If you put a teapot of boiling water on the kitchen table, it will gradually cool down. However, its temperature is not expected to fall below that of the table. It is precisely this everyday experience that illustrates one of the fundamental laws of physics — the second law of thermodynamics — which states that the entropy of a closed natural system must increase over time. Or, more simply put: Heat can flow by itself only from a warmer to a colder object, and not the other way round.

Cooling below room temperature

The results of a recent experiment carried out by the research group of Prof. Andreas Schilling in the Department of Physics at the University of Zurich (UZH) appear at first sight to challenge the second law of thermodynamics. The researchers managed to cool a nine-gram piece of copper from over 100°C to significantly below room temperature without an external power supply. “Theoretically, this experimental device could turn boiling water to ice, without using any energy,” says Schilling.

Creating oscillating heat currents

To achieve this, the researchers used a Peltier element, a component commonly used, for example, to cool minibars in hotel rooms. These elements can transform electric currents into temperature differences. The researchers had already used this type of element in previous experiments, in connection with an electric inductor, to create an oscillating heat current in which the flow of heat between two bodies perpetually changed direction. In this scenario, heat also temporarily flows from a colder to a warmer object so that the colder object is cooled down further. This kind of “thermal oscillating circuit” in effect contains a “thermal inductor.” It functions in the same way as an electrical oscillating circuit, in which the voltage oscillates with a constantly changing sign.

Laws of physics remain intact

Until now, Schilling’s team had only operated these thermal oscillating circuits using an energy source. The researchers have now shown for the first time that this kind of thermal oscillating circuit can also be operated “passively,” i.e. with no external power supply. Thermal oscillations still occurred and, after a while, heat flowed directly from the colder copper to a warmer heat bath with a temperature of 22°C, without being temporarily transformed into another form of energy. Despite this, the authors were also able to show that the process does not actually contradict any laws of physics. To prove it, they considered the change in entropy of the whole system and showed that it increased with time — fully in accordance with the second law of thermodynamics.

Potential application still a long way off

Although the team recorded a difference of only about 2°C compared to the ambient temperature in the experiment, this was mainly due to the performance limitations of the commercial Peltier element used. According to Schilling, it would be possible in theory to achieve cooling of up to -47°C under the same conditions, if the “ideal” Peltier element — yet to be invented — could be used: “With this very simple technology, large amounts of hot solid, liquid or gaseous materials could be cooled to well below room temperature without any energy consumption.”

The passive thermal circuit could also be used as often as desired, without the need to connect it to a power supply. However, Schilling admits that a large-scale application of the technique is still a long way off. One reason for this is that the Peltier elements currently available are not efficient enough. Furthermore, the current set-up requires the use of superconducting inductors to minimize electric losses.

Established perceptions challenged

The UZH physicist considers the work more significant than a mere “proof-of-principle” study: “At first sight, the experiments appear to be a kind of thermodynamic magic, thereby challenging to some extent our traditional perceptions of the flow of heat.”

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Conducting the study was a multi-institutional research team funded in part by the Gulf of Mexico Research Initiative, a 10-year independent program established through a $500 million financial commitment from BP. The team began sampling soon after the spill was finally contained, and continue their work today. Their most-recent article — in Estuaries and Coasts — reports on the first six and a half years of sampling post-spill.

Lead author on the study is John Fleeger, an emeritus professor at LSU. Co-authors are Rita Riggio, Irving Mendelssohn, Qianxin Lin, and Aixin Hou of LSU; David Johnson of William & Mary’s Virginia Institute of Marine Science; Donald Deis of Atkins North America; Kevin Carman of the University of Nevada-Reno; Sean Graham of Nicholls State University; and Scott Zengel of Research Planning, Inc.

Johnson, an assistant professor at VIMS and expert in salt marsh invertebrates, says “Our study highlights the crucial role that plants play in the recovery of important links in the Gulf of Mexico’s coastal food web.” Those links ultimately connect to the fish and shellfish that support the region’s economy and culture.

Two plants dominate healthy Gulf Coast salt marshes — the smooth cordgrass Spartina alterniflora and the black needlerush Juncus roemerianus. Also abundant on the marsh surface are single-celled, plant-like organisms that scientists collectively refer to as benthic microalgae, while a suite of small invertebrates — amphipods, copepods, nematodes, snails, worms, and others — swim, hop, and crawl among the grass blades or burrow in the underlying root zone.

