Thursday, February 16, 2017

Rigor and Reproducibility

 
Rigor and Reproducibility

A recent report found that the published findings from 89% of preclinical studies in the field of cancer research – in particular, those deemed ‘landmark’ studies – were unable to be confirmed by other scientists [1]. The authors concluded that despite competent, well-intentioned researchers, an alarming number of these inconsistencies were due to limitations in the design and execution of the initial studies, including the use of problematic endpoint measures and biased analysis of data.

This report is not unique to one particular research field, but is in fact representative of a growing awareness within the greater scientific community of the critical need for robust and unbiased experimental design [2-5]. The National Institutes of Health (NIH), one of the world’s leading medical research centers and major provider of extramural biomedical research funds, recently implemented new grant application instructions intended to enhance reproducibility through rigor and transparency [6]. Research grant applicants must now address four areas of focus:  scientific premise, scientific rigor, biological variables, and authentication.

The NIH defines scientific rigor as “the strict application of the scientific method to ensure robust and unbiased experimental design, methodology, analysis, interpretation and reporting of results.” This standard has been particularly difficult to achieve for imaging-based studies, which conventionally depend heavily on manual methods for collecting and analyzing data. This time-consuming approach frequently offers limited quantitative results due to small sample size and qualitative endpoints. Additionally, subjective selection criteria and lack of standardization across methods and instrumentation impedes reproducibility and transparency.

Recent advances in automated imaging systems have provided a great leap forward in our ability to conduct robust imaging-based biomedical research. The efficient and consistent acquisition of high quality images by BioTek’s Lionheart FX and Cytation automated imagers, across large numbers of conditions and replicates, allows researchers to meet the high standards set for reproducibility and transparency. Furthermore, the routine application of powerful Gen5 processing and analysis tools across entire image sets enables robust and meaningful quantitative results. 

Access to innovative technology and the prospect of life-improving therapeutics on the horizon make this an exciting time to be a scientist. Successfully translating biomedical research into clinical advances requires rigorous and reproducible approaches.  In our rapidly evolving scientific landscape, the need to demonstrate the relevance and benefit of sound scientific research is as important now as ever before.

  1. Begley, C.G. and L.M. Ellis, Drug development: Raise standards for preclinical cancer research. Nature, 2012. 483(7391): p. 531-3.
  2. Begley, C.G., A.M. Buchan, and U. Dirnagl, Robust research: Institutions must do their part for reproducibility. Nature, 2015. 525(7567): p. 25-7.
  3. Begley, C.G. and J.P. Ioannidis, Reproducibility in science: improving the standard for basic and preclinical research. Circ Res, 2015. 116(1): p. 116-26.
  4. Kretser, A., D. Murphy, and J. Dwyer, Scientific integrity resource guide: Efforts by federal agencies, foundations, nonprofit organizations, professional societies, and academia in the United States. Crit Rev Food Sci Nutr, 2017. 57(1): p. 163-180.
  5. Pusztai, L., C. Hatzis, and F. Andre, Reproducibility of research and preclinical validation: problems and solutions. Nat Rev Clin Oncol, 2013. 10(12): p. 720-4.
  6. Health, N.I.o. https://grants.nih.gov/reproducibility/index.htm. 2017.


By, BioTek Instruments, Joe Clayton, PhD., Principal Scientist

Tuesday, November 1, 2016

Anxiety and the U.S. Election

U.S. election season is reaching its zenith as we find ourselves in an endless tug of war between local and national political candidates, each vying for our vote. Emotions, including anxiety, are running high among candidates, electorates, and even from other countries that are not involved in the election process. As scientists, we appreciate the calming effects of facts and data, so we decided to detach from the day-to-day election buzz to take a look at how our instruments are being used to characterize anxiety in the laboratory.

Modulation of Anxiety and Fear via Distinct Intrahippocampal Circuits
In characterizing mouse hippocampal sub-regions, these researchers demonstrate separate regions of interest that are specific to suppressing anxiety and fear. Total RNA from these regions was measured using the Synergy HT Multi-Mode Reader.

