Sensing neuronal activity with light

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For years, neuroscientists have been trying to develop tools that would allow them to clearly view the brain’s circuitry in action—from the first moment a neuron fires to the resulting behavior in a whole organism. To get this complete picture, neuroscientists are working to develop a range of new tools to study the brain. Researchers at Caltech have developed one such tool that provides a new way of mapping neural networks in a living organism.

The work—a collaboration between Viviana Gradinaru (BS ’05), assistant professor of biology and biological engineering, and Frances Arnold, the Dick and Barbara Dickinson Professor of Chemical Engineering, Bioengineering and Biochemistry—was described in two separate papers published this month.

When a neuron is at rest, channels and pumps in the cell membrane maintain a cell-specific balance of positively and negatively charged ions within and outside of the cell resulting in a steady membrane voltage called the cell’s resting potential. However, if a stimulus is detected—for example, a scent or a sound—ions flood through newly open channels causing a change in membrane voltage. This voltage change is often manifested as an action potential—the neuronal impulse that sets circuit activity into motion.

The tool developed by Gradinaru and Arnold detects and serves as a marker of these voltage changes.

“Our overarching goal for this tool was to achieve sensing of neuronal activity with light rather than traditional electrophysiology, but this goal had a few prerequisites,” Gradinaru says. “The sensor had to be fast, since action potentials happen in just milliseconds. Also, the sensor had to be very bright so that the signal could be detected with existing microscopy setups. And you need to be able to simultaneously study the multiple neurons that make up a neural network.”

The researchers began by optimizing Archaerhodopsin (Arch), a light-sensitive protein from bacteria. In nature, opsins like Arch detect sunlight and initiate the microbes’ movement toward the light so that they can begin photosynthesis. However, researchers can also exploit the light-responsive qualities of opsins for a neuroscience method called optogenetics—in which an organism’s neurons are genetically modified to express these microbial opsins. Then, by simply shining a light on the modified neurons, the researchers can control the activity of the cells as well as their associated behaviors in the organism.

Gradinaru had previously engineered Arch for better tolerance and performance in mammalian cells as a traditional optogenetic tool used to control an organism’s behavior with light. When the modified neurons are exposed to green light, Arch acts as an inhibitor, controlling neuronal activity—and thus the associated behaviors—by preventing the neurons from firing.

However, Gradinaru and Arnold were most interested in another property of Arch: when exposed to red light, the protein acts as a voltage sensor, responding to changes in membrane voltages by producing a flash of light in the presence of an action potential. Although this property could in principle allow Arch to detect the activity of networks of neurons, the light signal marking this neuronal activity was often too dim to see.

To fix this problem, Arnold and her colleagues made the Arch protein brighter using a method called directed evolution—a technique Arnold originally pioneered in the early 1990s. The researchers introduced mutations into the Arch gene, thus encoding millions of variants of the protein. They transferred the mutated genes into E. coli cells, which produced the mutant proteins encoded by the genes. They then screened thousands of the resulting E. coli colonies for the intensities of their fluorescence. The genes for the brightest versions were isolated and subjected to further rounds of mutagenesis and screening until the bacteria produced proteins that were 20 times brighter than the original Arch protein.

A paper describing the process and the bright new protein variants that were created was published in the September 9 issue of the Proceedings of the National Academy of Science.

“This experiment demonstrates how rapidly these remarkable bacterial proteins can evolve in response to new demands. But even more exciting is what they can do in neurons, as Viviana discovered,” says Arnold.

In a separate study led by Gradinaru’s graduate students Nicholas Flytzanis and Claire Bedbrook, who is also advised by Arnold, the researchers genetically incorporated the new, brighter Arch variants into rodent neurons in culture to see which of these versions was most sensitive to voltage changes—and therefore would be the best at detecting action potentials. One variant, Archer1, was not only bright and sensitive enough to mark action potentials in mammalian neurons in real time, it could also be used to identify which neurons were synaptically connected—and communicating with one another—in a circuit.

The work is described in a study published on September 15 in the journal Nature Communications.

“What was interesting is that we would see two cells over here light up, but not this one over there—because the first two are synaptically connected,” Gradinaru says. “This tool gave us a way to observe a network where the perturbation of one cell affects another.”

However, sensing activity in a living organism and correlating this activity with behavior remained the biggest challenge. To accomplish this goal Gradinaru’s team worked with Paul Sternberg, the Thomas Hunt Morgan Professor of Biology, to test Archer1 as a sensor in a living organism—the tiny nematode worm C. elegans. “There are a few reasons why we used the worms here: they are powerful organisms for quick genetic engineering and their tissues are nearly transparent, making it easy to see the fluorescent protein in a living animal,” she says.

After incorporating Archer1 into neurons that were a part of the worm’s olfactory system—a primary source of sensory information for C. elegans—the researchers exposed the worm to an odorant. When the odorant was present, a baseline fluorescent signal was seen, and when the odorant was removed, the researchers could see the circuit of neurons light up, meaning that these particular neurons are repressed in the presence of the stimulus and active in the absence of the stimulus. The experiment was the first time that an Arch variant had been used to observe an active circuit in a living organism.

Gradinaru next hopes to use tools like Archer1 to better understand the complex neuronal networks of mammals, using microbial opsins as sensing and actuating tools in optogenetically modified rodents.

“For the future work it’s useful that this tool is bifunctional. Although Archer1 acts as a voltage sensor under red light, with green light, it’s an inhibitor,” she says. “And so now a long-term goal for our optogenetics experiments is to combine the tools with behavior-controlling properties and the tools with voltage-sensing properties. This would allow us to obtain all-optical access to neuronal circuits. But I think there is still a lot of work ahead.”

One goal for the future, Gradinaru says, is to make Archer1 even brighter. Although the protein’s fluorescence can be seen through the nearly transparent tissues of the nematode worm, opaque organs such as the mammalian brain are still a challenge. More work, she says, will need to be done before Archer1 could be used to detect voltage changes in the neurons of living, behaving mammals.

And that will require further collaborations with protein engineers and biochemists like Arnold.

“As neuroscientists we often encounter experimental barriers, which open the potential for new methods. We then collaborate to generate tools through chemistry or instrumentation, then we validate them and suggest optimizations, and it just keeps going,” she says. “There are a few things that we’d like to be better, and through these many iterations and hard work it can happen.”

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Wireless sensor transmits tumor pressure

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A novel sensor that can wirelessly relay pressure readings from inside a tumor has been developed by researchers.

Wireless interstitial fluid pressure sensor shown to scale on a dime. Photo Credit: Image courtesy of National Institute of Biomedical Imaging and Bioengineering

The interstitial pressure inside a tumor is often remarkably high compared to normal tissues and is thought to impede the delivery of chemotherapeutic agents as well as decrease the effectiveness of radiation therapy. While medications exist that temporarily decrease tumor pressure, identifying the optimal window to initiate treatment—when tumor pressure is lowest—remains a challenge. With support from NIBIB, researchers at Purdue University have developed a novel sensor that can wirelessly relay pressure readings from inside a tumor.

Contents under Pressure

Tumors, like healthy tissues, need oxygen and nutrients to survive. In order to accommodate the demands of a growing tumor, blood vessels from surrounding tissue begin to grow into the tumor. Yet, unlike normal tissue, these newly formed blood vessels are disorganized, twisty, and leaky. It’s thought that the high pressure observed in tumors is a result of these abnormal blood vessels, which leak fluid and proteins into the area between tumor cells, known as the interstitial space.

In normal tissues, tightly regulated differences in pressure pull nutrients out of a tissue’s blood vessels and into the interstitial space, where they can be taken up by cells. Medications travelling through the blood also rely on these pressure differences in order to reach cells. When pressure in the interstitial space increases — as is the case in many tumors — medications are less apt to leave blood vessels. As a result, patients who have tumors with high interstitial pressure often receive a less than adequate dose of chemotherapy or other types of anti-cancer drugs. In addition, high interstitial pressure can also contribute to low oxygen levels in tumors. Because radiation therapy requires the presence of oxygen to be effective, tumors with high interstitial pressure are often less receptive to radiation therapy.

