In the study, reported on December 14, 2022, in Science Advances, the researchers found that a particularly harmful, chemically modified form of an inflammatory immune protein called complement C3 was present at much higher levels in the brains of women who had died with the disease, compared to men who had died with the disease. They also showed that estrogen — which drops in production during menopause — normally protects against the creation of this form of complement C3.

“Our new findings suggest that chemical modification of a component of the complement system helps drive Alzheimer’s, and may explain, at least in part, why the disease predominantly affects women,” says study senior author Stuart Lipton, MD, PhD, professor and Step Family Foundation Endowed Chair in the Department of Molecular Medicine at Scripps Research and a clinical neurologist in La Jolla, California.

The study was a collaboration with a team led by Steven Tannenbaum, PhD, Post Tenure Underwood-Prescott Professor of Biological Engineering, Chemistry and Toxicology at MIT.

Alzheimer’s, the most common form of dementia that occurs with aging, currently afflicts about six million people in the U.S. alone. It is always fatal, usually within a decade of onset, and there is no approved treatment that can halt the disease process, let alone reverse it. The shortcomings of treatments reflect the fact that scientists have never fully understood how Alzheimer’s develops. Scientists also don’t know fully why women account for nearly two-thirds of cases.

Lipton’s lab studies biochemical and molecular events that may underlie neurodegenerative diseases, including the chemical reaction that forms a modified type of complement C3 — a process called protein S-nitrosylation. Lipton and his colleagues previously discovered this chemical reaction, which happens when a nitric oxide (NO)-related molecule binds tightly to a sulfur atom (S) on a particular amino acid building-block of proteins to form a modified “SNO-protein.” Protein modifications by small clusters of atoms such as NO are common in cells and typically activate or deactivate a target protein’s functions. For technical reasons, S-nitrosylation has been more difficult to study than other protein modifications, but Lipton suspects that “SNO-storms” of these proteins could be a key contributor to Alzheimer’s and other neurodegenerative disorders.

For the new study, the researchers used novel methods for detecting S-nitrosylation to quantify proteins modified in 40 postmortem human brains. Half of the brains were from people who had died of Alzheimer’s, and half were from people who hadn’t — and each group was divided equally between males and females.

In these brains, the scientists found 1,449 different proteins that had been S-nitrosylated. Among the proteins most often modified in this way, there were several that have already been tied to Alzheimer’s, including complement C3. Strikingly, the levels of S-nitrosylated C3 (SNO-C3) were more than six-fold higher in female Alzheimer’s brains compared to male Alzheimer’s brains.

The complement system is an evolutionarily older part of the human immune system. It consists of a family of proteins, including C3, that can activate one another to drive inflammation in what is called the “complement cascade.” Scientists have known for more than 30 years that Alzheimer’s brains have higher levels of complement proteins and other markers of inflammation, compared to neurologically normal brains. More recent research has shown specifically that complement proteins can trigger brain-resident immune cells called microglia to destroy synapses — the connection points through which neurons send signals to one another. Many researchers now suspect that this synapse-destroying mechanism at least partly underlies the Alzheimer’s disease process, and loss of synapses has been demonstrated to be a significant correlate of cognitive decline in Alzheimer’s brains.

Why would SNO-C3 be more common in female brains with Alzheimer’s? There has long been evidence that the female hormone estrogen can have brain-protective effects under some conditions; thus, the researchers hypothesized that estrogen specifically protects women’s brains from C3 S-nitrosylation — and this protection is lost when estrogen levels fall sharply with menopause. Experiments with cultured human brain cells supported this hypothesis, revealing that SNO-C3 increases as estrogen (?-estradiol) levels fall, due to the activation of an enzyme that makes NO in brain cells. This increase in SNO-C3 activates microglial destruction of synapses.

“Why women are more likely to get Alzheimer’s has long been a mystery, but I think our results represent an important piece of the puzzle that mechanistically explains the increased vulnerability of women as they age,” Lipton says.

