General

A Roadmap for PROTAC Development

Posted on by Kari Kenefick

PROTACs or Proteolysis-Targeting Chimeras are an emerging tool in protein degradation studies, potentially suited to any need involving the removal of a specific protein. These small-molecule chimeras are exciting due to: 1) their target specificity; and 2) their ability to enable target destruction versus target inhibition. Here we discuss a paper that presents a roadmap for PROTAC development.

PROTAC components: target protein ligand, E3 ligase and linker, these are components in the described roadmap for PROTAC development.
PROTAC components: target protein ligand, E3 ligase and linker.

Destruction/Inhibition: Is There a Difference?
An analogy that microbiologists (and wrestlers or anyone that has ever spent time in a locker room shower) would understand, is fungicidal versus fungistatic compounds. A fungicidal compound kills fungus. A fungistatic compound just slows the fungus down a bit.

A small-molecule inhibitor attaches to its target protein, but for how long? What inhibitor testing must be done to determine how long the inhibition lasts?

On the other hand, a small-molecule agent that causes protein degradation first targets the protein of interest, then attaches ubiquitin to that target. Once a protein is marked with ubiquitin, it’s a dead man. E3 ligase must be involved, but if the ubiquitin is added by E3, the end is near. Next stop, Hades.

This ubiquitinated protein is headed to the proteasome and proteins that go there don’t come back. Ubiquitination was called the ‘molecular kiss of death’ when this discovery was awarded the Nobel prize in Chemistry in 2004.

About PROTACs

PROTACs are degrader molecules composed of three parts: 1) a ligand that is specific for the target protein; 2) a ligand for E3 ligase; and 3) a linker molecule that connects the two ligands. The E3 ligase is one of three enzymes that can add ubiquitin to a cellular component, but only ubiquitins added by the E3 ligase cause targeting to the proteasome (Zoppi et al.).

Continue reading “A Roadmap for PROTAC Development”

2019: International Year of the Periodic Table

Posted on by Jordan Villanueva
Periodic table of the elements

The periodic table is one of the most pivotal and enduring tools of modern science. It’s seen from the inside covers of elementary science textbooks to the walls of chemistry labs all around the world. To honor the 150th anniversary of its discovery, the United Nations General Assembly and UNESCO have declared 2019 to be the International Year of the Periodic Table of Chemical Elements.

As with all scientific progress, Dmitri Mendeleev’s periodic table was the result of decades—centuries, even—of research performed by scientists all over the world. Aristotle first theorized the existence of basic building blocks of matter over 2,500 years ago, which later were believed to be earth, air, fire and water. Alchemist Hennig Brand is credited with discovering phosphorus in the late 17th century, sparking chemists to begin pursuing these basic atomic elements.

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Light enters eyes and is transmitted to SCN and PHb.

Light: A Happy Pill for Dark Days?

Posted on by Kari Kenefick

Have you ever had a day where you feel exceptionally good? As intake on the world kind of good? You feel so much better than the previous couple of days that you stop to wonder why.

Then it dawns on you.

The sun is out. It’s been cloudy for the past week but now—SUNSHINE.

You go out to lunch or for a walk just to take in those rays. Sure, it feels warmer than your darkened office space, but it’s the light rather than warmth that’s making a difference.

You purposely don’t wear sunglasses and it feels like the light is coming in through your eyes and massaging that part of your brain that is your happy zone. Are you imagining it or is the sun really affecting how you feel?

In a study reported in the September 2018 issue of Cell we learn that this is not a figment of your or my imagination (1). There is, in fact, a type of retinal cell that transports sunlight directly to the part of our brains that affects mood.

Eyes and the Body’s Master Clock

Circadian rhythms are innate time-keeping functions found in all multicellular organisms. This subject of the 2017 Nobel prize in Physiology or Medicine, circadian rhythms are fueled by daily light-dark cycles and are critical to the function of neurologic, immune, musculoskeletal and cardiac tissues (2). Nearly every mammalian cell is affected by circadian rhythms.

The human body has a circadian master clock, the suprachiasmatic nucleus or SCN. The SCN is a highly innervated tissue located in the hypothalamus (see image). It is connected directly to the retina by the optic nerve, and thus is influenced by external light and dark.

Light enters eyes and is transmitted to SCN and PHb.
Light enters the eyes and affects the SCN (physiologic effects), and as discussed in recent research, Fernandez et al. here, the perihabenular nucleus (behavioral effects). (Image in public domain.)

The retina of the eye is the light-gathering instrument for this organ. Historically, it’s been understood that the retina is composed of two cell types, rods and cones, that function in transmitting light and images to the optic nerve, which sends those signals to the brain.

