Red blood cells are amazing. They pick up oxygen from our lungs and carry it all over our body to keep us alive. The hemoglobin molecule in red blood cells transports oxygen by changing its shape in an all-or-nothing fashion. Four copies of the same protein in hemoglobin open and close like flower petals, structurally coupled to respond to each other. Using supercomputers, scientists are just starting to design proteins that self-assemble to combine and resemble life-giving molecules like hemoglobin. The scientists say their methods could be applied to useful technologies such as pharmaceutical targeting, artificial energy harvesting, ‘smart’ sensing and building materials, and more.

A science team did this work by supercharging proteins, which means that they changed the subunits of proteins, the amino acids, to give the proteins an artificially high positive or negative charge. Using proteins derived from jellyfish, the scientists were able to assemble a complex sixteen protein structure composed of two stacked octamers by supercharging alone, findings that were reported in January of 2019 in the journal Nature Chemistry.

The team then used supercomputer simulations to validate and inform these experimental results. Supercomputer allocations on Stampede2 at the Texas Advanced Computing Center (TACC) and Comet at the San Diego Supercomputer Center (SDSC) were awarded to the researchers through XSEDE, the Extreme Science and Engineering Discovery Environment funded by the National Science Foundation (NSF).

“We found that by taking proteins that don’t normally interact with each other, we can make copies that are either highly positively or highly negatively charged,” said study co-author Anna Simon, a postdoctoral researcher in the Ellington Lab of UT Austin. “Combining the highly positively and negatively charged copies, we can make the proteins assemble into very specific structured assemblies,” Simon said. The scientists call their strategy ‘supercharged protein assembly,’ where they drive defined protein interactions by combining engineered supercharged variants.

“We exploited a very well-known and basic principle from nature, that opposite charges attract,” added study co-author Jens Glaser. Glaser is an assistant research scientist in the Glotzer Group, Department of Chemical Engineering at the University of Michigan. “Anna Simon’s group found that when they mix these charged variants of green fluorescent protein, they get highly ordered structures. That was a real surprise,” Glaser said.

The stacked octamer structure looks like a braided ring. It’s composed of 16 proteins — two intertwined rings of eight that interact in very specific, discreet patches. “The reason why it’s so hard to engineer proteins that interact synthetically is that making these interacting patches and having them all line up right such that they’ll allow the proteins to assemble into bigger, regular structures is really hard,” explained Simon. They got around the problem by adding many positive and negative charges to engineer variants of green fluorescent protein (GFP), a well-studied ‘lab mouse’ protein derived from the Aequorea victoria jellyfish.

The positively charged protein, which they called cerulean fluorescent protein (Ceru) +32, had additional opportunities to interact with the negatively charged protein GFP -17. “By giving these proteins all these opportunities, these different places where they could potentially interact, they were able to choose the right ones,” Simon said. “There were certain patterns and interactions that were there, available, and energetically favored, that we didn’t necessarily predict beforehand that would allow them to assemble into these specific shapes.”

To get the engineered charged fluorescent proteins, Simon and co-authors Arti Pothukuchy, Jimmy Gollihar, and Barrett Morrow encoded their genes, including a chemical tag used for purification on portable pieces of DNA called plasmids in E. coli, then harvested the tagged protein that E. coli grew. The scientists mixed the proteins together. They initially thought the proteins might just interact to form large, irregularly structured clumps. “But then, what we kept on seeing was this weird, funny peak around 12 nanometers, that was a lot smaller than a big clump of protein, but significantly bigger than the single protein,” Simon said.

They measured the size of the particles that formed using a Zetasizer instrument at the Texas Materials Institute of UT Austin, and verified that the particles contained both cerulean and GFP proteins Förster Resonance Energy Transfer (FRET), which measures the energy transfer between different colored fluorescent proteins produce fluorescence in response to different energies of light to see if they are close together. Negative stain electron microscopy identifed the specific structure of the particles, conducted by the group of David Taylor, assistant professor of molecular biosciences at UT Austin. It showed that the 12 nm particle consisted of a stacked octamer composed of sixteen proteins. “We found that they were these beautifully shaped flower-like structures,” Simon said. Co-author Yi Zhou from Taylor’s group of UT Austin increased the resolution even further using cryo-electron microscopy to reveal atomic-level details of the stacked octamer.