He and his colleagues now hope to conduct further experiments with de-nitrosylating compounds — which remove the SNO modification — to see if they can reduce pathology in animal models of Alzheimer’s and eventually in humans.

Cluster headaches are short but extremely painful headaches that can occur many days, or even weeks, in a row. The headaches can last anywhere from 15 minutes to three hours.

“Around the world, headaches have an incredibly negative impact on people’s quality of life, both economically and socially,” said study author Caroline Ran, PhD, of the Karolinska Institutet in Stockholm, Sweden. “Our results show that people with cluster headaches not only have an increased risk of other illnesses, those with at least one additional illness missed four times as many days of work due to sickness and disability than those with just cluster headaches. They also have a higher chance of a long-term absence from work.”

The study involved 3,240 people with cluster headaches from age 16-64 in Sweden who were compared to 16,200 people who were similar in age, sex and other factors. The majority were men, which is common with cluster headache.

Researchers looked at work records and disability benefits to determine how many days during a year people were absent from work due to sickness and disability.

Among those with cluster headaches, 92%, or 2,977 people, had at least one additional illness. Of those without cluster headaches, 78%, or 12,575 people, had two or more illnesses.

Of those with cluster headaches, more women had additional illnesses than men, 96% and 90% respectively.

The average number of days a person was absent due to sickness and disability was nearly twice as high among people with cluster headaches with 63 days compared to those without cluster headaches with 34 days.

People with cluster headaches and at least one additional illness had four times as many absence days compared to people with cluster headaches who did not have an additional illness.

“Increasing our understanding of the other conditions that affect people with cluster headache and how they impact their ability to work is very important,” added Ran. “This information can help us as we make decisions on treatments, prevention and prognoses.”

A limitation of the study was that information on personal data, such as smoking, alcohol consumption and BMI, which could affect the occurrence of diseases, was not available.

The study was supported by Swedish Research Council, Swedish Brain Foundation and Mellby Gård, Region Stockholm, Märta Lundkvist Foundation and Karolinska Institutet Research Funds.

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The trials were conducted under the Partnership for Research on Ebola Vaccination (PREVAC) international consortium. PREVAC’s partner organizations include the U.S. National Institutes of Health (NIH), the French National Institute of Health and Medical Research and the London School of Hygiene & Tropical Medicine. The National Institute of Allergy and Infectious Diseases (NIAID), part of NIH, sponsored the trial in Liberia and Mali. In Liberia, the study was done in collaboration with the Liberia Ministry of Health through the Partnership for Research on Ebola Virus in Liberia program. In Mali, it was conducted in collaboration with the University Clinical Research Center and the Center for Vaccine Development-Mali.

The trials began enrollment in 2017, were conducted simultaneously and shared a placebo arm. A total of 1,400 adults and 1,401 children aged 1 year to 17 years old were randomized to receive two injections of either placebo or Ebola vaccine in one of three regimens. The Ebola vaccine regimens were Ad26.ZEBOV (supplied by Johnson & Johnson) followed eight weeks later with a booster dose of MVA-BN-Filo vaccine (supplied by manufacturer Bavarian Nordic); two doses of rVSVΔG-ZEBOV-GP (manufactured by Merck Sharp & Dohme Corp) separated by eight weeks; or one dose of the Merck vaccine followed eight weeks later by a placebo injection.

Antibody responses were observed by day 14 after the first injection of either Ad26.ZEBOV or rVSVΔG-ZEBOV-GP vaccine. The researchers say this finding is notable because vaccines against Ebola virus disease are typically administered during an outbreak and so information about how rapidly a vaccine produces an antibody effect is of potential use in efforts to protect at-risk populations. However, it is not currently known what level of antibody response reliably correlates with vaccine-induced protection against Ebola virus infection or disease. As no participants contracted Ebola virus disease during the trial, the investigators were not able to assess protection from disease.

The researchers cite several strengths of the trials, including outstanding retention of volunteers throughout the course of trial, achieved with continuing community engagement and ongoing trust-building efforts.

