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AI approach outperformed human experts in identifying cervical precancer

A research team led by investigators from the National Institutes of Health and Global Good has developed a computer algorithm that can analyze digital images of a woman’s cervix and accurately identify precancerous changes that require medical attention. This artificial intelligence (AI) approach, called automated visual evaluation, has the potential to revolutionize cervical cancer screening, particularly in low-resource settings.

To develop the method, researchers used comprehensive datasets to “train” a deep, or machine, learning algorithm to recognize patterns in complex visual inputs, such as medical images. The approach was created collaboratively by investigators at the National Cancer Institute (NCI) and Global Good, a project of Intellectual Ventures, and the findings were confirmed independently by experts at the National Library of Medicine (NLM). The results appeared in the Journal of the National Cancer Institute on January 10, 2019. NCI and NLM are parts of NIH.

“Our findings show that a deep learning algorithm can use images collected during routine cervical cancer screening to identify precancerous changes that, if left untreated, may develop into cancer,” said Mark Schiffman, M.D., M.P.H., of NCI’s Division of Cancer Epidemiology and Genetics, and senior author of the study. “In fact, the computer analysis of the images was better at identifying precancer than a human expert reviewer of Pap tests under the microscope (cytology).”

The new method has the potential to be of particular value in low-resource settings. Health care workers in such settings currently use a screening method called visual inspection with acetic acid (VIA). In this approach, a health worker applies dilute acetic acid to the cervix and inspects the cervix with the naked eye, looking for “aceto whitening,” which indicates possible disease. Because of its convenience and low cost, VIA is widely used where more advanced screening methods are not available. However, it is known to be inaccurate and needs improvement.

Automated visual evaluation is similarly easy to perform. Health workers can use a cell phone or similar camera device for cervical screening and treatment during a single visit. In addition, this approach can be performed with minimal training, making it ideal for countries with limited health care resources, where cervical cancer is a leading cause of illness and death among women.

To create the algorithm, the research team used more than 60,000 cervical images from an NCI archive of photos collected during a cervical cancer screening study that was carried out in Costa Rica in the 1990s. More than 9,400 women participated in that population study, with follow up that lasted up to 18 years. Because of the prospective nature of the study, the researchers gained nearly complete information on which cervical changes became precancers and which did not. The photos were digitized and then used to train a deep learning algorithm so that it could distinguish cervical conditions requiring treatment from those not requiring treatment.

Overall, the algorithm performed better than all standard screening tests at predicting all cases diagnosed during the Costa Rica study. Automated visual evaluation identified precancer with greater accuracy (AUC=0.91) than a human expert review (AUC=0.69) or conventional cytology (AUC=0.71). An AUC of 0.5 indicates a test that is no better than chance, whereas an AUC of 1.0 represents a test with perfect accuracy in identifying disease.

“When this algorithm is combined with advances in HPV vaccination, emerging HPV detection technologies, and improvements in treatment, it is conceivable that cervical cancer could be brought under control, even in low-resource settings,” said Maurizio Vecchione, executive vice president of Global Good.

The researchers plan to further train the algorithm on a sample of representative images of cervical precancers and normal cervical tissue from women in communities around the world, using a variety of cameras and other imaging options. This step is necessary because of subtle variations in the appearance of the cervix among women in different geographic regions. The ultimate goal of the project is to create the best possible algorithm for common, open use.

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Experimental antibody 'cocktail' protects animals from three deadly Ebola viruses

Scientists from academia, industry, and government have developed a combination of monoclonal antibodies (mAbs) that protected animals from all three Ebola viruses known to cause human disease. Their work is described in two companion studies published online in the journal Cell Host & Microbe.

The mAb “cocktail,” called MBP134, is the first experimental treatment to protect monkeys against Ebola virus (formerly known as Ebola Zaire), as well as Sudan virus and Bundibugyo virus, and could lead to a broadly effective therapeutic, according to the authors.

