Characterizing Compound Binding in Cell-Free Systems

Dioxins (e.g., 2,3,7,8-Tetrachlorodibenzo-p-dioxin, TCDD) and related compounds (DRCs) are persistent environmental pollutants that gradually accumulate through the food chain, mainly in the fatty tissues of animals. Dioxins are highly toxic and can cause reproductive and developmental problems, damage the immune system, interfere with hormones and also cause cancer. This broad range of toxic and biological effects of DRCs is mostly mediated by the aryl hydrocarbon receptor (AHR).

In animal cells, DRCs bind to AHR in the cytoplasm and then translocate into the nucleus, where they affect the transcription of multiple target genes, including xenobiotic-metabolizing enzymes, such as CYP1A isozymes. AHR is also involved in immune system maintenance, protein degradation and cell proliferation.

The jungle crow (Corvus macrorhynchos) has been considered a suitable indicator for monitoring environmental chemicals such as DRCs. While mammals only have one AHR form, avian species have multiple AHR isoforms such as AHR1 and AHR2. To unveil the functional diversity of multiple avian AHR isoforms in terms of their contribution to responses to DRCs a recent study by Kim et al. investigated the molecular and functional characteristics of jungle crow AHR isoforms, cAHR1 and jcAHR2 (1).

cAHR1 and jcAHR2 proteins were synthesized using AHR proteins were synthesized using the TnT Quick-Coupled Reticulocyte Lysate System  to examine whether these jcAHRs have the potential to bind to TCDD. TCDD-binding affinity of the in vitro-expressed jcAHR protein was analyzed using the velocity sedimentation assay with a sucrose gradient.

The results demonstrate that both jcAHR1and jcAHR2 are capable of binding to TCDD.

Reference
Kim, E-Y (2019) The aryl hydrocarbon receptor 2 potentially mediates cytochrome P450 1A induction in the jungle crow (Corvus macrorhynchos). Ecotoxicology and Environmental Safety 171. 99–111

Are We Doing Enough to Stop Candida Auris Infections?

Image of C. auris on plate.
The creamy colonies of C. auris look innocuous. Don’t be fooled. Photo by Shawn Lockhart – Centers for Disease Control, Public Domain, https://commons.wikimedia.org/w/index.php?curid=54680002

Life in the 21st century is full of electronic devices and apps purported to make life easier. Many of us can binge watch movies, videos and news on our phones. There are wireless headphones, electric bicycles, self-operating vacuum cleaners, wine in boxes with taps—and so much more.

This life is, however, not without challenges.

In the event that you or yours ends up in the hospital, the stay could be complicated by an unplanned, unwanted and potentially lethal infection.

No thanks to the yeast, Candida auris. Continue reading “Are We Doing Enough to Stop Candida Auris Infections?”

Cancer is a Scourge Most Ancient

Reconstruction of Pappochelys rosinae or grandfather turtle. Attribution: Rainer Schoch [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)], via Wikimedia Commons
The first words that come to mind when people hear “Triassic” are likely dinosaur or maybe ginkgo or possibly even phytoplankton, but probably not cancer. For all that we have learned about how cancer develops based on the efforts of numerous research scientists, this disease is not solely a modern affliction. In fact, cancer has deep roots in the past. From several thousand year old human mummies to fossils millions of years old, cancer has left evidence of its presence in the historical record. Yes, even in the era of dinosaurs, cancer existed.

Cancer is usually a soft-tissue-based malady, but occasionally, it can also be found on bones, altering the bone’s surface and leaving unmistakable signs. Alterations like those observed on the femur of a 240-million-year-old shell-less stem-turtle found in modern-day Germany and described in JAMA Oncology. Continue reading “Cancer is a Scourge Most Ancient”

Help the Environment through…Gaming?

Did you know that April is Earth Month? While you should be good to the planet every day, this month you should be extra good. Maybe buy it a nice pair of socks or something. Compliment it on its majestic mountains. Or, you could compete to see who can be the best at being nice to the planet, like we’re doing here at Promega with our Green Go Challenge.

Green Go Challenge bingo sheet
Get it? It’s like bingo, but green.

Continue reading “Help the Environment through…Gaming?”

Dual-Luciferase or Dual-Glo Luciferase Assay System? Which one should I choose for my reporter assays?