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But what happens with the sequences not selected by nature? And what possibilities lie in constructing entirely new sequences to make novel, or de novo, proteins bearing little resemblance to anything in nature?

That’s the terrain Princeton University’s Hecht Lab works in. And recently, their curiosity for designing their own sequences paid off.

They discovered the first known de novo protein that catalyzes, or drives, the synthesis of quantum dots. Quantum dots are fluorescent nanocrystals used in electronic applications from LED screens to solar panels.

Their work opens the door to making nanomaterials in a more sustainable way by demonstrating that protein sequences not derived from nature can be used to synthesize functional materials — with pronounced benefits to the environment.

Quantum dots are normally made in industrial settings with high temperatures and toxic, expensive solvents — a process that is neither economical nor environmentally friendly. But Hecht Lab researchers pulled off the process at the bench using water as a solvent, making a stable end-product at room temperature.

“We’re interested in making life molecules, proteins, that did not arise in life,” said Professor of Chemistry Michael Hecht, who led the research with Greg Scholes, the William S. Tod Professor of Chemistry and chair of the department. “In some ways we’re asking, are there alternatives to life as we know it? All life on earth arose from common ancestry. But if we make lifelike molecules that did not arise from common ancestry, can they do cool stuff?

“So here, we’re making novel proteins that never arose in life doing things that don’t exist in life.”

The team’s process can also tune nanoparticle size, which determines the color quantum dots glow, or fluoresce, in. That holds possibilities for tagging molecules within a biological system, like staining cancer cells in vivo.

“Quantum dots have very interesting optical properties due to their sizes,” said Yueyu Yao, co-author on the paper and a fifth-year graduate student in the Hecht Lab. “They’re very good at absorbing light and converting it to chemical energy — that makes them useful for being made into solar panels or any sort of photo sensor.

“But on the other hand, they’re also very good at emitting light at a certain desired wavelength, which makes them suitable for making LED screens.”

And because they’re small — comprised of only about 100 atoms and maybe 2 nanometers across — they’re able to penetrate some biological barriers, making their utility in medicines and biological imaging especially promising.

The research, “A de novo protein catalyzes the synthesis of semiconductor quantum dots,” was published this week in the Proceedings of the National Academy of Sciences (PNAS).

Why use de novo proteins?

“I think using de novo proteins opens up a way for designability,” said Leah Spangler, lead author on the research and a former postdoc in the Scholes Lab. “A key word for me is ‘engineering.’ I want to be able to engineer proteins to do something specific, and this is a type of protein you can do that with.

“The quantum dots we’re making aren’t great quality yet, but that can be improved by tuning the synthesis,” she added. “We can achieve better quality by engineering the protein to influence quantum dot formation in different ways.”

Based on work done by Sarangan Chari, Hecht Lab senior chemist and a corresponding author, the team used a de novo protein it designed named ConK to catalyze the reaction. Researchers first isolated ConK in 2016 from a large combinatorial library of proteins. It’s still made of natural amino acids, but it qualifies as “de novo” because its sequence doesn’t have any similarity to a natural protein.

Researchers found that ConK enabled the survival of E. coli in otherwise toxic concentrations of copper, suggesting it might be useful for metal binding and sequestration. The quantum dots used in this research are made out of cadmium sulfide. Cadmium is a metal, so researchers wondered if ConK could be used to synthesize quantum dots.

Their hunch paid off. ConK breaks down cysteine, one of the 20 amino acids, into several products, including hydrogen sulfide. That acts as the active sulfur source that will then go on to react with the metal cadmium. The result is CdS quantum dots.

“To make a cadmium sulfide quantum dot, you need the cadmium source and the sulfur source to react in solution,” said Spangler. “What the protein does is make the sulfur source slowly over time. So, we add the cadmium initially but the protein generates the sulfur, which then reacts to make distinct sizes of quantum dots.”

