To help meet this challenge, researchers from the School of Science at IUPUI are developing a new biosensor with the potential to achieve the speed and efficiency required for the future of COVID-19 testing.

The work was recently reported in Applied Material Interfaces, a journal of the American Chemical Society. It is led by Rajesh Sardar, a professor of chemistry and chemical biology in the School of Science, and Adrianna Masterson, a graduate student in Sardar’s lab at the time of the study.

“Everyone is chasing high-throughput testing; this type of high-speed analysis is essential to the future of the fight against COVID-19,” Sardar said. “There are many advantages to our technology in particular: It’s fast, efficient, accurate and unprecedentedly sensitive.”

In terms of speed, the COVID-19 test from Sardar’s lab can currently analyze samples from 96 individuals in under three hours, he said. In terms of efficiently, the system requires only 10 microliters of blood.

By comparison, a typical blood panel order by a primary-care physician collects 10 milliliters of blood — more than 1,000 times more.

The sensor also works with other sample types, such as saliva, Sardar said. But the study was conducted using blood since it’s the most complex bodily fluid and therefore the best indicator of a sensor’s accuracy. All test samples were obtained from the Indiana Biobank, which provided 216 blood samples, including 141 samples from patients with COVID-19 and 75 healthy control samples.

Based upon a blind analysis, IUPUI researchers found their biosensor’s accuracy rate was 100 percent and its specificity rate was 90 percent. In other words, the sensor never reported a false negative and only reported a false positive in 1 out of 10 samples. For the purposes of public safety, Sardar said the absence of false negatives is more important than false positives, because a person with a false negative may unknowingly infect others, whereas a person with a false positive is not a danger.

Additionally, Sardar said the sensor was found to be highly accurate at measuring the body’s COVID-19 antibody concentration. This is because it detects not only the virus’s spike protein but also the proteins created by the body to protect against the virus — immunoglobin G, or IgG.

He also said the ability to measure COVID-19 antibodies is significant because many COVI9-19 antibody tests currently approved under the FDA’s emergency use authorization don’t provide specific antibody counts, despite the fact that this number indicates the strength of a person’s immunity to infection.

“Accurately measuring patients’ immunity levels will be critical to protecting against COVID-19 going forward,” Sardar said. “This can be seen clearly in our current state of affairs, as variants like omicron — and, most recently, BA.5 — are infecting even fully vaccinated and boosted individuals.”

To achieve its results, Sardar’s lab’s biosensor uses chemically synthesized gold triangular nanoprisms, which provide a uniquely powerful optical response to even minuscule amounts of IgG. It also means the sensor can detect antibodies in the earliest stages of infection.

The work, which began in the early days of the pandemic, builds upon initial promising results published in June 2021. Next, Sardar aims to further refine the technology, with a goal of eventually being able to process 384 samples in less than an hour — or 5,000 samples per day, if used in a larger testing center.

“This research is about preparing for the future,” said Sardar, who is also a researcher with the Indiana University Melvin and Bren Simon Comprehensive Cancer Center. “The H1N1 strain of the flu is nearly 100 years old. I expect the coronavirus will also be with us a long time. Looking ahead, we need to come up with ways to measure many people’s infections, or risks of infection, quickly, easily and efficiently in order to stay one step ahead of the virus.”

This work was supported in part by the NIH’s National Center for Advancing Translational Science though a grant from the Indiana CTSI.

Protein homeostasis, or proteostasis, is the set of processes that maintain cellular proteins in a functional state, and eliminate damaged proteins that cannot be repaired. Ribosomes are multi-protein molecular machines that synthesize proteins, and mutations in genes that encode ribosomal proteins not only impair protein synthesis but also disrupt proteostasis, leading to chronic proteotoxic stress. That stress, in turn, has a host of cellular consequences and results in delayed development and other irregularities.

To better understand the disorders caused by such disruption of proteostasis, the authors compared gene expression in normal versus ribosomal protein mutant flies during the pupal stage of development. Unexpectedly, they found that a group of genes encoding six gustatory (taste) receptors, called Gr64 genes, were upregulated in the mutant cells.

This finding was unexpected because the Gr64 receptors had previously been known to be present in adult fly neurons, where they help the animal taste sugars, fatty acids, and glycerol. In ribosomal protein mutant flies with just one (rather than two) working copies of the Gr64 genes, there was an increase in cell death via a process called apoptosis. Complete elimination of the Gr64 genes induced multiple morphological defects in the ribosomal mutants, but had little to no effect in cells with normal ribosomal proteins.

