Bioluminescence

Neurons’ Role in FBP2 Regulation

Posted on by Kari Kenefick

Neuronal extracellular vesicles (NEVs) play a significant role in the communication between neurons and astrocytes, particularly by influencing metabolic processes such as glycolysis and lactate production. NEVs carry signaling molecules that affect the expression, degradation and oligomeric state of fructose 1,6-bisphosphatase 2 (Fbp2) in astrocytes, altering their metabolism (1).

Basic Backstory on CNS Architecture
The central nervous system (CNS) is composed of an intricate cellular communications complex, divided generally into neurons and glial cells. Neurons form the electrical signaling network, with dendrites receiving and integrating signals via chemical synapses, and axons, some up to 1 meter in length, rapidly transmitting the signals.

Glial cells, including astrocytes, microglia and other cells, interact with neuronal cells to sustain this network. For example, glial cells regulate synapse formation and provide metabolic support to promote CNS homeostasis. Glial cell dysfunction contributes to most neural diseases and can even drive neurodegenerative processes (2).

What are Neuronal Extracellular Vesicles (NEVs)?
NEVs are formed by neurons via endocytosis and are released into the extracellular space where they interact with astrocytes. These transport vesicles carry a variety of molecules, including proteins and RNA, that influence cellular processes in recipient astrocytes.

NEV and Astrocyte Interactions
Fbp2 is an important enzyme involved in glycogen synthesis that also has nonenzymatic functions, including support of neuronal processes like long-term potentiation (LTP). LTP underlies synaptic strength and plasticity and is important in both learning and memory formation.

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Visualize Protein:Protein Interactions with Bioluminescence Imaging

Posted on by Simon Moe

If you’re familiar with bioluminescence, you’ve probably used it in plate-based experiments to track various biological processes. You understand it provides distinct advantages over traditional fluorescence assays, particularly when it comes to sensitivity. However, there’s always that one nagging question: how representative is the signal on a cell-to-cell level?

Traditional approaches to decipher cell-to-cell signal rely on complex, time-intensive measures that only approximated the findings acquired through bioluminescence. That’s where the GloMax® Galaxy Bioluminescence Imager comes in. This new tool will enhance your ability to visualize proteins using NanoLuc® technology, going beyond simple numeric outputs to reveal what’s happening in individual cells.

NanoLuc® technology is well-known for its ability to assist in detecting subtle protein interactions in complex biological systems. This bright luminescent enzyme enables a much broader linear range than fluorescence, improving detection of small changes in protein activity, such as proteins interacting. Microplate readers measuring NanoLuc® assays rely on signal generated from many cells. This results in an approximation of what is occurring biologically. Truly validating those luminescent readings within a cell population has been challenging—until now. The GloMax® Galaxy allows you to perform bioluminescence imaging, moving beyond the numbers, offering the power to visualize protein interactions directly.

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The Marvel of Malate: A Crucial Component in Cellular Energy Metabolism

Posted on by Simon Moe

Today’s blog written by guest author Kim Haupt.

Cellular energy metabolism is a complex biological process that relies on a suite of metabolites, each with distinct roles to maintain. Malate is one of these metabolites and is essential for maintaining cellular function through important roles in both energy production and redox homeostasis. In this blog, we highlight malate’s diverse roles and uncover some of its connections to human disease. 

Illustration of energy metablism in cell.
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The Battle of Shiloh’s Angel’s Glow: Fact, Civil War Legend or Modern Myth? 

Posted on by Kelly Grooms

It sounds like the script for a Hollywood movie. The story, first appearing in 2001, begins with a purported civil war legend from the Battle of Shiloh. The legend said that the wounds of some soldiers glowed (faintly) in the dark. Soldiers with these glowing wounds were more apt to survive, giving the phenomenon the name “Angels Glow”. The story ends with two curious teenagers solving the mystery using their science fair project. They identify infection by the bioluminescent bacteria Photorhabdus luminescens (formerly Xenorhabdus luminescens) as the likely cause of the glowing wounds. P. luminescens produces bacteriocins (antimicrobial peptides), which the teenagers attribute to helping keep other infections at bay, resulting in the improved survival rate for the soldiers whose wounds glowed.

The teenagers win. The mystery is solved. The credits roll. 

Except life (and science) is rarely as simple as a summer block buster. 

Cannon at sunset on a civil war battlefield
The Battle of Shiloh took place in Hardin County Tennessee on April 6th and 7th, 1862.
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Expert Insights: A Look Forward at Multiplexing for in vivo Bioluminescence Imaging

Posted on by Simon Moe

Bioluminescent in vivo imaging tools

NanoLuc, NLuc

With advancements made over the past few decades, the future of in vivo bioluminescence imaging (BLI) continues to gain momentum. In vivo BLI provides a non-invasive way to image endogenous biological processes in whole animals. This provides an easier method to assess relevant systems and functions. Unlike fluorescent imaging, BLI relies on a combination of enzymes and substrates to produce light, greatly reducing background signal (Refaat et al., 2022). Traditional fluorescent tags are also quite large and may interfere with normal biological function. In vivo BLI research has been around for quite some time, primarily utilizing Firefly luciferase (Luc2/luciferin). A recent advancement was the creation of the small and bright NanoLuc® luciferase (NLuc). Promega offers an wide portfolio of NLuc products that provide ways to study genes, protein dynamics, and protein:protein interactions. To fully grasp the power of these tools, I interviewed several key investigators to determine their perspectives on the future of in vivo BLI. I was specifically interested in their thoughts on NLuc multiplexing potential with Firefly (FLuc), and future research areas. These two investigators are Dr. Thomas Kirkland, Sr. Scientific Investigator at Promega, and Dr. Laura Mezzanotte, Associate Professor at Erasmus MC.