Computational modeling refined the measurements of how the proteins were arranged into a clear picture of the beautiful, flower-like structure, according to Jens Glaser. “We had to come up with a model that was complex enough to describe the physics of the charged green fluorescent proteins and present all the relevant atomistic details, yet was efficient enough to allow us to simulate this on a realistic timescale. With a fully atomistic model, it would have taken us over a year to get a single simulation out of the computer, however fast the computer was,” Glaser said.

They simplified the model by reducing the resolution without sacrificing important details of the interactions between proteins. “That’s why we used a model where the shape of the protein is exactly represented by a molecular surface, just like the one that’s measured from the crystallographic structure of the protein,” Glaser added.

“What really helped us turn this around and improve what we were able to get out of our simulations was the cryo-EM data,” said Vyas Ramasubramani, a graduate student in chemical engineering at the University of Michigan. “That’s what really helped us find the optimal configuration to put into these simulations, which then helped us validate the stability arguments that we were making, and hopefully going forward make predictions about ways that we can destabilize or modify this structure,” Ramasubramani said.

The scientists required lots of compute power to do the calculations on the scale that they wanted.

“We used XSEDE to basically take these huge systems, where you have lots of different pieces interacting with each other, and calculate all of this at once so that when you start moving your system forward through some semblance of time, you could get an idea for how it was going to evolve on somewhat real timescales,” Ramasubramani said. “If you tried to do the same kind of simulation that we did on a laptop, it would have taken months if not years to really approach understanding whether or not some sort of structure would be stable. For us, not being able to use XSEDE, where you could use essentially 48 cores, 48 compute units all at once to make these calculations highly parallel, we would have been doing this much slower.”

The Stampede2 supercomputer at the TACC contains 4,200 Intel Knights Landing and 1,736 Intel Skylake X compute nodes. Each Skylake node has 48 cores, the basic unit of a computer processor. “The Skylake nodes of the Stampede2 supercomputer were instrumental in achieving the performance that was necessary to compute these electrostatic interactions that act between the oppositely-charged proteins in an efficient manner,” Glaser said. “The availability of the Stampede2 supercomputer was at just the right point in time for us to perform these simulations.”

Initially, the science team tested their simulations on the Comet system at the SDSC. “When we were first figuring out what kind of model to use and whether this simplified model would give us reasonable results, Comet was a great place to try these simulations,” Ramasubramani said. “Comet was a great testbed for what we were doing.”

Looking at the bigger scientific picture, the scientists hope that this work advances understanding of why so many proteins in nature will oligomerize, or join together to form more complex and interesting structures.

“We showed that there doesn’t need to be a very specific, pre-distinguished set of plans and interactions for these structures to form,” Simon said. “This is important because it means that maybe, and quite likely we can take other sets of molecules that we want to make oligomerize and generate both positively charged and negatively charged variants, combine them, and have specifically ordered structures for them.”

Natural biomaterials like bone, feathers, and shells can be tough yet lightweight. “We think supercharged protein assembly is an easier way to develop the kind of materials that have exciting synthetic properties without having to spend so much time or having to know exactly how they’re going to come together beforehand,” Simon said. “We think that will accelerate the ability to engineer synthetic materials and for discovery and exploration of these nanostructured protein materials.”

The study was published online March 28 in the journal Development and Psychopathology.

“One common reason children show up at doctors’ offices is intestinal complaints,” said Nim Tottenham, a professor of psychology at Columbia and senior author on the study. “Our findings indicate that gastrointestinal symptoms in young children could be a red flag to primary care physicians for future emotional health problems.”

Scientists have long noted the strong connection between the gut and brain. Previous research has demonstrated that a history of trauma or abuse has been reported in up to half of adults with irritable bowel syndrome (IBS), at a prevalence twice that of patients without IBS.

