Laboratory Focus August/September 2016

Pharmaceutical

Clinical

Chemical

w w w. l a b o r a t o r y f o c u s . c a When Work Hurts: Taking the pain out of the job Page 8

food

environment

August/September 2016 Volume 20, Number 3

Emerging in Vivo Electrophysiology Methods in Neuroscience Research Page 10

R&D News.......................... 1 Appointments..................... 6 Pharma Notes..................... 7 New Products................... 16 Calendar........................... 17 App Reviews...................... 18

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Canada invests in poultry anti-microbial development

MISSISSAUGA, ON – A Canadian biotechnology company has been awarded a $3.4 million government investment to help develop a new line of anti-microbial feed additives to help control disease outbreaks in poultry flocks AbCelex, which focuses on developing livestock food additives that help improve animal health

and food safety, is developing a line of innovative non-antibiotic, non-hormonal additives that are specifically targeted at Campylobacter and Salmonella, two of the most common food-borne bacteria that infect poultry.

Healthier poultry

The new anti-microbials – called

‘nanobodies’ – will result in healthier poultry and improve food safety.

The project will be conducted in collaboration with the International Vaccine Centre at the University of Saskatchewan, the University of Toronto and Colorado Quality Research Inc. Funding for the project comes from the AgriInnovation Program (Research and Development Stream) as part of the Growing Forward 2 agricultural policy framework.

Reducing the use of antibiotics

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“These innovations will reduce the use of antibiotics and result in safer food, a healthier population and a more productive agricultural economy,” stated Navdeep Bains,

Minister of Innovation, Science and Economic Development.

Dr. Saeid Babaei, president and CEO of AbCelex Technologies added his company is appreciative of the funding.

“With this visionary contribution and other strategic investments, AbCelex Technologies is well positioned to advance its platform technology and develop nextgeneration biological products needed for improved human health and a reduction in healthcare costs as a result of food contamination,” he said.

To see this story online visit http://www.laboratoryfocus.ca/ canada-invests-in-poultry-antimicrobial-development/

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Vaccine developed for devastating pig virus SASKATOON, SK – In less than a year, University of Saskatchewan (U of S) scientists have developed and tested a prototype vaccine that could protect the North American

swine industry from a virus that has killed more than eight million pigs and cost more than $400 million in lost income since 2013.

The Porcine Epidemic Diar-

rhea Virus (PEDV) hit the U.S. in 2013 and spread to Canada in 2014. It was first discovered in Europe, and has become increasingly problematic in Asian countries. Occurring

only in pigs, PEDV can kill up to 100 per cent of infected piglets. PEDV is a coronavirus, a virus group which Continued on page 3

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August/September 2016

news Continued from page 2 includes important emerging human diseases such as SARS and MERS. Using its new containment Level 3 facility, the U of S Vaccine and Infectious Disease Organization-International Vaccine Centre (VIDO-InterVac) quickly launched a vaccine development project.

“Our new facility, InterVac, provided us with the containment infrastructure to develop a vaccine and demonstrate it protected up to 100 per cent of the piglets,” said Dr. Volker Gerdts, VIDOInterVac’s research director.

The successful vaccine results triggered the interest of several animal health companies, including Huvepharma, which has partnered with VIDOInterVac to develop the technology for commercial production in North America.

“This is an exciting partnership with a world-class organization,” said Dr. Boris Gavrilov, senior scientist for biologics

to help protect piglets from a recent PEDV outbreak.

“This is a perfect example of why InterVac was constructed – it is one of the only facilities available internationally with the capacity to conduct vaccine development and testing on this scale for emerging infectious diseases,” said VIDO-InterVac director Andrew Potter. “It helps Canada remain prepared to quickly respond to outbreaks like this.”

The PEDV vaccine development project has been supported by a variety of funders including the Government of Saskatchewan (ADF), Sask Pork, and the Canadian Swine Health Network. development at Huvepharma. “Our goal is to have the vaccine available for commercial use as soon as possible to help stop producer losses.”

With the support of the swine industry, the vaccine is now undergoing field testing in Saskatchewan, as well as in Manitoba where it is being used

To see this story online visit http://www.laboratoryfocus.ca/ vaccine-developed-for-devastatingpig-virus/

Nanorobots that target cancerous tumours with precision – Administering anti-cancer drugs redefined

MONTRÉAL, QC – Researchers from Polytechnique Montréal, Université de Montréal and McGill University have just achieved a spectacular breakthrough in cancer research by developing new nanorobotic agents capable of navigating through the bloodstream to administer a drug specifically targeting the active cancerous cells of tumours.

This scientific breakthrough has just been published in the prestigious journal Nature Nanotechnology in an article titled “Magneto-aerotactic bacteria deliver drug-containing nanoliposomes to tumour hypoxic regions.” The article notes the results of the research done on mice, which were success-

fully administered nanorobotic agents into colorectal tumours. This way of injecting medication ensures the optimal targeting of a tumour and avoids jeopardizing the integrity of organs and surrounding healthy tissues. As a result, the drug dosage that is highly toxic for the human organism could be significantly reduced.

“These legions of nanorobotic agents were actually composed of more than 100 million flagellated bacteria – and therefore self-propelled – and loaded with drugs that moved by taking the most direct path between the drug’s injection point and the area of the body to cure,” explains

professor Sylvain Martel, holder of the Canada Research Chair in Medical Nanorobotics and director of the Polytechnique Montréal Nanorobotics Laboratory, who heads the research team’s work. “The drug’s propelling force was enough to travel efficiently and enter deep inside the tumours.”

When they enter a tumour, the nanorobotic agents can detect in a wholly autonomous fashion the oxygen-depleted tumour areas, known as hypoxic zones, and deliver the drug to them. This hypoxic zone is created by the substantial consumption of oxygen by rapidly proliferative tumour cells. Hypoxic zones are known to be resistant to most therapies, including radiotherapy.

But gaining access to tumours by taking paths as minute as a red blood cell and crossing complex physiological micro-environments does not come without challenges. So Professor Martel and his team used nanotechnology to do it.

Bacteria with compass

To move around, bacteria used by Professor Martel’s team rely on two natural systems. A kind of compass created by the synthesis of a chain of magnetic nanoparticles allows them to move in the direction of a magnetic field, while a sensor measuring oxygen concentration enables them to reach and remain in the tumour’s active regions. By harnessing these two transportation systems and by exposing the bacteria to a computer-controlled magnetic field, researchers showed that these

bacteria could perfectly replicate artificial nanorobots of the future designed for this kind of task.

“This innovative use of nanotransporters will have an impact not only on creating more advanced engineering concepts and original intervention methods, but it also throws the door wide open to the synthesis of new vehicles for therapeutic, imaging and diagnostic agents,” professor Martel adds. “Chemotherapy, which is so toxic for the entire human body, could make use of these natural nanorobots to move drugs directly to the targeted area, eliminating the harmful side effects while also boosting its therapeutic effectiveness.”

The work by professor Martel was supported by the Québec consortium for drug discovery (CQDM), the Canada Research Chairs, the Natural Sciences and Engineering Research Council of Canada (NSERC), the Research Chair in Nanorobotics of Polytechnique Montréal, Mitacs, the Canada Foundation for Innovation (CFI) and the National Institutes of Health (NIH).

Montréal’s Jewish General Hospital, the McGill University Health Centre (MUHC), the Institute for Research in Immunology and Cancer (IRIC), and the Rosalind and Morris Goodman Cancer Research Centre also took part in this research work.

To see this story online visit http://www.laboratoryfocus.ca/ legions-of-nanorobots-target-cancerous-tumours-with-precision-administering-anti-cancer-drugs-redefined/

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McMaster researchers overcome obstacle holding back a technological revolution HAMILTON, ON – Imagine an electronic newspaper that you could roll up and spill your coffee on, even as it updates itself before your eyes.

It’s an example of the technological revolution that has been waiting to happen, except for one major problem that, until now, scientists have not been able to resolve. Researchers at McMaster University have cleared that obstacle by developing a new way to purify carbon nanotubes – the smaller, nimbler semiconductors that are expected to replace silicon within computer chips and a wide array of electronics.

“Once we have a reliable source of pure nanotubes that are not very expensive, a lot can happen very quickly,” says Alex Adronov, a professor of Chemistry at McMaster whose research team has developed a new and potentially cost-efficient way to purify carbon nanotubes.

Carbon nanotubes – hair-like structures that are one billionth of a metre in diameter but thousands of times longer – are tiny, flexible conductive nano-scale materials, expected to revolutionize computers and electronics by replacing much larger siliconbased chips.

A major problem standing in the way of the new technology, however, has been untangling metallic and semiconducting carbon nanotubes, since both are created simultaneously in the process of producing the microscopic structures, which typi-

cally involves heating carbon-based gases to a point where mixed clusters of nanotubes form spontaneously as black soot. Only pure semiconducting or metallic carbon nanotubes are effective in device applications, but efficiently isolating them has proven to be a challenging problem to overcome. Even when the nanotube soot is ground down, semiconducting and metallic nanotubes are knotted together within each grain of powder.

Both components are valuable, but only when separated and researchers around the world have spent years trying to find effective and efficient ways to isolate carbon nanotubes and unleash their value.

While previous researchers had created polymers that could allow semiconducting carbon nanotubes to be dissolved and washed away, leaving metallic nanotubes behind, there was no such process for doing the opposite: dispersing the metallic nanotubes and leaving behind the semiconducting structures.

