But research has shown that the seeds of Alzheimer’s are planted years — even decades — earlier, long before the cognitive impairments surface that make a diagnosis possible. Those seeds are amyloid beta proteins that misfold and clump together, forming small aggregates called oligomers. Over time, through a process scientists are still trying to understand, those “toxic” oligomers of amyloid beta are thought to develop into Alzheimer’s.

A team led by researchers at the 91̽�� has developed a laboratory test that can measure levels of amyloid beta oligomers in blood samples. As they report in a published Dec. 9 in the Proceedings of the National Academy of Sciences, their test — known by the acronym SOBA — could detect oligomers in the blood of patients with Alzheimer’s disease, but not in most members of a control group who showed no signs of cognitive impairment at the time the blood samples were taken.

However, SOBA did detect oligomers in the blood of 11 individuals from the control group. Follow-up examination records were available for 10 of these individuals, and all were diagnosed years later with mild cognitive impairment or brain pathology consistent with Alzheimer’s disease. Essentially, for these 10 individuals, SOBA had detected the toxic oligomers before symptoms surfaced.

“What clinicians and researchers have wanted is a reliable diagnostic test for Alzheimer’s disease — and not just an assay that confirms a diagnosis of Alzheimer’s, but one that can also detect signs of the disease before cognitive impairment happens. That’s important for individuals’ health and for all the research into how toxic oligomers of amyloid beta go on and cause the damage that they do,” said senior author , a 91̽��professor of bioengineering and faculty member in the UW . “What we show here is that SOBA may be the basis of such a test.”

SOBA, which stands for soluble oligomer binding assay, exploits a unique property of the toxic oligomers. When misfolded amyloid beta proteins begin to clump into oligomers, they form a structure known as an alpha sheet. Alpha sheets are not ordinarily found in nature, and past research by Daggett’s team showed that alpha sheets tend to bind to other alpha sheets. At the heart of SOBA is a synthetic alpha sheet designed by her team that can bind to oligomers in samples of either cerebrospinal fluid or blood. The test then uses standard methods to confirm that the oligomers attached to the test surface are made up of amyloid beta proteins.

The team tested SOBA on blood samples from 310 research subjects who had previously made their blood samples and some of their medical records available for Alzheimer’s research. At the time the blood samples had been taken, the subjects were recorded as having no signs of cognitive impairment, mild cognitive impairment, Alzheimer’s disease or another form of dementia.

SOBA detected oligomers in the blood of individuals with mild cognitive impairment and moderate to severe Alzheimer’s. In 53 cases, the research subject’s diagnosis of Alzheimer’s was verified after death by autopsy — and the blood samples of 52 of them, which had been taken years before their deaths, contained toxic oligomers.

SOBA also detected oligomers in those members of the control group who, records show, later developed mild cognitive impairment. Blood samples from other individuals in the control group who remained unimpaired lacked toxic oligomers.

Daggett’s team is working with scientists at , a 91̽��spinout company, to develop SOBA into a diagnostic test for oligomers. In the study, the team also showed that SOBA easily could be modified to detect toxic oligomers of another type of protein associated with Parkinson’s disease and Lewy body dementia.

“We are finding that many human diseases are associated with the accumulation of toxic oligomers that form these alpha sheet structures,” said Daggett. “Not just Alzheimer’s, but also Parkinson’s, type 2 diabetes and more. SOBA is picking up that unique alpha sheet structure, so we hope that this method can help in diagnosing and studying many other ‘protein misfolding’ diseases.”

Daggett believes the assay has further potential.

“We believe that SOBA could aid in identifying individuals at risk or incubating the disease, as well as serve as a readout of therapeutic efficacy to aid in development of early treatments for Alzheimer’s disease,” she said.

Lead author on the study is Dylan Shea, a doctoral student in the 91̽��Department of Bioengineering’s Molecular Engineering Program. Co-authors are Elizabeth Colasurdo of the VA Puget Sound Health Care System; , a 91̽��research assistant professor of physiology and biophysics; , a student in the 91̽��Medical Scientist Training Program; Dr. , a 91̽��assistant professor of neurology; Dr. , a 91̽��professor of laboratory medicine and pathology; , a professor of neurosciences at the University of California, San Diego; Dr. , assistant professor of neurological surgery at the UW; and Ge Li and Dr. , both of the 91̽��Department of Psychiatry and Behavioral Sciences and the VA Puget Sound Health Care System. The research was funded by the National Institutes of Health, the Washington Research Foundation and the Northwest Mental Illness Research, Education and Clinical Center.

For more information, contact Daggett at daggett@uw.edu.

Membranes are crucial to our cells. Every cell in your body is enclosed by one. And each of those cells contains specialized compartments, or organelles, which are also enclosed by membranes.

Membranes help cells carry out tasks like breaking down food for energy, building and dismantling proteins, keeping track of environmental conditions, sending signals and deciding when to divide.

Biologists have long struggled to understand precisely how membranes accomplish these different types of jobs. The primary components of membranes — large, fat-like molecules called lipids and compact molecules like cholesterol — make great barriers. In all but a few cases, it’s unclear how those molecules help proteins within membranes do their jobs.