Cells bearing ribosomal protein mutants are at a disadvantage compared to normal cells, and are often eliminated in developing tissue where both occur. Mutants carrying only one set of Gr64 genes were even worse off, the authors found, losing out to normal cells at an even greater rate. When the level of Gr64 proteins was variably and quantitatively reduced, the team showed that the most direct effect of reduction was on the function of the proteasome and autophagosome, two different routes by which damaged proteins are removed and recycled.

The connection between promotion of proteostasis and the sense of taste is likely through the molecular mechanism of the Gr64 proteins, which regulate calcium flow; changes in calcium levels are used as a signal transducer in sensory cells, and also regulate multiple proteostatic processes, including both proteosome function and autophagy. Intriguingly, dysregulated and out-of-place gustatory and olfactory receptors have been detected in the affected brain tissue in several human diseases characterized by loss of protein homeostasis, including Alzheimer’s disease and Parkinson’s disease.

“Our work suggests that gustatory receptors play a previously unrecognized role in maintaining protein homeostasis during development in the fly,” Piddini said, “and points more generally toward the connection between calcium-modulating proteins and disorders of protein aggregation and proteotoxic stress.”

Piddini adds, “What I find truly fascinating about our findings is that they underscore the little we understand about how cells adapt to cope with proteotoxic stress. Our experimental strategy could be used further as a way to search and discover additional new proteostasis genes.”

Coauthor Michael Baumgartner concludes, “These findings emphasize an important theme in cell biology: context is everything. It’s easy to see the name ‘gustatory receptor’ and think that’s the end of the story, but evolution can and will repurpose almost anything if it ends up being advantageous. This highlights the possibility that the seemingly hyper-specialized gustatory receptors might be more flexible than previously appreciated.”

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Their findings publish in the July 21, 2022 online issue of PLOS Pathogens.

The research involved building plasmids genetically altered to contain bits of genetic material specifically intended to target a vulnerability in the SARS-CoV-2 virus’s spike protein, a portion of the virus critical to binding and infecting cells. Plasmids are small, circular DNA molecules from bacteria that are physically separate from chromosomal DNA and can replicate independently. They can be used by scientists to transfer genetic material from one cell to another, after which the introduced genetic material can replicate in the receiving cell.

The approach, said senior author Maurizio Zanetti, MD, professor of medicine at UC San Diego School of Medicine and head of the Laboratory of Immunology at UC San Diego Moores Cancer Center, points to the possibility of a more durable, and more broadly effective, COVID-19 vaccine.

“The details are complicated, but the fundamentals are simple,” said Zanetti. “They are based on well-known and proven principles and methods.”

COVID-19 mRNA vaccines, such as those by Pfizer and Moderna, are the result of decades of previous research and development. The pandemic added new urgency, focus and resources. These vaccines promised a faster way to people, though not without significant challenges, such as the need of an ultralow temperature cold chain.

The resulting mRNA vaccines have fundamentally altered the course of the pandemic, dramatically mitigating the severity of disease, hospitalizations and deaths. But notably, said Zanetti, they do little at blocking transmission of the virus. Case rates still rise and fall with the emergence of viral variants.

“The goal at the beginning wasn’t to stop the disease,” said Zanetti. “It was to mitigate the consequences, to reduce COVID’s severity and outcomes. The vaccines have done that. Vaccinated persons tend not to get as sick. They don’t require hospitalization as often. Death rates are down. All of this has greatly reduced pressures on health systems and society, which is a good thing.”

But the ever-evolving nature of the SARS-CoV-2 virus has revealed that the vaccines’ efficacy varies, depending upon variant, often diminishing. The Alpha variant, for example, proved more contagious than the “wild-type” strain that originated in Wuhan, China. The Delta variant was more transmissible than Alpha and Omicron more than Delta. Though the vaccines continue to provide substantial protection against severe disease, the antibodies they induce are consistently less powerful at neutralizing the virus, thus the increased transmission. SARS-CoV-2 continues to be an unrelenting global public health threat.

Zanetti said the newest work emphasizes “quality over quantity,” seeking the induction of antibodies preferentially blocking virus binding to its cell receptor and transmission. This results in a more focused antibody response with the vaccine.

“In the early days of COVID vaccine development, it was about generating a broad, robust immune response,” Zanetti said. “But it was a scattered approach. The vaccines response targeted many epitopes (parts of the virus that the host’s immune system recognizes) and it resulted in an immune response that was largely noise. Most of the resulting antibodies produced didn’t affect the virus’s ability to infect.”

