by Rob Mitchum

The brain is a privileged organ, afforded protections denied to all the other organs of the body. Though the circulatory system functions much the same way above and below the neck, using blood to exchange nourishment for waste with cells, the exchange is conducted under much heavier security in the central nervous system. This TSA for the brain and spinal cord is known as the blood-brain barrier, and its role is protecting the fragile, irreplaceable cells of the nervous system from external disease and the body’s own immune weapons.

While we can all be thankful for the unceasing service of the blood-brain barrier (sometimes abbreviated as BBB), many scientists are interested in figuring out how it can be breached. Many neurological diseases, including multiple sclerosis and stroke, can be attributed in part to breakdowns of the BBB. Drugs designed to treat brain disease must also find a way through the BBB’s strong defenses to get to their desired targets.

That made the blood-brain barrier the perfect subject for this year’s Chicago Symposium on Translational Neuroscience, an annual gathering of neurologists and laboratory neuroscientists from the University of Chicago Medicine and other institutions. This year, the Neuro contingent was joined by UChicago’s young Institute for Molecular Engineering in presenting the conference, underscoring that the topic is very much an engineering problem: how do you build a blood-brain barrier, and how do you selectively knock it down?

The day’s first speaker, Richard Daneman of the University of California, San Francisco, explained what the field currently knows about the unique properties of the BBB. In most of the body, the capillaries of the circulatory system are “leaky,” he said, allowing many molecules to pass between the cells and the blood through the cracks between the endothelial cells that make up the blood vessel walls. But in the BBB, those cells form a tight seal, and molecules are transported into and out of the vessels by highly selective transporters instead of passive diffusion. Daneman illustrated these defenses with an experiment where blue dye is injected into an animal, which is later dissected. While organs such as the kidney or liver take on a distinct blue hue, the brain and spinal cord remain free from the dye, which cannot penetrate the barrier.

Daneman is interested in “barriergenesis,” how the BBB is constructed during development. Previously, researchers hypothesized that brain cells called astrocytes were responsible for building the BBB. But using mice and rat models, Daneman’s laboratory determined that this defense system is already in place during embryonic stages, before astrocytes first appear. Instead, Daneman’s experiments pointed to another cell group called pericytes as critical architects of the BBB. When the genes for forming pericytes were knocked out in a mouse line, the BBB did not form its tight seal…as indicated by the appearance of blue brains after a dye injection.

Now Daneman’s lab is digging into the molecular signals that construct the BBB during development and maintain its integrity throughout life. Some of those experiments involve taking the endothelial cells that form the BBB out of their native habitat to study them on the lab bench, the subject of a talk from Eric Shusta of the University of Wisconsin - Madison. Endothelial cells only make up about one-tenth of one percent of the cells in the brain, Shusta said, and don’t form the tight seals characteristic of the BBB in the lab dish unless they are co-cultured with other neural cell types.

Shusta’s laboratory has tackled these problems using the hot prospects of laboratory science: pluripotent stem cells. When researchers in his group decided to try to differentiate stem cells into BBB-like endothelial cells, Shusta said “I thought they were a little bit crazy, but the initial experiments worked.” What’s more, with neural stem cells, the researchers could also generate other nervous system cell types that might play an important role in barriergenesis. That experimental set-up can now be used to test for the cellular factors that build the BBB and as well as assess different drugs’ ability to pass through the barrier, without the constraints of having to endlessly harvest scarce endothelial cells.

“From one rat brain we can get about 6 to 12 filters, but from one vial of stem cells, we can easily get ten thousands of filters,” Shusta said. “We think that we can keep optimizing this, and hopefully make an impact in developmental and drug screening applications.”

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by Rob Mitchum

A common feature of pharmacies and organic grocery stores is the aisle of natural remedies, featuring bottle upon bottle of herbs, extracts, and oils that promise a wide range of medical benefits. For legal reasons, the health claims made by these products are often fuzzy, boasting of vague antioxidant or anti-inflammatory activity. But online, the compounds are touted as a cure-all for everything from the common cold to depression to cancer, despite often scarce scientific evidence to support such claims. In many cases, scientists aren’t even sure what these compounds do on a biological level, limiting their usefulness in the clinic even if an anti-disease effect could be conclusively demonstrated.

