Our skin, respiratory system and gastrointestinal tract are continually bombarded by environmental challenges from potential pathogens like SARS-CoV-2. Yet, these exposures do not often cause illness because our immune system protects us. The human immune system is complex. It has both rapid, non-specific responses to injury and disease as well as long-term, pathogen-specific responses. Understanding how the immune response works helps us understand how some pathogens get past it and how to stop that from happening. It also provides key information to help us develop safe and effective vaccines.
The immune response involves two complementary pathways: Innate Immunity and Adaptive Immunity. Innate immunity is non-specific, rapid and occurs quickly after an injury or infection. As a result of the innate immune response, cytokines (small signaling molecules) are secreted to recruit immune cells to an injury or infection site. Innate immunity does not develop “memory” of an antigen or confer long-term immunity.
The immune response involves to complementary pathways: Innate Immunity and Adaptive Immunity.
Unlike innate immunity, adaptive immunity is both antigen-dependent and antigen-specific, meaning that adaptive immune response requires the presence of a triggering antigen—something like a spike protein on the surface of a virus. The adaptive immune response is also specific to the antigen that triggers the response. The adaptive immune response takes longer to develop, but it has the capacity for memory in the form of memory B and T cells. This memory is what enables a fast, specific immune response (immunity) upon subsequent exposure to the antigen.
Loss of smell (olfaction) is a commonly reported symptom of COVID-19 infection. Recently, Bilinska, et al. set out to better understand the underlying mechanisms for loss of smell resulting from SARS-CoV-2 infection. In their research, they used in situ hybridization to investigate the expression of TMPRSS2, a SARS-CoV-2 viral entry protein in olfactory epithelium tissues of mice.
Jonathan Campbell, PhD, asked me to write that he is taller and a bit more handsome than most scientists. I will neither confirm nor deny those assertions, but I will acknowledge that Dr. Campbell has a unique way of describing his recent collaborations and research on metabolism and Type 2 diabetes.
“The rest of the world has been thinking that it’s almost like the emperor has no clothes,” he says. “But we’re the guys who came right in and said ‘Hm, that dude’s naked.’”
On March 13, only a few days before the COVID-19 pandemic caused widespread shutdowns in Wisconsin, Jon visited the Promega headquarters in Madison, Wisconsin to meet with R&D scientists and discuss opportunities for new technologies. Over the course of a few hours, Jon and his collaborator Matthew Merrins, PhD, demonstrated how their research challenges dogma and could fundamentally change our understanding of postprandial metabolism. For five decades, the paradigm of glucose control focused on a model that positioned insulin and glucagon as diametrically opposing forces to raise or lower glycemia. As Jon states, things did not always add up.
“For years, everybody has been saying ‘Glucagon is the antithesis of insulin,’ right? Insulin is a good guy. It makes glucose come down. Glucagon is a bad guy. It makes glucose go up. And these two are in this cosmic battle against each other over the control of glycemia. Well, we asked, ‘Why do the beta cells that secrete insulin have glucagon receptors?’ And as you follow the breadcrumbs, you find that these two things are actually working in cooperation. Without that cooperation, the whole thing falls apart,” Jon says.
The Incretin Effect
In addition to exploring the complex biology of glucagon, Jon’s lab studies the Incretin Effect, a mechanism by which the gut influences the secretion of insulin in the pancreas. Past research revealed that rises in blood-glucose matched closely whether glucose was administered orally or intravenously. However, the amount of insulin secreted was 3—4 times higher following oral intake. This is a result of the actions of GLP1 and GIP, the two major human incretins. GLP1 and GIP bind to G-protein coupled receptors in the beta cells of the pancreas to induce insulin secretion. Insulin then acts to promote glucose uptake, reducing glycemia. Many researchers believe that dysfunction of the incretin mechanisms contributes to the reduced insulin secretion seen in individuals with Type 2 diabetes.
“If we can understand the mechanisms of the incretin effect,” Jon says, “We may be able to understand the pathophysiology driving Type 2 diabetes. My hope is that people are going to realize that diabetes is not just a glucose disease. Maybe we have been looking at this too much from a glucose-centric viewpoint. Clearly, glucose is a big problem with diabetes, but it’s not just glucose. This is a metabolic disease, and in order to understand how to fix a metabolic disease, you need to look at all the metabolites and the way overall metabolism is dysregulated.”
Research on the incretin effect has already supported the development of two new classes of drugs for Type 2 diabetes: GLP1R agonists and DPP4 inhibitors (DPP4 is an enzyme that degrades GLP1).
“We collaborate with industry quite a bit, especially pharmaceuticals. We are helping them understand the mechanism of action by which their drugs may work, and that funding has allowed us to expand and grow our program a lot in our first five years. I like to bridge that line between basic and translational science—translating basic science into the clinic.”
The Search for New Technology
Jon wasn’t visiting Promega in mid-March with the goal of seeing the world before COVID-19-related travel restrictions were announced. He’s constantly looking for new collaborations in which both parties can bring something unique to the table. Jon was one of the first to try the new Lumit™ Insulin and Glucagon Immunoassays, which he says are easier to use and have produced better results in his work with glucagon than radioimmunoassays or ELISAs.
