Tuesday, December 12, 2023

New tool to analyze blood platelets holds major medical potential

Graphic image shows a blood clot forming in an artery. The white, spikey platelets are amid red and white blood cells. Activated platelets use their spkes like "arms" to grip onto one another and to stringy chains of proteins in the blood called fibrin, forming clumps that also bind up red and white blood cells.

By Carol Clark

A novel technique to test platelet function within a person’s blood sample is faster, easier and more precise than methods currently in use, an experimental study shows. 

Nature Biomedical Engineering published the research, led by scientists at Emory University. The researchers demonstrated the proof-of-concept for the technique, which provides the first detailed look at the molecular forces generated by activated platelets in patient blood samples. 

The study results show that the technology holds the potential to assess the effects of antiplatelet drugs on individuals and to gain a clearer picture of bleeding risks for patients undergoing cardiopulmonary bypass surgery. 

The technique requires only about a drop of blood to run tests, compared to the tablespoon needed for current assays. This ultrasensitivity may make the technology a valuable tool for the diagnosis of babies suffering from rare, congenital platelet disorders. 

The breakthrough is based on synthetic-DNA tension probes developed more than a decade ago in the laboratory of Khalid Salaita, professor in Emory’s Department of Chemistry and in the Wallace H. Coulter Department of Biomedical Engineering at Emory and Georgia Tech. 

‘A scientist’s dream’ 

The tension probes can detect cellular forces on the magnitude of just a few piconewtons, or about a billion times less than the weight of a paper clip. The researchers found a way to amplify the signal of the probes by tapping the power of an enzyme known as CRISPR-associated 12a. The mechanical signal is then detected using a plate-reader, a tool already routinely used in clinical testing. 

“This project started out of basic curiosity,” says Salaita, co-corresponding author. “We wanted to know whether we could measure the tiniest forces exerted by cells. It’s exciting that we are now building on this basic curiosity to develop diagnostic tools to help patients. It’s a scientist’s dream.” 

First author of the paper is Yuxin Duan, an American Heart Association postdoctoral fellow in the Salaita lab. 

Roman Sniecinski, a professor in Emory School of Medicine’s Department of Anesthesiology and a leading expert in the field of perioperative coagulation, is co-corresponding author of the paper.

“Platelet function in general is important and yet the current tools that we have to measure it are relatively primitive,” Sniecinski says. “This new technique offers an easier, faster and cheaper way to measure platelet function, while also providing us with key information that we didn’t have before.” 

Co-authors include: Fania Szlam, a senior associate in the Sniecinski lab; Yuesong Hu, a graduate student in the Salaita lab; Renhao Li, a professor in Emory School of Medicine’s Department of Pediatrics, Hematology/Oncology; Wenchun Chen, a postdoctoral fellow in Emory School of Medicine; and Yonggang Ke, an associate professor in the Coulter Department of Biomedical Engineering at Emory and Georgia Tech. 

The importance of platelets 

Platelets are colorless, disc-shaped blood-cell fragments whose job is to bind at the site of an injured blood vessel to stop the bleeding. In some cases, however, platelets may not function optimally. When platelets are weak, or less active than optimal, the blood may not clot properly leading to uncontrolled bleeding. But if platelets are “hyperactive” they may become too sticky and cause spontaneous blood clots that can lead to heart attack or stroke. 

Regulating platelet function is especially critical to people at higher risk for some conditions. Antiplatelet drugs, such as clopidogrel, ticagrelor and even aspirin, are among the most commonly prescribed medications in the United States. In some patients, however, these drugs may not work well and adjustments in doses or changing to another drug might better help prevent heart attacks. 

During cardiac surgery, platelet function becomes even more dysregulated. The operating-room team must perform a balancing act of making blood not clot during cardiopulmonary bypass, then using procoagulant interventions, including transfusions of platelets, to stop the bleeding when the surgical procedure is finished. This can be difficult because the use of the cardiopulmonary bypass machine can stress and weaken blood platelets. 

“For decades, people have written in the scientific literature about this problem of platelet dysfunction during cardiac surgery,” Sniecinski says, “but it’s really difficult to measure it with the tools that we’ve been using. And since we haven’t been able to measure platelet function well, that’s made it difficult to study it in effective ways.” 

