Key Features of Graphene Technology

Following years of research under the European Graphene Flagship project, ICN2 spearheaded in collaboration with the University of Manchester the development of EGNITE (Engineered Graphene for Neural Interfaces), a novel class of flexible, high-resolution, high-precision graphene-based implantable neurotechnology. The results published today in Nature Neurotechnology aim to contribute with innovative technologies to the blooming landscape of neuroelectronics and brain-computer interfaces.

EGNITE builds on the vast experience of its inventors in fabrication and medical translation of carbon nanomaterials. This innovative technology based on nanoporous graphene integrates fabrication processes standard in the semiconductor industry to assemble graphene microelectrodes of a mere 25 µm in diameter. The graphene microelectrodes exhibit low impedance and high charge injection, essential attributes for flexible and efficient neural interfaces.

Preclinical Validation of Functionality

Preclinical studies by various neuroscience and biomedical experts that partnered with ICN2, using different models for both the central and peripheral nervous system, demonstrated the capacity of EGNITE in recording high-fidelity neural signals with exceptional clarity and precision and, more importantly, afford highly targeted nerve modulation. The unique combination of high-fidelity signal recording and precise nerve stimulation offered by EGNITE technology represents a potentially critical advancement in neuroelectronic therapeutics.

This innovative approach addresses a critical gap in neurotechnology, which has seen little advancement in materials over the last two decades. The development of EGNITE electrodes has the capacity to place graphene at the forefront of neurotechnological materials.

International Collaboration and Scientific Leadership

The technology presented today builds on the legacy of the Graphene Flagship, a European initiative that during the last decade strived to advance European strategic leadership in technologies that rely on graphene and other 2D materials. Behind this scientific breakthrough is a collaborative effort led by ICN2 researchers Damià Viana (now at INBRAIN Neuroelectronics), Steven T. Walston (now at University of Southern California), and Eduard Masvidal-Codina, under the guidance of ICREA Jose A. Garrido, leader of the ICN2Advanced Electronic Materials and Devices Group, and ICREA Kostas Kostarelos, leader of the ICN2Nanomedicine Lab and the Faculty of Biology, Medicine & Health at the University of Manchester (UK). The research has had the participation of Xavier Navarro, Natàlia de la Oliva, Bruno Rodríguez-Meana and Jaume del Valle, from the Institute of Neurosciences and the Department of Cellular Biology, Physiology and Immunology of the Universitat Autònoma de Barcelona (UAB).

The collaboration includes the contribution from leading national and international institutions, such as the Institut de Microelectrònica de Barcelona — IMB-CNM (CSIC), the National Graphene Institute in Manchester (UK), and the Grenoble Institut des Neurosciences — Université Grenoble Alpes (France) and the University of Barcelona. The technology integration into the standard semiconductor fabrication processes has been performed at the Micro and Nanofabrication cleanroom of the IMB-CNM (CSIC), under the supervision of CIBER researcher Dr Xavi Illa.

Clinical Translation: Next Steps

The EGNITE technology described in the Nature Nanotechnology article has been patented and licensed to INBRAIN Neuroelectronics, a spin-off based in Barcelona from ICN2 and ICREA, with support from IMB-CNM (CSIC). The company, also a partner in the Graphene Flagship project, is leading the translation of the technology into clinical applications and products. Under the direction of CEO Carolina Aguilar, INBRAIN Neuroelectronics is gearing up for the first-in-human clinical trials of this innovative graphene technology.

The industrial and innovation landscape on semiconductor engineering in Catalonia, where ambitious national strategies plan to build state-of-the-art facilities to produce semiconductor technologies based on emerging materials, offer an unprecedented opportunity to accelerate the translation of such results presented today into clinical applications.

Closing Remarks

The Nature Nanotechnology article describes an innovative graphene-based neurotechnology that can be upscaled using established semiconductor fabrication processes, holding the potential for a transformative impact. ICN2 and its partners continue to advance and mature the described technology with the aim to translate it into a real efficacious and innovative therapeutic neurotechnology.

