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.
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.
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.
Continue reading “Expert Insights: A Look Forward at Multiplexing for in vivo Bioluminescence Imaging”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.
Continue reading “Cell-Based Target Engagement and Functional Assays for NLRP3 Inhibitor Profiling Help Identify Successes and Failures”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.
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.
Continue reading “Monoamine Oxidase and Mental Health: From Psychedelics to Diet”
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.
Continue reading “Glowing Testimonies: A Review of NanoLuc® Use in Model Organisms”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.
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.
Continue reading “Illuminating the Brain with a New Bioluminescence Imaging Substrate”Traditional approaches for protein degrader compound screening like Western blotting can be laborious, time consuming and cannot be streamlined with automation. By implementing a high-throughput, automated workflow that uses our CRISPER/Cas9 knock-in cell lines, live-cell bioluminescent assays and sensitive GloMax® Discover microplate readers, our custom assay services offer protein degradation profiling at an accelerated rate.
To do this, we collaborated with HighRes® Biosolutions, to develop an automated system that can screen up to 100 384-well plates each day, generating roughly 40,000 data points with minimal hands-on work.
Learn how bioluminescent tools like HiBiT and NanoBRET™ technology can help you answer key questions in your targeted protein degradation research.
An important step of building this system is to integrate four GloMax® Discover microplate readers into the automated system using instrument’s built-in SiLA2 communication driver. The driver software makes it easy to connect the microplate readers with HighRes® Biosolution’s robotic components.
Check out our setup in the video below.
The first monoclonal antibody (mAb) was produced in a lab 1975, and the first therapeutic mAb was introduced in the United States to prevent kidney transplant rejection in 1986. The first mAb used in cancer treatment the anti-CD20 mAb, rituximab, was used to treat non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. Today therapeutic mAbs have become a mainstay of cancer, autoimmune disease, and metabolic disease therapies and include HERCEPTIN® used to treat certain forms of breast cancer, Prolia used to treat bone loss in post-menopausal women, and Stelara used to treat autoimmune diseases like psoriatic arthritis and severe Crohn disease, among many others. Therapeutic mAbs bind targets with high specificity and affinity and they can recruit effector cells to drive target elimination through mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), making them highly specific, effective therapies.