‘A small part of the picture’ 

Aggregometry is a standard tool currently used to assess platelet function. It measures the speed and degree at which platelets in a blood sample clump together, or aggregate. 

“This data provides only a small part of the picture of platelet function and it’s not the most interesting part,” Sniecinski says. 

When a platelet gets activated, he explains, it changes its morphology and grows tiny pseudo “arms.” Platelets use these arms to grip onto chains of proteins in the blood called fibrinogen to form clots. 

“Aggregometry tells you that platelets are clumping together,” Sniecinski says. “But it doesn’t tell you about their level of activation — the amount of force they’re using to hold on to other coagulation proteins, as well as each other.” 

Amplifying the signal 

The Salaita lab is a leader in visualizing and measuring the mechanical forces applied by cells using tension probes made from synthetic strands of double-stranded DNA tethered to a surface. 

The double-strands of DNA can be programmed to bind to platelet cells. When the cells bind and apply force to the anchored DNA, the DNA splits into two strands, leaving one strand stuck to the surface. The resulting physical tug is converted into a fluorescent signal. 

A major challenge to reading this signal, however, is that these physical tugs are faint, fleeting and infrequent. They require a microscope to detect them. 

During the COVID-19 pandemic , the enzyme CRISPR 12a, or Cas12a, came to the fore as a diagnostic tool for SARS-CoV-2 virus. Bacteria use Cas12a to defend against phages, or viruses that attack bacteria. The Cas12a enzyme can be loaded with single-stranded “guide” RNA that is programmed to bind to a complementary single-stranded DNA. The enzyme then reacts to the single-stranded DNA by destroying other single-stranded DNA surrounding it. 

The Salaita lab decided to combine Cas12a with its tension probes to see if the enzyme would amplify the signal for the mechanical forces exerted by blood platelets. The lab developed what it calls the Mechano-Cas12a Assisted Tension Sensor, or MCATS. 

“It worked like gangbusters,” Salaita says. 

“Cas12a is quiet and inactive if it doesn’t see its target,” he explains. “But as soon as it sees a specific single-strand DNA, it goes bananas and starts destroying any single-stranded DNA it comes across. This activation generates a massive fluorescence signal output.” 

MCATS is precise and ultrasensitive, able to measure cellular traction forces generated by as few as 2,000 platelets within a sample. And the resulting signal is robust enough to measure via a conventional fluorometer — a tool commonly used in routine blood tests. 

MCATS also works with a plate reader, an instrument designed to handle dozens of samples simultaneously, for the kind of high-throughput readout needed to conduct research. 

Testing its clinical potential 

To test the efficacy of MCATS at measuring the activity of platelet function, the researchers drew blood samples from healthy volunteer donors. They first validated that the MCATS response was sensitive to the mechanical forces of platelets. 

They next added to the healthy blood samples different antiplatelet drugs, ranging from over-the-counter aspirin to a panel of different prescription medications. The MCATS results showed that the antiplatelet therapies reduced the mechanical activity of platelets by an amount similar to the reduction observed in aggregometry. 

The researchers also received permission to take blood samples for investigation from seven patients pre- and post-cardiopulmonary bypass surgery. The results showed that the MCATS readings for the platelet activity of each individual patient’s sample correlated to their likelihood to need platelet transfusions to minimize bleeding after surgery. 

The researchers are now enrolling participants in a prospective study to further explore MCATS as a diagnostic tool. People diagnosed with a platelet disorder will have their blood samples tested pre- and post-treatment to assess how well a therapy is working. 

“The bottom line is that MCATS is a whole new way to measure platelet function using a really tiny sample,” Sniecinski says. “It’s telling us something specific that we haven’t been able to measure before and that can give us a new way to understand what’s going on with platelet dysfunction and the best methods for controlling it.” 

“Blood work up gives you a basic readout of your health based on data like platelet count and metabolic concentrations,” Salaita adds. “Now we’re adding information about the mechanics of platelets. That’s like getting a whole new dial on your dashboard for monitoring your health.” 

Work on the current paper was funded by the National Institutes of Health, the National Science Foundation and the American Heart Association.