Over 20 Ebola virus outbreaks have occurred since the first outbreak was documented in 1976 in the Democratic Republic of Congo, or DRC (formerly called Zaire). The 2013-2016 Ebola epidemic in Western Africa — the largest outbreak to date — sickened more than 28,000 people and caused more than 11,000 deaths. An ongoing outbreak in the eastern Kivu region of DRC is already the second largest on record, according to the World Health Organization.

No Ebola virus medical countermeasures have been approved by the U.S. Food and Drug Administration. An experimental vaccine and several experimental therapeutics — including three based on mAbs — are being studied in the field. Despite their promise, all target only a single Ebola virus (Zaire) and are ineffective against the other two.

“Developing a single treatment that could potentially be used for patients suffering from all the different types of Ebola viruses is an enormous advancement in the field,” commented John M. Dye, Ph.D. of the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID), one of the authors.

Citing growing evidence of the value of monoclonal antibodies for treating even the most virulent infections, Dye added, “This discovery has implications not only for the treatment of Sudan and Bundibugyo viruses, but for newly emerging Ebola viruses as well.”

The two mAbs that make up MBP134 were previously discovered by the same research team in the blood of a human survivor of the 2013-2016 outbreak in Western Africa and were shown to target key sites of vulnerability shared by Ebola viruses.

In the first study, a team led by Kartik Chandran, Ph.D., of the Albert Einstein College of Medicine (Einstein) engineered one of the mAbs to improve its activity against Sudan virus. They demonstrated that this enhanced mAb could work especially well with the second naturally occurring mAb to block infection by all three viruses and protect guinea pigs against both Ebola virus and Sudan virus. Additional modification of both mAbs to harness the power of “natural killer” immune cells enhanced MBP134’s broad protective efficacy in guinea pigs even further.

In the second study, a team led by Dr. Zachary A. Bornholdt, Ph.D., of Mapp Biopharmaceutical Inc. (MappBio) evaluated the MBP134 cocktail in large animal models that mimic Ebola virus disease in humans more closely. They found that a single low dose of MBP134 could protect monkeys against all three Ebola viruses associated with human disease, even when treatment was begun 4-7 days after the animals were infected.

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Madariaga virus spreads to Haiti

Madariaga virus (MADV), or South American eastern equine encephalitis virus (EEEV), has — until now — been found primarily in animals of South and Central America, with the first human outbreak occurring in Panama in 2010. Now, scientists writing in PLOS Neglected Tropical Diseases report the identification of MADV in eight children in Haiti in 2015 and 2016.

Madariaga is a mosquito-borne disease transmitted to humans from animals including horses, mice, rats and bats. Little is known about its epidemiology or life cycle. While MADV can cause serious encephalitis, surveys in Panama after the 2010 outbreak found that between 2 to 5 percent of the population in the areas affected by MADV had evidence of prior infection, suggesting that mild or asymptomatic infections are common.

Glenn Morris of the University of Florida and colleagues maintain a surveillance program at a school clinic in Haiti and, since 2014, have collected blood samples of children presenting to the clinic with acute febrile illness. In April 2015, one such sample — collected from an 8-year-old girl in the Gressier/Leogane region of Haiti — tested positive for MADV. The virus was then cultured from an additional seven patients from the same cohort throughout 2016.

Symptoms from the patients most closely resembled those seen in dengue fever infection, and no patients had encephalitis. All the strains isolated were very similar, and using available information on the genetic sequence of the MADV cultured from the patients, the team was able to hypothesize that the virus was introduced to Haiti from Panama sometime between October 2012 and January 2015.

“Our data indicate that this virus, which has the potential for causing serious illness, has recently been introduced into Haiti, and raises the possibility that it might move into other parts of the Caribbean or North America,” the researchers say.

