“The secretome orchestrates the subtle and complex processes of embryonic development, maintains the function of individual organs, and coordinates organ activity through inter-organ communication,” said McMahon, chair of the Department of Stem Cell Biology and Regenerative Medicine at USC. “However, it can be challenging to track which cells are secreting proteins, and which cells are being targeted.”

To address this challenge, co-first authors Rui Yang and Amanda S. Meyer at USC and Ilia A. Droujinine formerly at Harvard Medical School and now at Scripps Research generated mice with a built-in system for labeling and tracking the secretome.

“This built-in system is like a “passport system in the body, as we are identifying where proteins are coming from and where they’re going,” said Droujinine, who is a Scripps Research fellow and principal investigator in the Department of Molecular Medicine.

To establish this system, the research team started by genetically editing mouse embryonic stem cells to encode an enzyme engineered in Alice Ting’s laboratory at Stanford University. The enzyme, BirA*G3, indiscriminately tags nearby proteins with the vitamin B7 derivative, biotin. The reason that biotin is a useful tag is because it binds strongly the protein streptavidin, so that biotin-tagged proteins can be separated from other proteins in the cell and blood through a readily available technology called “streptavidin affinity purification.”

The next step was to ensure that BirA*G3 specifically tagged only the proteins of the secretome. These proteins originate in a structure within the cell known as the endoplasmic reticulum, or ER. Some of these proteins remain in the ER, while others are either secreted or incorporated into the cell’s surrounding membrane.

To specifically label only these proteins, BirA*G3 was itself tagged with four extra protein building blocks, or peptides. These four peptides, known as KDEL, served as a signal to the cell to retain BirA*G3 in the ER. So when biotin was added, BirA*G3 only added biotin labels to the proteins traveling through the ER.

Using these genetically edited stem cells, the team then produced transgenic mice. When these mice were given biotin, the scientists were able to demonstrate BirA*G3 labeling and identify the secretomes for a number of organs including the brain, liver and kidney. The scientists also identified proteins that were secreted anyway, even though they weren’t marked with the typical signal indicating the cell should secrete them.

These transgenic mice enable scientists to label and study specific cell types and organs, facilitating studies of inter-organ communication.

“Our new mouse model demonstrates enhanced labeling efficiency of the secreted and ER proteins in specific cell types, providing a valuable resource for mapping and profiling the secretome,” said Meyer, who is a PhD candidate in the McMahon Lab at the Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC.

Yang, a postdoctoral fellow in the McMahon Lab, added: “Our mouse model will facilitate a deeper understanding of the secretome’s critical role in development, as well as in healthy and diseased adult states.”

The mice are now in a public repository, so that other scientists can benefit from this new technology.

“Given the central role of key secreted proteins such as insulin, there is a great deal of interest in identifying novel secreted proteins,” said McMahon. “Genome studies are suggesting that many new proteins remain to be characterized. We’re looking forward to a deep dive into this area now that we have validated the technology.”

Additional co-authors include: Jinjin Guo and Jill A. McMahon from USC; Namrata D. Udeshi, Dominique K. Carey, Charles Xu, and Steven A. Carr from the Broad Institute of Harvard and MIT; Yanhui Hu and David Rocco from Harvard Medical School; Norbert Perrimon from Harvard Medical School and the Howard Hughes Medical Institute; Qiao Fang from the University of Toronto; Jihui Sha and James Wohlschlegel from UCLA; Shishang Qin from Peking University; and Alice Y. Ting from the Chan Zuckerberg Biohub and Stanford University.

Temperature affects the span of human behavior, from eating and activity levels to sleep-wake cycles. We may have a harder time sleeping in the summer and be slow to get out of bed on colder mornings. But the link between sensory neurons and neurons that control this cycle are not understood completely.

Northwestern University neurobiologists have found a few clues about what’s happening. In a new study, published today (Aug. 17) in the journal Current Biology, researchers found that fruit flies are pre-programmed to take a nap in the middle of the day. A follow-up to their 2020 Biology paper that identified a brain thermometer only active in cold weather, the new paper explores a similar “thermometer” circuit for hot temperatures.

