Fluorescent noise in understanding and treating diseases

After being diagnosed with cancer, Dr. Tim Lei changed his research focus from pure optics to biomedical applications. Since then, he has sought to apply his research techniques towards “Advanced fluorescence microscopy and linear and nonlinear spectroscopy techniques in biomedical applications.” By filtering through noise, Lei was able to determine the movement of proteins in cells using fluorescent dyes, characterizing the difference in protein motion between healthy and infected cells.

One of the studies Lei has been doing involves transport of phosphates throughout the body. Phosphates are components of the structure of DNA and RNA. They also make up ATP and ADP, which are used in making and storing energy in cells. When proteins are constructed, they are formed in such a way that they perform only one function. A phosphate molecule can activate or deactivate a protein’s function through a process called Phosphorylation. Hydroxyapatite, whose chemical formula contains calcium and phosphates, is the primary mineral component of bone, which makes bones a huge reservoir for calcium and phosphate in the body.

Every day, an average person will consume 1200 milligrams of phosphate. Most is absorbed through the intestines and the rest enters the bloodstream. The kidney filters most of the phosphorus in the body, roughly 800 milligrams. Those who develop kidney disease have a hard time extracting extra phosphate from the blood stream. The extra phosphate builds up as plaque in the arteries and can lead to a heart attack, which is why many with kidney disease die from heart disease.

Lei’s research looks specifically at the kidney and how it filters phosphate from the body. The blood reaches a collection of capillaries called the Glomerulus, which pass filtered material to the Bowman’s Capsule. There they pass through the proximal tube, which is where the kidney regulates phosphate concentration through active transport. This process requires two isoformic cotransport proteins that reside on microvilli covering the Brush Border membrane called NaPi-2a and NaPi-2c respectively. NaPi-2a mediates seventy percent of the reabsorption from the plasma filtrate and NaPi-2c mediates the rest. To study the transport process, Lei uses fluorescent dyes that are encoded as proteins and tagged onto the end of the protein you want to study. This is inserted into the cell and the cell will replicate the protein and the fluorescent dye. Using spectroscopy techniques, Lei was able to track the concentration of each cotransport protein in a variety of situations.

Lei examined the internalized mechanics of each cotransport protein. He gave mice kidney disease and analyzed their kidney membranes, graphing the concentrations of each cotransport protein on the surface of the membrane over time. The NaPi-2a extraction is relatively fast with an intensity that dropped significantly over thirty minutes while the NaPi-2c protein’s intensity dropped at roughly four hours.

Lei argues what many consider just noise on fluorescent spectroscopy results is actually proteins moving around. Using a correlation calculation, Lei found the NaPi-2a diffusion increased after twenty minutes of hormone treatment while the NaPi-2c diffusion increased after sixty minutes of hormone treatment. Therefore the internalization mechanisms of the two proteins are indeed different.

Lei also examined the possibility of NaPi-2a and NaPi-2c interacting with each other. He examined the proteins using cross correlation. In short, he measured one protein’s movements with one detector and the other protein’s movements with another detector and performed a cross correlation measurement. Because there was no increase in the calculation, Lei concluded the two proteins do not travel together.

By understanding how transport proteins work, even in areas that are just considered noise, kidney disease can be understood better. Lei has shown that the optical techniques he and others are using can be applied to areas other than kidney disease.
Lei is an assistant professor in the Electrical Engineering Department at the University of Colorado at Denver with adjunct positions in the Bioengineering, Physiology and Biophysics, and Medicine departments at the university’s Anschutz Medical Campus.



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