This work was accomplished through a close partnership between the groups of Professor Akif Tezcan and Assistant Professor Mark Herzik, both in UC San Diego’s Department of Chemistry and Biochemistry. While Tezcan has long studied nitrogenase, Herzik provided the cryoEM expertise needed to carry out the research.

“This is a very important advance in terms of biological nitrogen fixation as well as structural biology, in general,” stated Tezcan. “To be able to obtain atomic-level-resolution pictures of an enzyme as dynamic and complex as nitrogenase in action is extremely exciting. It opens the doors to fully understanding the mechanism of this enigmatic enzyme, which has preoccupied researchers for decades.”

Understanding the significance of these cryoEM images requires understanding the tremendous global importance of nitrogen fixation. All organisms require “fixed” sources of nitrogen for the biosynthesis of life’s building blocks such as proteins and DNA. However, most living organisms do not possess the nitrogenase enzyme and cannot metabolize atmospheric nitrogen into a bioprocessable form.

Nitrogenase was the essentially the only source of fixed nitrogen in the biosphere until the advent of the Haber-Bosch process — the industrial procedure for converting atmospheric nitrogen to ammonia — more than a hundred years ago. Industrially produced ammonia is largely used for fertilizers, and its advent revolutionized agricultural practices in the first half of the 20^th century. The Haber-Bosch process has often been cited as the driving factor behind the world’s population explosion over the past century, having “turned air into bread.”

However, the Haber-Bosch process is very energy-intensive, requiring temperatures exceeding 400°C and high pressures of hydrogen gas. An estimated 1-2% of all global energy production is consumed by the Haber-Bosch process. The process also raises environmental concerns including leaching of nitrates into groundwater and higher emissions of the greenhouse gas nitrous oxide.

A key question that drives biological nitrogen-fixation research is the contrast between nitrogenase and the Haber-Bosch process. How does the enzyme catalyze nitrogen reduction at ambient temperatures and pressure whereas the industrial process requires such extreme conditions?

“If we can understand the mechanism of nitrogenase, we may not only figure out why nature evolved it to be such a complex enzyme, but we might also uncover design principles for ammonia production in a more cost-effective and environmentally friendly fashion,” stated Tezcan.

Although much is known about the structure of nitrogenase, until now, no one has been able to acquire atomic-resolution images of the enzyme while “turning over” or in the process of catalyzing atmospheric nitrogen into ammonia, largely due to technological limitations.

Although scientists can obtain atomic-resolution images of proteins using X-ray crystallography, this method requires the proteins to be fixed in place within a crystal — stationary in a sense — which means that it cannot capture nitrogenase in action. Nitrogenase catalysis requires different parts of the enzyme to associate with each other and then come apart several times to make a single ammonia molecule from nitrogen. The process is anything but stationary.

CryoEM not only allows researchers to capture the structures of proteins without requiring them to be fixed in crystals but, thanks to recent advances in hardware and data processing, do so with atomic resolution. Such high resolution is needed to visualize the small changes associated with enzymatic catalysis.

These advances led Tezcan and graduate student Hannah Rutledge to consider using cryoEM to study nitrogenase in catalytic action. And for this, they sought the help of resident cryoEM expert, Mark Herzik, and his group members Brian Cook and Hoang Nguyen.

“This was both an exciting and technologically challenging project to pursue, during the pandemic no less. Although cryoEM is a very capable technique, few studies have reported on enzymes as they undergo catalysis. The critical insights and technological developments in this study not only pave the way for future explorations of the nitrogenase mechanism but enzymes in general,” stated Herzik.

Herzik and Rutledge worked together closely to prepare hundreds of cryoEM samples. Because nitrogenase is oxygen-sensitive, the samples were prepared in an anaerobic glove box, then quickly transferred and frozen within seconds to prevent any degradation. In the end the team collected over 15,000 videos capturing over 20 million individual molecules at varying stages of catalysis.

It took the teams almost a year to sort through several terabytes of data: they discarded low-quality images, then identified and classified all the particles. Eventually they were able to get the first atom-resolution images of nitrogenase in the middle of turnover.

The cryoEM structures revealed several unexpected features of nitrogenase which were previously not observed in X-ray structures. Importantly, the new observations provide new mechanistic hypothesis for nitrogenase catalysis. Tezcan and Herzik hope to collaborate for many years to come to test these hypotheses and understand the catalytic mechanism of nitrogenase in detail.