“The role of trauma in increasing vulnerability to both gastrointestinal and mental health symptoms is well established in adults but rarely studied in childhood,” said study lead author Bridget Callaghan, a post-doctoral research fellow in Columbia’s psychology department. In addition, she said, animal studies have demonstrated that adversity-induced changes in the gut microbiome — the community of bacteria in the body that regulates everything from digestion to immune system function-influence neurological development, but no human studies have done so.

“Our study is among the first to link disruption of a child’s gastrointestinal microbiome triggered by early-life adversity with brain activity in regions associated with emotional health,” Callaghan said.

The researchers focused on development in children who experienced extreme psychosocial deprivation due to institutional care before international adoption. Separation of a child from a parent is known to be a powerful predictor of mental health issues in humans. That experience, when modeled in rodents, induces fear and anxiety, hinders neurodevelopment and alters microbial communities across the lifespan.

The researchers drew upon data from 115 children adopted from orphanages or foster care on or before approximately they were 2 years old, and from 229 children raised by a biological caregiver. The children with past caregiving disruptions showed higher levels of symptoms that included stomach aches, constipation, vomiting and nausea.

From that sample of adoptees, the researchers then selected eight participants, ages 7 to 13, from the adversity exposed group and another eight who’d been in the group raised by their biological parents. Tottenham and Callaghan collected behavioral information, stool samples and brain images from all the children. They used gene sequencing to identify the microbes present in the stool samples and examined the abundance and diversity of bacteria in each participant’s fecal matter.

The children with a history of early caregiving disruptions had distinctly different gut microbiomes from those raised with biological caregivers from birth. Brain scans of all the children also showed that brain activity patterns were correlated with certain bacteria. For example, the children raised by parents had increased gut microbiome diversity, which is linked to the prefrontal cortex, a region of the brain known to help regulate emotions.

“It is too early to say anything conclusive, but our study indicates that adversity-associated changes in the gut microbiome are related to brain function, including differences in the regions of the brain associated with emotional processing,” says Tottenham, an expert in emotional development.

More research is needed, but Tottenham and Callaghan believe their study helps to fill in an important gap in the literature.

“Animal studies tell us that dietary interventions and probiotics can manipulate the gut microbiome and ameliorate the effects of adversity on the central nervous system, especially during the first years of life when the developing brain and microbiome are more plastic,” Callaghan says. “It is possible that this type of research will help us to know if and how to best intervene in humans, and when.”

Callaghan and Tottenham are currently working on a larger-scale study with 60 children in New York City to see if their findings can be replicated. They expect the results later this year.

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According to a new study, however, polygenic scores developed by studying Europeans do a better job at predicting disease risk for people of European ancestry than for those of other ancestries.

Researchers from the Broad Institute of MIT and Harvard and Massachusetts General Hospital (MGH) led a team that used large-scale genetic data from UK Biobank to develop prediction scores for height, body mass index, T2D, and certain other traits and diseases.

The researchers found that polygenic scores, calculated based on data from UK Biobank, had a 4.5 times higher prediction accuracy for people of European ancestry than those of African ancestry, and two times higher accuracy than those of East Asian ancestry.

“From a clinical context, this means that current polygenic scores are significantly better in predicting the risk of common diseases for people of European ancestry than those of African ancestry,” said Alicia Martin, the lead author of the study and an affiliate of the Program in Medical and Population Genetics and the Stanley Center for Psychiatric Research at the Broad Institute.

Martin, who is now an instructor in investigation at MGH, started this work while she was a postdoctoral researcher in the lab of Mark Daly, institute member and co-director of the Program in Medical and Population Genetics at the Broad Institute.

These findings are published in Nature Genetics.

With advances in genome sequencing technology, studies in people of European ancestry have grown rapidly in the last few years, while the proportion of non-Europeans in these genomic studies have stagnated since 2014, the authors report. As of 2016, 80 percent of participants in genetic studies are of European descent, even though Europeans constitute only 16 percent of the world population.