Window of Opportunity

Results from recent clinical trials and studies in animals suggest that a class of anti-cancer drugs called angiogenesis inhibitors may be able to temporarily reduce interstitial pressure and improve the efficacy of chemotherapy and radiation treatments. Angiogenesis inhibitors prevent the growth of new blood vessels and have long been investigated as a way to stop tumor growth. Recently, it has been hypothesized that there is a brief window after these drugs are given in which blood flow to tumors is actually normalized. This window provides an opportunity to more efficiently deliver chemotherapeutic drugs and radiation therapy.

However, because efficient methods for measuring interstitial tumor pressure are lacking, determining the optimal time to begin chemotherapy or radiation treatment within this normalization window remains a challenge.

“Right now, the only option for measuring pressure is to stick a needle inside the tumor. That’s not practical for clinical applications,” says Babak Ziaie, Ph.D, director of the Biomedical Microdevices Laboratory at Purdue University.

A Wireless Pressure Sensor

After conversations with radiation oncologists with whom he collaborates, Ziaie decided to take on the challenge of creating a tumor pressure sensor. He was enticed by the novelty of the project. “No one had done this before,” said Ziaie. “No one was working on it or even attempting it.”

With support from NIBIB, Ziaie and his research team created a novel sensor that can be implanted into a tumor to wirelessly transmit interstitial fluid pressure readings. The sensor is an adaptation of a technology developed in the 1950s called the Guyton capsule, which is a perforated capsule that, once implanted, allows interstitial fluid to flow through it. Subsequent insertion of a needle into the capsule provides direct access to the interstitial fluid for pressure measurements.

Using special microfabrication techniques, Ziaie created a miniaturized wireless pressure sensor and combined it with a Guyton-like capsule so that it could generate interstitial pressure readings without the use of a needle and that could be read remotely.

Recently, Ziaie and his team tested the device by implanting it into pancreatic tumors in mice and were able to show a decrease in interstitial tumor pressure following administration of an angiogenic inhibitor.

“This is a great example of the power of convergence science,” said Tiffani Lash, PhD, program director for sensor technologies at NIBIB. “Integrating knowledge from the life and physical sciences with engineering concepts can help solve important clinical problems. It’s about thinking creatively to generate novel ways to treat disease.”

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Scientists discover an on/off switch for aging cells

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Scientists at the Salk Institute have discovered an on-and-off “switch” in cells that may hold the key to healthy aging. This switch points to a way to encourage healthy cells to keep dividing and generating, for example, new lung or liver tissue, even in old age.

Photo Credit: Courtesy of the Salk Institute for Biological Studies

In our bodies, newly divided cells constantly replenish lungs, skin, liver and other organs. However, most human cells cannot divide indefinitely–with each division, a cellular timekeeper at the ends of chromosomes shortens. When this timekeeper, called a telomere, becomes too short, cells can no longer divide, causing organs and tissues to degenerate, as often happens in old age. But there is a way around this countdown: some cells produce an enzyme called telomerase, which rebuilds telomeres and allows cells to divide indefinitely.

In a new study published September 19th in the journal Genes and Development, scientists at the Salk Institute have discovered that telomerase, even when present, can be turned off.

“Previous studies had suggested that once assembled, telomerase is available whenever it is needed,” says senior author Vicki Lundblad, professor and holder of Salk’s Ralph S. and Becky O’Connor Chair. “We were surprised to discover instead that telomerase has what is in essence an ‘off’ switch, whereby it disassembles.”

Understanding how this “off” switch can be manipulated–thereby slowing down the telomere shortening process–could lead to treatments for diseases of aging (for example, regenerating vital organs later in life).

Lundblad and first author and graduate student Timothy Tucey conducted their studies in the yeast Saccharomyces cerevisiae, the same yeast used to make wine and bread. Previously, Lundblad’s group used this simple single-celled organism to reveal numerous insights about telomerase and lay the groundwork for guiding similar findings in human cells.

“We wanted to be able to study each component of the telomerase complex but that turned out to not be a simple task,” Tucey said. Tucey developed a strategy that allowed him to observe each component during cell growth and division at very high resolution, leading to an unanticipated set of discoveries into how–and when–this telomere-dedicated machine puts itself together.

Every time a cell divides, its entire genome must be duplicated. While this duplication is going on, Tucey discovered that telomerase sits poised as a “preassembly” complex, missing a critical molecular subunit. But when the genome has been fully duplicated, the missing subunit joins its companions to form a complete, fully active telomerase complex, at which point telomerase can replenish the ends of eroding chromosomes and ensure robust cell division.

Surprisingly, however, Tucey and Lundblad showed that immediately after the full telomerase complex has been assembled, it rapidly disassembles to form an inactive “disassembly” complex — essentially flipping the switch into the “off” position. They speculate that this disassembly pathway may provide a means of keeping telomerase at exceptionally low levels inside the cell. Although eroding telomeres in normal cells can contribute to the aging process, cancer cells, in contrast, rely on elevated telomerase levels to ensure unregulated cell growth. The “off” switch discovered by Tucey and Lundblad may help keep telomerase activity below this threshold.

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Neuroscientists identify key role of language gene

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Neuroscientists have found that a gene mutation that arose more than half a million years ago may be key to humans’ unique ability to produce and understand speech.

Researchers from MIT and several European universities have shown that the human version of a gene called Foxp2 makes it easier to transform new experiences into routine procedures. When they engineered mice to express humanized Foxp2, the mice learned to run a maze much more quickly than normal mice.

The findings suggest that Foxp2 may help humans with a key component of learning language — transforming experiences, such as hearing the word “glass” when we are shown a glass of water, into a nearly automatic association of that word with objects that look and function like glasses, says Ann Graybiel, an MIT Institute Professor, member of MIT’s McGovern Institute for Brain Research, and a senior author of the study.

“This really is an important brick in the wall saying that the form of the gene that allowed us to speak may have something to do with a special kind of learning, which takes us from having to make conscious associations in order to act to a nearly automatic-pilot way of acting based on the cues around us,” Graybiel says.

Wolfgang Enard, a professor of anthropology and human genetics at Ludwig-Maximilians University in Germany, is also a senior author of the study, which appears in the Proceedings of the National Academy of Sciences this week. The paper’s lead authors are Christiane Schreiweis, a former visiting graduate student at MIT, and Ulrich Bornschein of the Max Planck Institute for Evolutionary Anthropology in Germany.

All animal species communicate with each other, but humans have a unique ability to generate and comprehend language. Foxp2 is one of several genes that scientists believe may have contributed to the development of these linguistic skills. The gene was first identified in a group of family members who had severe difficulties in speaking and understanding speech, and who were found to carry a mutated version of the Foxp2 gene.

In 2009, Svante Pääbo, director of the Max Planck Institute for Evolutionary Anthropology, and his team engineered mice to express the human form of the Foxp2 gene, which encodes a protein that differs from the mouse version by only two amino acids. His team found that these mice had longer dendrites — the slender extensions that neurons use to communicate with each other — in the striatum, a part of the brain implicated in habit formation. They were also better at forming new synapses, or connections between neurons.

Pääbo, who is also an author of the new PNAS paper, and Enard enlisted Graybiel, an expert in the striatum, to help study the behavioral effects of replacing Foxp2. They found that the mice with humanized Foxp2 were better at learning to run a T-shaped maze, in which the mice must decide whether to turn left or right at a T-shaped junction, based on the texture of the maze floor, to earn a food reward.

The first phase of this type of learning requires using declarative memory, or memory for events and places. Over time, these memory cues become embedded as habits and are encoded through procedural memory — the type of memory necessary for routine tasks, such as driving to work every day or hitting a tennis forehand after thousands of practice strokes.

Using another type of maze called a cross-maze, Schreiweis and her MIT colleagues were able to test the mice’s ability in each of type of memory alone, as well as the interaction of the two types. They found that the mice with humanized Foxp2 performed the same as normal mice when just one type of memory was needed, but their performance was superior when the learning task required them to convert declarative memories into habitual routines. The key finding was therefore that the humanized Foxp2 gene makes it easier to turn mindful actions into behavioral routines.

The protein produced by Foxp2 is a transcription factor, meaning that it turns other genes on and off. In this study, the researchers found that Foxp2 appears to turn on genes involved in the regulation of synaptic connections between neurons. They also found enhanced dopamine activity in a part of the striatum that is involved in forming procedures. In addition, the neurons of some striatal regions could be turned off for longer periods in response to prolonged activation — a phenomenon known as long-term depression, which is necessary for learning new tasks and forming memories.

