Thursday, March 16, 2017

Digital PCR found effective and reproducible in a research study

21 independent labs quantified a rare single nucleotide variant using digital PCR (dPCR) with high reproducibility between labs, demonstrating the method's potential for routine clinical testing, according to a study published in Analytical Chemistry.

The research study is the first to examine the reproducibility of digital PCR between such a large number of laboratories at different geographic locations. The findings illustrate the inherent potential advantages of dPCR in measuring clinically important mutations, including greater reproducibility and less variability than real-time quantitative PCR (qPCR). In addition, the findings highlight the ability of dPCR to detect relevant rare sequence variants, according to the authors.

"Digital PCR lends itself to clinical testing where precise and reproducible results are critical not only for interlab data comparability, but also for dynamic testing in the same lab to track disease progression," said George Karlin-Neumann, co-author and Director of Scientific Affairs at Bio-Rad's Digital Biology Group.

The method permits precise and accurate quantification of nucleic acid concentration by minimizing the variability from common sources of error that can influence qPCR results. These sources include PCR inhibitors that can alter assay efficiency and skew standard curves. Digital PCR also provides absolute quantification of target nucleic acids without the need to establish standard curves, a major source of variability in qPCR.

The blinded study was part of the BioSITrace project, coordinated by the Laboratory of the Government Chemist (LGC), a private life sciences measurement and testing company and the UK's designated National Measurement Laboratory for chemical and biomeasurement. Researchers in 21 laboratories across North America and Europe were asked to use dPCR to quantify copy number concentrations and fractional abundance of a KRAS mutant DNA with a single nucleotide variation (G12D) in the presence of excess wild-type DNA (down to ~0.2% minor allele frequency). The laboratories performed the experiments using Bio-Rad's Droplet Digital™ PCR (ddPCR™) technology, on either a QX100™ or a QX200™ Droplet Digital PCR System from Bio-Rad. Droplet Digital PCR is Bio-Rad's proprietary method for performing digital PCR in which a sample is divided into 20,000 water-oil emulsion droplets.

Cancer-derived mutations that differ only slightly from a wild-type sequence present in a sample in large excess are particularly difficult to detect and quantify using qPCR, especially when fractional abundance of the mutant is less than 5%.

The results of all 21 laboratories agreed with each other within a rigorously defined margin of error. Though three laboratories initially reported differing results, further analysis revealed methodological error attributed to misclassification of positive and negative droplets rather than measurement error. When the data were reanalyzed according to the recommended guidelines, they generated results consistent with the other 18 labs, validating the potential of dPCR for clinical testing applications.

Citation: Whale, Alexandra S., Alison S. Devonshire, George Karlin-Neumann, Jack Regan, Leanne Javier, Simon Cowen, Ana Fernandez-Gonzalez, Gerwyn M. Jones, Nicholas Redshaw, Julia Beck, Andreas W. Berger, Valérie Combaret, Nina Dahl Kjersgaard, Lisa Davis, Frederic Fina, Tim Forshew, Rikke Fredslund Andersen, Silvia Galbiati, Álvaro González Hernández, Charles A. Haynes, Filip Janku, Roger Lacave, Justin Lee, Vilas Mistry, Alexandra Pender, Anne Pradines, Charlotte Proudhon, Lao H. Saal, Elliot Stieglitz, Bryan Ulrich, Carole A. Foy, Helen Parkes, Svilen Tzonev, and Jim F. Huggett. "International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant." Analytical Chemistry 89, no. 3 (2017): 1724-733.
Adapted from press release by Bio-Rad.

Friday, March 10, 2017

Research on Allulose, a rare naturally occuring sugar show promise as healthy alternative to Sucrose

Animal research have shown that low calorie natural sugars like allulose could help regulate glucose levels. Researchers are now trying to find out how it works, and their findings are reported in Journal of Agricultural and Food Chemistry.

Sucrose is the natural sweetener what everyone refer to when sugar is on the ingredient list. Allulose, which is 70 percent as sweet as sucrose, and other rare sugars also can be found in fruits and vegetables in very small amounts. Recently researchers discovered an industrial way to produce allulose in large quantities from high-fructose corn syrup. Past studies have suggested that allulose can help control weight gain and control glucose levels. Tomoya Shintani and colleagues wanted to confirm these results and understand how it works.

