The answer, according to archaeologists from the University of Colorado Boulder: It’s complicated. In a new study, the team drew on a wide range of evidence — from medical studies of modern equestrians to records of human remains across thousands of years.

The researchers concluded that horseback riding can, in fact, leave a mark on human skeletons, such as by subtly altering the shape of the hip joint. But those sorts of changes on their own can’t definitively reveal whether people have ridden horses during their lives. Many other activities, even sitting for long periods of time, can also transform human bones.

“In archaeology, there are vanishingly few instances in which we can tie a particular activity unequivocally to skeletal changes,” said Lauren Hosek, lead author of the study and an assistant professor in the Department of Anthropology at CU Boulder.

She and her colleagues reported their findings Sept. 20 in the journal Science Advances.

The results may have implications for researchers who study the origins of when humans first domesticated horses — and also cast doubt on a long-standing theory in archaeology known as the Kurgan hypothesis.

The first equestrians

The research lies at the center of what is among the old debates in archaeology, said William Taylor, a co-author of the new study and curator of archaeology at the CU Museum of Natural History.

He explained that the earliest, incontrovertible evidence of humans using horses for transport comes from the region around the Ural Mountains of Russia. There, scientists have uncovered horses, bridles and chariots dating back to around 4,000 years ago.

But the Kurgan hypothesis, which emerged in the early 20th century, argues that the close relationship between humans and horses began much earlier. Proponents believe that around the fourth millennium B.C., ancient humans living near the Black Sea called the Yamnaya first began galloping on horseback across Eurasia. In the process, the story goes, they may have spread a primordial version of the languages that would later evolve into English, French and more.

“A lot of our understanding of both the ancient and modern worlds hinges on when people started using horses for transportation,” Taylor said. “For decades, there’s been this idea that the distribution of Indo-European languages is, in some way, related to the domestication of the horse.”

Recently, scientists have pointed to human remains from the Yamnaya culture dating back to about 3500 B.C. as a key piece of evidence supporting the Kurgan hypothesis. These ancient peoples, the group argued, showed evidence of wear and tear in their skeletons that likely came from riding horses.

Hips can lie

But, in the new study, Hosek and Taylor argue that the story isn’t so simple.

Hosek has spent a lot of time poring over human bones to learn lessons about the past. She explained that the skeleton isn’t static but can shift and change shape over an individual’s lifetime. If you pull a muscle, for example, a reaction can emerge at the site where the muscle attaches to the underlying bone. In some cases, the bone can become more porous or raised ridges may form.

Reading those sorts of clues, however, can be murky at best. The hip joint is one example.

Hosek noted that when you flex your legs at the hip for long periods of time, including during long horse rides, the ball and socket of the hip joint may rub together along one edge. Over time, that rubbing can cause the round socket of the hip bone to become more elongated, or oval in shape. But, she said, other activities can cause the same kind of elongation.

Archaeological evidence shows that humans used cattle, donkeys and even wild asses for transport in some areas of western Asia centuries before they first tamed horses. Ancient peoples likely yoked these beasts of burden to pull carts or even smaller, two-wheeled vehicles that looked something like a chariot.

“Over time, this repetitive, intense pressure from that kind of jostling in a flexed position could cause skeletal changes,” Hosek said.

She’s seen similar changes, for example, in the skeletons of Catholic nuns from the 20th century. They never rode horses, but did take long carriage rides across the American West.

Ultimately, Hosek and Taylor say that human remains on their own can’t be used to put a date on when people first started riding horses — at least not with currently available science.

“Human skeletons alone are not going to be enough evidence,” Hosek said. “We need to couple that data with evidence coming out of genetics and archaeology and by looking at horse remains, too.”

Taylor added that the picture doesn’t look good for the Kurgan hypothesis:

“At least for now, none of these lines of evidence suggest that the Yamnaya people had domestic horses.”

Their tool, the Framework for Narrative Storylines and Impact Classification (FRNSIC), can help decision-makers explore many plausible futures and identify consequential scenario storylines — or descriptions of what critical futures might look like — to help planners better address the uncertainties and impacts presented by climate change. They reported their findings Sept. 19 in the journal Earth’s Future.

“One of the ways states like Colorado are preparing for the future is by making plans for how things might evolve based on the available science and inputs from various stakeholders,” said Antonia Hadjimichael, assistant professor in the Department of Geosciences at Penn State and lead author of the study. “This scenario planning process recognizes that planning for the future comes with many uncertainties about climate and water needs. So, planners have to consider different possibilities, such as a high-warming or a low-warming scenario.”