Together, these changes help to “tune” the brain differently to adapt it to speech and language acquisition, the researchers believe. They are now further investigating how Foxp2 may interact with other genes to produce its effects on learning and language.

This study “provides new ways to think about the evolution of Foxp2 function in the brain,” says Genevieve Konopka, an assistant professor of neuroscience at the University of Texas Southwestern Medical Center who was not involved in the research. “It suggests that human Foxp2 facilitates learning that has been conducive for the emergence of speech and language in humans. The observed differences in dopamine levels and long-term depression in a region-specific manner are also striking and begin to provide mechanistic details of how the molecular evolution of one gene might lead to alterations in behavior.”

The research was funded by the Nancy Lurie Marks Family Foundation, the Simons Foundation Autism Research Initiative, the National Institutes of Health, the Wellcome Trust, the Fondation pour la Recherche Médicale, and the Max Planck Society.

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15 years of carbon dioxide emissions on Earth mapped

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World leaders face multiple barriers in their efforts to reach agreement on greenhouse gas emission policies. And, according to Arizona State University researchers, without globally consistent, independent emissions assessments, climate agreements will remain burdened by errors, self-reporting and the inability to verify emissions progress.

This image shows global fossil fuel carbon dioxide emissions as represented by the Fossil Fuel Data Assimilation System. Photo Credit: Gurney lab

Now, an international research team led by ASU scientists has developed a new approach to estimate CO2 emissions from burning fossil fuels – one that provides crucial information to policymakers. Called the “Fossil Fuel Data Assimilation System,” or FFDAS, this new system was used to quantify 15 years of CO2 emissions, every hour, for the entire planet – down to the city scale. Until now, scientists have estimated greenhouse gas emissions at coarser scales or used less reliable techniques.

Researchers unveiled the new system in an article published Sept. 10 in the Journal of Geophysical Research.

The FFDAS uses information from satellite feeds, national fuel accounts and a new global database on power plants to create high-resolution planetary maps. These maps provide a scientific, independent assessment of the planet’s greenhouse gas emissions – something policymakers can use and the public can understand.

“With this system, we are taking a big step toward creating a global monitoring system for greenhouse gases, something that is needed as the world considers how best to meet greenhouse gas reductions,” said Kevin Robert Gurney, lead investigator and associate professor in ASU’s School of Life Sciences. “Now we can provide all countries with detailed information about their CO2 emissions and show that independent, scientific monitoring of greenhouse gases is possible.”

The research team combined information from space-based “nighttime lights,” a new population database, national statistics on fuel use, and a global database on power plants to create a CO2 emissions map broken down by hour, year and region.

“The accuracy of the FFDAS results is confirmed by independent, ground-based data in the United States,” said Salvi Asefi-Najafabady, lead author of the report and postdoctoral researcher at ASU. “This makes us confident that the system is working well and can provide useable, policy-salient information.”

“This is an incredibly helpful tool for national and international policymakers and the public to get a grasp of whether strategies to reduce greenhouse gases are effective,” said Jennifer Morgan, director of the Climate and Energy Program at World Resources Institute. “It serves as a complementary approach to current bottom-up accounting methodologies. No longer will there be a delay in understanding the latest GHG trends.”

The FFDAS showed surprising detail on global emissions before and after the Global Financial Crisis, with portions of the U.S., Europe and India recovering sooner and more dramatically. The multiyear results also showed the dramatic rise of CO2 emissions in China and South Asia. Hence, the sub-national details offer insights into economic activity at scales for which traditional economic data has been limited.

“It used to take years to assemble all the statistics on CO2 emissions,” said Peter Rayner, lead investigator from the University of Melbourne, Australia. “With this system, once the satellite data is flowing, we can update our emissions maps each year. It gives a quick check on efforts to limit climate change.”

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Simple Method Turns Skin Cells Into White Blood Cells

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The fast and safe technique developed at the Salk Institute circumvents problems that have hindered regenerative medicine.

Scanning electron micrograph of T lymphocyte (right), a platelet (center) and a red blood cell (left)

For the first time, scientists have turned human skin cells into transplantable white blood cells, soldiers of the immune system that fight infections and invaders. The work, done at the Salk Institute, could let researchers create therapies that introduce into the body new white blood cells capable of attacking diseased or cancerous cells or augmenting immune responses against other disorders.

The work, as detailed in the journal Stem Cells, shows that only a bit of creative manipulation is needed to turn skin cells into human white blood cells.

“The process is quick and safe in mice,” says senior author Juan Carlos Izpisua Belmonte, holder of Salk’s Roger Guillemin Chair. “It circumvents long-standing obstacles that have plagued the reprogramming of human cells for therapeutic and regenerative purposes.”

Those problems includes the long time—at least two months—and tedious laboratory work it takes to produce, characterize and differentiate induced pluripotent stem (iPS) cells, a method commonly used to grow new types of cells. Blood cells derived from iPS cells also have other obstacles: an inability to engraft into organs or bone marrow and a likelihood of developing tumors.

The new method takes just two weeks, does not produce tumors, and engrafts well.

“We tell skin cells to forget what they are and become what we tell them to be—in this case, white blood cells,” says one of the first authors and Salk researcher Ignacio Sancho-Martinez. “Only two biological molecules are needed to induce such cellular memory loss and to direct a new cell fate.”

Belmonte’s team developed the faster technique (called indirect lineage conversion) and previously demonstrated that these approaches could be used to produce human vascular cells, the ones that line blood vessels. Rather than reversing cells all the way back to a stem cell state before prompting them to turn into something else, such as in the case of iPS cells, the researchers “rewind” skin cells just enough to instruct them to form the more than 200 cell types that constitute the human body.

The technique demonstrated in this study uses a molecule called SOX2 to become somewhat plastic—the stage of losing their “memory” of being a specific cell type. Then, researchers use a genetic factor called miRNA125b that tells the cells that they are actually white blood cells.

The researchers are now conducting toxicology studies and cell transplantation proof-of-concept studies in advance of potential preclinical and clinical studies.

“It is fair to say that the promise of stem cell transplantation is now closer to realization,” Sancho-Martinez says.

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Scientists map white matter connections within the human brain

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Roughly 100 trillion connections between neurons make it possible for the brain to function. Psychology Professor Brian Wandell’s group has devised a technique for mapping these connections with greater accuracy than ever before.

Two different algorithms produce two very different estimates of the shape of the same white matter connection in Franco Pestilli’s brain. The LiFE software, developed in the Wandell Lab, aims to produce more precise estimates. Photo Credit: Wandell Lab / Stanford

To see, think or feel, the 100 billion neurons in our brain must exchange messages. These are transmitted over some 100 trillion specialized connections, known collectively as the “connectome.” Most connections are extremely short, carrying information a few hundred-thousandths of an inch between nearby neurons. But many important connections are much longer, winding as much as a foot from one end of the brain to the other.

Scientists at Stanford University have developed a mathematical and computational technology that allows researchers to more accurately map the large, long connections within the white matter tissue of living human brains. The methodology is called LiFE, for linear fascicle evaluation. The work is detailed online in Nature Methods.

To test the new method, lead author Franco Pestilli analyzed MRI scans of two such connections within his own brain. In one analysis, the two structures – the arcuate fasciculus, which is involved in reading and language, and the corticospinal tract, which plays a role in motor coordination – appeared short and fairly smooth. In the other, the ridged tendrils within the tracts spread far longer and wider.

“Previously, scientists had no method for deciding which of the two representations of the human brain is correct,” said Pestilli, a research associate in the laboratory of Brian Wandell, a professor of psychology at Stanford. “As a result, different research groups using the same data often came to different conclusions. The new technology provides a mathematical analysis and open-source software to decide which of the two estimates is better.”

Many candidate representations of the human connectome can be created using available fiber tracking technology. LiFE interprets these pathways and uses them to simulate synthetic MRI signals. Candidate connectomes can be evaluated to find the one that generates a synthetic signal that most closely matches the actual data.

“I like to think about each candidate connectome as a prototype,” Pestilli said. “We can learn much by building many prototypes and finding the one that best represents the measured MRI signal. Once we have this optimized prototype, we can more accurately study the function of the actual pathways.”

The National Institutes of Health has made mapping the human brain an important scientific objective because of its importance in predicting healthy development and critical brain function. For example, in a series of papers over the years, the Wandell lab has shown that the properties of the brain connections differ between good and poor readers, particularly in one specific pathway, the arcuate fasciculus.