Now, Adronov’s research group has managed to reverse the electronic characteristics of a polymer known to disperse semiconducting nanotubes – while leaving the rest of the polymer’s structure intact. By so doing, they have reversed the process, leaving the semiconducting nanotubes behind while making it possible to disperse the metallic nanotubes.

The researchers worked closely with experts and equipment from

Artistic rendition of a metallic carbon nanotube being pulled into solution, in analogy to the work described by the Adronov group. (photo by Alex Adronov) McMaster’s Faculty of Engineering and the Canada Centre for Electron Microscopy, located on the university’s campus.

“There aren’t many places in the world where you can do this type of interdisciplinary work,” Adronov says.

The next step, he explains, is for his team or other researchers to exploit the discovery by finding a way to develop even more efficient polymers

FedDev Ontario invests in BEAM L-R- BEAM Directors, Drs. John Brennan, Christopher Oelkrug & Jonathan Bramson Photo by Ron Scheffler for McMaster University.

HAMILTON, ON – The federal government is investing almost $12-million to develop McMaster’s new Biomedical Engineering and Advanced Manufacturing centre.

Navdeep Bains, Canada’s Minister of Innovation, Science and Economic Development made the announcement at the site of the new Centre, joined by McMaster leaders and other officials.

The state-state-of-the-art Fraunhofer Project Centre for Biomedical Engineering and Advanced Manufacturing (BEAM) research facility, located at McMaster Innovation Park, will be home to several of McMaster’s leading researchers working on the development of novel technologies for eye care, point-of-care medical devices and cancer treatments.

The overall value of McMaster’s BEAM project is more than $33-million with significant investments from the University, Fraunhofer IZI, the Government of Ontario and the City of Hamilton, as well as other organiza-

and scale up the process for commercial production.

The research is described in the cover story of Chemistry – A European Journal.

To see this story online visit http://www.laboratoryfocus.ca/ mcmaster-researchers-resolve-aproblem-that-has-been-holdingback-a-technological-revolution/ tions and private sector partners.

The project is expected to create at least 74 full-time jobs, produce 35 new industrial collaborations and bring together several partners, including small businesses and multi-national enterprises, university-based researchers, and the German-based Fraunhofer Institute for Cell Therapy and Immunology IZI. The Centre will also help commercialize new products, accelerate the growth of small businesses by providing them with access to facilities, expertise and global value chains. It could also attract other businesses and talent to the region, diversifying Hamilton’s economy.

The 20,000 square foot state-ofthe-art facility is slated to open late next year.

To see this story online visit http://www.laboratoryfocus.ca/ feddev-ontario-invests-in-beam/

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Laboratory Focus August/September 2016

Study examining tailored anti-platelet heart medication after coronary stent lands grant TORONTO, ON – Researchers at the Peter Munk Cardiac Centre, with expertise running large clinical trials, and at Mayo Clinic, are leading the Tailored Antiplatelet Therapy to Lessen Outcomes after Percutaneous Coronary Intervention (TAILOR-PCI) study.

The study has received $7 million in additional funding from the National Heart, Lung and Blood Institute (NHLBI) of the National Institutes of Health (NIH).

“The significance and potential farreaching impact of a clinical trial of this scope will push the boundaries about the benefits that personalized medicine for patients who undergo a coronary stent procedure can provide,” says Dr. Barry Rubin, medical director, Peter Munk Cardiac Centre, University Health Network (UHN).

“This is the largest trial to evaluate a personalized molecular medicine approach to patients with heart disease that has even been carried out.”

When it began in 2013, TAILOR-PCI included study teams at 15 hospitals

from Canada, the U.S, and South Korea, with a goal of enrolling 5,270 patients. Now, some 29 medical centres are participating, with more expected.

Launched by Mayo Clinic Center for Individualized Medicine and the Department of Cardiovascular Diseases at Mayo Clinic, in collaboration with the Peter Munk Cardiac Centre, the

UBC research aims to help Canadian flax farmers

UBC researcher Michael Deyholos stands in front of the flax plants he studies. Photo: UBC OKANAGAN, BC – A University of British Columbia professor’s flax research could one day help Canadian farmers grow a car fender. In a recent study, UBC researcher Michael Deyholos identified the genes responsible for the bane of many Canadian flax farmers’ existence; the fibres in the plant’s stem.

“These findings have allowed us to zero in the genetic profile of the toughest part of this plant and may one day help us engineer some of that toughness out,” says Deyholos, a biology professor at UBC’s Okanagan campus. “With further research, we might one day be able to help farmers make money off a waste material that wreaks havoc on farm equipment and costs hundreds of hours and thousands of dollars to deal with.”

As part of his research, Deyholos and his former graduate student at

the University of Alberta dissected thousands of the plant’s stem under a microscope in order to identify which genes in the plant’s make up were responsible for the growth of the stem, and which weren’t.

Due to the length of the Canadian prairie’s growing season, where flax is grown, farmers typically burn the stems, known as flax straw, as opposed to harvesting the material. In many European countries, flax straw is used as an additive in paper, plastics and other advanced materials such as those used in the production of automobiles.

Currently, Canadian flax is used only for the value of its seeds, which can be eaten or broken down into flaxseed oil. Flaxseed oil is used in the manufacturing of paints, linoleum, and as a key element in the manufacturing of packaging materials and plastics.

According to the Flax Council of Canada, Canada is one of the largest flax producers in the world with the nation’s prairie provinces cultivating 816,000 tonnes of the plant in 2014/15 on 1.6 million acres of land.

Deyholos’ research was recently published in the journal Frontiers of Plant Science.

To see this story online visit http://www.laboratoryfocus.ca/ ubc-research-aims-to-help-canadianflax-farmers/

Applied Health Research Centre at the University of Toronto, and Spartan Bioscience, the study involves the randomized comparison of Plavix (clopidogrel bisulfate) and Brilinta (ticagrelor), based on genetic testing that identifies resistance to ticagrelor in patients undergoing coronary balloon angioplasty.

“This is a multinational collaboration designed to inform clinical practice, in its truest sense,” says Dr. Michael Farkouh, cardiologist, Chair, Peter Munk Centre of Excellence in Multinational Clinical Trials and one of two principal investigators of TAILOR-PCl. The other principal investigator is Dr. Naveen Pereira, cardiologist at the Mayo Clinic. “We are delighted to partner with the Mayo Clinic in this effort,” says Dr. Farkouh.

The study is examining whether prescribing heart medication based on a patient’s CYP2C19 genotype will help prevent heart attack, stroke, unstable angina, and cardiovascular death in patients who undergo percutaneous coronary intervention (PCI). A PCI,

news commonly known as an angioplasty, is a non-surgical procedure that opens up narrowed or blocked blood vessels in the heart. Anti-platelet medication has been shown to reduce the risk of heart attack, unstable angina, stroke and cardiovascular death after stent placement by reducing the possibility of blood clots around the site of the surgical incision. The current standard of care after angioplasty is to prescribe clopidogrel for one year.

“Today, we do this regardless of a person’s individual genotype, even though we have known for several years that variation in the CYP2C19 gene may diminish the benefit from the drug,” says Dr. Pereira. “What we don’t know — and why there is such confusion in the cardiovascular community — is whether these genetic differences affect long-term clinical outcomes.”

To see this story online visit http://www.laboratoryfocus.ca/ study-examining-tailored-anti-platelet-heart-medication-after-coronarystent-lands-grant/

UBC takes steps toward examining the DNA of the world’s wine regions VANCOUVER, BC – University of British Columbia researchers are one step closer to identifying the biological personalities of the world’s greatest wines.

In a recent study Dan Durall and Mansak (Ben) Tantikachornkiat developed a technique that combines a process to identify the full spectrum of DNA in yeast and bacteria samples with a technique that distinguishes between live and dead micro-organisms.

“Since only live micro-organisms are relevant in the various stages of fermentation as they relate to the senses, this study provides some of the important tools that will be necessary to determine why different types of wine taste and smell as they do,” says Durall, an associate professor of biology at UBC’s Okanagan campus.

“While more research needs to be conducted, these findings could also lead to the identification and elimination of micro-organisms that are responsible for spoilage.”

In undertaking the study, the pair used a number of different kinds of yeast and bacteria specimens, including those typically found in wine fermentations.

Key in the development of the new scientific technique was the use of a light-sensitive dye, propidium monoazide, which binds to dead DNA and prevents it from being detected. This allows scientists to identify and focus on the more relevant aspects of a test sample.

“This technique allows us to quickly and accurately monitor in one experiment what previously could have taken multiple experiments and months of trial and error,” says Tantikachornkiat. “This will inevitably make research in this area faster, cheaper and more efficient.

“The next stages of research will focus this technique on different types of wine making methods to see how they change microorganisms that affect the final wine product.”

Durall and Tantikachornkiat’s research was recently published in the International Journal of Food Microbiology.

To see this story online visit http://www.laboratoryfocus.ca/ ubc-takes-steps-toward-examining-the-dna-of-the-worlds-wineregions/

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August/September 2016 Laboratory Focus www.laboratoryfocus.ca

Appointments

Rob Baker has taken the reins as McMaster’s new vice-president Research. Baker previously served as McMaster’s Dean of Science, coming to McMaster from the University of Toronto in July 2013.

A behavioural ecologist, Baker has more than 30 years of research experience of his own, much of it spent trying to better understand

art comes to the NRC from the Treasury Board of Canada Secretariat, where he held several senior positions, most recently associate secretary. He has also served in a number of leadership positions in the innovation, science and economic development portfolio. Previously, he was the assistant vice-president of research at Dalhousie University. Stewart replaces outgoing president John McDougall, who has decided to retire from the public service. Ms. Maria Aubrey will continue acting as president of the NRC until Mr. Stewart begins in late August.