Confused womanI’ve got a set of experiments planned that, if all goes well, will provide me with the answer I have been seeking for months. Plus, my supervisor is eagerly awaiting the results because she needs the data for a grant application, so I don’t want to mess it up. However, I am faced with a choice for my firefly and Renilla luciferase reporter assays: Do I use the Dual-Luciferase® Reporter Assay System or Dual-Glo® Luciferase Assay System? What’s the difference? How do I decide which to use? I’m so confused! Help!

Sound familiar? Not to worry! The choice is not difficult once you know how these assays work and how they differ.
Continue reading “Dual-Luciferase or Dual-Glo Luciferase Assay System? Which one should I choose for my reporter assays?”

For the Birds: Knitting Nests for Baby Birds Might Just Help Your Health To

It seems that spring has finally come to Southern Wisconsin. The snow has melted. Most days it is warm enough you can go outside without a parka, hat and mittens. The tree buds are starting to swell. And that traditional oracle of spring, the American robin (Turdus migratorius), has been spotted in trees and yards—along with its less friendly cousin, the red winged black bird (Agelaius phoeniceus).

While spring brings the return of migratory birds, it also brings an increase in the number of rescued baby birds flooding into local wildlife rescues and humane societies. When the babies come to these centers, they need a warm, soft, breathable and washable home that resembles the nest they were hatched in.

It turns out that knitted or crocheted nests are a perfect solution. The nests aren’t just used for baby birds; baby rabbits, squirrels, bats, ferrets and racoons are just a few additional animals that benefit. And the best part is, you could be improving your own health while you create those cozy nests. Continue reading “For the Birds: Knitting Nests for Baby Birds Might Just Help Your Health To”

When Good Proteins Go Bad

Ribbon model of p53 protein bound to DNA molecule.
Ribbon model of p53 protein bound to DNA molecule.

Following what feels like an exceptionally long and brutal winter, I for one couldn’t be happier about the arrival of Spring and the way it makes everything seem brighter and brand-new. Soaking in the soul-warming sunshine. Reveling in the sweet melody of chirping birds. Watching the earth literally coming alive again with greenery. And for those of us who love and are enthralled by scientific discoveries like myself, the report of a recent shiny new discovery in the world of cancer research is equally as day-brightening and spirit-lifting.

To suppress tumors or to not suppress tumors: that is the question.

In the world of oncology, the protein known as p53 has long proven itself to be a primary target of interest. p53 operates as a tumor suppressor protein, often lauded as the “guardian of the human genome”, due to its dedication to governing controlled cell division and assessing damaged DNA. There are a number of cellular stressors that can wreak havoc on your DNA, including exposure to ultraviolet light or radiation, oxygen deficiency (hypoxia), and contact with hazardous chemicals.

Consider a normal-functioning p53 protein as the quality control person in a production factory. The p53 protein evaluates the products, DNA, coming down the line and determines an appropriate course of action for those that do not meet the quality standards.

Let’s say some less-than-quality DNA comes down the pipe. If the DNA is not too severely injured, p53 will alert and activate additional genes to repair the damage. However, if the products coming through are too marred to repair, p53 will shut down the whole factory, if you will, by signaling for the cell to self-destruct via apoptosis. In doing so, p53 effectively impedes tumor development by inhibiting the ability for flawed DNA to further divide.

So, it would seem like p53 has proven itself to be an undeniably upstanding citizen of the protein variety, right? The unfortunate truth of the matter is p53 balances delicately on a double-edged sword, establishing itself as the veritable Dr. Jekyll and Mr. Hyde of the cellular world: usually unquestionably good, but sometimes unspeakably evil. Continue reading “When Good Proteins Go Bad”

What’s for Dinner? Saving the Planet One Meal at a Time

On March 13, 2019, a bomb cyclone hit Denver, Colorado. With this storm the Denver area experienced a 24 millibar (mbar) drop in atmospheric pressure in a 24-hour period. At about the same time, several cities in Colorado recorded >24 mbar drops in pressure, including a 35.6mbar drop reported at Pueblo, Colorado!

The intensity of the pressure drop indicates storm severity and this storm was a record-breaker for Denver and surrounding communities.

No location on planet Earth has gone unaffected by new extremes in weather. 2018 was the hottest year on record for countries on every continent. In the past 4 months we’ve experienced record-breaking rainfall and droughts, wildfires, high temperatures and snowfall.

Food for Climate Extremes?
Climate change news is sometimes so bleak as to be overwhelming. But good scientific research and climate change-ameliorating efforts are all around us—I was encouraged to read recently that simply changing what we eat can benefit the environment. There are better food choices available to most of us, no matter what diet or cooking style we favor. Continue reading “What’s for Dinner? Saving the Planet One Meal at a Time”

Lynch Syndrome Awareness Day: What is Lynch Syndrome?