Hadjimichael said that both the scientific community and decision makers around the world often turn to scenarios to describe what conditions may look like in the future, but this approach may regard only a few possibilities and discount other alternatives.

These scenario planning approaches often feature a relatively small number of scenarios — for example what drought conditions might look like under different levels of warming — and may fail to capture the complexity of all the factors involved.

Alternatively, scientists use a technique called exploratory modeling, where models simulate thousands to millions of possible futures to discover which are consequential. But this approach is often not practical for use by decision makers, the scientists said.

“We wanted to provide something in the middle,” Hadjimichael said. “We wanted to create something that bridges the two — that considers the complexities but also boils it down to something that’s a little more actionable and a little less daunting.”

Their tool, FRNSIC, uses exploratory modeling first to investigate a large number of hypothesized plausible future conditions. It then uses that data to classify and identify relevant and locally meaningful storylines, the scientists said.

“Our approach essentially explores plausible future impacts and then says, ‘for this stakeholder, this is the storyline that would matter the most — and then for this other stakeholder, there is a different storyline they should be worried about,” Hadjimichael said. “It’s adding a little bit more pluralism and a little bit more nuance into how planning scenarios are established.”

In the Colorado River basin, decision makers face a complex set of factors, including how to supply enough water for growing populations and farmers while ensuring their state is not using more than their allowed share of the river’s flow, Hadjimichael said.

“The problem is there is not a single criterion that captures everybody and what they care about,” she said. “Maybe you have a very large farm, and maybe I have a very small farm. And maybe we grow different things. It’s hard to use a single factor to find out scenarios that would make us all happy, or make us all unhappy.”

The storylines produced by FRNSIC can be used in future work in the Colorado River basin — for example, how drought events are impacted when populations adapt and make changes.

“This allows policymakers to explore different states the world and helps review how different interventions might affect the basin under each storyline,” Hadjimichael said. “These drought scenarios can be used to illuminate potential consequences, and therefore be used in negotiations or when asking stakeholders for their input.”

Also contributing were Patrick Reed, professor at Cornell University; Julianne Quinn, assistant professor at the University of Virginia; and Chris Vernon, geospatial scientist, and Travis Thurber, software engineer, at Pacific Northwest National Laboratory

The U.S. Department of Energy, Office of Science, as part of research in MultiSector Dynamics, in the Earth and Environmental System Modeling Program supported this research.

“Lasso peptides are interesting because they are basically linear molecules that have been tied into a slip knot-like shape,” said Susanna Barrett, a graduate student in the Mitchell lab (MMG). “Due to their incredible stability and engineerability, they have a lot of potential as therapeutics. They have also been shown to have antibacterial, antiviral, and anti-cancer properties.”

Lasso peptides are ribosomally synthesized and post-translationally modified molecules. The peptide chains are formed from joining amino acids together in the form of a string, which is done by the ribosome. Two enzymes, a peptidase and a cyclase, then collaborate to convert a linear precursor peptide into the distinctive knotted lasso structure. Since their discovery over three decades ago, scientists have been trying to understand how the cyclase folds the lasso peptide.

“One of the major challenges of solving this problem has been that the enzymes are difficult to work with. They are generally insoluble or inactive when you attempt to purify them,” Barrett said.

One rare counterexample is fusilassin cyclase, or FusC, which the Mitchell lab characterized in 2019. Former group members were able to purify the enzyme, and since then, it has served as a model to understand the lasso knot-tying process. Yet, the structure of FusC remained unknown, making it impossible to understand how the cyclase interacts with the peptide to fold the knot.

In the current study, the group used the artificial intelligence program AlphaFold to predict the FusC protein structure. They used the structure and other artificial intelligence-based tools, like RODEO, to pinpoint which cyclase active site residues were important for interacting with the lasso peptide substrate.

“FusC is made up of approximately 600 amino acids and the active site contains 120. These programs were instrumental to our project because they allowed us to do ‘structural studies’ and whittle down which amino acids are important in the active site of the enzyme,” Barrett said.

They also used molecular dynamics simulations to computationally understand how the lasso is folded by the cyclase. “Thanks to the computing power of Folding@home, we were able to collect extensive simulation data to visualize the interactions at the atomic level,” said Song Yin, a graduate student in the Shukla lab. “Before this study, there were no MD simulations of the interactions between lasso peptides and cyclases, and we think this approach will be applicable to many other peptide engineering studies.”

From their computational efforts, the researchers found that among different cyclases, the backwall region of the active site seemed to be especially important for folding. In FusC, this corresponded to the helix 11 region. The researchers then carried out cell-free biosynthesis where they added all the cell components that are necessary for the synthesis of the lasso peptides to a test tube with enzyme variants that had different amino acids in the helix 11 region. Ultimately, they identified a version of FusC with a mutation on helix 11 that could fold lasso peptides which cannot be made by the original cyclase. This data confirms the model for lasso peptide folding that the researchers developed with their computational approaches.