Dr. Gary Newton, (CNW Group/Sinai Health System)

Rob Baker

the evolution of animal behaviour.

He has also spent many years as a university administrator: first as chair of biology at the University of Toronto in Mississauga, and as associate dean of science at that campus. On U of T’s St. George campus he served as chair of the Department of Zoology, chair of Ecology and Evolutionary Biology and, in the Faculty of Arts and Science, vice-dean of Graduate Education and Research and vice-dean, Research and Infrastructure.

ChroMedX Corp. has named Ash Kaushal as its new president and CEO, effective immediately.

Mr. Kaushal has over 25 years of experience in product development in the medical, defence, safety and nuclear industries. He has also held vice president and senior level management positions with several Canadian and British corporations throughout his career including NIR Science Corp and NIR Diagnostics Corp in Campbellvile, ON; CME Telemetrix in Waterloo; Indal Technologies in Mississauga, ON and several companies based in England, UK. Kaushal takes over from Wayne Maddever who has stepped down as an officer and director of ChroMedX Corp.

Dr. Gary Newton, currently chief medical strategy officer and Physician-in-Chief of Sinai Health

System, will assume the role of president and CEO of Sinai Health System, effective October 3, 2016.

Dr. Newton is a graduate of the University of Toronto Medical School and has been on faculty at the University of Toronto since 1996. He completed internal medicine at the University of Toronto and training in adult cardiology at the University of Ottawa Heart Institute. Additionally, Dr. Newton served as the head of the Division of Cardiology, University Health Network and Mount Sinai Hospital, from 2009 through 2013. He was appointed Physician-in-Chief at Mount Sinai Hospital in November 2013 and assumed his current role in January 2015.

Vaxil Bio has hired Dr. Limor Chen as the company’s new vice president. Dr. Chen is a highly experienced scientist, having been a senior researcher and team leader within the Special Projects Division at Israel’s largest pharmaceutical company, Teva. Most recently, Dr. Chen worked as head of business development for an Israeli biotech, which was successful in listing on the NASDAQ. Dr. Chen earned his Ph.D. at Israel’s Weizmann Institute of Science, in the laboratory of prof. Ruth Arnon, the developer of Teva’s blockbuster Copaxone against Multiple Sclerosis. He was a Research Fellow at the MD Anderson Cancer Center in Houston, and at the Sunnybrook Research Institute in Toronto.

The National Research Council of Canada (NRC) has named Iain Stewart as its new president effective August 24, 2016. Stew-

ProMIS Neurosciences has appointed Dr. Johanne Kaplan to the post of chief development officer. Dr. Kaplan joins ProMIS following 24 years at Genzyme, where she held positions of increasing responsibility culminating in her decade-long tenure as VP of Research.

ProNAi Therapeutics appoints Dr. Christian Hassig as senior vice president of research. In his new role he will lead ProNAi’s research activities from discovery research through to early candidate development. Dr. Hassig has a strong track record in both discovery research and preclinical development across multiple therapeutic areas and has pioneered lead discovery projects against innovative targets in oncology, from target discovery through to IND. He was formerly the vice president of Drug Discovery at the Sanford Burnham Prebys Medical Discovery Institute, having previously served as its director of Drug Discovery from 2010 to 2015. Prior to this, Dr. Hassig served as the Department head of Biology at Kalypsys, Inc., a privately owned biopharmaceutical company specializing in small molecule drug discovery and development, and held multiple roles within its Biology and Lead Discovery departments.

Dr. Christine Williams has joined OICR as deputy director and vice-president, outreach. Previously she was the chief mission officer for the Canadian Cancer Society and prior to that she was the national vice-president, research at the Society. She holds a PhD in Immunology from the University of Toronto and completed postdoctoral training at Massachusetts General Hospital, where she studied the

Dr. Christine Williams molecular pathways involved in the development of leukemia and lymphoma in children.

TRIUMF announces that Dr. Jens Dilling will become Associate Laboratory Director for its Physical Sciences Division (ALD-Physical Sciences), effective September 2016. In this role he will work closely with TRIUMF’s Accelerator Division to strengthen isotope delivery, enabling leading edge science and discovery and preparing the lab for its new flagship facility – the Advanced Rare IsotopE Laboratory (ARIEL). A nuclear physicist and an expert in trapping radioactive isotopes, Dilling is no stranger to TRIUMF having first been recruited to the lab’s Science Division (now Physical Sciences Division) in 1995. Prior to his new role, he served as deputy and department head for nuclear physics, deputy division head of the Science Division, and most recently as the interim Associate Laboratory Director for the Physical Sciences Division. Dilling received both his undergraduate degree (Diplom) and doctorate (PhD) in physics from the University of Heidelberg in Germany. As an expert on ion traps, Dilling has been involved in experiments around the world, including at facilities at ISOLDE, CERN, and SHIP at GSI. Dilling designed, built, and operated the TITAN (TRIUMF’s Ion Trap for Atomic and Nuclear science) system at TRIUMF’s ISAC accelerator complex. Dilling also serves on many national and international committees, and received the CAP-Vogt Award for outstanding contributions to subatomic physics research in 2013.

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Laboratory Focus August/September 2016

Pharma Notes BioPharma Services (Toronto, ON) reports it has acquired key human resource assets along with a list of validated assays from Bioanalytical Laboratory Services (BLS) (Toronto, ON). Through the deal, BLS assets will be combined with BioPharma’s existing bioanalytical lab, while expanding the latter’s service offerings. Also, effective immediately, Dr. Hughes will be appointed to the BioPharma executive team as the vice-president of Lab Operations.

The US Food and Drug Administration (FDA) has given Trillium Therapeutics Inc. (Toronto, ON) the go-ahead to initiate a Phase 1 clinical trial for its lead drug candidate, TTI-621 (SIRPaFc), in solid tumours and mycosis fungoides. Trillium is developing TTI-621 as a novel checkpoint inhibitor of the innate immune system, and the drug is currently being evaluated in an ongoing Phase 1 dose escalation study in patients with relapsed or refractory hematologic malignancies. Patient enrollment is anticipated to commence by year end.

Patient enrollment is complete in ProMetic Life Sciences’ (Laval, QC) Phase 2/3 clinical trial for congenital plasminogen deficient patients. The trial is a requirement for qualifying for the accelerated regulatory approval pathway with the Food and Drug Administration (FDA). Prometic’s Plasminogen is a naturally occurring protein that is synthesized by the liver and circulates in the blood. Activated plasminogen, plasmin, is a fundamental component of the fibrinolytic system and is the main enzyme involved in the lysis of blood clots and clearance of extravasated fibrin. It has been shown in studies to be vital in wound healing, cell migration, tissue remodeling, angiogenesis and embryogenesis. ProMetic has received an Orphan Drug Designation by the FDA and the European Commission for the US and the European markets respectively for this product.

Zymeworks Inc. (Vancouver, BC) has licensed a new drug discovery platform from Innovative Targeting Solutions (ITS) (Vancouver, BC) as part of a collaboration agreement between the two parties. Zymeworks plans to integrate ITS’ proprietary HuTARG™ platform to help identify and develop therapeutics directed towards challenging disease targets, while accelerating the development of its bi-specific and multi-functional biologics and drug conjugates.

HuTARG™ is a first-in-class protein engineering platform and represents

a disruptive technology in the field. It is the first fully mammalian technology that generates antibody diversity in vitro via RAG1/RAG2 mediated V(D)J recombination, allowing for the generation and affinity maturation of highly potent protein-based biologics. Zymeworks will pay a technology licensing fee and ITS will receive, on a per product basis upon successful development and commercialization, up to USD$65 million in clinical and commercial milestones and low single-digit royalties on net sales.

Additional financial terms were not disclosed.

Helix BioPharma Corp. (Aurora, ON), an immuno-oncology company developing drug candidates for the prevention and treatment of cancer, has reached an agreement in principle with the National Research Council of Canada (NRC) to collaborate on various immuno-oncology initiatives. The agreement spells out a framework that would enable Helix to finalize a master agreement with

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NRC to develop new therapeutics for cancer immunotherapies and allows for discussion on a potential license of an antibody previously obtained from NRC under a material transfer agreement. L-DOS47 is Helix’s first immunoconjugate based drug candidate in development based on the company’s DOS47 platform technology, which is designed to use an innovative approach to modify the micro-environmental conditions of cancer cells in a manner that leads to their destruction.

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August/September 2016 Laboratory Focus www.laboratoryfocus.ca

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b y C athy B ouwers, Com m u nic ations Sp ec ial ist, Canad ian So ciet y fo r Medical L abo rat o ry Science

When Work Hurts:

Taking the pain out of the job

F

or many people, aches and pains are considered all in a day’s work. The idea that humans need to adapt to the demands of the work is an antiquated concept that no longer has a place in today’s workforce.

Ergonomics is a familiar term often associated with office work. However, ergonomics is not just about adjusting the height of a chair, or any other work implement. It’s about creating a work environment that is productive while focusing on the human element of the work.

The Association of Canadian Ergonomists defines ergonomics as the “scientific discipline concerned with interactions among humans and other elements of a system (e.g. the tools, equipment, products, tasks, organization, technology, and environment)”. The laboratory setting has unique challenges to the adaptability of the environment versus the humans working in the environment. In an office setting, it’s common to find ergonomically correct desks, chairs, hand and back supports. These items are relatively inexpensive and easy to find in local office supply stores. Laboratory equipment is another story.

