Penquite, Cara (MU-Student)

Pew Charitable Trusts may be best known for its non-partisan think tank subsidiary that focuses on demographic and social science issues, but its mission to improve public policy and inform bleeds over into support for science.

David Mendoza — a Bond Life Sciences Center principal investigator and associate professor of plant sciences in the College of Agriculture, Food and Natural Resources — received a $200,000 grant for biomedical sciences from the Pew Charitable Trusts.

“To have people you need plants, so if we manage to engineer better and more nutritious plants, the chances of improving human health are high,” Mendoza said.

Mendoza was one of six pairs of scientists awarded grants for collaboration. Mendoza will team up with Clarissa Nobile, an associate professor at the University of California Merced, to look at plant-microbe interactions within the context of iron intake. Both Mendoza and Nobile are previous Pew scholars/ fellows, making them eligible for this Pew Innovation Fund grant to work together on cutting-edge science.

Most of Mendoza’s work involves growing plants in the lab, looking at how plants manage essential nutrients including heavy metals like iron. With this grant, he plans to take the next step to grow plants in a less sterile environment and exploring the role of the microbiome on nutrient acquisition.

Bacteria and other microscopic organisms surrounding plants are known as the microbiome. These microorganisms don’t just sit passively around the plant, they interact and affect plant’s health and behavior.

“That communication between plants and microorganisms led to the discovery of growth-promoting bacteria, which could be used as biofertilizers,” Mendoza said. “We [want to] find which bacteria really helps the plant to fight against pathogens, then instead of spraying them [with fertilizers], maybe we can just spike them with the right combination of bacterial communities.”

Mendoza’s expertise lies in plant nutrition, and Nobile brings the microbiology expertise. Nobile will send collections of bacteria known as synthetic communities, or SynComs, for the Mendoza lab to use on plants to study how they affect plant nutrition.

“Pew people are eager collaborators, so Clarissa replied immediately, and said, ‘yes, absolutely,’ and we got into a Zoom call,” Mendoza said. “We wrote the grant together, and we submitted together.”

The grant allows for flexibility during the research process. If Mendoza and Nobile start their research on one path and discover another path they want to pursue, they are allowed to change their plans.

“You get judged by the idea in general,” Mendoza said. “So, we have a lot of flexibility when it comes to execution and changes, if we have to. It’s like a priming type of award to get you to the next stage.”

Getting to the next stage of Mendoza’s project would mean learning more about how SynComs can improve plant nutrition.

“It was not about cancer, it was not about a disease, it’s about plant nutrition for human nutrition,” Mendoza said.

Pew Innovation Fund 2022 projects range from cancer and neurology to schizophrenia and animal regeneration. Find out more about all six 2022 collaborations at pewtrusts.org.

By Cara Penquite | Bond LSC

Kamlendra Singh sat down in his fourth floor office as a Bond LSC Investigator for the first time on September first after nearly 14 years at the Bond Life Sciences Center studying HIV, COVID-19 and how the right molecule can interact to fight disease.

Designer compounds are Singh’s specialty, and as a new principal investigator he directs four projects on microscopic treatments for various diseases. He will also continue his work as Molecular Interactions Core Director alongside his new role.

“Everyone [in the center] is such a tight community … That’s why this building is a benefit to be here,” Singh said.

Bond LSC director Walter Gassmann recalls Singh’s arrival at the center as an assistant research professor.

“He developed independent ideas very quickly, but there was no tenure track position for him,” Gassmann said. “That changed in January of this year.”

As a research center, the LSC does not have tenure-track positions and partners with academic units for those. Once Singh obtained a tenure-track position in the Department of Veterinary Pathobiology, faculty members in the LSC voted Singh into the LSC Investigator position after he presented his research plan during a chalk talk.

“He’s done it. He’s done great work, and he deserves it,” Gassmann said.

As Singh pivots into his new role, he tackles major projects designing molecules as solutions to diseases that may pave the way for future treatment options.

Antiquated Antivirals

A drug is only as good as its ability to avoid resistance.

That’s the case with HIV, which is manageable with medication but evolves and mutates to avoid getting caught. Singh’s job is to be smarter.

Singh’s idea is to find new HIV antivirals that block the virus from infecting host cells. That goal could lead to novel antivirals that work for mutated HIV subtypes in underdeveloped countries.

“Most of the HIV treatments are developed where there is money, that is in developed countries,” Singh said.

HIV treatments are often made for the version of the virus that exists in first-world countries where the research takes place. Singh, on the contrary, concentrates his efforts on overlooked strains of HIV that often exist in underdeveloped countries, trying to understand the structural components of the virus to improve the treatment.

“We have to develop the drugs such that they work everywhere, but to do that you have to know the mechanism first,” Singh said.

Correcting Concentrations

Too much of a good thing can become bad, especially in living organisms. That’s where Singh’s second project focuses, tackling copper concentrations.

Too much copper facilitates cancer growth and spread. Singh works with Bond LSC researcher Michael Petris to find ways to hijack molecules that transport copper in and out of the cell. With funding from the National Cancer Institute, they have already found two potential molecules.

“Copper is a trace metal, and if it accumulates too much into the cell … then those cells become cancerous cells,” Singh said.

Singh’s preliminary trials sifted through millions of drug candidates and then tested 10 compounds to find one that could bind to the copper transporters and block copper’s path into the cell. Out of the initial 10, two were successful.

“That’s a lot. In this kind of drug discovery if you can get two out of a million that’s a success,” Singh said.