The genome or DNA of an organism incorporates the blueprint for proteins and orchestrates the production of new cells. Aiming to combat pathogens, cure genetic diseases or achieve other positive effects, molecular biological CRISPR technologies are being used to specifically alter or silence genes and inhibit protein production.

One of these molecular biological tools is CRISPRi (from “CRISPR interference”). CRISPRi blocks genes and gene expression without modifying the DNA sequence. As with the CRISPR-Cas system also known as “gene scissors,” this tool involves a ribonucleic acid (RNA), which serves as a guide RNA to direct a nuclease (Cas). In contrast to gene scissors, however, the CRISPRi nuclease only binds to the DNA without cutting it. This binding results in the corresponding gene not being transcribed and thus remaining silent.

Until now, it has been challenging to predict the performance of this method for a specific gene. Researchers from the Würzburg Helmholtz Institute for RNA-based Infection Research (HIRI) in cooperation with the University of Würzburg and the Helmholtz Artificial Intelligence Cooperation Unit (Helmholtz AI) have now developed a machine learning approach using data integration and artificial intelligence (AI) to improve such predictions in the future.

The approach

CRISPRi screens are a highly sensitive tool that can be used to investigate the effects of reduced gene expression. In their study, published today in the journal Genome Biology, the scientists used data from multiple genome-wide CRISPRi essentiality screens to train a machine learning approach. Their goal: to better predict the efficacy of the engineered guide RNAs deployed in the CRISPRi system.

“Unfortunately, genome-wide screens only provide indirect information about guide efficiency. Hence, we have applied a new machine learning method that disentangles the efficacy of the guide RNA from the impact of the silenced gene,” explains Lars Barquist. The computational biologist initiated the study and heads a bioinformatics research group at the Würzburg Helmholtz Institute, a site of the Braunschweig Helmholtz Centre for Infection Research in cooperation with the Julius-Maximilians-Universität Würzburg.

Supported by additional AI tools (“Explainable AI”), the team established comprehensible design rules for future CRISPRi experiments. The study authors validated their approach by conducting an independent screen targeting essential bacterial genes, showing that their predictions were more accurate than previous methods.

“The results have shown that our model outperforms existing methods and provides more reliable predictions of CRISPRi performance when targeting specific genes,” says Yanying Yu, PhD student in Lars Barquist’s research group and first author of the study.

The scientists were particularly surprised to find that the guide RNA itself is not the primary factor in determining CRISPRi depletion in essentiality screens. “Certain gene-specific characteristics related to gene expression appear to have a greater impact than previously assumed,” explains Yu.

The study also reveals that integrating data from multiple data sets significantly improves the predictive accuracy and enables a more reliable assessment of the efficiency of guide RNAs. “Expanding our training data by pulling together multiple experiments is essential to create better prediction models. Prior to our study, lack of data was a major limiting factor for prediction accuracy,” summarizes junior professor Barquist. The approach now published will be very helpful in planning more effective CRISPRi experiments in the future and serve both biotechnology and basic research. “Our study provides a blueprint for developing more precise tools to manipulate bacterial gene expression and ultimately help to better understand and combat pathogens,” says Barquist.

The results at a glance

• Gene features matter: The characteristics of targeted genes have a significant impact on guide RNA depletion in genome-wide screens.

• Data integration improves predictions: Combining data from multiple CRISPRi screens significantly improves the accuracy of prediction models and enables more reliable estimates of guide RNA efficiency.

• Designing better CRISPRi experiments: The study provides valuable insights for designing more effective CRISPRi experiments by predicting guide RNA efficiency, enabling precise gene-silencing strategies.

Funding

The study was supported by funds from the Bavarian State Ministry of Science and Art through the bayresq.net research network.