The major obstacle to determining how a natural remedy works (or doesn’t) is the difficulty in assessing the totality of its effects upon a cell, rather than just the effect on one particular factor at a time. But a technique recently invented by University of Chicago researchers to monitor the activity of hundreds of proteins at once allowed scientists to assess the anti-cancer potential of one natural remedy aisle staple: beehive propolis.

“A typical problem in bringing some of these herbal remedies into the clinic is that nobody knows how they act, nobody knows the mechanism, and therefore researchers are typically very hesitant to add them to any pharmaceutical treatment strategy,” said Richard Jones, assistant professor in the Ben May Department for Cancer Research and Institute for Genomics and Systems Biology. “Now we’ll actually be able to systematically demonstrate the parts of cell physiology that are affected by these compounds.”

Beehive propolis is a sticky resin that honeybees use to patch up holes and fill cracks in their hives. Natural remedy suppliers sell this “bee glue” in the form of capsules or liquid extract, touting its abilities to boost immunity, fight off infections, and soften skin. According to anecdotal reports, the substance has been used for centuries to treat sore throats, allergies, and burns, or for less medicinal purposes such as car wax and instrument polish.

Chih-Pin Chuu, at the time a post-doctoral researcher in Jones’ laboratory, wanted to examine whether the active compound in beehive propolis — called caffeic acid phenethyl ester, or CAPE — was effective against cancer cells. Testing concentrations of CAPE that you would expect to find in the blood after a person swallowed a propolis capsule, Chuu found the compound successfully inhibited the growth of early-stage prostate cancer cell in culture dish experiments. Subsequent experiments on mice implanted with human prostate cancer cells confirmed CAPE’s anti-cancer effect, and hinted at a mechanism.

“If you feed CAPE to mice daily, their tumors will stop growing. After several weeks, if you stop the treatment, the tumors will begin to grow again at their original pace,” Jones said of the results, published in the journal Cancer Prevention Research. “So it doesn’t kill the cancer, but it basically will indefinitely stop prostate cancer proliferation.”

That activity suggests CAPE could be a promising co-treatment alongside a chemotherapy drug that targets the cells. But if CAPE were to truly make the crossover from holistic remedy to clinical option, the scientists would also have to demonstrate how the compound freezes cancer cells in a non-proliferative state. Enter the micro-western array, the innovative proteomics technique first described in 2010 by Jones and colleagues.

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By Rob Mitchum

Brucella abortus is a particularly pesky pathogen. Frequently infecting cattle in many countries around the world, the bacterium causes the most common zoonotic infection, usually passing from animal to humans through ingestion of unpasteurized dairy products. While the infection, known as brucellosis or undulant fever, is rarely deadly, it can cause assorted flu-like symptoms including high fever, muscle pain, weight loss, and in severe cases, meningitis and encephalitis. These debilitating effects made it appealing to both the United States and Soviet Union for the development of biological weapons during the Cold War. Although these “brucella bombs” have long since been destroyed, the bacterium remains a concern around the world.

“It’s a substantial human health problem on a global level,” said Sean Crosson, assistant professor of biochemistry and molecular biology at the University of Chicago Biological Sciences. “The World Health Organization predicts that there are a half million cases annually, and it may be higher than that. It’s hard to know, because it’s more of a problem in the developing world where it’s difficult to quickly diagnose.”

Crosson’s laboratory is one of the few in the United States studying the bacterium and its surprisingly evasive behavior. The infection is very difficult to treat, requiring two different antibiotics to be taken for nearly two months — not an easy task in countries without easy access to pharmacies and the proper storage of the heat-sensitive drugs. Even when this rigorous treatment is followed under ideal conditions, the relapse rate for the disease is still high. When the Crosson lab searched through the genome of the bacterium, they found a potential reason for this resilience: a genetic poison and antidote pair usually thought of as a bacterial self-destruct mechanism.