“People like Promega scientists say they have a new technology, and they’re looking for someone to try it out it in real-world situations. I don’t have that kind of technology, but I know how to apply it, so there’s a lot of value there. It’s a no-brainer to talk to people about how we can find synergy when the two of us both bring something like that to the table. For some applications, the Lumit™ assays are blowing out whatever we can do, and they’re also incredibly easy to use. So that was a significant improvement in our workflow.”
When asked what he hopes to accomplish in the next few years, Jon similarly points to innovative technology and techniques.
“We have to say, ‘What’s the next innovative step forward, and what new tools can we bring?’ We need to figure out new ways to interrogate the systems that we’re interested in. Then we can start to strip away new biology. If we ask the right question and we answer definitively, we’ll end up with three more questions. Which is great, because we’ll always have more work to do.”
Lumit™ Immunoassays provide a simple and fast alternative to conventional immunoassay methods including sandwich ELISAs and Western blots. Learn more here.
Working on diabetes research? Read more about Promega assays to measure insulin activity in real time.
Today’s blog is written by Technical Services Scientist, Joliene Lindholm, PhD.
Many of us have come back to the lab after a summer of field work or a vacation break, but there is usually someone checking in on the lab to make sure the gel electrophoresis box did not completely overflow with dead bugs and the water baths are not completely overrun with exciting new algae. Maybe this was just because I worked in an older building in an entomology department, but why do insects like running buffer so much? Some labs have been completely shut down for months at this point or maybe just a few essential people have been in keeping stocks and colonies going. Some labs have adapted to the new normal and developed guidelines to keep researchers safe while still doing essential work in the lab. See how the Promega Scientific Applications group has maintained this balance.
Here are a few tips from what I learned in managing a lab after a period of field work to get back into the swing of things:
Studying protein function in live cells is limited by the tools available to analyze the expression and interactions of those proteins. Although mass spectrometry and antibody-based protein detection are valuable technologies for protein analysis, both methods have drawbacks that limit the range of targets and contexts in which proteins can be investigated.
Mass spectrometry is often poor at detecting low-abundance proteins. Antibody-based techniques require high quality, specific antibodies, which can be difficult to impossible to acquire. Both methods require cell lysis, preventing real-time analysis and limiting the physiological relevance, and both methods can be limiting for higher-throughput analysis. While plasmid-based overexpression of tagged target proteins simplifies detection and can allow for real time analysis, protein levels don’t typically resemble endogenous levels. Overexpression also has the potential to create experimental artifacts or limit the dynamic range of an observed response.
While their findings showed that this method provides efficient and specific tagging of endogenous proteins, the research was limited to just five different proteins within a single signaling pathway in two cell lines. This left unanswered questions about whether this approach was scalable, had broader applications and how accurately the natural biology of the cells was represented.
Today’s blog is written by Malynn Utzinger, Director of Integrative Practices, and Tim Weitzel, ESI Architect.
Optimism sometimes gets a bad rap. Many people associate optimism with a sugar-coating of reality or a blind faith that all will be well. Worse, there are brands of optimism which imply that we have the power to transform the conditions of our lives through positive thinking alone, regardless of their deep-seated and/or systemic origins. However, evolving research and our own field work suggests that psychologically mature optimism, a pillar of emotional and social intelligence (ESI), is not only useful as a life-orientation, it is the gateway to self-actualization.
Transcription is the production of RNA from a DNA sequence. It’s a necessary life process in most cells. Transcription performed in vitro is also a valuable technique for research applications—from gene expression studies to the development of RNA virus vaccines.
During transcription, the DNA sequence is read by RNA polymerase to produce a complimentary, antiparallel RNA strand. This RNA strand is called a primary transcript, often referred to as an RNA transcript. In vitro transcription is a convenient method for generating RNA in a controlled environment outside of a cell.
In vitro transcription offers flexibility when choosing a DNA template, with a few requirements. The template must be purified, linear, and include a double stranded promoter region. Acceptable template types are plasmids or cloning vectors, PCR products, synthetic oligos (oligonucleotides), and cDNA (complimentary DNA).
In vitro transcription is used for production of large amounts of RNA transcripts for use in many applications including gene expression studies, RNA interference studies (RNAi), generation of guide RNA (gRNA) for use in CRISPR, creation of RNA standards for quantification of results in reverse-transcription quantitative PCR (RT-qPCR), studies of RNA structure and function, labeling of RNA probes for blotting and hybridization or for RNA:protein interaction studies, and preparation of specific cDNA libraries, just to name a few!
In vitro transcription can also be applied in general virology to study the effects of an RNA virus on a cell or an organism, and in development and production of RNA therapeutics and RNA virus vaccines. The large quantity of viral RNA produced through in vitro transcription can be used as inoculation material for viral infection studies. Viral mRNA transcripts, typically coding for a disease-specific antigen, can be quickly created through in vitro transcription, and used in the production of vaccines and therapeutics.