In a published Jan. 25 in the Proceedings of the National Academy of Sciences, a team at the 91̽�� looked at phase separation in budding yeast — the same single-celled fungus of baking and brewing fame — and reports that living yeast cells can actively regulate a process called phase separation in one of their membranes. During phase separation, the membrane remains intact but partitions into multiple, distinct zones or domains that segregate lipids and proteins. The new findings show for the first time that, in response to environmental conditions, yeast cells precisely regulate the temperature at which their membrane undergoes phase separation. The team behind this discovery suggests that phase separation is likely a “switch” mechanism that these cells use to govern the types of work that membranes do and the signals they send.

“Previous work showed that these domains can be seen in the membranes of living yeast cells,” said lead author Chantelle LeveilIe, a 91̽��doctoral student in chemistry. “We asked: If it’s important for a cell to have these domains, then if we change the cell’s environment — by growing them at different temperatures — would the cell ‘care’ and devote energy to maintaining phase separation in its membranes? The clear answer is yes, it does!”

Past research has shown that when sugar is plentiful, the yeast cell’s vacuole — an important organelle for storage and signaling — grows large and its membrane appears uniform under a microscope. But when food supplies dwindle, the vacuole undergoes phase separation, with many round zones appearing in the organelle’s membrane.

In this new study, Leveille and her co-authors — 91̽��chemistry professor , 91̽��biochemistry professor and Caitlin Cornell, previously a 91̽��doctoral student in chemistry — sought to understand whether yeast can actively regulate phase separation. Leveille grew yeast at their typical laboratory temperature of 86 F with plenty of food. After the food dwindled, the yeast cell vacuole membranes underwent phase separation, as expected. When Leveille briefly raised the temperature in the yeast’s environment by about 25 degrees Fahrenheit, the domains disappeared. Then Leveille grew yeast at a cooler temperature — 77 F instead of the normal 86 F — and discovered that the domains disappeared about 25 degrees above this new temperature. When she grew yeast in still colder conditions, at 68 F, phase separation yet again disappeared about 25 degrees higher than their growth temperature.

These experiments showed that the yeast cells always maintained phase separation in the vacuole membrane until the temperature rose about 25 degrees above their growth temperature.

“We think this is a clear sign that yeast cells are engineering the vacuole membrane in different environmental conditions to maintain this consistent state of phase separation,” said Leveille.

Phase separation in the vacuole membrane likely serves an important purpose in yeast, she added.

“This result suggests that membrane phase separation for yeast is likely a two-way door,” said Leveille. “For example, if the cells ever found food again, they would want to go back to their original state. Yeast do not want to get too far away from the transition.”

Future research could identify other membrane components that affect the vacuole membrane’s ability to phase separate, as well as the consequences of its phase separation. Biologists have known that, when the domains appear in the yeast vacuole’s membrane, the cell stops dividing. These two events may be linked because the yeast vacuole’s membrane contains two complexes of proteins that are important for cell division. When the complexes are far apart, cell division stops.

“Phase separation in the vacuole occurs right when the yeast cell needs to stop dividing because its food supply has run out,” said Merz. “One idea is that phase separation is the mechanism that the yeast cell ‘uses’ to separate these two protein complexes and stop cell division.”

In cells from yeast to humans, protein complexes embedded in membranes affect cell behavior. If additional research shows that phase separation in the yeast vacuole regulates cell division, it would likely be the first rigorous example of cell regulation through this once-overlooked property of membranes.

“Phase separation could be a common, reversible mechanism to modulate many, many types of cellular properties,” said Keller.

Cornell is now a postdoctoral researcher at the University of California, Berkeley. The research was funded by the National Institutes of Health and the National Science Foundation.

For more information, contact Keller at slkeller@uw.edu and Merz at merza@uw.edu.

Many people consult their friends and neighbors before making a big decision. It turns out that cells also consult their neighbors — in the human body.

Scientists and physicians have long known that immune cells migrate to the site of an infection, which individuals experience as inflammation — swelling, redness and pain. Now, researchers at the 91̽�� and Northwestern University have uncovered evidence that this gathering is not just a consequence of immune activation. Immune cells count their neighbors before deciding whether or not the immune system should kick into high gear.

Understanding how to influence inflammation and activate an immune response could lead to new therapies to treat chronic autoimmune diseases or to mobilize the immune system to help fight cancer.

“This is a previously unrecognized aspect of immune function,” said , a professor of chemical and biological engineering at Northwestern. “The cells make a coordinated decision. They don’t uniformly activate but instead collectively decide how many cells will activate, so that together, the system can fend off a threat without dangerously overreacting.”

“A key part of this work relied on the development of new computational models to interpret our experiments and elucidate how cells perform calculations to make coherent decisions,” said , an assistant professor of biology and chemical engineering at the UW.

Bagheri and Leonard co-led the , which was published Feb. 13 in Nature Communications.

The body’s immune system works constantly to maintain a delicate balance. When a threat is introduced, the system needs to respond strongly enough to fight off infection or disease, but not so strongly that it causes harm.

