Drug delivery systems control the time and place therapeutics are released inside the body. An essential component of many drug delivery systems is the molecule that links an antibody — which seeks out a target such as cancer cells — to a drug designed to destroy the target. The linker must not only keep the drug tethered to the antibody as it travels to a targeted cell, it must properly release the drug inside the cell, in the right place at the right time.

Biomolecular engineers gather clues about linkers’ effectiveness by testing them in cell-free environments — model fluids that don’t contain all the complex interactions typically found in whole, living cells. They’re easier to study, but aren’t always as accurate, potentially leading to linker failure in subsequent testing.

A research team led by Chris Alabi, associate professor of chemical and biomolecular engineering, has developed a method that employs fluorescent probes to see and measure the rate at which linkers successfully release drugs in living cells. The research, “Responsive Antibody Conjugates Enable Quantitative Determination of Intracellular Bond Degradation Rate,” published Oct. 8 in the journal Cell Chemical Biology.

“Right now, pharmaceutical companies make a ton of linkers and then see which functions best for a particular application by having to test each one. It’s a shotgun approach,” Alabi said. “With our technique, they can now make an informed decision based on actual intracellular numbers, before they put the drug system together.”

The probes consist of two fluorescent dyes that render each other invisible when they’re tethered together with the antibody as part of the delivery system. When the linker breaks and untethers the dyes, they become visible using a microscope, signaling the drug has been released inside the cell.

These probes were developed in Alabi’s lab and offer engineers an accurate way to measure the rate at which a linker’s chemical bond breaks from the delivery system while inside the cell.

“Once we know what the timing is for different linker bonds and cell processes, then we can say, ‘OK, for drug A connected to antibody B, this is how long it will take, so if we want to treat disease C, we should use this linker,'” Alabi said.

To demonstrate the probe, Alabi and his team engineered an antibody construct with a disulfide linker known to work well within the HER2 protein, a highly valued target for breast cancer treatment. Once the delivery system reached the protein, the team was able to reliably measure intracellular processes, such as the kinetics and half-life of the disulfide linker.

“For the biomolecular engineers and companies that have a target in mind, we can … make the drug discovery process that much faster because we now know something about how long it’s going to take to release the drug,” said Alabi, who hopes to demonstrate the technique further through partnerships with pharmaceutical companies.

The probes can also be used by chemical biologists to learn more about intracellular processes, such as the specific agents responsible for cleaving linkers, Alabi said.

The work was supported by funds from Cornell and the Nancy and Peter Meinig Investigator Fellowship.

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Materials provided by Cornell University. Original written by Syl Kacapyr. Note: Content may be edited for style and length.

Insulin is a hormone that plays a central role in precisely controlling levels of circulating glucose — the essential fuel used by cells -. Diabetes affects more than 30 million Americans according to the Centers for Disease Control and Prevention (CDC). In type II diabetes — the most common form of the disease — the cells of the body become inefficient at responding to insulin and as a consequence, glucose in circulation can become dangerously high (hyperglycemia) while the pancreas cannot produce enough insulin to compensate. In type I diabetes, the beta cells, which are the only cells in the body that produce insulin, are destroyed by the immune system resulting in complete lack of the hormone.

Current treatments include the administration of drugs that enhance the production of insulin by pancreatic beta cells, or direct injection of insulin to supplement the naturally produced supply. In both cases, regulation of blood glucose becomes a manual process, with drug or insulin intervention conducted after periodic readings of glucose levels, often leading to spikes and valleys that can have harmful long-term effects.

The researchers sought to develop a new way to amplify insulin production while maintaining the important real-time link between the release of insulin and concentration of glucose in the bloodstream. They accomplished this by taking advantage of ‘optogenetics’, an approach relying on proteins that change their activity on demand with light. Pancreatic beta cells were engineered with a gene that encodes a photoactivatable adenylate cyclase (PAC) enzyme. The PAC produces the molecule cyclic adenosine monophosphate (cAMP) when exposed to blue light, which in turn cranks up the glucose-stimulated production of insulin in the beta cell. Insulin production can increase two- to three-fold, but only when the blood glucose amount is high. At low levels of glucose, insulin production remains low. This avoids a common drawback of diabetes treatments which can overcompensate on insulin exposure and leave the patient with harmful or dangerously low blood sugar (hypoglycemia).