“Changes in temperature have a strong effect on behavior in both humans and animals, and offer animals a cue that is time to adapt to the changing seasons,” said Marco Gallio, associate professor of neurobiology in the Weinberg College of Arts and Sciences. “The effect of temperature on sleep can be quite extreme, with some animals deciding to sleep off an entire season — think of a hibernating bear — but the specific brain circuits that mediate the interaction between temperature and sleep centers remain largely unmapped.”

Gallio led the study and said fruit flies are a particularly good model to study big questions like “why do we sleep,” and “what does sleep do for the brain” because they don’t attempt to disrupt instinct in the same way humans do when we pull all-nighters, for example. They also allow researchers to study the influence of external cues like light and temperature on cellular pathways.

Cells that stay on longer

The paper is the first to identify “absolute heat” receptors in fly head, which respond to temperatures above about 77 degrees Fahrenheit — the fly’s favorite temperature. As it turns out, the common laboratory fruit fly (Drosophila) has colonized nearly the entire planet by forming a close association with humans. Not surprisingly, its favorite temperature also matches that of many humans.

Just as they expected based on the results of their previous paper on cold temperature, researchers found that brain neurons receiving information about heat are part of the broader system that regulates sleep. When the hot circuit, which runs parallel to the cold circuit, is active, the target cells that promote midday sleep stay on longer. This results in an increase in midday sleep that keeps flies away from the hottest part of the day.

The study was enabled by a 10-year initiative that produced the first completed map of neural connections in an animal (a fly), called the connectome. With the connectome, researchers have access to a computer system that tells them all possible brain connections for each of the fly’s ~100,000 brain cells. However, even with this extremely detailed road map, researchers still need to figure out how information in the brain goes from point A to B. This paper helps fill that gap.

The different circuits for hot versus cold temperatures make sense to Gallio because “hot and cold temperatures can have quite different effects on physiology and behavior,” he said. This separation may also reflect evolutionary processes based on heat and cold cycles of the Earth. For example, the possibility that brain centers for sleep may be directly targeted in humans by a specific sensory circuit is now open to be investigated based on this work.

Next steps

Next, Gallio’s team hopes to figure out the common targets of the cold and hot circuit, to discover how each can influence sleep.

“We identified one neuron that could be a site of integration for the effects of hot and cold temperatures on sleep and activity in Drosophila,” said Michael Alpert, the paper’s first author and a post-doctoral researcher in the Gallio lab. “This would be the start of interesting follow-up studies.”

Gallio added that the team is interested in looking at the long-term effects of temperature on behavior and physiology to understand the impact of global warming, looking at how adaptable species are to change.

“People may choose to take an afternoon nap on a hot day, and in some parts of the world this is a cultural norm, but what do you choose and what is programmed into you?” Gallio said. “Of course, it’s not culture in flies, so there actually might be a very strong underlying biological mechanism that is overlooked in humans.”

The paper is titled “A thermometer circuit for hot temperature adjusts Drosophila behavior to persistent heat.” Work in the Gallio lab is supported by the National Institutes of Health (grants R01NS086859 and R21EY031849), a Pew Scholars Program in the Biomedical Sciences and a McKnight Technological Innovations in Neuroscience Awards. The all-Northwestern team includes Gallio, Alpert, Hamin Gil and Alessia Para.

The paper coauthored by Caroline Freund, economist and dean of the School of Global Policy and Strategy, finds that 3D printing changed production processes, but supply chains remained intact. The study is the first to examine the impact 3D printing has on trade.

Published in the Journal of International Economics, the paper looks at the production of hearing aids — a good most commonly produced by 3D printing.

The results reveal that the shift to 3D printing led to a doubling or near doubling in producers’ exports after five years and the technology was the main cause for the rise in exports.

Freund and co-coauthors also examined 35 other products, such as running shoes, aircraft parts and prosthetic limbs that are increasingly being 3D printed and they found similar patterns.

“The technology is a boon, not a curse to trade,” Freund said. “A country’s exports of hearing aids increased more than trade in other similar goods following the adoption of 3D printing by manufacturers there. The new production technology in combination with trade means that consumers around the world suffering hearing loss are benefitting from better and often cheaper hearing aids.”

One reason behind the expansion is that printing hearing aids in high volumes requires a large investment in technology and machinery. The countries that were early innovators — Denmark, Switzerland and Singapore — dominate exports of the good, while middle-income economies such as China, Mexico and Vietnam also have been able to substantially increase their market shares.