In a new account of exquisitely preserved microfossils, researchers at UC Santa Barbara and McGill University revealed that eukaryotic organisms had already evolved into a diverse array of forms even 1.64 billion years ago. The paper, published in the journal Papers in Paleontology, recounts an assemblage of eukaryotic fossils from an era early in the group’s evolutionary history. The authors describe four new taxa, as well as evidence of several advanced characteristics already present in these early eukaryotes.

“These are among the oldest eukaryotes that have ever been discovered,” explained lead author Leigh Anne Riedman, an assistant researcher in UCSB’s Department of Earth Science. “Yet, even in these first records we’re seeing a lot of diversity.”

Eukarya forms one of the major domains of life, encompassing the plant, animal and fungi clades, as well as all other groups whose cells have a membrane-bound nucleus, like protists and seaweeds. Many scientists had thought early eukaryotes were all fairly similar during the late Paleoproterozoic, and that diversification took place around 800 million years ago. But Riedman and her co-authors found fossils of a delightfully diverse, and complex, cast of characters in rock nearly twice as old.

Scientists knew from previous studies that eukaryotes had evolved by this time, but their diversity in this era was poorly understood. So Riedman headed to the Outback in late 2019. Within one week, she had collected about 430 samples from eight cores drilled by a prospecting company; they now reside in the library of the Northern Territory Geological Survey. The two cores used for this study spanned roughly 500 meters of stratigraphy, or 133 million years, with around 15 million years of significant deposition.

Riedman returned to the United States with shale and mudstone: remnants of an ancient coastal ecosystem that alternated between shallow, subtidal mudflats and coastal lagoons. A dip in hydrofluoric acid dissolved the matrix rock, concentrating the precious microfossils which she then analyzed under the microscope.

“We were hoping to find species with interesting and different characteristics to their cell walls,” Riedman said. She hoped that these features could shed light on what was happening within the cells during this time period. Reaching any conclusions about the cellular interior would require a great deal of sleuthing, though, since the fossils preserve only the exterior of the cells.

The researchers were surprised by the diversity and complexity preserved in these fossils. They recorded 26 taxa, including 10 previously undescribed species. The team found indirect evidence of cytoskeletons, as well as platy structures that suggest the presence of internal vesicles in which the plates were formed — perhaps ancestral to Golgi bodies, present in modern eukaryotic cells. Other microbes had cell walls made of bound fibers, similarly suggestive of the presence of a complex cytoskeleton.

The authors also found cells with a tiny trapdoor, evidence of a degree of sophistication. Some microbes can form a cyst to wait out unfavorable environmental conditions. In order to emerge, they need to be able to etch an opening in their protective shell. Making this door is a specialized process. “If you’re going to produce an enzyme that dissolves your cell wall, you need to be really careful about how you use that enzyme,” Riedman said. “So in one of the earliest records of eukaryotes, we’re seeing some pretty impressive levels of complexity.”

Many people in the field had thought this ability emerged later, and the evidence for it in this assemblage further emphasizes how diverse and advanced eukaryotes were even at this early juncture. “The assumption has always been that this is around the time that eukaryotes appeared. And now we think that people just haven’t explored older rocks,” said co-author Susannah Porter, an Earth science professor at UC Santa Barbara.

This paper is part of a larger project investigating early eukaryote evolution. Riedman and Porter want to know in what environments early eukaryotes were diversifying, why they were there, when they migrated to other places, and what adaptations they needed in order to fill those new niches.

A big part of this effort involves understanding when different characteristics of eukaryotes first arose. For instance, the authors are quite interested to learn whether these organisms were adapted to oxygenated or anoxic environments. The former would suggest that they had an aerobic metabolism, and possibly mitochondria. Every modern eukaryote that’s been found descends from ancestors that possessed mitochondria. This suggests that eukaryotes acquired the organelle very early on, and that it provided a significant advantage.

Riedman and Porter are currently working on a fresh account of eukaryote diversity through time. They’ve also collected even older samples from Western Australia and Minnesota. Meanwhile, their geochemist collaborators at McGill are spearheading a study on oxygen levels and preferred eukaryote habitats, aspects that could shed light on their evolution.

