Author: Michele Arduengo, PhD

Finding Space for Passion: Interview with Promega Quality Assurance Scientist, Matt Hanson

Posted on by Michele Arduengo, PhD
QA Senior Scientist Matt Hanson
QA Senior Scientist Matt Hanson

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”

A NanoBRET™ Biosensor for GPCR:G protein Interaction with the Kinetics and Temporal Resolution of Patch Clamping

Posted on by Michele Arduengo, PhD

Electrophysiologists are talented scientists/artists who see into the events of the cell with amazing detail.
Electrophysiology experiments provide a view into the cell with amazing detail. The paper reviewed here describes a molecular reporter biosensor (NanoBRET) that can offer the same kind of temporal and spatial resolution traditionally reserved for extremely labor-intensive experiments like patch clamp analysis.

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”

Do you want to build a snowman? Developing and optimizing a qPCR assay to detect ice-nucleating activity

Posted on by Michele Arduengo, PhD
Snowflakes---MA-400x600

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”

NanoBiT™ Assay: Transformational Technology for Studying Protein Interactions Named a Top 10 Innovation of 2015

Posted on by Michele Arduengo, PhD

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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.

Rewriting the Histone Code: Searching for Treatments for Stage IV Thyroid Cancers

Posted on by Michele Arduengo, PhD

Chromatin fiberOften 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”

Summer Friday Blog: This Week We Travel to Hawaii and Peshtigo, WI to Learn about Firestorms

Posted on by Michele Arduengo, PhD

Fire-whirlThis week’s video takes us to a forest fire on Mauna Kea, a dormant volcano on the island of Hawaii. One of the firefighters captured this amazing video of a fire whirl that erupted as the air temperature near the ground grew very hot. Fire whirls like this one are caused by extreme heat rising from the ground rather than a confluence of atmospheric events, but they can be be every bit as destructive as atmospheric tornadoes and cause a forest fire to continue to burn out of control.

There are written records of fire tornadoes including several that developed after lightning struck an oil storage facility near San Luis Obispo, CA, USA in 1926. In 2003, scientists confirmed true fire tornadoes in Australia associated with the Canberra fires. In this case the fire tornadoes produced damage consistent with the intensity of an F2 tornado. In The Great Pestigo Fire in 1871, the town of Peshtigo, WI, may well have been consumed by fire tornadoes. Dry weather conditions and slash and burn farming practices contributed to this devastating fire (as they did the more famous Chicago fire that occurred on the same day). Strong winds carried a forest fire into the mill town of Peshtigo, WI, and researchers theorize that cut timber and wooden structures of the town fueled such intense heat that a massive fire whirls formed, consuming the town. You can read some compelling stories about the Peshtigo fire here and here.

Understanding the conditions under which firestorms and fire tornadoes form hopefully will lead to a better understanding of how forest and brush fires spread and allow scientists and fire control experts to develop more effective methods of control.

If We Could But Peek Inside the Cell …Quantifying, Characterizing and Visualizing Protein:Protein  Interactions

Posted on by Michele Arduengo, PhD
14231183 WB MS Protein Interactions Hero Image 600x214

Robert Hooke first coined the term “cell” after observing  plant cell walls through a light microscope—little empty chambers, fixed in time and space. However,  cells are anything but fixed.

Cells are dynamic: continually responding to a shifting context of time, environment, and signals from within and without. Interactions between the macromolecules within cells, including proteins, are ever changing—with complexes forming, breaking up, and reforming in new ways. These interactions provide a temporal and special framework for the work of the cell, controlling gene expression, protein production, growth, cell division and cell death.

Visualizing and measuring protein:protein interactions at the level of the cell without perturbing them is the goal of every cell biologist.

A recent article by Thomas Machleidt et al. published in ACS Chemical Biology, describes a new technology that brings us closer to being able to realize that goal.