In addition, hearing aids are lightweight products, which makes them fairly cheap to ship internationally. The same is true for the other products the authors examined — lighter products are associated with more trade growth.

These results are based on comparisons of the growth of the 3D printed products to other similar goods. The authors also accounted for trends and other factors that could skew the data.

“Policymakers often view 3D printing as a means to shorten supply chains when in fact it is more likely to enhance trade and reshape supply chains,” said Freund, former global director of Trade, Investment and Competitiveness at the World Bank.

While the analysis of 3D printing’s impact on trade is positive, it has the possibility of being short-lived. If 3D printers become more accessible to local producers or even consumers in some sectors, production could be more localized, hindering development opportunities through trade.

The study “Is 3D printing a threat to global trade? The trade effects you didn’t hear about” is coauthored by Alan Mulabdic, economist for the Equitable Growth, Finance and Institutions’ Chief Economist’s Office at the World Bank, and Michele Ruta, lead economist at the World Bank.

Story Source:

Materials provided by University of California – San Diego. Original written by Christine Clark. Note: Content may be edited for style and length.

“No previous study has looked at this important coronavirus enzyme at physiological (or body) temperature,” said Daniel Keedy, a structural biologist at the City University of New York (CUNY), who conducted the study in collaboration with scientists at the U.S. Department of Energy’s Brookhaven National Laboratory.

Most structures to date come from frozen samples — far from the temperatures at which the molecules operate within living cells. “If you are working at physiological temperature, you should get a more realistic picture of what’s happening during an actual infection, because that’s where biology happens,” Keedy said.

In addition, he added, the team used temperature as a tool. “By turning that knob and seeing how the protein reacts, we can learn about its mechanics — how it physically works.

Deciphering Mpro’s structure

The protein in question is the main protease (Mpro) of SARS-CoV-2, the virus that causes COVID-19. Like all proteases, it’s an enzyme that cuts other proteins. In many viral infections, including COVID-19, infected cells initially produce a virus’s functional proteins as one single connected protein chain. Proteases cut the pieces apart so the individual proteins can make and assemble themselves into new copies of the virus. Finding a drug to disable Mpro could put the brakes on COVID-19.

To study the enzyme’s structure, the researchers used a technique called x-ray crystallography at Brookhaven Lab’s National Synchrotron Light Source II (NSLS-II). NSLS-II is a DOE Office of Science user facility that produces bright beams of x-rays. Shining those x-rays on a crystallized sample of a biological molecule can reveal the three-dimensional arrangement of atoms that make up the molecule.

Studying samples that aren’t frozen can be a challenge.

“The higher the temperature, the greater the chances the x-rays will damage the crystal,” explained study co-author Babak Andi, who operates NSLS-II’s Frontier Macromolecular Crystallography (FMX) beamline.

“To minimize the damage, we rotate and move the crystal linearly as it moves through the x-rays. That distributes the x-ray dose over the whole length of the crystal,” he said.

He noted that the small size of the x-ray beam at NSLS-II makes it possible to keep the beam focused on even the smallest dimension of the crystal — an edge measuring 10 to 20 millionths of a meter or smaller — as it rotates.

“In addition, the FMX detector and other systems operate so fast, we can collect one full dataset in just 10-15 seconds per sample, with good enough quality to solve a structure before significant x-ray damage occurs.”

From frozen to physiological

The scientists used FMX to collect the first-ever series of Mpro crystallographic data at five different temperatures, ranging from cryogenic (-280 degrees Fahrenheit) to what is often referred to as “room temperature” in x-ray crystallography (~39°F) to physiological (98°F). They also studied the room-temperature crystal under high humidity. Then they fed the data into a special type of computer simulation to identify many possible atomic-level arrangements under each set of conditions.

The results revealed subtle conformational changes, including increased flexibility in parts of the protein at higher temperatures. The team also saw some features that were unique to the enzyme under physiological conditions.

Most of the changes were not directly in the enzyme’s “active site” — the part that is directly involved in cutting other proteins. Instead, they were in parts of the enzyme more distant from that location. But the data suggest that these distant sites could affect the active site through a sort of remote-control mechanism that’s common in biological systems, Keedy said. Disabling even those distant locations could potentially block the enzyme’s function.