“These results are a directive to go look for older material, older eukaryotes, because this is clearly not the beginning of eukaryotes on Earth,” Riedman said.

The value of wide beaches and dunes

Beaches and dunes are vital to society. They protect us from flooding, are an important source for our drinking water, and are an important habitat for numerous plant and animal species. They are also indispensable when we spend a day at the beach or walk through the dunes. When beaches and dunes are given enough space, they can fulfill all these functions. But, if dune areas become too narrow, natural flood protection, drinking water extraction and biodiversity are threatened. Yet beaches and dunes are increasingly squeezed by infrastructure on the one hand and rising sea levels on the other, write researchers from the Netherlands Institute for Sea Research, University of Groningen, Utrecht University, TU Delft and the Dutch Forestry Commission in the leading journal Nature Communications.

A quarter million measurements

To map this coastal squeeze, the researchers combined measurement data from previous work of TU Delft with Open Street Map data. They then measured the straight-line distance from the coastline to the nearest paved road or building. They took this measurement every kilometer, along all the sandy beaches in the world, yielding a total of 235,469 measurements.

Less than 400 meters

These measurements showed that human infrastructure is generally located very close to the sea. On average, the first building or paved road is found at 390 meters from the sea. In the densely populated Netherlands this is even at 210 meters, and in France the space is even tighter with 30 meters from the sea. Of all continents, Europe appears to have the most trapped beaches and dunes with an average distance of 130 meters, while Oceania is the least squeezed with an average distance of 2.8 km.

Rising sea levels

In the future, coastal squeeze is likely to increase worldwide. Rising sea levels will further narrow the space between buildings and the sea. In a natural situation, beaches and dunes would migrate inland, but buildings and roads impede this process. Therefore, researchers expect that 23 to 30 percent of beaches and dunes will be washed away or drowned by 2100.

Protection

Hopeful is the important role of nature protection. The researchers found that, when dune areas have a protected status, buildings and roads are found to be four times more distant than in unprotected areas. However, currently only 16% of the world’s sandy coasts are protected. The researchers therefore emphasize that it is important to better protect beaches and dunes.

“When people restrict the amount of food that they eat, they typically think it might affect their digestive tract or fat buildup, but not necessarily about how it affects the brain,” said Kenneth Wilson, Ph.D., Buck postdoc and first author of the study, published online on January 11, 2024 in Nature Communications. “As it turns out, this is a gene that is important in the brain.”

The team additionally demonstrated a detailed cellular mechanism of how dietary restriction can delay aging and slow the progression of neurodegenerative diseases. The work, done in fruit flies and human cells, also identifies potential therapeutic targets to slow aging and age-related neurodegenerative diseases.

“We found a neuron-specific response that mediates the neuroprotection of dietary restriction,” said Buck Professor Pankaj Kapahi , Ph.D., co-senior author of the study. “Strategies such as intermittent fasting or caloric restriction, which limit nutrients, may enhance levels of this gene to mediate its protective effects.”

“The gene is an important brain resilience factor protecting against aging and neurological diseases,” said Buck Professor Lisa Ellerby, Ph.D., co-senior author of the study.

Understanding variability in response to dietary restriction

Members of the team have previously shown mechanisms that improve lifespan and healthspan with dietary restriction, but there is so much variability in response to reduced calories across individuals and different tissues that it is clear there are many yet to be discovered processes in play. This project was started to understand why different people respond to diets in different ways.

The team began by scanning about 200 strains of flies with different genetic backgrounds. The flies were raised with two different diets, either with a normal diet or with dietary restriction, which was only 10% of normal nutrition. Researchers identified five genes which had specific variants that significantly affected longevity under dietary restriction. Of those, two had counterparts in human genetics.

The team chose one gene to explore thoroughly, called “mustard” (mtd) in fruit flies and “Oxidation Resistance 1” (OXR1) in humans and mice. The gene protects cells from oxidative damage, but the mechanism for how this gene functions was unclear. The loss of OXR1 in humans results in severe neurological defects and premature death. In mice, extra OXR1 improves survival in a model of amyotrophic lateral sclerosis (ALS).