Acute Stress Dysregulates the LPP ERP Response to Emotional Pictures and Impairs Sustained Attention: Time-Sensitive Effects (direct pdf download)
When ascertaining the effects of acute stress on subsequent emotion processing following an acute stressor, the ELx800 Absorbance Reader was used to measure salivary cortisol levels in female undergraduate students.

Anxiety, Depression, Stress, and Cortisol Levels in Mothers of Children Undergoing Maintenance Therapy for Childhood Acute Lymphoblastic Leukemia
Salivary cortisol levels were also measured, this time using a PowerWave 340, in mothers of healthy children, and also mothers of children with leukemia as part of a study to compare anxiety, depression and stress among these groups.

Regulator of Calcineurin 1 Modulates Expression of Innate Anxiety and Anxiogenic Responses to Selective Serotonin Reuptake Inhibitor Treatment
Using mice, regulator of calcineurin 1 (RCAN1) is positively identified as influencing expression of anxiety-related and selective serotonin reuptake inhibitor-related behaviors, which makes it a possible therapeutic target for anxiety. Calcineurin phosphatase activity in mouse prefrontal cortexes was measured using a Synergy 2.

We certainly feel less anxious now, but if those weren’t enough to calm you this election cycle, you can try a distraction technique. Here’s a picture of a basket of kittens. Enjoy!

Kittens

By, BioTek Instruments

Monday, October 10, 2016

CRISPR: From editing to imaging

Since the discovery of the CRISPR (clustered regularly interspaced short palindromic repeats) pathway, the evolution of the gene editing technology has been astounding. Applications now include not only an easily implemented method for genetic modification but adaptation for gene knockdown, generation of allelic series for control of differential expression, an alternative method for generation of inducted pluripotent stem cells, up-regulation for genetic screening and more recently loci imaging by creation of fluorescent protein:dCas9 conjugates to investigate nuclear and chromatin structure and dynamics with real time imaging1. While not without limitations the current applications show how quickly a technology can be adopted and further adapted to meet the needs of a plethora of investigators. In a similar fashion, the range of applications performed on BioTek’s expanding range of Automated Cell Imagers and Cell Imaging Multi-Mode Readers has been broadened. In a few short years the applications group and customer base have performed a wide range of assays such as wound-healing, proliferation, 3D cell culture and label-free cell counting, just to name a few. As the application space evolves so does the need for instrumentation that can provide flexible and affordable solutions. BioTek’s range of imaging capable instruments has certainly filled the bill. Wouldn’t automated loci imaging be interesting...

dCaspp9-Loci-imaging

For further details of the imaging application space visit the BioTek website: http://www.biotek.com/resources/

For a video of live cell imaging by Drost, J. et. al.: http://www.nature.com/nature/journal/v521/n7550/fig_tab/nature14415_SV4.html

1. Drost, Jarno; van Jaarsveld, Richard H.; Ponsioen, Bas; Zimberlin, Cheryl; van Boxtel, Ruben; Buijs, Arjan et al. (2015): Sequential cancer mutations in cultured human intestinal stem cells. In Nature 521 (7550), pp. 43–47.

By: BioTek Instruments, Peter J. Brescia Jr., MSc, MBA

Monday, September 19, 2016

The Evolution and Domestication of Yeast

Anyone who knows me is aware that I’m a beer guy. While I’m a cell biologist by training one of my passions for quite some time is beer; whether it be tasting or brewing the beverage. In a recent article in the journal Cell, a group led by geneticist Kevin Verstrepen at the University of Leuven sequenced the genomes from 157 strains of Saccharomyces cerevisiea used to make ale, wine, sake, and bread [1]. When beer and science come together only good things can happen.

Yeast has been used to make fermented beverages for thousands of years. A 5,000-year-old Sumerian tablet describes an ancient party where ingredients to make fermented beverages known as beer today were used. Probably the first record of a kegger party! Since that time strains of S. cerevisiea have been developed to meet different needs.