Continue reading “If We Could But Peek Inside the Cell …Quantifying, Characterizing and Visualizing Protein:Protein  Interactions”

Targeting MYC: The Need to Study Protein:Protein Interactions in Cells

Posted on by Michele Arduengo, PhD
Crystal Structure of MYC MAX Heterodimer bound to DNA ImageSource=RCSB PDB; StructureID=1nkp; DOI=http://dx.doi.org/10.2210/pdb1nkp/pdb;
Crystal Structure of MYC MAX Heterodimer bound to DNA ImageSource=RCSB PDB; StructureID=1nkp; DOI=http://dx.doi.org/10.2210/pdb1nkp/pdb;

In 1982, picked up because of its homology to chicken virus genes that could transform cells, MYC became one of the first human genes identified that could drive cellular transformation (1,2). Since that time countless laboratories have prodded and poked the human MYC gene, the MYC protein, their homologs in other animal models, and their transforming viral counterparts.

MYC is a transcription factor and forms heterodimers with a required protein partner, MAX, before binding to the E box sequences of DNA regulatory regions (3). MYC regulates gene expression of many targets through interactions with a host of proteins, often referred to as the MYC Interactome (2).  In fact, MYC is estimated to bind 10–15% of the genome, and it regulates the expression of genes that  are transcribed by by each of the three RNA polymerases (2).

MYC plays a central role in regulating cell growth, proliferation, apoptosis, differentiation and transformation, acting as a central integrator of cellular signals. MYC is tightly regulated at multiple levels from gene expression to protein stability. Dysregulation (usually upregulation) of the amount and stability of Myc protein is observed in many human cancers. Even in cancers in which MYC is not directly involved in transforming cells, its normal expression is often required to support the extracellular matrix and/or vascularization necessary for tumor growth and formation (4).

Because MYC is such a central player cancer pathology, it is an attractive target for cancer therapeutics  (2) .

Continue reading “Targeting MYC: The Need to Study Protein:Protein Interactions in Cells”

Thawing Out to Sing: The Story of the Wood Frog

Posted on by Michele Arduengo, PhD

Wood Frog_Northern WisconsinOne of the hallmarks of the arrival of Spring in Wisconsin is the cacophony of evening croaks and calls from the Spring Peepers and Chorus frogs. Indeed frogs and toads are ubiquitous around the globe, and many of us who have become life scientists (even those of us who have relegated ourselves to the world of macromolecules, cell signaling networks, and nucleic acids) probably spent some time in our childhood chasing and catching frogs.

But what happens to those frogs and toads over the harsh winter months in places like Wisconsin? Well, their strategies are species-dependent, but at least some of them overwinter by freezing, and the story of one species, the Wood Frog, is quite amazing. Think about it. It freezes from the inside out. No heart beat, no circulation, completely dormant. Then in response to some unknown signal (day length? temperature? angle of the sun?), bodily functions slowly resume. What kind of cell signaling cascade controls that response?

Here is a video from NOVA about the Wood Frog and its amazing deicing event. The next time you are out on a Spring or Summer evening and you hear a chorus of frogs calling, you can think about the incredible molecular story behind the event and be even more impressed!

A NOVA Video about the Wood Frog:

 

Get Ready to Celebrate Pi!

Posted on by Michele Arduengo, PhD

26562342_lWhat will you be doing on 3/14/15 at precisely 9:26:53? Twice the clocks will align with the first few digits of my favorite irrational and transcendental number (3.141592653…) or Ď€. Over 1 trillion digits to the right of the decimal point have been calculated, and they still go on, never making a pattern, never repeating.

Take any circle, that blueberry pie that your grandmother baked would be a good choice. Measure the circumference and divide by the diameter and you will have Pi, the mathematical constant that represents the ratio of circumference to diameter in a circle. You probably encountered Pi in your early mathematics education when learning the formula for the area of a circle (2Ď€r2).

In celebration of Pi, here are a few Pi diversions to brighten your day. Continue reading “Get Ready to Celebrate Pi!”