It is the start of summer here in Wisconsin, so it’s time for some Friday Fun (#FridayFun) blog posts on Promega Connections. To kick us off, I have scraped the internet for a few good and groaner, G-rated science jokes.
So, here it goes, a few jokes to send you smiling (or shaking your head) into your weekend.
![Fig 4. Four point MMOA screen for tideglusib and GW8510. Time dependent inhibition was evaluated by preincubation of TbGSK3β with 60 nM tideglusib and 6 nM GW-8510 with 10μM and 100μM ATP. (A). Tideglusib [60 nM] in 10μM ATP. (B). GW8510 [60 nM] in 10μM ATP. (C.) Tideglusib [60 nM] at 100μM ATP. (D.) GW8510 [60 nM] at 100μM ATP. All reactions preincubated or not preincubated with TbGSK3β for 30 min at room temperature. Experiments run with 10μM GSM peptide, 10μM ATP, and buffer. Minute preincubation (30 min) was preincubated with inhibitor, TbGSK3β, GSM peptide, and buffer. ATP was mixed to initiate reaction. No preincubation contained inhibitor, GSM peptide, ATP, and buffer. The reaction was initiated with TbGSK3β. Reactions were run at room temperature for 5 min and stopped at 80°C. ADP formed was measured by ADP-Glo kit. Values are mean +/- standard error. N = 3 for each experiment and experiments were run in duplicates. Control reactions contained DMSO and background was determined using a zero time incubation and subtracted from all reactions. Black = 30 min preincubation Grey = No preincubation.](https://www.promegaconnections.com/wp-content/uploads/2016/04/journal.pntd_.0004506.g004-243x300.png)
Time dependent inhibition was evaluated by preincubation of TbGSK3β with 60 nM tideglusib and 6 nM GW-8510 with 10μM and 100μM ATP. (A). Tideglusib [60 nM] in 10μM ATP. (B). GW8510 [60 nM] in 10μM ATP. (C.) Tideglusib [60 nM] at 100μM ATP. (D.) GW8510 [60 nM] at 100μM ATP. All reactions preincubated or not preincubated with TbGSK3β for 30 min at room temperature. Black = 30 min preincubation Grey = No preincubation.
Understanding the MMOA for a small-molecule inhibitor can play a major role in optimizing a drug’s development. The way a drug actually works–the kinetics of binding to the target molecule and how it competes with endogenous substrates of that target–ultimately determines whether or not a a candidate therapeutic can be useful in the clinic. Drugs that fail late in development are extremely costly.
Drug research and discovery for neglected tropical diseases suffer from a lack of a large commercial market to absorb the costs of late-stage drug development failures. It becomes very important to know as much as possible, simply and quickly, about MMOA for candidate molecules for these diseases that are devastating to large populations.
One such neglected topical disease is Human African trypanosomiasis (HAT, also known as sleeping sickness). Continue reading “Increasing Drug Research and Development Efficiency Using a 4-point Screening Method to Determine Molecular Mechanism of Action”
Imagine for a moment this conversation between a senior graduate student and his dissertation adviser:
“Everybody’s doing it. Physicists and computer scientists do it all the time. And even Carol Greider has done it, and she’s a Nobel laureate.”
“Yes,” his adviser from her work, “she is a Nobel laureate; she can take that risk. But, I don’t have tenure, and I am still working on my first NIH grant. You don’t have a degree yet. None of these things—your PhD, the grant renewal, my promotion—come without publications in a peer-reviewed journal, and most peer-reviewed journals in our field, at the least the ones that count for grant renewals and promotion, don’t allow publication of previously released data.”
“But why let the publishers decide what is good science—why not let the scientific community decide and crowd source the review?”
“I agree, but I also want a future. We write the paper and submit it. So do your homework, let’s go to a journal with a short turnaround time, open review, and a reputation for publishing good science.”
Open Data and the Biological Sciences
The debate over prepublication in biology is raging. Prepublication is the standard in physics, computer science, math, and economics to get results publicly available quickly for scientific commentary, and it doesn’t seem to interfere with career advancement and grant renewals. Is there a good reason that the same practice isn’t followed in the life/biological sciences?
Continue reading “Prepublication: Everybody’s Doing It?”
