Cielo and Sapphire aid in understanding osteoarthritis at the University of Delaware

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Customer Spotlight: Sofia Gonzalez, graduate student in Parreno Lab at University of Delaware (UD)

Students from the Parreno Lab at the University of Delaware (UD) use the Cielo qPCR System and Sapphire Biomolecular Imager to help develop novel actin-based therapeutics to combat degenerative diseases, such as osteoarthritis.

Since the release of this interview, the Sapphire has been succeeded by the Sapphire FL, which was designed to be the flexible choice in bringing precise quantitation of nucleic acids and proteins. Learn more.

Cartilage is the flexible tissue around the ends of bones at the joints that two bones to smoothly glide over one another. Osteoarthritis is one of the most common forms of arthritis and affects millions of people worldwide1. It is a degenerative joint disease that causes progressive damage to cartilage. For those affected, osteoarthritis can often lead to pain, stiffness, and ultimately loss of joint function.

Actin is a protein that plays an important role in the formation and maintenance of cartilage. When chondrocytes (the cells responsible for the maintenance of cartilage) are exposed to the cytokines associated with osteoarthritis, actin polymerization is activated2. Due to this, Principal Investigator and UD Assistant Professor Dr. Justin Parreno suspects there could be a connection between actin signaling and osteoarthritis.

Gene expression and cartilage

UD Graduate student Sofia Gonzalez is investigating the expression of an important lubricating molecule secreted by cartilage, called proteoglycan-4 (PRG-4). Gonzalez is interested in learning how to stimulate PRG-4 production; her findings could potentially prevent the onset of arthritis. However, her research path wasn’t always clear.

Initially, Gonzalez’s project began with studying hip cartilage from an ex-vivo mouse model of osteoarthritis after it was cultured for two days. She would then use the Cielo for qPCR to determine gene expression and note any changes along the way. Gene expression can tell us how a cell is functioning at a given time and is a process that allows a cell to respond to its environment. A specific protein can only be produced when its gene is turned on.

What is gene expression?

It was shortly after her initial observations that Gonzalez discovered something fascinating: the actin cytoskeleton (outer layer of cells responsible for maintaining cell structure) was changing and reorganizing itself. What she was noticing meant the gene expression changes were correlated with the cytoskeletal changes. Gonzalez has since characterized what the actin cytoskeleton looks like in chondrocytes and how it is arranged in correlation with PRG-4 expression. She has been able to emulate osteoarthritis and disease-like changes ex-vivo with mouse femoral heads. With her discovery, she seeks to evaluate PRG-4 expression in the natural environment. 

The Cielo's Popularity in the Lab

Justin Parreno and lab members using Azure Cielo Manager qPCR software
Justin Parreno and other lab members using Azure Cielo Manager qPCR software

The Parreno Lab currently has about 20 members working to understand the regulation of biological phenomena by the cytoskeleton. The students periodically use analysis of gene expression in their projects, which makes the Cielo a popular and often fully booked machine due to its ability to do semi-quantitative and quantitative PCR. Gene expression is so demanded in the Parreno lab that learning to use the Cielo is one of the first skills a student joining the lab learns. With its simple interface and reliability, Gonzalez said using it is a good way to build confidence in new students.

Gene Expression as Easy as ABC

We are so confident that you will be impressed with the ease of use and performance of the Cielo that we'll arrange to send your lab a system to use for one week, without any obligation, absolutely FREE. See if your lab qualifies today.

Using the Sapphire for high-throughput screening and In-cell Westerns

In addition to using the Cielo, the Parreno lab also uses a Sapphire for a variety of projects, because they rely on the crisp and clear images it produces. The Sapphire is used to develop high-throughput screens with in-cell Westerns in 96-well plates (pictured below), with the aim of using the fluorescent intensity of proteins to determine protein regulation in chondrocytes. In-cell Westerns are useful in this case, as they allow the user to quantify proteins in cultured cells in situ to assess the effects of interventions, such as drugs, on protein levels without further manipulating the cells.

In cell western screen for Type II Collagen imaged using Azure Sapphire Biomolecular Imager
An in-cell Western scanned using the Sapphire provided by doctoral student Stephanie Richardson-Solorazno of the Parreno Lab. Eleven drugs were screened for their ability to promote cartilage deposition in cells.

In-cell Westerns quantify proteins that serve as biomarkers of disease progression. Used in research, ICWs give students and researchers the ability to assess potential therapeutics for anti-disease activity. The Sapphire is capable of in-cell westerns due to its sensitivity and speed. With four fluorescent channels including two NIR channels, the Sapphire facilitates multiplex detection like a pro.

Want to know more about in-cell Western blots? Read about them here.

Looking to the future

While the Parreno Lab has made significant progress in understanding osteoarthritis, much work remains to be done. Lab members are looking at how to quantify PRG-4 at the protein level, but she needs to develop a system to get enough protein to be able to detect it. Using mice presented the challenge of working with small bones, so the team switched to bovine cells, but are continuing to develop new assays for detecting protein in mice.

Since this interview was conducted, Gonzalez has finished her graduate degree, but her lab mates are continuing to research the biologies behind actin and cartilage and the diseases that are associated to develop novel therapeutic options for a better tomorrow.

Stay up to date with the Parreno Lab at UDEL by following their X: @ParrenoJustin

Read more customer spotlights:

SOURCES

  1. “Osteoarthritis (OA) | Arthritis.” CDC, https://www.cdc.gov/arthritis/basics/osteoarthritis.htm. Accessed 09 February 2023
  2. “Parreno Lab.” Parreno Lab, https://parrenolab.com/research/. Accessed 11 February 2023.

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