He plans to continue searching for viable compounds that could be used as cancer treatment, but his compounds may also be multi-purposeful.

Certain bacteria have similar compounds to the copper transporters, and just as viruses mutate to avoid antivirals, bacteria rarely go down without a fight.

Resistance bacteria mutate to survive antibiotic treatments, but Singh’s compounds may bind to compounds in the bacteria and have antibacterial roles.

“So, now we want to develop antibiotics using those drugs but making subtle changes. We want to make it specific for bacteria,” Singh said.

Viral Inhibitors

In the face of the COVID-19 pandemic, Singh’s third project tackles another set of viruses.

“The idea was to discover an inhibitor that not only blocks this coronavirus, but it can also be used for other viruses,” Singh said.

Tackling a diverse portfolio of viruses with a single method sounds impossible with vast differences between each pathogen. Singh’s solution — look for a  common compound.

The enzyme RNA polymerase might be just that.

It’s role is to copy the virus’ genetic material to create more virus within the host. By targeting RNA polymerase, Singh hopes to prevent the virus from replicating.

“Not only can it be effective against Coronavirus … but other viruses, let’s say Zika virus for example,” Singh said.

Returning to Roots

Singh’s fourth project brings him back to his postdoctoral work with a fresh perspective and decades of more experience. He turns to DNA polymerases now with the goal of cancer treatment.

Similar to RNA polymerase in viruses, DNA polymerase’s job is to replicate the genetic material for new cells in humans.

Every day, cells replicate to replace damaged or dead cells. Think about your skin — skin cells are constantly exposed to the elements and suffer wear and tear. When skin cells die, nearby cells divide to create new cells in their place. To create new cells, the first step is to duplicate the genetic material in a cell to be used in the new cell.

But with cancer cells, DNA polymerase replicates faulty genetic material and creates more cancer cells. If DNA polymerase can be stopped in cancer cells, the faulty genome will not be replicated.

“So that’s a brand new project, and I’m really excited about it,” Singh said.

By Cara Penquite | Bond LSC

Gene-editing is the pinnacle of a biologist’s toolbox, but often left unexplained it seems more magic than science.

Growing rice from a small cluster of cells to 4-foot stalks can take six-months or more of planning and careful nurture. But how do scientists change the intricate genetic material in each cell of the plants?

The CRISPR-Cas9 gene editing tool changes a plant’s DNA. As Ph.D. student Ajay Gupta knows firsthand through work altering plants for the Bing Yang lab.

“CRISPR is relatively new. It’s like 10 years old only and still we are working to modify it and improve it in plants,” Gupta said.

The process starts with a few cells from seeds capable of maturing into any type of cell to form a cell cluster, known as a callus.

But how do scientists insert instructions into a cell?

In the Yang lab, they use particle bombardment to insert DNA, fundamental instructions used to build everything within a cell. These inserted DNA strands tell the cells how to build the CRISPR tools to change their own DNA.

To shoot the DNA molecules into the cells, researchers use high pressure helium gas operated gene gun to bombard the cells with DNA coated gold particles.

Once the DNA instructions are in the cell, they build the CRISPR-Cas9 tool using the plant’s genetic machinery.

CRISPR-Cas9 has two parts: the Cas9 protein and an RNA molecule that only binds to the DNA section scientists want to change. This RNA strand guides the Cas9 to the correct location.

But the parts of the guide RNA do two things. First, the spacer part — usually about 20 base pairs only binds to the section of DNA researchers want it to. Second, a part called the scaffold — nearly four times the size of the spacer is required for attaching to the Cas9.

When the spacer of guide RNA binds to the DNA, the Cas9 enzyme acts like a pair of scissors, cutting that section of DNA.

Now we have DNA broken in two, and the plant’s built-in repair system starts its job. However, this repair system is flawed, and it usually messes up while rebuilding the DNA’s bases, or building blocks. Its errors vary every time, so many different mutations are possible.

“The repair machinery which is employed is not perfect,” Gupta said. “When it repairs those double-strand breaks, it causes some errors. The error could be a single base, and it could be 10 bases or it could be a thousand bases. So that is unpredictable.”

Think of it this way: imagine you have a ten-page instruction manual, and you rip out pages two and three. You could replace them with blank, yellow or red pieces of paper and the book would have ten pages again, but the instruction manual can’t be used again. Just like that, it doesn’t matter which error happens, because any mutated change in the gene results in it not working.

“This is how the normal CRISPR-Cas9 works, when you just want to shut that gene down,” Gupta said.

This puts the gene’s instructions out of commission and is commonly called a knockout gene. That helps scientists determine what the particular gene does. They then compare “normal” plants to plants with the knocked out gene to determine what happens differently.

Now the waiting happens. Researchers grow the plants using different hormones to mature the callus cells into roots and stems.

“Once they have shoots and roots then you transfer them to soil and grow the complete plants out of that,” Gupta said.

When the plants become adults, the researchers check the knocked out changes by extracting its DNA in a process known as genotyping.

While traditional CRISPR-Cas9 is used by researchers to learn more about plant genomes, Gupta and others in the Yang lab also work to edit parts of DNA and replace it with specific mutations. This process is known as prime editing.

“The exciting part about this [prime editing] is that theoretically it can do anything,” Gupta said.

Prime editing takes traditional CRISPR-Cas9 editing a step further, and the Yang lab’s research into it opens the door for future collaborations with labs around the world in applied and basic sciences.