Domestication of industrial yeasts
Figure 1.  Representation of the history and domestication of yeast used form making beer and other types of alcohol as revealed through genotypic and phenotypic analysis. Credit: Gallone and Steensels et al Cell 2016

The researchers dated the earliest cultivated yeast strains to the 1500s, which is likely a consequence of beer production in Europe moving from pubs into monasteries (Fig 1). As these early brewers fine-tuned their recipes, they also selected for favorable yeast strains. Domesticated yeasts have a greater capacity to metabolize sugar, fewer distasteful byproducts, and weaker reproductive abilities, compared to their wild-type cousins.

What is interesting is that the industrial yeast used today came from only a few ancestral strains. Five large groups separated out genetically, with strains mainly clustered together according to their industrial purpose. Geographic boundaries further divided each category: in one grouping of beer yeast, for example, the strains from Belgium and Germany were closely related, but separate from those in the UK and US [2].

As a brewer I know that the flavor of the beer depends greatly on the yeast. While many different beers can be made with the same strain using different grain mixtures, some beers that have very specific traits, such as the smoky clove and banana flavoring of German Hefeweizen, require specific strains. Hefeweizen requires the production of the compound 4-vinyl guaiacol (4-VG) in order to impart these unique flavors. These same flavors are considered flaws in other beer types. The genomes of the strains used to make Hefeweizen contain stretches of DNA, including the genes that make 4-VG, that seem to originate from wine yeast. It has been speculated that these strains emerged when an ale strain hybridized with a wine-making yeast, regaining the capacity to make the clove-smelling chemical.

Another point is that wine yeasts, which share their origins with beer yeast, show fewer signs of domestication. "This is probably because wine yeasts are only used to ferment grape juice once a year, and survive in and around the winery for the rest of the year, where they may interbreed with feral yeasts," Brigida Gallone and Jan Steensels of the University of Leuven told Scientific American.  "In that sense, beer yeasts are like dogs, completely 'tamed' and adapted to their relation with humans, whereas wine yeasts resemble the wilder character of cats." [3].

Me, I’m a beer guy who likes cats...

References
  1. B. Gallone and J. Steensels et.al (2016) Domestication and Divergence of Saccharomyces cerevisiea Beer Yeasts, Cell, 166(6):1397-1410.
  2. http://phys.org/news/2016-09-beer-yeasts-dogs-wine-cats.html#jCp
  3. http://www.scientificamerican.com/article/ale-genomics-how-humans-tamed-beer-yeast/

By: BioTek Instruments, Paul Held, PhD., Laboratory Manager

Tuesday, July 26, 2016

Gen5 3.0: Growing for the Future


It’s an exciting time here at the BioTek Headquarters in Winooski, Vermont!  We just had a groundbreaking ceremony for an expansion to our manufacturing department that will allow us to better serve our growing global customers for years to come; we just launched our newest imaging product, the Lionheart FX Automated Live Cell Imager and our new Gen5 3.0 Microplate Reader and Imager Software.  All of these are significant components to BioTek’s future, but I want to focus for a moment on the new Gen5 3.0 software. 

Gen5 screen

It was back in 2011 that BioTek took its previous big leap forward in Microplate Reader software, moving from Gen5 version 1.0 to Gen5 version 2.0.  At that time the Gen5 software only controlled BioTek plate readers; we did not offer any imaging systems back then. What a transition it’s been over the last five years!  Since 2011 BioTek has added a number of new plate readers, including the Cytation and Lionheart FX imaging systems and has seen computer technology in the market more than double in capability.  Because of all these factors, BioTek decided over a year ago to develop our next generation version of Gen5 in order to stay at the forefront of technological capabilities, while also ensuring we develop market-leading software tools for our customer base.  We are excited that half a decade after Gen5 2.0 was released; we have launched our next big version, Gen5 3.0.  This version significantly expands capabilities across the entire user workflow in order to meet and exceed customer demands for years to come.

As a central component to so many of BioTek’s multi-mode and single mode plate reader and imaging instruments, it took a phenomenal cross-functional team effort to complete Gen5 3.0.  All the various groups at BioTek have done an amazing job at orchestrating the product requirements, development, validation/testing, manufacturing and launch activities. 

Gen5

The whole team at BioTek is excited to get behind this new Gen5 3.0 software.  We hope that our current and future customers will use it to make discoveries that help advance our scientific understanding and ultimately improve our world! 