“When it comes to immune responses, it’s the difference between life and death,” Leonard said. “If your body over-responds to a bacterial infection, then you could die from septic shock. If your body doesn’t respond enough, then you could die from rampant infection. Staying healthy requires the body to strike a balance between these extremes.”

Bagheri, Leonard and their teams wanted to better understand how the immune system makes these types of decisions.

“It’s especially interesting because the immune system is decentralized,” said lead author Joseph Muldoon, a graduate student at Northwestern University who is co-advised by Bagheri and Leonard. “Immune cells are individual agents that need to work together, and nature has come up with a solution for how they can get on the same page. Cells arrive at different activation states, but in such a way that, on the whole, the population response is calibrated.”

To explore this phenomenon, the researchers examined macrophages, a type of immune cell that is part of the first line of defense for combatting infection and disease. They observed how macrophages responded to a chemical produced by bacteria — a red flag that alerts the body to the presence of infection — using techniques that enabled the researchers to watch individual cells’ responses over time. They then used computational models to help interpret and explain these observations.

Immune cells were ‘counting’ how many of them had gathered to determine how much the system should react.

“Over time, the cells observe their surroundings to get a sense of their neighbors,” Muldoon said. “Each cell becomes poised to respond as a high activator or not. Now that we know there’s this additional layer controlling the immune system, it opens up a whole avenue to study whether there are new targets for immunomodulation.”

The researchers believe this information could be used to develop improved cancer immunotherapies or treatments for autoimmune diseases, help design better drugs and guide the engineering of advanced cell-based therapies.

Co-author on the paper is Yishan Chuang, who conducted this work as a graduate student at Northwestern. The research was funded by the National Institutes of Health and Northwestern University.

For more information, contact Bagheri at nbagheri@uw.edu.

Adapted from a by Amanda Morris at Northwestern University.

A team of chemical engineers has developed a new way to produce medicines and chemicals and preserve them using portable “biofactories” embedded in water-based gels known as hydrogels. The approach could help people in remote villages or on military missions, where the absence of pharmacies, doctor’s offices or even basic refrigeration makes it hard to access critical medicines and other small-molecule compounds.

The team — led by , a professor of chemical engineering at the University of Texas, and , an assistant professor of chemistry at the 91̽�� — describes the new approach in a published Feb. 4 in Nature Communications.

“We expect these developments to afford new technologies for on-demand production of small-molecule and peptide products in the future,” said Nelson, who is also a faculty member in the 91̽��, in a accompanying the paper. “This technology will be especially applicable in remote or isolated areas where space and resources are limited, which could include manned space missions or personalized medications.”

Their system effectively embeds microbial biofactories — cells engineered to overproduce a product — into the solid scaffold of a hydrogel, allowing for portability and optimized production. It is the first hydrogel-based system to organize both individual microbes and consortia for in-the-moment production of high-value chemical feedstocks, used for processes such as fuel and pharmaceutical production. Products can be produced within a couple of hours to a couple of days.

“Many of the chemicals, fuels, nutraceuticals and pharmaceuticals we use rely on traditional fermentation technology,” said Alper. “Our technology addresses a strong limitation in the fields of synthetic biology and bioprocessing, namely the ability to provide a means for both on-demand and repeated-use production of chemicals and antibiotics from both mono- and co-cultures.”

As a crosslinked polymer, the hydrogel used in this work can be 3D printed or manually extruded. The gel material, along with the cells inside, can flow like a liquid and then harden upon exposure to UV light. The resulting polymer network is large enough for molecules and proteins to move through it, but the space is too small for cells to leak out.

The team also found that by freeze-drying the hydrogel system, it can effectively preserve the fermentation capacity of the biofactories until needed in the future. The result of the freeze-drying somewhat resembles an ancient mummy, shriveled up but well-preserved. To revive the hydrogel, the researchers simply added water, sugar and other basic nutrients, and the cells could resume production just as effectively as before the preservation process.

One of the novel applications enabled by this platform is the ability to combine multiple different organisms, called consortia, together in a way that outperforms traditional, large-scale bioreactors. In particular, this system enables a plug-and-play approach to combining and optimizing chemical production. For example, if one set of enzymes works best in the bacteria E. coli, while the other works best in budding yeast, the two organisms can work together to more efficiently go straight to the product. The research team tested both of these organisms.

This platform has the added benefit of multitasking, keeping different types of cells separated while they grow, preventing one from taking over and killing off the others. Likewise, by testing a range of temperatures, the team could control the dynamics of the system, keeping the growth of multiple cell types balanced.

Finally, the team showed continuous, repeated use of the system — with yeast cells — over the course of an entire year without a decrease in yields, indicating the sustainability of the process over time.

Medicines such as antibiotics have a fixed shelf life and require specific storage conditions. The portability of a biofactory that can synthesize these molecules makes the hydrogel system especially useful in remote places that don’t have access to refrigeration to store medications. It would also be a small and compact way to maintain access to several medications and other essential chemicals when there is no access to a pharmacy or a store, like during a military mission or a mission to Mars. Although not quite there yet, the possibilities are promising.

“This technology can be applied to a wide range of products and cell types. We see engineers and scientists being able to plug and play with different consortia of cells to produce diverse products that are needed for a specific scenario,” Alper said. “That’s part of what makes this technology so exciting.”

