Alternatives to animal testing have long been explored when it comes to studying the safety of various chemical compounds for use in food, medicine and cosmetics. With the advent of three-dimensional (3D) cell culture to create organoids, more relevant human organoid models are being explored as one way to provide safe and effective compound testing while minimizing the need for testing in animals. The international project Physiologically Anchored Tools for Realistic nanOmateriaL hazard aSsessment (PATROLS) led by the Swansea University Medical School aims to establish a battery of innovative, next-generation safety testing tools that can more accurately predict the effects of engineered nanomaterial (ENM) exposure in humans and the environment.
One project being researched by Samantha Llewellyn, a research assistant at Swansea University, is developing predictive physiologically relevant 3D liver models for ENM safety assessment. By having a model to evaluate realistic ENM exposures, a researcher can study liver function, hepatic metabolism and microtissue cell viability after acute (24 hours) or prolonged (several days) exposure. A microtissue model for assessing ENM hepatotoxicity needs to mimic primary hepatocytes and be amenable to assays used to test cell viability and metabolism.
The right tools for testing this 3D liver model include the bioluminescent-based CellTiter-Glo® 3D Viability and P450-Glo® Assays. When creating organoids, having reagents that can penetrate to the center of the dense and complex 3D liver spheroids is important so that the cell viability readout encompasses the entire microtissue. The CellTiter-Glo® 3D Viability Assay accomplishes this task, providing accurate assessment of 3D tissue cell health. Measuring cytochrome P450 (CYP450) activity is necessary for studying liver function. The P450-Glo® Assays have the flexibility to assess CYP450 activity while preserving the liver spheroids; thus, researchers can gather more data from a single experiment.
The importance of Samantha Llewellyn’s research as part of PATROLs is establishing a 3D liver model that could evaluate realistic ENM exposures and reduce the need for animal testing. Bioluminescent assays for assessing cell health and liver enzyme function are necessary to reach this goal.
Science is the practice of figuring out how things work and then using that knowledge to further our understanding or to create tools that can solve problems facing the world. Bioluminescent tools and assays are examples of science doing all these things. Bioluminescence is the light-yielding (luminescence) chemical reaction that is used by many lifeforms. When fireflies flicker in the twilight, they are using bioluminescence to flash on and off. Chemically, bioluminescence happens when an enzyme called luciferase acts on a light-emitting compound, luciferin, in the presence of adenosine triphosphate (ATP), magnesium and oxygen.
For scientists, bioluminescence can serve as a tool to help them understand many cellular functions. Since few animal or plant cells produce their own light, there is little to no background signal (light) to be concerned about. This lack of background means that all light coming from the sample can be measured. In fact, bioluminescence is often a preferred tool for scientists because it does not require an external light source or special filters, which are required for fluorescence-based technologies.
Promega scientists have developed bioluminescent tools and assays to support leading edge scientific research for decades, beginning in 1990 with the Luciferase biosensor technology based on firefly luciferase. Luciferase is a wonderful tool for studying how enzymes work because its output (light) is so easy to measure: samples are placed into a special instrument called a luminometer, and the amount of light being produced (Relative Light Units) is recorded. Bioluminescence technology can be configured to measure a variety of cellular biology, ranging from cell health to enzyme activity down to the specific event of turning a gene on or off. The advent of new techniques for genetic manipulation, along with an enhanced understanding of bioluminescence and the discovery and engineering of better luciferases, enables science to use bioluminescence in even more unique ways.
Since its invention in 1983, the polymerase chain reaction (PCR) has become a fundamental technology in life science laboratories across the world. Much of the technological innovation is driven by quantitative PCR and digital PCR (1); however, endpoint PCR remains a workhorse technology for applications such as gene cloning, mutagenesis and detection of microbial pathogens. Variations on the basic endpoint PCR method—for example, the use of multiplexed, fluorescently labeled primers followed by capillary electrophoresis to analyze the amplified DNA fragments—are popular in forensic DNA analysis and cell line authentication.
The COVID-19 pandemic has created an urgent need for PCR-based diagnostic testing for SARS-CoV-2. Most of these diagnostic tests use real-time, reverse-transcription quantitative PCR (RT-qPCR). However, RT-qPCR can be challenging for routine use in developing countries and in laboratories with limited access to real-time PCR thermal cyclers. A recent study described an endpoint PCR method for SARS-CoV-2 detection to address these limitations (2).
Inflammation, a process that was meant to defend our body from infection, has been found to contribute to a wide range of diseases, such as chronic inflammation, neurodegenerative disorders—and more recently, COVID-19. The development of new tools and methods to measure inflammation is crucial to help researchers understand these diseases.
Cytokines—small signaling molecules that regulate inflammation and immunity—have recently become the focus of inflammation research due to their role in causing severe COVID-19 symptoms. In these severe cases, the patient’s immune system responds to the infection with uncontrolled cytokine release and immune cell activation, called the “cytokine storm”. Although the cytokine storm can be treated using established drugs, more research is needed to understand what causes this severe immune response and why only some patients develop it.