The link between brain aging, neurodegeneration and lifespan

To figure out how a gene that is active in neurons affects overall lifespan, the team did a series of in-depth tests. They found that OXR1 affects a complex called the retromer, which is a set of proteins necessary for recycling cellular proteins and lipids. “The retromer is an important mechanism in neurons because it determines the fate of all proteins that are brought into the cell,” said Wilson. Retromer dysfunction has been associated with age-related neurodegenerative diseases that are protected by dietary restriction, specifically Alzheimer’s and Parkinson’s diseases.

Overall, their results told the story of how dietary restriction slows brain aging by the action of mtd/OXR1 in maintaining the retromer. “This work shows that the retromer pathway, which is involved in reusing cellular proteins, has a key role in protecting neurons when nutrients are limited,” said Kapahi. The team found that mtd/OXR1 preserves retromer function and is necessary for neuronal function, healthy brain aging, and lifespan extension seen with dietary restriction.

“Diet is influencing this gene. By eating less, you are actually enhancing this mechanism of proteins being sorted properly in your cells, because your cells are enhancing the expression of OXR1,” said Wilson.

The team also found that boosting mtd in flies caused them to live longer, leading researchers to speculate that in humans excess expression of OXR1 might help extend lifespan. “Our next step is to identify specific compounds that increase the levels of OXR1 during aging to delay brain aging,” said Ellerby.

“Hopefully from this we can get more of an idea of why our brains degenerate in the first place,” said Wilson.

“Diet impacts all the processes in your body,” he said. “I think this work supports efforts to follow a healthy diet, because what you eat is going to affect more than you know.”

Kapahi is founder and a member of the scientific advisory board at Juvify Bio. The other authors have no conflicts of interest.

“You can put the carbon nanofibers into cement to strengthen the cement,” said Jingguang Chen, a professor of chemical engineering at Columbia with a joint appointment at Brookhaven Lab who led the research. “That would lock the carbon away in concrete for at least 50 years, potentially longer. By then, the world should be shifted to primarily renewable energy sources that don’t emit carbon.”

As a bonus, the process also produces hydrogen gas (H₂), a promising alternative fuel that, when used, creates zero emissions.

Capturing or converting carbon

The idea of capturing CO₂ or converting it to other materials to combat climate change is not new. But simply storing CO₂ gas can lead to leaks. And many CO₂ conversions produce carbon-based chemicals or fuels that are used right away, which releases CO₂ right back into the atmosphere.

“The novelty of this work is that we are trying to convert CO₂ into something that is value-added but in a solid, useful form,” Chen said.

Such solid carbon materials — including carbon nanotubes and nanofibers with dimensions measuring billionths of a meter — have many appealing properties, including strength and thermal and electrical conductivity. But it’s no simple matter to extract carbon from carbon dioxide and get it to assemble into these fine-scale structures. One direct, heat-driven process requires temperatures in excess of 1,000 degrees Celsius.

“It’s very unrealistic for large-scale CO₂ mitigation,” Chen said. “In contrast, we found a process that can occur at about 400 degrees Celsius, which is a much more practical, industrially achievable temperature.”

The tandem two-step

The trick was to break the reaction into stages and to use two different types of catalysts — materials that make it easier for molecules to come together and react.

“If you decouple the reaction into several sub-reaction steps you can consider using different kinds of energy input and catalysts to make each part of the reaction work,” said Brookhaven Lab and Columbia research scientist Zhenhua Xie, lead author on the paper.

The scientists started by realizing that carbon monoxide (CO) is a much better starting material than CO₂ for making carbon nanofibers (CNF). Then they backtracked to find the most efficient way to generate CO from CO₂.

Earlier work from their group steered them to use a commercially available electrocatalyst made of palladium supported on carbon. Electrocatalysts drive chemical reactions using an electric current. In the presence of flowing electrons and protons, the catalyst splits both CO₂ and water (H₂O) into CO and H₂.

