In recent years, scientists have been able to refine their molecular tools to resurrect ancient DNA from human graves and determine that yes, Yersinia pestis was the causative agent for the Black Death in the 14th century and the Plague of Justinian in the 6th century. As more and more human graves have been uncovered, their DNA has revealed many secrets that scientists even ten years ago were unable to discover. With the ability to sequence entire genomes of bacteria that died with their hosts hundreds and even thousands of years ago, researchers are exploring the rise and possible spread of Y. pestis. Each new member sequence adds to the Y. pestis family tree, pinpointing the origin of this bacteria as it diverged from its ancestor Y. pseudotuberculosis. Peering into the past, scientists have been able to track down a strain of Y. pestis from individuals in a Swedish passage grave that is basal to known strains and that the authors of a Cell article suggest has interesting implications.
This pathogenic journey into history started by analyzing ancient DNA data sets from the teeth of individuals present in a communal passage grave in Gökhem parish, located in western Sweden, for any disease-causing microbial sequences that might be present. Y. pestis was flagged in one 20-year-old female dated 4,867–5,040 years ago. The bacterial sequences from this individual, named Gok2, were more closely aligned with Y. pestis than the Y. pseudotuberculosis reference genome. Continue reading “Expanding the Plague Family Tree: Yersinia pestis in the Neolithic”
Ebola virus (EBOV) and Marburg virus (MARV) are two closely-related viruses in the family Filoviridae. Filoviruses are often pathogenic, causing hemorrhagic fever disease in human hosts. The Ebola outbreak of 2014 caught the world by surprise by spreading so quickly and severely that public health organizations were unprepared. The devastating outcome was a total of over 11,000 deaths by the time the outbreak ended in 2016. Research that provides further understanding of filoviruses and their potential for transmission is important in preventing future outbreaks from occurring. But what if the outbreak comes from a virus we’ve never seen before?
All in the viral family
A recent study published in the journal Nature Microbiology provides evidence of a newly identified filovirus species. Using serum samples taken from bats, a well-known host for filoviruses, Yang et al. isolated and identified viral RNA for an unclassified viral genome sequence using next generation sequencing analysis. This new virus genome sequence was organized with the same open reading frames as other filoviruses, encoding for nucleoprotein (NP), viral protein 35 (VP35), VP40, glycoprotein (GP), VP30, VP24, and RNA-dependent RNA polymerase (L). This new genome sequence shared up to 54% of the nucleotide sequences for the filovirus species Lloviu virus (LLOV), EBOV and MARV, with MARV being the most similar. Their analysis suggested that this novel virus should be classified within the Filoviridae family tree as a separate genus, Dianlovirus, and was named Měnglà virus (MLAV).
One hundred years ago, the world was taking its first deep breaths as it celebrated the end of World War I. The Armistice of Compiègne, was signed on November 11,1918, officially ending the four-year long conflict, which claimed the lives of more than 8 million soldiers (1). What the world didn’t yet realize was that they had been battling a far deadlier enemy in the hospitals and at home than any army the soldiers faced on the fields of war.
During the last year of the war, a deadly influenza virus rampaged around the globe leaving between 50 and 100 million dead in its wake.
The boys were coming in with colds and a headache and they were dead within two or three days. Great big handsome fellows, healthy men, just came in and died. There was no rejoicing in Lille the night of the Armistice. Sister Catherine Macfie from her post at casualty clearing station no. 11 at St André near Lille, France (2).
We have published 130 blogs here at Promega this year (not including this one). I diligently reviewed every single one and compiled a list of the best 8.5%, then asked my coworkers to vote on the top 5 out of that subset. Here are their picks:
Today NASA’s InSight lander will touch down on Mars. InSight, which launched on May 5, is NASA’s first Mars landing since the Curiosity rover in 2012. The lander will begin a two-year mission to study Mars’ deep interior, gathering data that will help scientists understand the formation of rocky planets, including Earth.
While every spacecraft that reaches Mars offers more knowledge of the Red Planet, a lot of the excitement is fueled by hopes that someday these missions will bring humans to Mars and enable us to start colonies there. While this goal seems very distant, tremendous progress is being made. Scientists around the globe are making incremental discoveries that will lead to the advances necessary to make colonization of Mars a reality.