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Bacteria help discover human cancer-causing proteins

A team led by researchers at Baylor College of Medicine and the University of Texas at Austin has applied an unconventional approach that used bacteria to discover human proteins that can lead to DNA damage and promote cancer. Reported in the journal Cell, the study also proposes biological mechanisms by which these proteins can cause damage to DNA, opening possibilities for future cancer treatments.

“Our cells make protein carcinogens,” said co-corresponding author Dr. Susan M. Rosenberg, Ben F. Love Chair in Cancer Research and professor of molecular and human genetics, of molecular virology and microbiology and of biochemistry and molecular biology at Baylor. “Cancer is a disease of mutations. A normal cell that has accumulated several mutations in particular genes becomes likely to turn into a cancer cell.”

Mutations that cause cancer can be the result of DNA damage. External factors such as tobacco smoke and sunlight can damage DNA, but most DNA damage seems to result from events that occur within cells and is mediated by cellular components, including proteins. Despite the importance of these events, they have not been studied extensively.

“One way proteins can cause DNA damage is by being overproduced, which is a relatively frequent cellular event,” said Rosenberg, who also is leader of the Cancer Evolvability Program at the Dan L Duncan Comprehensive Cancer Center at Baylor. “In this study, we set out to uncover proteins that, when overproduced by the cell, cause damage to DNA in ways that can lead to cancer.”

To uncover these DNA “damage-up” proteins, the researchers took an unconventional approach. They searched for proteins that promote DNA damage in human cells by looking at proteins that, when overproduced, would cause DNA damage in the bacterium E. coli.

“Although bacteria and people are different, their basic biological processes are similar, so with this approach we thought we might find common mechanisms of DNA damage that could be relevant to cancer,” Rosenberg said.

“This was a wild idea,” said Rosenberg, and was possible because of funding from two sources aimed at trying high-risk strategies that, if successful, would have high impact: a National Institutes of Health Director’s Pioneer Award and a gift from the W.M. Keck Foundation, among many other grants to the 16-lab team.

E. coli reveals novel candidates for cancer-promoting proteins

The researchers genetically modified bacteria so they would fluoresce red when DNA was damaged. Then, they overexpressed each of the 4,000 genes present in E coli individually and determined which ones made bacteria glow red.

“We uncovered an extensive and varied network of proteins that, when overproduced, alter cells in ways that lead to DNA damage,” Rosenberg said. “Some of these proteins are, as expected, involved in DNA processing or repair, but, surprisingly, most are not directly connected to DNA. For instance, some of the DNA damage-up proteins participate in the transport of molecules across the cell membrane.”

When the researchers looked for human protein relatives of the DNA “damage-up” proteins they had found in bacteria, they identified 284. Interestingly, they determined that these human proteins are linked to cancer more often than random sets of proteins. In addition, the proteins’ RNAs, an indicator of protein production, predicted mutagenesis in tumors and poor patient prognosis. When the researchers overproduced these proteins in human cells in the lab, half of the proteins triggered DNA damage and mutation.

“We showed that E. coli can help to identify DNA damage-up proteins and mechanisms of action in human cells quickly and inexpensively. Some of the proteins and their mechanisms were known to be involved in cancer, but many others were not suspected of being in the cancer-causing list,” said co-corresponding author Dr. Christophe Herman, professor of molecular and human genetics and molecular virology and microbiology at Baylor College of Medicine and member of the Dan L Duncan Comprehensive Cancer Center.

“I think it is extraordinary to identify so many ways DNA can be damaged. This study is opening up new avenues for discoveries of novel mechanisms that protect our genomes and how their dysfunction can alter the integrity of our DNA and cause cancer,” said co-corresponding author Dr. Kyle M. Miller, associate professor of molecular biosciences at the University of Texas at Austin and member of the Dan L Duncan Comprehensive Cancer Center at Baylor. “It is yet another example of the power of model organisms to uncover basic biological processes that can shine a light on how human cells and cancer work.”