The team studied these organisms by measuring their abundance and biomass in heavily oiled, moderately oiled, and oil-free areas of Louisiana’s Barataria Bay, using both surface plots and shallow cores. Sampling took place at roughly 6-month intervals between 2011 and 2016.

The researchers’ early sampling showed that nearly all the plants in heavily oiled areas died, while benthic microalgae and burrowing invertebrates suffered significant reductions. Their later sampling showed that marsh recovery was led by benthic microalgae and Spartina — which began to show significant above-ground growth within two to three years.

Importantly, it was only after Spartina started its comeback that recovery of the invertebrate community began in earnest. “Plants are the foundation of salt marshes,” explains Johnson. “Marsh grasses facilitate colonization by burrowing invertebrates; fuel the food web, provide animal habitat, bind the soil, and slow water flow. Without plants there is no marsh, and there is no marsh recovery following a spill without plants leading the way.”

If you plant it, they will come

The team’s findings have important implications for responding to any future spills. Fleeger says “our findings indicate that mitigation strategies for any future spills should include the planting of foundation species such as Spartina.”

Mendelssohn, a VIMS alumnus (M.A. ’73), says that foundation species “enhance recovery by providing habitat and reducing sediment erosion.” Over the longer term, he says, “plant growth enhances recovery by improving soil quality. Plants generate organic matter that accumulates belowground, while their roots and rhizomes release oxygen, bind sediments, and increase sediment volume. Breakdown of plant tissues also provides nutrients that further stimulate plant growth and beneficial microbial processes in the marsh.”

A slow road to full recovery

Tempering the promise of marsh recovery via planting of grasses such as Spartina is the team’s discovery that heavily oiled marsh sites remained less healthy than moderately oiled and oil-free sites more than 6 years after the Deepwater Horizon spill. Heavily oiled sites still had elevated concentrations of oil and its breakdown products, and showed slower growth of black needlerush, lower production of plant detritus and below-ground organic matter, and altered soil density. Populations of worms, juvenile snails, and other small invertebrates had also failed to fully recover.

Particularly troubling was the continued rarity of the polychaete worm Manayunkia aestuarina. One of the most abundant single species in the invertebrate community, this tube dweller is important to the health of marsh sediments, and plays a key role in the marsh food web as a major prey item for crabs, shrimp, and fish. “The near absence of this species could indicate significant alteration of ecological function at heavily oiled sites,” says Johnson.

Also troubling is that projecting the observed pace of mash recovery into the future suggests that complete recovery at moderately and heavily oiled sites will likely take much longer than a decade. This is slower than reported in many previous studies of oil spills and their impacts on the marsh community.

“Previous work shows that oil spills in salt marshes can impact bottom-dwelling invertebrates for more than four decades,” says Fleeger. “Long-term exposure to oil and its breakdown products may also decrease the sensitivity and resilience of these organisms to future spills,” he adds.

On a brighter note, a previous study by Johnson and colleagues suggests that fertilization of Spartina plantings can enhance growth of both its stems and roots, thus aiding marsh recovery in the long term. “We’re starting to see the salt marsh in the Gulf of Mexico rebound,” says Johnson, “but it will likely be a decade or more before we see complete recovery.”

The Mueller Lab created a framework using the tremendous influx of new genome sequences as a training resource for undergraduates interested in learning genome annotation. This framework was published online in PLOS Computational Biology on April 3, 2019.

What is genome annotation, and why is it important?

After researchers determine the sequence of the millions of base pairs of DNA in an organism’s genome, they need to figure out two things: which DNA segments are genes that encode proteins, and what are those proteins’ functions. This process of identifying genes and predicting their functions is called genome annotation.

“The prediction of genes and their functions is what most biologists are interested in. That’s where most understanding of biological processes is happening,” says Prashant Hosmani, a Bioinformatics Analyst in the Mueller Lab and first author on the paper.

A genome is annotated by comparing its sequence to gene sequences from other related organisms. The most accurate method of genome annotation is manual curation, where a person does the analysis. In contrast, utilizing a computer program to identify genes and their functions is faster but is sometimes less accurate.

“Manual annotation is very time-intensive and thus expensive,” said Surya Saha, Senior Bioinformatics Analyst in the Mueller Lab and the project coordinator. “The trick is to do both: first use automatic annotation, and then focus on genes and biochemical pathways of interest and annotate them manually.”