Tables, benches and workstations are highly customized to the work.

They can be expensive and limited by supplier availability. Ergonomics in the laboratory becomes a question of immediate expense, rather than long- term health and wellness of the employees.

We humans are highly adaptable and flexible. We are willing to bend, twist, reach and contort our bodies in an effort to get the job done. Often time, this is when injury occurs. Injuries to muscles, tendons, ligaments, nerves, blood vessels, and joints of the neck, shoulders, arms, wrists, legs, and back are called musculoskeletal injuries (MSI) or musculoskeletal disorders (MSD). Both terms are used interchangeably, but they do refer to two origins of discomfort.

An injury can occur from a sudden occurrence, such as lifting something too heavy or over time from repeated exposure to the same movement pattern, as is the case in repetitive strain injuries (RSIs). MSI or MSD are one of the most common workplace injuries. According to Health Canada, MSI claims are the largest amongst health care workers. “Musculoskele-

tal injuries (MSI), which account for the greatest number of time-loss injuries among healthcare workers, occur due to such factors as equipment inadequacies and poorly configured patient rooms, as well as work organizational factors such as high work demands, inadequate staffing, poor work morale and low social support.”

The unfortunate truth is that MSIs and MSDs are as common as they are preventable. They often occur with tasks that are repetitive or cause overexertion of the muscles. When looking at typical laboratory work, these two elements are abundant.

Abigail Overduin, Ergonomist for the University of British Columbia, instructs a course specifically for laboratory ergonomics. She says the laboratory is a unique workplace environment that may require special attention when considering ergonomic adjustments.

“Anything that is repetitive, particularly if awkward postures or force is involved, such as with pipetting, increases the risk of injury,” says Overduin, “as well as any task that requires the same body position for long periods of time, such as sitting or standing at a microscope.”

A 1994 Swedish study, published in Applied Ergonomics, found there was an increased risk of hand and shoulder ailments associated with pipetting for more than 300 hours per year. The average lab technician has been found to pipette 495 hours per year. It’s no wonder that another study published in 2005 found that “90 per cent of users who pipette continuously for more than 60 minutes reported hand complaints”.

Although neck and back strain are considered part of the group of MSI ailments, they are a specific cause for concern in a lab setting. Microscope work uses multiple fine motor movements for long periods of time. Strain and stress on muscles in the eyes, neck, shoulders, hands and back can add up over time.

“Another risk is when we change the task from one extreme to another very quickly,” says Overduin. “If someone is sitting for a long time, then gets up, lifts something heavy, say a centrifuge router, they are at risk for injury. The body hasn’t had time to adjust to the new demand.”

There are elements that individuals can control within their work environment that can help reduce their risk of

When standing at a task for a longer period of time, try using an anti-fatigue mat to stand on or a footstool to alternate your feet and change your body position. When working at a table/bench, ensure your elbows are at a neutral height, being too high or too low will affect your neck and shoulders. Consider raising or lowering the work you are doing to help maintain correct body position.

injury. Ensuring arms, neck and back are kept in a neutral position throughout a task will help eliminate tension and soreness. If standing for longer than two hours, take a short break to sit down to alleviate back, knee and feet soreness. The information at the end of this article can provide some simple reminders to consider with performing various tasks in the lab.

Ask your manager for devices that will help make your workstation more comfortable. Things like cushions to cover sharp corners of tables, adjustable chairs or anti-fatigue mats. These are relatively inexpensive and are readily available and easy to add to a workplace. Depending on the work

you need to do, you can even ask to try different brands or styles of equipment, such as pipettes. There are pipettes that require less force, helping to reduce muscle fatigue. Workplace ergonomics is not a one size- fits-all solution. There should be open conversations happening to work on solutions that help everyone.

“Workplace culture plays a role in health,” says Overduin, “no one wants to be seen as weak or unable to do their job because they are uncomfortable or in pain.” She also suggests that the mentality of ‘work is pain’ can hold an entire organization back from taking necessary steps to help their employees.

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Laboratory Focus August/September 2016

Feature When using small hand tools in a repetitive motion, it’s important to use a neutral grip to protect your wrist and forearms. Be sure to use tools that don’t require excessive force and take breaks and vary tasks to allow muscle tension to dissipate before continuing work.

When sitting at a microscope, keep your back supported in a more upright position to reduce the strain to keep your body close to the eyepiece. Adjust the eyepiece closer or further from you to promote neutral neck postures. Consider moving equipment around, such as a computer keyboard, to keep your arm close to your body and minimize awkward positions.

When sitting at the computer, ensure your back is supported in a slight reclining posture. Elbows and neck should be in a neutral position. Keep monitors at eye level and if using multiple monitors, keep them close together with the one you use most often positioned towards the midline in front of you to minimize head movement.

“The idea that the pain will go away, or it will get better is the wrong thinking. People need to speak up sooner if they are experiencing discomfort while working.”

By waiting too long to get help, employees risk having acute injuries that turn to chronic problems. Employers need to look beyond the upfront costs of ergonomic accommodations to longer term benefits. It may cost a little bit more now to change the types of pipettes used, but it may decrease the number of sick days or lost time due to hand injuries over time.

Even with all the proper ergonomic concepts and equipment in place there are just some jobs that will affect the body more than others. As long as people are mindful of their posture it can go a long way to prevent larger issues.

No need for an elaborate or possibly embarrassing stretching routine.

Overduin suggests just straightening up, coming back to a neutral position and moving just a couple minutes every hour at work will help counter the effects of a certain position on

the body. A more elaborate stretching routine may be done at home. She also recommends being aware of the demands of a task before beginning.

“Understand the workflow and have the proper tools close-by. Don’t improvise because you think it will only take a minute, it will compromise your posture and could result in injury.”

There are several reputable resources available for anyone looking for more information about workplace ergonomics. Many larger organizations, hospitals, universities, private labs, may have an in-house department that can assess the laboratory for potential risks.

These departments are often part of Human Resources, Health and Safety or Risk Management. If you work in an organization that doesn’t have these services, there are public services available. The Association for Canadian Ergonomists’ website lists ergonomists across Canada that are available to provide workplace assessments. The most important tool in protecting your physical health at

work is being educated about the possible risks involved and taking the corrective action to reduce those risks. It could be something as simple as having an open conversation with a manager, or speaking to your organization’s staff ergonomist. The main goal is to keep you working pain-free for many years.

References: 1. Health Canada. Trends in workplace injuries, illnesses and policies in healthcare across Canada. 2004. Online http:// www.hc-sc.gc.ca/ hcs-sss/pubs/ nurs-infirm/2004-hwi-ipsmt/ index-eng.php#f10Association of Canadian Ergonomists http:// www.ace-ergocanada.ca/ 2. McLean, L., Tingley, M., Scott, R.N., Rikards, J. Computer terminals work and the benefit of microbreaks. Applied Ergonomics, 3. 32 (2001) 225-237. http://www. udel.edu/PT/PT%20Clinical%20 Services/journalclub/caserounds/02_03/nov02/McleanL.pdf 4. Asundi, Krishna R., Bach, Joel M., and Rempel, David H. Thumb force and muscle loads are influenced by the design of a mechanical pipette and by pipet-

ting tasks. Human Factors, vol.

47 no. 1 (2005) 67-76.

5. Almby, Bo, Björksen, Marianne Gerner and Jansson, Ejvor Sassarinis. Hand and shoulder ailments among laboratory technicians using modern plunger-operated pipettes. Applied Ergonomics, vol.

25 no. 2 (1994) 88-94.

Acknowledgments:

The CSMLS would like to thank the following for their contribution for this article.

The staff at Hamilton Health Sciences, Juravinksi Hospital laboratory for their contribution.

Victoria McDonald and Andrea Tjahja for their assistance and The Public Services Health and Safety Association for use of their material.

This article originally appeared in the Canadian Journal of Medical Laboratory Science, Vol. 75 no. 2 2013.

To see this story online visit http://laboratory focus.ca/when-work-hurtstaking-the-pain-out-of-the-job/

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August/September 2016 Laboratory Focus www.laboratoryfocus.ca

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b y Jeffrey Du chem in

Emerging New in Vivo Electrophysiology Methods in Neuroscience Research Introduction Since the original study from Renshaw, Forbes & Morrison in the 1940s recording the activity of neurons in the brain of anesthetized cats,1 the electrophysiology technique has always had an essential place in neuroscience research for the understanding of the main central nervous system (CNS) functions (language, motor control, cognition, etc.) as well as related CNS disorders: mental/neurological disorders, substance abuse and alcohol-related issues, neurodegenerative disease related with the aging of the population, spinal cord injuries, etc.

At the beginning of neuroscience research, brain research belonged to many different areas that differed in methodology and targets: the morphological, the physiological and the psychological. Nowadays, scientific and technological researchers, from molecular to behavioral levels, understand the benefits of developing brain research in a really interdisciplinary way. Indeed, research is more and more based on the convergence of different interconnected scientific sectors.

In that context, in vivo electrophysiology research is not an exception. For instance, a parallel electrophysiological recording and behavior monitoring of freely moving animals is essential for a better understanding of the neural mechanisms underlying behavior and intrinsic brain processes.

It is worth noting that the increase in importance of such multidisciplinary approaches in today’s research would not be possible without rapid improvements in electrophysiology techniques. As an example, the use of wireless in vivo electrophysiology techniques is one of the innovations that is making it easier to combine electrophysiology and behavior in laboratory animals.