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Cell-Based Target Engagement and Functional Assays for NLRP3 Inhibitor Profiling Help Identify Successes and Failures

Posted on by Kari Kenefick

Identifying Inflammasome Inhibitors: What’s Missing
The NLRP3 inflammasome is implicated in a wide range of diseases. The ability to inhibit this protein complex could provide more precise, targeted relief to inflammatory disease sufferers than current broad-spectrum anti-inflammatory compounds, potentially without side effects.

Studies of NLRP3 inflammasome inhibitors have relied on cell-free assays using purified NLRP3. But cell-free assays cannot assess physical engagement of the inhibitor and target in the cellular micro-environment. Cell-free assays cannot show if an NLRP3 inhibitor enters the cell, binds the target and how long the inhibitor binding lasts.

Cell-based assays that interrogate the physical interaction of the NLRP3 target and inhibitor inside cells are needed.

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The Power of Pyruvate, A Pivotal Player in Cellular Energy Metabolism

Posted on by Simon Moe

Today’s blog written by guest author Kendra Hanslik.

In the intricate dance of cellular processes that sustain life, pyruvate emerges as a central figure. It plays a crucial role in the energy production saga. This small molecule is the linchpin between glycolysis and the citric acid cycle, linking the breakdown of glucose to the production of adenosine triphosphate (ATP). In this article, we explore pyruvate’s origins, multifaceted roles, and its association with various diseases.

Illustration of energy metablism in cell showing the mitochondria where pryruvate is metabolized.
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Monoamine Oxidase and Mental Health: From Psychedelics to Diet

Posted on by Simon Moe
Kiwi fruit are thought to contain compounds that naturally inhibit monoamine oxidase

Public awareness of mental disorders has increased over the past decade. Post-traumatic stress disorder (PTSD), anxiety and depression are both debilitating and complex to approach therapeutically. Recent research has begun exploring monoamine oxidase (MAO) enzymes as potential treatment options. MAO enzymes are responsible for the metabolism of monoamine neurotransmitters in the central nervous system, such as serotonin and dopamine (Jones & Raghanti, 2021). Abnormal levels of these neurotransmitters within the nervous system are a key characteristic of several neurological conditions. Thus, exploring MAO regulation may help our understanding of these complex clinical conditions.

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Glowing Testimonies: A Review of NanoLuc® Use in Model Organisms

Posted on by Kerstin Hurd
NanoLuc®

Model organisms are essential tools in the pursuit of understanding biological processes, elucidating the mechanisms of diseases, and developing potential treatments and therapies. Use of these organisms in scientific research has paved the way for groundbreaking discoveries across various fields of biology. In particular, non-mammalian models can be valuable due to characteristics such as rapid life cycles, low cost, and amenability to use with advanced genetic tools, including bioluminescent reporters such as NanoLuc® Luciferase.

NanoLuc® is a small (19.1 kDa) luciferase enzyme originating from deep sea shrimp that is 100x brighter than firefly or Renilla luciferase. It utilizes a furimazine substrate to produce its bright glow-type luminescence. In the decade following its development, the NanoLuc® toolbox has expanded to include NanoBiT® complementation, NanoBRET™ energy transfer methods, and new reagents such as the Nano-Glo® Fluorofurimazine In Vivo Substrate (FFz) which was designed for in vivo detection of NanoLuc® Luciferase, NanoLuc® fusion proteins or reconstituted NanoBiT® Luciferase. In addition to the aqueous-soluble reagents increased substrate bioavailability in vivo, with fluorofurimazine, NanoLuc® and firefly luciferase can be used together in dual-luciferase molecular imaging studies.

Here we spotlight some recent research that demonstrates how the expanded NanoLuc® toolbox can be adapted to use in non-mammalian models, shedding new light on fundamental biological processes and advancing our understanding of complex mechanisms in these diverse organisms.

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Illuminating the Brain with a New Bioluminescence Imaging Substrate

Posted on by Kari Kenefick

Bioluminescence imaging is a powerful tool for non-invasive studies of the effect of treatments on cells and tissues. The luminescent signal is strong, and can be used in vivo, enabling repeated observations over time, allowing longitudinal study of cellular changes for hours or days. Bioluminescence imaging can be used in live animals over varying periods of time, without interfering with normal cellular processes.

Fluorescence imaging is also used in cellular studies. Although it can provide a stronger signal than luminescence, fluorescence requires light for excitation, and thus its in vivo use is limited at a tissue or cell depth greater than 1mm.

NanoLuc® Luciferase. Small, bright and now useful in brain bioluminescence imaging.

In addition, autofluorescence can be an issue with fluorescence imaging, as cellular components and surrounding proteins and cells can fluoresce when exposed to light. Autofluorescence can result in high background signals, making it difficult to distinguish true fluorescence from background.

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