The paper outlines a set of logical steps to begin an undergraduate annotation program from the ground up. When students first join the project, they are trained by team leaders and expert annotators on the tools of the trade.

Throughout the project, students keep careful notes of their research and results, ultimately compiling them into a report about the biochemical pathway of interest and the member gene families, which may be published. Indeed, this method has been used to generate a peer-reviewed publication with more than 20 undergraduate authors.

“Working is one thing, and receiving an acknowledgement for that work is also really important,” says Hosmani. “That acts as a real motivation for the students.”

Other student benefits include working with international collaborators, networking, practicing communication and peer review skills, and gaining valuable insights into career options. Undergraduates may also receive research or capstone project credits for their work, which increases their commitment to the project. More and more science-based graduate programs also require knowledge of bioinformatics, so these skills will prove valuable in many fields.

In the end, the researchers gain high-quality genome annotations for any species — not just plants — which offer a better understanding of how the organism functions, ultimately benefitting society in many fields, such as agriculture, biofuels and medicine.

The authors hope that other institutions will adapt and build upon this framework, no matter their size, access to resources or annotation goals. To make the framework easy to use, the authors designed their figures and tables to be stand-alone and printer-friendly for easy reference.

“Anybody who has a research problem, a sequenced genome, and interested students can implement a system by building off our workflow,” said Saha.

Part of the answer, it turns out, may lie in the skin.

New evidence points to a key role for a molecular switch called VGLL3. Three years ago, a team of University of Michigan researchers showed that women have more VGLL3 in their skin cells than men.

Now, working in mice, they’ve discovered that having too much VGLL3 in skin cells pushes the immune system into overdrive, leading to a “self-attacking” autoimmune response. Surprisingly, this response extends beyond the skin, attacking internal organs too.

Writing in JCI Insight, the team describes how VGLL3 appears to set off a series of events in skin that trigger the immune system to come running — even when there’s nothing to defend against.

“VGLL3 appears to regulate immune response genes that have been implicated as important to autoimmune diseases that are more common in women, but that don’t appear to be regulated by sex hormones,” says Johann Gudjonsson, M.D., Ph.D., who led the research team and is a professor of dermatology at the U-M Medical School. “Now, we have shown that over-expression of VGLL3 in the skin of transgenic mice is by itself sufficient to drive a phenotype that has striking similarities to systemic lupus erythematosus, including skin rash, and kidney injury.”

Effects of excess VGLL3

Gudjonsson worked with co-first authors Allison Billi, M.D., Ph.D., and Mehrnaz Gharaee-Kermani, Ph.D., and colleagues from several U-M departments, to trace VGLL3’s effects.

They found that extra VGLL3 in skin cells changed expression levels of a number of genes important to the immune system. Expression of many of the same genes is altered in autoimmune diseases like lupus.

The gene expression changes caused by excess VGLL3 wreaked havoc in the mice. Their skin becomes scaly and raw. Immune cells abound, filling the skin and lymph nodes. The mice also produce antibodies against their own tissues, including the same antibodies that can destroy the kidneys of lupus patients.

The researchers don’t yet know what causes female skin cells to have more VGLL3 to begin with. It may be that over evolutionary time females have developed stronger immune systems to fight off infections — but at the cost of increased risk for autoimmune disease if the body mistakes itself for an invader.

The researchers also don’t know what triggers might set off extra VGLL3 activity. But they do know that in men with lupus, the same VGLL3 pathway seen in women with lupus is activated.

Many of the current therapies for lupus, like steroids, come with unwanted side effects, from increased infection risk to cancer. Finding the key factors downstream of VGLL3 may identify targets for new, and potentially safer, therapies that could benefit patients of both sexes.

Lupus, which affects 1.5 million Americans, can cause debilitating symptoms, and current broad-based treatment with steroids can make patients far more vulnerable to infections and cancer.

Patients’ role in future research

Their colleague and senior coauthor Michelle Kahlenberg, M.D., of the U-M Division of Rheumatology, is now recruiting patients with lupus for a study sponsored by U-M’s A. Alfred Taubman Medical Research Institute that could provide answers to these questions and more.

