The researchers will present their results today at the spring meeting of the American Chemical Society (ACS).

Each year, the World Anti-Doping Agency (WADA) publishes a list of substances, including steroids, that athletes are prohibited from using. However, it can be difficult to distinguish an athlete’s natural or “endogenous” steroids from synthetic “exogenous” ones administered to boost performance.

And regulatory bodies face another challenge: “As quickly as we develop methods to look for performance-enhancing drugs, clandestine labs develop new substances that give athletes a competitive advantage,” says Christopher Chouinard, Ph.D., the project’s principal investigator. Those designer drugs evade detection if testing labs don’t know to look for their specific chemical structures.

Chouinard’s team at Florida Institute of Technology is trying to outsmart cheaters with an assay that can differentiate endogenous and exogenous steroids and can also anticipate the structure of new compounds that might show up in athletes’ urine samples.

Currently, testing labs analyze samples using tandem mass spectrometry (MS) and gas or liquid chromatography. These approaches break up molecules in the sample and separate the fragments, yielding spectra that can reveal the identity of the original, intact compounds. But it can be tough to differentiate molecules with minor structural differences — including isomers — that distinguish endogenous steroids from exogenous ones, such as the synthetic anabolic steroids athletes take to build muscle.

To accentuate those differences, Chouinard pairs MS with ion mobility (IM) spectrometry, a separation technique he learned as a graduate student with Richard Yost, Ph.D., at the University of Florida. Yost’s team and others found that the differences between isomers could be made even more apparent if the molecules in a sample were modified prior to IM-mass spec analysis by reacting them with other compounds. After Chouinard set up his own lab in 2018, he applied this technique by reacting steroid samples with ozone or acetone in the presence of ultraviolet light — reactions already well-established among researchers who study lipid isomers, but new in the anti-doping arena.

Last year, Chouinard’s team reported they had successfully used these reactions with IM-MS to improve isomer separation, identification and quantification for a few steroids in sample solutions. Now, the researchers report they have tested this technique in urine against nearly half the prohibited steroids on WADA’s list and have shown it can successfully characterize and identify these compounds. They also showed the method can characterize and identify banned glucocorticoids, such as cortisone, that improve athletic performance by suppressing inflammation from injuries. Detection limits are below one nanogram per ml.

In addition to tracking down known dopants, the team wants to be able to find newly created illicit steroids not yet known to WADA. With Florida Institute of Technology collaborators including Roberto Peverati, Ph.D., they are developing computational modeling and machine learning techniques to try to predict the structure, spectra and other characteristics of these molecules. “If we can develop methods to identify any theoretical steroids in the future, we could dramatically reduce doping because we would be able to detect these new species immediately, without the lag time that’s been associated with anti-doping testing over the last 40 years,” Chouinard says.

Though the assays themselves are quick, simple and inexpensive, IM instruments are costly, with a price ranging up to roughly a million dollars, Chouinard notes. However, he adds, with the support of anti-doping funding organizations like the Partnership for Clean Competition (PCC), more labs might be willing to foot that bill, so long as the method offers a significant advantage in detection and deterrence.

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The researchers will present their results today at the spring meeting of the American Chemical Society (ACS).

If developed successfully, a fish-oil based polyurethane could help meet the immense need for more sustainable plastics, says Francesca Kerton, Ph.D., the project’s principal investigator. “It is important that we start designing plastics with an end-of-life plan, whether it’s chemical degradation that turns the material into carbon dioxide and water, or recycling and repurposing.”

To make the new material, Kerton’s team started out with oil extracted from the remains of Atlantic salmon, after the fish were prepared for sale to consumers. “I find it interesting how we can make something useful, something that could even change the way plastics are made, from the garbage that people just throw out,” says Mikhailey Wheeler, a graduate student who is presenting the work at the meeting. Both Kerton and Wheeler are at Memorial University of Newfoundland (Canada).

The conventional method for producing polyurethanes presents a number of environmental and safety problems. It requires crude oil, a non-renewable resource, and phosgene, a colorless and highly toxic gas. The synthesis generates isocyanates, potent respiratory irritants, and the final product does not readily break down in the environment. The limited biodegradation that does occur can release carcinogenic compounds. Meanwhile, demand for greener alternatives is growing. Previously, others have developed new polyurethanes using plant-derived oils to replace petroleum. However, these too come with a drawback: The crops, often soybeans, that produce the oil require land that could otherwise be used to grow food.

Leftover fish struck Kerton as a promising alternative. Salmon farming is a major industry for coastal Newfoundland, where her university is located. After the fish are processed, leftover parts are often discarded, but sometimes oil is extracted from them. Kerton and her colleagues developed a process for converting this fish oil into a polyurethane-like polymer. First, they add oxygen to the unsaturated oil in a controlled way to form epoxides, molecules similar to those in epoxy resin. After reacting these epoxides with carbon dioxide, they link the resulting molecules together with nitrogen-containing amines to form the new material.

But does the plastic smell fishy? “When we start the process with the fish oil, there is a faint kind of fish smell, but as we go through the steps, that smell disappears,” Kerton says.

Kerton and her team described this method in a paper last August, and since then, Wheeler has been tweaking it. She has recently had some success swapping out the amine for amino acids, which simplifies the chemistry involved. And while the amine they used previously had to be derived from cashew nut shells, the amino acids already exist in nature. Wheeler’s preliminary results suggest that histidine and asparagine could fill in for the amine by linking together the polymer’s components.