“Our work has significant implications both in basic biological fields and in clinical research,” Rosenberg said. “We provide a previously unknown understanding of the diverse mechanisms that can generate DNA damage leading to cancer. In the future, this finding may lead to new ways to identify people who are likely to develop cancer so that strategies to prevent it, slow it down or catch it early can be used.”

The study’s two co-first authors were students earning their doctorates: Dr. Jun Xia at Baylor College of Medicine and Dr. Liya Chiu at the University of Texas at Austin.

For a complete list of all the contributors and their affiliations and the financial support of this study, visit the journal Cell.

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A human model to test implants for cataract surgery

Researchers at the University of East Anglia in collaboration with Hoya Surgical Optics have improved a laboratory model that simulates cataract surgery on human donor eyes.

Their latest human model allows evaluation of artificial intraocular lens (IOL) implants under conditions that better reflect the post-surgical environment.

A cataract is a clouding of the eye’s lens and affects millions worldwide. At present, the only way of treating cataract is with surgery and it is estimated that by 2020, more than 30 million such operations will be performed annually.

During cataract surgery, the eye’s cloudy natural lens is removed and replaced with an IOL.

Initially, outcomes from cataract surgery are superb, but a haziness known as Posterior Capsule Opacification (PCO) can develop in a significant number of patients following surgery.

At the moment the only treatment for PCO is laser surgery — which is expensive and not without risks. There is therefore a need for better understanding the physiological events driving PCO and to better manage the condition.

The human capsular bag model was pioneered at UEA in the 1990s and has progressively evolved with time.

Lead researcher Prof Michael Wormstone, from UEA’s School of Biological Sciences, said: “Our model now mimics the transient nature of inflammation that patients experience after cataract surgery. It is more reflective of clinical events and allows comparative evaluation of different types of IOLs.

“We believe this latest model will allow assessment of current commercial IOLs and will aid the development of next generation lenses.”

The team used this clinically relevant model to assess the influence of two commercial market-leading IOLs (Alcon AcrysofTM and Hoya VivinexTM) on PCO management.

Their findings showed that cell growth on the posterior capsule was reduced, light-scatter in the central visual axis was found to be lower and growth on the IOL surface was significantly reduced with the Hoya VivinexTM IOL relative to the Alcon AcrysofTM.

The research team conclude that “our model system predicts that the Hoya VivinexTM is better able to manage events leading to PCO than the Alcon AcrysofTM IOL.”

The research was funded by Hoya Surgical Optics and The Humane Research Trust.

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Targeting an RNA-binding protein to fight aging

As we age, our bodies undergo biological changes that cause a decline in the function of our cells and tissues. However, most studies attempting to identify molecules involved age-related dysfunctions have focused only on mechanisms based on mRNA transcription, a very important step in gene expression, but nonetheless only part of the complex regulatory mechanisms in our cells.

Scientists led by Johan Auwerx’s lab at EPFL, have taken a different route, and studied the link between aging and RNA-binding proteins (RBPs), which bind mRNA molecules and regulate their fate after gene transcription. They have published their findings in Molecular Cell.

The scientists first screened cells from old animals to identify any RBPs that change upon aging. The screening showed that one particular protein, Pumilio2 (PUM2), was highly induced in old animals. PUM2 binds mRNA molecules containing specific recognition sites. Upon its binding, PUM2 represses the translation of the target mRNAs into proteins.

Using a systems genetics approach, the researchers then identified a new mRNA target that PUM2 binds. The mRNA encodes for a protein called Mitochondrial Fission Factor (MFF), and is a pivotal regulator of mitochondrial fission — a process by which mitochondria break up into smaller mitochondria. Having high levels of MFF also allows the clearance of broken up, dysfunctional mitochondria, a process called mitophagy.

The study found that this newly identified PUM2/MFF axis is dysregulated upon aging. Evidence for this came from examining muscle and brain tissues of old animals, which were found to have more PUM2, and, consequently, fewer MFF proteins. This leads to a reduction of mitochondrial fission and mitophagy, and without the ability to chop up and remove smaller mitochondria, the aged tissues start accumulating bigger and unhealthy organelles.