“You can think of Mpro as a kind of folded ribbon, made up of two identical halves (forming dimers) that bind together in a symmetrical way, kind of like a handshake,” Keedy said. The center of this handshake region (the “dimer interface”) links to the active site through a flexible loop region of the protein.

As Keedy explained, the scientists found that at higher temperatures, “the grip of the ‘handshake’ changes — the two components readjust their grip a little bit. This tells us that, when the virus is infecting us, there may be some kind of communication via this loop between the dimer interface and the active site,” Keedy said.

The path to drug design

“We see subtle changes in structure in this study, but drug design is dependent on subtle changes — less than a billionth of a meter here, less than a billionth of a meter there,” Keedy noted.

Other studies have shown that small drug-like molecules can bind to the enzyme at some of distant locations identified in this new work.

“If we could perfect these molecules, optimize them, elaborate on them, tweak them, then we could potentially have a new foothold for altering the function of the enzyme — not at the active site, as essentially all of the antivirals for this protein are currently targeting, but at a different site through a different mechanism,” Keedy said. “Our findings set up the inspiration for exploring this idea.”

The role of water

Exploring the enzyme at elevated humidity also mimics the physiological conditions within water-filled cells — and may provide additional clues to guide drug design.

“For these studies, after selecting the crystal we want to study, we put a special sleeve over it to keep it from drying out,” said NSLS-II’s Babak Andi. “Then, when we put the sample in the beamline for x-ray data collection, we remove the sleeve and blow a continuous stream of air at 99.5% humidity over the crystal as we collect the data.”

The results revealed specific water molecules that bind loosely to the enzyme, including one near the active site, only under the high-humidity condition.

“Those water molecules give you a hint where inhibitors may bind,” said Andi.

His group has been studying a range of potential drug-like molecules that appear to replace weakly bound water molecules when they bind near Mpro’s active site. That work is reported on in a paper recently published in Scientific Reports from the Nature portfolio of journals.

Both scientists were grateful for the collaborative spirit of the entire team, which included additional scientists at both Brookhaven Lab and CUNY, as well as Diamond Light Source in the United Kingdom.

“It was very important to be able to have the remote collaboration to make this project work, where we had people onsite at the beamline, and people offsite who could do the computer modeling,” Keedy said. “It’s one example of many during the pandemic of the scientific community coming together.”

This research was supported by the DOE Office of Science through the National Virtual Biotechnology Laboratory (NVBL) with funding from the Coronavirus CARES Act and by Laboratory Directed Research and Development program funding for COVID-19 research at Brookhaven Lab. The FMX beamline (17-ID-2) at NSLS-II is funded by the Office of Science (BES). Daniel Keedy is supported by the National Institutes of Health (NIH, R35 GM133769). The Center for BioMolecular Structure (CBMS) at Brookhaven Lab’s NSLS-II, which includes the FMX beamline, is funded by the National Institutes of Health, National Institute of General Medical Sciences, and by the DOE Office of Science (BER).

The A1R-CT peptide the scientists developed, which can be administered through a nasal spray, holds promise for tamping down the uncontrolled electrical activity that is common after traumatic brain injury, stroke and which affects more than half of individuals with Alzheimer’s, says Dr. Qin Wang, neuropharmacologist and founding director of the Program for Alzheimer’s Therapeutics Discovery at the Medical College of Georgia at Augusta University.

The fact that it can be delivered through the nose indicates the peptide’s potential as a new seizure rescue medication as well, to help interrupt, for example, a seizure cluster, where disabling seizures are occurring back-to- back, says Wang, corresponding author of the study in the journal JCI Insight.

A1R-CT works by inhibiting neurabin, a protein that helps ensure that the protective mechanism itself, which tamps down the hyperexcitability of neurons that disrupts normal communication and produces seizures, doesn’t overdo, she says.

The peptide was named after the protective adenosine 1 receptor on the surface of neurons, which gets activated by adenosine, a chemical made mostly in the brain by neuron-supporting glial cells in response to hyperexcitability.

“This is a powerful receptor to then silence the neurons,” Wang says. This natural, calming relationship also is known to block electrical activity that can result in an irregular heartbeat. In fact, an injectable form of adenosine is used to treat a very high heart rate.

“But the A1 receptor itself has to be regulated because if it’s activated too much, you will fall asleep,” says Wang. “The neurons try to make sure everything stays in control and in most of us, it works pretty well. We don’t fall asleep at our desk. We don’t have seizures,” she says, noting that caffeine blocks the A1 receptor.