Researchers found that transplanting the engineered pancreatic beta cells under the skin of diabetic mice led to improved tolerance and regulation of glucose, reduced hyperglycemia, and higher levels of plasma insulin when subjected to illumination with blue light.

“It’s a backwards analogy, but we are actually using light to turn on and off a biological switch,” said Emmanuel Tzanakakis, professor of chemical and biological engineering at the School of Engineering at Tufts University and corresponding author of the study. “In this way, we can help in a diabetic context to better control and maintain appropriate levels of glucose without pharmacological intervention. The cells do the work of insulin production naturally and the regulatory circuits within them work the same; we just boost the amount of cAMP transiently in beta cells to get them to make more insulin only when it’s needed.”

The blue light simply flips the switch from normal to boost mode. Such optogenetic approaches utilizing light-activatable proteins for modulating the function of cells are being explored in many biological systems and have fueled efforts toward the development of a new genre of treatments.

“There are several advantages to using light to control treatment,” said Fan Zhang, graduate student in Tzanakakis’ lab at Tufts and first author of the study. “Obviously, the response is immediate; and despite the increased secretion of insulin, the amount of oxygen consumed by the cells does not change significantly as our study shows. Oxygen starvation is a common problem in studies involving transplanted pancreatic cells.”

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“Our 15-year research project aimed to build a hologram printer with all the advantages of previous technologies while eliminating known drawbacks such as expensive lasers, slow printing speed, limited field of view and unsaturated colors,” said research team leader Yves Gentet from Ultimate Holography in France. “We accomplished this by creating the CHIMERA printer, which uses low-cost commercial lasers and high-speed printing to produce holograms with high-quality color that spans a large dynamic range.”

In The Optical Society (OSA) journal Applied Optics, the researchers describe the new printer, which creates holograms with wide fields of view and full parallax on a special photographic material they designed. Full parallax holograms reconstruct an object so that it is viewable in all directions, in this case with a field of view spanning 120 degrees.

The printer can create holograms from 3D computer generated models or from scans acquired with a dedicated scanner developed by the researchers. The high-quality holograms can even be used as masters to produce holographic copies.

Building a better printer

When developing the new hologram printer, the researchers carefully studied two previously developed holographic printer technologies to understand their advantages and drawbacks.

“The companies involved in developing the first two generations of printers eventually faced technical limitations and closed,” said Gentet. “Our small, self-funded group found that it was key to develop a highly sensitive photomaterial with a very fine grain rather than use a commercially available rigid material like previous systems.”

The CHIMERA printer uses red, green and blue low-power commercially available continuous wave lasers with shutters that adjust the exposure for each laser in a matter of milliseconds. The researchers also created a special anti-vibrating mechanical system to keep the holographic plate from moving during the recording.

Holograms are created by recording small holographic elements known as hogels, one after another using three spatial light modulators and a custom designed full-color optical printing head that enables the 120-degree parallax. After printing, the holograms are developed in chemical baths and sealed for protection.

The hogel size can be toggled between 250 and 500 microns and the printing rate adjusted from 1 to 50 hertz (Hz). For example, if a hogel size of 250 microns is used, the maximum printing speed is 50 Hz. At this speed it would take 11 hours to print a hologram measuring 30 by 40 centimeters, about half of the time it would take using previous systems based on pulsed lasers.

High brightness and clarity

The researchers used the new technology to print holograms that measured up to 60 by 80 centimeters showing various color objects including toys, a butterfly and a museum object.

“The new system offers a much wider field of view, higher resolution and noticeably better color rendition and dynamic range than previous systems,” said Gentet. “The full-color holographic material we developed provides improved brightness and clarity while the low-power, continuous wave lasers make the system easy to use.”

The researchers say that as technology improves, especially 3D software, it may be possible to expand their hologram printing approach to medical or other advanced applications.

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Dr. Markus Hardt, Assistant Member of the Staff at Forsyth and co-author of the study, is an expert in mass spectrometry and salivary diagnostics. His team helped analyze the protein samples from humans, chimpanzees and gorillas for the study.

“For this study, we were interested in how human evolution impacts saliva and how food sources may have influenced human development, and whether that’s reflected in saliva as well,” Hardt said.