“This is only the beginning,” stated Tezcan. “We have a picture of the whole enzyme now, not just one specific part, during catalytic action. This will really open the floodgates to further research in understanding how nitrogenase works and, potentially, down the road, developing more efficient processes for producing fixed nitrogen.”

Funding provided by National Institutes of Health grants R01-GM099813, R35-GM138206 and T32-GM008326; NASA grant 80NSSC18M0093; and the Searle Scholars Program. CryoEM experiments were conducted at UC San Diego’s CryoEM facilities as well as the Stanford-SLAC Cryo-EM Center.

Malaria is still the deadliest parasitic disease worldwide, with approximately 241 million cases and over half a million deaths annually. It is caused by a single-celled parasite called Plasmodium, which is transmitted between people by the female Anopheles mosquito when they bite to take blood.

In this new study, published in PLOS Biology, scientists have uncovered the crucial roles of a group of motor proteins named kinesins during the parasite life cycle.

The research, led by Rita Tewari, Professor of Parasite Cell Biology in the University’s School of Life Sciences, has shown the significance of kinesins in basic cellular processes needed for malaria parasite development, multiplication and invasion, most importantly within the mosquitoes that transmit the parasite.

Kinesins are molecular motor proteins that use energy from the hydrolysis of adenosine triphosphate (ATP — a universal store of energy in all cells), and function in various cellular processes. They are involved in transport, cell division, cell polarity and cell motility.

This latest study showed that of the nine kinesins present in the parasite genome, eight are required for the various functions of cell proliferation to cell movement in the mosquito host which was very surprising.

Researchers at the University of Nottingham have studied the location and function of all kinesins in live parasite cells at various stages of development, both in the mosquitoes which transmit the disease, and in the host where it causes disease. These proteins are important potential drug targets, hence the importance of this study in the search for new intervention targets.

Professor Tewari said: “This is an important genome-wide study and an essential resource for studying the various morphologically distinct parasite cells involved in parasite transmission. It shows how these important motors proteins are involved in forming molecular tracks for movement, multiplication, and transmission.”

Dr Zeeshan, who is the first author of the paper, said: “This is a comprehensive study on parasite molecular motors. It was very challenging to capture the dynamics of these proteins in live parasite cells within mosquitoes. Most importantly, we could study the formation of the male gamete (sperm), which involves a rapid multiplication process that completes within 10-12 min after the female mosquito has ingested blood from an infected host. Multiple kinesins are involved in efficient production of male gametes and deletion of kinesin genes halts parasite transmission, a discovery that can be explored further for drug discovery.

“In addition, we found one motor protein, kinesin-13, which is essential for parasite multiplication in all stages of the life cycle.”

The study was carried out in collaboration with several scientists; Tony Holder at the Francis Crick Institute, London; Prof Carolyn Moores at Birbeck College; Profs Sue Vaughan and David Ferguson at Oxford Brookes; Prof Mathieu Brochet and Ravish Raspa at the University of Geneva; and Prof Karine Le Roch and Steven Abel at the University of California. This study demonstrates the power of multidisciplinary science and how networking and collaboration lead greater global understanding in science. The work was funded by BBSRC, MRC, CRUK, Wellcome Trust, NIH and NIAID.

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On Little Saltee, a small island off the coast of Ireland, the researchers attached tiny trackers to the feathers of Manx shearwaters. The aim of the study, published in Proceedings of the Royal Society B, was to understand how underwater visibility affects seabirds’ ability to forage for fish and other prey. It is the first study to examine the impact of ocean clarity on seabirds’ diving abilities.

Climate change is leading to the oceans becoming cloudier

Jamie Darby, a marine ecologist in the School of Biological Environmental and Earth Sciences and the MaREI centre at UCC, and lead author of the study, stated “The chemical and physical properties of the planet’s oceans are changing at an unnatural rate, bringing about challenges for marine life. One consequence of climate change is that large areas of our oceans are becoming cloudier.”

Darby and the research team investigated the diving patterns of the black and white Manx shearwaters in relation to local environmental conditions like cloud cover and water clarity. Over 5000 different dives were recorded and using publicly available databases, and a range of relevant information about weather patterns and ocean conditions were amassed.

Struggling to find food

The study found that the birds dove deeper when sunlight could penetrate further underwater, suggesting that visibility is key to their ability to dive for food. As the planet warms and the ocean becomes cloudier this finding is important because it means that seabirds will have to overcome this challenge.

“Our findings support the idea that the birds needed sufficient sunlight to be able to forage at depth. While this study examined one particular seabird. The results can be extended to other animals. Many visually-dependent predators could find themselves struggling to find food as human activities continue to make the oceans murkier” stated Jamie Darby.