When someone is admitted to a hospital for an illness, the hope is that medical care and treatment will help them them feel better. However, nosocomial infections—infections acquired in a health-care setting—are becoming more prevalent and are associated with an increased mortality rate worldwide. This is largely due to the misuse of antibiotics, allowing some bacteria to become resistant. Furthermore, when an antibiotic wipes out the “good” bacteria that comprise the human microbiome, it leaves a patient vulnerable to opportunistic infections that take advantage of disruptions to the gut microbiota.
One such bacteria, Clostridium difficile, is of growing concern world-wide since it is resistant to many different antibiotics. When a patient is treated with an antibiotic, C. difficile can thrive in the intestinal tract without other bacteria populating the gut. C. difficile infection is the leading cause of antibiotic-associated diarrhea. While symptoms can be mild, aggressive infection can lead to pseudomembranous colitis—a severe inflammation of the colon which can be life-threatening.
Have you ever thought about plant viruses? Unless you’re a farmer or avid gardener, probably not. And yet, for many people the battle against agricultural viruses never ends. Plant viruses cause billions of dollars in damage every year and leave millions of people food insecure (1–2), making viruses a major barrier to meeting the United Nations’ global sustainable development goal of Zero Hunger by 2030.
At the University of Western Australia, Senior Research Fellow Dr. Laura Boykin is using genomics and supercomputing to tackle the problem of viral plant diseases. In a recent study, Dr. Boykin and her colleagues used genome sequencing to inform disease management in cassava crops. For this work, they used the MinION, a miniature, portable sequencer made by Oxford Nanopore Technologies, to fully sequence the genomes of viruses infecting cassava plants.
Cassava (Manihot esculenta) is one of the 5 most important calorie sources worldwide (3). Over 800 million people rely on cassava for food and/or income (4). Cassava is susceptible to a group of viruses called begomoviruses, which are transmitted by whiteflies. Resistant cassava varieties are available. However, these resistant plants are usually only protected against a small number of begomoviruses, so proper deployment of these plants means farmers must know both whether their plants are infected and, if so, the strain of virus that’s causing the infection. Continue reading “Moving Towards Zero Hunger, One Genome at a Time”
Tradeoffs are a constant source of challenge in any research lab. To get faster results, you will probably need to use more resources (people, money, supplies). The powerful lasers used to do live cell imaging may well kill those cells in the process. Purifying DNA often leaves you to choose between purity and yield.
Working with biologics also involves a delicate balancing act. Producing compounds in biological models rather than by chemical synthesis offers many advantages, but it is not without certain challenges. One of those tradeoffs results from scaling up; the more plasmid that is produced, the greater probability of endotoxin contamination.
In general, people like to know that their food is what the label says it is. It’s a real bummer to find out that beef lasagna you just ate was actually horsemeat. Plus, there are many religious, ethical and medical reasons to be cognizant of what you eat. Someone who’s gluten intolerant and Halal probably doesn’t want a bite of that BLT.
Labels don’t always accurately reflect what is in food. So how do we confirm that we are in fact buying crab, and not whitefish with a side of Vibrio contamination?
For the most part, it comes down to separation science. Scientists and technicians use various chromatographic methods, such as gas chromatography, liquid chromatography, and mass spectrometry, to separate the complex mixture of molecules in food into individual components. By first mapping out the molecular profile of reference samples, they can then take an unknown sample and compare its profile to what it should look like. If the two don’t match up, an analyst would assume that the unknown is not what it claims to be. Continue reading “Of Mice and Microbes: The Science Behind Food Analysis”
Recently, I had the opportunity to attend a fascinating symposium held at Promega featuring conservationist Steward Brand, where he described some of the projects developed by his foundation, Revive & Restore.
The organization’s mission is to apply emerging biotechnology techniques to endangered and extinct species with the intent to increase genetic diversity, provide disease resistance and facilitate adaptation to changing climates. Although the overall message of enhancing biodiversity through the application of new genetic technology was inspiring, the project that resonated most for me was related to the plight of horseshoe crabs.
Horseshoe crabs, often referred to as living fossils, include four extant species with origins dating back about 450 million years. Although they look like crabs, they belong to their own subphylum and are more closely related to spiders. When horseshoe crabs spawn, they leave their usual habitat on the ocean floor and migrate to shore in large numbers. As a result, they have been exploited for bait and fertilizer for decades.