“We can much more reliably identify the arcuate fasciculus in a child who is struggling to read and understand whether or not that connection might be the source of the difficulty,” Wandell said.

Understanding the human connectome will allow clarifying the fundamental organizing principles of structure and function of the human brain. This will allow us to understand the mechanisms behind some of the most important diseases affecting individuals and society. For example, multiple sclerosis, Alzheimer’s disease and schizophrenia have all been regarded as brain diseases affecting the connectome.

An overall effort of the Wandell lab involves sharing data and computational methods with the broader scientific community. This work, which was funded by a grant from the National Science Foundation, supports that effort by providing data and a complete implementation of the method through the Stanford Digital Repository and GitHub.

“We hope other investigators use the code and improve it,” Pestilli said. “Code and data sharing can be a very important contribution as we try to understand the human brain.”

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Scientists revert human stem cells to pristine state

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Researchers at EMBL-EBI have resolved a long-standing challenge in stem cell biology by successfully ‘resetting’ human pluripotent stem cells to a fully pristine state, at point of their greatest developmental potential. The study, published in Cell, involved scientists from the UK, Germany and Japan and was led jointly by EMBL-EBI and the University of Cambridge.

Resetting transcription factor control circuitry toward ground-state pluripotency in Human. Photo Credit: Takashima et al., Cell (2014)

Embryonic stem (ES) cells, which originate in early development, are capable of differentiating into any type of cell. Until now, scientists have only been able to revert ‘adult’ human cells (for example, liver, lung or skin) into pluripotent stem cells with slightly different properties that predispose them to becoming cells of certain types. Authentic ES cells have only been derived from mice and rats.

“Reverting mouse cells to a completely ‘blank slate’ has become routine, but generating equivalent naïve human cell lines has proven far more challenging,” says Dr Paul Bertone, Research Group Leader at EMBL-EBI and a senior author on the study. “Human pluripotent cells resemble a cell type that appears slightly later in mammalian development, after the embryo has implanted in the uterus.”

At this point, subtle changes in gene expression begin to influence the cells, which are then considered ‘primed’ towards a particular lineage. Although pluripotent human cells can be cultured from in vitro fertilised (IVF) embryos, until now there have been no human cells comparable to those obtained from the mouse.

Wiping cell memory

“For years, it was thought that we could be missing the developmental window when naïve human cells could be captured, or that the right growth conditions hadn’t been found,” Paul explains. “But with the advent of iPS cell technologies, it should have been possible to drive specialised human cells back to an earlier state, regardless of their origin – if that state existed in primates.”

Taking a new approach, the scientists used reprogramming methods to express two different genes, NANOG and KLF2, which reset the cells. They then maintained the cells indefinitely by inhibiting specific biological pathways. The resulting cells are capable of differentiating into any adult cell type, and are genetically normal.

The experimental work was conducted hand-in-hand with computational analysis.

“We needed to understand where these cells lie in the spectrum of the human and mouse pluripotent cells that have already been produced,” explains Paul. “We worked with the EMBL Genomics Core Facility to produce comprehensive transcriptional data for all the conditions we explored. We could then compare reset human cells to genuine mouse ES cells, and indeed we found they shared many similarities.”

Together with Professor Wolf Reik at the Babraham Institute, the researchers also showed that DNA methylation (biochemical marks that influence gene expression) was erased over much of the genome, indicating that reset cells are not restricted in the cell types they can produce. In this more permissive state, the cells no longer retain the memory of their previous lineages and revert to a blank slate with unrestricted potential to become any adult cell.

Unlocking the potential of stem cell therapies

The research was performed in collaboration with Professor Austin Smith, Director of the Wellcome Trust-Medical Research Council Stem Cell Institute.

“Our findings suggest that it is possible to rewind the clock to achieve true ground-state pluripotency in human cells,” said Professor Smith. “These cells may represent the real starting point for formation of tissues in the human embryo. We hope that in time they will allow us to unlock the fundamental biology of early development, which is impossible to study directly in people.”

The discovery paves the way for the production of superior patient material for translational medicine. Reset cells mark a significant advance for human stem cell applications, such as drug screening of patient-specific cells, and are expected to provide reliable sources of specialised cell types for regenerative tissue grafts.

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The interactive brain

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Amy Griffin, associate professor of psychological and brain sciences at the University of Delaware, has received a five-year, $1.78 million grant from the National Institute of Mental Health to support her research into the brain mechanisms of working memory.

A neuroscientist, Griffin has been interested for some time in the interaction between the prefrontal cortex, located at the front of the brain, and the hippocampus, a region in the temporal lobe of the brain. When the two areas fail to work together, that failure appears to be correlated with deficits in working memory, a condition that commonly occurs in schizophrenia, general anxiety and other psychiatric disorders.

The hippocampus is the portion of the brain responsible for memory, while the prefrontal cortex controls executive function, a term that includes such cognitive abilities as problem-solving, planning and abstract thinking.

“These are two areas of the brain that are far apart, but their oscillations [rhythmic activities] are synchronized,” Griffin said. “When one area is active, so is the other.”

Working memory, sometimes called short-term memory, is “the kind of memory that fails when you walk into a room and forget why you came there,” she said.

When the oscillations in the hippocampus and prefrontal cortex are out of sync, deficits of working memory occur. In those cases, Griffin said, “both regions are active, but they’re not talking to each other.” The mechanism that causes that lack of communication has not been well explored, and her research will seek to do that.

Griffin and her research team plan to conduct two types of experiments. One will inhibit activity in a brain region called the nucleus reuniens, a region that is hypothesized to synchronize the hippocampus and prefrontal cortex and is expected to cause impairments with working memory. In the other experiment, researchers will activate the nucleus reuniens to increase synchrony, hoping to learn if that improves working memory.

The research will employ a cutting-edge technique called optogenetics, a process that uses proteins to make neurons sensitive to light and then uses light to control them.

“Optogenetics is becoming a common technique,” Griffin said. “It’s a way to study these processes on a millisecond timescale.”

A 2013 article in the journal Nature Neuroscience said optogenetics “is transforming the field of neuroscience. For the first time, it is now possible to use light to both trigger and silence activity in genetically defined populations of neurons with millisecond precision.”

Griffin, using a rat model, will inject the light-sensitizing substance — a harmless virus — into the nucleus reuniens and then use a laser to inhibit or activate this brain region. The rats then perform tasks that assess their working memory. Synchronization between the hippocampus and prefrontal cortex will also be recorded, with the prediction that the degree of the working memory impairment will be correlated with reductions in synchrony.

“Our experiments will not be interfering with the activities of the hippocampus or the prefrontal cortex within themselves,” Griffin said. “We want to affect only the ability of the structures to talk to each other.”

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Researchers Create World’s Largest DNA Origami

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Researchers from North Carolina State University, Duke University and the University of Copenhagen have created the world’s largest DNA origami, which are nanoscale constructions with applications ranging from biomedical research to nanoelectronics.

Scaffolded DNA origami utilizes numerous chemically synthesized, short DNA strands (staple strands) to direct the folding of a larger, biologically derived strand of DNA (scaffold strand). Molecular recognition (base pairing, i.e., A binds to T and G binds to C) directs the DNA to self-assemble into a specific structure as programed by the staple strand sequences. Unique staple strands produce a molecular pegboard with single-digit nanometer site-specificity precision. The atomic force microscopy image (right) demonstrates the final origami structure. Image credit: Alexandria Marchi.

“These origami can be customized for use in everything from studying cell behavior to creating templates for the nanofabrication of electronic components,” says Dr. Thom LaBean, an associate professor of materials science and engineering at NC State and senior author of a paper describing the work.

DNA origami are self-assembling biochemical structures that are made up of two types of DNA. To make DNA origami, researchers begin with a biologically derived strand of DNA called the scaffold strand. The researchers then design customized synthetic strands of DNA, called staple strands. Each staple strand is made up of a specific sequence of bases (adenine, cytosine, thaline and guanine – the building blocks of DNA), which is designed to pair with specific subsequences on the scaffold strand.

The staple strands are introduced into a solution containing the scaffold strand, and the solution is then heated and cooled. During this process, each staple strand attaches to specific sections of the scaffold strand, pulling those sections together and folding the scaffold strand into a specific shape.