Related:

Tuesday, December 5, 2023

Building boom boosts malaria-carrying, invasive mosquito in Ethiopia

Sampling for mosquito larva in the water of a manmade pit at a construction site made of simple earth walls covered in plastic sheeting. 

A malaria-carrying mosquito that thrives in urban environments is moving into Africa where a construction boom may be one factor helping the newcomer feel at home.

Lancet Planetary Health published the findings on the ecology of the invasive Anopheles stephensi mosquito led by Gonzalo Vazquez-Prokopec, a professor in Emory University’s Department of Environmental Sciences. 

The invasion of stephensi poses a major threat to urban populations in Africa, where malaria has primarily been a rural disease. While most of the limited data available on stephensi in Africa has been gathered during the rainy season, this study focused on the city of Jigjiga in eastern Ethiopia during the peak of the dry season. 

Stephensi was first detected in Jigjiga in 2018 and has persisted there despite harsh dry seasons of around three rainless months. “We found that during this period of low-water availability, Anopheles stephensi is primarily exploiting habitats associated with construction,” says Vazquez-Prokopec, a leading expert in the disease ecology of urban mosquitos. 


Related:



Monday, November 20, 2023

Birds set foot near South Pole in Early Cretaceous, Australian tracks show

Emory's Anthony Martin, left, at the site of the discovery with Melissa Lowery, a local volunteer fossil hunter who was the first to spot most of the tracks. (Photo by Ruth Schowalter)

The discovery of 27 avian footprints on the southern Australia coast — dating back to the Early Cretaceous when Australia was still connected to Antarctica — opens another window onto early avian evolution and possible migratory behavior. 

PLOS ONE published the discovery of some of the oldest, positively identified bird tracks in the Southern Hemisphere, dated to between 120 million and 128 million years ago. 

“Most of the bird tracks and body fossils dating as far back as the Early Cretaceous are from the Northern Hemisphere, particularly from Asia,” says Anthony Martin, first author of the study and a professor in Emory University’s Department of Environmental Sciences. “Our discovery shows that there were many birds, and a variety of them, near the South Pole about 125 million years ago.” 

Read the full story here. 

Related: 

Tell-toe toes point to oldest-known bird tracks from Australia 

Paleontologist explores a billion years of animals breaking up rocks, bones, shells and wood

Friday, November 10, 2023

NSF funds holistic approach to help farmers adapt to climate change

"There is already a lot of work on what climate change may mean for agriculture in general," says Emily Burchfield, Emory assistant professor of environmental sciences. "But what climate change means for an individual farm must be filtered through issues particular to that farm."

By Carol Clark

The National Science Foundation (NSF) awarded Emily Burchfield, Emory assistant professor of environmental sciences, $1.6 million to lead efforts to identify emerging pressures on agriculture in Georgia, Iowa and Ohio and to develop predictive models to help farmers and policymakers weather these changes. 

“In a nutshell, we’re trying to understand what climate change will mean for agriculture in these three states,” Burchfield says. “We’ll be integrating biophysical projections based on environmental data with insights gathered from farmers and agricultural experts.” 

The goal is to develop possible scenarios for the impacts of climate change — along with the evolving technical, socioeconomic and political landscapes in each state — for how and where crops could be grown over the next 30 to 40 years. The researchers will create a public, online tool to allow farmers and policymakers to explore the possible futures of agriculture at regional and state levels and to support their efforts to manage these scenarios. 

The grant is part of the NSF Dynamics of Integrated Socio-Environmental Systems Program (DISES). 

“Traditionally, the NSF has mainly split programs into the social sciences and the natural sciences but DISES is one of their newer programs that joins the two, looking at how nature affects people and people affect nature,” Burchfield says. “Coupling human and natural systems in theoretical frameworks allows us to take on some of the grand challenges that we’re facing, like climate change and food and water security.” 

Burchfield is principal investigator for the project, which also includes researchers from Arizona State University, Ohio State University and the University of Nebraska, Lincoln. 

A range of agricultural systems 

While the two main crops in both Iowa and Ohio are corn and soy, agriculture in Georgia is far more diverse. The state leads the nation in the production of peanuts, pecans, blueberries and spring onions and is also a leading producer of cotton, watermelon, peaches, cucumbers, sweet corn, bell peppers, tomatoes, cantaloupes, rye and cabbage. 