But removing PUM2 from the muscles of old mice can reverse this. “We used the CRISPR-Cas9 technology to specifically target and inactivate the gene encoding for Pum2 in the gastrocnemius muscles of old rodents,” says Davide D’Amico, first author of the paper. “Reducing Pum2 levels, we obtained more MFF protein and increased mitochondrial fragmentation and mitophagy. Notably, the consequence was a significant improvement of the mitochondrial function of the old animals.”

The same mechanism is conserved also in the nematode C. elegans, where the protein PUF-8 is also induced upon aging. “Depleting the gene for PUF-8 from old worms is sufficient to improve mitochondrial function and to increase lifespan,” says Johan Auwerx. “This work is an example of how a multi-omics and cross-species approach can unveil new genes associated with aging.”

RNA-binding proteins are also linked to neuromuscular degenerative diseases, and often aggregate in pathological granules. “We discovered that PUM2 tends to condensate into particles that bind and trap Mff mRNA upon aging,” says D’Amico. “These observations require further studies, but clearly show that RNA-binding proteins could be promising targets in aging and age-related dysfunctions.”

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New role for brain's support cells in controlling circadian rhythms

Astrocytes, ‘caretaker’ cells that surround and support neurons in the brain, play a much more important role in circadian rhythms, the body’s 24-hour internal clock, than previously understood.

The Medical Research Council (MRC)-funded study published today in the journal Science, found that these star-shaped cells, previously thought of as just supporting neurons in regulating circadian rhythms, can actually lead the tempo of the body’s internal clock and have been shown for the first time to be able to control patterns of daily behaviour in mammals.

The findings of the new study could pave the way for new treatments to be exploited when circadian rhythms are disrupted, which can cause jet lag and sleep disorders, as well as contribute to a range of health conditions, from psychiatric disorders to dementia, diabetes and cancer.

Circadian rhythms are well known for their role in maintaining human health and although many different types of cells across the body have been found to have their own internal clock, the timing of these clocks is chiefly controlled by the suprachiasmatic nucleus (SCN), a small brain region in the hypothalamus which acts as the master clock responsible for regulating daily behaviour.

This new study, led by the MRC’s Laboratory of Molecular Biology (LMB) in Cambridge, used microscopic imaging to observe the detailed internal molecular clock timing of the astrocytes and neurons of the SCN. Surprisingly, this showed that although both types of cell have their own circadian clocks, they are differently regulated and were seen to be active at different times of the day. This delicate interplay was found to be critical in keeping the entire SCN clockwork ticking.

Following this initial discovery, the scientists found mice genetically altered to silence their internal body clock showed disruption to their SCN function and behaviour but, unexpectedly, found that the restoration of a genetically functional clock in astrocytes alone enabled the mice to regulate their daily activity. This meant that even when astrocytes were the only cell in an animal with a working internal clock, there were still observed patterns of daily behaviour of mice. When the researchers compared this pattern of behaviour to mice whose neuronal clocks were working, they found that the period of regulated activity in the SCN was approximately one hour shorter, which was also reflected by the mouse behaviour, showing that astrocytes were capable of controlling animal behaviour to their own cell-specific tune.

The study also revealed that glutamate, a neurotransmitter in the brain and central nervous system, acted as the chemical signal used to convey time cues from the working astrocytes of the SCN to their clockless neuronal partners.

“The discovery that astrocytes can be as effective as neurons in generating and transmitting a circadian timing signal across an animal really surprised us,” said Dr Marco Brancaccio, a Dementia Lecturer and UK Dementia Research Institute Fellow at Imperial College London and lead author of the paper, previously at the MRC’s LMB when this research was undertaken. “We knew from previous research that these cells played a role in circadian clocks, but we had no idea they could restart the circadian function of neurons. This adds a totally new and unanticipated dimension to the neurobiology of circadian body clocks and suggests some exciting avenues for future research and the potential to develop treatments.”