Drug research and development is a complex and expensive process that begins with initial screening steps of candidate chemical compounds, and compounds that appear to have the desired potency against a specific cellular target or pathway are further evaluated. Candidate compounds that fail late in development or during clinical trials because of off-target effects are costly, and can be dangerous. Therefore drug developers not only need to ensure that a candidate compound is effective as a therapy, but also they need to predict any potential undesirable side effects due to off-target activities as early as possible in the drug discovery and development process. Continue reading “Making Drug Discovery More Efficient: Predicting Drug Side Effects in Early Screening Efforts”
I remember one particular encounter with “mystery meat” when I was in college. I was walking along the serving line at the dining hall, and when I came to the entrée, I asked the server, “What is it?”
She replied quite succinctly, “Don’t know. Got beef in it.” I passed on the entrée that night, settling for salad and bread.
I would probably not have be a good candidate for membership in the Explorers Club.
The Explorers Club, founded in New York City in 1904, is a professional society that champions the cause of field research (1). The member list is impressive, including Teddy Roosevelt, the American President responsible for setting aside many of the most treasured public lands in the United States so that explorers have fields for research and wild places for adventures, Neil Armstrong, the first man to set foot on the moon, and Don Walsh and Jacques Piccard, the two men who descended into the Mariana’s trench to explore the deepest part of the ocean, among others.
In addition to a membership list that reads like a who’s who of science and exploration, The Explorers Club also has an annual dinner that for many years has popularized a menu of “exotic” foods (at least exotic foods from the point of view of the typical Midwest United States pallet). One of the club’s most celebrated dinners took place on January 13, 1951.
Continue reading “What’s for Dinner? Mystery Meat Served at the 47th Explorer’s Club Annual Dinner Finally Identified”
When he was a kid, Matt Hanson would disappear into the basement for an entire day and emerge later with a completed model of the USS Constitution or a completed robot or a new rocket (he still makes model rockets). Design and how things fit together have always fascinated him, so a career in science was a natural fit as well.
Today Matt is a Quality Control Supervisor/QA Senior Scientist at Promega Corporation at the Madison, WI, USA, campus. He has been with Promega for 5 years now.
After completing his undergraduate studies in molecular biology, a masters in zoology where he focused on cell biology, and a PhD in developmental biology and immunology, Matt was fortunate to pursue a successful and rewarding career as an Associate Staff Scientist in the Department of Surgery at the University of Wisconsin-Madison. His work focused on diabetes and transplantation biology.
So why did Matt join the scientific staff at Promega?
Continue reading “Finding Space for Passion: Interview with Promega Quality Assurance Scientist, Matt Hanson”
I confess that I struggled through biophysics, and my Bertil Hille textbook Ion Channels of Excitable Membranes lies neglected somewhere in a box in my basement (I have not tossed it into the recycle bin—I can’t bear too, I spent too much time bonding with that book in graduate school).
My struggles in that graduate class and my attendance at the seminars of my grad school colleagues who were conducting electrophysiological studies left me with a sincere awe and appreciation of both the genius and the artistry required to produce nice electrophysiology data. The people who are good at these experiments are artists—they have the golden touch when it comes to generating that megaohm seal between a piece of cell membrane and a finely pulled glass pipette. And, they are brilliant scientists, they really understand the physics, the chemistry and the biology of the cells they study from a perspective that very few scientists ever develop.
Electrophysiology data, which often demonstrate the gating of a single channel protein in response to a single stimulus in real time–ions crossing a membrane through a single protein–are amazing for their ability, unlike virtually any other experimental data for the story they can tell about what is going on in a cell in real time under physiological conditions.
So when I read the paper recently published by Mashuo et al. in Science Signaling “Distinct profiles of functional discrimination among G proteins determine the action of G protein-coupled receptors”, this sentence really caught my attention:
When constructs were ectopically expressed in HEK 293T/17 cells, we obtained very similar kinetics for the GPCR-driven responses between NanoBRET™ biosensors and the patch clamp recordings.
They continue:
Indeed, the activation rates that we observed were very similar to those of GPCR-stimulated GIRKs [G protein-coupled, inwardly rectifying K+ channel] in native cells, suggesting that the conditions of this assay closely match the in vivo setting. This finding further demonstrates the ability of the system to resolve the fast, physiological relevant kinetics of GPCR signaling.
A reporter biosensor that can resolve events similarly to patch clamping?! Amazing. Continue reading “A NanoBRET™ Biosensor for GPCR:G protein Interaction with the Kinetics and Temporal Resolution of Patch Clamping”
Over the last few months we have published several blogs about qPCR—from basic pointers on avoiding contamination in these sensitive reactions to a collection of tips for successful qPCR. Today we look in depth at a paper that describes the design and and optimization of a qPCR assay, and in keeping with the season of winter in the Northern hemisphere, it is only fitting that the assay tests for the abundance and identity of ice-nucleating bacteria.