Agriculture contributes nearly $70 billion annually to Georgia’s economy and one in seven Georgians works in agriculture, forestry or related fields, according to the Georgia Farm Bureau. 

“Compared to other parts of the country, Georgia is incredibly diverse not just in terms of what is grown in the state but in terms of who grows it,” Burchfield says. “A lot of exciting changes are happening in the state — citrus production is moving into South Georgia. And the biggest organic farm east of the Mississippi is located in Georgia, producing carrots.” 

While California currently produces the bulk of the nation’s produce, that state is facing significant challenges for water availability, Burchfield notes. “Georgia has a unique opportunity to expand its fresh-produce production to help meet future demand,” she says. “We want to provide farmers the resources they need to capitalize on such trends.” 

Building tools for the future of farming 

Burchfield’s research combines spatial-temporal social and environmental data to understand the future of food security in the United States, including the consequences of a changing climate. 

For the current project, the researchers will draw from available climate, soil and land-use data to create biophysical models for how changes in climate will affect where and how particular crops can be grown. These models will be integrated with data gathered from surveys and focus groups conducted with agricultural experts, climatologists and farmers working the land throughout Georgia, Iowa and Ohio. 

The project aims to get input from a diverse range of farmers growing different crops and using different management practices. 

“There is already a lot of work on what climate change may mean for agriculture in general,” Burchfield says. “But what climate change means for an individual farm must be filtered through issues particular to that farm. So many dimensions that matter deeply to farmers are not included in policy discussions about agriculture.” 

Farmers will be asked what information and resources they need to sustain their operations and to adapt to climate change. “We want to understand the vision that farmers have for the future of their farms,” Burchfield says. “What would they would like to see happen? What do they see as the barriers and bridges to achieving that vision?” 

The public, online tool that the researchers develop will include interactive maps for crop forecasts by region. It will also provide information to guide policymakers and to help farmers adapt to the changes ahead. 

“It’s impossible to accurately say exactly what’s going to happen in the future,” Burchfield says. “But combining biophysical data with an understanding of the technical, economic and political changes emerging in each of these states, along with the expertise of our farmers, will allow us to forecast trends for how suitable particular regions will be for growing certain crops. The bottom line is we are pulling together the best information available to give a sense of the emergent opportunities in the state for agriculture as well as the emergent challenges.” 

Related:

Emory breaking new ground for climate-smart agriculture

Climate change on course to hit U.S. Corn Belt especially hard

Thursday, November 9, 2023

New antimicrobial shuts down bacterial growth without harming human cells

Graphic shows how the KKL-55 molecule (in red) inhibits trans-translation, a process that bacteria cells use for quality control during protein synthesis.

By Carol Clark

Scientists have shown how a molecule with broad-spectrum antibiotic activity works by disabling a process vital to bacterial growth without affecting the normal functioning of human cells. mBio, a journal of the American Society for Microbiology, published the work, led by researchers at Emory University and Pennsylvania State University. 

The molecule, known as KKL-55, is one of a suite of recently identified molecules that interfere with a key bacterial mechanism known as trans-translation, essentially shutting down the ability of bacteria to grow. 

“We’re opening a promising pathway for the development of new antibiotics to treat drug-resistant infections,” says Christine Dunham, co-corresponding author of the paper and a professor in Emory’s Department of Chemistry and the Emory Antibiotic Resistance Center. 

Kenneth Keiler, a professor in the Department of Biochemistry and Molecular Biology at Pennsylvania State, is co-corresponding author of the paper. 

First authors are Ha An Nguyen, who did the work as an Emory chemistry PhD candidate and has since graduated and works at Memorial Sloan Kettering, and Neeraja Marathe, a graduate student at Pennsylvania State. 

A growing global threat 

Antimicrobial-resistant infections have long been a public health threat. The situation grew even worse during the COVID-19 pandemic with increased antibiotic use and less prevention actions, according to the U.S. Centers for Disease Control and Prevention (CDC). 

The CDC estimates that at least 2.8 million antimicrobial-resistant infections continue to occur in the United States each year, killing more than 35,000 people. Globally, the World Health Organization projects that these infections will cause up to 10 million deaths annually by 2050 if new antibiotics are not developed. 