The benefits of a multidisciplinary approach are not only scientific (e.g. direct correlation between the observed phenomena, higher specificity in the collected data in terms of their cause-effect relation) but there is also ethical collateral. Indeed, by improving the data quality and quantity, a multidisciplinary approach assists in the effort of reducing the number of animals needed to obtain scientifical-

ly valid data as well as in minimizing stress by promoting the use of lessinvasive methods allowing the running of experiments in progressively more natural conditions. This is particularly important in the context of the current higher pressure directed toward neuroscience researchers by ethical committees.

In this perspective, it is expected that new demand for electrophysiology instruments in the life science or clinical markets will continue to grow with new application areas, related to a more multidisciplinary/integrated analysis of the CNS functions by itself and of its relation with the whole organism.

Neurological diseases also continue to affect one out of five people in the world, which is also driving the increase of research funding in this space. Continually evolving refinements in in vivo electrophysiology methodology will undoubtedly aid neuroscience researchers in getting high-quality, physiologically relevant data and contribute to ground-breaking discoveries that may, ultimately, lead to new therapeutic strategies.

Wireless in vivo electrophysiology and behavior The living brain is essentially an electrical organ at the interface of the external world. Consequently, most of our knowledge regarding the neuronal correlates of behavior would come from the concomitant study of the electrical activity of the brain. In this context, the oldest and most frequently used technique to study the living brain is the in vivo extracellular recording.

The combination of electrophysiology with the measurement of behavior in awake/freely moving laboratory animals is gaining more sense with the progressive evolution of techniques, especially in the improvements made in the neurophysiology of extracellular recording with a trend toward miniaturization, increased neuronal sampling and data collection. For these recordings to be “ecologically” significant, animals need to be awake and behaving in small, indoor environments defined by the laboratory context.

In this context, the advent of commercially available wireless tech-

nology2 for running in vivo electrophysiology recording in laboratory animals is a significant advancement in neuroscience research. It offers the possibility of running long-term, hands-off multidisciplinary measurements in freely moving animals.

The in vivo wireless electrophysiology method provides a great number of benefits with respect to the traditional tethered recording techniques.

For instance, wireless recording of many signals (field potential, spike, electroencephalogram, electrocorticogram, electromyogram, temperature, etc.) is demonstrated to be less stressful and more relevant physiologically. Crucially, it is able to circumvent various practical issues inherent with the tethered recordings, which includes cable twisting, the external force and visual distraction arising from the cable itself or, for instance in the particular context of a social interaction study, the entanglement of the cables or ease of chewing damage to the cable by the cage mate.

In addition to their rapid set-up times, wireless systems can be eas-

Figure 1

Example of the components of the standard TBSI in vivo wireless recording system used in small laboratory animals.

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Laboratory Focus August/September 2016

Feature Figure 2

Example of miniaturization of the MCS wireless headstages and the variability of their field of application, here used in fishes and birds. ily utilized and moved between the wide variety of experimental arenas and behavioral tests used in neuroscience in small laboratory animals for the study of the CNS functions and related diseases: locomotor activity/ exercise and coordination (open-field test, rotarod, treadmill), pain sensitivity (tail-flick test, hot-plate), anxiety (plus maze), emotion and memory (fear conditioning test, radial maze, T or Y maze, water maze), addiction and cognition (operant boxes, selfadministration boxes), social interaction, etc.

Technically, in the wireless in vivo electrophysiology systems, the neural signal emitted by the neurons is collected by brain-implanted electrodes that are associated with a battery alimented head-mounted system (headstage) that is used as a transmitter, driving the signal wirelessly to a special receiver. The signal is then interfaced to data acquisition software for storage, further filtering and analysis (Figure 1).

The current headstages available in the market are already a wonder of electronic engineering; interfacing stimulations and signals from different sensors. For instance, the W2100 wireless system developed in Germany by Multi Channel Systems (MCS) features a level-based construction making custom-built headstages possible. This kind of headstage consists of levels for amplification and Analog/Digital (A/D) conversion, electrical or optical stimulation and many more that are currently under development. Of note is that the recorded signals are converted into digital data already on the headstage. Therefore, the signal-to-noise ratio is much better and most importantly, independent from the distance between sender and receiver. This kind of headstage permits flexible long-term experiments in large environments.

The signal received from the electrodes implanted in the brain can also be transmitted through an analog (radio frequency – RF) signal. This is

done with the headstage provided

by Triangle BioSystems International (TBSI, US), with the advantage of providing a very high data-rate recording (50 kHz/channel) for maximum precision and time-resolution in the recording of the neuron activity, which is in the submillisecond regime.

All wireless systems depend on the associated battery lifetime, whose duration is proportional to the size of the battery. The average nominal battery life of a wireless system can be several hours. The battery life can be extended by turning off data acquisition at some user-defined period of time, allowing the battery life to be distributed over a longer period. Some solutions also exist for the continuous recording of neural data using far-field inductive power technology, such as the one developed by TBSI. This is particularly important for experiments evaluating the diurnal and nocturnal variations of neural activity such as sleeping, chronobiology or epilepsy studies.

Wireless electrical and optical (optogenetics) stimulation3 of the neurons can also be used to complement the recording techniques and are used to artificially challenge the neuronal activity to answer specific questions raised by the experimental context.

The in vivo wireless electrophysiology technique has been widely validated and is available for use in virtually all laboratory animals from mice to monkeys, even in birds and fish (Figure 2). Of note, its greatest use so far has been in rodents, the most frequently used in neuroscience research.

New perspectives in multi-site recording/stimulation and implantable headstages What would the next steps be in the improvement of in vivo electrophysiology methods? Different avenues are being explored; among them are multi-site recordings/stimulation and implantable headstages.

“Multi-site recordings/stimulation” refers to the capacity to record neural activity in different locations of the brain and/or to stimulate one region of the brain and simultaneously record in another region. These techniques would provide more flexibility in the study of neural activity in a specific network in association with the behavior correlates.

Implantable wireless devices will be a significant accomplishment for recording and stimulation both in the cortical and peripheral nervous system. The implantable devices can be designed to be packaged in a miniaturized capsule and implanted in the animal’s gut in surgery. After recovery, the animal can proceed to deliv-

er recordings or receive stimulation in a manner that is as close as possible to natural behavior. Although there is a risk of the package budging with certain organs, the risk is far less of an issue than having a tether or external device on the animal’s head. This technique may provide an additional step into multidisciplinary research, not only for improving the experimental conditions (e.g., minimizing animal stress) but also in providing new information on CNS functions and their relation to the whole organism.

For instance, implantable stimulation and recording electrophysiology technologies can open up new research markets centered around peripheral nerve applications. These new markets lie beyond the brain or central nerve electrophysiology that is possible using the existing headmounted technologies: for example, new research areas with digestive, bladder and respiration nerve-related research. An additional very important prospect would be related to robotics and new artificial interface developments for amputees.

Overall, the continuing advances in the field of electrophysiology are opening up a host of new opportunities for scientific researchers to learn more about how living organisms work.

References: 1. Renshaw B, Forbes A, Morrison BR. Activity of isocortex and hippocampus: electrical studies with micro-electrodes. J Neurophysiol. 1940;3:74-105.

2. Fan D, Rich D, Holtzman T, et al. A wireless multi-channel recording system for freely behaving mice and rats. PLoS One. 2011;6(7):e22033.

3. Rossi MA, Go V, Murphy T, Fu Q, Morizio J, Yin HH. A wirelessly controlled implantable LED system for deep brain optogenetic stimulation. Front Integr Neurosci. 2015 Feb 10;9:8.

Jeffrey Duchemin is President and Chief Executive Officer of Harvard Bioscience, Inc., a global developer, manufacturer and marketer of a broad range of solutions to advance life science.

To see this story online visit http://laboratory focus.ca/emerging-new-in-vivoelectrophysiology-methods-inneuroscience-research

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b y Anthony J . S a por ita , P h.D., Se nior Scie ntist, Mil l ipor e Sigm a

Bird’s eye view of intracellular signal transduction pathways Using customizable MILLIPLEX® map Cell Signaling Phospho/Total 2-Plex assays Abstract A wide range of phosphorylation events are critical for cellular processes, including growth and differentiation. Cells must be able to respond to extracellular and intracellular cues to modulate activation or repression of specific proteins. The Luminex xMAP® platform enables multiplex analysis of both circulating and intracellular proteins, including phosphoproteins. Luminex xMAP® technology is a bead-based assay that enables the simultaneous measurement of concentrations of multiple proteins in a single sample using Luminex instrumentation to acquire and analyze resulting data.

MILLIPLEX® MAP Cell Signaling Phospho/Total 2-Plex assays enable the simultaneous detection of both phosphorylated and total protein in the same well. Assays for phosphorylated and total Akt1, Akt2, Akt3, (pan) Akt, CREB, ERK/MAPK 1/2, IRS1, JNK, mTOR, p38, and STAT3 are currently available (Table 1). Furthermore, MILLIPLEX® MAP Cell Signaling Phospho/Total 2-Plex assays can be combined, or “plexed,” together in a customizable panel, permitting the simultaneous study of multiple phosphorylated and total signaling proteins in the same well (Figure 1).

In this study, we use MILLIPLEX® MAP Cell Signaling Phospho/Total 2-Plex assays to monitor the responses of several signal transduction pathways to three compounds: a growth stimulus (IGF-1), a kinase inhibitor (LY294002), and a differentiation agent (retinoic acid). Our findings indicate that these assays can sensitively detect even minor changes in protein phosphorylation and expression to determine which signaling proteins are impacted by a given treatment.