Drawing of the retina with rods and cones, some nervous tissues.
Some parts of the retina. Light enters the eye (from left) and passes through to the rods and cones. Here a chemical change converts the light to nerve signals. Image-based on drawing by Ramón y Cajal, 1911 and licensed under Wikimedia commons.

Studies by Hattar et al. in the early 2000s identified another cell found in the retina, the melanopsin-containing intrinsically photoactive retinal ganglion cells (ipRGCs) as the transmitter of circadian light signals (3). Through this direct connection to the SCN, the circadian master clock, the ipRGCs can influence a wide range of light-dependent functions independent of image processing (4).

Now Fernandez et al. have identified multiple types of ipRGCs. They showed that ipRGCs that mediate the effects of light on learning work via the SCN, while the pathway for light influencing emotions is different.

They discovered a new target of ipRGC cells, the perihabenular nucleus (PHb). The PHb is a newly recognized thalamic region of the brain. The authors showed that the connection between light and mood is regulated by ipRGCs through the PHb versus the SCN. They show that the PHb is integrated into other mood-regulating centers of the thalamic region.

In Conclusion

Daylight, and lack thereof, does affect both our mood and our ability to learn. In this 2018 report, we have learned that the pathways for these effects are distinct, and gain an understanding of a new thalamic region by which the light and mood actions occur. This information could influence the development of better drugs and/or therapies for major depressive disorders.

For those of us with seasonal affective disorder, the evidence is undeniable—lack of light can cause issues, from sleep-wake problems, to mood and learning issues.

And while we can’t create sunshine, a special lamp or lightbox may help to gain some full-spectrum light. To learn more about how to choose such a lamp and when to use it, see this Mayo clinic article for details.

References

  1. Fernandez, D.C. et al. (2018) Light affects mood and learning through distinct retinal pathways. Cell 175, 71–84.
  2. Ledford, H. and Callaway, E. (2017) Circadian clock scoops Nobel prize. Nature 550, 18.
  3. Hattar, S. et al. (2002) Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity. Science 295, 1065–70.
  4. Hattar, S. et al. (2003) Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice. Nature 424(6944)76–81.

A Healthier Kind of Blues

Posted on by Kari Kenefick

We are in the midst of a very intense time of the year, with holidays and seasonal celebrations like Thanksgiving (recently past), Hanukkah this week and Christmas a mere two-plus weeks away.

Wrap that up with a New Year’s celebration and “Wham”—more friends, family and food/alcohol than one normally enjoys in a three-month period.

Yet it can also be the season of SAD—seasonal affective disorder when the amount of daylight decreases daily, and for those of us in the northern latitudes, cold weather intensifies. We’re eating more, getting less sunshine and quite probably less exercise. Hibernation is great for bears, not so good for humans.

It’s the wintertime blues. For myself and many, once the solstice passes and day length starts to increase, mood improves. But noticeable day-length increases don’t really occur here until mid-February. That’s a long time to feel the blues.

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Learning New Things About mtDNA Inheritance from a Four-Year-Old Boy and a Tenacious Team of Scientists

Posted on by Kelly Grooms

We inherit our cells’ mitochondria from our mother. These energy-producing organelles are present in large numbers in most cells, meaning that cells can contain thousands of copies of the DNA associated with the mitochondria (mtDNA)—all passed on wholly from our mother. New evidence suggests, however, that this cannon principle of maternal-only inheritance of mtDNA might need to be refined. And it all started with a four-year-old boy.

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How To Make Medicine on Mars

Posted on by Darcia Schweitzer

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.

NASA's InSight lander approaching Mars.
Image credit: NASA/JPL-Caltech

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.

I had the pleasure of meeting one team of scientists doing just this—eight high school students from iGEM Team Navarra BG. I met the team and their advisors at the 2018 iGEM Giant Jamboree, where they presented their synthetic biology project, BioGalaxy, as part of the iGEM competition. The problem they aimed to solve is key to helping humans stay on Mars for an extended period of time—how do you take everything you need when there isn’t enough room on the spacecraft? Continue reading “How To Make Medicine on Mars”

Dear Tech Serv, Thank You!

Posted on by Johanna Lee

It’s that time of year again. Time to be thankful and show gratitude for those special people in your life. The undergrad who does the dishes, the labmate who shares their buffers when yours runs out, the collaborator that sends you data on a Saturday… Take a moment this week to say thank you, or send them an email to show your appreciation.

Today, we want to thank our Technical Services team. They work hard to help researchers choose the right assay for their needs, understand results and troubleshoot technical problems. They strive to provide the best service for those in need. Many on the receiving end have sent thankful messages:

“I deeply appreciate the help you have been and the email you just sent. I think with the information here, I may have sorted out an issue that has plagued our lab for the past few months.”

“Cannot tell you how grateful I am–you’ve been a tremendous help.”

