Cytotoxicity Testing of 9,667 Tox21 Compounds using Two Real-Time Assays by Promega

A recent paper in PLOS One demonstrated real-time cytotoxicity profiling of approximately 10,000 chemical compounds in the Tox21 compound library, using two Promega assays, RealTime-Glo™ MT Cell Viability Assay and CellTox™ Green Cytotoxicity Assay. This is exciting to me, a science writer working at Promega; exciting because it’s tricky figuring out how to write about the utility of our products without sounding like an evangelist.

I don’t know about you, but I tend to shut out evangelists and their messages.

Instead of me telling you about real-time viability and cytotoxicity assays from Promega, here is an example of their use in Tox21 chemical compound library research.

What is the Tox21 compound library?
As described in the article by Hsieh, J-H. et al. (2017) in PLOS One:
“The Toxicology in the 21st Century (Tox21) program is a federal collaboration among the National Institutes of Health, including the National Toxicology Program (NTP) at the National Institute of Environmental Health Sciences and the National Center for Advancing Translational Sciences, the Environmental Protection Agency, and the Food and Drug Administration. Tox21 researchers utilize a screening method called high throughput screening (HTS) that uses automated methods to quickly and efficiently test chemicals for activity across a battery of assays that target cellular processes. These assays are useful for rapidly evaluating large numbers of chemicals to provide insight on potential human health effects.” Continue reading

Searching for Secrets in Single Cells

There has been a lot of effort recently to perform whole genome sequencing, for humans and other species. The results yield new frontiers of data analysis that offer a lot of promise for groundbreaking scientific discoveries.

One objective of human genome sequencing has been to identify sources of disease and new therapeutic targets. This movement has opened the door to create personalized medicine for cancer, whereby the genetic makeup of an individual’s tumors can be used to determine the most effective drug intervention to administer.

Interest in studying the characteristics unique to individual cells seems obvious when considering the function of healthy cells versus tumor cells, or brain cells compared to heart cells. What has surprised scientists is the realization that two cells in the same tissue can be more different from each other, genetically, than from a cell in another organ.

For example, a small number of brain cells with a specific mutation can lead to some forms of epilepsy while healthy people may also carry cells with these mutations, but too few to cause disease. The lineage of a cell, where it came from and what events shaped its development, ultimately determines what diseases can exist.

Continue reading

All Aglow in the Ocean Deep

 

Fascinating bioluminescent creature floating on dark waters of the ocean. Polychaete tomopteris.

Today’s blog comes to you from the Promega North America Branch Office.

In nature, the ability to “glow” is actually quite common. Bioluminescence, the chemical reaction involving the molecule luciferin, is a useful adaptation for many lifeforms. Fireflies, mushrooms and creatures of the ocean deep use their internal lightshows to cope with a variety of situations. Used for hunting, communicating, ridding cells of oxygen, and simply surviving in the darkness of the ocean depths, bioluminescence is one of nature’s more flashy, and advantageous traits.

In new research published in April in the journal Scientific Reports, MBARI researchers Séverine Martini and Steve Haddock found that three-quarters of all sea animals make their own light.  The study reviewed 17 years of video from Monterey Bay, Calif in oceans that descended to 2.5 miles, to determine the commonality of bioluminescence in the deep waters.

Martini and Haddock’s observations concluded that 76 percent off all observed animals produced some light, including 97 to 99.7 cnidarians (jellyfish), half of fish, and most polychaetes (worms), cephalopods (squid), and crustaceans (shrimp).

Most of us are familiar with the fabled anglerfish, the menacing deep-sea creature known for attracting ignorant prey with a glowing lure attached to their head. As you descend below 200 meters, where light no longer penetrates, you will be surprised at the unexpected color display of the oceans’ sea life. Bioluminescence is not simply an exotic phenomenon, but an important ecological trait that the oceans’ sea creatures have wholeheartedly adopted to cope with complete darkness. Continue reading

Revealing Time of Death: The Microbiome Edition

Forensic analysts have long sought precision when determining time of death. While on crime scene investigation television shows, the presence of insects always seems to reveal when a person died, there are many elements to account for, and the probable date may still not be accurate. Insects arrive days after death if at all (e.g., if the body is found indoors or after burial), and the stage of insect activity is influenced by temperature, weather conditions, seasonal variation, geographic location and other factors. All this makes it difficult to estimate the postmortem interval (PMI) of a body discovered an unknown time after death. One way to make estimating PMI less subjective would be to have calibrated molecular markers that are easy to sample and are not altered by environmental variabilities.