“The new research narrows the focus to a part of the viral spike specifically involved in the virus’s ability to infect that appears to be evolutionarily conserved,” said co-senior author Aaron F. Carlin, MD, PhD, assistant professor in the Division of Infectious Diseases and Global Public Health at UC San Diego Health. In other words, the site doesn’t change with new variants, and represents a persisting site of vulnerability and a reliable vaccine target.

How it works

Zanetti and colleagues built plasmids containing immunogens — molecules that cause B lymphocytes to create antibodies — that were specifically designed to display a knob of the spike protein that is part of the receptor binding motif or RBM. Specifically, these were amino acid residues that act like keys to unlock the cell door. The keys and lock don’t change.

B lymphocytes are part of the immune system. They are prodigious producers of antibodies created to respond and protect against specific antigens or unwanted substances in the body, such as viruses. The average B lymphocyte can spit out 1,000 antibody molecules per second, an incredibly robust production if it is the right antibody for the job.

Zanetti and colleagues cloned the selected spike protein amino acids into a plasmid DNA so that, when injected into the spleen of mice, the introduced immunogen molecules would provoke the production of neutralizing antibodies specifically tuned to the targeted nob on the RBM of the virus protein spike. The researchers then tested their approach on mice with variants of the original SARS-CoV-2 strain (Beta, Delta and Omicron) and found that the immune response was similar across all variants.

“We were a bit lucky in picking our target on the spike,” said Zanetti, “though it was also the result of experience and intuition. I’ve been doing this for 30 years. Earlier experiments by others had suggested this might be a ‘supersite.’ I followed my instincts.”

Zanetti said translating these findings into a vaccine suitable for clinical trials will be “an uphill battle.” There is much invested in current approaches, and it’s a considerable leap from mouse studies to human clinical trials.

But the promise of a consistently effective and easy to administer vaccine is irresistible.

“DNA is very stable. The new ideas for delivery include a pill that survives the digestive system and releases the plasmid DNA to be picked up by B lymphocytes that seem to possess an ancestral property for taking up plasmid DNA. Alternatively, the DNA can be formulated for delivery to the upper airways by suitable formulation for inhalation. Many other researchers and I have investigated and pursued this basic idea before in other ways. It’s time to try it with COVID.”

Co-authors include: Gonzalo Almanza, Alex E. Clark, Valentina Kouznetsova, Andrea Castro, Igor F. Tsigelny, Sandra Leibel, William Bray and Aaron F. Carlin, all at UC San Diego; Eduardo Olmedillas and Erica Ollmann Saphire, La Jolla Institute for Immunology; Yan Wu, Capital Medical University, Beijing, China; and George F. Gao, Chinese Academy of Sciences and Chinese FDA.

A team of researchers including UTHealth Houston faculty members Aaron M. Gusdon, MD, assistant professor in the Vivian L. Smith Department of Neurosurgery with McGovern Medical School at UTHealth Houston; H. Alex Choi, MD, associate professor in the department as well as the Department of Neurology; and Louise D. McCullough, MD, PhD, professor and Roy M. and Phyllis Gough Huffington Distinguished Chair in the Department of Neurology, conducted a multi-site, randomized, double-blind, placebo-controlled, adaptive Phase 2 trial evaluating the safety and efficacy of an anti-inflammatory compound, called OP-101, in patients with severe COVID-19. The results of the trial were published today in Science Translational Medicine.

In the trial, 24 patients classified as having severe COVID-19 across five clinical sites in the U.S. were randomized to receive a single intravenous dose of placebo or OP-101 at 2, 4, or 8 mg/kg. All patients received standard of care, including corticosteroids.

“OP-101 is a novel nanotherapeutic compound that specifically targets activated macrophages and microglia, the primary immune cell in the brain,”said Gusdon, who was first author on the study. “Due to its excellent safety profile, we were excited to offer this therapy to these critically ill patients at Memorial Hermann Hospital.”

Hyperinflammation triggered by SARS-CoV-2 is a major cause of disease severity in COVID-19. OP-101 was found to be better than a placebo at decreasing inflammatory markers, as well as better at reducing markers of neurological injury, including neurofilament light chain and glial fibrillary acidic protein.

Additionally, risk for the composite outcome of mechanical ventilation or death at 30 or 60 days after treatment was 71% for patients receiving the placebo, but just 18% for patients in the pooled OP-101 treatment arms. At 60 days after treatment, 3 of 7 patients given placebo and 14 of 17 patients treated with OP-101 survived.

