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

Pollinator-Plant Interactions, Neanderthal Teeth, Desiccated Tardigrades and Blood Typing: Science News This Week

Keeping up with the pace of scientific discoveries being published each week can be difficult. Here I share a few scientific publications that piqued my interest over the past week:

Pollinators influence evolution of plant traits

Brassica rapa cv. By I, KENPEI [GFDL (http://www.gnu.org/copyleft/fdl.html), CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/) or CC BY-SA 2.5-2.0-1.0 (http://creativecommons.org/licenses/by-sa/2.5-2.0-1.0)], via Wikimedia Commons

Brassica rapa cv. By I, KENPEI [GFDL (http://www.gnu.org/copyleft/fdl.html), CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/) or CC BY-SA 2.5-2.0-1.0 (http://creativecommons.org/licenses/by-sa/2.5-2.0-1.0)], via Wikimedia Commons

To explore the plant-pollinator relationship, researchers studied field mustard, a relative of oilseed rape, under the influence of three pollination conditions: by hand, by bumblebee and by hoverfly. After nine generations, the plants were visually changed. The ones pollinated by bumblebees were taller than the original plant; the ones pollinated by hoverflies, shorter. In addition, the bumblebee-pollinated field mustard developed more fragrant floral compounds and more UV-reflecting petals while the hoverfly-pollinated plants became more self-pollinated. While this experimental was done in isolation from other plants, the research suggests a pollinator can influence the traits evolved by a plant.

Read the Nature Communications research article.

Calculus from Neanderthals reveal diet and probable self-medication

The calcified plaque on teeth of five Neanderthal skulls was scraped, PCR amplified and sequenced to examine what could be learned of diet, behavior and disease. One specimen was eliminated because the DNA did not amplify, one due to environmental contamination, leaving two specimens from Spain and one from Belgium that were used for analysis. The Belgian individual had rhinoceros, sheep and mushrooms caught in its teeth while the Spanish Neanderthals consumed mushrooms, pine nuts, forest moss, and poplar as well as plant fungus. The last two items were of interest because these sequences were found in the Neanderthal suffering from a dental abscess. Poplar contains the active ingredient in aspirin and the fungus was Penicillium from which the first antibiotic was derived. Researchers also compared the bacterial sequences of oral microbes across hominid species and sequenced a draft genome of the 48,000-year-old oral bacterium Methanobrevibacter oralis subsp. neandertalensis.

Read the research article in Nature.

The desiccation tolerance of water bears explained

Scanning electron micrograph of an adult tardigrade (water bear). By Goldstein lab - tardigrades (originally posted to Flickr as water bear) [CC BY-SA 2.0 (http://creativecommons.org/licenses/by-sa/2.0)], via Wikimedia Commons

Adult tardigrade (water bear). By Goldstein lab – tardigrades (originally posted to Flickr as water bear) [CC BY-SA 2.0 (http://creativecommons.org/licenses/by-sa/2.0)], via Wikimedia Commons

The microscopic tardigrades are a creature that inspire microbiologists and others with their cuteness (hence the nickname water bears) and their resilience under dry conditions. However, little was known why they can survive desiccation. New research reveals that unlike other organisms that use sugar to resist drying, tardigrades use disordered proteins to protect itself. These proteins lack stable 3D structures and form glass-like protection under desiccation. Not surprisingly, these proteins are called tardigrade-specific intrinsically disordered proteins or TDPs. By transferring TDPs into yeast, researchers were able to increase yeast tolerance to drying as well as enhance survival.

Read a summary of the research in The Scientist (contains link to research article).

Blood type determined in 30 seconds using a paper-based assay

Matching blood type usually involves centrifuging blood samples to test both red blood cells and plasma, and takes about 30 minutes. However, a rapid test would be useful in emergencies while an alternate test for those without the funds for lab facilities would be beneficial. What about paper infused with dye that could show blood type in seconds, no centrifugation needed? In fact, researchers have developed a paper-based assay that uses microliter volumes of whole blood to determine blood type with a visual indicator. Using immobilized antibodies and a green dye, the blood will clump in the presence of an antibody that is recognized, turning the paper blue to show it has the marker for A (left side of chip) or B (right side of chip). Type AB will have both markers while type O has neither, turning the paper brown on both sides of the chip. Rare blood types and five Rhesus markers can also be analyzed using this paper-based chip assay, starting with a small sample of whole blood.

Read a summary of the research and watch a video of the paper assay chip in Science (contains link to research article).

Probing RGS:Gα Protein Interactions with NanoBiT Assays

gpcr_in_membrane_on_white2When I was a post-doc at UT Southwestern, I was fortunate to interact with two Nobel prize winners, Johann Deisenhofer and Fred Gilman.  Johann once helped me move a -80°C freezer into his lab when we lost power in my building. I once replaced my boss at small faculty mixer with a guest speaker and had a drink with Fred Gilman and several other faculty members from around the university. Among the faculty, one professor had a cell phone on his belt, an odd sight in 1995. Fred Gilman asked him what it was and why he had it. It was so his lab could notify him of good results anytime of the day. Fred laughed and told him to get rid of it – if it’s good data, it will survive until morning.

I was reminded of this story when I read a recent paper by Bodle, C.R. et al (1) about the development of a NanoBiT® Complementation Assay (2) to measure interactions of Regulators of G Protein Signaling (RGS) with Gα proteins in cells. (Fred Gilman was the first to isolate a G protein and that led to him being a co-recipient of the Nobel Prize in 1994). The authors created over a dozen NanoBiT Gα:RGS domain pairs and found they could classify different RGS proteins by the speed of the interaction in a cellular context. The interactions were readily reversible with known inhibitors and were suitable for high-throughput screening due to Z’ factors exceeding 0.5. The study paves the way for future work to identify broad spectrum RGS domain:Gα inhibitors and even RGS domain-specific inhibitors. This is the second paper applying NanoBiT Technology to GPCR studies (3).

A Little Background…
A primary function of GPCRs is transmission of extracellular signals across the plasma membrane via coupling with intracellular heterotrimeric G proteins. Upon receptor stimulation, the Gα subunit dissociates from the βγ subunit, initiating the cascade of downstream second messenger pathways that alter transcription (4). The Gα subunits are actually slow GTPases that propagate signals when GTP is bound but shutdown and reassociate with the βγ subunit when GTP is cleaved to GDP. This activation process is known as the GTPase cycle. G proteins are extremely slow GTPases. Continue reading