“After treatment for brucellosis, there’s still a 15 percent chance of relapse,” said Brook Heaton, a Committee on Microbiology graduate student in Crosson’s lab. “This relapse is something that is obviously problematic, but also interesting when thinking of toxin-antitoxin systems, which are thought to play a role in persistence of infection.”

Toxin-antitoxin systems have been discovered in a variety of bacterial species, and studied primarily for their role in bacterial quality control. The tiny toxin and antitoxin genes were initially found on plasmids, small bits of bacterial DNA separate from the chromosomes. The two genes are typically transcribed together, producing both the toxin and antitoxin proteins simultaneously. Under normal conditions, the two proteins would stick together, nullifying the toxic activity like a sword in a sheath. But in this case, the antitoxin “sheath” degrades faster than the toxic “sword.” So if gene transcription stops for some reason or the cell divides and the daughter cell does not properly inherit a copy of the plasmid, then the self-destruct process begins.

But over the past decade, scientists started to find toxin-antitoxin genes on the chromosomes themselves, throwing the quality control theory into doubt.

“What exactly these things are doing is mysterious,” Crosson said. “They’ve been implicated in bacterial stress response, they get activated by certain stressors, and if they’re missing toxin-antitoxin genes then bacterial cells may not survive as well in certain environments. But the real biological function of toxin-antitoxin genes is still unclear.”

In a paper recently published by The Journal of Biological Chemistry, Heaton and her colleagues examined one toxin-antitoxin system from Brucella abortus with a wide variety of scientific techniques, from genetic manipulation and cell culture experiments carried out at the Ricketts Regional Biocontainment Laboratory at Argonne to biophysical analyses of protein structure. Functionally, when the toxin (named BrnT) was unleashed without its antitoxin (named BrnA), protein synthesis screeched to a halt and the bacterial cells stopped growing within an hour. But reports of the bacterial massacre were greatly exaggerated — subsequent activation of the antitoxin, even hours later, resurrected the cells to a normal, healthy state.

“On the surface, when you express the toxin, it looks almost like all the cells are dying,” Crosson said. “99.999% of the cells appear to die. But they’re not actually dead.”

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The rise of optogenetics — where flashes of light can manipulate brain activity and behavior — have excited scientists looking for more precise ways of manipulating cells and their components in the laboratory and the clinic. Two papers published this month by University of Chicago laboratories explore new methods with great scientific potential of controlling cells through light.  We’ll cover them in two installments.

Many researchers hope that the light-activated cell technologies currently being used in laboratories will eventually cross the threshold into the clinic. The ability of optogenetics to relieve anxiety in lab rats could someday inform a similar human treatment, where psychiatric symptoms are reduced through a light-delivery implant that stimulates the right brain regions and neurotransmitters on command. But most methods that tweak cell activity with light require some form of genetic manipulation to work, and researchers have so far encountered steep barriers to effectively and safely performing gene therapy in humans.

One potential workaround would be to use a method of light activation that requires no genetic tinkering: infrared light. These long-wavelength light spectra are already used for a variety of purposes, from night vision devices to weather observation to detecting fraudulent paintings. In biology, scientists found that infrared light is capable of exciting cells that discharge electricity, such as neurons or heart cells, prompting optimism about medical uses for this less complicated form of photo-activity.

“If you’re thinking about clinical applications, already the requirement of a light fiber to deliver the light stimulus is a hurdle,” said Mikhail Shapiro, a Miller Research Fellow at the University of California, Berkeley. “If you add on top of that the need for gene therapy or using caged neurotransmitters or other light-sensitive chromophore molecules, you’re adding an additional layer of complications. I think part of the appeal of the infrared approach is all you need is the light.”