The standard for DNA origami has long been limited to a scaffold strand that is made up of 7,249 bases, creating structures that measure roughly 70 nanometers (nm) by 90 nm, though the shapes may vary.

However, the research team led by LaBean has now created DNA origami consisting of 51,466 bases, measuring approximately 200 nm by 300 nm.

“We had to do two things to make this viable,” says Dr. Alexandria Marchi, lead author of the paper and a postdoctoral researcher at Duke. “First we had to develop a custom scaffold strand that contained 51 kilobases. We did that with the help of molecular biologist Stanley Brown at the University of Copenhagen.

“Second, in order to make this economically feasible, we had to find a cost-effective way of synthesizing staple strands – because we went from needing 220 staple strands to needing more than 1,600,” Marchi says.

The researchers did this by using what is essentially a converted inkjet printer to synthesize DNA directly onto a plastic chip.

“The technique we used not only creates large DNA origami, but has a fairly uniform output,” LaBean says. “More than 90 percent of the origami are self-assembling properly.”

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Building Replacement Kidneys in the Lab

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Regenerative medicine researchers at Wake Forest Baptist Medical Center have addressed a major challenge in the quest to build replacement kidneys in the lab. Working with human-sized pig kidneys, the scientists developed the most successful method to date to keep blood vessels in the new organs open and flowing with blood. The work is reported in journal Technology.

Anthony Atala, M.D., director and professor at the Wake Forest Institute for Regenerative Medicine

“Until now, lab-built kidneys have been rodent-sized and have functioned for only one or two hours after transplantation because blood clots developed,” said Anthony Atala, M.D., director and professor at the Wake Forest Institute for Regenerative Medicine and a senior author on the study. “In our proof-of-concept study, the vessels in a human-sized pig kidney remained open during a four-hour testing period. We are now conducting a longer-term study to determine how long flow can be maintained.”

If proven successful, the new method to more effectively coat the vessels with cells (endothelial) that keep blood flowing smoothly, could potentially be applied to other complex organs that scientists are working to engineer, including the liver and pancreas.

The current research is part of a long-term project to use pig kidneys to make support structures known as “scaffolds” that could potentially be used to build replacement kidneys for human patients with end-stage renal disease. Scientists first remove all animal cells from the organ – leaving only the organ structure or “skeleton.” A patient’s own cells would then be placed in the scaffold, making an organ that the patient theoretically would not reject.

The cell removal process leaves behind an intact network of blood vessels that can potentially supply the new organ with oxygen. However, scientists working to repopulate kidney scaffolds with cells have had problems coating the vessels and severe clotting has generally occurred within a few hours after transplantation.

The Wake Forest Baptist scientists took a two-pronged approach to address this problem. First, they evaluated four different methods of introducing new cells into the main vessels of the kidney scaffold. They found that a combination of infusing cells with a syringe, followed by a period of pumping cells through the vessels at increasing flow rates, was most effective.

Next, the research team coated the scaffold’s vessels with an antibody designed to make them more “sticky” and to bind endothelial cells. Laboratory and imaging studies — as well as tests of blood flow in the lab – showed that cell coverage of the vessels was sufficient to support blood flow through the entire kidney scaffold.

The final test of the dual-approach was implanting the scaffolds in pigs weighing 90 to 110 pounds. During a four-hour testing period, the vessels remained open.

“Our cell seeding method, combined with the antibody, improves the attachment of cells to the vessel wall and prevents the cells from being detached when blood flow is initiated,” said In Kap Ko, Ph.D., lead author and instructor in regenerative medicine at Wake Forest Baptist.

The scientists said a long-term examination is necessary to sufficiently conclude that blood clotting is prevented when endothelial cells are attached to the vessels.

The scientists said if the new method is proven successful in the long-term, the research brings them an important step closer to the day when replacement kidneys can be built in the lab.

“The results are a promising indicator that it is possible to produce a fully functional vascular system that can deliver nutrients and oxygen to engineered kidneys, as well as other engineered organs,” said Ko.

Using pig kidneys as scaffolds for human patients has several advantages, including that the organs are similar in size and that pig heart valves – removed of cells – have safety been used in patients for more than three decades.

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Nuclear waste eaters

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Although bacteria with waste-eating properties have been discovered in relatively pristine soils before, this is the first time that microbes that can survive in the very harsh conditions expected in radioactive waste disposal sites have been found. The findings are published in the ISME (Multidisciplinary Journal of Microbial Ecology) journal.

The disposal of our nuclear waste is very challenging, with very large volumes destined for burial deep underground. The largest volume of radioactive waste, termed ‘intermediate level’ and comprising of 364,000m3 (enough to fill four Albert Halls), will be encased in concrete prior to disposal into underground vaults. When ground waters eventually reach these waste materials, they will react with the cement and become highly alkaline. This change drives a series of chemical reactions, triggering the breakdown of the various ‘cellulose’ based materials that are present in these complex wastes.

One such product linked to these activities, isosaccharinic acid (ISA), causes much concern as it can react with a wide range of radionuclides – unstable and toxic elements that are formed during the production of nuclear power and make up the radioactive component of nuclear waste. If the ISA binds to radionuclides, such as uranium, then the radionuclides will become far more soluble and more likely to flow out of the underground vaults to surface environments, where they could enter drinking water or the food chain. However, the researchers’ new findings indicate that microorganisms may prevent this becoming a problem.

Working on soil samples from a highly alkaline industrial site in the Peak District, which is not radioactive but does suffer from severe contamination with highly alkaline lime kiln wastes, they discovered specialist “extremophile” bacteria that thrive under the alkaline conditions expected in cement-based radioactive waste. The organisms are not only superbly adapted to live in the highly alkaline lime wastes, but they can use the ISA as a source of food and energy under conditions that mimic those expected in and around intermediate level radwaste disposal sites. For example, when there is no oxygen (a likely scenario in underground disposal vaults) to help these bacteria “breath” and break down the ISA, these simple single-cell microorganisms are able to switch their metabolism to breath using other chemicals in the water, such as nitrate or iron.

The fascinating biological processes that they use to support life under such extreme conditions are being studied by the Manchester group, as well as the stabilizing effects of these humble bacteria on radioactive waste. The ultimate aim of this work is to improve our understanding of the safe disposal of radioactive waste underground by studying the unusual diet of these hazardous waste eating microbes.

One of the researchers, Professor Jonathan Lloyd, from the University’s School of Earth, Atmospheric and Environmental Sciences, said: “We are very interested in these Peak District microorganisms. Given that they must have evolved to thrive at the highly alkaline lime-kiln site in only a few decades, it is highly likely that similar bacteria will behave in the same way and adapt to living off ISA in and around buried cement-based nuclear waste quite quickly.

“Nuclear waste will remain buried deep underground for many thousands of years so there is plenty of time for the bacteria to become adapted. Our next step will be to see what impact they have on radioactive materials. We expect them to help keep radioactive materials fixed underground through their unusual dietary habits, and their ability to naturally degrade ISA.”

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Cell Smashes and Rebuilds Its Own Genome

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Life can be so intricate and novel that even a single cell can pack a few surprises, according to a study led by Princeton University researchers.

A study led by Princeton University researchers found that the pond-dwelling, single-celled organism Oxytricha trifallax (above) has the remarkable ability to break its own DNA into nearly a quarter-million pieces and rapidly reassemble those pieces when it’s time to mate. This elaborate process could provide a template for understanding how chromosomes in more complex animals such as humans break apart and reassemble, as can happen during the onset of cancer. (Image by John Bracht, American University, and Robert Hammersmith, Ball State University)

The pond-dwelling, single-celled organism Oxytricha trifallax has the remarkable ability to break its own DNA into nearly a quarter-million pieces and rapidly reassemble those pieces when it’s time to mate, the researchers report in the journal Cell. The organism internally stores its genome as thousands of scrambled, encrypted gene pieces. Upon mating with another of its kind, the organism rummages through these jumbled genes and DNA segments to piece together more than 225,000 tiny strands of DNA. This all happens in about 60 hours.

The organism’s ability to take apart and quickly reassemble its own genes is unusually elaborate for any form of life, explained senior author Laura Landweber, a Princeton professor of ecology and evolutionary biology. That such intricacy exists in a seemingly simple organism accentuates the “true diversity of life on our planet,” she said.