By: BioTek Instruments, Caleb Foster, Product Manager, Development

Friday, July 8, 2016

So What Do You Do For Work?

For several years I have struggled with a very specific problem when meeting new people.  The problem is how to effectively answer the inevitable question: So what do you for work?

I struggle answering this because my standard answer of “I work with a great group of people in the Service Department at BioTek Instruments” just doesn’t do justice to the pride I have in all the things that my colleagues accomplish every week.

Typically after I provide my answer I don’t get a follow up about service.  I do receive an inquiry for more information about BioTek the company and I do have a good response about the innovative instruments we manufacture at BioTek.  I understand why people rarely ask about Service.  When anyone hears Service, the perception comes to mind of a call center where someone takes notes about a problem and then dispatches a technician to drive out and fix the broken product. 

Since I work in the Service Department, I desperately want to share the dynamic nature of service and how well my colleagues support customers and each other.  However all of this information might be a bit much for the unsuspecting stranger.  I could dangerously be that guy.  You know, the one who is asked what time it is and responds with how to make a watch. 

Well I thought this would be a good opportunity to share the short version of what is bouncing around in my head, dying to get out while I utter the words “I work with a great group of people in the Service Department at BioTek Instruments”.

Like most people, I dread calling technical support due to the maze of automated responses to find a real human voice.  When I get to the end of the maze, that human voice usually doesn’t resolve my issue in the timely fashion.  At BioTek there is no maze. Phone calls and emails are directly answered by engineers and scientists stationed at BioTek's headquarters.  We provide the most effective way to solve a problem by having experts directly accessible to the customer.  Most of the problems they solve are helping customers with their instrument settings, software parameters or research process so the pace of break-through science can take place at laboratories all over the world.  Oh, and if by chance this team doesn’t know the answer, within about 500 feet of their desks are BioTek’s: Senior Service Engineers, Application Scientists and R&D Engineers.  So when a scientist who is looking for a better way to improve cancer treatment calls us about his instrument settings, we take care of it.

I am just getting started about the world of BioTek Service.  We have scientists and engineers around the world responsible for installing products at the laboratory and then training the scientists using the products.  This team will also qualify the products, provide service maintenance and even complete the infrequent repair if needed.  They travel by plane, car, train and even bicycle to customer sites.  Some of our best service representatives work at service centers at various international locations. Every team has specialized gear to make sure the products perform at factory specifications.  If any team ever has a problem, they have direct contact to the experts back at headquarters.

Every Service representative doesn’t just start working with customers from day one on their own.  BioTek’s Service Department has an infrastructure committed to supporting the mission of world class service.  Service Engineers work with BioTek Product Development teams to enhance product design to ensure serviceability and reliability.  We also have a dedicated team of technical writers in the Service Department focused on creating thorough and up to date technical documentation for our team.  A great Service Manual is the most valuable tool a service representative can have.

When parts are needed, we make sure we get it on time.  Our team works with the warehouse and manufacturing so we can get the part built and shipped on time anywhere in the world. 

All of this activity and success relies heavily on training.  Due to BioTek’s investment in innovation, multiple new products are released every year, as well as improvements to existing products.  This requires BioTek’s service experts to train fellow service representatives all around the world. 

Training is a constant activity for our Service team.  We bring our technical experts, products and tools across the globe as well as host training sessions back at our headquarters.  This significant investment in training is critical to our success.  As I write this, our gear that was shipped from Vermont to Barcelona, Spain for training last week, is on its way to Seoul, South Korea for our next training event.  I expect everything will arrive back in Vermont just in time for the next training.

Barcelona Service Training
Training goes beyond enhancing the knowledge and skill of our teams so we can resolve customer requests effectively.  Training brings together representatives from all over the globe, building a healthy, diverse community of professionals that support each other whenever they have chance. 

Well, I can certainly keep going on but I promised a short version.  Besides, I think need to get back to work with those great group of people in the Service Department.