However, there was one caveat underlying the promise of infrared cell excitation: nobody was exactly sure how it worked. That was the question that Shapiro set out to tackle during a postdoctoral scholarship in the laboratory of Francisco Bezanilla, Lillian Eichelberger Cannon Professor of Biochemistry and Molecular Biophysics at the University of Chicago Biological Sciences. Over several months of experiments, recently published in an article in Nature Communications, Shapiro and his colleagues searched for the mechanism by which infrared light excites cells…eventually settling on a surprising conclusion.

“I think the interesting thing here is it’s a case where the use of the technology leaps ahead of the understanding of how it works,” Shapiro said. “But if you’re implanting things into your body and stimulating and you don’t know how it works, how comfortable can you really be with that? Are you making little holes in these cells that are exciting them?”

Zapping a cell with infrared light produces an inward current, a sign that the cell is depolarizing. In a neuron, those types of inward currents are created by the opening of ion channels, allowing positive ions such as sodium to come flooding into the cell and pushing it toward the firing of an action potential. So Shapiro, Bezanilla, and collaborators Kazuaki Homma, Sebastian Villareal, and Claus-Peter Richter started their experiments by testing infrared light on Xenopus oocytes — frog egg cells that serve as a common laboratory cell model — injected with DNA for different types of ion channels. Frustratingly, the experiments did not initially provide a simple answer.

“We tested the infrared light, and found out that regardless of what you injected, it was exactly the same response,” Bezanilla said. “Then we said, maybe we don’t need to inject anything. Sure enough, if you don’t inject anything you get exactly the same response again.”

The failure to find an ion channel sent the researchers back to basics. Infrared’s long wavelengths are capable of locally exciting molecules, producing very rapid increases in temperature. So Shapiro measured the effect of the infrared laser on water held at the same distance as their cells, and found that his beams did indeed have a heating effect. What’s more, the inward current caused by infrared light correlated with the rate of this temperature change. The results indicated that the cellular component sensitive to infrared light wasn’t a complex protein, but plain old water.

“It was very challenging, because the type of responses we were getting were very unusual,” Bezanilla said. “But when we started looking at the temperature changes, that really gave us some clue what was happening here. You don’t need to put in a special molecule, because the molecule that you use to produce the effect is the water. You don’t have to genetically engineer anything, you just apply the infrared in the right place and you get the effect.”

Further experiments determined how these temperature changes could produce cell excitation.

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The rise of optogenetics — where flashes of light can manipulate brain activity and rat behavior — have excited scientists looking for more precise ways of manipulating cells and their components in the laboratory and the clinic. Two papers published this month by University of Chicago laboratories explore new methods with great scientific potential of controlling cells through light  We’ll cover them in two installments.

Many important cellular functions are principally the result of factors being in the right place at the right time. The complex pathways that handle important jobs such as cell growth or migration can have hundreds of components that must arrive at the same location within the busy cellular environment. As such, biologists would like the ability to move proteins to different parts of the cell at will, giving them control over the staging of these pathways and their functions. With a new system that draws upon the work of several University of Chicago laboratories, researchers have figured out how to achieve this power with light.

Like many good ideas in science, the collaboration that created TULIPs began with a question posed at a bar. At the annual Molecular Biosciences retreat in Galena, Illinois, Devin Strickland and Tobin Sosnick challenged their colleague Michael Glotzer with a provocative question.

“They asked, ‘If you could control any protein you wanted, what would you control?’,” recalled Glotzer, Professor of Molecular Genetics & Cell Biology at the University of Chicago.

Strickland, then a graduate student in the laboratory of Sosnick, Professor and Chair of Biochemistry & Molecular Biophysics, were looking for applications of  their strategy of using light to control the activity of a specific protein. Glotzer realized that the system could bring two proteins together in a cell, whenever and wherever researchers wanted. The result was an exciting new technique, called TULIPs, published this month in Nature Methods.

The acronym TULIPs stands for TUnable, Light-controlled Interacting Protein tags. Instead of  the light-activated ion channels or gene transcription used by other optogenetic methods, the TULIPs use a LOV domain, a light-sensitive component from a protein plants use to detect and grow toward sunlight. By attaching different proteins to this domain and another known as an ePDZ “clamp,” the researchers built a customizable way of precisely manipulating cellular signals in both space and time.