Billi, a resident in dermatology, notes that when she speaks with patients who come to Michigan Medicine’s dermatology clinics for treatment of the skin problems lupus can cause, she has to acknowledge the limits of current treatment. Even so, she says, patients are eager to take part in studies by contributing skin and DNA samples that could lead to new discoveries about their condition.

“Many patients are frustrated that they’ve had to try multiple therapies, and still nothing is working well,” she says. “To be able to tell them that we’re working on a mouse that has the same disease as them, and that we need their help, brings out their motivation and interest in research. They know that it’s a long game, and they’re in for it.”

Now, Salk Institute researchers have found that GUN1 — a gene that integrates numerous chloroplast-to-nucleus retrograde signaling pathways — also plays an important role in how proteins are made in damaged chloroplasts, which provides a new insight into how plants respond to stress. The paper was published in the Proceedings of the National Academy of Sciences (PNAS) on April 15, 2019, and may help biologists breed plants that can better withstand environmental stressors.

“Climate change holds the potential to affect our food system dramatically. When plants are stressed, like in a drought, they produce lower crop yields. If we understand how plants respond to stress, then perhaps we can develop a way to increase their resistance and keep food production high,” says Salk Professor Joanne Chory, director of the Plant Molecular and Cellular Biology Laboratory and senior author of the paper.

In plant cells, structures called chloroplasts convert energy from sunlight into chemical energy (photosynthesis). Normally, the nucleus of the cell transmits information to the chloroplasts to maintain steady energy production. However, in a stressful environment, chloroplasts send an alarm back to the cell nucleus using retrograde signaling (creating a chloroplast-to-nucleus communication feedback loop). This SOS prompts a response that helps regulate gene expression in the chloroplasts and the nucleus to optimize energy production from sunlight.

Previously, the Chory lab identified a group of genes, including GUN1, that influence other genes’ expression in the cell when the plant experiences stress. GUN1 accumulates under stressful conditions but the exact molecular function of GUN1 has been difficult to decipher, until now.

“Plants often experience environmental stressors, so there must be a chloroplast-to-nucleus communication pathway that helps the plant know when to conserve energy when injury occurs,” says Xiaobo Zhao, first author and postdoctoral fellow in Chory’s lab. “GUN1 turns out to play a big role in this.”

To understand how GUN1 regulates chloroplast-to-nucleus communication, the scientists observed plants with functional and nonfunctional GUN1 under pharmacological treatments that could damage chloroplasts. In plants without GUN1, gene expression changed, as did RNA editing in chloroplasts. (RNA editing is a modification of the RNA that changes the identity of nucleotides, so that the information in the mature RNA differs from that defined in the genome, altering the instructions for making proteins.) Some areas of RNA had more editing and other locations had less editing — suggesting that GUN1 plays a role in regulating chloroplast RNA editing.

After further analysis, the team unexpectedly found that GUN1 partners with another protein, MORF2 (an essential component of the plant RNA editing complex), to affect the efficiency of RNA editing during chloroplast-to-nucleus communication in damaged chloroplasts. Greater activity of MORF2 led to widespread editing changes as well as defects in chloroplast and leaf development even under normal growth conditions (see image). During periods of stress and injury, MORF2 overproduction also led to disruption of chloroplast-to-nucleus communication.

“Taken together, these findings suggest a possible link between chloroplast-to-nucleus communication and chloroplast RNA editing, which are important regulatory functions for flowering plants, especially during stress,” says Chory, Howard Hughes Medical Institute investigator and holder of the Howard H. and Maryam R. Newman Chair in Plant Biology.

Next, the researchers plan to examine the mechanism of how the RNA editing changes in chloroplasts activate signals that can be relayed to the nucleus, and how these modifications alter the ability of the plant to respond to stress.

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CRISPR edits a person’s genome by cutting the DNA at a specific location. The challenge is to ensure the tool doesn’t also make cuts elsewhere along the DNA — damage referred to as “off-target effects,” which could have unforeseen consequences.

In a study to be published tomorrow in the journal Science, the two first authors, Beeke Wienert and Stacia Wyman, found a new way to approach the problem.

“When CRISPR makes a cut, the DNA is broken,” says Wienert, PhD, who began the work in Jacob E. Corn’s IGI laboratory and who is now a postdoctoral scholar in Bruce R. Conklin’s laboratory at Gladstone. “So, in order to survive, the cell recruits many different DNA repair factors to that particular site in the genome to fix the break and join the cut ends back together. We thought that if we could find the locations of these DNA repair factors, we could identify the sites that have been cut by CRISPR.”