UK Biobank is one of the largest publicly available genetic data sets. It contains information for half a million people, about 94 percent of whom are of European ancestry. Fewer than 10 percent are of African, South Asian, East Asian, and Hispanic or Latino ancestry.

However, Martin and her team also developed separate polygenic scores using data from the BioBank Japan Project, an East Asian data set, and found that scores calculated from this data set were almost 50 percent more accurate in predicting disease risk for East Asians than scores based on UK Biobank data.

“This further confirms that risk predictors are more precise if they are drawn from genetic data derived from a similar ancestry,” Martin said. “It is crucial that researchers should recruit more minority populations in future genetic studies and also make data from such studies accessible and open. Failure to do this will lead to further inequities in our healthcare system.”

In recent years, Sekar Kathiresan, an institute member and director of the Cardiovascular Disease Initiative at the Broad Institute, and his colleagues have advanced research in polygenic scoring, increasing their predictive power tremendously, and they are working to implement clinically meaningful risk predictors.

“Though health disparities are currently related to social determinants of health rather than genetic testing, it will be important for the biomedical community to ensure that all ethnic groups have access to genetic risk prediction of comparable quality,” said Kathiresan, who is also director of the Center for Genomic Medicine at MGH and a professor of medicine at Harvard Medical School. “This will require undertaking or expanding large genomic studies in non-European ethnic groups.”

This study was funded by the National Institutes of Health.

Then, tiny glass beads began to fall like birdshot from the heavens. The rain of glass was so heavy it may have set fire to much of the vegetation on land. In the water, fish struggled to breathe as the beads clogged their gills.

The heaving sea turned into a 30-foot wall of water when it reached the mouth of a river, tossing hundreds, if not thousands, of fresh-water fish — sturgeon and paddlefish — onto a sand bar and temporarily reversing the flow of the river. Stranded by the receding water, the fish were pelted by glass beads up to 5 millimeters in diameter, some burying themselves inches deep in the mud. The torrent of rocks, like fine sand, and small glass beads continued for another 10 to 20 minutes before a second large wave inundated the shore and covered the fish with gravel, sand and fine sediment, sealing them from the world for 66 million years.

This unique, fossilized graveyard — fish stacked one atop another and mixed in with burned tree trunks, conifer branches, dead mammals, mosasaur bones, insects, the partial carcass of a Triceratops, marine microorganisms called dinoflagellates and snail-like marine cephalopods called ammonites — was unearthed by paleontologist Robert DePalma over the past six years in the Hell Creek Formation, not far from Bowman, North Dakota. The evidence confirms a suspicion that nagged at DePalma in his first digging season during the summer of 2013 — that this was a killing field laid down soon after the asteroid impact that eventually led to the extinction of all ground-dwelling dinosaurs. The impact at the end of the Cretaceous Period, the so-called K-T boundary, exterminated 75 percent of life on Earth.

“This is the first mass death assemblage of large organisms anyone has found associated with the K-T boundary,” said DePalma, curator of paleontology at the Palm Beach Museum of Natural History in Florida and a doctoral student at the University of Kansas. “At no other K-T boundary section on Earth can you find such a collection consisting of a large number of species representing different ages of organisms and different stages of life, all of which died at the same time, on the same day.”

In a paper to appear next week in the journal Proceedings of the National Academy of Sciences, he and his American and European colleagues, including two University of California, Berkeley, geologists, describe the site, dubbed Tanis, and the evidence connecting it with the asteroid or comet strike off Mexico’s Yucatan Peninsula 66 million years ago. That impact created a huge crater, called Chicxulub, in the ocean floor and sent vaporized rock and cubic miles of asteroid dust into the atmosphere. The cloud eventually enveloped Earth, setting the stage for Earth’s last mass extinction.

“It’s like a museum of the end of the Cretaceous in a layer a meter-and-a-half thick,” said Mark Richards, a UC Berkeley professor emeritus of earth and planetary science who is now provost and professor of earth and space sciences at the University of Washington.