Saliva as a window into disease

Hardt is interested in saliva composition because it may serve as a noninvasive diagnostic fluid for certain oral and systemic diseases. He contributes to the Human Salivary Proteome Wiki, a database of more than 1,000 unique human saliva proteins open to the public and research community.

In other research, Hardt is developing a new sampling technique involving a paper strip that collects tiny amounts of saliva from specific salivary glands in the mouth. This method would be ideal for people with Sjogren’s syndrome, an autoimmune disease that causes dry mouth and dry eyes in more than 4 million Americans. Very little is known about how Sjogren’s syndrome impacts individual glands because there hasn’t been a way to take such focused samples.

“This new method could give a window into looking at the individual glands,” Hardt said.

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In a study published in the October 15 issue of Ecological Modelling, a team of University of Tennessee researchers along with a colleague from the University of Florida model how a chimeric Frog virus 3 (FV3)-like ranavirus, also known as RCV-Z2, can spread rapidly throughout a population of North American wood frog (Lithobates sylvaticus) tadpoles.

Ranaviruses are globally emerging pathogens that affect amphibians, reptiles and fish, and they are threatening the diversity of our ecological systems, says Matt Gray, professor in the UT Institute of Agriculture Center for Wildlife Health. Gray is the founding and past director of the Global Ranavirus Consortium — an organization dedicated to creating partnerships to combat the global emergence of ranaviruses. “In our previous work, we found that RCV-Z2 is a recombinant ranavirus that has DNA from a strain in North America and one from Europe and Asia. We think these viruses mixed DNA in a bullfrog farm in southern Georgia — the outcome was a highly virulent hybrid virus! The point of this modeling effort was to demonstrate how this evolved virus with eastern hemisphere DNA can infect and spread in a widely distributed amphibian species,” says Gray. “The news is not good.”

The paper’s title says it all: “A highly invasive chimeric ranavirus can decimate tadpole populations rapidly through multiple transmission pathways.”

Led by two post-doctoral research associates in UT’s National Institute for Mathematical and Biological Synthesis, known as NIMBioS, who teamed with Gray and others from the UTIA Center for Wildlife Health and College of Veterinary Medicine, the study determined that transmission of the RCV-Z2 can occur efficiently through direct contact of hosts, necrophagy (feeding on corpses), and by waterborne transmission.

Their model predicts significant risks to amphibian diversity in North America, which can translate into broader biodiversity issues for other species. Previous studies have already demonstrated that this chimeric ranavirus can be transmitted to mosquitofish, turtles and multiple amphibian species.

In 2019, the Georgia Department of Natural Resources provided funds to UTIA and UF to look for the pathogen in wild amphibian populations near the original outbreak site where RCV-Z2 was isolated. The team collected more than 390 samples during last summer in wetlands in the Alapaha River watershed and are testing them for RCV-Z2. “The goal of this surveillance effort is to determine if RCV-Z2 is in the wild, and if so, if virulence remains high,” Gray indicates.

If RCV-Z2 is detected, the team can use their simulations to direct disease intervention strategies, such as management activities that reduce host contacts or environmental persistence of the pathogen.

It’s depressing to note the authors say that targeting only one transmission pathway is unlikely to thwart an invasion of the ranavirus. Also, no response on the part of wildlife management agencies and others interested in conservation is not an option if the diversity of native amphibians is to be preserved.

Additionally, Gray recommends a clean trade program for amphibians and other wildlife. “Currently, wildlife in trade fly under the radar because health certificates that verify animals are pathogen free are not required unlike livestock. As a consequence, wildlife and their pathogens are being moved around the globe, creating opportunities for genetic mixing in captive facilities and spillover to wild populations.”

These experiments exploring rice production in future climate conditions show rice yields could drop about 40 percent by 2100 — with potentially devastating consequences in parts of the world that rely on the crop as a basic food source. What’s more, changes to soil processes due to increased temperatures will cause rice to contain twice as much toxic arsenic than the rice consumed today. The research was published Nov. 1 in Nature Communications.