“How enzymes tie a lasso knot is a fascinating question. This study provides a first glimpse of the biophysical interactions responsible for producing this unique structure,” said Diwakar Shukla, an associate professor of chemical and biomolecular engineering.

“We also showed that these molecular contacts are the same in several different cyclases across different phyla. Even though we have not tested every system, we believe it’s a generalizable model,” Barrett said.

Collaborating with the San Diego-based company Lassogen, the researchers showed that the new insights can guide cyclase engineering to generate lasso peptides that otherwise cannot be made. As a proof-of-concept, they engineered a different cyclase, called McjC, to efficiently produce a potent inhibitor of a cancer-promoting integrin.

“The ability to generate lasso peptide diversity is important for optimizing drugs,” said Mark Burk, CEO of Lassogen. “The enzymes from nature do not always allow us to produce the lasso peptides of interest and the ability to engineer lasso cyclases greatly expands the therapeutic utility of these amazing molecules.”

“Our work would not have been possible without access to powerful computing and recent advances in artificial intelligence and cell-free biosynthetic methods,” said Douglas Mitchell, John and Margaret Witt Professor of Chemistry. “This work is an extraordinary example of how interdisciplinary collaborations are catalyzed at the Carl R. Woese Institute for Genomic Biology. I am grateful to the MMG theme at IGB and our external colleagues at Lassogen for their participation in solving this complicated problem.”

Most recently, the team applied this new computational tool to analyze pollutants in seawater in Southern California. The team swiftly captured the chemical profiles of coastal environments and highlighted potential sources of pollution.

“We are interested in understanding how such pollutants get introduced in the ecosystem,” said Daniel Petras, an assistant professor of biochemistry at UC Riverside, who led the research team. “Figuring out which molecules in the ocean are important for environmental health is not straightforward because of the ocean’s sheer chemical diversity. The protocol we developed greatly speeds up this process. More efficient sorting of the data means we can understand problems related to ocean pollution faster.”

Petras and his colleagues report in the journal Nature Protocols that their protocol is designed not only for experienced researchers but also for educational purposes, making it an ideal resource for students and early-career scientists. This computational workflow is accompanied by an accessible web application with a graphical user interface that makes metabolomics data analysis accessible for non-experts and enables them to gain statistical insights into their data within minutes.

“This tool is accessible to a broad range of researchers, from absolute beginners to experts, and is tailored for use in conjunction with the molecular networking software my group is developing,” said coauthor Mingxun Wang, an assistant professor of computer science and engineering at UCR. “For beginners, the guidelines and code we provide make it easier to understand common data processing and analysis steps. For experts, it accelerates reproducible data analysis, enabling them to share their statistical data analysis workflows and results.”

Petras explained the research paper is unique, serving as a large educational resource organized through a virtual research group called Virtual Multiomics Lab, or VMOL. With more than 50 scientists participating from around the world, VMOL is a community-driven, open-access community. It aims to simplify and democratize the chemical analysis process, making it accessible to researchers worldwide, regardless of their background or resources.

“I’m incredibly proud to see how this project evolved into something impactful, involving experts and students from across the globe,” said Abzer Pakkir Shah, a doctoral student in Petras’ group and the first author of the paper. “By removing physical and economic barriers, VMOL provides training in computational mass spectrometry and data science and aims to launch virtual research projects as a new form of collaborative science.”

All software the team developed is free and publicly available. The software development was initiated during a summer school for non-targeted metabolomics in 2022 at the University of Tübingen, where the team also launched VMOL.

Petras expects the protocol will be especially useful to environmental researchers as well as scientists working in the biomedical field and researchers doing clinical studies in microbiome science.

“The versatility of our protocol extends to a wide range of fields and sample types, including combinatorial chemistry, doping analysis, and trace contamination of food, pharmaceuticals, and other industrial products,” he said.

Petras received his master’s degree in biotechnology from the University of Applied Science Darmstadt and his doctoral degree in biochemistry from the Technical University Berlin. He did postdoctoral research at UC San Diego, where he focused on the development of large-scale environmental metabolomics methods. In 2021, he launched the Functional Metabolomics Lab at the University of Tübingen. In January 2024 he joined UCR, where his lab focuses on the development and application of mass spectrometry-based methods to visualize and assess chemical exchange within microbial communities.

The findings, published Sept. 20 in the journal Science Immunology, offer a mechanistic understanding for how cells respond to heat and could explain how chronic inflammation contributes to the development of cancer.