To test their idea, the researchers studied a panel of different DNA repair factors. They found that one of them, called MRE11, is one of the first responders to the site of the cut. Using MRE11, the scientists developed a new technique, named DISCOVER-Seq, that can identify the exact sites in the genome where a cut has been made by CRISPR.

“The human genome is extremely large — if you printed the entire DNA sequence, you would end up with a novel as tall as a 16-story building,” explains Conklin, MD, senior investigator at Gladstone and deputy director at IGI. “When we want to cut DNA with CRISPR, it’s like we’re trying to remove one specific word on a particular page in that novel.”

“You can think of the DNA repair factors as different types of bookmarks added to the book,” Conklin adds. “While some may bookmark an entire chapter, MRE11 is a bookmark that drills down to the exact letter than has been changed.”

Different methods currently exist to detect CRISPR off-target effects. However, they come with limitations that range from producing false positive results to killing the cells they’re examining. In addition, the most common method used to date is currently limited to being used in cultured cells in the laboratory, excluding its use in patient-derived stem cells or animal tissue.

“Because our method relies on the cell’s natural repair process to identify cuts, it has proven to be much less invasive and much more reliable,” says Corn, PhD, who now runs a laboratory at ETH Zurich. “We were able to test our new DISCOVER-Seq method in induced pluripotent stem cells, patient cells, and mice, and our findings indicate that this method could potentially be used in any system, rather than just in the lab.”

The DISCOVER-Seq method, by being applied to new cell types and systems, has also revealed new insights into the mechanisms used by CRISPR to edit the genome, which will lead to a better understanding of the biology of how this tool works.

“The new method greatly simplifies the process of identifying off-target effects while also increasing the accuracy of the results,” says Conklin, who is also a professor of medical genetics and molecular pharmacology at UC San Francisco (UCSF). “This could allow us to better predict how genome editing would work in a clinical setting. As a result, it represents an essential step in improving pre-clinical studies and bringing CRISPR-based therapies closer to the patients in need.”

Researchers from the University of Virginia School of Engineering are employing neutron-imaging techniques at Oak Ridge National Laboratory to probe lithium-ion batteries and obtain insights into the electrochemical characteristics of the batteries’ materials and structures. Their research, published in the Journal of Power Sources, focused on tracking the lithiation and delithiation — or charge and discharge — processes in lithium-ion battery electrodes using thin and thick sintered samples of two electroactive materials, lithium titanate and lithium cobalt oxide.

Understanding how the lithium moves in battery electrodes is important in designing batteries that can charge and discharge at faster rates. In some batteries this is the slowest process, which means that improving the lithium movement through the electrodes could result in batteries that can be recharged much quicker.

“When electrodes are relatively thick, transport of lithium ions through the porous material and separator architecture can limit charge and discharge rates,” said Gary Koenig, an associate professor of chemical engineering at UVA Engineering. “To develop methods of improving lithium ion transport through an electrode’s porous void regions filled with electrolyte, we need to first be able to track the transport and distribution of the ions within a cell during the charge and discharge processes.”

According to Koenig, other techniques such as high-resolution X-ray diffraction can provide detailed structural data during electrochemical processes, but this method typically averages relatively large volumes of the material. Similarly, X-ray phase imaging can visualize salt concentrations in battery electrolytes, but the technique requires a special spectrochemical cell and can only access composition information between the electrode regions.

To obtain detailed information across a wider area, the researchers conducted their studies using neutrons at the cold neutron imaging beamline at Oak Ridge’s High Flux Isotope Reactor.

“Lithium has a large absorption coefficient for neutrons, which means that neutrons passing through a material are highly sensitive to its lithium concentrations,” said Ziyang Nie, lead author and graduate student in Koenig’s group. “We demonstrated we could use neutron radiographs to track in situ lithiation in thin and thick metal oxide cathodes inside battery cells. Because neutrons are highly penetrating, we did not have to build custom cells for the analysis and were able to track the lithium across the entire active region containing both electrodes and electrolyte.”