To find out researchers gave three groups of rats plain water, water with high-fructose corn syrup and water with rare-sugar syrup containing glucose, fructose, allulose and other rare sugars for 10 weeks. The rats drinking rare-sugar syrup infused water gained less weight, had less abdominal fat, and had lower blood glucose and insulin levels compared to the high-fructose corn syrup group. The research showed that rats fed with rare-sugar syrup infused water had increased levels of glucokinase in liver cells. Glucokinase is an enzyme that reduces blood-sugar levels by helping convert glucose to its stored form, glycogen.

Although more research is needed, the scientists say, the findings suggest that rare sugars could be a good alternative sweetener.

Citation: Shintani, Tomoya, Takako Yamada, Noriko Hayashi, Tetsuo Iida, Yasuo Nagata, Nobuaki Ozaki, and Yukiyasu Toyoda. "Rare Sugar Syrup Containing d-Allulose but Not High-Fructose Corn Syrup Maintains Glucose Tolerance and Insulin Sensitivity Partly via Hepatic Glucokinase Translocation in Wistar Rats." Journal of Agricultural and Food Chemistry, 2017. doi:10.1021/acs.jafc.6b05627.
Adapted from press release by American Chemical Society.

Tuesday, February 28, 2017

Microgravity and cellular adaptation

Based on real-time readings on the ISS conducted by ESA astronaut Samantha Cristoforetti, University of Zurich scientists Oliver Ullrich and Cora Thiel can now reveal that cells are able to respond to changes in gravitational conditions very quickly and continue functioning. The research team used the alleged oxidative burst an old evolutionary mechanism to exterminate bacteria via defense cells to understand how rat cells responded to changes in gravity.

Picture of experiment equipment. Credit: C. Thiel und Airbus DS
With the help of centrifuges, Cristoforetti altered the gravitational conditions on the ISS, that enabled the research team on earth to track how the cells reacted.

"Although the immune defense collapsed as soon as zero gravity hit, to our surprise the defense cells made a full recovery within 42 seconds." For Ullrich and Thiel, the direct evidence of a rapid and complete adaptation to zero gravity in less than a minute begs the question as to whether previous cell changes measured after hours or days were also the result of an adaptation process.

"It seems paradoxical," says Thiel: "Cells are able to adapt ultra-rapidly to zero gravity. However, they were never exposed to it in the evolution of life on Earth. Therefore, the results raise more questions regarding the robustness of life and its astonishing adaptability." In any case, as far as Ullrich is concerned the result of the ISS experiment is good news for manned space flight: "There's hope that our cells are able to cope much better with zero gravity than we previously thought."

Citation: Thiel, Cora S., Diane De Zélicourt, Svantje Tauber, Astrid Adrian, Markus Franz, Dana M. Simmet, Kathrin Schoppmann, Swantje Hauschild, Sonja Krammer, Miriam Christen, Gesine Bradacs, Katrin Paulsen, Susanne A. Wolf, Markus Braun, Jason Hatton, Vartan Kurtcuoglu, Stefanie Franke, Samuel Tanner, Samantha Cristoforetti, Beate Sick, Bertold Hock, and Oliver Ullrich. "Rapid adaptation to microgravity in mammalian macrophage cells." Scientific Reports 7, no. 1 (2017). doi:10.1038/s41598-017-00119-6.
Adapted from press release by the University of Zurich.

Saturday, February 25, 2017

Novel and unique DNA vaccine to fight effectively against cancer antigen

Scientists at The Wistar Institute and Inovio pharmaceuticals, Inc. have devised a unique deoxyribonucleic acid (DNA) vaccine approach through molecular design to boost the immune responses induced against one of the most important cancer antigen targets. Research results were published in the journal Molecular Therapy.

Cancer immunotherapy approaches, designed to harness the body's natural immune defenses to focus on and kill cancer cells, are showing great promise for cancer treatment and prevention. DNA vaccines can induce immunity through the delivery by an intramuscular injection of a sequence of synthetically designed DNA that contains the instructions for the immune cells in the body to become activated and target a particular antigen against which an immunologic response is wanted.