Enter endotoxins, an indicator for bacterial contamination in biologicals, drugs and medical devices. U.S. Food & Drug Administration regulations dictate that finished products be tested for the presence of endotoxins. These pyrogenic compounds, found in the cell wall of Gram-negative bacteria, can cause fever and affect a wide range of biological activity, possibly leading to aseptic shock and death. The most common method for testing is the gel clot and Limulus Amebocyte Lysate (LAL) Test.
I first learned about the LAL test during graduate school, where it was presented as a ubiquitous and standard requirement for testing bacterial contamination in injectable drugs. I remember being fascinated that horseshoe crabs (Limulus sp.), contain a substance that could be used to detect endotoxins. Although the instructors mentioned the need to collect blood from horseshoe crabs in order to produce the test, the method or scale of this harvest wasn’t discussed, nor were the true costs of using this method of endotoxin testing.
The LAL test has served as a faster, more inexpensive replacement for the rabbit pyrogens test for the past 35 years. Every year during mating season horseshoe crabs move to shallow water, where they are removed in huge numbers. (To get an idea of scale for the harvest and read a much more comprehensive investigation of the issue, check out this article in The Atlantic, which features an archive photo of Delaware Bay horseshoe crab harvest from 1928—for fertilizer, not pharmaceutical testing.)
After collection, the crabs end up in a lab where up to 30% of their blood is drained from a needle stuck in tissue around their heart. The LAL is extracted from the blood and can yield a product worth up to $15,000/quart. In order to avoid recollection, the crabs are returned to the ocean far from the shore where they were collected a few days before. Although it’s estimated that only 10-30% of these crabs die as a result of the process, there are indications that the horseshoe crab population and their ecosystems are impacted in other ways.
Researchers at the University of New Hampshire and Plymouth State University used accelerometers attached to recently bled female horseshoe crabs to test the hypothesis that harvesting for LAL was affecting their ability to spawn. While the research supported previous estimates with a death rate of 18%, females were found to be less likely to mate after being bled.
During his talk, Brand shared results from a study still in review that confirm the effect of over-harvesting Limulus on the survival of long distance migratory shorebirds. These birds synchronize their migration with horseshoe crab spawning, which provides a needed feast of eggs before the homestretch of their journey. Along with other ecosystem threats from climate change, the accelerated decline in the horseshoe crab population and dependency of migratory birds will likely to lead to a devastating ecological domino effect.
Fortunately, a synthetic alternative to LAL, recombinant factor C (rFC), has been available for nearly 20 years. Alas, there has been no significant shift by pharmaceutical companies away from the test based on horseshoe crab blood. rFC was patented and licensed to one company, Lonza, which Brand posited as one reason for the reluctance of drug companies to adopt its use.
Obviously, relying on one source for an essential testing reagent with no competition to temper cost is quite unattractive. But that argument has less bearing now that the patent is scheduled to expire in a few months, opening the door for additional manufacturers and creating an economic incentive for switching to the synthetic test.
Another reason may be that implementing a new test would require additional resources to validate the synthetic test for products that are already being tested with the LAL. Since the LAL has been specified in FDA guidance documents on endotoxin testing for decades, quality standards for existing products are based on the LAL, limiting momentum to change.
If both tests offered the same benefits, these arguments would make sense; however, research by one of the discoverers of rFC, Jeak Ling Ding of the National University of Singapore, shows that in many respects rFC is more efficacious than LAL. Since the raw material for the LAL test depends on an organism, there is seasonal variation in the components of the processed blood that must be taken into account. The reaction of the LAL also depends on a cascade of multiple compounds that can be affected by temperature, pH and proteins—leaving the test vulnerable to false positive results.
Although Eli Lilly is the only pharmaceutical company to date to use rFC in place of LAL, It seems the tide may be turning. According to Brand, others are interested in making the transition. It seems foolish not to, given the source for LAL shows signs of dwindling due to overexploitation. Perhaps pharmaceutical companies are beginning to see the value of a “slower/better” philosophy (the cornerstone of the Long Now Foundation, another brainchild of Brand’s), rather than “faster/cheaper.” I have certainly gained a new perspective on endotoxin testing and a deep appreciation for the work of Brand and his foundation.
Does your organization use the LAL test? What is preventing you from switching to the synthetic alternative? Let us know!