“It’s one of nature’s early attempts to become more complex despite staying small in the sense of being unicellular,” Landweber said. “There are other examples of genomic jigsaw puzzles, but this one is a leader in terms of complexity. People might think that pond-dwelling organisms would be simple, but this shows how complex life can be, that it can reassemble all the building blocks of chromosomes.”

From a practical standpoint, Oxytricha is a model organism that could provide a template for understanding how chromosomes in more complex animals such as humans break apart and reassemble, as can happen during the onset of cancer, Landweber said. While chromosome dynamics in cancer cells can be unpredictable and chaotic, Oxytricha presents an orderly step-by-step model of chromosome reconstruction, she said.

“It’s basically bad when human chromosomes break apart and reassemble in a different order,” Landweber said. “The process in Oxytricha recruits some of the same biological mechanisms that normally protect chromosomes from falling apart and uses them to do something creative and constructive instead.”

Gertraud Burger, a professor of biochemistry at the University of Montreal, said that the “rampant and diligently orchestrated genome rearrangements that take place in this organism” demonstrate a unique layer of complexity for scientists to consider when it comes to studying an organism’s genetics.

“This work illustrates in an impressive way that the genetic information of an organism can undergo substantial change before it is actually used for building the components of a living cell,” said Burger, who is familiar with the work but had no role in it.

“Therefore, inferring an organism’s make-up from the genome sequence alone can be a daunting task and maybe even impossible in certain instances,” Burger said. “A few cases of minor rearrangements have been described in earlier work, but these are dilettantes compared to [this] system.”

Burger added that the work is “extremely comprehensive as to the experimental techniques employed and analyses performed.” The project is one of the first complex genomes to be sequenced using Pacific Biosciences (PacBio) technology that reads long, single molecules.

Oxytricha already stands apart from other microorganisms, Landweber said. It is a large cell, about 10 times the size of a typical human cell. The organism also contains two nuclei whereas most single-celled organisms contain just one. A cell’s nucleus regulates internal activity and, typically, contains the cell’s DNA as well as the genes that are passed along during reproduction.

An individual Oxytricha cell, however, keeps its active DNA in one working nucleus and uses the second to store an archive of the genetic material it will pass along to the next generation, Landweber said. The genome of this second nucleus — known as the germ-line nucleus — undergoes the dismantling and reconstruction to produce a new working nucleus in the offspring.

Oxytricha uses sex solely to exchange DNA rather than to reproduce, Landweber said — like plant cuttings, new Oxytricha populations spawn from a single organism. During sex, two organisms fuse together to share half of their genetic information. The object is for each cell to replace aging genes with new genes and DNA parts from its partner. Together, both cells construct new working nuclei with a fresh set of chromosomes. This rejuvenates them and diversifies their genetic material, which is good for the organism, Landweber said.

“It’s kind of like science fiction — they stop aging by trading in their old parts,” she said.

It’s during this process that the scrambled genes in the germ-line nucleus are sorted through to locate the roughly 225,000 small DNA segments that each mate uses to reconstruct its rejuvenated chromosomes, the researchers found. Previous work in Landweber’s lab — a 2012 publication in Cell and a 2008 paper in the journal Nature — showed that millions of noncoding RNA molecules from the previous generation direct this undertaking by marking and sorting the DNA pieces in the correct order.

Also impressive is the massive scale of Oxytricha’s genome, Landweber said. A 2013 paper from her lab in PLoS Biology reported that the organism contains approximately 16,000 chromosomes in the active nucleus; humans have only 46. Most of Oxytricha’s chromosomes contain just a single gene, but even those genes can get hefty. A single Oxytricha gene can be built up from anywhere between one to 245 separate pieces of DNA, Landweber said.

The exceptional genetics of Oxytricha protect its DNA, so that mainly healthy material is passed along during reproduction, Landweber said. It’s no wonder then that the organism can be found worldwide munching on algae.

“Their successful distribution across the globe has something to do with their ability to protect their DNA through a novel method of encryption, then rapidly reassemble and transmit robust genes across generations,” Landweber said.

Landweber worked with, from her lab, first author Xiao Chen and Derek Clay, two graduate students in molecular biology, co-first author John Bracht and Aaron Goldman, postdoctoral fellows in ecology and evolutionary biology, and David Perlman, director of the molecular biology mass spectrometry facility. Other researchers on the paper are from the University of South Florida, the University of Bern in Switzerland, Indiana University, the Benaroya Research Institute at Virginia Mason in Seattle, and the Icahn School of Medicine at Mount Sinai.

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How the brain finds what it’s looking for

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Despite the barrage of visual information the brain receives, it retains a remarkable ability to focus on important and relevant items. This fall, for example, NFL quarterbacks will be rewarded handsomely for how well they can focus their attention on color and motion – being able to quickly judge the jersey colors of teammates and opponents and where they’re headed is a valuable skill. How the brain accomplishes this feat, however, has been poorly understood.

Now, University of Chicago scientists have identified a brain region that appears central to perceiving the combination of color and motion. They discovered a unique population of neurons that shift in sensitivity toward different colors and directions depending on what is being attended – the red jersey of a receiver headed toward an end zone, for example. The study, published Sept. 4 in the journal Neuron, sheds light on a fundamental neurological process that is a key step in the biology of attention.

“Most of the objects in any given visual scene are not that important, so how does the brain select or attend to important ones?” said study senior author David Freedman, PhD, associate professor of neurobiology at the University of Chicago. “We’ve zeroed in on an area of the brain that appears central to this process. It does this in a very flexible way, changing moment by moment depending on what is being looked for.”

The visual cortex of the brain possesses multiple, interconnected regions that are responsible for processing different aspects of the raw visual signal gathered by the eyes. Basic information on motion and color are known to route through two such regions, but how the brain combines these streams into something usable for decision-making or other higher-order processes remained unclear.

To investigate this process, Freedman and postdoctoral fellow Guilhem Ibos, PhD, studied the response of individual neurons during a simple task. Monkeys were shown a rapid series of visual images. An initial image showed either a group of red dots moving upwards or yellow dots moving downwards, which served as an instruction for which specific colors and directions were relevant during that trial. The subjects were rewarded when they released a lever when this image later reappeared. Subsequent images were composed of different colors of dots moving in different directions, among which was the initial image.

Dynamic neurons

Freedman and Ibos looked at neurons in the lateral intraparietal area (LIP), a region highly interconnected with brain areas involved in vision, motor control and cognitive functions. As subjects performed the task and looked for a specific combination of color and motion, LIP neurons became highly active. They did not respond, however, when the subjects passively viewed the same images without an accompanying task.

When the team further investigated the responses of LIP neurons, they discovered that the neurons possessed a unique characteristic. Individual neurons shifted their sensitivity to color and direction toward the relevant color and motion features for that trial. When the subject looked for red dots moving upwards, for example, a neuron would respond strongly to directions close to upward motion and to colors close to red. If the task was switched to another color and direction seconds later, that same neuron would be more responsive to the new combination.

“Shifts in feature tuning had been postulated a long time ago by theoretical studies,” Ibos said. “This is the first time that neurons in the brain have been shown to shift their selectivity depending on which features are relevant to solve a task.”

Freedman and Ibos developed a model for how the LIP brings together both basic color and motion information. Attention likely affects that process through signals from higher-order areas of the brain that affect LIP neuron selectivity. The team believes that this region plays an important role in making sense of basic sensory information, and they are trying to better understand the brain-wide neuronal circuitry involved in this process.

“Our study suggests that this area of the brain brings together information from multiple areas throughout the brain,” Freedman said. “It integrates inputs – visual, motor, cognitive inputs related to memory and decision making – and represents them in a way that helps solve the task at hand.”

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Synthetic messenger boosts immune system

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Specific immune cells, known as T lymphocytes, have to be activated so that the body can develop long-term protection against infections. Previously, it was believed that this process only took place in the lymph nodes and the spleen. But now scientists from Klinikum rechts der Isar at Technische Universität München have discovered that T cells can also be activated in the liver – via a much faster, more direct signaling pathway. The findings, which have been published in Cell Reports, could lead to improvements in the formulation of vaccines.