“Basically nothing previously had been designed to control cell signaling that wasn’t engineered on a case-by-case basis,” said Strickland, now a postdoctoral researcher in Glotzer’s laboratory. “We started off wanting to be the first people to create an adaptable system.”

In the Nature Methods paper, the authors describe testing the system by attaching it to fluorescent proteins and applying it to Glotzer’s chosen target, a GTPase pathway that controls growth and genes related to mating in yeast. The experiments demonstrated both that the TULIPs worked, and that it could be easily adapted to manipulate a wide range of cellular signals.

“The idea was let’s solve this generic problem once, and then we’ll be able to reuse that solution in many different contexts,” Glotzer said. “Looking at lots of different biological phenomena, it’s pretty clear that a lot of functions correlate with changes in sub-cellular localization of specific proteins. So there’s almost no limit; you’re only limited by your imagination.”

[Light flashes on the right demonstrate the successful localization of fluorescent proteins attached to the TULIPs system. Video by Elizabeth Wagner]

The TULIP system is made up of two components that draw upon work done previously by the University of Chicago laboratories of Shohei Koide, who developed the ePDZ clamp, and Keith Moffat, who characterized the LOV domain and first suggested its use in the TULIP system.

“It’s really a nice story of how basic research can lead to things that aren’t really anticipated,” Strickland said.

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By Rob Mitchum

Alan Turing is best known as the father of the modern computer, a skillful World War II codebreaker, and a pioneer in the study of artificial intelligence. But in the last years before Turing’s death at age 41, he  aimed his genius at a different target: the then-stalled field of developmental biology. By the middle of the 20th century, many scientists had tried and failed to explain how a complex organism could form itself from a simple embryo originally made up of identical cells. One 19th century biologist, Hans Driesch, grew so frustrated with the problem that he gave up and wrote a text on vitalism, the doctrine that life cannot be explained by science alone.

In a 1952 paper called “The Chemical Basis of Morphogenesis,” Turing rushed headlong into this challenge, building a mathematical model of how patterned cells can be formed from non-patterned beginnings. It was Turing’s only published work on the topic; he died two years later. But in those 35 pages, he predicted elements of developmental biology that wouldn’t be discovered for 30 more years, coined a term that is central to the field today, and accidentally sparked a new sub-field of mathematical study for a bonus. In a recent Nature retrospective commemorating Turing’s 100th birthday, University of Chicago scientist John Reinitz wrote, “What Turing should receive credit for is opening the door to a new view of developmental biology…He was well ahead of his time.”

Reinitz’s own research is deeply indebted to Turing’s landmark paper. A professor with appointments in Statistics, Ecology & Evolution, and Molecular Genetics & Cell Biology, Reinitz’s laboratory studies how gene expression controls the development of the fruit fly Drosophila melanogaster. As part of those efforts, the laboratory has built several computational models of gene transcription and fly development, one of which is a specific example of a class of equations in Turing’s paper, Reinitz said in an interview about his essay.

Beyond that direct lineage, Reinitz admires the paper (”The article is just a pile of interesting ideas.”) and teaches it in his courses. But it wasn’t fully appreciated in the field of developmental biology until decades after its publication, when the role of DNA and the molecules that Turing preemptively named “morphogens” became more widely known in biology.

“When I was in grad school, this paper was circulating, and it was considered to be a sort of interesting but crazy paper,” Reinitz said. “It didn’t have anything about genes, and when I first saw it, it was really before any of these morphogens had actually been found. So it didn’t seem to have any direct bearing on actual experimental science.”

The core of the paper is a computational model — one of the first ever published, Reinitz said — that mathematically proved one could create complex patterns from a symmetrically organized cell. Early in development, the “pluripotent” cells of the embryo are each capable of developing into a wide range of cell types, from blood to skin to muscle to hair. Indeed, if an embryo is split in two early enough, it can form two entire organisms…as is the case with identical twins.