“This is the first time that it has been demonstrated that astrocytes, cells we’d previously overlooked as mere support cells, can actually control animal behaviour. This is a significant advance in the field of neuroscience,” added Dr Michael Hastings, Head of the Neurobiology Division at the MRC’s LMB and senior author of the paper.

Dr Joanna Latimer, Head of Neurosciences and Mental Health at the MRC, said: “In recent years it has become increasingly clear that disruption of the body’s internal clock through shift-work, dementia and other neurological diseases can have a dangerous impact on our health and well-being. This research is an important step towards a better understanding of how the brain controls these circadian rhythms at a molecular and cellular level, an essential advance if we are to manage the impact of these conditions more effectively.”

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Using genetics of human fat cells to predict response to anti-diabetes drugs

Drugs called thiazolidinediones (TZDs) reverse insulin resistance in patients with type 2 diabetes by targeting the activity of a receptor protein. However, an array of side effects, including weight gain, edema, and high cholesterol, limits their use in the clinic.

In a new study published in Cell Stem Cell, a team of researchers led by Mitchell Lazar, MD, PhD, director of the Institute for Diabetes, Obesity, and Metabolism in the Perelman School of Medicine at the University of Pennsylvania, have demonstrated — using fat cells derived from human stem cells — that individual genetic variation can be used to predict whether the TZD rosiglitazone will produce the unwanted side effect of increasing cholesterol levels in certain individuals.

“Obesity has reached global epidemic proportions and is a major risk factor for type 2 diabetes, so any steps we can take toward understanding how patients respond to treatment is crucial,” Lazar said. “We found that genetic variation impacts how PPAR? [a receptor protein] interacts with the genome DNA in fat cells. This determines an individual’s responses to anti-diabetic drugs and has direct implications for developing personalized therapies for diabetes.”

PPAR? is required for the maturation of fat cells and is the target of TZDs, the only class of drugs that reverse insulin resistance associated with type 2 diabetes.

By studying the genome of fat cells derived from patients treated at Penn Medicine, Lazar’s team discovered a genetic variation that predicted whether rosiglitazone would increase expression of a gene called ABCA1, which regulates cholesterol levels. The variation does not appear in the protein-coding region of the ABCA1 gene, but is found in regions that code for molecules that regulate the level of ABCA1 expression. The team demonstrated the causal relationship between the genetic variation and increased ABCA1 expression by editing the variant from its inactive form to the active one using CRISPR/Cas9.

The ability of the active variant to predict whether rosiglitazone treatment will increase cholesterol was confirmed by studying 84 patients treated with the drug in Shanghai. While the overall effects of TZDs are clearly governed by many genes, the Penn study showed that individual genetic variations can predict effects of drugs on gene expression and metabolic physiology.

“Understanding the underlying mechanism driving a differential response to TZDs could inform personalized and precision approaches to treating type 2 diabetes,” Lazar said. “Doctors could test for this genetic variation and choose to avoid rosiglitazone treatment of diabetic patients whose genes predict that the drug would have a greater chance of increasing their serum cholesterol levels.”

These principles can be applied not only to TZDs but more broadly to other classes of drugs that work at non-coding regions of the genome, including those targeting steroid hormone receptors. The hope is to be able to predict which patients will have beneficial versus detrimental responses to drugs for tailoring drug therapy for individuals.

This work was supported by the Cox Medical Institute, the JPB Foundation, the National Institutes of Health (R01-DK049780, R01-DK098542, K08-DK094968, F32-DK116519), and the American Diabetes Association (1-18-PDF-132).

Penn coauthors include Wenxiang Hu, Chunjie Jiang, Dongyin Guan, Pieterjan Dierickx, David J. Steger, and Raymond E. Soccio.

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Researchers reveal active-state structure of popular drug target for blood pressure

Bringing a long quest to a satisfying conclusion, researchers have mapped the active-state structure of the angiotensin II type 1 receptor, the target of widely prescribed drugs to regulate blood pressure and kidney function.

