An Ambitious Endeavor: The Human Proteoform Project

On November 15, 2021, Science Advances announced the launch of The Human Proteoform Project. The ambitious project, led by the Consortium for Top-Down Proteomics, aims to address a critical next step in disease research. This means developing new technologies to outline a complete set of protein forms based on the ~20,000 genes in the human genome.

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Save Precious Time with Same-Well Multiplexing

Scientist performing a multi-well assay. Same-well multiplexing enables you to look at one event from several perspectives.

A graduate student believes he has mastered the art of “the assay”. No need to run duplicates, he knows exactly which one will get him the answers he needs right away.  

To challenge this, his PI proposes an exercise. He asks of the graduate student, “What happens when you treat cells with doxorubicin?”

The graduate student raises his cells, treats them accordingly, and decides to run a cell viability assay to determine their fate. He returns to the PI with the final verdict: his cells are dead.

The PI takes a look at the data and asks the graduate student to repeat the experiment with an additional assay for cytotoxicity―but the cytotoxicity assay shows that the cell membranes are intact, which only puzzles the graduate student. The PI asks him to run a third assay for apoptosis, and when the student does so, it becomes clear that the cells are dying.

The PI uses this opportunity to make his point: “Now do you see why I ask for more than one assay?”

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Seeing is Believing: How NanoLuc® Luciferase Illuminates Virus Infections

Artists interpretation of in vivo imaging of viral infections in mice using NanoLuc luciferase.

Wearing blue surgical gowns and white respirator hoods, research scientist Pradeep Uchil and post-doctoral fellow Irfan Ullah carry an anesthetized mouse to the lab’s imaging unit. Two days ago, the mouse was infected with a SARS-CoV-2 virus engineered to produce a bioluminescent protein. After an injection of a bioluminescence substrate, a blue glow starts to emanate from within the mouse’s nasal cavity and chest, visible to the imaging unit’s camera and Uchil’s eyes.

“We were never able to see this kind of signal with retrovirus infections.” Uchil is a research scientist at the Yale School of Medicine whose work focuses on the in vivo imaging of retroviral infections. Normally, the mouse would have to be sacrificed and “opened up” for viral bioluminescent signals from internal tissues to be imaged directly.

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3D Cell Culture Models: Challenges for Cell-Based Assays

3D Cell Culture Spheroid
3D Cell Culture Spheroid

In 3D cell culture models, cells are grown under conditions that allow the formation of multicellular spheroids or microtissues. Instead of growing in a monolayer on a plate surface, cells in 3D culture grow within a support matrix that allows them to interact with each other, forming cell:cell connections and creating an environment that mimics the situation in the body more closely than traditional 2D systems. Although 3D cultures are designed to offer a more physiologically accurate environment, the added complexity of that environment can also present challenges to experimental design when performing cell-based assays. For example, it can be a challenge for assay reagents to penetrate to the center of larger microtissues and for lytic assays to disrupt all cells within the 3D system.

Earlier this week Terry Riss, a Senior Product Specialist at Promega, presented a Webinar on the challenges of performing cell-based assays on microtissues in 3D cell culture. During the Webinar, Terry gave an overview of the different methods available for 3D cell culture, providing a description of the advantages of each. He then discussed considerations for designing and optimizing cell-based  assays for use in 3D culture systems, providing several  recommendations to keep in mind when performing cell viability assays on larger microtissue samples.

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Permeability Possibilities with the New NanoClick Assay

Peptide Predicament

For decades now, peptides have been a molecule of interest for drug discovery research. Peptides offer a unique opportunity for therapeutic intervention that closely mimics natural pathways, as many physiological functions utilize peptides as intrinsic signaling molecules. Macrocyclic peptides, in particular, have recently proven to be promising candidates for targeting intracellular protein–protein interactions (PPIs), an attractive but hard-to-reach therapeutic target for conventional small molecule and biological drugs.  

As with any opportunity, there are also challenges that accompany the peptide therapeutic development. Peptide ligands typically have poor membrane permeability, so thus far the majority of peptide therapeutics predominantly target extracellular proteins and receptors. There are also multiple mechanisms for cellular uptake of peptides, including both energy-dependent routes like endocytosis, and energy-independent, like passive diffusion or membrane translocation. Multiple mechanisms of cellular uptake paired with poor permeability makes engineering enough membrane permeability into peptides in order to advance them through drug discovery pipelines extremely difficult.  

There are other factors to consider in developing peptide therapeutics, such as solubility, protein/lipid binding and stability, which can also have an affect on the overall cytosolic concentration and, ultimately impact the ability of the peptide to effectively engage its desired intracellular targets.

With so many challenging factors, the ability to have a predictive, high-throughput assay to assess cell permeability, independent of the mechanism(s) of entry, would be a critical and invaluable tool to support peptide drug discovery research.

In a recent study published in ACS Chemical Biology, researchers sought to develop such a tool, and demonstrated a new application for Promega NanoBRET™ technology: the NanoClick assay.

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The NLRP3 Inflammasome: Flipping the Switch

It’s been just over 10 years since the world lost a pioneering immunologist and biochemist, Dr. Jürg Tschopp. He died tragically during a hiking trip in the Swiss Alps on March 22, 2011. A host of academic journals, including Science, Nature and Cell, paid tribute to Dr. Tschopp with obituaries that highlighted his many accomplishments in the fields of apoptosis and immunology.