“By the time we get to 2100, we’re estimated to have approximately 10 billion people, so that would mean we have 5 billion people dependent on rice, and 2 billion who would not have access to the calories they would normally need,” said co-author Scott Fendorf, the Terry Huffington Professor in Earth system science at Stanford University’s School of Earth, Energy & Environmental Sciences (Stanford Earth). “We have to be aware of these challenges that are coming so we can be ready to adapt.”

The researchers specifically looked at rice because it is grown in flooded paddies that help loosen the arsenic from the soil and make it especially sensitive to arsenic uptake. While many food crops today contain small amounts of arsenic, some growing regions are more susceptible than others. Future changes in soil due to higher temperatures combined with flooded conditions cause arsenic to be taken up by rice plants at higher levels — and using irrigation water with naturally occurring high arsenic exacerbates the problem. While these factors will not affect all global commodities in the same way, they do extend to other flood-grown crops, like taro and lotus.

“I just didn’t expect the magnitude of impact on rice yield we observed,” said Fendorf, who is also a senior fellow at the Stanford Woods Institute for the Environment. “What I missed was how much the soil biogeochemistry would respond to increased temperature, how that would amplify plant-available arsenic, and then — coupled with the temperature stress — how that would really impact the plant.”

A naturally occurring, semi-metallic chemical, arsenic is found in most soils and sediments, but is generally in a form that doesn’t get taken up by plants. Chronic exposure to arsenic leads to skin lesions, cancers, aggravation of lung disease and, ultimately, death. It is especially concerning in rice not only because of its global significance, but also because the low-allergen food is often introduced early to infants.

“I think this problem is also crucial for people that have young kids in our society,” said lead author E. Marie Muehe, a former postdoctoral scholar at Stanford and now at the University of Tübingen, Germany. “Because infants are a lot smaller than we are, if they eat rice, that means that they take up more arsenic relative to their body weight.”

Climate simulations

The researchers created future climate conditions in greenhouses based on estimates of a possible 5 degree Celsius temperature increase and twice as much atmospheric carbon dioxide by 2100, as projected by the Intergovernmental Panel on Climate Change.

While previous research examined the impacts of increasing temperature in the context of the global food crisis, this study was the first to account for soil conditions in combination with shifts in climate.

For the experiments, the group grew a medium-grain rice variety in soil from the rice-growing region of California. The greenhouses were controlled for temperature, carbon dioxide concentrations and soil arsenic levels, which will be higher in the future due to its buildup in soils from irrigating crops with arsenic-contaminated water, a problem that is worsened by over-pumping groundwater.

“We don’t often think about this, but soil is alive — it’s teeming with bacteria and a lot of different microorganisms,” Fendorf said. “It turns out those microorganisms determine whether the arsenic stays partitioned onto the minerals and away from the plants or comes off the minerals into the water phase.”

The researchers found that with increased temperatures, microorganisms destabilized more of the soil’s inherent arsenic, leading to greater amounts of the toxin in the soil water that is available for uptake by the rice. Once taken up, arsenic inhibits nutrient absorption and decreases plant growth and development, factors that contributed to the 40 percent decrease in yield the scientists observed.

Early warning, future planning

While the dramatic loss in production is a major cause for concern, the scientists are hopeful that this research will help producers find potential solutions for feeding the world.

“The good news is that given past advances in terms of the global community’s ability to breed varieties that can adapt to new conditions, along with revisions to soil management, I’m optimistic we can get around the problems observed in our study,” Fendorf said. “I’m also optimistic that as we continue to shine a light on the threats resulting from a 5 degree Celsius change, society will adopt practices to ensure we never reach that degree of warming.”

As next steps, Fendorf, co-author Tianmei Wang and Muehe hope to asses rice yields on a global scale by using remote sensing to pinpoint contaminated rice paddies in order to model future yields and arsenic contamination.

“This is most likely to be a problem where most rice is consumed, so we think about South and East Asia,” said Wang, a PhD candidate in Earth system science. “Especially for people like my dad — he consumes rice three times a day and he just cannot live without it.”

Fendorf is also a member of the Maternal & Child Health Research Institute (MCHRI). Researchers from the Bayreuth Center for Ecology and Environmental Research at the University of Bayreuth, Germany, are co-authors on the study.

The research was funded by the Marie Sklodowska Curie Action Fellowship of the European Commission, the U.S. Department of Energy, the National Institutes of Health, National Institute of General Medical Sciences.