A Tale of Two Toxins: the mechanisms of cell death in Clostridium difficile infections

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.

C. difficile causes disease by releasing two large toxins, TcdA and TcdB. Understanding the role these toxins play in colonic disease is important for treatment strategies. However, most published research data only report the effects of the toxins independently. A 2016 study demonstrated a method of comparing the toxins side-by-side using the same time points and cell assays to investigate the role each toxin plays in the cell death that leads to disease of the colon. Continue reading

Moving Towards Zero Hunger, One Genome at a Time

Farmer and a pile of cassava bulbs.

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

Overcoming Challenges When Scaling Antibody Production

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.

Robot performing autosamplingWorking 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.

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Of Mice and Microbes: The Science Behind Food Analysis

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

Evaluating the Costs of Endotoxin Testing

http://www.eniscuola.net/en/mediateca/king-crab/

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!

The Bacteria that are Good for Us

Chains of StreptococciSalmonella. Streptococcus. Shigella. The most well-known bacteria are those that cause disease. Our relationship with them is one of combat. With good reason, we look for ways to avoid encountering them and to eliminate them when we do meet.

But not all bacteria are bad for us. Of course we have known for years that we are colonized by harmless bacteria, but recently, studies on the human microbiome have revealed many surprising things about these bacterial tenants. Studies are showing that the teeming multitudes of organisms living in and on the human body are not just harmless bystanders, but complex, interrelated communities that can have profound effects on our health.

Three studies published last week in Science add more to the growing body of microbiome surprises, showing that certain gut bacteria are not only good for us, but may even be required for the effectiveness of some anti-cancer immunotherapies.

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Elephant Endotheliotropic Herpesvirus—A Tiny Virus Threatens the World’s Elephants

My favorite ice-breaker of all time is: “List one fact about you that no one would guess”. It is my favorite because I have an awesome answer (if I do say so myself). My go-to answer is: I spent a summer working with elephants.

It was the summer before I graduated from college, and it was really only one elephant, a five-year-old African elephant named Connie. Connie was intelligent, curious and mischievous—her favorite game with me was trying to untie my shoelaces (hint: double knotting is important). Working with her was one of the most amazing experiences of my life and left me with an abiding love for these creatures.

Elephant Endotheliotropic Herpesviruses threaten captive breeding programs. Copyright.

Understandably, I was excited last year when one of my fellow bloggers wrote about Promega helping support the work of Virginia Riddle Pearson, who was working to identify and track strains of elephant endotheliotropic herpesvirus (EEHV) in African Elephant populations. EEHV is associated with the lethal elephant hemorrhagic disease (EHD) (1). This disease is a serious threat to the captive breeding programs of these endangered creatures. Between 1962 and 2007, it accounted for 58% of the deaths of North American captive-born Asian elephants between 4 months and 15 years of age (1). These deaths include the first Asian elephant calves born at the National, Oakland and Bronx Zoos. EHD also claimed the first live-born Asian elephant calves conceived by artificial insemination in both North America and Europe. Continue reading

CRISPR: Gene Editing and Movie Madness

There are new developments in genetics coming to light every day, each with the potential to dramatically change life as we know it. The increasingly controversial gene editing system, dubbed CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), is at the root of it all. Harnessed for use in genome editing in 20131, CRISPR has given hope to researchers looking to solve various biological problems. It’s with this technology that researchers anticipate eventually having the means to genetically modify humans and rid society of genetic disorders, such as hemophilia. While this is not yet possible, the building blocks are steadily being developed. Most recently, two groundbreaking studies concerning CRISPR have been released to the public. Continue reading

Predicting the Future with Dirty Diapers

Microbiome research is booming right now, with more and more evidence that our personal health and environment are shaped and influenced by the microbes we harbor and encounter. One area of study I find particularly interesting is how the microbiome we acquire at birth affects our long-term health.

A flood of new findings have emerged related to infant microbiome research, leaving parents like me scratching their heads about whether the secrets to our children’s future health may exist in the seemingly endless stream of dirty diapers we change.

The human microbiome evolves and develops in utero and then during and after delivery is colonized by bacteria encountered during exposure to the external environment. The initial composition of microbes an infant is populated with influences their lifelong microbiome signature and can be influenced by many factors along the way, including the microbiome community of the mother, use of antibiotics or other antibacterial substances, breastfeeding, C-section birth. These variables have been correlated with disruption of the infant microbiome and associated with differences in cognitive development and the development of disease, such as asthma and allergies.

In general, these correlations are discovered by taking a fecal sample from an infant and analyzing the DNA sequences of the bacteria present. The microbiome composition of the individual is then compared against different individual characteristics (such as presence or absence of a disease) at the time of the sample and/or at later points in time. Finally researchers look for statistically significant patterns among individuals with similar characteristics or microbiome communities. This type of study can reveal associations between the microbiome and individual traits, but further experiments are needed to show causation. 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