For the second step, the scientists turned to a heat-activated thermocatalyst made of an iron-cobalt alloy. It operates at temperatures around 400 degrees Celsius, significantly milder than a direct CO₂-to-CNF conversion would require. They also discovered that adding a bit of extra metallic cobalt greatly enhances the formation of the carbon nanofibers.

“By coupling electrocatalysis and thermocatalysis, we are using this tandem process to achieve things that cannot be achieved by either process alone,” Chen said.

Catalyst characterization

To discover the details of how these catalysts operate, the scientists conducted a wide range of experiments. These included computational modeling studies, physical and chemical characterization studies at Brookhaven Lab’s National Synchrotron Light Source II (NSLS-II) — using the Quick X-ray Absorption and Scattering (QAS) and Inner-Shell Spectroscopy (ISS) beamlines — and microscopic imaging at the Electron Microscopy facility at the Lab’s Center for Functional Nanomaterials (CFN).

On the modeling front, the scientists used “density functional theory” (DFT) calculations to analyze the atomic arrangements and other characteristics of the catalysts when interacting with the active chemical environment.

“We are looking at the structures to determine what are the stable phases of the catalyst under reaction conditions,” explained study co-author Ping Liu of Brookhaven’s Chemistry Division who led these calculations. “We are looking at active sites and how these sites are bonding with the reaction intermediates. By determining the barriers, or transition states, from one step to another, we learn exactly how the catalyst is functioning during the reaction.”

X-ray diffraction and x-ray absorption experiments at NSLS-II tracked how the catalysts change physically and chemically during the reactions. For example, synchrotron x-rays revealed how the presence of electric current transforms metallic palladium in the catalyst into palladium hydride, a metal that is key to producing both H₂ and CO in the first reaction stage.

For the second stage, “We wanted to know what’s the structure of the iron-cobalt system under reaction conditions and how to optimize the iron-cobalt catalyst,” Xie said. The x-ray experiments confirmed that both an alloy of iron and cobalt plus some extra metallic cobalt are present and needed to convert CO to carbon nanofibers.

“The two work together sequentially,” said Liu, whose DFT calculations helped explain the process.

“According to our study, the cobalt-iron sites in the alloy help to break the C-O bonds of carbon monoxide. That makes atomic carbon available to serve as the source for building carbon nanofibers. Then the extra cobalt is there to facilitate the formation of the C-C bonds that link up the carbon atoms,” she explained.

Recycle-ready, carbon-negative

“Transmission electron microscopy (TEM) analysis conducted at CFN revealed the morphologies, crystal structures, and elemental distributions within the carbon nanofibers both with and without catalysts,” said CFN scientist and study co-author Sooyeon Hwang.

The images show that, as the carbon nanofibers grow, the catalyst gets pushed up and away from the surface. That makes it easy to recycle the catalytic metal, Chen said.

“We use acid to leach the metal out without destroying the carbon nanofiber so we can concentrate the metals and recycle them to be used as a catalyst again,” he said.

This ease of catalyst recycling, commercial availability of the catalysts, and relatively mild reaction conditions for the second reaction all contribute to a favorable assessment of the energy and other costs associated with the process, the researchers said.

“For practical applications, both are really important — the CO₂ footprint analysis and the recyclability of the catalyst,” said Chen. “Our technical results and these other analyses show that this tandem strategy opens a door for decarbonizing CO₂ into valuable solid carbon products while producing renewable H₂.”

If these processes are driven by renewable energy, the results would be truly carbon-negative, opening new opportunities for CO₂ mitigation.

This research was supported by the DOE Office of Science (BES). The DFT calculations were performed using computational resources at CFN and at the National Energy Research Scientific Computing Center (NERSC) at DOE’s Lawrence Berkeley National Laboratory. NSLS-II, CFN, and NERSC are DOE Office of Science user facilities.