The impact of fever temperatures on cells is a relatively understudied area, said Jeff Rathmell, PhD, Cornelius Vanderbilt Professor of Immunobiology and corresponding author of the new study. Most of the existing temperature-related research relates to agriculture and how extreme temperatures impact crops and livestock, he noted. It’s challenging to change the temperature of animal models without causing stress, and cells in the laboratory are generally cultured in incubators that are set at human body temperature: 37 degrees Celsius (98.6 degrees Fahrenheit).

“Standard body temperature is not actually the temperature for most inflammatory processes, but few have really gone to the trouble to see what happens when you change the temperature,” said Rathmell, who also directs the Vanderbilt Center for Immunobiology.

Graduate student Darren Heintzman was interested in the impact of fevers for personal reasons: Before he joined the Rathmell lab, his father developed an autoimmune disease and had a constant fever for months on end.

“I started thinking about what an increased set point temperature like that might do. It was intriguing,” Heintzman said.

Heintzman cultured immune system T cells at 39 degrees Celsius (about 102 degrees Fahrenheit). He found that heat increased helper T cell metabolism, proliferation and inflammatory effector activity and decreased regulatory T cell suppressive capacity.

“If you think about a normal response to infection, it makes a lot of sense: You want effector (helper) T cells to be better at responding to the pathogen, and you want suppressor (regulatory) T cells to not suppress the immune response,” Heintzman said.

But the researchers also made an unexpected discovery — that a certain subset of helper T cells, called Th1 cells, developed mitochondrial stress and DNA damage, and some of them died. The finding was confusing, the researchers said, because Th1 cells are involved in settings where there is often fever, like viral infections. Why would the cells that are needed to fight the infection die?

The researchers discovered that only a portion of the Th1 cells die, and that the rest undergo an adaptation, change their mitochondria, and become more resistant to stress.

“There’s a wave of stress, and some of the cells die, but the ones that adapt and survive are better — they proliferate more and make more cytokine (immune signaling molecules),” Rathmell said.

Heintzman was able to define the molecular events of the cell response to fever temperatures. He found that heat rapidly impaired electron transport chain complex 1 (ETC1), a mitochondrial protein complex that generates energy. ETC1 impairment set off signaling mechanisms that led to DNA damage and activation of the tumor suppressor protein p53, which aids DNA repair or triggers cell death to maintain genome integrity. Th1 cells were more sensitive to impaired ETC1 than other T cell subtypes.

The researchers found Th1 cells with similar changes in sequencing databases for samples from patients with Crohn’s disease and rheumatoid arthritis, adding support to the molecular signaling pathway they defined.

“We think this response is a fundamental way that cells can sense heat and respond to stress,” Rathmell said. “Temperature varies across tissues and changes all the time, and we don’t really know what it does. If temperature changes shift the way cells are forced to do metabolism because of ETC1, that’s going to have a big impact. This is fundamental textbook kind of stuff.”

The findings suggest that heat can be mutagenic — when cells that respond with mitochondrial stress don’t properly repair the DNA damage or die.

“Chronic inflammation with sustained periods of elevated tissue temperatures could explain how some cells become tumorigenic,” Heintzman said, noting that up to 25% of cancers are linked to chronic inflammation.

“People ask me, ‘Is fever good or bad?'” Rathmell added. “The short answer is: A little bit of fever is good, but a lot of fever is bad. We already knew that, but now we have a mechanism for why it’s bad.”

The research was supported by the National Institutes of Health (grants R01DK105550, R01HL136664, R01CA217987, R01HL118979, R01AI153167, R01CA245134, T32AI112541, T32DK101003, T32AR059039, K00CA253718), Lupus Research Alliance, Waddell Walker Hancock Cancer Discovery Fund, and National Science Foundation.

Several years ago, MIT researchers showed that administering a series of escalating doses of an HIV vaccine over a two-week period could help overcome a part of that challenge by generating larger quantities of neutralizing antibodies. However, a multidose vaccine regimen administered over a short time is not practical for mass vaccination campaigns.

In a new study, the researchers have now found that they can achieve a similar immune response with just two doses, given one week apart. The first dose, which is much smaller, prepares the immune system to respond more powerfully to the second, larger dose.

This study, which was performed by bringing together computational modeling and experiments in mice, used an HIV envelope protein as the vaccine. A single-dose version of this vaccine is now in clinical trials, and the researchers hope to establish another study group that will receive the vaccine on a two-dose schedule.