Co-lead authors on the paper are 91̽��graduate student Trevor Johnston and University of Texas graduate student Shuo-Fu Yuan. Co-authors are James Wagner and Xiunan Yi at the University of Texas, former 91̽��postdoctoral researcher Abhijit Saha, and 91̽��graduate student Patrick Smith. The research was funded by the Camille and Henry Dreyfus Foundation, the 91̽�� and the Royalty Research Fund.

For more information contact Nelson at alshakim@uw.edu.

Adapted from a by the University of Texas news office.

But how did a lifeless environment on the early Earth supply this key ingredient?

“For 50 years, what’s called ‘the phosphate problem,’ has plagued studies on the origin of life,” said first author , a 91̽�� research assistant professor of Earth and space sciences.

The problem is that chemical reactions that make the building blocks of living things need a lot of phosphorus, but phosphorus is scarce. A new 91̽��study, published Dec. 30 in the Proceedings of the National Academy of Sciences, finds an answer to this problem in certain types of lakes.

The focuses on carbonate-rich lakes, which form in dry environments within depressions that funnel water draining from the surrounding landscape. Because of high evaporation rates, the lake waters concentrate into salty and alkaline, or high-pH, solutions. Such lakes, also known as alkaline or soda lakes, are found on all seven continents.

The researchers first looked at phosphorus measurements in existing carbonate-rich lakes, including in California, in Kenya and in India.

While the exact concentration depends on where the samples were taken and during what season, the researchers found that carbonate-rich lakes have up to 50,000 times phosphorus levels found in seawater, rivers and other types of lakes. Such high concentrations point to the existence of some common, natural mechanism that accumulates phosphorus in these lakes.

Today these carbonate-rich lakes are biologically rich and support life ranging from microbes to Lake Magadi’s famous flocks of flamingoes. These living things affect the lake chemistry. So researchers did lab experiments with bottles of carbonate-rich water at different chemical compositions to understand how the lakes accumulate phosphorus, and how high phosphorus concentrations could get in a lifeless environment.

The reason these waters have high phosphorus is their carbonate content. In most lakes, calcium, which is much more abundant on Earth, binds to phosphorus to make solid calcium phosphate minerals, which life can’t access. But in carbonate-rich waters, the carbonate outcompetes phosphate to bind with calcium, leaving some of the phosphate unattached. Lab tests that combined ingredients at different concentrations show that calcium binds to carbonate and leaves the phosphate freely available in the water.

“It’s a straightforward idea, which is its appeal,” Toner said. “It solves the phosphate problem in an elegant and plausible way.”

Phosphate levels could climb even higher, to a million times levels in seawater, when lake waters evaporate during dry seasons, along shorelines, or in pools separated from the main body of the lake.

“The extremely high phosphate levels in these lakes and ponds would have driven reactions that put phosphorus into the molecular building blocks of RNA, proteins, and fats, all of which were needed to get life going,” said co-author , a 91̽��professor of Earth & space sciences.

The carbon dioxide-rich air on the early Earth, some four billion years ago, would have been ideal for creating such lakes and allowing them to reach maximum levels of phosphorus. Carbonate-rich lakes tend to form in atmospheres with high carbon dioxide. Plus, carbon dioxide dissolves in water to create acid conditions that efficiently release phosphorus from rocks.

“The early Earth was a volcanically active place, so you would have had lots of fresh volcanic rock reacting with carbon dioxide and supplying carbonate and phosphorus to lakes,” Toner said. “The early Earth could have hosted many carbonate-rich lakes, which would have had high enough phosphorus concentrations to get life started.”

Another recent by the two authors showed that these types of lakes can also provide abundant cyanide to support the formation of amino acids and nucleotides, the building blocks of proteins, DNA and RNA. Before then researchers had struggled to find a natural environment with enough cyanide to support an origin of life. Cyanide is poisonous to humans, but not to primitive microbes, and is critical for the kind of chemistry that readily makes the building blocks of life.

The research was funded by the Simons Foundation’s Collaboration on the Origins of Life.

For more information, contact Toner at 267-304-3488 or toner2@uw.edu and Catling at 206-543-8653 or dcatling@uw.edu.

Theberge’s research probes the chemical signals that cells use to communicate with one another. The organization of our bodies, with different types of cells taking on discrete functions, depends on this biochemical language.

“We’re alive because our cells can exchange chemical messages in appropriate ways,” said Theberge, who is also an affiliate assistant professor of urology at the UW. “All cells — human cells, microbes — utilize chemical signals to deliver information and influence the properties of other cells.”

Theberge focuses on the chemical messages released by cells, which diffuse out into the environment — be it a body or a colony of microbes — and are picked up by other groups of cells. To measure these signals and characterize their effects, scientists need precision: experimental systems that will let researchers set up a population of cells, identify the types and precise amounts of chemicals the cells release, how they diffuse through the environment, which chemical messages are picked up by other groups of cells and their effects.

Theberge and her collaborators — which include Erwin Berthier, a 91̽��affiliate assistant professor of chemistry and co-founder of the medical device company Tasso, Inc. — develop and manufacture laboratory tools to make these precise measurements possible. These include microscale plastic and gel-based dividers, which can partition commonly used cell culture plates or the surface of a slide into more complex arrangements of compartments. These allow researchers to grow different populations of cells in close proximity and sample the types of chemical signals that pass between them.

