The Laboratory Technology Keeping Sport Clean
28 Sep 2016
Rio de Janeiro - Closing ceremony of the 2016 Olympic Games in the Maracanã
(Fernando Frazão/Agência Brasil)
Author bio: Lisa Thomas BS, MBA, serves as senior director, Clinical, Forensic, Toxicology Markets, Chromatography and Mass Spectrometry, for Thermo Fisher Scientific. As a former bench chemist, Lisa has moved quickly from research to industry. She has developed and globally marketed scientific solutions, medical devices, software, and professional services which span numerous markets.
Pressure to perform
Over the past few weeks, the eyes of the world have been on the Olympic and Paralympic athletes striving for gold in Rio. But it hasn’t just been the competitors in the spotlight – the fallout from the reported cheating at the Sochi games
means anti-doping authorities are under huge pressure to catch those who disregard the World Anti-Doping Agency (WADA) mantra: play true.
With the range of available performance-enhancing drugs wider than ever before, it’s vital that the technology and infrastructure used to beat the cheats responds to the ever-changing methods used by competitors who go to extreme – and illegal – lengths to be “the best”.
Catch me if you can
Athletes are randomly tested throughout their sporting season and during major competitions. Blood, and more commonly urine samples, are tested against a list of over 500 identified prohibited substances, spanning 11 drug categories. With such a broad spectrum of banned drugs to test for, accredited laboratories are using approximately 20 different procedures to analyse aliquots of an athlete’s “A” sample.
Anabolic steroids, synthetic derivatives of testosterone that allow athletes to train harder and build muscle more rapidly, are among the most prevalent classes of performance-enhancing drugs. To address sensitivity, triple quadrupole gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectroscopy (LC-MS) are routinely used to accurately quantify the body’s levels of testosterone (T) and epitestosterone (E) – a naturally occurring inactive stereoisomer of testosterone that does not affect performance. Testosterone administered externally does not affect epitestosterone levels, meaning T/E ratios can be used to identify doping. For most, a T/E ratio of 1-to-1 is normal, but WADA set their standardised T/E limit at 4-to-1 to account for natural variation. Athletes testing outside this limit are further evaluated using techniques like carbon isotope ratio mass spectrometry (IRMS) interfaced to GC or GC-MS systems, which is used to determine whether the testosterone present in urine is endogenous (natural) or exogenous (synthetically derived).
On top of their game
For a major sporting event like the Olympics, it’s not just the athletes who have to be on their ‘A game’. Handling over 6000 samples in just 10 days, the anti-doping laboratory also needs to be running at ultra-peak performance.
Testosterone and other dopants can be detected using ultra-high resolution Orbitrap mass spectrometers, capable of screening for large numbers of drugs rapidly. By analysing the way molecules fragment, compound identities can be confirmed beyond reasonable doubt. Such instruments have become the gold standard for accurate mass analysis, and have been routinely used since 2004 to screen all incoming athlete samples for suspect compounds as well as specifically for banned peptide hormones, which increase the number of oxygen-carrying red blood cells. More recently, this technology has been used to test for substances like recombinant insulin, which slows down muscle degradation, and growth hormones that stimulate cell regeneration.
Laboratory information management systems (LIMS), such as Thermo Scientific SampleManager LIMS, are used to integrate the fleet of instruments required for testing, allowing processes to be automated and data to be stored in central repositories, like the Anti-Doping Administration & Management System(ADAMS). With athletes’ reputations on the line, such technology infrastructure maintains the highest levels of integrity and accountability while ensuring efficient access to data.
Those using illegal methods will always try to be one step ahead of the authorities. It’s therefore essential that new ways of avoiding detection are dealt with quickly. Following lessons learned from the Sochi Winter Olympics, where sample-swapping is alleged to have taken place, human identification techniques traditionally used by forensic scientists are beginning to be used to match samples to athletes.
New strategies like the steroidal module of Athlete Biological Passports, introduced by WADA in 2014, monitor athletes’ physiological indicators over careers, making it easier to identify sudden biomarker fluctuations that may indicate use of performance-enhancing drugs.
With the enduring appeal that performance-enhancing drugs hold for some athletes, the problem of doping in sport is unlikely to disappear any time soon. But with best-in-class technology and the latest testing methods, anti-doping laboratories will remain at the forefront of the fight to ensure global sport remains fair.
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Life in Science – Improving Crystal Quality
27 Sep 2016
An Interview with Professor Naomi Chayen, Head of the Crystallization Group in Computational and Systems Medicine, Imperial College London
Winner of prestigious awards, including Innovator of the Year, and nicknamed the ‘Crystallization Guru’, Professor Naomi Chayen tells us a little about her career and the work her lab is doing to help advance the crystallization field.Q: What originally led you to become interested in science and structural biology in particular?