Despite being specific for cancer cells, cancer tumor-associated antigens generally trigger weak immune responses as a result of they're recognized as self-antigens and also the body has in place natural mechanisms of immune acceptance, or "Tolerance", that forestall autoimmunity and also additionally limit the efficacy of cancer vaccines. This is often the case of Wilm's tumor gene 1 (WT1), a cancer tumor antigen that's overexpressed in many varieties of cancer and possibly plays a key role in driving tumor development. vaccine approaches against WT1 thus far haven't appeared promising because of immune tolerance leading to poor immune responses against cancers expressing WT1.

Wistar scientists have developed a unique WT1 deoxyribonucleic acid (DNA) vaccine employing a strategically changed DNA sequence that tags the WT1 as foreign to the host immune system breaking tolerance in animal models.

"This is an important time in the development of anti-DNA cancer immune therapy approaches. This team has developed an approach that may play an important role in generating improved immunity to WT1 expressing cancers,"said David B. Weiner, Ph.D., Executive Vice President and Director of the Vaccine Center at The Wistar Institute and the W.W. Smith Charitable Trust Professor in Cancer Research, and senior author of the study."These immune responses represent a unique tool for potentially treating patients with multiple forms of cancer. Our vaccine also provides an opportunity to combine this approach with another immune therapy approach, checkpoint inhibitors, to maximize possible immune therapy impact on specific cancers."

The team lead by Weiner has optimized the dna vaccine employing a artificial dna sequence for WT1 that, while maintaining a very high similarity with the native sequence, contains new modified sequences that differ from native WT1 in an attempt to render it more recognizable by the host immune system. The novel WT1 vaccine was superior to a more traditional native WT1 vaccine because it was able to break immune tolerance and induce long-term immune memory. Significantly, the vaccine also stimulated a therapeutic anti-tumor response against leukemia in mice.

Citation: Walters, Jewell N., Bernadette Ferraro, Elizabeth K. Duperret, Kimberly A. Kraynyak, Jaemi Chu, Ashley Saint-Fleur, Jian Yan, Hy Levitsky, Amir S. Khan, Niranjan Y. Sardesai, and David B. Weiner. "A Novel DNA Vaccine Platform Enhances Neo-antigen-like T Cell Responses against WT1 to Break Tolerance and Induce Anti-tumor Immunity." Molecular Therapy, 2017. doi:10.1016/j.ymthe.2017.01.022.
Research funding: Inovio Pharmaceuticals, Inc. Basser Center for BRCA/Abramson Cancer CenterWeiner, W.W. Smith Charitable Trust Professorship for Cancer Research.
Adapted from press release by The Wistar Institute.

Wednesday, February 22, 2017

I-Wire, a new Heart-on-a-Chip device to study biomechanical properties of heart

Scientists at Vanderbilt University have created a 3D organ-on-a-chip that can mimic the biomechanical properties of the heart. The device and the results of initial experiments are reported in the journal Acta Biomaterialia (for links see below).

View of the cardiac fiber in the I-Wire device at two levels of magnification.
Credit: VIIBRE Vanderbilt University
The unique aspect of the new device, which represents about two-millionths of a human heart, is that it controls the mechanical force applied to cardiac cells.  This allows the researchers to reproduce the mechanical conditions of the living heart in addition to its electrical and biochemical environment.

"We created the I-Wire Heart-on-a-Chip so that we can understand why cardiac cells behave the way they do by asking the cells questions, instead of just watching them," said Gordon A. Cain University Professor John Wikswo, who heads up the project. "We believe it could prove invaluable in studying cardiac diseases, drug screening and drug development, and, in the future, in personalized medicine by identifying the cells taken from patients that can be used to patch damaged hearts effectively."

The I-Wire device consists of a thin thread of human cardiac cells 0.014 inches thick (about the size of 20-pound monofilament fishing line) stretched between two perpendicular wire anchors. The amount of tension on the fiber can be varied by moving the anchors in and out, and the tension is measured with a flexible probe that pushes against the side of the fiber. The fiber is supported by wires and a frame in an optically clear well that is filled with a liquid medium like that which surrounds cardiac cells in the body. The apparatus is mounted on the stage of a powerful optical microscope that records the fiber's physical changes. The microscope also acts as a spectroscope that can provide information about the chemical changes taking place in the fiber. A floating microelectrode also measures the cells' electrical activity.