Materials and Methods Cell Culture. All cell lines used in this study were obtained from ATCC and cultured according to the supplier’s recommended protocols. For serum-

starvation experiments, cells were cultured in serum-free media for three hours prior to the addition of the drug. Cells were lysed and samples collected according to the MILLIPLEX® MAP Cell Signaling Buffer and Detection Kit (Cat. No. 48-602) instructions.

Immunoassay. MILLIPLEX® MAP Cell Signaling Phospho/Total 2-Plex assays were conducted in 96-well plates according to protocol. Briefly, plates were washed with assay buffer prior to addition of 25 μL of sample (20 μg cell lysate unless otherwise indicated) and 25 µL of 1X magnetic capture beads. Assays were incubated overnight at 4 °C. The following day, plates were washed twice with 100 µL assay buffer, then incubated in 25 μL 1X biotinylated detection antibody cocktail for one hour. Detection antibody was removed and streptavidin-phycoerythrin (SAPE) was added for 15 minutes. Cell Signaling Amplification buffer was added to SAPE, and plates were incubated for an additional 15 minutes. All incubation steps were carried out on a plate shaker at medium speed. As-

Table 1

Growth factor or cytokine activation of membrane receptors

Figure 1 + IRS1

+ P

+

JAK MAPKKK

PI3K Akt

MKK

P

mTORP

Stress

STAT3 P

ERK

P

JNK

P

CREB

P

p38

Overview of MILLIPLEX® MAP Cell Signaling Phospho/Total 2-plex Assays. These assays monitor activation of intracellular pathways in response to a variety of stimuli. This streamlined model illustrates some of the core components of the PI3K/Akt, JAK/STAT, and MAPK pathways. Proteins included in the MILLIPLEX® MAP Cell Signaling Phospho/Total 2-Plex assay portfolio are shaded dark blue. Upstream signaling intermediates are shaded grey. The MAPKKK and MKK proteins responsible for activation of each of the MAPK pathways (ERK, JNK, and p38) have been grouped together using generalized terminology to simplify the illustration.

MILLIPLEX® MAP assays used

Panel Name Akt1 Phospho/Total – 2 Plex Akt2 Phospho/Total – 2 Plex Akt3 Phospho/Total – 2 Plex Akt Phospho/Total – 2 Plex CREB Phospho/Total - 2 Plex Erk/MAPK 1/2 Phospho/Total - 2 Plex IRS1 Phospho/Total - 2 Plex JNK Phospho/Total - 2 Plex mTOR Phospho/Total - 2 Plex p38 Phospho/Total - 2 Plex STAT3 Phospho/Total - 2 Plex

P

Phosphorylation Site Ser473 Ser474 Ser472 Ser473/Ser474/Ser472 Ser133 Thr185/Tyr187 Ser636 Thr183/Tyr185 Ser2448 Thr180/Tyr182 Tyr705

Cat. No. 48-631MAG 48-632MAG 48-633MAG 48-618MAG 48-628MAG 48-619MAG 48-626MAG 48-622MAG 48-625MAG 48-624MAG 48-623MAG

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Figure 2 2A.

2B.

Unstimulated Cell Lysates

20000 16000

20000 MFI

Single 2-Plex

12000

Single 2-Plex

15000

8000

10000

4000

5000

0

0

pC RE CR B E pJ B NK JN pp K 38 p p A 38 kt Ak 1 pA t1 kt Ak 2 pA t2 kt Ak 3 pI t3 RS IR 1 pm S1 T m OR pS TOR TA ST T3 AT 3

pC RE CR B E pJ B NK JN pp K 38 p p A 38 kt Ak 1 pA t1 kt Ak 2 pA t2 kt Ak 3 pI t3 RS 1 IR pm S1 TO m R pS TOR TA ST T3 AT 3

MFI

Stimulated Cell Lysate Controls

25000

2C. 25000

y = 1.0105x - 78.241 R² = 0.99492

2-Plex MFI

20000

STAT3 pSTAT3

15000

tAkt2 CREB

10000 mTOR

pCREB

5000

pJNK

pAkt3 pp38

0

0

JNK

pAkt1

pIRS1 pmTOR

IRS1

tAkt1

p38

tAkt3

pAkt2

5000

10000

15000

20000

25000

Single-plex MFI

Plex-ability of phospho and total antibody pairs. 2A) MFI values in unstimulated cell lysates. HeLa unstimulated cell lysates (Cat. No. 47-205) were used for the measurement of Phospho/Total CREB, JNK, p38, Akt1, Akt2, Akt3, IRS1, and STAT3. HepG2 unstimulated cell lysate (Cat. No. 47-239) was used for the measurement of Phospho/Total mTOR. 2B) MFI values in stimulated cell lysates. HeLa: IFNα (Cat. No. 47-226) was used for the measurement of Phospho/Total STAT3. HeLa: TNFα+CalA (Cat. No. 47-230) was used for the measurement of Phospho/ Total CREB. HeLa:HS/Ars (Cat. No. 47-211) was used for the measurement of Phospho/Total p38 and JNK. HepG2:Insulin (Cat. No. 47-227) was used for the measurement of Phospho/Total mTOR. MCF7:IGF-1 (Cat. No. 47-216) was used for the measurement of Phospho/Total IRS1.

HEK293:Serum (Cat. No. 47-233) was used for the measurement of Phospho/Total Akt1, Akt2, and Akt3. 2C) Correlation of single-plex vs.

2-plex MFI values in stimulated cell lysates. The data from Figure 2B was plotted to compare the MFI values obtained by 2-plex assays with those obtained when a “single-plex” assay was run for each total or phosphoprotein.

Figure 3 3A. Phospho/Total ratio (16-plex)

1.6

y = 0.9102x + 0.0137 R² = 0.99775

1.2

JNK

1.0 STAT3

0.8 0.6

Akt2

mTOR

0.4

CREB

p38

0.2

Akt1 IRS1

0 0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Phospho/Total ratio (2-plex)

3B.

1.0

A549 NIH3T3 HEK293

0.8 0.6

ERK

1.5 Phospho/Total ratio

Phospho/Total ratio

3C.

Akt3

1.2

0.4 0.2 0

A549 NIH3T3 HEK293

1.0

0.5

0 2-plex

16-plex

2-plex

16-plex

Plex-ability of combined 2-plex assays. 3A) Correlation of 2-plex versus 16-plex measurements in stimulated cell lysates. HepG2:Insulin was used as a stimulated cell lysate for Phospho/Total JNK, mTOR, STAT3, Akt1, and Akt2. HeLa:TNFα+CalA was used as a stimulated cell lysate for Phospho/Total CREB, p38, and IRS1. The phospho/total (P/T) ratio was calculated by dividing the MFI of the phosphoprotein over the MFI of the total protein. The reported 16-plex values include MFI data averaged from separate 16-plex assays containing either Akt1 (Cat. No. 48-631MAG), Akt2 (Cat. No. 48-632MAG), or (pan) Akt (Cat. No. 48-618MAG) beads and detection antibody. 2B and 2C) Phospho/Total ERK and Phospho/Total Akt3 assays are affected by multiplexing. The Phospho/Total ERK and Phospho/Total Akt3 assays displayed MFI values that experienced fluctuation when moving from 2-plex to 16-plex. The phospho/total protein ratios for ERK and Akt3 were analyzed in three cell lysates: A549:Camptothecin (Cat. No. 47-218), NIH3T3:Anisomycin (Cat. No. 47-219), and HEK293:Serum (Cat. No. 47-233). While the absolute phospho/total ratio for both proteins changed, the overall phosphorylation pattern for each analyte was preserved.

say plates were read and analyzed on a Luminex 200™ system and mean fluorescence intensity (MFI) data was collected.

The names and catalog numbers for the 2-plex assays used in this report are provided in Table 1. Individual (unpooled) capture beads and detection antibodies were used to compare “single-plex” and 2-plex MFI measurements of total and phosphoprotein.

Each phospho/total 2-plex assay is compatible with other phospho/total 2-plexes with the exception of the Akt isoform assays. Three highly homologous Akt isoforms exist, with different physiological roles and patterns of expression.1 Each Akt isoform phospho/ total 2-plex assay detects the intended isoform with high specificity. These assays can be successfully plexed with all other (non-Akt) 2-plex kits, but not with one another. As such we independently tested the customizable 16-plex assay (8 phospho/total 2-plex assays) with each of the Akt isoform and pan-AKT kits.

Results and Conclusions Proteins can be regulated at the level of total protein expression and by phosphorylation events that may modulate protein activity. Consequently, the ability to measure both total protein and phosphoprotein within the same sample in an immunoassay is advantageous. Further, being able to identify the relative proportion of protein existing in the phosphorylated state enables researchers to normalize their data. In an immunoassay format, however, it is crucial that the antibody pairs for the total protein and phosphorylated epitope do not compete or interfere with one another. To demonstrate the feasibility of this approach, we compared MFI measurements for phosphoproteins and total proteins run either individually or in a 2-plex format, using the unstimulated and stimulated cell lysates associated with each particular kit as controls.

Total protein and phosphoprotein MFIs were nearly identical when tested in either a “single-plex” or 2-plex assay (Figure 2A and 2B).

This pattern was consistently observed whether assessing unstimulated or stimulated cell lysates, demonstrating that both the single-plex and 2-plex assays had similar sensitivities. When MFI values were compared for individual analytes, the data showed a striking correlation (R2=0.9949) regardless of signal intensity (Figure 2C).