Classic linear mathematics can’t explain how one generic cell can produce so many unique descendants. So Turing’s model employed a “mathematical trick,” using the interplay of two diffusing factors (Turing’s “morphogens”) to produce the temporary instability necessary for a pattern to form. That these morphogens had never been observed in scientific experiments at the time he published was beside the point; Turing simply wanted to show that pattern-making could be done with a minimum of elements.

“I think that one of the things that’s seriously misunderstood about the paper is that a lot of people read it and think it’s making specific predictions about biological systems,” Reinitz said. “The main thing he was concerned about was just demonstrating that you could form patterns from non-patterns. He wanted to show with chemistry that you can have patterns form spontaneously.”

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By Rob Mitchum

Cells, like people, are not perfect. If a cell’s primary responsibility is to produce proteins, then it makes a remarkable amount of mistakes in that job, with some studies estimating that an error appears in as many as 1 out of every 5 proteins. Defective proteins can be a serious problem — scientists are learning that many aging-related neurological illnesses, such as ALS, Alzheimer’s, and Parkinson’s disease, are caused in part by faulty proteins clumping together into neuron-killing aggregates. Cells have a quality inspection and trash disposal system in place to neutralize these toxic defects, but that’s an expensive way to deal with a problem with a simpler solution: why don’t cells just make better proteins?

That question has fascinated D. Allan Drummond, assistant professor of biochemistry and molecular biophysics at the University of Chicago Biological Sciences, since his graduate work in the laboratory of Frances Arnold at CalTech studying directed evolution. His work on the high error rate of protein production, otherwise known as a cell’s “translational infidelity,” led to Drummond’s inclusion in the 2012 class of Sloan Research Fellows, an award recognizing young scholars with exciting ideas across various scientific fields. Drummond’s previous research has revealed some promising clues about why protein errors, in some cases, may be a good thing for a cell, and he thinks the field is on the verge of real progress thanks to technical advances.

“There previously existed no method at all to assess and measure the true infidelity of translation,” Drummond said. “Now we have higher quality mass spectrometry, which allows you to do essentially do for proteins what DNA sequencing does for genomes. Mass spectrometry is now sensitive enough to detect mistranslated proteins on a large scale.”

One hypothesis that Drummond would like to tackle with that technology is the idea that cells are unexpectedly crafty in how they deal with their protein error rate. Experiments have shown that the errors are a necessary sacrifice cells will make to increase the speed of protein translation. Cells with more stringent quality control grow more slowly, which under the highly competitive conditions of natural selection, can be a fatal luxury. So if the cells must endure a fairly high error rate in order to keep up with the Joneses, the least they can do is pick and choose the best places to put those errors.

Some proteins, like the enzymes used in glycolysis, are kept at high levels in cells, with as many as a million copies floating around at any one time. Other proteins, such as the transcription factors, are nowhere near as plentiful, with only tens or hundreds of copies on hand. If a cell could somehow channel its error rate toward the second group of proteins and away from the first, it would be much better off, Drummond said.

“It’s going to be devastating in the case of the glycolytic enzymes, because the misfolded products alone will be more abundant than most proteins in the cell. It’s spamming the cell with all sorts of garbage,” Drummond said. “If you put errors in a low abundance protein, the amount of misfolding you’re going to get is probably negligible.”

Fortunately, cells do have a way to selectively disperse their protein errors. Proteins are built out of amino acids, directed by RNA codons that correspond to the original DNA recipe. But each amino acid is associated with multiple codons, and some codons are more error-prone than others. Analyses by Drummond’s laboratory found that the higher-fidelity codons that make fewer errors are used more often in building high expression proteins, while the less reliable codons appear more frequently in rarer proteins.

But that strategy begs the question: why would a cell ever use anything but the most reliable codons? Drummond has a provocative answer that challenges whether all mistakes are created equal.

“The most exciting idea is that these are not errors,” Drummond said. “They’re so frequent, they’re believed to be present at such high levels, that it is almost inconceivable that cells have not become addicted to the presence of some of them. In a sense, they could be using a single DNA sequence to make multi-functional proteins.”