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From 2012 to 2019, the researchers monitored elephants’ movements through six wildlife corridors using of motion-detected camera traps in two different human-dominated landscapes: the townships of Kasane and Kazungula, and the farming villages of the Chobe Enclave, both located in the Chobe District.

The study shows that various land-use seemingly affects when elephants use wildlife corridors on an hourly basis. Elephants in agricultural areas largely moved through the corridors predominantly nocturnally, when humans are less active, compared to the urban corridors, where humans and elephants actively mostly overlap.

‘This is the first study of this type that takes an in-depth look at comparing how varying land-use affects elephant movements and their use of wildlife corridors,’ states lead author Tempe Adams of EWB. Increasing human development and changes in land-use restrict wildlife’s access to resources. It also increases competition and conflict between animals and people. This is one of the largest conservation challenges facing the future of elephants in both Africa and Asia. The designation and maintenance of wildlife corridors is a fundamental wildlife management tool to allow both development and conservation to continue.

‘This was a great opportunity to link our work on examining human pressures on biodiversity at Radboud University with the conservation work being done by Elephants Without Borders,’ says Marlee Stevens, assistant professor at the ?Department of Environmental Science of Radboud University and one of the authors of the paper. ‘Our results show that elephants alter their behaviour in human-modified landscapes, but their response varies depending on the human disturbance.’

The study highlights the need to consider wildlife daily activity patterns in and around cities, towns, and farming areas, for conservation and environmental management planning. ‘What is truly remarkable is that we found elephants do not perceive all human development the same way, but they are adjusting their behaviours to adapt to the variations and human pressure,’ says Adams. ‘It also further highlights the need for transparent documentation of human pressure within and around protected wildlife areas, which is critical to assist in the conservation of species’.

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In their analysis researchers from the University of East Anglia (UEA), the French Centre National de la Recherche Scientifique (CNRS) and the OACIS initiative of the Prince Albert II of Monaco Foundation, challenge the widely held view that restoring areas such as mangroves, saltmarsh and seagrass can remove large amounts of carbon dioxide (CO₂) from the atmosphere.

The findings of their review, published today in the journal Frontiers in Climate, identify seven reasons why carbon accounting for coastal ecosystems is not only extremely challenging but risky.

These include the high variability in carbon burial rates, vulnerability to future climate change, and fluxes of methane and nitrous oxide. The authors, who also looked at information on restoration costs, warn that extra measurements can reduce these risks, but would mean much higher costs.

However, they stress that blue carbon habitats should still be protected and, where possible, restored, as they have benefits for climate adaptation, coastal protection, food provision and biodiversity conservation.

Lead author Dr Phil Williamson, honorary reader in UEA’s School of Environmental Sciences, said: “We have looked into the processes involved in carbon removal and there are just too many uncertainties. The expected climate benefits from blue carbon ecosystem restoration may be achieved, yet it seems more likely they will fall seriously short.

“If you want to have extra carbon removal, you need extra habitat, and the scope for restoration is limited. Many of these sites have been built on, for coastal settlement, tourism and port development.

“Nevertheless, we believe that every effort should be made to halt, and wherever possible reverse, the worldwide loss of coastal vegetation. That’s because blue carbon habitats are more than carbon stores — they also provide storm protection, support biodiversity and fisheries, and improve water quality.”

The sediments beneath mangrove forests, tidal saltmarshes and seagrass meadows are rich in organic carbon, accumulated and stored over many hundreds of years.

Many recent studies and reviews have favourably identified the potential for these coastal blue carbon ecosystems to provide a natural climate solution in two ways: by conservation, reducing the greenhouse gas emissions arising from the loss and degradation of such habitats, and by restoration, to increase carbon dioxide drawdown and its long-term storage.

This new review focuses on the latter, assessing the feasibility of achieving quantified and secure carbon removal (negative emissions) through the restoration of coastal vegetation.

Increasingly businesses and states have pledged to offset their emissions by restoring these ecosystems through carbon credits, assuming reliable knowledge on how much CO₂ they will remove in future from the atmosphere.

However, Dr Williamson and co-author Prof Jean-Pierre Gattuso, of CNRS and the OACIS initiative of the Prince Albert II of Monaco Foundation, say the policy problem is more subtle. That is, CO₂ removal using coastal blue carbon restoration has questionable cost-effectiveness when considered only as a climate mitigation action, either for carbon-offsetting or for inclusion in countries’ Nationally Determined Contributions, which set out their efforts to reduce emissions and adapt to the impacts of climate change under the Paris Agreement.

