Lipson gave an very entertaining lecture about taking the grunt out of science by sifting through data and looking for the laws that govern how something behaves. He described how his software tool Eureqa can examine experimental data and derive the simplest mathematical formula to describe the behavior. Finally, the conference was closed out in an unconventional manner by Bruce Sterling, a science fiction novelist and co-founder of the cyberpunk movement in that genre, where he provided a glimpse into the future, as he sees it.
If you haven't considered attending LabAutomation, we fully recommend it!
R. Graham Cooks gave a stimulating opening plenary session tracking the evolution of mass spectrometry focusing on the 100 years of development of ion sources and mass analyzers culminating in his vision of portable mass spectrometers. Kei Koizumi gave an overview of OSTP and hinted at levels of Federal R&D investment in the years to come. Hod Lipson gave an very entertaining lecture about taking the grunt out of science by sifting through data and looking for the laws that govern how something behaves. He described how his software tool Eureqa can examine experimental data and derive the simplest mathematical formula to describe the behavior. Finally, the conference was closed out in an unconventional manner by Bruce Sterling, a science fiction novelist and co-founder of the cyberpunk movement in that genre, where he provided a glimpse into the future, as he sees it.
If you haven't considered attending LabAutomation, we fully recommend it!
Tuesday, January 26, 2010
Federal Research and Development Investments by the Obama Administration
Kei Koizumi opened the second day of LabAutomation 2010 with a lecture concerning the role the Obama Administration has played in increased R&D funding in 2009 and plans for both 2010 and beyond. Koizumi is the Assistant Director for Federal R&D of the Office of Science and Technology Policy (OSTP). He detailed the Recovery Act of 2009 which provided an additional $10.4B for NIH-based funding. As of today, approximately $6B of these monies has been awarded and efforts are accelerating to distribute the remaining funds as soon as possible.
Koizumi indicated that through 2016, the National Science Foundation, Department of Energy and National Institute of Standards and Technology will see their annual budgets doubled. The premise behind this is to foster basic science. NIH funding will remain essentially flat at about $30B annually, where it has been since 2003. Koizumi indicated that the new Director of NIH, Francis Collins has defined specific investment areas to be pursued in 2010 and beyond. They include:
- Genomics and high throughput technologies for disease research
- Translation of basic research into treatments, especially for diseases with limited market potential
- Science for health care reform
- Global health initiatives
- Reinvigorating health care
Some of these future developments will be touched on during the President’s State of the Union Address tomorrow where Koizumi specified that Obama will reiterate his belief in and support for Federal R&D investment. We will also wait with anticipation for the initial announcement for the 2011 Federal R&D budget this coming Monday
Do you agree with where the R&D investments are being made?
Opening Plenary Lecture, LabAutomation 2010
R. Graham Cooks from Purdue University gave the opening plenary lecture at LabAutomation 2010. This annual conference, organized by the Association of Laboratory Automation (ALA), has been characterized by steady growth in both exhibitors and delegates for a number of years, largely due to the quality of speakers, such as Graham Cooks.
Cooks has been at the forefront of mass spectrometry (MS) development for a number of decades. He is most well known for his work with Paul ion traps, noted for their simplicity of design, small size and ability for MSn. In his talk, he gave an overview of MS development since J.J. Thomson first developed a mass spectrometer in his Cavendish labs over a 100 years ago. The time line of development traced the development of ion sources from electron impact to electrospray (ESI) and matrix-assisted laser desorption (MALDI); and mass analyzers from sector instruments to fourier transform – ion cyclotron resonance (FT-ICR).
It was clear from the talk that Cooks believes that MS-detection is in a golden age and is still developing. He pointed to his work with new ambient ionization sources, such as desorption electrospray ionization (DESI) and compact mass analyzers based on ion traps that weigh only 11 lbs. He explained that both can be used as the basis for a portable MS that could be used for applications such as testing athletes for performance enhancing substances right on the field before start of play!
Can you believe it?
Cooks has been at the forefront of mass spectrometry (MS) development for a number of decades. He is most well known for his work with Paul ion traps, noted for their simplicity of design, small size and ability for MSn. In his talk, he gave an overview of MS development since J.J. Thomson first developed a mass spectrometer in his Cavendish labs over a 100 years ago. The time line of development traced the development of ion sources from electron impact to electrospray (ESI) and matrix-assisted laser desorption (MALDI); and mass analyzers from sector instruments to fourier transform – ion cyclotron resonance (FT-ICR).
It was clear from the talk that Cooks believes that MS-detection is in a golden age and is still developing. He pointed to his work with new ambient ionization sources, such as desorption electrospray ionization (DESI) and compact mass analyzers based on ion traps that weigh only 11 lbs. He explained that both can be used as the basis for a portable MS that could be used for applications such as testing athletes for performance enhancing substances right on the field before start of play!
Can you believe it?
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