Factors Influencing Compound Potency in Biochemical and Cellular Assays

Late in 2017, a group here at Promega launched an exciting new assay, the NanoBRET™ Target Engagement (TE) Intracellular Kinase Assay.

It’s easy for me to call this assay exciting; I was an editor on the project team. But judging by the reviews on the SelectScience® web site, others are excited about NanoBRET™ Target Engagement Intracellular Kinase Assay too.

A review of the NanoBRET TE Kinase assay from SelectScience® .

A review of the NanoBRET TE Kinase assay from SelectScience® .

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Millions of Pickles, Pickles in the Sea

For a few years beginning late in 2013, warmer ocean conditions in the eastern Pacific prompted the appearance of unexpected species and toxic algal blooms that devastated others. When temperatures cooled in 2017, the marine ecosystems seemed to be returning to normal. Except for the pyrosomes. Although these previously rare organisms did start to wash up on beaches during the periods of warming, they began to appear by the millions from Oregon to Alaska that spring.

Pyrosomes

Photo by Steven Grace.

Some combination of ideal conditions led pyrosomes to multiply, dominate the ocean surface and wash up on beaches along the US and Canadian Pacific Coasts. Pyrosomes typically exist offshore, far below the surface in warm, tropical waters all over the world. Their sudden proliferation in other areas is likely due to the warm, Pacific ocean “blob,” although atypical sea currents and changes in pyrosome diet have been offered as other possible explanations.

While the appearance of pyrosomes impeded the efforts of fisherman by clogging nets and filling hooks, greater ecological effects have yet to be observed. As we celebrate World Oceans Month, pyrosomes offer a mesmerizing example of the astounding biological diversity our oceans have to offer and, perhaps, a cautionary tale of the impact climate change can have on those marine lifeforms.

The pyrosome species common in the NE Pacific, Pyrosoma atlanticum, goes by a few other colorful names. Each name reveals something captivating about these creatures. Commonly called “sea pickles” due their size, shape and bumpy texture (like a transparent cucumber), these are not single organisms, but colonies formed by hundreds or thousands of individual multicellular animals call zooids.

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High-Throughput Purification with Experts Included

Implementing automated nucleic acid purification or making changes to your high-throughput (HT) workflow can be complicated and time-consuming. There are also many barriers to success such as challenging samples types and maintaining desirable downstream results that can add to the stress, not to mention actually getting the robotic instrumentation to do what you want it to. All of this makes it easy to understand why many labs avoid automating or own expensive instrumentation that goes unused. Continue reading

When School is just a Memory: Science after Graduation

Happy graduation! Whether you graduated last week or twenty years ago, the experience is roughly the same. As soon as you arrive on the far side of the stage, empty diploma folder under your arm, hand still sticky from the Dean’s sweaty handshake, the reality of post-academic life sets in. Perhaps grad school is on the horizon for some and others might be busy prepping for med school. For some of us, though, our years of formal education end after four and we run off to rejoice in our newfound freedom. No more exams, group projects, late nights writing papers, disapproving professors, supervisors and mentors – done with that life forever! We didn’t even bother with the GRE, MCAT, LSAT or a single “Why [insert school]” essay. Now it’s off to enjoy the Real World, which will definitely be better than college.

I’ve found, in my one year of post-college life, that sometimes you can miss academic life. You’ll occasionally look back and think, “I didn’t know how good I had it.” In particular, those of us with a pure love of learning can find ourselves unsatisfied with our prospective learning opportunities or lack thereof. We spent college soaking up mountains of knowledge–and not just from textbooks. University life gives you access to free talks from eminent thought leaders, unrestricted access to myriad scientific journals, and plenty of people around who are eager to argue about that day’s lecture in Cell Biology or Neuroscience. After college, it’s tough to fill that void.

I work at Promega (obviously), a biotech company, so I still have access to journals and there are plenty of brilliant scientists around me. However, I’m still looking for more opportunities to learn and grow. I may be out of school, but the love of science never goes away. Here are a few of my tips for everyone receiving their hard-earned science degree this spring.

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Wonders of the Conscious and Unconscious Mind—What I Learned from the International Forum on Consciousness

When Heather Berlin was 5 years old, she realized that, at some point in the future, she was going to die. This disturbed her so much that she couldn’t sleep all night. The next morning, she asked her father where she could store all her thoughts so they could live on after she died. There’s no way to do that, said her physician father. “What can I do to make this happen?” she asked. “Maybe become a psychiatrist?” said her dad. Decades later, she became an Assistant Professor of Psychiatry at Icahn School of Medicine at Mount Sinai. Her research focuses on interactions of the brain and mind, with the goal of treating and preventing psychiatric and neurological disorders.

Dr. Heather Berlin told this story at the International Forum on Consciousness held at the BioPharmaceutical Technology Center Institute in Madison, Wisconsin last week. This annual forum gathers scientists from around the world, all interested in understanding how our conscious and unconscious minds work. This year, the forum focused on the newest research and technology for detecting and measuring consciousness. As someone with limited knowledge in this field, my mind was blown by how much researchers have learned so far about consciousness. (No, we can’t store our thoughts in a box…yet.) Here are a few takeaways: Continue reading

From Dating Apps to In Vitro Fertilization, the Challenges to Saving the Endangered Northern White Rhino

In April of 2017 a profile appeared on the dating app Tinder. Describing himself as “One of a kind”, the poster was 43 years old, not in great physical shape, and yet so sought after he required around the clock body guards. His name was Sudan, and he was the last living male northern white rhino. His keepers at the Ol Pejeta Conservancy in Kenya weren’t expecting Sudan to find love. They were hoping to raise awareness of the species’ dire situation and money for the research and development of an in vitro fertilization (IVF) method for rhinos.