“If we use these ecosystems for carbon offsets in a major way, expecting that they would remove up to, say, 100 gigatonnes of carbon dioxide over the period 2025-2100, but find they only remove 10 or maybe just one gigatonne of CO₂, then climate tipping points could be crossed, with really serious consequences,” said Dr Williamson.

“If, however, such ecosystems are restored to protect biodiversity, and we find that they also remove several gigatonnes of CO₂, then that would be a bonus, assuming other means are used for climate mitigation.

“Restoration should therefore be in addition to, not as a substitute for, near-total emission reductions. Where coastal blue ecosystems restoration projects are carried out primarily for carbon removal, they need to include comprehensive long-term monitoring to verify that the intended climate benefits are being achieved.”

Prof Gattuso said: “Many important issues relating to the measurement of carbon fluxes and storage have yet to be resolved, affecting certification and resulting in potential over-crediting.

“The restoration of coastal blue carbon ecosystems is nevertheless highly advantageous for climate adaptation, coastal protection, food provision and biodiversity conservation. Such action can therefore be societally justified in very many circumstances, based on the multiple benefits that such habitats provide at the local level.”

Our cells are subject to mechanical forces exerted on a microscopic scale, triggering biological signals essential to many cell processes involved in the normal functioning of our body or in the development of diseases.

For example, the feeling of touch is partly conditional on the application of mechanical forces on specific cell receptors (the discovery of which was this year rewarded by the Nobel Prize in Physiology or Medicine). In addition to touch, these receptors that are sensitive to mechanical forces (known as mechanoreceptors) enable the regulation of other key biological processes such as blood vessel constriction, pain perception, breathing or even the detection of sound waves in the ear, etc.

The dysfunction of this cellular mechanosensitivity is involved in many diseases — for example, cancer: cancer cells migrate within the body by sounding and constantly adapting to the mechanical properties of their microenvironment. Such adaptation is only possible because specific forces are detected by mechanoreceptors that transmit the information to the cell cytoskeleton.

At present, our knowledge of these molecular mechanisms involved in cell mechanosensitivity is still very limited. Several technologies are already available to apply controlled forces and study these mechanisms, but they have a number of limitations. In particular, they are very costly and do not allow us to study several cell receptors at a time, which makes their use very time-consuming if we want to collect a lot of data.

DNA origami structures

In order to propose an alternative, the research team led by Inserm researcher Gaëtan Bellot at the Structural Biology Center (Inserm/CNRS/Université de Montpellier) decided to use the DNA origami method. This enables the self-assembly of 3D nanostructures in a pre-defined form using the DNA molecule as construction material. Over the last ten years, the technique has allowed major advances in the field of nanotechnology.

This enabled the researchers to design a “nano-robot” composed of three DNA origami structures. Of nanometric size, it is therefore compatible with the size of a human cell. It makes it possible for the first time to apply and control a force with a resolution of 1 piconewton, namely one trillionth of a Newton — with 1 Newton corresponding to the force of a finger clicking on a pen. This is the first time that a human-made, self-assembled DNA-based object can apply force with this accuracy.

The team began by coupling the robot with a molecule that recognizes a mechanoreceptor. This made it possible to direct the robot to some of our cells and specifically apply forces to targeted mechanoreceptors localized on the surface of the cells in order to activate them.

Such a tool is very valuable for basic research, as it could be used to better understand the molecular mechanisms involved in cell mechanosensitivity and discover new cell receptors sensitive to mechanical forces. Thanks to the robot, the scientists will also be able to study more precisely at what moment, when applying force, key signaling pathways for many biological and pathological processes are activated at cell level.

“The design of a robot enabling the in vitro and in vivo application of piconewton forces meets a growing demand in the scientific community and represents a major technological advance. However, the biocompatibility of the robot can be considered both an advantage for in vivo applications but may also represent a weakness with sensitivity to enzymes that can degrade DNA. So our next step will be to study how we can modify the surface of the robot so that it is less sensitive to the action of enzymes. We will also try to find other modes of activation of our robot using, for example, a magnetic field,” emphasizes Bellot.

[1] Also contributed to this research: the Institute of Functional Genomics (CNRS/Inserm/Université de Montpellier), the Max Mousseron Biomolecules Institute (CNRS/Université de Montpellier/ENSCM), the Paul Pascal Research Center (CNRS/Université de Bordeaux) and the Physiology and Experimental Medicine: Heart-Muscles laboratory (CNRS/Inserm/Université de Montpellier).