The study, published online Jan. 10 in Cell, was conducted by researchers in the Blavatnik Institute at Harvard Medical School and colleagues at Duke University Medical Center.

When the hormone angiotensin attaches to this receptor, it constricts blood vessels to raise blood pressure and stimulates the adrenal glands to retain salt in the kidneys. An estimated 5 percent of adults in the United States take angiotensin II receptor blockers to lower blood pressure, treat heart failure or prevent kidney failure or stroke.

Despite the receptor’s critical roles in the body and its popularity as a drug target, researchers have struggled to answer the question: How does it activate?

“Millions of people take drugs that turn this receptor off, and it has been studied very thoroughly, but because of technological limitations, people couldn’t see what it looks like when it’s turned on,” said Andrew Kruse, associate professor of biological chemistry and molecular pharmacology at HMS and co-senior author of the paper with Robert Lefkowitz of Duke.

“Our study provides a framework for interpreting a lot of data that’s been collected over the years about the receptor’s function, helping to turn disparate pieces into a single coherent model,” Kruse said.

The new findings also offer clues about how drugs might be developed that activate, rather than block, the angiotensin II receptor or other receptors in highly controlled ways.

Twist and shout

Like many proteins embedded in the cell membrane, the angiotensin II receptor twists into different shapes depending on whether it’s inactive or bound to one protein versus another.

Researchers who want to study a receptor in a specific shape, or conformation, often try to generate an antibody with a complementary shape that will hold the receptor still. Then they can crystallize it, bombard it with X-rays and translate the resulting image into a 3D atomic structure.

Lefkowitz’s group tried the standard tactic — inoculating a llama to stimulate its immune system to produce the needed antibodies — but each attempt proved unsuccessful.

“The receptor would flip-flop between all these different conformations,” said Lefkowitz. “We tried for at least six years to develop an antibody that would stabilize it, and we failed at every turn.”

A tool developed by the Kruse lab in 2018 provided the key. Using yeast instead of llamas, the researchers had generated a library of 500 million artificial nanobodies (small antibodies) to help structural biologists.

One of the nanobodies did exactly what Lefkowitz needed.

“Andrew’s new technique, combined with lots of hard, collaborative effort, finally made it possible,” he said.

“This is an example of a good collaboration, where we each provided something unique,” Kruse agreed. “We offered our nanobody library and expertise in crystallizing G protein-coupled receptors like the angiotensin II receptor, while the Lefkowitz lab brought their strengths in receptor biochemistry and pharmacology.”

Conor McMahon, a postdoctoral researcher in the Kruse lab and co-first author of the paper, together with Laura Wingler and Dean Staus in the Lefkowitz lab, added, “This demonstrates the utility of our nanobody library, and it’s particularly exciting to see it succeed in a case where llama immunization was unsuccessful.”

Details revealed

Capturing the receptor’s intricately detailed crystal structure revealed how it binds to angiotensin, how changes on the portion of the receptor outside the cell triggers changes on the portion inside the cell, and more.

“Many of the conformational changes were unique compared to those we’d seen before,” said Kruse.

Kruse and team also hope the study will help provide a path to answering questions about the mysterious phenomenon known as biased agonism, in which a protein that binds to a receptor activates one pathway preferentially, rather than activating two or more pathways equally.

This study was supported by the National Institutes of Health (grants R01HL16037 and 5DP5OD021345), the Mandel Center for Hypertension and Atherosclerosis at Duke University, the Vallee Foundation and the Smith Family Foundation. Lefkowitz is an investigator with the Howard Hughes Medical Institute.

Lefkowitz is a founder and stockholder of Trevena, Inc., and is a director of Lexicon Pharmaceuticals. Kruse is a founder of Ab Initio Biotherapeutics, Inc.