A: I took a degree in pharmacy as I wanted a vocational subject. I never actually worked as a pharmacist since I was offered a PhD studentship in Biochemistry. I carried on pursuing research when structural biology, of which I knew nothing about at the time, came my way. With trepidation, I took the plunge thus gaining exciting new horizons to my science and life such as receiving awards from Royalty, working with Russian astronauts, media interviews, commercialisation and more…
There has been no looking back since, and three decades on I am still here with the same enthusiasm and vigour trying to come up with new innovations and ideas all the time. Q: What have some of your most rewarding achievements been so far?
A: I would say that for me, the most rewarding achievement is making a difference to the field by developing a variety of novel methods for obtaining successful crystals that have led to structure determinations of numerous proteins including membrane proteins and large macromolecular complexes that had previously failed to crystallize using conventional techniques. Translating my scientific research into practical applications has enhanced the impact of the research.
Another satisfying aspect is leading multidisciplinary research - especially when unconnected fields are combined, resulting in breakthroughs. For example, tying together research on bone tissue regeneration or biosensor research to the crystallization of macromolecules. Q: Aside from science, what are some of your interests and passions?
A: I am passionate about skiing. It is a unique activity that enables one to keep improving but at the same time to switch off totally and relax. I also love travelling and exploring new places and cultures.Q: Can you tell us a little about your lab's research directions?
A: Research in my lab has two main strands which are interrelated: The first is developing a fundamental understanding of the crystallization process and exploiting this to design practical methodology (including high-throughput methods) for producing high quality crystals of medical and industrial interest. The second, is crystallizing target proteins for structure determination and rational drug design. At the moment we are working on the crystallization of proteins related to cancer, HIV, diabetes and heart disease.Q: What are some of the challenges faced during crystallization of proteins?
A: Getting no crystals at all, obtaining tiny, low quality crystals, phase separation or amorphous precipitate, and most frustrating: attaining large, beautiful crystals that do not diffract a single spot!Q: What are nucleants, and how can they help the crystallization process?
A: Nucleants are materials that induce nucleation and formation of crystals. Nucleants can be made of protein or non-protein materials. They help the crystallization process by serving as an anchor or template for the protein molecules to stick to and gather around. Nucleants can be used at the screening stage to facilitate the initial appearance of crystals and also at the optimisation stage of crystallisation to aid in the improvement of crystal quality. Two nucleants have so far been commercialised, ‘Naomi’s Nucleant (2009) and ‘Chayen Reddy MIP’ (2016) and further products are in the pipeline.Q: Can you tell us more about the potential of carbon nanomaterials as nucleants?
A: The carbon nanomaterials harness the power of graphene. This so-called 'wonder material' is formed of a single layer of atoms, and has been proposed for a wide range of technologies from touch screens to satellites. For the purpose of designing nucleants, polymers are attached to the graphene; the polymers appear to define small pockets on the graphene surface, a bit like a baking tray. These grafted molecules help attract the proteins, and confine them in small regular clusters. This helps them to nucleate a crystal which can be analysed using X-ray diffraction.
Q: Based on your experiences, do you have any advice for those considering embarking on a career in science?
A: We need scientists for progress in every field. My advice for those considering embarking on a career in science is don’t be afraid of failure, persevere, use your imagination and make it fun!
And if you can, choose the environment and the people that you work with carefully. From a personal point of view, having a superb environment to work in at Imperial College and a great team enables me to be productive and to enjoy the work.
You can find out more about Professor Chayen and the work being carried out in her lab here https://www.imperial.ac.uk/people/n.chayen
Professor Chayen was speaking to Anna MacDonald, Editor for Technology Networks.
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Fostering Biomanufacturing Collaborations
23 Sep 2016
Throughout 2016 and 2017, Merck is relaunching its global network of biomanufacturing science training centers, providing customers with end-to-end process development support and training.
We spoke to Daniel Stamm, Head of Global Process Solutions Commercial at Merck, to learn more about the new M Lab™ Collaboration Centers and the benefits they offer customers.
AB: Can you tell me more about the training offered at M Lab?
DS: Our approach with the M Lab™ Collaboration Centers involves partnering with customers to ensure they have the support and technical expertise they need to be successful in their manufacturing processes for the long term. In our M Lab™ Collaboration Centers, we not only partner with customers to trouble-shoot and solve specific problems – we also offer formal training courses. Both hands-on training and classroom-style theory courses are offered in our M Lab™ Collaboration Centers around the world and, in many cases, our team of experts has delivered customized training directly at the customer site. The curricula may vary from region to region so we encourage our customers to let us know what type of education they need most so we can deliver the right mix of educational programs where and when they are needed.
AB: Why does Merck feel it’s important to have hands on facilities?