Northern white rhino

With only two remaining, can we save the northern white rhino? © Matt Caldwell / 123RF Stock Photo.

Northern white rhinos used to range over all or parts of Uganda, Sudan, Chad, the Democratic Republic of the Congo and the Central African Republic. In the 1960s there were an estimated 2,360 northern white rhinos left in the wild (1). Civil unrest in the region made conservation difficult, and by 2003 poaching and other pressures had reduced the number of northern white rhinos living in the wild to four individuals living in the Garamba National Park in the Democratic Republic of the Congo. There has been no sign of that wild population since 2007 (2), and they are considered extinct in the wild as of 2008. Continue reading

Kinase Drug R & D: Helping Your Inhibitor Make the Cut

Finding the best inhibitor for your kinase doesn’t have to be a long trip.

A recent paper in Journal of Medicinal Chemistry, “Discovery of GDC-0853: A Potent, Selective and Noncovalent Bruton’s Tyrosine Kinase Inhibitor in Early Clinical Development” (1) details some elegant work in chemical modification and extensive testing during exploration of inhibitors for BTK. As a warmup to the article, here is a brief BTK backstory.

BTK (Bruton Tyrosine Kinase): Importance in Health and Disease 

Bruton’s tyrosine kinase (BTK) was initially identified as a mediator of B-cell receptor signaling in the development and functioning of adaptive immunity. More recent and growing evidence supports an additional role for BTK in mononuclear cells of the innate immune system, especially dendritic cells and macrophages. For example, BTK functions in receptor-mediated recognition of infectious agents, cellular maturation and recruitment processes, and Fc receptor signaling. BTK has recently been identified as a direct regulator of a key innate inflammatory machinery, the NLRP3 inflammasome (2). Continue reading

HaloTag Application: Fluorescence Under Stress

diagram illustrating the mechanism of the stress reporterCellular stress is associated with global misfolding and aggregation of the endogenous proteome. Monitoring stress-induced abnormalities remains one of the major technical challenges facing established sensors. Misfolded monomers induced by mild stresses, however, remain largely invisible with current sensors.

In a recent publication (1) Fares and colleagues describe a new sensor based upon a fluorescent molecular rotor that is conjugated to a Halo mutant (AgHalo). In non-stressed cells, the AgHalo sensor remains largely folded, and is fluorescent when misfolded. The fluorescent molecular rotor, when conjugated to purified AgHalo to form the proteome stress sensor, is able to report on urea-induced partially unfolded (misfolded) conformations with a higher fluorescent increase than the previously reported fluorophore-based sensors. Heat-induced misfolding is also effectively monitored by the fluorescence change of the sensor that is based on fluorescent molecular rotor, but not the solvatochromic fluorophore. The unique feature of the fluorescent molecular rotor makes the new generation of the AgHalo proteome sensor more sensitive to misfolded conformations that are primarily induced by mild proteome stress. Further, the new sensor exhibits a higher fluorescence signal when detecting soluble and insoluble protein aggregates that are induced by more severe proteome stress. These data collectively suggest that thermo-labile Halo conjugated with a fluorescent molecular rotor serves as a suitable sensor to detect a wide range of proteome stress conditions.

Literature Cited

Fares, M. et al. (2018) A Molecular Rotor-Based Halo-Tag Ligand Enables a Fluorogenic Proteome Stress Sensor to Detect Protein Misfolding in Mildly Stressed Proteome. Bioconjugate Chem 29, 215–24.

Introduction to the Laboratory: A New Workshop in Partnership with the UW-Madison Center for Educational Opportunity

During the week of March 26, 2018, while many students were having fun and relaxing during Spring Break, others were busy doing extra lab work at the BTC Institute.  This four-day workshop was designed to provide an introduction to the molecular biology laboratory for students affiliated with the Center for Educational Opportunity (CeO) on the UW Madison campus.  As noted on its web site: “CeO promotes access to resources, academic achievement and personal growth for students whose parents have not received a four-year degree, students who meet specific federal family income guidelines, and students with documented disabilities.”

It is well known that first-generation college students, women and students of color persist in STEM fields at lower rates than the general population. This interferes with the creation of a diverse STEM talent pool, in turn needed to ensure diverse problem-solving perspectives.

Further, STEM fields are often seen as being stressful, given their competitive learning environments.  This may be especially discouraging for students from racial/ethnic minorities who may not have as many mentors and role models to turn to.

Group photo of attendees

Introduction to the Laboratory attendees

This workshop aimed to give students an experience that would strengthen their skills and confidence as they continue to pursue scientific paths. In addition to laboratory work, students discussed the importance of clear communication in written and oral presentations, were required to work as partners to experience teamwork, and were encouraged to use reflection and lab reporting as ways to internalize what they learned throughout the week. Continue reading

From Antarctica to Mars: Growing Food in Extreme Conditions

Even those of us with the greenest thumbs are baffled by the idea of growing food in Antarctica. From my tiny desk plant to my neighbor’s cabbage patch, plants generally have the same requirements: soil, sun and water. At the southern end of the planet, however, those are all scarce commodities. Nonetheless, on April 5, 2018, the team managing the EDEN-ISS greenhouse at Neumayer III announced that they had harvested 8 pounds of salad greens, 18 cucumbers and 70 radishes. This project has implications beyond just Antarctica, from moderate climates on Earth to future Mars missions. Continue reading