“While we pursue our own biological hypotheses, we’re also focused on exporting the technologies we’ve developed to other laboratories,” said Theberge. “We really want these tools to be available and used widely.”

Depending on the arrangement of compartments, signals can diffuse horizontally between cell populations separated by short walls, through vertical stacks of cells or other arrangements. Theberge and her team design their cell culture devices with the physics of fluidics in mind. They precisely control the position of liquids in their devices via capillary forces — the passive forces that allow fluids to flow.

Theberge has also put these tools to work. She has started more than 20 collaborations since joining the 91̽��faculty in 2016. The tools she and her group have developed are being used to identify cellular signals involved in testis development and male infertility, communication between epithelial and endothelial cells in kidneys and the immune system signals involved in inflammation. Some of these experiments study chemical signals present in tissue samples from patients, including a collaboration with the .

Her group has also been working on molecular methods to accurately quantify the amount of different types of chemicals that are received by individual cells.

“That will give us information not just on the type of signal reaching a cell, but how signal strength and origin can affect cell communication,” said Theberge.

Theberge earned a bachelor’s degree in chemistry from Williams College and a doctoral degree in chemistry from the University of Cambridge. Prior to joining the 91̽��faculty, she was a postdoctoral researcher at the University of Wisconsin-Madison. According to the Office of Research at the UW, Theberge is the 11th faculty member to earn a Packard Fellowship, and the fourth overall from the Department of Chemistry, after Brandi Cossairt, and former 91̽��faculty member Younan Xia.

###

For more information, contact Theberge at abt1@uw.edu.

These cells faced a chemical conundrum. They needed particular ions from the soup in order to perform basic functions. But those charged ions would have disrupted the simple membranes that encapsulated the cells.

A team of researchers at the 91̽�� has solved this puzzle using only molecules that would have been present on the early Earth. Using cell-sized, fluid-filled compartments surrounded by membranes made of fatty acid molecules, the team discovered that amino acids, the building blocks of proteins, can stabilize membranes against magnesium ions. Their results set the stage for the first cells to encode their genetic information in RNA, a molecule related to DNA that requires magnesium for its production, while maintaining the stability of the membrane.

The findings, Aug. 12 in the , go beyond explaining how amino acids could have stabilized membranes in unfavorable environments. They also demonstrate how the individual building blocks of cellular structures — membranes, proteins and RNA — could have co-localized within watery environments on the ancient Earth.

“Cells are made up of very different types of structures with totally different types of building blocks, and it has never been clear why they would come together in a functional way,” said co-corresponding author Roy Black, a 91̽��affiliate professor of chemistry and bioengineering. “The assumption was just that — somehow — they did come together.”

Black after a career at Amgen for the opportunity to fill in the crucial, missing details behind that “somehow.” He teamed up with , a 91̽��professor of chemistry and an expert on membranes. Black had been inspired by the observation that fatty acid molecules can self-assemble to form membranes, and hypothesized that these membranes could act as a favorable surface to assemble the building blocks of RNA and proteins.

“You can imagine different types of molecules moving within the primordial soup as fuzzy tennis balls and hard squash balls bouncing around in a big box that is being shaken,” said Keller, who is also co-corresponding author on the paper. “If you line one surface inside the box with Velcro, then only the tennis balls will stick to that surface, and they will end up close together. Roy had the insight that local concentrations of molecules could be enhanced by a similar mechanism.”

The team that the building blocks of RNA preferentially attach to fatty acid membranes and, surprisingly, also stabilize the fragile membranes against detrimental effects of salt, a common compound on Earth past and present.

The team hypothesized that amino acids might also stabilize membranes. They used a variety of experimental techniques — including light microscopy, electron microscopy and spectroscopy — to test how 10 different amino acids interacted with membranes. Their experiments revealed that certain amino acids bind to membranes and stabilize them. Some amino acids even triggered large structural changes in membranes, such as forming concentric spheres of membranes — much like layers of an onion.

“Amino acids were not just protecting vesicles from disruption by magnesium ions, but they also created multilayered vesicles — like nested membranes,” said lead author Caitlin Cornell, a 91̽��doctoral student in the Department of Chemistry.

The researchers also discovered that amino acids stabilized membranes through changes in concentration. Some scientists have hypothesized that the first cells may have formed within shallow basins that went through cycles of high and low concentrations of amino acids as water evaporated and as new water washed in.

The new findings that amino acids protect membranes — as well as prior results showing that RNA building blocks can play a similar role — indicate that membranes may have been a site for these precursor molecules to co-localize, providing a potential mechanism to explain what brought together the ingredients for life.

Keller, Black and their team will turn their attention next to how co-localized building blocks did something even more remarkable: They bound to each other to form functional machines.

“That is the next step,” said Black.

Their ongoing efforts are also forging ties across disciplines at the UW.