According to the researchers, the I-Wire system can be used to characterize how cardiac cells respond to electrical stimulation and mechanical loads and can be implemented at low cost, small size and low fluid volumes, which make it suitable for screening drugs and toxins. Because of its potential applications, Vanderbilt University has patented the device. Unlike other heart-on-a-chip designs, I-Wire allows the researchers to grow cardiac cells under controlled, time-varying tension similar to what they experience in living hearts.

To demonstrate the I-Wire's value in determining the effects that different drugs have on the heart, the scientists tested its response with two drugs known to affect heart function in humans: isoproterenol and blebbistatin. Isoproterenol is a medication used to treat bradycardia (slow heart rate) and heart block (obstruction of the heart's natural pacemaker). Blebbistatin inhibits contractions in all types of muscle tissue, including the heart.

According to Veniamin Sidorov, the research assistant professor at the Vanderbilt Institute for Integrative Biosystems Research and Education (VIIBRE) who led its development, the device faithfully reproduces the response of cardiac cells in a living heart.

1. Sidorov, Veniamin Y., Philip C. Samson, Tatiana N. Sidorova, Jeffrey M. Davidson, Chee C. Lim, and John P. Wikswo. "I-Wire Heart-on-a-Chip I: Three-dimensional cardiac tissue constructs for physiology and pharmacology." Acta Biomaterialia 48 (2017): 68-78. doi:10.1016/j.actbio.2016.11.009.
2. Schroer, Alison K., Matthew S. Shotwell, Veniamin Y. Sidorov, John P. Wikswo, and W. David Merryman. "I-Wire Heart-on-a-Chip II: Biomechanical analysis of contractile, three-dimensional cardiomyocyte tissue constructs." Acta Biomaterialia 48 (2017): 79-87. doi:10.1016/j.actbio.2016.11.010.

Research funding: National Institutes of Health, National Science Foundation, Defense Threat Reduction Agency, American Heart Association, Department of Veterans Affairs.
Adapted from press release by Vanderbilt University. 

Tuesday, February 21, 2017

Research shows rise in global life expectancy by 2030

The study, led by scientists from Imperial College London in collaboration with the World Health Organization, analyzed long-term data on mortality and longevity trends to predict how life expectancy will change in 35 industrialized countries by 2030. Nations in the study included both high-income countries, such as the USA, Canada, UK, Germany, Australia, and emerging economies such as Poland, Mexico, and the Czech Republic. The researchers chose countries in the study as they all had reliable data on deaths since at least 1985.

Posterior distribution of projected change in life expectancy at birth from 2010 to 2030.
Posterior distribution of projected change in life expectancy at birth from 2010 to 2030.
Credit: The Lancet.
The study, published in The Lancet and funded by the UK Medical Research Council, revealed all nations in the study can expect to see an increase in life expectancy by 2030. The results also found that South Koreans may have the highest life expectancy in the world in 2030. The UK's average life expectancy at birth for women will be 85.3 years in 2030.

Professor Ezzati, lead researcher from the School of Public Health at Imperial explained that South Korea's high life expectancy may be due to a number of factors including good nutrition in childhood, low blood pressure, low levels of smoking, good access to healthcare, and uptake of new medical knowledge and technologies.

French women and Swiss men were predicted to have the highest life expectancies at birth in Europe in 2030, with an average life expectancy of 88.6 years for French women and nearly 84 years for Swiss men.

The results also revealed that the USA is likely to have the lowest life expectancy at birth in 2030 among high-income countries. The nation's average life expectancy at birth of men and women in 2030 (79.5 years and 83.3 years), will be similar to that of middle-income countries like Croatia and Mexico. The research team thinks this may be due to a number of factors including a lack of universal healthcare, as well as the highest child and maternal mortality rate, homicide rate and obesity among high-income countries.

Along with the US, other countries who may see only small increases in life expectancy by 2030 included Japan, Sweden, and Greece, while Macedonia and Serbia were projected to have the lowest life expectancies at birth for women and men respectively in 2030.