In 2002, a team led by Dr. Tschopp at the University of Lausanne, Switzerland, was studying the role of the proinflammatory cytokine interleukin 1 beta (IL-1β). This cytokine is produced in the cytoplasm as an inactive precursor (pro-IL-1β). It is cleaved by caspase-1 to the active form, but the exact process by which caspase-1 itself is activated was unknown at the time. Several members of the caspase family contain a conserved region known as the caspase recruitment domain or CARD, and it was proposed that this domain was essential to caspase activation.

Based on similarity to another protein containing an N-terminal CARD motif (Apaf-1) that is involved in activation of caspase-9, the researchers examined the roles of a family of proteins known as NALP1, NALP2 and NALP3 (1). In particular, they were interested in NALP1, which is involved in the immune response. Unlike Apaf-1, NALP1 contains a CARD motif at the C terminus, while the N terminus contains a related motif known as a pyrin-like domain (PYD). The research team had previously showed that the PYD region of NALP1 interacted with an adapter protein known as PYCARD or ASC, which also contains an N-terminal PYD and C-terminal CARD.

The results of the team’s in vitro binding, activation and immunodetection studies showed that a multi-unit protein complex is responsible for caspase activation, and they called this complex the “inflammasome” (1). It is composed of caspase-1, caspase-5, PYCARD/ASC and NALP1.

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Immune Checkpoint Bioassays Strengthen Cancer Research in the Development of New Therapies

This post was written by guest blogger, Nicole Werner, Product Management Support at Promega GmbH.

“You have cancer.” – a statement that fundamentally changes life in a second. After the first shock, the insight often arises: “If only I had stopped smoking sooner!”

Lung cancer, while not the leading cause of death worldwide, is the leading preventable cause of death in developed countries. According to the WHO, eight million people die each year as a result of smoking, including one million as a result of passive smoking [1]. Currently, 80% of those affected die within the next 13 months after diagnosis [1]. New therapeutic approaches, such as treatment with immune checkpoint inhibitors, bring hope.

Promega supports research in this area with the high-precision tools needed to develop this new form of therapy.

Artistic 3D rendering  of Immune checkpoint signaling. Immune checkpoint bioassays enable researcher to characterize  therapeutic antibodies trageting these pathways.
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Overcoming Challenges to Detect Apoptosis in 3D Cell Structures

This blog is written by guest author, Maggie Bach, Sr. Product Manager, Promega Corporation.

Researchers are increasingly relying on cells grown in three-dimensional (3D) structures to help answer their research questions. Monolayer, or 2D cell culture, was the go-to cell culture method for the past century. Now, the need to better represent in vivo conditions is driving the adoption of 3D cell culture models. Cells grown in 3D structures better mimic tissue-like structures, better exhibit differentiated cellular functions, and better predict in vivo responses to drug treatment.

Switching to 3D cell culture models comes with challenges. Methods to interrogate these models need to be adaptable and reliable for the many types of 3D models. Some of the most popular 3D models include spheroids grown in ultra-low attachment plates, and cells grown in an extracellular matrix, such as Matrigel® from Corning. Even more complex models include medium flow over the cells in microfluidic or organ-on-a-chip devices. Will an assay originally developed for cells grown in monolayer perform consistently with various 3D models? How is measuring a cellular marker different when cells are grown in 3D models compared to monolayer growth?

Close up of cells in 3D culture apparatus. 3D Cell Structures Provide Challenges for Measuring Markers of Cellular Activitiy
3D Cell Structures Provide Challenges for Measuring Markers of Cellular Activitiy
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Bringing Cutting Edge Technologies to Academic Researchers Through the Academic Access Program

This post was written by guest blogger Iain Ronald, Director Academic/Government Market Segment at Promega.

My back story is similar to most of you reading this blog, high school education, undergraduate degree then onto a postgraduate degree. However, over 25 years ago during my undergraduate study, I was fortunate enough to work in the lab of Professor Ray Waters studying DNA damage in S. cerevisiae as a model organism and at the time PCR was cutting-edge technology and the PCR license was in full effect. However, there was one company that was fighting the good fight to democratize PCR for the good of the scientific community, Promega.

I became enamored with Promega then, and the next steps in my career were taken with a view to working at this company who, for all intents and purposes, seemed to really care about the progression of science beyond self-aggrandizement.

Now that I am working at Promega in a position where I can bring benefit to our academic community, I have pondered what I can do to equal the disruptive attitude I observed in this company all those years ago when they were fighting the then “big tech” for the enablement of the scientific community. 

Reporter bioassays are one of hte many offerings of the academic access program.
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How Promega Helped Our Lab Scale Up Drug Discovery for Bloodborne Pathogens

This blog was written by Sebastien Smick, Research Technician in Dr. Jacquin Niles’ laboratory at Massachusetts Institute of Technology (MIT)

Our lab is heavily focused on the basic biology and drug discovery of the human bloodborne pathogen Plasmodium falciparum, which causes malaria. We use the CRISPR/Cas9 system, paired with a TetR protein fused to a native translational repressor alongside a Renilla luciferase reporter gene, to conditionally knock down genes of interest to create modified parasites. We can then test all kinds of compounds as potential drug scaffolds against these gene-edited parasites. Our most recent endeavor, one made possible by Promega, is a medium-low throughput robotic screening pipeline which compares conditionally-activated or-repressed parasites against our dose-response drug libraries in a 384-well format. This process has been developed over the past few years and is a major upgrade for our lab in terms of data production. Our researchers are working very hard to generate new modified parasites to test. Our robots and plate readers rarely get a day’s rest!

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