“The 91̽�� is an unusually good place to make discoveries because of the enthusiasm of the scientific community to work collaboratively to share equipment and ideas across departments and fields,” said Keller. “Our collaborations with the Drobny Lab and the Lee Lab were essential. No single laboratory could have done it all.”

Co-authors are , 91̽��professor of chemistry; , 91̽��associate professor of medicinal chemistry; 91̽��postdoctoral researchers Mengjun Xue and Helen Litz in the Department of Chemistry, and James Williams in the Department of Medicinal Chemistry; 91̽��graduate students Zachary Cohen in the Department of Chemistry and Alexander Mileant in the Biological Structure, Physics and Design Graduate Program; and 91̽��undergraduate alumni Andrew Ramsay and Moshe Gordon. The research was funded by NASA, the National Institutes of Health and the National Science Foundation.

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For more information, contact Black at blackr5@uw.edu or 206-713-4603 and Keller at slkeller@uw.edu or 206-543-9613.

Grant numbers: NNX17AK86G, R01-GM0999989, T32GM008268, T32GM007750, MCB-1402059

In patients with chronic lymphocytic leukemia (CLL), the rate of disease growth varies widely. In a new study from the Dana-Farber Cancer Institute, the Broad Institute of MIT and Harvard, Massachusetts General Hospital and the 91̽��, scientists report that CLL growth is apt to follow one of three trajectories: relentlessly upward, steadily level or something in between. The particular course the disease takes is tightly linked to the genetic makeup of the cancer cells, particularly the number of growth-spurring “driver” mutations they contain.

The , published online May 29 in the journal , contains a further insight: Genetic changes that occur very early in CLL development exert a powerful influence on the growth pattern the CLL cells will ultimately take. This raises the possibility that physicians may one day be able to predict the course of the disease by its molecular features at the time of diagnosis.

“Our findings provide a framework not only for understanding the differing patterns of CLL growth in patients but also for exploring the basic biological mechanisms that underlie these differences,” said Dr. of Dana-Farber, the Broad Institute and Brigham and Women’s Hospital, who is co-corresponding author with of the Broad Institute and Massachusetts General Hospital. “Ultimately, we’d like to be able to tie the genotype of the disease — the particular genetic abnormalities in a patient’s cancer cells — to its phenotype, or how the cancer actually behaves.”

CLL is a useful model for studying the pace of cancer growth because it progresses at widely different rates from one patient to another, said Wu. In many patients, it persists at a low level for many years before advancing to the point where treatment is necessary. In others, it progresses so rapidly that treatment is required shortly after diagnosis.

To see if there were different patterns of CLL growth among patients, researchers drew on data from 107 patients diagnosed with the disease. Beginning at diagnosis, each patient underwent periodic blood tests to track disease progress over the succeeding months and years, and continued until the disease reached a stage where treatment would begin. Each test consisted of a white blood cell count, which served as a proxy measure of CLL: the greater the number of white cells within a blood sample, the greater the burden of the disease. The tests were conducted over a period ranging from two years in one patient to 19 years in another.

The serial testing allowed researchers to calculate growth rates over time for CLL in each patient. They used a statistical model to determine if the rates were consistent with various patterns of cancer growth.

“We found that some cases of CLL show exponential growth, in which it expands without any apparent limit, while other cases show ‘logistic’ growth, in which it plateaus at a fairly consistent level,” said co-lead author , a 91̽��assistant professor of applied mathematics.

Cases that didn’t fit either category were classified as indeterminate.

To explore whether genetic differences were at the root of these divergent growth patterns, the researchers performed whole-exome sequencing on several CLL samples collected from each patient prior to receiving therapy. Whole-exome sequencing provides a letter-by-letter readout of the regions of DNA that encode for cellular proteins.

They found that exponentially growing CLL typically carried a large number of driver mutations — those that confer a competitive advantage in growth — and quickly reached the stage where treatment was called for. In contrast, logistically growing CLL had fewer genetic alterations and fewer types of alterations and progressed relatively slowly toward the level that requires treatment. Seventy-five percent of patients with exponential growth eventually warranted treatment; by comparison, 21% of those with logistic growth and 67% of those with indeterminate growth eventually required treatment.

By analyzing patients’ serial blood samples collected over a period of time, researchers found that exponential CLL not only grows faster but also evolves faster, spinning off new subtypes of cancer cells, each with a particular set of genetic abnormalities. Whole-exome sequencing revealed that exponential CLL is marked by a great variety of tumor cell types and subtypes, while logistic CLL is marked by a relatively less diverse collection of tumor cells.

The information from whole-exome sequencing further enabled researchers to discover the growth rates of those subpopulations of cells within each patient’s leukemia that could be identified on the basis of a subset of mutations, some of them putative driver mutations. These measurements clearly revealed that many of the mutations, which were suspected to be centrally involved in CLL growth, did in fact provide subpopulations with preferential growth acceleration compared to populations lacking these putative drivers. Their results further indicate that the eventual course of CLL growth is inscribed in the genes of tumor cells early in the disease’s development.

“If the course of the disease isn’t altered by therapeutic treatment, the rate and pattern of CLL growth over time seems to ‘play out’ according to a predetermined set of genetic instructions,” said Wu.