This research was supported by the National Science Foundation MRSEC program (DMR-2011750), the Princeton University Writing Center, and the Canadian Institute for Advanced Research. The research was also supported by NSF grant MCB-1947720 to MH.

“The Paleocene-Eocene Thermal Maximum is the closest thing we have in the geologic record to anything like what we’re experiencing now and may experience in the future with climate change,” said Lee Kump, professor of geosciences at Penn State. “There has been a lot of interest in better resolving that history, and our work addresses important questions about what triggered the event and the rate of carbon emissions.”

The scientists analyzed core samples from a well-preserved record of the PETM near the Maryland coast using astrochronology, a technique for dating sediments against orbital patterns that occur over tens to hundreds of thousands of years, known as Milankovitch cycles.

They found the shape of Earth’s orbit, or eccentricity, and the wobble in its rotation, or precession, favored hotter conditions at the onset of the PETM and that these orbital configurations together may have played a role in triggering the event.

“An orbital trigger may have led to the carbon release that caused several degrees of global warming during the PETM as opposed to what’s a more popular interpretation at the moment that massive volcanism released the carbon and triggered the event,” said Kump, the John Leone Dean in the College of Earth and Mineral Sciences.

The findings, published in the journal Nature Communications, also indicated the onset of the PETM lasted about 6,000 years. Previous estimates have ranged from several years to tens of thousands of years. The timing is important to understand the rate at which carbon was released into the atmosphere, the scientists said.

“This study allows us to refine our carbon cycle models to better understand how the planet reacts to an injection of carbon over these timescales and to narrow down the possibilities for the source of the carbon that drove the PETM,” said Mingsong Li, assistant professor in the School of Earth and Space Sciences at Peking University and a former assistant research professor of geosciences at Penn State who is lead author on the study.

A 6,000-year onset, coupled with estimates that 10,000 gigatons of carbon were injected into the atmosphere as the greenhouse gases carbon dioxide or methane, indicates that about one and a half gigatons of carbon were released per year.

“Those rates are close to an order of magnitude slower than the rate of carbon emissions today, so that is cause for some concern,” Kump said. “We are now emitting carbon at a rate that’s 5 to 10 times higher than our estimates of emissions during this geological event that left an indelible imprint on the planet 56 million years ago.”

The scientists conducted a time series analysis of calcium content and magnetic susceptibility found in the cores, which are proxies for changes in orbital cycles, and used that information to estimate the pacing of the PETM.

Earth’s orbit varies in predictable, calculable ways due to gravitational interactions with the sun and other planets in the solar system. These changes impact how much sunlight reaches Earth and its geographic distribution and therefore influence the climate.

“The reason there’s an expression in the geologic record of these orbital changes is because they affect climate,” Kump said. “And that affects how productive marine and terrestrial organisms are, how much rainfall there is, how much erosion there is on the continents and therefore how much sediment is carried into the ocean environment.”

Erosion from the paleo Potomac and Susquehanna rivers, which at the onset of the PETM may have rivaled the discharge of the Amazon River, carried sediments to the ocean where they were deposited on the continental shelf. This formation, called the Marlboro Clay, is now inland and offers one of the best-preserved examples of the PETM.

“We can develop histories by coring down through the layers of sediment and extracting specific cycles that are creating this story, just like you could extract each note from a song,” Kump said. “Of course, some of records are distorted and there are gaps — but we can use the same types of statistical methods that are used in apps that can determine what song you are trying to sing. You can sing a song and if you forget half the words and skip a chorus, it will still be able to determine the song, and we can use that same approach to reconstruct these records.”

Timothy Bralower, professor of geosciences at Penn State, also contributed to this research.

Other contributors were James Zachos, distinguished professor at the University of California Santa Cruz; William Rush, a postdoctoral associate at Yale University and the Cooperative Institute for Research in Environmental Science at the University of Colorado Boulder; and Jean Self-Trail and Marci Robinson, research geologists at the Florence Bascom Geoscience Center, United States Geological Survey.

The National Key R&D Program of China and the Heising-Simons Foundation provided funding for this work.