Prof. Percy Knolle (right) and his research group investigate the local regulation of immune responses in the liver. Photo Credit: A. Heddergott / TUM

When a pathogen attacks a healthy cell in the body, T lymphocytes are tasked with identifying and destroying the infected cell. Scientists know that they undergo a “training program” for this task in the lymph nodes or the spleen. “Programming cells” play a key role here, presenting pathogen constituents to the T lymphocytes. This is how the T lymphocytes learn to recognize these components and become specialized “killer” cells. Research teams led by Prof. Percy Knolle from Klinikum rechts der Isar and the University of Bonn and Prof. Stefan Rose-John from the University of Kiel have now discovered a second site where T lymphocytes can be programmed to attack pathogens.

The researchers identified the liver as an immunological organ where special tissue cells act as “programming cells” to prepare the T lymphocytes for their battle against infected cells. They were also able to decrypt the exact molecular code used by these tissue cells to equip the T lymphocytes with special infection-fighting capabilities. “This new mechanism is particularly interesting because the T cells are activated directly and very rapidly – in just 18 hours instead of 72,” explains Percy Knolle.

Synthetic messenger as new adjuvant

The key to all this is a natural messenger from the interleukin family: IL-6/sIL-6R. It only becomes effective when the two individual components (IL-6 and sIL-6R) combine. Previously, scientists were only aware of a role for the substance in regeneration and the development of inflammatory responses. But now the research teams have shown that the T lymphocytes in the liver engage in direct contact with the “programming cells” found there, and that IL-6/sIL-6R very efficiently activates the T cells.

Once the researchers had uncovered this new mechanism, they were able to use a synthetic designer messenger developed by Stefan Rose-John, known as Hyper-IL-6, for the targeted stimulation of T lymphocytes. In this construct, the two individual components which are normally separated are directly and firmly linked to each other. “In combination with the familiar stimulation mechanisms of the T lymphocytes, Hyper-IL-6 can be used to effect a specific ‘hyper stimulation’ of T lymphocytes for particular therapeutic purposes. This could be an important step in helping us improve vaccine formulations,” adds Percy Knolle.

As well as components of the pathogen, current vaccines also contain substances known as adjuvants. After a few intermediate steps, adjuvants indirectly activate the T lymphocytes and help to build up immunity. “We are placing high hopes on hyper-IL-6 as an effective new adjuvant capable of activating T cells directly and thus more rapidly. This could for example help us tackle chronic bacterial or viral infections which have not previously responded to vaccinations,” he concludes.

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Artificial cells take their first steps: Movable cytoskeleton membrane fabricated for first time

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Using only a few ingredients, the biophysicist Prof. Andreas Bausch and his team at the Technische Universität München (TUM) have successfully implemented a minimalistic model of the cell that can change its shape and move on its own. They describe how they turned this goal into reality in the current edition of the academic journal Science, where their research is featured as cover story.

Artificial minimal model of cell deformations. The encapsulated bio-molecules form a layer on the membrane that actively drives continuous motion. Photo Credit: Keber, Loiseau, Sanchez, Bausch/TUM

Cells are complex objects with a sophisticated metabolic system. Their evolutionary ancestors, the primordial cells, were merely composed of a membrane and a few molecules. These were minimalistic yet perfectly functioning systems.

Thus, “back to the origins of the cell” became the motto of the group of TUM-Prof. Andreas Bausch, who is member of the cluster of excellence “Nanosystems Initiative Munich (NIM)” and his international partners. Their dream is to create a simple cell model with a specific function using a few basic ingredients. In this sense they are following the principle of synthetic biology in which individual cellular building blocks are assembled to create artificial biological systems with new characteristics.

The vision of the biophysicists was to create a cell-like model with a biomechanical function. It should be able to move and change its shape without external influences. They explain how they achieved this goal in their latest publication in Science.

The magic ball

The biophysicists’ model comprises a membrane shell, two different kinds of biomolecules and some kind of fuel. The envelope, also known as a vesicle, is made of a double-layered lipid membrane, analogous of natural cell membranes. The scientists filled the vesicals with microtubules, tube-shaped components of the cytoskeleton, and kinesin molecules. In cells, kinesins normally function as molecular motors that transport cellular building blocks along the microtubules. In the experiment, these motors permanently push the tubules alongside each other. For this, kinesins require the energy carrier ATP, which was also available in the experimental setup.

From a physical perspective, the microtubules form a two-dimensional liquid crystal under the membrane, which is in a permanent state of motion. “One can picture the liquid crystal layer as tree logs drifting on the surface of a lake,” explains Felix Keber, lead author of the study. “When it becomes too congested, they line up in parallel but can still drift alongside each other.”

Migrating faults

Decisive for the deformation of the artificial cell construction is that, even in its state of rest, the liquid crystal must always contain faults. Mathematicians explain these kinds of phenomena by way of the Poincaré-Hopf theorem, figuratively also referred to as the “hairy ball problem.” Just as one can’t comb a hairy ball flat without creating a cowlick, there will always be some microtubules that cannot lay flat against the membrane surface in a regular pattern. At certain locations the tubules will be oriented somewhat orthogonally to each other – in a very specific geometry. Since the microtubules in the case of the Munich researchers are in constant motion alongside each other due to the activity of the kinesin molecules, the faults also migrate. Amazingly, they do this in a very uniform and periodic manner, oscillating between two fixed orientations.

Spiked extensions

As long as the vesicle has a spherical shape, the faults have no influence on the external shape of the membrane. However, as soon as water is removed through osmosis, the vesicle starts to change in shape due to the movement within the membrane. As the vesicle loses ever more water, slack in the membrane forms into spiked extensions like those used by single cells for locomotion.

In this process, a fascinating variety of shapes and dynamics come to light. What seems random at first sight is, in fact, following the laws of physics. This is how the international scientists succeeded in deciphering a number of basic principles like the periodic behavior of the vesicles. These principles, in turn, serve as a basis for making predictions in other systems.

“With our synthetic biomolecular model we have created a novel option for developing minimal cell models,” explains Bausch. “It is ideally suited to increasing the complexity in a modular fashion in order to reconstruct cellular processes like cell migration or cell division in a controlled manner. That the artificially created system can be comprehensively described from a physical perspective gives us hope that in the next steps we will also be able to uncover the basic principles behind the manifold cell deformations.”

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Scientists call for investigation of mysterious cloud-like collections in cells

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About 50 years ago, electron microscopy revealed the presence of tiny blob-like structures that form inside cells, move around and disappear. But scientists still don’t know what they do — even though these shifting cloud-like collections of proteins are believed to be crucial to the life of a cell, and therefore could offer a new approach to disease treatment.

Jeffrey Toretsky, MD, professor in the department of oncology and pediatrics at Georgetown Lombardi Comprehensive Cancer Center

In the Journal of Cell Biology, two researchers are issuing a call to investigators from various backgrounds, from biophysics to cell biology, to focus their attention on the role of these formations— for which they coin a new unifying term “assemblages.”

“I want to know what these assemblages are doing in Ewing sarcoma, the disease I concentrate on — and I would think all other researchers who study human biology would want to know their functions in both health and disease,” says Jeffrey Toretsky, MD, professor in the department of oncology and pediatrics at Georgetown Lombardi Comprehensive Cancer Center.

So Toretsky partnered with co-author Peter Wright, PhD, professor in the department of integrative structural and computational biology at The Scripps Research Institute in La Jolla, Calif., to pull together all the biophysics and protein biochemistry knowledge available on assemblages into a review article. Toretsky also called on the expertise of chemists and physicists from Georgetown University.

The authors say these assemblages are often, but not always, made up of proteins that are intrinsically disordered, meaning that they do not assume a specific shape in order to fit like a lock and key onto other proteins. These intrinsically disordered proteins seem to find each other and then form into gel-like assemblages — a process called “phase separation” — that can trap and interact with other proteins and even RNA, biological molecules that help decode and regulate genes.

When their work is done — whatever that is — the assemblages dissolve, Toretsky says.

“It is only in the last five years that researchers have begun recognizing that proteins without fixed structures may have important transitional properties that change based upon their local abundance in cells,” he says.

Toretsky suspects that if these assemblages play a role in disease, they could be targeted with a small molecule. “Current drug-discovery dogma suggests that it is very hard to make a small molecule to prevent two structured proteins from interacting. However, small molecules have a greater likelihood of disrupting intrinsically disordered protein-protein interactions,” he says.

“This review links together very basic biologic phenomena of protein interaction with the potential for new drug discovery,” Toretsky says. “It’s an exciting challenge.”