The data shows that OP-101 was well tolerated in the critically ill patient population and could serve as an effective treatment for patients hospitalized with COVID-19.

“Although this was a small-dose escalation trial, there was clearly a strong signal toward benefit at both acute and chronic timepoint,” Gusdon said. “The possibility that this therapy could also benefit patients with other diseases that lead to systemic inflammatory responses, including various forms of brain injury, is extremely exciting.”

OP-101 is a nanotherapeutic compound that has previously been evaluated in several animal models of inflammatory disease and has shown superior anti-inflammatory and anti-oxidant effects.

The COVID-19 pandemic has resulted in more than 300 million people infected globally, with more than 5 million deaths.

Sujatha Kannan, MD, professor in the Department of Anesthesiology and Critical Care Medicine at Johns Hopkins University School of Medicine in Baltimore, Maryland, was senior author on the study. Other co-authors from Johns Hopkins University included Nauder Faraday, MD, and Rangaramanujam M. Kannan, PhD, both also with the medical school; and Derek K. Ng, PhD, with the Bloomberg School of Public Health.

Also contributing to the study were John S. Aita, MD, with Avera McKennan Hospital in Sioux Falls, South Dakota; Sunil Kumar, MD, with Broward Health Medical Center in Fort Lauderdale, Florida; Ishan Mehta, MD, with Emory University School of Medicine in Atlanta, Georgia; Jeffery L. Cleland, PhD, with Ashvattha Therapeutics, Inc. in San Francisco, California; and Keith Robinson, MD, with Syneos Health in Morrisville, North Carolina.

McCullough is also a faculty member with The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences.

The resident’s enjoyment of the session and the benefit for their well-being is real. But the animal is not. It’s a robotic pet with synthetic fur and programmed movements and sounds. But researchers are finding that robotic pets can be useful in therapy, without some of the disadvantages and unpredictability of real animals.

In a paper published in the Canadian Journal of Recreation Therapy, University of Utah researcher Rhonda Nelson and graduate student Rebecca Westenskow developed a protocol for using robotic pets with older adults with dementia. The protocol uses a low-cost robotic pet, establishes ideal session lengths, and identifies common participant responses to the pets to aid in future research.

“Our protocol had questions like: Would you like to scratch the dog behind his ears? Would you like to pet him? Would you like to brush him?” says Nelson, an assistant professor in the Department of Occupational and Recreational Therapies. “And then we were evaluating how people responded to those different cues so that we could then provide some guidelines to people on how to have the most beneficial actions with these animals.”

An affordable robotic pet

Nelson has watched the development of robotic pets for the past decade, intrigued by the potential to use them therapeutically in long-term and geriatric care settings. But until recently the price was prohibitive. “Having been a therapist myself and training our students to work as therapists, I’m very aware that most facilities would never be able to purchase them.”

But with the introduction of Ageless Innovation’s Joy For All Companion pets in 2015, priced at under $150, widespread use of robotic pets as therapy “animals” seemed within reach. Robotic pets can get around many of the risks and drawbacks of live animals in long-term care settings. Many facilities don’t allow personal pets because of allergies, the potential for bites or scratches and other reasons.

Researchers have already begun to study how people with dementia interact with robotic pets, Nelson notes, but haven’t yet developed a unified protocol to give, say, assisted living staff a plan to gain the most benefit from the pets’ use through directed interaction.

“There was very little information on what people were doing with the pets,” Nelson says. “So without that guidance, it’s just a toy. And what do you do with it?”

Observing interactions

Prior to the COVID-19 pandemic, the researchers met with five people between 82 and 87 years old living in long-term care facilities who experienced severe cognitive impairment. In two sessions of 30 minutes each, the researchers brought out either a robotic dog or a cat (participants’ choice) in a pet carrier.

“Many participants leaned toward the [robotic pet] as it was taken out of the pet carrier,” the researchers noted, “then instinctively reached for it and began petting, rubbing or scratching the pet when first introduced.”

Throughout the session, the researchers asked questions, both about the participants’ experiences with past pets and about interacting with the current robotic pet. “Did they have dogs or cats?” Nelson says, giving examples of typical questions. “What were their names? Did they keep them indoors or outdoors? What types of food did they eat?”

The researchers carefully observed the responses of the participants to the pets. The robotic pets moved and made sounds, which Nelson says helped the participants engage with them.

“When the dog would bark they would say things like, ‘Oh, are you trying to tell me something?'” she says. “Or they would comment on the cat purring and would say things like, ‘Wow, you must really be happy! I feel you purring.’ One of the activities that people responded to the most was brushing the animals.”