While antibiotics can save lives, any time they are used they can also contribute to the problem of resistance. Bacteria keep evolving new weapons as a defense against drugs, even as scientists work on developing new strategies to disarm bacteria. 

Cross-toxicity, or harmful effects on humans, is another key drawback of some of the drugs used in a last-ditch effort to kill antibiotic-resistant bacteria. 

Avoiding cross-toxicity 

Dunham and Keiler are avoiding the problem of cross-toxicity by focusing on the inhibition of a mechanism unique to bacteria — trans-translation. This mechanism is vital to the proper functioning of the bacterial ribosome. 

Keiler, a molecular geneticist and biochemist, first identified trans-translation in bacteria and is an expert in how it functions. Dunham, a structural biologist, is an expert in the human ribosome. She uses advanced biochemistry and structural biology techniques to understand the mechanics of molecular interactions. 

“Our individual areas of expertise mesh well for this project,” Dunham says. “By collaborating, we are able to take the science further, faster.” 

A cellular protein factory 

The ribosome is an elaborate macromolecular machine within a cell that operates like a factory to manufacture proteins. Proteins are the machines that make cells run while nucleic acids such as DNA and RNA store the blueprints for life. The ribosome is made mostly of RNA, which does not just store information but can also act as an enzyme, catalyzing chemical reactions. 

In a human cell, messenger RNA (mRNA), containing the instructions for manufacturing a protein, originates in the nucleus. While still in the nucleus, mRNA undergoes an elaborate quality-control process. It must pass inspection before getting exported to translate the information it contains into a protein. 

“A lot of mRNAs have defects,” Dunham says. “Human cells have efficient ways to test mRNAs and ultimately remove the defective ones.” 

Bacterial cells, however, have no nucleus or organized center for quality control. 

“Bacteria wants to grow, grow and grow, which requires the ribosome to make a lot of proteins,” Dunham says. “But when mRNA has defects, there is little to no quality control. When the ribosome encounters a defective mRNA protein, synthesis gets stalled.” 

The trans-translation process “rescues” ribosomes stalled due to such defects, in order to maintain proper protein synthesis and cell viability in bacteria. 

How KKL-55 works 

Using a high-throughput screening process, the Keiler lab has identified dozens of molecules that inhibit trans-translation in bacteria. 

For the current paper, the researchers focused on understanding how one of these molecules, KKL-55, performs this trick. They used the high-powered structural biology technique of X-ray crystallography to capture KKL-55 in action as it interacted with a protein required for translation. 

The results showed how KKL-55 blocks trans-translation by binding to elongation factor thermos-unstable (EF-Tu). EF-Tu is a protein that interacts with transfer RNA molecules, which play a key role in protein synthesis, and also transfer-messenger RNA, an RNA molecule required for the trans-translation pathway. 

“We got lucky,” Dunham says. “There are dozens of steps involved in the process that KKL-55 could have inhibited and we might have had to test for each one. But the results are clear-cut. It shuts down trans-translation right at the beginning by preventing EF-Tu from binding to tmRNA.” 

Determining the mechanism by which a molecule works to inhibit bacteria is a critical step toward developing a new antibiotic for clinical use. The next step is to test the efficacy of KKL-55 to treat a bacterial infection in a mouse model. 

In 2021, the research team published their finding that a group of trans-translation inhibitors known as acylaminooxadiazoles clear multiple-drug-resistant Neisseria gonorrhoeae infection in mice after a single oral dose. That work is now advancing to clinical trials. 

Dozens more trans-translation inhibitors await the team’s investigation. Each represents a potential new weapon to help humans stay on top in the arms race with drug-resistant bacteria. 

Co-authors of the current paper include Alexandra Nagy (a former National Institutes of Health FIRST Institutional Research and Academic Career Development postdoctoral fellow at Emory who is now at Earlham College); as well as John Alumasa and Michael Vazquez (both from Pennsylvania State University). The work was funded by the National Institutes of Health, the National Institute of General Medical Sciences. 

Related:

Images of enzymes in action reveal secrets of antibiotic-resistant bacteria 

Biochemist Dunham shifts the frame on proteins