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The above story is based on materials provided by Georgetown University Medical Center.

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Memory in silent neurons

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BIOENGINEER.ORG http://bioengineer.org/memory-silent-neurons/

According to a generally-accepted model of synaptic plasticity, a neuron that communicates with others of the same kind emits an electrical impulse as well as activating its synapses transiently.

This is a group of neurons. Photo Credit: EPFL/Human Brain Project

This electrical pulse, combined with the signal received from other neurons, acts to stimulate the synapses. How is it that some neurons are caught up in the communication interplay even when they are barely connected? This is the chicken-or-egg puzzle of synaptic plasticity that a team at UNIGE is aiming to solve.

When we learn, we associate a sensory experience either with other stimuli or with a certain type of behavior. The neurons in the cerebral cortex that transmit the information modify the synaptic connections that they have with the other neurons. According to a generally-accepted model of synaptic plasticity, a neuron that communicates with others of the same kind emits an electrical impulse as well as activating its synapses transiently. This electrical pulse, combined with the signal received from other neurons, acts to stimulate the synapses. How is it that some neurons are caught up in the communication interplay even when they are barely connected? This is the crucial chicken-or-egg puzzle of synaptic plasticity that a team led by Anthony Holtmaat, professor in the Department of Basic Neurosciences in the Faculty of Medicine at UNIGE, is aiming to solve.

The results of their research into memory in silent neurons can be found in the latest edition of Nature.

Learning and memory are governed by a mechanism of sustainable synaptic strengthening. When we embark on a learning experience, our brain associates a sensory experience either with other stimuli or with a certain form of behavior. The neurons in the cerebral cortex responsible for ensuring the transmission of the relevant information, then modify the synaptic connections that they have with other neurons. This is the very arrangement that subsequently enables the brain to optimize the way information is processed when it is met again, as well as predicting its consequences.

Neuroscientists typically induce electrical pulses in the neurons artificially in order to perform research on synaptic mechanisms.

The neuroscientists from UNIGE, however, chose a different approach in their attempt to discover what happens naturally in the neurons when they receive sensory stimuli. They observed the cerebral cortices of mice whose whiskers were repeatedly stimulated mechanically without an artificially-induced electrical pulse. The rodents use their whiskers as a sensor for navigating and interacting; they are, therefore, a key element for perception in mice.

An extremely low signal is enough

By observing these natural stimuli, professor Holtmaat’s team was able to demonstrate that sensory stimulus alone can generate long-term synaptic strengthening without the neuron discharging either an induced or natural electrical pulse. As a result – and contrary to what was previously believed – the synapses will be strengthened even when the neurons involved in a stimulus remain silent.In addition, if the sensory stimulation lasts over time, the synapses become so strong that the neuron in turn is activated and becomes fully engaged in the neural network. Once activated, the neuron can then further strengthen the synapses in a forwards and backwards movement. These findings could solve the brain’s “What came first?” mystery, as they make it possible to examine all the synaptic pathways that contribute to memory, rather than focusing on whether it is the synapsis or the neuron that activates the other.

The entire brain is mobilized

A second discovery lay in store for the researchers. During the same experiment, they were also able to establish that the stimuli that were most effective in strengthening the synapses came from secondary, non-cortical brain regions rather than major cortical pathways (which convey actual sensory information). Accordingly, storing information would simply require the co-activation of several synaptic pathways in the neuron, even if the latter remains silent. These findings may also have important implications both for the way we understand learning mechanisms and for therapeutic possibilities, in particular for rehabilitation following a stroke or in neurodegenerative disorders. As professor Holtmaat explains: “It is possible that sensory stimulation, when combined with another activity (motor activity, for example), works better for strengthening synaptic connections”. The professor concludes: “In the context of therapy, you could combine two different stimuli as a way of enhancing the effectiveness.”

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The above story is based on materials provided by University of Geneva.

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A new way to diagnose malaria

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Using magnetic fields, technique can detect parasite’s waste products in infected blood cells.

Red blood cells from a patient infected with Plasmodium falciparum. Photo Credit: Osaro Erhabor

Over the past several decades, malaria diagnosis has changed very little. After taking a blood sample from a patient, a technician smears the blood across a glass slide, stains it with a special dye, and looks under a microscope for the Plasmodium parasite, which causes the disease. This approach gives an accurate count of how many parasites are in the blood — an important measure of disease severity — but is not ideal because there is potential for human error.

A research team from the Singapore-MIT Alliance for Research and Technology (SMART) has now come up with a possible alternative. The researchers have devised a way to use magnetic resonance relaxometry (MRR), a close cousin of magnetic resonance imaging (MRI), to detect a parasitic waste product in the blood of infected patients. This technique could offer a more reliable way to detect malaria, says Jongyoon Han, a professor of electrical engineering and biological engineering at MIT.

“There is real potential to make this into a field-deployable system, especially since you don’t need any kind of labels or dye. It’s based on a naturally occurring biomarker that does not require any biochemical processing of samples” says Han, one of the senior authors of a paper describing the technique in the Aug. 31 issue of Nature Medicine.

Peter Rainer Preiser of SMART and Nanyang Technical University in Singapore is also a senior author. The paper’s lead author is Weng Kung Peng, a research scientist at SMART.

Hunting malaria with magnets

With the traditional blood-smear technique, a technician stains the blood with a reagent that dyes cell nuclei. Red blood cells don’t have nuclei, so any that show up are presumed to belong to parasite cells. However, the technology and expertise needed to identify the parasite are not always available in some of the regions most affected by malaria, and technicians don’t always agree in their interpretations of the smears, Han says.

“There’s a lot of human-to-human variation regarding what counts as infected red blood cells versus some dust particles stuck on the plate. It really takes a lot of practice,” he says.

The new SMART system detects a parasitic waste product called hemozoin. When the parasites infect red blood cells, they feed on the nutrient-rich hemoglobin carried by the cells. As hemoglobin breaks down, it releases iron, which can be toxic, so the parasite converts the iron into hemozoin — a weakly paramagnetic crystallite.

Those crystals interfere with the normal magnetic spins of hydrogen atoms. When exposed to a powerful magnetic field, hydrogen atoms align their spins in the same direction. When a second, smaller field perturbs the atoms, they should all change their spins in synchrony — but if another magnetic particle, such as hemozoin, is present, this synchrony is disrupted through a process called relaxation. The more magnetic particles are present, the more quickly the synchrony is disrupted.

“What we are trying to really measure is how the hydrogen’s nuclear magnetic resonance is affected by the proximity of other magnetic particles,” Han says.

For this study, the researchers used a 0.5-tesla magnet, much less expensive and powerful than the 2- or 3-tesla magnets typically required for MRI diagnostic imaging, which can cost up to $2 million. The current device prototype is small enough to sit on a table or lab bench, but the team is also working on a portable version that is about the size of a small electronic tablet.

After taking a blood sample and spinning it down to concentrate the red blood cells, the sample analysis takes less than a minute. Only about 10 microliters of blood is required, which can be obtained with a finger prick, making the procedure minimally invasive and much easier for health care workers than drawing blood intravenously.

“This system can be built at a very low cost, relative to the million-dollar MRI machines used in a hospital,” Peng says. “Furthermore, since this technique does not rely on expensive labeling with chemical reagents, we are able to get each diagnostic test done at a cost of less than 10 cents.”

Tracking infection

Hemozoin crystals are produced in all four stages of malaria infection, including the earliest stages, and are generated by all known species of the Plasmodium parasite. Also, the amount of hemozoin can reveal how severe the infection is, or whether it is responding to treatment. “There are a lot of scenarios where you want to see the number, rather than a yes or no answer,” Han says.

In this paper, the researchers showed that they could detect Plasmodium falciparum, the most dangerous form of the parasite, in blood cells grown in the lab. They also detected the parasite in red blood cells from mice infected with Plasmodium berghei.

This new technique is “more sensitive, less error-prone, and requires less blood sample as compared to the standard blood-smear protocol,” says Donhee Ham, a professor of electrical engineering at Harvard University who was not part of the research team. “I think there is a strong potential here, and I look forward to its further development for reliable field deployment.”

The researchers are launching a company to make this technology available at an affordable price. The team is also running field tests in Southeast Asia and is exploring powering the device on solar energy, an important consideration for poor rural areas.

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The above story is based on materials provided by MIT News Office, Anne Trafton.

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