Richards and Walter Alvarez, a UC Berkeley Professor of the Graduate School who 40 years ago first hypothesized that a comet or asteroid impact caused the mass extinction, were called in by DePalma and Dutch scientist Jan Smit to consult on the rain of glass beads and the tsunami-like waves that buried and preserved the fish. The beads, called tektites, formed in the atmosphere from rock melted by the impact.

Tsunami vs. seiche

Richards and Alvarez determined that the fish could not have been stranded and then buried by a typical tsunami, a single wave that would have reached this previously unknown arm of the Western Interior Seaway no less than 10 to 12 hours after the impact 3,000 kilometers away, if it didn’t peter out before then. Their reasoning: The tektites would have rained down within 45 minutes to an hour of the impact, unable to create mudholes if the seabed had not already been exposed.

Instead, they argue, seismic waves likely arrived within 10 minutes of the impact from what would have been the equivalent of a magnitude 10 or 11 earthquake, creating a seiche (pronounced saysh), a standing wave, in the inland sea that is similar to water sloshing in a bathtub during an earthquake. Though large earthquakes often generate seiches in enclosed bodies of water, they’re seldom noticed, Richards said. The 2011 Tohoku quake in Japan, a magnitude 9.0, created six-foot-high seiches 30 minutes later in a Norwegian fjord 8,000 kilometers away.

“The seismic waves start arising within nine to 10 minutes of the impact, so they had a chance to get the water sloshing before all the spherules (small spheres) had fallen out of the sky,” Richards said. “These spherules coming in cratered the surface, making funnels — you can see the deformed layers in what used to be soft mud — and then rubble covered the spherules. No one has seen these funnels before.”

The tektites would have come in on a ballistic trajectory from space, reaching terminal velocities of between 100 and 200 miles per hour, according to Alvarez, who estimated their travel time decades ago.

“You can imagine standing there being pelted by these glass spherules. They could have killed you,” Richards said. Many believe that the rain of debris was so intense that the energy ignited wildfires over the entire American continent, if not around the world.

“Tsunamis from the Chicxulub impact are certainly well-documented, but no one knew how far something like that would go into an inland sea,” DePalma said. “When Mark came aboard, he discovered a remarkable artifact — that the incoming seismic waves from the impact site would have arrived at just about the same time as the atmospheric travel time of the ejecta. That was our big breakthrough.”

At least two huge seiches inundated the land, perhaps 20 minutes apart, leaving six feet of deposits covering the fossils. Overlaying this is a layer of clay rich in iridium, a metal rare on Earth, but common in asteroids and comets. This layer is known as the K-T, or K-Pg boundary, marking the end of the Cretaceous Period and the beginning of the Tertiary Period, or Paleogene.

Iridium

In 1979, Alvarez and his father, Nobelist Luis Alvarez of UC Berkeley, were the first to recognize the significance of iridium that is found in 66 million-year-old rock layers around the world. They proposed that a comet or asteroid impact was responsible for both the iridium at the K-T boundary and the mass extinction.

The impact would have melted the bedrock under the seafloor and pulverized the asteroid, sending dust and melted rock into the stratosphere, where winds would have carried them around the planet and blotted out the sun for months, if not years. Debris would have rained down from the sky: not only tektites, but also rock debris from the continental crust, including shocked quartz, whose crystal structure was deformed by the impact.

The iridium-rich dust from the pulverized meteor would have been the last to fall out of the atmosphere after the impact, capping off the Cretaceous.

“When we proposed the impact hypothesis to explain the great extinction, it was based just on finding an anomalous concentration of iridium — the fingerprint of an asteroid or comet,” said Alvarez. “Since then, the evidence has gradually built up. But it never crossed my mind that we would find a deathbed like this.”

Key confirmation of the meteor hypothesis was the discovery of a buried impact crater, Chicxulub, in the Caribbean and off the coast of the Yucatan in Mexico, that was dated to exactly the age of the extinction. Shocked quartz and glass spherules were also found in K-Pg layers worldwide. The new discovery at Tanis is the first time the debris produced in the impact was found along with animals killed in the immediate aftermath of the impact.