Alzheimer’s often is accompanied by seizures because the characteristic buildup of the proteins amyloid and tau in the brain disrupts communication between neurons, creates increased oxidative stress and resulting inflammation, and in response to the altered dynamic, neurons can become hyperexcited, she says. “In Alzheimer’s there are so many things that go wrong,” she says. Seizures can precede the cognitive decline in Alzheimer’s, and definitely contribute to it, Wang says.

A1 receptor’s activation by adenosine in this type of hyperactive scenario makes it seem like a logical treatment target for seizures. But the fact that it is so pervasive throughout the body, including in the heart, lungs and kidneys, makes potential extensive side effects likely.

Back to neurons’ desire for homeostasis, Wang and her colleagues were the ones who found that the protein neurabin, which appears to be primarily present in the brain, provides that balance to prevent hyperactivity of the A1 receptor.

The fact neurabin is primarily in the brain, means that altering its activity should not have the potential body-wide impact of directly altering A1 receptor activity, Wang says.

“Neurabin is a brake so it doesn’t do too much,” Wang says. “But now we need to remove it to unleash A1’s power.”

So, they set to work developing the peptide that could instead interfere with the A1 receptor and neurabin’s interaction and so enable more of the natural protective, seizure-reducing benefit.

A1 receptor activation tamps down the excited state of the neurons by modulating ion channels — proteins in the cell membrane that allow passage through the cell of other proteins — which help generate electrical signals. A result is so-called hyperpolarization, which means the neuron is less likely to fire an electrical signal. “The more polarized the neurons are, the harder it is for them to get excited,” Wang says.

A1 receptor activation also decreases the release of glutamate, a neurotransmitter produced by neurons that excites neurons. It also provides additional benefit to neurons by providing some protection from inadequate oxygen and blood supplies, which may occur in the case of an injury. The scientists have noted a dramatic reduction in death of neurons in their Alzheimer’s model, for example, with the use of their peptide.

Now they’ve shown that inhibiting neurabin — either by reducing it directly or with their peptide — enables increased action by A1C to reduce excessive electrical activity in the brain. They’ve shown the peptide is effective in both a mouse model of severe seizures and seizures in an Alzheimer’s mouse model. And It’s effective when directly injected into the brain or via nasal spray.

The scientists opted to look at nasal spray delivery to fully explore the peptide’s potential clinical benefit. They found a similar robust response in both the seizure and Alzheimer’s models.

Looking further at the impact of targeting neurabin, they found that mice with a neurabin deficiency had significantly shorter, less severe seizures and they all survived. Those with the normal neurabin levels intact experienced seizures lasting for up to 30 minutes and about 10% of the mice died shortly afterward. Blocking A1 receptor resulted in more severe seizures in the neurabin-deficient mice and increased the death rate to more than 50%.

Next steps include additional exploration of ideal doses and delivery times for specific conditions the peptide may be used to treat.

The scientific team also continues to tweak the peptide to ensure it functions optimally, and is pursuing funding needed to pursue clinical trials.

Wang, a Georgia Research Alliance Eminent Scholar, came to MCG in April 2021 from the University of Alabama at Birmingham, where she began the studies of A1 receptor and peptide development. She continues extensive collaboration with her colleagues at UAB on the studies who are coauthors on the new paper. First author Dr. Shalini Saggu is now also a faculty member in the MCG Department of Neuroscience and Regenerative Medicine.

Epileptic seizures are common after a traumatic brain injury; a stroke, which is considered an acquired brain injury; and with chronic neurodegenerative diseases including Alzheimer’s.

As much as 64% of the some 50 million individuals with Alzheimer’s experience seizures, the scientists write. Patients can experience generalized tonic-clonic seizures, in which they fall down, shake and become unresponsive. Also, focal onset seizure, which tend to be shorter and may include repetitive movement of the arms or legs, lip smacking and chewing.

Seizures are uncontrolled in about 40% of people, which indicates an urgent need for novel therapies, the scientists write, and current therapies tend to be less effective in individuals with Alzheimer’s. Left uncontrolled, seizures can produce brain damage and cognitive impairment.

Adenosine also is a building block of our DNA and a component of the cell fuel ATP.

The research was supported by the National Institutes of Health.