In the Literature

Blogs about published peer-reviewed scientific studies.

H7N9 Influenza Virus: A Perfect Pathogen?

Posted on by Kari Kenefick
Artist’s rendition of a virus particle.

It’s late October and here in Wisconsin, like many of you, we are experiencing a change of seasons, with the associated drop in temperatures, changes in leaf color and later this week, Halloween.

Another thing that comes with fall is the start of cold and flu season. By “flu”, I mean influenza, caused by avian influenza viruses of the H-N type. Recent research results by teams at UWI-Madison and in Japan, makes the coming influenza season potentially more scary than usual.

In a recent Cell Host & Microbe paper, M. Imai et al. study a seemingly more virulent version of H7N9 avian influenza virus that is startling in its ability to spread from infected to healthy animal models. Based on a current epidemic of H7N9, human-to-human transmission with this strain is increasing.

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Evaluating DNA Quantity and Quality in FFPE Tumor Samples After Prolonged Storage Using the ProNex® DNA QC Assay

Posted on by Johanna Lee

When tumors are surgically removed from cancer patients, the tumor samples are often stored as formalin-fixed and paraffin-embedded (FFPE) tissue blocks. In many cases, tumor samples need to be analyzed several years after diagnosis in order to develop target treatments. But what happens to the DNA after years of storage in FFPE blocks? How well is the DNA preserved?

Scientists in France tried to answer this question in a recent study published in Virchows Arch. The authors extracted DNA from 46 FFPE tumor samples of lung, colon and the urothelial tract, all stored between 4–6 years at room temperature. They then compared the quantity and quality of the DNA to DNA that had been extracted before storage. Using common fluorimetry and qPCR methods, the authors found that the total amount of DNA extracted decreased by half. In addition, the percentage of amplifiable DNA decreased from 56% to only 15% after prolonged storage. Continue reading “Evaluating DNA Quantity and Quality in FFPE Tumor Samples After Prolonged Storage Using the ProNex® DNA QC Assay”

Tick, Tock! The Molecular Basis of Biological Clocks

Posted on by Jordan Villanueva

A long time ago, before the rise of humans, before the first single celled organisms, before the planet even accumulated atmospheric oxygen, Earth was already turning, creating a 24-hour day-night cycle. It’s no surprise, then, that most living things reflect this cycle in their behavior. Certain plants close their leaves at night, others bloom exclusively at certain times of day. Roosters cock-a-doodle-doo every morning, and I’m drowsy by 9:00 pm every night. These behaviors roughly align with the daylight cycles, but internally they are governed by a set of highly conserved molecular circadian rhythms.

Jeffrey Hall, Michael Rosbash and Michael Young were awarded the 2017 Nobel Prize in Physiology/Medicine for their discoveries relating to molecular circadian rhythms. The official statement from the Nobel Committee reads, “…this year’s Nobel laureates isolated a gene that controls the normal daily biological rhythm. They showed that this gene encodes a protein that accumulates in the cell during the night, and is then degraded during the day. [They exposed] the mechanism governing the self-sustaining clockwork inside the cell.” What, then, does this self-sustaining clockwork look like? And how does it affect our daily lives (1)?

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Analysis of a biosimilar mAb using Mass Spectrometry

Posted on by Gary Kobs

Several pharmaceutical companies have biosimilar versions of therapeutic mAbs in development. Biosimilars can promise significant cost savings for patients, but the unavoidable differences
between the original and thencopycat biologic raise questions regarding product interchangeability. Both innovator mAbs and biosimilars are heterogeneous populations of variants characterized by differences in glycosylation,oxidation, deamidation, glycation, and aggregation state. Their heterogeneity could potentially affect target protein binding through the F´ab domain, receptor binding through the Fc domain, and protein aggregation.

As more biosimilar mAbs gain regulatory approval, having clear framework for a rapid characterization of innovator and biosimilar products to identify clinically relevant differences is important. A recent reference (1) applied a comprehensive mass spectrometry (MS)-based strategy using bottom-up, middle-down, and intact strategies. These data were then integrated with ion mobility mass spectrometry (IM-MS) and collision-induced unfolding (CIU) analyses, as well as data from select biophysical techniques and receptor binding assays to comprehensively evaluate biosimilarity between Remicade and Remsima.

The authors observed that the levels of oxidation, deamidation, and mutation of individual amino acids were remarkably similar. they found different levels of C-terminal truncation, soluble protein aggregates, and glycation that all likely have a limited clinical impact.  Importantly, they identified more than 25 glycoforms for each product and observed glycoform population differences.

Overall the use of mass spectrometry-based analysis provides rapid and robust analytical information vital for biosimilar development. They demonstrated the utility of our multiple-attribute monitoring workflow using the model mAbs Remicade and Remsima and have provided a template for analysis of future mAb biosimilars.

1. Pisupati, K. et. al. (2017) A Multidimensional Analytical Comparison of Remicade and the Biosimilar Remsima. Anal. Chem 89, 38–46.

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

Posted on by Kari Kenefick

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.”

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Searching for Secrets in Single Cells

Posted on by Darcia Schweitzer

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.

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All Aglow in the Ocean Deep

Posted on by Promega
Fascinating bioluminescent organisms 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.

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Revealing Time of Death: The Microbiome Edition

Posted on by Sara Klink

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.

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

Posted on by Kari Kenefick

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 its 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 over 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).

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

Posted on by Kyle Hooper
13305818-cr-da-nanoluc-application_ligund

Live 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.

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