“And now we have this magnificent and completely unexpected site that Robert DePalma is excavating in North Dakota, which is so rich in detailed information about what happened as a result of the impact,” Alvarez said. “For me, it is very exciting and gratifying!”

Tektites

Jan Smit, a retired professor of sedimentary geology from Vrije Universiteit in Amsterdam in The Netherlands who is considered the world expert on tektites from the impact, joined DePalma to analyze and date the tektites from the Tanis site. Many were found in near perfect condition embedded in amber, which at the time was pliable pine pitch.

“I went to the site in 2015 and, in front of my eyes, he (DePalma) uncovered a charred log or tree trunk about four meters long which was covered in amber, which acted as sort of an aerogel and caught the tektites when they were coming down,” Smit said. “It was a major discovery, because the resin, the amber, covered the tektites completely, and they are the most unaltered tektites I have seen so far, not 1 percent of alteration. We dated them, and they came out to be exactly from the K-T boundary.”

The tektites in the fishes’ gills are also a first.

“Paddlefish swim through the water with their mouths open, gaping, and in this net, they catch tiny particles, food particles, in their gill rakers, and then they swallow, like a whale shark or a baleen whale,” Smit said. “They also caught tektites. That by itself is an amazing fact. That means that the first direct victims of the impact are these accumulations of fishes.”

Smit also noted that the buried body of a Triceratops and a duck-billed hadrosaur proves beyond a doubt that dinosaurs were still alive at the time of the impact.

“We have an amazing array of discoveries which will prove in the future to be even more valuable,” Smit said. “We have fantastic deposits that need to be studied from all different viewpoints. And I think we can unravel the sequence of incoming ejecta from the Chicxulub impact in great detail, which we would never have been able to do with all the other deposits around the Gulf of Mexico.”

“So far, we have gone 40 years before something like this turned up that may very well be unique,” Smit said. “So, we have to be very careful with that place, how we dig it up and learn from it. This is a great gift at the end of my career. Walter sees it as the same.”

“Plant breeding and other agricultural technologies have contributed considerably to hunger reduction during the last few decades,” says Matin Qaim, an agricultural economist at the University of Göttingen and one of the article’s authors. But the resulting high intensity in the use of agrochemicals has also caused serious environmental problems. Future technologies need to reduce the negative environmental footprint and make agriculture more resilient to climate stress. Predictions suggest that small farms in Africa and Asia will suffer especially from the effects of climate change.

“Genome editing allows us to develop crop plants that are more resistant to pests and diseases and more tolerant to drought and heat,” says Shahid Mansoor from the National Institute for Biotechnology and Genetic Engineering in Pakistan. This can help to reduce crop losses and chemical pesticide sprays. In genome editing, certain DNA sequences are changed or “switched off” in a very precise way without foreign genes being introduced. Hence, genome-edited crops are different from transgenic genetically modified organisms (GMOs). “The new methods are already being used in various cereals and also to improve neglected food crops such as pulses or local vegetables,” Mansoor explains.

“We should be careful not to repeat the mistakes that were made with GMOs,” says Qaim. “The limited public acceptance and the high regulatory hurdles for transgenic GMOs have contributed to a concentration of biotech developments in only a few major crops and in the hands of only a few multinationals. We need more diversity and more competition,” adds Qaim. “Genome-edited crops do not contain foreign genes; as the breeding techniques are more precise, these crops are as safe as conventionally bred crops. Hence, genome-edited crops should not be regulated as if they were transgenic GMOs.”

In Europe, regulations for genome-edited crops are still being debated. In July 2018, the EU Court of Justice ruled that these crops would fall under the existing GMO law, which is disappointing according to the authors of this position paper. “This will hold up future applications” says Qaim. The regulation of new breeding technologies in Europe also has a major impact on developing countries, carrying the risk that the enormous potential of genome editing for food security cannot be fully harnessed, the researchers fear.

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