Comparing the lithiation process in thin and thick electrodes is essential to aid understanding of the effects of heterogeneity — local variations in mechanical, structural, transport and kinetic properties — on battery life and performance. Local heterogeneity can also result in non-uniform battery current, temperatures, state of charge and aging. Typically, as the thickness of an electrode increases, so do the detrimental effects of heterogeneity on battery performance. Yet, if thicker anodes and cathodes could be used in batteries without impacting other factors, it would help increase energy storage capacities.

For the initial experiments, the thin electrode samples had thicknesses of 0.738 mm for lithium titanate and 0.463 mm for lithium cobalt oxide, while the thick lithium titanate and lithium cobalt oxide samples were 0.886 mm and 0.640 mm, respectively.

“Our immediate goal is to develop a model to help us understand how modifying the structure of an electrode, such as changing how the material is oriented or distributed, could improve ion transport properties,” Koenig said. “By imaging through each sample at different points in time, we were able to create 2-D maps of lithium distribution. In the future, we plan to rotate our samples within the neutron beam to provide 3-D information that will reveal in more detail how heterogeneity impacts ion transport.”

“We found that they do interact socially and are making more sounds than previously thought,” she says. “Their vocal repertoire is very diverse.”

The findings of May Collado are her colleagues were published in the journal PeerJ on April 18.

The Araguaian dolphins, also called botos, are a difficult animal to study. They are hard to find in the first place, and while the waters of the Araguaia and Tocatins rivers are clear, it is challenging to identify individuals because the dolphins are skittish and hard to approach.

Luckily, Gabriel Melo-Santos, a biologist from the University of St Andrews in Scotland and leader of the project, found a fish market in the Brazilian town of Mocajuba where the botos regularly visit to be fed by people shopping there. The clear water and regular dolphin visits provided a unique opportunity to get a close look at how the animals behave and interact, and to identify and keep track of various individuals.

The team used underwater cameras and microphones to record sounds and interactions between the dolphins at the market, and took some genetic samples. They identified 237 different types of sounds the dolphins make, but even with 20 hours of recordings the researchers don’t believe they captured the animals’ entire acoustic repertoire. The most common sounds were short, two-part calls that baby dolphins made when they were approaching their mothers.

“It’s exciting; marine dolphins like the bottlenose use signature whistles for contact, and here we have a different sound used by river dolphins for the same purpose,” says May Collado. The river dolphins also made longer calls and whistles, but these were much rarer, and the reasons for them are not yet clear. But there is some indication that whistles serve the opposite purpose than in bottlenose dolphins, with the botos using them to maintain distance rather than for group cohesion.

The acoustic characteristics of the calls are also interesting; they fall somewhere between the low-frequency calls used by baleen whales to communicate over long distances, and the high-frequency ones used by marine dolphins for short distances. May Collado speculates that the river environment may have shaped those characteristics.

“There are a lot of obstacles like flooded forests and vegetation in their habitat, so this signal could have evolved to avoid echoes from vegetation and improve the communication range of mothers and their calves,” she says.

May Collado and her colleagues next want to study whether the same diversity of communication is seen in other populations of Araguaian river dolphins that are less accustomed to humans, and compare them to their relatives elsewhere in South America. The Araguaian dolphins are closely related to two other species, the Bolivian river dolphin and Amazon river dolphin; the Araguaian dolphins were only described as a separate species in 2014, and that classification is still under debate. But there seems to be a large amount of variation in the repertoire of sounds each species makes.

The Amazon dolphins in Ecuador, studied by May Collado in 2005, are generally very quiet. “We need more information on these other species and more populations,” she says. “Why is one population chattier than others and how do these differences shape their social structure?”

May Collado says the work could help researchers gain clearer understanding of how communication evolved in marine mammals. Similar calls have been reported in pilot whales and killer whales, for example, and the similarities and differences between different species could help tease out which signals evolved first, and why.

The river dolphins are evolutionary relics, represented by just a few species around the world, and they diverged from other cetaceans much earlier than other dolphins. So these calls may have arisen first in river dolphins, then later evolved in marine dolphins into whistles and calls but in a different social context. Or was there a change in the function of the calls, with this kind of sound being used for group identity in killer whales, and individual identity in river dolphins? The calls may also have other functions in addition to identity, perhaps indicating group identity, or providing information on emotional state.

“We can’t say what the evolutionary story is yet until we get to know what sounds are produced by other river dolphins in the Amazon area, and how that relates to what we found,” she says. “We now have all these new questions to explore.”

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