In one case, though, the session proceeded in silence. The participant had difficulty communicating their thoughts but stayed focused on the robotic dog throughout. By the end of the session, the participant seemed to develop a connection with the robotic animal, saying “I like that dog. When he likes me.”

Nelson is often asked if the participants with cognitive decline understand that the robotic pets are not alive. In this study, she says, they all seemed aware that it was not a live animal.

“Interestingly enough one of our participants was a retired veterinarian,” she says. “So I was very intrigued to see how he would interact with it.” He chose to have both the robotic dog and cat on his lap at the same time. “We would never tell somebody that it was live if they asked. We would be honest with them. We usually introduce it as ‘Would you like to hold my dog’ and people react to it or respond to it in a way that’s meaningful for them.”

Initial recommendations

Unfortunately, data collection was cut short by the COVID-19 pandemic. But the researchers were able to draw some conclusions.

All of the participants enjoyed the activity, with several saying they liked it “very much.” One participant didn’t like the sounds the pet made, which was easily remedied by turning off the sound — not an option for a live animal.

The questions that spurred the most response related to personal reminiscences and directions for interacting with the pet.

A common, yet unprompted behavior, the researchers’ report, was communication with the pet. “Several participants used comments, sounds, specific inflections and facial expressions spontaneously with the pets,” the researchers wrote. “Some participants imitated the animal sounds made by the [pet] and repositioned the pet to look at its face or make eye contact.”

Although more research is needed to determine the optimal session length, the researchers noted that the 30-minute sessions in the study were sufficient. Nelson also hopes to explore how people with varying levels of cognitive decline respond to the pets, as well as how they can be used in a group setting.

The study found that the most meaningful interactions and the most enjoyable experiences came when the participant self-directed the session.

“In recreational therapy, we always talk about providing person-centered care,” Nelson says. So it’s not really about what I think about an activity. If somebody enjoys it and it brings happiness to them, then it’s really about what they think about it.”

Why does interacting with robotic pets provide such an enjoyable experience?

“People in long-term care facilities are in a position where everybody provides care to them,” Nelson says, “and to be in the role where you are nurturing something else, or you are the caregiver I think is also psychologically very comforting for people to feel like, even though they know that it’s not live, they’re the person who’s giving love and compassion to something, and it’s responding.”

“We found that giving the rats a multidrug antibiotic cocktail resulted in significant and sex-specific changes in both the stool, or large intestine, and the small bowel. For example, greater loss of the diversity of the microbes in both stool and small bowel was observed in the male rodents than in the females,” said Ruchi Mathur, MD, the study’s principal investigator.

“Changes in the diversity of the gut microbiome could have a negative impact. Previous studies of intestinal health have demonstrated that overall microbial diversity promotes vitality and resilience, often making for a healthier gut,” said Mathur, an endocrinologist.

In the controlled study, investigators from the Cedars-Sinai Medically Associated Science and Technology (MAST) Program compared the composition of the gut microbes of the male and female rats before, during and after treatment with broad-spectrum antibiotics, including vancomycin, ampicillin, metronidazole, and neomycin.

“We followed the rats for a period after the antibiotics were stopped and found that many of the sex-specific changes we observed during treatment remained. The constellation of microbes in the gut did not return to their original, pre-antibiotic compositions in either sex during the duration of the study,” said research associate Gonzalo Parodi, first author of the paper.

Some studies have identified sex-specific effects of antibiotics on the large bowel (stool) microbiome in mice. But Cedars-Sinai investigators emphasize that this is the first study to also examine changes in the small bowel microbiome induced by antibiotics and to also use male and female rats that had no significant differences in microbiome composition prior to treatment.

“It is difficult to tease out the impact of the drugs if the lab animals’ gut microbiomes are different to begin with. We used rats with similar gut profiles right out of the gate. This allowed us to pinpoint changes in the microbiomes of the male and female rats that could be attributed specifically to the impact of the antibiotics during and after exposure to the medicines,” said Mark Pimentel, MD, director of the MAST program and a co-author of the study.

More studies are needed using different combinations of antibiotics and exposure times. Investigators say if these findings hold true for humans, the research could have implications for the way antibiotics are prescribed.

“Sex is a biological variable, and like any variable, it needs to be taken into consideration in basic and medical research. Currently, we consider factors such as kidney function and weight when dosing medications for patients. Depending on the results of further research, specifically in humans, the sex of patients may one day be an important consideration when prescribing antibiotics,” said Mathur.

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