Bacterial communities called microbiomes have been frequently in the news. The influence of these microbes encompass living creatures and the environment. Not surprisingly, research has focused on the influence of microbiomes on humans. For example, changes in gut microbiome seem to affect human health. Intriguingly, microbiomes may also be a key to determining time of death. The National Institute of Justice (NIJ) has funded several projects focused on the forensic applications of microbiomes. One focus involves the necrobiome, the community of organisms found on or around decomposing remains. These microbes could be used as an indicator of PMI when investigating human remains. Recent research published in PLOS ONE examined the bacterial communities found in human ears and noses after death and how they changed over time. The researchers were interested in developing an algorithm using the data they collected to estimate of time of death. Continue reading

In Healthy Eating Less is More: The Science Behind Intermittent Fasting

Mix a love of eating with a desire to live a long, healthy life what do you get? Probably the average 21st century person looking for a way to continue enjoying food despite insufficient exercise and/or an age-related decline in caloric needs.

Enter intermittent fasting, a topic that has found it’s way into most news sources, from National Institutes of Health (NIH) and Proceedings of the National Academy of Sciences publications to WebMD and even the popular press. For instance, National Public Radio’s “The Salt” writers have tried and written about their experiences with dietary restriction.

While fasting has enjoyed fad-like popularity the past several years, it is not new. Fasting, whether purposely not eating or eating a restricted diet, has been practiced for 1,000s of years. What is new is research studies from which we are learning the physiologic effects of fasting and other forms of decreased nutrient intake.

You may have heard the claims that fasting makes people smarter, more focused and thinner? Researchers today are using cell and animal models, and even human subjects, to measure biochemical responses at the cellular level to restricted nutrient intake and meal timing, in part to prove/disprove such claims (1,2). Continue reading

Making BRET the Bright Choice for In vivo Imaging: Use of NanoLuc® Luciferase with Fluorescent Protein Acceptors

13305818-cr-da-nanoluc-application_ligundLive animal in vivo imaging is a common and useful tool for research, but current tools could be better. Two recent papers discuss adaptations of BRET technology combining the brightness of fluorescence with the low background of a bioluminescence reaction to create enhanced in vivo imaging capabilities.

The key is to image photons at wavelengths above 600nm, as lower wavelengths are absorbed by heme-containing proteins (Chu, J., et al., 2016 ). Fluorescent protein use in vivo is limited because the proteins must be excited by an external light source, which generates autofluorescence and has limited penetration due to absorption by tissues. Bioluminescence imaging continues to be a solution, especially firefly luciferase (612nm emission at 37°C), but its use typically requires long image acquisition times. Other luciferases, like NanoLuc, Renilla, and Gaussia, etc. either do not produce enough light or the wavelengths are readily absorbed by tissues, limiting their use to near- surface imaging.

The two papers discussed here illustrate how researchers have combined NanoLuc® luciferase with a fluorescent protein to harness bioluminescent resonance energy transfer (BRET) for brighter in vivo imaging reporters. Continue reading

Preventing Viral Infection by Blocking Cellular Receptors with a Tethered Antibody

Cross section of mature HIV. Copyright David S. Goodsell, The Scripps Research Institute.

Cross section of mature HIV. Copyright David S. Goodsell, The Scripps Research Institute.

Finding a way to neutralize or block infection by HIV has long been pursued by viral researchers. Various treatments have been developed, driven by the need to find effective drugs to manage HIV in infected individuals. The ultimate goal is to develop a vaccine to prevent HIV from even taking hold in the body. With all of our knowledge about the cellular receptors HIV needs to enter the cell, there has to be a method to prevent a viral particle from binding and being internalized. Many researchers are pursuing neutralizing antibodies to the virus as one method. What about antibodies that target the cellular receptor recognized by the virus? In a recently published article in Proceedings of the National Academy of Sciences, antibodies to cellular receptors for rhinovirus and HIV were tethered to the plasma membrane and tested for the ability to prevent infection. Continue reading

Making a Case for Basic Research Funding

The value of public funding for “basic” versus “applied” research has long been questioned. To address this debate, the authors of a recent report in Science performed a large-scale evaluation of the value of public investment in biomedical research. After analyzing the relationship between the U.S. National Institutes of Health (NIH) grants and private patents, they found that distinguishing research as basic or applied is not useful in determining the productivity of grant funding.