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Men and women remember pain differently

Scientists increasingly believe that one of the driving forces in chronic pain — the number one health problem in both prevalence and burden — appears to be the memory of earlier pain. Research published today/this week in Current Biology suggests that there may be variations, based on sex, in the way that pain is remembered in both mice and humans.

The research team, led by colleagues from McGill and University of Toronto Mississauga, found that men (and male mice) remembered earlier painful experiences clearly. As a result, they were stressed and hypersensitive to later pain when returned to the location in which it had earlier been experienced. Women (and female mice) did not seem to be stressed by their earlier experiences of pain. The researchers believe that the robust translational nature of the results, from mice to men, will potentially aid scientists to move forward in their search for future treatments of chronic pain.

It was a discovery that came as a total surprise.

Robust results in mice and men

“We set out to do an experiment looking at pain hypersensitivity in mice and found these surprising differences in stress levels between male and female mice,” explains Jeffrey Mogil, the E.P. Taylor Professor of Pain Studies in McGill’s Department of Psychology and Alan Edwards Centre for Research on Pain who is the senior author on the study. “So we decided to extend the experiment to humans to see whether the results would be similar. We were blown away when we saw that there seemed to be the same differences between men and women as we had seen in mice.”

“What was even more surprising was that the men reacted more, because it is well known that women are both more sensitive to pain than men, and that they are also generally more stressed out,” explains Loren Martin, the first author on the paper and an Assistant Professor of Psychology at the University of Toronto Mississauga.

Creating memories of pain in humans and mice

In experiments with both humans and mice, the subjects (41 men and 38 women between the ages of 18-40 in the case of humans) were taken to a specific room (or put in a testing container of a certain shape — depending on the species) where they experienced low levels of pain caused by heat delivered to their hind paw or forearm. Humans rated the level of pain on a 100-point scale and mice “rated” the pain by how quickly they moved away from the heat source. Immediately following this initial experience of low-level pain, subjects experienced more intense pain designed to act as Pavlovian conditioning stimuli. The human subjects were asked to wear a tightly inflated blood pressure cuff and exercise their arms for 20 minutes. This is excruciating and only seven of the 80 subjects rated it at less than 50 on a 100-point scale. Each mouse received a diluted injection of vinegar designed to cause a stomach ache for about 30 minutes.

In order to look at the role that memory plays in the experience of pain, the following day the subjects returned to either the same or a different room, or to the same or a different testing container. Heat was once again applied to their arms or hind paws.

When (and only when) they were taken into the same room as in the previous test, the men rated the heat pain higher than they did the day before, and higher than the women did. Similarly, male, but not female mice returning to the same environment exhibited a heightened heat pain response, while mice placed in a new and neutral environment did not.

“We believe that the mice and the men were anticipating the cuff, or the vinegar, and, for the males, the stress of that anticipation caused greater pain sensitivity,” says Mogil. “There was some reason to expect that we would see increased sensitivity to pain on the second day, but there was no reason to expect it would be specific to males. That came as a complete surprise.”

Blocking memories makes the pain go away

In order to confirm that pain was increased due to memories of previous pain, the researchers interfered with memory by injecting the brains of male mice with a drug called ZIP that is known to block memory. When the researchers then ran the pain memory experiment, these mice showed no signs of remembered pain.

“This is an important finding because increasing evidence suggests that chronic pain is a problem to the extent that you remember it , and this study is the first time such remembered pain has been shown using a translational — both rodent and human subject — approach,” says Martin, who is also the Tier II Canada Research Chair in Translational Pain Research. “If remembered pain is a driving force for chronic pain and we understand how pain is remembered, we may be able help some sufferers by treating the mechanisms behind the memories directly.”

Mogil echoes this optimism, “This research supports the idea that the memory of pain can affect later pain.” He adds, “I think it is appropriate to say that further study of this extremely robust phenomenon might give us insights that may be useful for future treatment of chronic pain, and I don’t often say that! One thing is for sure, after running this study, I’m not very proud of my gender.”