By: BioTek Instruments, Sean Jordan, Service Director

Tuesday, June 21, 2016

Using Automated Imaging and Cellular Analysis to Increase Throughput and Objective Result Generation in Genotoxicity Assays


Genotoxicity refers to the potential of in vivo or ex vivo agents, such as cancer-causing cellular mechanisms, environmental compounds, or chemical molecules to induce damage or alterations to DNA, genes and chromosomes. Testing for potential inducers that cause, or treatments that can repair DNA damage has implications for a number of life science research areas, including oncology, environmental, and cosmetic research. Several in vitro genotoxicity tests have been developed, and among the more popularly performed tests are the Ames test which uses histidine negative Salmonella bacterial strains; the micronucleus test where treated or test cells are visually examined for the presence and frequency of micronuclei created due to toxicity, the single cell gel electrophoresis (SCGE) or comet assay where fragmented (damaged) and undamaged DNA create a comet-like configuration in a gel upon electrophoresis and staining; and the gamma H2AX (γ-H2AX) assay where nuclear foci created from antibody labeled phosphorylated histone 2AX are a biomarker for genotoxic exposure.

The desire to perform genotoxicity tests in a higher throughput format has increased as a direct relationship to the incorporation of some of these tests to develop personalized medicines or to move away from the use of animal models. This is witnessed by the growing number of companies offering standardized "off-the-shelf" plates, kits, and reagents to perform these assays. In addition, contract research organizations (CROs) are now offering their services in this area to increase throughput.

While methods to improve throughput have advanced recently, the means to perform these higher throughput assays has in many ways stagnated. Imaging is still performed manually in many situations. Analysis can also be a daunting task, particularly in the case of the comet assay where manual hunting for the “correct” comet is performed, images are taken, and the process is then repeated…comet after comet, and well after well. Not only is this process extremely time consuming, but also builds in a great deal of subjectivity into the analysis. BioTek's Cytation™ 5 and Lionheart™ FX automated imaging systems and Gen5™ Microplate Reader and Imager Software can provide easy-to-use solutions to both automate the image capture process and create objective analyses of genotoxicity assays.

The first example shown below illustrates imaging and analysis of the comet assay. Following fluorescent staining, the Cytation 5 Cell Imaging Multi-Mode Reader was used to automatically image the samples. Unlike manual methods, simple and advanced inclusion/exclusion criteria were programmed into the Gen5 Microplate Reader and Imager Software to automatically place object masks around each separate comet head and tail to eliminate anomalies and other false objects that might be mistakenly included in manual analyses (Figure 1). The total fluorescence intensity in the tail portion of the comet, in relation to the total comet fluorescence, is used to calculate “Percent DNA in the Tail”. These calculations can then be combined with object size data to calculate the “Comet Tail Moment”.
Automated comet analyses to determine (A.) comet head and (B.) comet tail in relation to the comet head.
Figure 1. Automated comet analyses to determine (A.) comet head and (B.) comet tail in relation to the comet head.
The second example illustrates a nuclear stain imaging and analysis of the γ-H2AX assay. Immunofluorescence using a primary antibody specific to γ-H2AX and a Cy-5 labeled secondary antibody is initially performed followed by automated imaging. Two separate cellular analyses are then carried out using Gen5 software. The first to determined the average area of a single labeled phosphorylation site, or foci. The second to place object masks around individual nuclei using primary analysis parameters, and then secondary parameters to place masks around individual foci within each nuclei (Figure 2). The combined analyses allow for a number of metrics to measure DNA damage to be generated, such as labeled foci coverage area and calculated foci number per nuclei.
Automated γ-H2AX analyses to determine (A.) cellular nuclei per image and (B.) labeled foci within each nuclei.
Figure 2. Automated γ-H2AX analyses to determine (A.) cellular nuclei per image and (B.) labeled foci within each nuclei.
By incorporating automated imaging and analysis many of the headaches and limitations of performing in vitro genotoxicity assays can be eliminated. Countless hours do not have to be spent in front of a manual imager and objectively generated results have greater comparability from experiment to experiment. We encourage you to contact BioTek to find out more about how these assays can be performed.

By: BioTek Instruments, Brad Larson, Principal Scientist