“You are super sharp and caught critical errors in my protocol (the calculation and dilution errors you referenced below). While few of my colleagues run kinase assays, I did consult 6 of them, and none caught the errors you did. You’re clearly an expert and I truly appreciate how you’ve tailored everything for my ‘beginner’ level.”

“Wow, I cannot thank you enough! You have NO idea how helpful this is! You guys are absolutely great.”

Here’s one heart-warming story we had to share in which Tech Serv helped a group of students turn frowns into smiles.

In April, Tech Serv received a message from a professor from a university in Michigan regarding an issue with the pGEM Vector System. He was teaching a cell and molecular biology course and his students were unable to generate any colonies. “I have a very disappointed group of seniors on my hands. Please see the photo attached. All those sad faces trying to exude how hard they’ve worked with nothing to show for it. Any insight would be greatly appreciated,” he wrote.

“I understand the frustation of a kit that is not working, the students look so sad!” replied the Tech Serv team. Turns out, the cells may have been past expiration or subjected to repeated freeze thaws that caused the cells to lose competence. Tech Serv sent them a replacement kit with a photo of the team for encouragement.

“We greatly appreciate you replacing what we have and aim to turn those frowns into happy faces before graduation,” the professor replied.

Two weeks later, they got their colonies and wrote back: “It worked very well! We were able to make the most of this and they experienced a very good exercise in troubleshooting. I would say the group would view all that happened as a success. Thank you, we will continue to order from Promega as you’ve always proven to be a very client-friendly company!”

Nothing brings more happiness to the Tech Serv team than your success, so don’t hesitate to contact them with any questions you may have. They’re here to help.

Thanks, Tech Serv!

The Five Steps to miRNA Profiling

Posted on by Kelly Grooms

MicroRNAs (miRNAs) are small, non-coding RNAs that play a role in regulating cancer by acting as both tumor suppressors and oncogenes. Ranging in size from 18–25 nucleotides, miRNAs function in feedback mechanisms to regulate many cellular processes including cell proliferation, apoptosis, cell signaling and tumorigenesis (1).

Not surprisingly, dysregulation of miRNA expression can have serious repercussions. For example, miRNAs are dysregulated in almost all human cancers (1). Because of the potential to influence cancer growth and development, there is growing interest in miRNA profiling to identify possible biomarkers for cancer diagnosis or prognosis, as well as potential therapeutic targets (1).

Growing interest in miRNAs as both biomarkers of disease and therapeutic targets drives the need for fast and effective methods for miRNA profiling. Profiling miRNA targets follows a relatively simple workflow: sample selection, RNA extraction, RNA QC and quantitation, RNA profiling and data analysis (2,3). So what happens at each step?

Five steps of miRNA profiling
The Five Steps of miRNA Profiling

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Could Your Appendix Predispose You to Parkinson’s Disease?

Posted on by Kari Kenefick

Image of span of vagal nerve, humans.
The vagal nerve could serve as conduit for transit of alpha-synuclein from appendix to brain.

Since about 2000 we’ve learned a lot about the bacteria in our guts. We’ve learned that the right bacterial communities in our gastrointestinal system can make us feel better, think better and even help avoid obesity (1). My colleague Isobel has previously blogged about how certain gut bacteria can improve immunotherapy outcomes.

Conversely, the wrong bacteria in our guts can have negative consequences on health and cognition.

Along the way we’ve learned that gut bacterial flora can be influenced by what we eat, certain medications like antibiotics, and even stressful events. We now know that fermented foods like yogurt, sauerkraut, kombucha and that horrible-smelling stuff (kimchi) that another colleague eats are happy food for the good gut bacteria.

And you might guess that fried foods, saturated fats and certain carbohydrates can support the growth of gut bacteria that are doing us no favors when present in large quantities in our gastrointestinal system. Continue reading “Could Your Appendix Predispose You to Parkinson’s Disease?”

In a Perfect World, Bacteria Wins

Posted on by Jordan Villanueva

iGEM 2018 from aboveLast month, several of my Promega colleagues and I attended the 2018 iGEM Giant Jamboree in Boston, MA. This annual event is the culmination of the International Genetically Engineered Machines competition, in which 350+ teams of high school, undergraduate and graduate students use synthetic biology to solve a problem they see in the world.

The iGEM Giant Jamboree is the closest I have ever come to a scientific utopia. For four days, several thousand students from 45 countries come together to share their experiences and discuss ways that science can change the world. They present impressive projects with real-world applications including human diagnostics and alternative energy. Collaboration and open science are among the core tenets of iGEM, and it’s not unusual to see three or more countries represented on the Collaborators slide at the end of a presentation. Each project also contains a public engagement component, which many teams fulfill with educational programs or partnerships with underrepresented communities. Continue reading “In a Perfect World, Bacteria Wins”