DS: A hands-on approach makes it easier to move discussions between the lab and other collaboration and learning areas within the center with minimal gowning and re-gowning. Customers can bring their own samples to the center to work on a specific challenge in a real-time environment with the support of our seasoned technical experts. Although we offer a rich variety of online information, product finding apps, scale up tools, etc., many of our customers have told us that there is no substitute for a hands-on experience.
AB: How have the centers evolved since they were established in 1995?
DS: Previously known as biomanufacturing science training centers, our new state-of-the-art M Lab™ Collaboration Centers provide a simulated manufacturing environment for experiments, troubleshooting, and problem solving, in a non-GMP setting. These centers foster education and full end-to-end process development support. Customers participate in product demonstrations, hands-on training and proof-of-concept work, as well as apply best practices and new approaches to develop, optimize and scale-up processes and simplify global technology transfer.
AB: How will your customers benefit from this relaunch?
DS: We have created a unique and inspiring setting where customers can explore new ways to increase productivity and improve processes in close collaboration with our team of experts. Each M Lab™ Collaboration Center welcomes customers and collaboration partners from government, academia, regulatory bodies and industry associations, offering application best practices and knowledge sharing tailored to address the specific needs of local pharmaceutical manufacturers. For those who cannot travel to a center, we can arrange virtual visits and tours. We can also conduct remote training and troubleshooting sessions. In addition, we offer a wealth of supporting information, application notes and educational videos on our website www.merckmillipore.com/mlab.
Daniel Stamm was speaking to Ashley Board, Managing Editor at Technology Networks.
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What is Vimentin?
12 Sep 2016
What is Vimentin?
There are three main kinds of cytoskeletal filaments: microfilaments, microtubules, and intermediate filaments in Eukaryotic cells. Intermediate filaments help to provide structure to cells, and are involved in cell movement. Vimentin, is a 57kDa class III intermediate filament protein and often found in mesenchymal cells in eukaryotes, or cells that contain a distinct nucleus, and predominately expressed in developing embryo and in cells.
Vimentin intermediate filaments are generally present in mesenchymal cells. Vimentin is found not only in eukaryotic cells, but also in bacteria, where it helps to form the cytoskeleton. Vimentin is encoded by the VIM gene and has 466 amino acids. The VIM gene is highly conserved in vertebrates, The VIM gene is also involved in the immune response, and controls the transport of low-density lipoprotein (LDL)-derived cholesterol from a lysosome to the site of esterification.
Function of Vimentin
Vimentin plays a significant role in holding the organelles in the cytosol, it has a flexible nature, allowing it to respond to mechanical stress. By interacting with other structural proteins, like microtubules, it makes the cell rigid and sturdy. Studies performed on cells without vimentin found that they were functional, but very easily damaged when exposed to pressure. It was found that cells without Vimentin are extremely delicate when disturbed with a micropuncture. Vimentin is attached to the nucleus, endoplasmic reticulum, and mitochondria, either laterally or terminally. As an organizer of a number of critical proteins, Vimentin is involved in attachment, migration, and cell signaling.
The primary function of Vimentin is to maintain cellular integrity, stabilize cytoskeleton interactions and provide resistance to avoid cell damage. Vimentin also has an important clinical significance as a tumor marker. All intermediate filament proteins are expressed in a highly developmentally-regulated fashion; Vimentin is the major cytoskeletal component of mesenchymal cells. Vimentin is widely expressed and highly conserved and is constitutively expressed in mesenchymal cells. Because of this, Vimentin is often used as a marker of mesenchymally-derived cells or cells undergoing an epithelial-to-mesenchymal transition (EMT) during both normal development and metastatic progression. Vimentin IF proteins have been implicated in many aspects of cancer initiation and progression, including tumorigenesis, epithelial-to-mesenchymal transition, and the metastatic spread of cancer.
VIM antibodies are usually used as a marker in histopathological diagnosis, and often with keratin to distinguish epithelial and mesenchymal tumors, such as identification of malignant melanoma, lymphoma and thymoma; VIM antibodies and leukocyte common antigen are also used to distinguish lymphoma and other mesenchymal tumors. VIM antibodies and myogenic cell markers are often used to identifiy myogenic and fibrous tumors, etc.
Creative Diagnostics offer a wide range of Vimentin related products such as Vimentin proteins, antibodies, ELISA kits, hybridomas, engineered antibodies and cDNA products for use in common research applications, including ELISA, Flow Cytometry, Immunocytochemistry, Immunofluorescence, Immunohistochemistry, Immunoprecipitation, Intracellular Staining by Flow Cytometry, Peptide ELISA, Sandwich ELISA, Simple Western, Western Blot. To find more about the Vimentin antibodies, you can visit the Creative Diagnostics site.
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