Lynch Syndrome is a hereditary condition caused by germline mutations that inactivate at least one of the major DNA mismatch repair (MMR) genes. Individuals with Lynch Syndrome have an elevated risk of developing several cancers, especially colorectal, uterine and endometrial. Approximately 1 in 279 individuals in the United States is Lynch-positive, but most people are unaware of their status.

Lynch Syndrome results in highly elevated risks of several cancers.

Lynch Syndrome can be diagnosed following screening by microsatellite instability (MSI) analysis or immunohistochemistry (IHC) for the MMR proteins. For some patients, MMR gene sequencing is as easy as an oral “swish.” However, the genetic basis of Lynch Syndrome and its clinical relevancy are relatively recent discoveries. Long before modern sequencing methods simplified testing and diagnosis, a seamstress in Ann Arbor, Michigan correctly predicted her own Lynch Syndrome status based only on her family history. Talking with Dr. Alfred Scott Warthin in the late 19th century, she said that since so many of her family members had died of several specific cancers, she believed that she would follow the same path. Several years later, she unfortunately proved herself right.

Dr. Warthin took interest in the story and began studying the woman’s family. At the time of their conversation, five of her nine siblings had already been diagnosed with uterine, stomach or “abdominal” cancer. Warthin concluded that the family, which he dubbed “Cancer Family G,” did, in fact, have a predisposition to cancer. Warthin and other researchers continued studying the family for several decades. They found that cancers of the colon, uterus and stomach were most common, and that many members of the family were diagnosed at extraordinarily young ages.

In the 1970s, Dr. Henry T. Lynch organized a family reunion for Cancer Family G and subsequently published a report on “Cancer Family Syndrome.” By this time, 95 members of the family had developed one of the expected cancers. Dr. Lynch still didn’t have the technology to determine the molecular basis of the disease, but he noticed that it followed an autosomal dominant inheritance pattern.

In the mid-1990s, three labs simultaneously discovered microsatellite instability and its connection to colorectal cancer. It had been established in bacteria and yeast that inactivating mutations in DNA mismatch repair genes resulted in mutations in microsatellite sequences, so several labs began racing to clone the human homologs of the DNA MMR genes. Within a few months, two labs had cloned the MSH2 gene and found mutations that were present in members of Lynch-positive families who developed cancer.

Around this time, the name “Lynch Syndrome” was adopted to apply to families carrying germline mutations in a gene associated with the condition. Further research established four genes (MSH2, MLH1, MSH6, PMS2) as “Lynch Syndrome Genes,” and researchers began working on guidelines for diagnostic testing (See “The History of Lynch Syndrome” below for further reading).

Today, over two decades later, many researchers are pushing for the adoption of universal tumor screening for Lynch Syndrome. One of the widely recommended screening method is MSI analysis. MSI-H status indicates that certain sections of DNA called microsatellites have become unstable because the major mismatch repair genes that correct errors during DNA replication are not functioning properly. MSI status can influence treatment decisions, based on the 2015 discovery that MSI-H tumors respond well to immunotherapy drugs (1).

It’s also important knowledge for a patient’s family. Lynch-associated cancers are among the most preventable, so individuals who know they are Lynch-positive can work with their healthcare providers to develop robust strategies for prevention and surveillance. As one Lynch-positive mother said to her Lynch-positive son, “Your knowledge is power, and it’s going to keep you healthy and safe.”

March 22, 2019 is Lynch Syndrome Awareness Day, and we’re encouraging everyone to join the fight against colorectal cancer. Visit our website to learn more about Lynch Syndrome and colorectal cancer.

 

 

Further reading:

Take the pledge to fight colorectal cancer: During Colorectal Cancer Awareness Month, we’re donating $10 to Fight CRC for every person who takes the pledge.

Life with Lynch Syndrome: Read about what a Lynch Syndrome diagnosis means for Carrie Ketcham and her family

Dreaming of Universal Tumor Screening: Learn how cancer genetic counselor Heather Hampel is advocating for universal tumor screening

The History of Lynch Syndrome: Dr. C. Richard Boland and Dr. Henry T. Lynch provide a broad review of Lynch Syndrome research, starting over a hundred years ago.

 

 

References

(1) Le, D.T. et al. (2015) PD-1 Blockade in Tumors with Mismatch-Repair DeficiencyNew Engl. J. Med. 372, 2509–20.