Almost 90% of the human genome is transcribed into RNA, but only 3% is ultimately translated into a protein. Some non-translated RNA is thought to be useless, while some play a significant yet often mysterious role in cancer and other diseases. Despite its abundance and biological significance, RNA is rarely the target of therapeutics.
“We say it’s undruggable, but I would say that ‘not-yet-drugged’ is a better way to put it,” says Amanda Garner, Associate Professor of Medicinal Chemistry at the University of Michigan. “We know that RNA biology is important, but we don’t yet know how to target it.”
Amanda’s lab develops systems to study RNA biology. She employs a variety of approaches to analyze the functions of different RNAs and study their interactions with proteins. Her lab recently published a paper describing a novel method for studying RNA-protein interactions (RPI) in live cells. Amanda says that with the right tools, RPI could become a critical target for drug discovery.
“It’s amazing that current drugs ever work, because they’re all based on really old approaches,” Amanda says. “This isn’t going to be like developing a small molecule kinase inhibitor. It’s a whole new world.”
As a student, I wasted so much time wondering which assay would work best or muddling through problems on my own. I wish I had known I could reach out to get help from another knowledgeable scientist: A Technical Services Scientist!
On May 21st, 2021 we celebrate National Endangered Species Day. This day helps raise awareness and increase knowledge of endangered species and wildlife, in hopes to save them. We have been lucky enough to collaborate with organizations and partners to help save species that were on the brink of extinction. Take a look at some species that are hoping for a second chance to survive and thrive.
Kit Elizabeth Ann the Black-Footed Ferret
In February 2018, resurrection efforts began for the then endangered black-footed ferret. With the help of the U.S. Fish and Wildlife Service, Revive and Restore, partners ViaGen Pets & Equine, San Diego Zoo Global, and the Association of Zoos and Aquariums, the successful cloning of a black-footed ferret was announced in February 2021. “Elizabeth Ann” was cloned from Willa, a female ferret that died in 1988, using somatic cell nuclear transfer (SCNT). Elizabeth Ann’s genetic variants reveal a lot of much-needed hope for the genetic diversity of wild ferrets. Check out the full story on Elizabeth Ann’s journey here!
A tiny worm called Onchocerca lupi can make life uncomfortable for both humans and their best friends. This thread-like nematode is found in the eyes or under the skin of infected animals. Historically, diagnosis required skin biopsy or surgical removal of ocular tissue, but a recent study demonstrates a new non-invasive diagnostic tool for infection by Onchocerca lupi in dogs.
Wastewater surveillance of SARS-CoV-2 is an increasingly common method for monitoring the spread of COVID-19 within a community. As researchers and public health officials around the world are working together to set up wastewater surveillance systems, there is an urgent need to establish standard SARS-CoV-2 detection methods.
A key leader in this new field is Dr. Gloria Sanchez. She is a tenured scientist at the Institute of Agrochemistry and Food Technology, a center within the Spanish National Research Council. Before the COVID-19 pandemic, her team focused on detecting human enteric viruses in food and water. But soon, detecting SARS-CoV-2 in wastewater became their main focus.
This blog was written by guest author, Amy Landreman, PhD.
Drug repurposing, identifying new uses for approved or investigational drugs, is an attractive strategy when looking for new disease treatments. Because the compounds have already gone through some level of pre-clinical optimization and safety testing, this approach can reduce risk, reduce cost, and speed up the timeline for further drug development. An additional benefit of this approach is that it can result in new biological insights or a better understanding of disease mechanisms since these compounds usually already have some level of mechanistic characterization. Indeed, there are now a number of compound collections openly available specifically for the purpose of facilitating drug repurposing efforts. For example, the ReFRAME (Repurposing, Focused Rescue, and Accelerated Medchem) library is a collection of 12,000 compounds developed by Scripps Research Center and has been screened to identify novel candidate therapeutics for Cryptosporidium infection (1). The Broad Institute also offers a drug repurposing hub that contains an annotated collection of over 7,000 compounds.
Drug repurposing libraries, although often smaller than novel compound small molecule libraries, are designed for implementation into high-throughput screening workflows in order to efficiently triage compounds for the desired result. Effective compound screens require assays that can be scaled to 384 or 1536-well microplate formats and implemented in batch or continuous processing workflows. The firefly luciferase reaction has been leveraged to create many assays that are well-suited to these types of high-throughput screening approaches. In particular, the generation of “Glow” assays that have stable luminescent signals and homogenous assay design is a good fit. The signal stability allows for multi-plate processing and because the reagent is added directly to cells in culture, pre-processing steps are eliminated allowing for automated workflows. Assay reagents such as the CellTiter-Glo® Cell Viability Assay and the ADP-Glo™ Kinase Assay are commonly used in screening efforts including those done with repurposing libraries. In addition, there are several firefly luciferase reporter assay reagents such as Steady-Glo® and Bright-Glo™ Luciferase Assays that have been optimized for high-throughput detection of firefly luciferase activity making them well-suited to repurposing screens.
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