Transcriptional activation of genes within the nucleus of eukaryotic cells occurs by a variety of mechanisms. Typically, these mechanisms rely on the interaction of regulatory proteins (transcriptional activators or repressors) with specific DNA sequences that control gene expression. Upon DNA binding, regulatory proteins also interact with other proteins that are part of the RNA polymerase II transcriptional complex.
One type of transcriptional activation relies on inducing a conformational change in chromatin, the DNA-protein complex that makes up each chromosome within a cell. In a broad sense, “extended” or loosely wound chromatin is more accessible to transcription factors and can signify an actively transcribed gene. In contrast, “condensed” chromatin hinders access to transcription factors and is characteristic of a transcriptionally inactive state. Acetylation of lysine residues in histones—the primary constituents of the chromatin backbone—results in opening up the chromatin and consequent gene activation. Disruption of histone acetylation pathways is implicated in many types of cancer (1).
As scientists, we can do science forever. The beauty about science is that the questions never end – we can keep asking, and every time we find an answer, we have a new direction to pursue. But it’s very important to know when it’s time to write up your results.
Publishing may be connected to leaving or transitioning your position, but at all times you should be thinking, “What is my end goal? What is the big question I want to answer? What are the questions the field has about my research?” As you reach milestones and make discoveries, whether big or small, consider whether you will have a complete and compelling story to tell in the end.
Monitoring the use of performance-enhancing substances among athletes is complex and the requirements for tests and assays that detect use of such substances have changed significantly over the last few decades.
The haematological (blood) module of Athlete Biological Passport was adopted December 1, 2009 (ABP) by the World Anti-Doping Agency. The module sets out standard protocols to monitor doping of professional athletes by looking at changes in biological parameters, without relying on the detection of illegal compounds in body fluids. Such biological methods eliminate the need to develop and validate a test to detect every new compound that can be used for doping. The current version of the ABP, adopted in 2014, also adds monitoring of certain steroid use indicators from urine samples.
Blood doping which aims at increasing red blood cells so that more oxygen can be transported to muscles to increase stamina or performance is particularly difficult to detect. There are typically three ways that it is accomplished: use of erythropoietin (EPO) or synthetic oxygen carriers and blood transfusions. While transfusions of large volumes of blood or use of EPO can be detected, microdosing EPO or transfusing smaller volumes of packed red blood cells is much harder to detect.
Nicolas Leuenberger and colleagues at the Swiss Laboratory for Doping Analysis have developed a method to detect blood doping. In addition to addressing the detection of blood doping, his laboratory is also concerned about easing the transport and storage requirements for samples and ensuring that sample collection does not adversely affect athlete performance.
Improving Collection and Storage of Blood Samples
Because sample collection and storage are so critical to accurate test results, any new assays developed to detect blood doping benefit from ease of collection and storage. The Leuenberger laboratory investigated the use of the TAP™ Push Button collection device, which is billed as a simple method for blood collection that is easy to use and eliminates the need for painful needle sticks or finger pricks that can affect athlete performance. After TAP collection, 20µl of blood from the device was placed on to filter paper and dried (dried blood samples; DBS), which are much easier to store and transport from collection site to laboratory.
An RNA Biomarker for Blood Doping
Blood withdrawal and autologous transfusion or recombinant human EPO injection stimulate erythropoiesis and immature red blood cells can be distinguished based on their gene expression profiles. One of the genes that is expressed by immature red blood cells is aminoleuvulinate synthase 2, a gene that encodes an enzyme ALAS2 involved in the synthesis of heme, a pathway active during RBC maturation. RNA transcripts are unstable and tend to degrade rapidly, so isolating linear RNA transcripts from a collected sample can be difficult. However circular RNAs (circRNAs) are a class of RNA molecule produced by the backsplicing of pre-mRNAs that are high in abundance, quite stable and maintain cell-type specific expression. The Leuenberger laboratory developed a method for measuring the linear and circular forms of ALAS2 RNA in DBS to monitor erythropoiesis.
One of the greatest challenges in developing this protocol was achieving efficient RNA extraction from only 20ul of dried blood. Leuenberger and his colleagues adopted a two-step purification; beginning with a phenol:chloroform extraction on the DBS followed by a further purification on the Maxwell® RSC automated instrument, using the Maxwell RSC miRNA Serum and Plasma kit. Switching from a manual to an automated method for the second step was crucial. It reduced chances of contamination as well reduced pipetting errors, without compromising good quality and yield of RNA therefore contributing to assay reproducibility. To normalize volumes within the blood spot, the protocol uses RNA produced by housekeeping genes. The work to automate the assay has been published in Bioanalysis.
This protocol is being tested to see if microdosing of EPO or small transfusions can also be detected by monitoring ALAS2 RNA expression in DBS. The Swiss laboratory of Doping Analysis is also in the process of developing a method to detect gene doping by isolating plasmid DNA from whole blood samples, using the Maxwell RSC.
Additionally, the collection and storage methods used have implications for the clinic, especially for patients that need routine blood monitoring. The ability to isolate circular RNAs shows promise in forensic applications to identify body fluids.