The UK's average life expectancy at birth for women will be 85.3 years in 2030. This places them at 21st in the table of 35 countries. The average life expectancy of a UK man meanwhile will be 82.5 years in 2030. This places them at 14th in the table of 35 countries.

The research also suggested the gap in life expectancy between women and men is closing. Professor Ezzati explained: "Men traditionally had unhealthier lifestyles and so shorter life expectancies. They smoked and drank more, and had more road traffic accidents and homicides. However as lifestyles become more similar between men and women, so does their longevity."

Professor Colin Mathers, co-author from the World Health Organization explained: "The increase in average life expectancy in high-income countries is due to the over-65s living longer than ever before. In middle-income countries, the number of premature deaths - i.e. people dying in their forties and fifties, will also decline by 2030." The team developed a new method to predict longevity, similar to the methods used for weather forecasting, which takes into account numerous different models for forecasting mortality and life expectancy.

Life expectancy is calculated by assessing the age at which people die across the whole population. For instance, if a country has high childhood mortality rate, this will make average national life expectancy much lower, as would a country in which many young people die of injuries and violence. The team developed a new method to predict longevity, similar to the methods used for weather forecasting, which takes into account numerous different models for forecasting mortality and life expectancy. All the predictions in the study come with some uncertainty range.

Professor Ezzati added that these results suggest we need to be thinking carefully about the needs of an ageing population: "The fact that we will continue to live longer means we need to think about strengthening the health and social care systems to support an ageing population with multiple health needs. This is the opposite of what is being done in the era of austerity. We also need to think about whether current pension systems will support us, or if we need to consider working into later life."

Citation: Kontis, Vasilis, James E Bennett, Colin D Mathers, Guangquan Li, Kyle Foreman, and Majid Ezzati. 2017. “Future Life Expectancy in 35 Industrialised Countries: Projections with a Bayesian Model Ensemble.” The Lancet, February. Elsevier.
Research funding: UK Medical Research Council, US Environmental Protection Agency.
Adapted from press release by Imperial College London.

Obesity genes: role of foraging gene in fruit fly

A University of Toronto study on fruit flies has uncovered a gene that could play a key role in obesity in humans. The paper published online this month in Genetics examines a "foraging gene" humans share in common with the flies, which plays multiple roles and is found in similar places, such as the nervous system, in the muscle and in fat.

"What our study does is nails the gene for being very important for the traits of moving, feeding and fat storage," says University Professor Marla Sokolowski of the Department of Ecology & Evolutionary Biology in U of T's Faculty of Arts & Science.

In nature, fruit flies called "Rovers" with high amounts of the gene tend to move a lot, eat very little and stay lean, while flies with low amounts of for called "Sitters" are the opposite. The same could apply to obesity in humans.

"So you could imagine if you are a fly, preferences for sugar, the tendency to store a lot of fat and the tendency to move less could all be contributing to the likelihood of being more obese if you have low levels of this gene, or to be leaner if you have higher levels."

Such similarities between species are known as orthologs, meaning they are genes that evolved from a common ancestor eons ago.

The study involved a technique called recombineering to manipulate DNA at the molecular level, so as to remove and then reinsert the gene in various doses to see the effects on behavior and metabolism.

"To be able to take a gene of this large size and complexity and put it back in the fly so that it works is almost at the edge of what is possible," Sokolowski says it's particularly interesting that one gene should have multiple roles in feeding and obesity in the body, a characteristic known as pleiotropy.

The next question would be how exactly it plays multiple roles. "Lots of genes have multiple roles, but the idea here is that this gene may be involved in the coordination of roles in traits important for feeding and obesity."

 "We don't know much about pleiotropy, or how it happens, or how it's regulated at the level of the molecules. So this sets the groundwork to be able to look at that in detail."

Citation: Allen, Aaron M., Ina Anreiter, Megan C. Neville, and Marla B. Sokolowski. "Feeding-Related Traits Are Affected by Dosage of the foraging Gene in Drosophila melanogaster." Genetics 205, no. 2 (2016): 761-73.
Research funding: Natural Sciences and Engineering Council of Canada, Canadian Institutes for Health Research.
Adapted from press release by the University of Toronto.