In other experiments, they have begun examining how readily the new material would likely break down once its useful life is over. Wheeler soaked pieces of it in water, and to speed up the degradation for some pieces, she added lipase, an enzyme capable of breaking down fats like those in the fish oil. Under a microscope, she later saw microbial growth on all of the samples, even those that had been in plain water, an encouraging sign that the new material might biodegrade readily, Wheeler says.

Kerton and Wheeler plan to continue testing the effects of using an amino acid in the synthesis and studying how amenable the material is to the microbial growth that could hasten its breakdown. They also intend to study its physical properties to see how it might potentially be used in real world applications, such as in packaging or fibers for clothing.

The researchers will present their results today at the spring meeting of the American Chemical Society (ACS). 

Each year in the U.S., at least 2.8 million people get antibiotic-resistant infections, leading to 35,000 deaths, according to the U.S. Centers for Disease Control and Prevention. “We knew from previous research that there were substances in Baltic amber that might lead to new antibiotics, but they had not been systematically explored,” says Elizabeth Ambrose, Ph.D., who is the principal investigator of the project. “We have now extracted and identified several compounds in Baltic amber that show activity against gram-positive, antibiotic-resistant bacteria.”

Ambrose’s interest originally stemmed from her Baltic heritage. While visiting family in Lithuania, she collected amber samples and heard stories about their medicinal uses. The Baltic Sea region contains the world’s largest deposit of the material, which is fossilized resin formed about 44 million years ago. The resin oozed from now-extinct pines in the Sciadopityaceae family and acted as a defense against microorganisms such as bacteria and fungi, as well as herbivorous insects that would become trapped in the resin.

Ambrose and graduate student Connor McDermott, who are at the University of Minnesota, analyzed commercially available Baltic amber samples, in addition to some that Ambrose had collected. “One major challenge was preparing a homogeneous fine powder from the amber pebbles that could be extracted with solvents,” McDermott explains. He used a tabletop jar rolling mill, in which the jar is filled with ceramic beads and amber pebbles and rotated on its side. Through trial and error, he determined the correct ratio of beads to pebbles to yield a semi-fine powder. Then, using various combinations of solvents and techniques, he filtered, concentrated and analyzed the amber powder extracts by gas chromatography-mass spectrometry (GC-MS).

Dozens of compounds were identified from the GC-MS spectra. The most interesting were abietic acid, dehydroabietic acid and palustric acid — 20-carbon, three-ringed organic compounds with known biological activity. Because these compounds are difficult to purify, the researchers bought pure samples and sent them to a company that tested their activity against nine bacterial species, some of which are known to be antibiotic resistant.

“The most important finding is that these compounds are active against gram-positive bacteria, such as certain Staphylococcus aureus strains, but not gram-negative bacteria,” McDermott says. Gram-positive bacteria have a less complex cell wall than gram-negative bacteria. “This implies that the composition of the bacterial membrane is important for the activity of the compounds,” he says. McDermott also obtained a Japanese umbrella pine, the closest living species to the trees that produced the resin that became Baltic amber. He extracted resin from the needles and stem and identified sclarene, a molecule present in the extracts that could theoretically undergo chemical transformations to produce the bioactive compounds the researchers found in Baltic amber samples.

“We are excited to move forward with these results,” Ambrose says. “Abietic acids and their derivatives are potentially an untapped source of new medicines, especially for treating infections caused by gram-positive bacteria, which are increasingly becoming resistant to known antibiotics.”

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COVID-19 has infected more than 18 million people and led to more than 700,000 deaths around the world. Emergency department presentation varies widely, suggesting that distinct clinical phenotypes exist and, importantly, that these distinct phenotypic presentations may respond differently to treatment.

In the new study, researchers analyzed electronic health records (EHRs) from 14 hospitals in the midwestern United States and from 60 primary care clinics in the state of Minnesota. Data were available for 7,538 patients with PCR-confirmed COVID-19 between March 7 and August 25, 2020; 1,022 of these patients required hospital admission and were included in the study. Data on each patient included comorbidities, medications, lab values, clinic visits, hospital admission information, and patient demographics.

Most patients included in the study (613 patients, or 60 percent) presented with what the researchers dubbed “phenotype II.” 236 patients (23.1 percent) presented with “phenotype I,” or the “Adverse phenotype,” which was associated with the worst clinical outcomes; these patients had the highest level of hematologic, renal and cardiac comorbidities (all p<0.001) and were more likely to be non-White and non-English speaking. 173 patients (16.9 percent) presented with “phenotype III,” or the “Favorable phenotype,” which was associated with the best clinical outcomes; surprisingly, despite having the lowest complication rate and mortality, patients in this group had the highest rate of respiratory comorbidities (p=0.002) as well as a 10 percent greater risk of hospital readmission compared to the other phenotypes. Overall, phenotypes I and II were associated with 7.30-fold (95% CI 3.11-17.17, p<0.001) and 2.57-fold (95% CI 1.10-6.00, p=0.03) increases in hazard of death relative to phenotype III.

The authors conclude that phenotype-specific medical care could improve COVID-19 outcomes, and suggest that future research is needed to determine the utility of these findings in clinical practice.

The authors add: “Patients do not suffer from COVID-19 in a uniform matter. By identifying similarly affected groups, we not only improve our understanding of the disease process, but this enables us to precisely target future interventions to the highest risk patients.”

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