Genetic research at the laboratoryThe $30 billion annual budget of the NIH makes it the largest source of life science funding in the world and provides a third of all US biomedical research and development. Although there has long been a strong argument for public investment in scientific research, attacks on the tangible benefits of this research persist. In particular, some opponents argue that “basic” research is too far removed from practical applications to be worthy of investment.

To quantify the effects of NIH funding for basic versus applied research, the authors looked at data from 365,380 grants awarded between 1980–2007 and compared their direct and indirect influence on patent filed. In particular, they decided to use patent-article citations as a measure of the influence of publicly funded science on commercial developments.

The researchers determined two ways in which research funded by the NIH could impact patenting; patents could be filed by the NIH-funded scientists or by private entities that cited research funded by NIH grants. This study found that roughly 10% of NIH grants were directly responsible for a patent while nearly a third of NIH grants had an indirect influence on patents. This indirect influence was attributed to articles associated with grant research that were later cited by a patent.

Delving deeper into the data, the authors found a similar pattern when looking at drugs brought to market that were associated with NIH grants; less than 1% of grants were directly linked to a patent associated with a drug, while 5% resulted in a publication cited by a patent for a drug. Despite public policies like the Bayh-Dole Act, that encourage academic researchers to file their own patents, the traditional route of applying public research to private patents continues to predominate.

For those that question the value of basic research and aim to steer public policy toward supporting applied research, this report makes a strong case against this way of thinking. The findings also suggest that using direct generation of patents as a metric for the return on investment of publicly funded biomedical research is not very useful since most of the effects of NIH research appear to be indirect.

In fact, the authors posit that basic research is just as productive as applied research in terms of patenting since the amount of grant research cited by private patents is much greater than the number of grants directly associated with patents. Perhaps it is time policy makers consider studies like this and forgo disseminating grant funds based on whether research is basic or applied.

The Randomness of Cancer

A major scientific study grabbed headlines recently, and the implications of its findings may affect many of us, if not all of us. In a paper published in Science by Cristian Tomasetti, Lu Li and Bert Vogelstein of Johns Hopkins University, the authors report that nearly two-thirds of known cancer causing mutations can be attributed to random mistakes that occur during DNA replication. In other words, the vast majority of these mutations occur in a spontaneous, uncontrollable way— it may not matter how you live your life, or what measures you take to decrease your chance of developing cancer. As the authors and the press put it, it really just comes down to luck.

gene-mutationDisturbing? For many, yes. It’s not easy to accept that one’s luck in activities such as winning the lottery may also apply to whether or not you will be touched by cancer. That is partly why this study is gaining so much attention.

As the authors explain in their publication, until now most cancer-causing mutations had been attributed to two major sources: inherited and environmental factors. But they found that a third kind of mutation, replicative (R) mutations that arise from unavoidable errors associated with DNA replication, account for 66 percent of mutations that drive cancer. Continue reading

Making the Switch from FRET to BRET: Applications of NanoLuc® Luciferase with Fluorescent Protein Acceptors for Sensing Cellular Events

A Bioluminescent Alternative

Fluorescence resonance energy transfer (FRET) probes or sensors are commonly used to measure cellular events. The probes typically have a matched pair of fluorescent proteins joined by a ligand-binding or responsive protein domain. Changes in the responsive domain are reflected in conformational changes that either bring the two fluorescent proteins together or drive them apart. The sensors are measured by hitting the most blue-shifted fluorescent protein with its excitation wavelength (donor). The resulting emission is transferred to the most red-shifted fluorescent protein in the pair, and the result is ultimately emission from the red-shifted protein (acceptor).

As pointed out by Aper, S.J.A. et al. below, FRET sensors face challenges of photobleaching, autofluorescence, and, in the case of exciting cyan-excitable donors, phototoxicity. Another challenge to using FRET sensors comes when employing optogenetic regulators to initiate the event you wish to monitor. Optogenetic regulators respond to specific wavelengths and initiate signaling. The challenge comes when the FRET donor excitation overlaps with the optogenetic initiation wavelengths. Researchers have sought to alleviate many of these challenges by exchanging the fluorescent donor for a bioluminescent donor, making bioluminescence resonance energy transfer (BRET) probes. In the three papers described below, the authors chose NanoLuc® Luciferase as the BRET donor due to its extremely bright signal. Continue reading