This data demonstrates that both the total protein and its phosphorylated epitope can be detected simultaneously and independently in the same sample, without competition or interference. In general, the to-

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Feature compare the 2-plex assay results with those of a customized 16-plex assay.

As expected, there were some differences in the MFI values observed for specific analytes when comparing the 2-plex and 16-plex assays.

However, analyzing the ratio of phosphoprotein to total protein demonstrated a strong, linear relationship between the measurement of most phospho/total analytes when transitioning from a 2-plex assay to a customized 16-plex assay (R2=0.9977, Figure 3A). Two phospho/total 2-plex assays (ERK and Akt3) experienced larger variations in MFI when transitioning to the customized 16-plex assay. Phospho-ERK experienced a reduction in MFI, with minimal change in total ERK, whereas both phosphoAkt3 and total Akt3 exhibited decreases in MFI signal in the customizable 16-plex assay format (data not shown). While these reductions in signal intensity may have disrupted the ratio of phosphoprotein to total protein, the 16-plex panel format did not interfere with the relative detection of these proteins when comparing cell lysates with varying degrees of pathway activation (Figure 3B, 3C). For example, HEK293:Serum lysate (Cat. No. 47-233) has a higher ratio of phospho-ERK/total ERK compared to A549:Camptothecin lysate (Cat. No. 47-218), regardless of whether a 2-plex assay or 16-plex assay was used. Thus, the general pattern of protein phosphorylation for ERK and Akt3 is preserved when integrating these assays into a customizable 16-plex panel.

Commercially available MILLIPLEX® MAP Cell Signaling lysates were then screened using the customizable 16-plex assay. This generated a profile in which the relative expression and phosphorylation of a signaling protein can be compared across a breadth of cell lines and treatments (Figure 4). While the

pCREB

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A4 A5 31 49 :EG : F HE Da cam K2 ud p 93 i:I L He :Se 4 ru He La:I m La FNα H :P He eLa pas La :TN e He :Un Fα La st : im H Hs He epG /Ars pG 2: H 2 Ins HU L-6 :TG VE 0:P Fβ C VD Ju :Ser r u Ju kat m rk :An a Ju Jurk t:H2 s rk a O2 at t:P NI :Un ac H st Ra 3T3 im m :An o Ra s:PV s tH D ea rt MFI

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Figure 4

Evaluating MILLIPLEX® MAP Cell Signaling lysates with a customizable 16-plex panel comprised of Phospho/Total 2-plex assays. Cell lysates were screened by 16-plex assay, and the total and phosphoprotein MFI measurements for each of the 2-plex assays were collected and illustrated in separate graphs.

Figure 5 7 Phospho/Total ratio (relative to unstim)

tal protein MFI was typically greater than the phosphoprotein MFI, which would be predicted as only a portion of the total protein will be phosphorylated. However, there are instances when the phosphoprotein MFI is greater than that of the total protein, as was observed with JNK. It is important to note that other factors besides protein abundance may affect MFI measurement, such as the presence of multiple isoforms or antibody pair avidity for a target.

Next we determined whether the individual 2-plex kits could be pooled to create a “customizable” multiplex assay capable of measuring multiple phosphoproteins and total proteins from several intracellular signal transduction pathways simultaneously. HeLa:TNFα+CalA (Cat. No. 47-230) and HepG2:Insulin (Cat. No. 47-227) were used as stimulated cell lysates to

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IGF dose response in MCF7. MCF-7 human breast cancer cells were serumstarved for 3 hours prior to addition of IGF-1 at the indicated concentrations for 15 minutes. Cell lysates were collected according to MILLIPLEX® MAP assay protocols and samples (20 μg/ well) were measured using a 16-plex assay composed of several phospho/total 2-plex assays. The ratio of phosphoprotein to total protein was calculated using median MFI values. These ratios were then normalized to the phospho/ total protein ratios from unstimulated MCF-7 cells.

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Laboratory Focus August/September 2016

feature Figure 6 6B.

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LY294002 preferentially inhibits Akt1 phosphorylation in MCF7 cells. 6A) MCF-7 cells express Akt1 and Akt2 but not Akt3. Lysates were collected from unstimulated MCF7 cells. Phospho/Total 2-plex assays for Akt1, Akt2, and Akt3 were used to measure the relative expression of the Akt isoforms. 6B) LY294002 preferentially inhibits Akt1 phosphorylation in response to IGF-1 in MCF7 cells. MCF7 cells were cultured in the presence or absence of 20 μM LY294002 for 20 minutes before the addition of IGF-1. Lysates were collected and subjected to analysis using a customizable 16-plex panel composed of the indicated phospho/total 2-plex assays.

uated by multiplex assay. RA-induced differentiation of SH-SY5Y cells was accompanied by phosphorylation of all three Akt isoforms, with Akt2 and Akt3 displaying the greatest induction (Figure 7). Multi-pathway analysis demonstrated co-induction of phospho- JNK in the RA-treated SHSY5Y cells, consistent with the role of JNK in RA-mediated differentiation.4 Phosphorylation of mTOR and STAT3 also increased in the RA-treated cells.

In summary, MILLIPLEX® MAP Cell Signaling Phospho/Total 2-Plex assays are a valuable research tool to study phosphoprotein expression and activation. Further, the “Plexability” of these 2-plex assays allows researchers to create customizable panels to get a broad overview of signaling events, or interrogate specific pathways of interest.

References

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SH-SY5Y differentiation is accompanied by activation of multiple signaling proteins. SH-SY5Y cells were treated with retinoic acid (RA) for 3 days to stimulate neuronal differentiation. After 3 days, cells cultured in the presence or absence of RA were collected and lysed according to MILLIPLEX® MAP assay protocols. MFI measurements were obtained from 16-plex panels using pooled 2-plex assays (Akt isoform 2-plex assays were run both individually and in separate 16-plex panels), and the ratio of phosphoprotein to total protein was calculated for each analyte.

majority of tested lysates originated from human cell lines, the inclusion of NIH3T3:Anisomycin (Cat. No. 47219) and rat heart lysates demonstrate that the MILLIPLEX® MAP Cell Signaling Phospho/Total 2-Plex assays can also effectively detect mouse and rat proteins. Analysis of the cell lysate profile illustrated both the variation in protein expression between cell lines and the differences in protein phosphorylation patterns in response to diverse stimuli.

To demonstrate the sensitivity of the customizable 16-plex panel, we examined changes in phosphorylation in a dose-response assay. MCF-7 human breast cancer cells were serumstarved for 3 hours, then treated with the indicated doses of IGF-1 for 15 minutes. Cell lysates were collected and the relative levels of phosphorylated proteins and total proteins were measured in a 16-plex panel comprising pooled reagents from mul-

tiple 2-plex assays. Not all proteins responded to the short IGF-1 stimulus; however, ERK, Akt, and JNK exhibited induction of phosphorylation.

In addition, phosphorylation of these proteins was sensitive to increasing concentrations of IGF-1 (Figure 5).

We then examined the effect of the PI3K inhibitor LY294002 on IGF1-induced phosphorylation. MCF-7 cells were serum-starved and pretreated with LY294002 for 20 minutes prior to the addition of IGF-1.

After a 15-minute IGF-1 stimulation, cells were lysed and analyzed by a 16-plex assay with either Akt1 or Akt2. In MCF-7 cells, Akt3 expression was undetectable (Figure 6A), consistent with published reports.2 Consequently, the phospho/total Akt3 2-plex assay was not included in the 16-plex analysis. Akt1, Akt2, ERK, and JNK exhibited >2-fold inductions of phosphorylation in response to IGF-1. Pre-treatment with 20 µM

LY294002 completely abrogated the IGF-induced phosphorylation of Akt1 and, to a lesser extent, attenuated the activation of Akt2 (Figure 6B). This result suggests a differential sensitivity of the Akt isoforms to PI3K inhibition. In contrast to the Akt isoforms, phospho-JNK and phospho-ERK were further stimulated

by LY294002, suggesting a potential compensatory feedback mechanism in the cross-talk between these intracellular signaling pathways.3 Selective activation of signal transduction pathways also plays an integral role in differentiation. SH-SY5Y cells were used as a model to study intracellular signaling in the context of neuronal differentiation. We were particularly interested in which Akt isoforms would be activated. Briefly, SH-SY5Y cells were treated with retinoic acid (RA) for three days to induce neuronal differentiation before cells were collected, lysed, and eval-

1. Gonzalez, E., & McGraw, T. E. (2009). The Akt kinases: Isoform specificity in metabolism and cancer. Cell Cycle, 8(16), 25022508.

2. Chin, Y., Yoshida, T., Marusyk, A., Beck, A., Polyak, K., & Toker, A. (2014). Targeting Akt3 Signaling in Triple-Negative Breast Cancer.

Cancer Research, 74, 964-973.

3. Mendoza, M., Er, E., & Blenis, J. (2011). The Ras-ERK and PI3KmTOR Pathways: Cross-talk and Compensation. Trends Biochem Sci., 36(6), 320-328.

4. Cheung, Y., Lau, W., Yu, M., Lai, C., Yeung, S., So, K., & Chang, R. (2009). Effects of all-transretinoic acid on human SH-SY5Y neuroblastoma as in vitro model in neurotoxicity research. Neurotoxicology, 30, 127-135.

Dr. Saporita has over 10 years of experience in protein biochemistry and assay development. He earned his Ph.D. from Northwestern University and received postdoctoral training at Washington University in Saint Louis. He has several peer-reviewed publications in the areas of cancer biology and cell signaling. As part of the Research & Development team at MilliporeSigma, Dr. Saporita designs new multiplex immunoassay panels for protein biomarker detection.