“The discovery that CLL growth accelerates in the presence of large numbers of driver mutations is compelling evidence that these mutations do, in fact, confer a growth advantage to cells — that they truly do ‘drive’ the disease,” said co-lead author Dr. of Dana-Farber, the Broad Institute and the Medical University of Vienna.

Bozic and co-lead authors and at the Broad Institute developed methods to jointly model possible phylogenetic relationships of cancer cell subpopulations — which are a description of each subpopulation’s history and relationships to each other during the evolution of the cancer — as well as integrate growth rates with subclone-specific genetic information.

“Combining clinical data with computational and mathematical modeling, we show that the growth of many CLLs seems to follow specific mathematical equations — exponential and logistic — each associated with distinct underlying genetics and clinical outcomes,” said Bozic. “Integrating tumor burden and whole-exome sequencing data allowed us to quantify the growth rates of different tumor subpopulations in individual CLLs, methodology that could potentially inform personalized therapy in the future.”

Co-authors of the study are: Kristen Stevenson, Oriol Olive, Reaha Goyetche, Stacey M. Fernandes, Jing Sun, Wandi Zhang and Donna Neuberg of Dana-Farber; Dr. Jennifer R. Brown of Dana-Farber and Brigham and Women’s Hospital; Laura Rassenti and Dr. Thomas J. Kipps of the Moores Cancer Center at the University of California, San Diego; Daniel Rosebrock, Amaro Taylor-Weiner, Chip Stewart, Alicia Wong and Carrie Cibulskis of the Broad Institute; Johannes G. Reiter, Jeffrey M. Gerold and Martin A. Nowak of Harvard University; Dr. John G. Gribben of the Barts Cancer Institute at the University of London; Dr. Kanti R. Rai of Hofstra North Shore-LIJ School of Medicine; and Michael J. Keating of the MD Anderson Cancer Center.

The study funded by the National Cancer Institute; the CLL Global Research Foundation; the National Heart, Lung, and Blood Institute; the European Union; and the Leukemia and Lymphoma Society.

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For more information, contact Bozic at ibozic@uw.edu.

Grant numbers: 5P01CA081534-14, 1R01CA155010-01A1, P01CA206978, U10CA180861, 1RO1HL103532-0, PIOF-2013-624924

Adapted from a by the Dana-Farber Cancer Institute.

Now, a team led by researchers at the 91̽�� has developed synthetic peptides that target and inhibit those small, toxic aggregates. As they report in a published April 19 in the , their synthetic peptides — which are designed to fold into a structure known as an — can block amyloid beta aggregation at the early and most toxic stage when oligomers form.

The team showed that the synthetic alpha sheet’s blocking activity reduced amyloid beta-triggered toxicity in human neural cells grown in culture, and inhibited amyloid beta oligomers in two laboratory animal models for Alzheimer’s. These findings add evidence to the growing consensus that amyloid beta oligomers — not plaques — are the toxic agents behind Alzheimer’s disease. The results also indicate that synthetic alpha sheets could form the basis of therapeutics to clear toxic oligomers in people, according to corresponding author , a 91̽��professor of and faculty member in the 91̽��.

“This is about targeting a specific structure of amyloid beta formed by the toxic oligomers,” said Daggett. “What we’ve shown here is that we can design and build synthetic alpha sheets with complementary structures to inhibit aggregation and toxicity of amyloid beta, while leaving the biologically active monomers intact.”

Cellular proteins assume many different 3D structures, usually by first folding into certain types of basic shapes. The alpha sheet is a nonstandard protein structure, discovered by Daggett’s group using computational simulations. The research team has previously shown that alpha sheets are associated with aggregation of amyloid beta. These and related findings indicate that, in nature, alpha sheets likely occur in only rare instances when proteins fold incorrectly and interact in ways that disrupt cellular function, leading to so-called “” diseases like Alzheimer’s.

In this new paper, Daggett and her team provide evidence that amyloid beta oligomers form an alpha sheet structure as they aggregate into longer strands and plaques. Critically, the team’s synthetic alpha sheets can actually block this aggregation by specifically binding and neutralizing the toxic oligomers.

Using both novel and conventional spectroscopic techniques, Daggett’s team observed the individual stages of development of amyloid beta clusters, from monomers to six- and 12-protein oligomers all the way up to plaques, in human neural cell lines. The researchers confirmed that the oligomer stages were most toxic to the neurons, which agrees with clinical reports of amyloid beta plaques in the brains of people who don’t have Alzheimer’s.

“Amyloid beta definitely plays a lead role in Alzheimer’s disease, but while historically attention has been on the plaques, more and more research instead indicates that amyloid beta oligomers are the toxic agents that disrupt neurons,” said Daggett.

In addition, the researchers designed and built small, synthetic alpha sheet peptides, each made up of just 23 amino acids, the building blocks of proteins. The synthetic peptides folded into a hairpin-like structure and are not toxic to cells. But the synthetic alpha sheets neutralized the amyloid beta oligomers in human neural cell cultures, inhibiting further aggregation by blocking parts of the oligomers involved in the formation of larger clumps.