Ice-nucleating bacteria are gram-negative bacteria that occur in the environment and are able to “catalyze” the formation ice crystals at warmer temperatures because of the expression of specific, ice-nucleating proteins on their outer membrane. Ice-nucleating bacteria are found in abundance on crop plants, especially grains, and are estimated to cause one-billion dollars in crop damage from frost in the United States alone.
In addition to their abundance on crop plants, ice-nucleating bacteria are also found on natural vegetation and have been isolated from soil, snow, hail, cloud water, in the air above crops under dry conditions and during rain fall. They have even been isolated from soil, seedlings and snow in remote locations in Antarctica. For the bacteria, ice nucleation may be a method to promote dissemination through rain and snow.
Although ice-nucleating bacteria have been isolated from clouds, ice and rain, little is known about their true contribution to precipitation or other events such as glaciation. Are such bacteria the only source of warm-temperature (above temperatures at which ice crystals form without a catalyst) ice nucleation? Can they trigger precipitation directly? What are the factors that trigger their release from vegetation into the atmosphere? Can we determine their abundance and variety in the environment?
Continue reading “Do you want to build a snowman? Developing and optimizing a qPCR assay to detect ice-nucleating activity”
For three out of the last four years, we have been honored to have one of our key technologies named a Top 10 Innovation by The Scientist. This year the innovative NanoBiT™ Assay (NanoLuc® Binary Technology) received the recognition. NanoBiT™ is a structural complementation reporter based on NanoLuc® Luciferase, a small, bright luciferase derived from the deep sea shrimp Oplophorus gracilirostris.
Using plasmids that encode the NanoBiT complementation reporter, you can make fusion proteins to “report” on protein interactions that you are studying. One of the target proteins is fused to the 18kDa subunit; the other to the 11 amino acid subunit. The NanoBiT™ subunits are stable, exhibiting low self-affinity, but produce an ultra-bright signal upon association. So, if your target proteins interact, the two subunits are brought close enough to each other to associate and produce a luminescent signal. The strong signal and low background associated with a luminescent system, and the small size of the complementation reporter, all help the NanoBiT™ assay overcome the limitations associated with traditional methods for studying protein interactions.
The small size reduces the chances of steric interference with protein interactions. The ultra bright signal, means that even interactions among proteins present in very low amounts can be detected and quantified–without over-expressing large quantities of non-native fusion proteins and potentially disrupting the normal cellular environment. And the NanoBiT™ assay can be performed in real time, in live cells.
The NanoBiT™ assay is already being deployed in laboratories to help advance understanding of fundamental cell biology. You can see how one researcher is already taking full advantage of this innovative technology in the video embedded below:
Visit the Promega web site to see more examples more examples how the NanoBiT™ assay can break through the traditional limitations for studying protein interactions in cells.
You can read the Top 10 article in The Scientist here.
Often a diagnosis of thyroid cancer is associated with a good prognosis and fairly straightforward surgical treatments to remove the tumor followed by radioactive iodine ablation. Such treatment works well in tumors that have not metastasized and retain enough of their thyroid cell “identity” that they can still accumulate radioactive iodine.
However, aggressive thyroid cancers, which often metastasize and recur, do not respond to standard treatments because they are generally too dedifferentiated to accumulate iodine, so alternative treatments are needed.
One approach is to look for compounds that will reverse dedifferentiation, making tumor cells more likely to take up and concentrate radioactive iodine regardless of their location in the body. One possible target to effect dedifferentiation is epigenetic modification of histone proteins.
Histone proteins are more than the structural components of the nucleosome that organizes the chromatin inside cells. Histone proteins are subject to a host of protein modifications on their N-terminal tails such as acetylation, phosphorylation, methylation, ubiquitination and ADP-ribosylation. These various modifications are seen as creating a “histone code” that is read by other proteins and protein complexes (1). This code regulates patterns of gene expression and activity for a cell—in part resulting in a differentiated phenotype. Previous studies have suggested that some histone deacetylase (HDAC) inhibitors (e.g., valproic acid) can reverse some of the dedifferentiation associated with aggressive cancers (2).
Jang, et al. in a recent paper (3) published in Cancer Gene Therapy synthesized a group of HDAC inhibitor analogs (AB1–AB13) and tested them for their ability to inhibit growth of three aggressive human thyroid cancer cell lines and induce partial re-differentiation to the thyroid cell phenotype. Continue reading “Rewriting the Histone Code: Searching for Treatments for Stage IV Thyroid Cancers”