To see this story online visit http://www. laboratoryfocus.ca/ birds-eye-view-ofintracellular-signal-transduction-pathways-usingcustomizable-milliplexmap-cell-signalingphosphototal-2-plex-assays/

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August/September 2016 Laboratory Focus www.laboratoryfocus.ca

New Products Osmometer The OsmoPRO® from Advanced Instruments is a 20-position micro sample osmometer designed specifically to address the changing needs of today’s busy laboratories. The device can be used in a variety of laboratory applications including clinical diagnostics, formulation development, bioprocess monitoring and process control, industrial, environmental, and finished product quality control. Ideal for sample-limited applications, OsmoPRO uses a small 20 μL sample size. Samples can be analyzed one at a time, or batch processed depending on the workflow demands. OsmoPRO uses the industry preferred freezing point depression method to deliver results in just two minutes. It is able to analyze a variety of complex aqueous mixtures including blood, serum, plasma, urine, cell culture media, drug formulations, and biological cell therapies. Time-saving features include the ability to run tests unattended, automated processing of up to 20 samples, and an intuitive touchscreen. Additionally, its integrated 2-D barcode scanner provides positive sample identification to reduce transcription errors. Data management and transfer can be handled via the on-board printer or by easily exporting the data using the Ethernet connection and multiple USB ports.

Web: www.aicompanies.com

Spectrophotometers Biochrom announces the release of two new products to its WPA Visible Spectrophotometer Range, the WPA S800+ and S1200+ spectrophotometers. The new WPA spectrophotometers are ideal for student use in busy teaching laboratories, as well as for use for applications in the clinical, life-science and industrial market. Both products are compact, lightweight and ergonomically designed. They also each include a large display that is easy to read and a simple user interface for rapid set up and analysis. The WPA S800+ is an entry level visible spectrophotometer for labs performing basic spectroscopy measurements. The WPA S800+ performs single wavelength measurements of absorbance, % transmission, concentration and has the ability to perform simple kinetics. The WPA S1200+ builds on the features of the S800+ with the addition of multiwavelength measurements and life science methods that include BCA, Bradford, Biuret and Lowry. Both instruments accept standard 10mm pathlength glass or disposable cuvettes. An optional test tube cell holder is available for test tubes that are between 10 and 18mm in diameter. A heated cell holder is also available for thermostatted measurements at 37°C. The cell holders are easy to remove and install making cleaning and decontamination simple.

Web: www.biochrom.co.uk

Glassware BrandTech introduces a new line of BLAUBRAND® class A, USP certified volumetric glassware from BRAND GMBH + CO KG. The line includes clear and amber volumetric flasks, graduated cylinders, bulb pipettes, and volumetric pipettes. The USP Volumetric instruments comply with Class A error limits required by the United States Pharmacopeia thus are a suitable choice for companies that are audited by the FDA or any other U.S. authority. They are also well suited for companies that are not audited but demand high quality glassware that meets Class A error limits.

Web: www.BrandTech.com

X-Ray Microscopes The new ZEISS FPX flat panel extension for the ZEISS Xradia Versa 500-series of 3D X-ray microscopes delivers large-sample, high throughput scanning with best-inclass image quality. Combined with the high resolution of ZEISS Xradia Versa X-ray microscopes (XRM), the new ZEISS FPX enhances imaging flexibility and creates workflow efficiencies with an all-in-one system for research work in areas such as the oil and gas, medical device industries, and for materials and life sciences. The ZEISS Xradia Versa with FPX enables researchers to scout large samples 2 to 5x faster to identify a region of interest (ROI), and then zoom to image areas at high resolution with the exclusive ZEISS Xradia Versa RaaD dual magnification microscope objectives that enable resolution at a distance. ZEISS FPX extends the ability for ZEISS Xradia Versa XRM to achieve full field of view, whole-sample imaging, up to five inches in diameter for samples 10x greater in volume with higher throughput.

Web: www.zeiss.com

Analytical Instrument Rigaku introduces its new XtaLAB mini II benchtop X-ray crystallography system for small molecule 3D molecular structure determination. The XtaLAB mini™ II system is a research grade, compact single crystal X-ray diffractometer designed to produce ready-topublish 3D structures with quality that exceeds IUCr publication standards. This next-generation instrument expands the capabilities of the XtaLAB mini by the addition of a low-noise, state-of-the-art hybrid pixel array detector as well as the full-featured CrysAlisPro software from Rigaku Oxford Diffraction. To the user, this means that poorly diffracting samples can now be measured more accurately and the set of software tools will be the same as with the top of the line diffractometers. As an ideal addition to any synthetic chemistry laboratory, new compounds can be rapidly analyzed as they are synthesized in the lab. The newly developed detector is based on hybrid photon counting technology (HPC) and the low noise, high dynamic range and fast frame rate allow highly precise, shutterless data collection, a technique that is effective at reducing data measurement time. The X-ray tube lifespan is extended by running at 600 W. To countervail running at lower power, a special curved monochromator is used to produce usable X-ray flux comparable to a standard X-ray system and provide the same quality data as a larger 3 kW X-ray system.

Web: www.rigaku.com

Fluorimetry Cuvettes BrandTech® Scientific’s fluorimetry cuvettes are precision molded with four optically-clear sides for fluorescence applications. Made from premium raw materials, and BRAND’s three decades of cuvette molding experience, they offer minimal cuvette-to-cuvette variance for optimal results. Available in PMMA/acrylic for the visible range, and a proprietary UV transparent polymer with virtually no autofluorescence for maximum signal/noise ratios down to 230nm. Packed in low-dust, non-scratching expanded polystyrene with an industry-standard 10mm light path.

Web: www.BrandTech.com

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app review Science Dictionary

International Mass Spectrometry Conference comes to Toronto After years of exclusively being held in Europe, the International Mass Spectrometry Conference (IMSC) made its North American debut in Toronto.

Hosted by the Canadian Society for Mass Spectrometry and the Canadian scientific community at the Metro Toronto Convention Centre (MTCC), the conference ran from Aug. 20 to 26 and drew more than 1,200 delegates. Opportunities were plentiful to network, as well as learn about the latest advances in the science of determining and characterizing ionized molecules, including those that are of importance to the environment, industry, health, and medicine.

The conference featured a dynamic week of daily scientific sessions, exhibitions, workshops, courses, and seminars. Individuals that have made significant contributions in the field also got their time in the spotlight with a special award portion to the event.

Most importantly, the conference not only brought the most recent findings in mass spectrometry science to the forefront, but also served as a stage for Canadian scientists to showcase their work.

“Canadian scientists have made significant contributions to mass spectrometry, and many of these advancements were made by Toronto-based scientists specifically,” said professor Michael Siu, vice president of Research and Innovation at the University of Windsor, who led the charge in bringing IMSC to Toronto as an Ambassador of the Leaders Circle. He adds that Toronto has one of the highest concentrations of mass spectrometers in Canada, with the Greater Toronto Area being home to two mass spectrometry manufacturers, SCIEX (now a division of Danaher) and IONICS (now a division of PerkinElmer).

These were just some of the reasons that he and others felt made Toronto an ideal place to host the conference. And already, hopes are high that the conference will return in future years.

From Farlex https://play.google.com/store/apps/details?id=com.farlex.dictionary. science&hl=en There are so many different industries, sectors and aspects to science in Canada. Biology, chemistry and physics have valuable intersections, but there can be gaps between experts of each branch. An astronomer, for instance, wouldn’t have the same knowledge as a marine biologist, just as a marine biologist wouldn’t fully understand the human heart as well as a cardiologist. The Science Dictionary

by Farlex is the bridge to this gap. With over 100,000 terms from multiple scientific dictionaries and encyclopaedias, users gain access to a wealth of definitions that are industry certified. All disciplines, from computer science to environmental science, are well documented and well represented. Particularly beneficial to the sciences, where spelling and pronunciations can be notoriously complex, is the voice search feature. Simply pronounce the word into a supported device and the dictionary will search itself. If the opposite is true, and you have found a written word but aren’t sure of its pronunciation, the app features 35,000 audio clips for American and British speakers. There are also “starts with”, “ends with” and “contains” advanced searchers if you only have a part of the word. All in all, Farlex’s science dictionary delivers a plethora of information – and quickly. The downsides are that the offline mode is quite impaired, swiping through ads can get tiresome.

But for a free app, it’s a decent trade-off.

Science Journal

From Google https://makingscience.withgoogle.com/science-journal Google’s new app, Science Journal, is a tool for doing science with your smartphone. You simply use the sensors in your phone or connect to external sensor source and conduct experiments on the world around you, including measuring sound, light, and more. As examples, you can use the microphone to investigate the sound around you or use the accelerometers to investigate movement. Additionally this Android app helps you visualize and graph this data in an easy-to-understand way. Science Journal offers two different ways of visualizing your measurements—meter mode and graph mode. Meter mode displays your data with a changing animation and a numerical value. Graph mode displays your data in a line graph. Within this mode you can zoom in to take a closer look at your graph and scroll back to see data from earlier observations. In terms of cons, the app doesn’t let you do a ton of measurements so far, but Google is working to expand its functionality. It’s also partnering with San Francisco’s Exploratorium to develop external kits that can be used in conjunction with the Science Journal app. Google also plans to expand the App to open source by end of summer. Users can also access the App’s online forum for troubleshooting questions.

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