The peptides also protected laboratory animals from toxic oligomer damage. In brain tissue samples from mice, the team observed an up to 82% drop in amyloid beta oligomer levels after treatment with a synthetic alpha sheet peptide. Administering a synthetic alpha sheet to living mice triggered a 40% drop in amyloid beta oligomer levels after 24 hours. In the common laboratory worm , another model for Alzheimer’s disease, treatment with synthetic alpha sheets delayed the onset of amyloid beta-induced paralysis. In addition, C. elegans worms showed signs of intestinal damage when they were fed bacteria that express amyloid beta. That damage was inhibited when the scientists first treated the bacteria with their synthetic alpha sheets.

Daggett’s team is continuing experiments with synthetic alpha sheets to engineer compounds that are even better at clearing amyloid beta oligomers. For the current study, the researchers also created a novel laboratory assay that uses a synthetic alpha sheet to measure levels of amyloid beta oligomers. They believe this assay could form the basis of a clinical test to detect toxic oligomers in people before the onset of Alzheimer’s symptoms.

“What we’re really after are potential therapeutics against amyloid beta and diagnostic measures to detect toxic oligomers in people,” said Daggett. “Those are the next steps.”

Lead author is , a 91̽��doctoral student in . Co-authors are 91̽��bioengineering undergraduate students , , and doctoral student Matthew Childers; and professor in the 91̽��Department of Chemistry; and associate professor with the University of Colorado Boulder; and with ; and and executive director with the . The research was funded by the National Institutes of Health, the 91̽��, the American Microscopy Society, the National Science Foundation and the Roskamp Institute.

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For more information, contact Daggett at daggett@uw.edu.

Grant numbers: R01GMS95808, R21AG049693

Cells — the building blocks of our bodies — are encapsulated by membranes. The same goes for the specialized compartments within our cells.

These membranes are extremely thin, oily films, containing proteins and fatty molecules called lipids. For decades, scientists have argued about how cell membranes organize and maintain distinct regions enriched in particular protein and lipid types. These regions are thought to influence cellular activities, such as the signaling that controls both normal cellular growth and the growth of cancerous cells.

In a paper in the , scientists at the 91̽�� show for the first time that the complex distribution of molecules within a membrane of a living yeast cell arises through demixing. Also known as phase separation, demixing is a simple physical process that is similar to the action that causes droplets of oil to separate from vinegar in a salad dressing.

“Cells have a toolbox with a variety of resources to help them complete a variety of tasks,” said senior author , a 91̽��professor of chemistry. “By teaming up with , a 91̽��professor of biochemistry and a yeast expert, we’ve shown that phase separation is one of those tools to shape membranes and their functions within a living system.”

The 91̽��researchers were inspired by pictures of a genetically engineered strain of yeast in which membrane proteins fluorescently glowed. The proteins lit up intracellular, membrane-bound compartments called vacuoles. The vacuoles looked like miniature green balls patterned with dark polka dots. Those polka dots, the researchers realized, looked nearly identical to membrane regions that arise from phase separation in two types of non-living systems: simple, artificial membranes created in a lab and membranes shed from cells under severe stress.

“The membranes of living systems contain many different types of fats, proteins and other molecules,” said co-lead author , a lecturer at 91̽��Tacoma who conducted this research when he was a 91̽��doctoral student in chemistry. “Each of these types of molecules harbors different physical and chemical properties with the potential to affect the properties of the membrane as a whole. We and other groups have hypothesized that this variety of molecules would allow membranes to phase separate by composition into discrete regions.”

First, the team discovered that the polka dots that appear on vacuole membranes can merge quickly. This behavior is consistent with fluid phases, just as droplets in a recently-shaken oil and vinegar salad dressing quickly coalesce when they collide. Next, the team found that phase separation in the membranes of yeast vacuoles depends on temperature. When the researchers warmed the yeast above 90 degrees Fahrenheit, the two liquid phases merged into one — the polka dots vanished. As the yeast cells were cooled back to room temperature, the phase-separated regions reappeared.

“Scientists had never previously shown that phase-separated liquids can co-exist in the membranes of living cells,” said co-lead author , a 91̽��doctoral student in chemistry. “To show that phase separation occurs, we had to reliably track the distribution of proteins within membranes, show that they formed regions like in artificial systems and that these regions would merge in response to changing environmental conditions.”

Now that the researchers have shown that living membranes can undergo phase separation, future work is needed to show how cells regulate phase separation. This could be through the action of genes, environmental conditions or a combination of factors.

“Our finding that phase separation can drive membrane organization in yeast suggests that similar processes may operate in other cells, including human cells,” said Merz. “Again, we see the power of model systems such as yeast, fruit flies and worms in our exploration of fundamental physiology. 91̽��has been at the forefront of yeast genetics and cell biology for over 60 years.”

“There is incredible potential here to unlock how different types of cells form and maintain unique structures — and how different structures are formed even within the same cell,” said Keller.

Caitlin Cornell, a 91̽��doctoral student in chemistry, is a co-author. The research was funded by the National Science Foundation and the National Institutes of Health.

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For more information, contact Keller at skeller@chem.washington.edu or 206-543-9613 and Merz at merza@uw.edu or 206-616-8308.

Grant numbers: DGE-1256082, MCB-1402059, T32GM008268, GM077349.