“By bringing together the physical and life sciences, we shed light on some basic immunological questions that helped develop this two-dose schedule to mimic the multiple-dose regimen,” says Arup Chakraborty, the John M. Deutch Institute Professor at MIT and a member of MIT’s Institute for Medical Engineering and Science and the Ragon Institute of MIT, MGH and Harvard University.

This approach may also generalize to vaccines for other diseases, Chakraborty notes.

Chakraborty and Darrell Irvine, a former MIT professor of biological engineering and materials science and engineering and member of the Koch Institute for Integrative Cancer Research, who is now a professor of immunology and microbiology at the Scripps Research Institute, are the senior authors of the study, which appears in Science Immunology. The lead authors of the paper are Sachin Bhagchandani PhD ’23 and Leerang Yang PhD ’24.

Neutralizing antibodies

Each year, HIV infects more than 1 million people around the world, and some of those people do not have access to antiviral drugs. An effective vaccine could prevent many of those infections. One promising vaccine now in clinical trials consists of an HIV protein called an envelope trimer, along with a nanoparticle called SMNP. The nanoparticle, developed by Irvine’s lab, acts as an adjuvant that helps recruit a stronger B cell response to the vaccine.

In clinical trials, this vaccine and other experimental vaccines have been given as just one dose. However, there is growing evidence that a series of doses is more effective at generating broadly neutralizing antibodies. The seven-dose regimen, the researchers believe, works well because it mimics what happens when the body is exposed to a virus: The immune system builds up a strong response as more viral proteins, or antigens, accumulate in the body.

In the new study, the MIT team investigated how this response develops and explored whether they could achieve the same effect using a smaller number of vaccine doses.

“Giving seven doses just isn’t feasible for mass vaccination,” Bhagchandani says. “We wanted to identify some of the critical elements necessary for the success of this escalating dose, and to explore whether that knowledge could allow us to reduce the number of doses.”

The researchers began by comparing the effects of one, two, three, four, five, six, or seven doses, all given over a 12-day period. They initially found that while three or more doses generated strong antibody responses, two doses did not. However, by tweaking the dose intervals and ratios, the researchers discovered that giving 20 percent of the vaccine in the first dose and 80 percent in a second dose, seven days later, achieved just as good a response as the seven-dose schedule.

“It was clear that understanding the mechanisms behind this phenomenon would be crucial for future clinical translation,” Yang says. “Even if the ideal dosing ratio and timing may differ for humans, the underlying mechanistic principles will likely remain the same.”

Using a computational model, the researchers explored what was happening in each of these dosing scenarios. This work showed that when all of the vaccine is given as one dose, most of the antigen gets chopped into fragments before it reaches the lymph nodes. Lymph nodes are where B cells become activated to target a particular antigen, within structures known as germinal centers.

When only a tiny amount of the intact antigen reaches these germinal centers, B cells can’t come up with a strong response against that antigen.

However, a very small number of B cells do arise that produce antibodies targeting the intact antigen. So, giving a small amount in the first dose does not “waste” much antigen but allows some B cells and antibodies to develop. If a second, larger dose is given a week later, those antibodies bind to the antigen before it can be broken down and escort it into the lymph node. This allows more B cells to be exposed to that antigen and eventually leads to a large population of B cells that can target it.

“The early doses generate some small amounts of antibody, and that’s enough to then bind to the vaccine of the later doses, protect it, and target it to the lymph node. That’s how we realized that we don’t need to give seven doses,” Bhagchandani says. “A small initial dose will generate this antibody and then when you give the larger dose, it can again be protected because that antibody will bind to it and traffic it to the lymph node.”

T-cell boost

Those antigens may stay in the germinal centers for weeks or even longer, allowing more B cells to come in and be exposed to them, making it more likely that diverse types of antibodies will develop.

The researchers also found that the two-dose schedule induces a stronger T-cell response. The first dose activates dendritic cells, which promote inflammation and T-cell activation. Then, when the second dose arrives, even more dendritic cells are stimulated, further boosting the T-cell response.

Overall, the two-dose regimen resulted in a fivefold improvement in the T-cell response and a 60-fold improvement in the antibody response, compared to a single vaccine dose.

“Reducing the ‘escalating dose’ strategy down to two shots makes it much more practical for clinical implementation. Further, a number of technologies are in development that could mimic the two-dose exposure in a single shot, which could become ideal for mass vaccination campaigns,” Irvine says.

The researchers are now studying this vaccine strategy in a nonhuman primate model. They are also working on specialized materials that can deliver the second dose over an extended period of time, which could further enhance the immune response.

The research was funded by the Koch Institute Support (core) Grant from the National Cancer Institute, the National Institutes of Health, and the Ragon Institute of MIT, MGH, and Harvard.