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By John Easton

Once considered the cause of cancer, a tiny organelle known as the “powerhouse of the cell” may soon spawn a new treatment.

In 1955, Otto Warburg, recipient of the 1931 Nobel Prize for Medicine or Physiology, attributed cancer to damage to the mitochondria, tiny structures within each cell that are involved in energy production, the manufacture of ATP. Because of irreversible damage to the mitochondria, he argued, tumor cells shifted from respiration to fermentation, a much less efficient method for producing ATP.

“What was formerly only qualitative has now become quantitative,” Warburg said during a Stuttgart lecture reprinted by Science. “What was formerly only probable has now become certain. The era in which the fermentation of cancer cells or its importance could be disputed is over, and no one today can doubt that we understand the origin of cancer cells if we know how their large fermentation originates.”

With those confident words, Warberg hoped to put an end to disputes about the many potential causes of cancer. “I should like to add, as a further argument,” he continued, “the fact that there is no alternative today… From this point of view, mutation and carcinogenic agents are not alternatives, but empty words.”

As new information became available, the words mutation and carcinogenic agents were gradually reinflated and the notion of mitochondrial damage as the root cause of all cancers lost favor. Interest in mitochondria shifted from oncologists to scientists interested in liver or muscle biology, especially cardiologists studying heart muscle.

But Stephen L. Archer, the Harold Hines Jr. Professor of Medicine at the University of Chicago Medicine, a cardiologist specializing in pulmonary hypertension, and Jalees Rehman, a German scientist who worked with Archer, got interested all over again in studying mitochondria after reading some of Warburg’s historical papers. Instead of causing cancer, they wondered, could mitochondria provide a target for cancer therapy?

Within each cell, mitochondria are perpetually splitting in two, a process called fission, and merging back into one, called fusion. Before a cell can divide, the mitochondria must increase their numbers through fission and separate into two piles, one for each cell.

This makes them a promising new target for cancer therapy. By manipulating two of the biochemical signals that regulate the numbers of mitochondria in cells, the researchers found they could shrink human lung cancers transplanted into mice, a discovery they reported in February in the journal FASEB.

By tipping the balance toward fusion and away from fission in rapidly dividing cancer cells, Archer and colleagues were able to dramatically reduce cell division and prevent the rapid cell proliferation that is a hallmark of cancer growth. Increasing production of the signal that promotes fusion caused tumors to shrink to one-third of their original size. Treatment with a molecule that inhibits fission reduced tumor size by more than half.

“By boosting the fusion signal or blocking the fission signal we were able to tip the balance the other way, reducing cancer cell growth and increasing cell death,” said Archer, senior author of the study. “We believe this provides a promising new approach to cancer treatment.”

“This could be a potential new Achilles’ heel for cancer cells,” said lead author, Rehman, now an associate professor of medicine and pharmacology at the University of Illinois at Chicago. “Many anticancer drugs target cell division. Our work shifts the focus to a distinct but necessary step: mitochondrial division. The cell division cycle comes to a halt if the mitochondria are prevented from dividing. This new therapy may be especially useful in cancers which become resistant to conventional chemotherapy that directly targets the cycle.”

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By Rob Mitchum

Among the most hyped cancer therapies for the future, microRNA looms large. While much smaller than the RNA produced by protein-coding genes, these tiny transcripts play an important regulatory role in cells by acting as a brake on the process of making proteins from genes. MicroRNAs bind to their relatives, the messenger-RNAs, which are used by the cell as a recipe for making new proteins. When this binding occurs, the protein builders (called ribosomes) can no longer attach to messenger-RNA, essentially halting production.

Because many kinds of cancers are caused by the excessive production of protein from various “oncogenes,” researchers have fixated upon the interrupting power of microRNAs as a potential targeted therapy. In theory, treating a patient with the right microRNA for their over-expressed oncogene could bring the out-of-control protein back to normal levels, preventing the unrestrained cellular growth that characterizes cancer. But a new discovery published this week in Nature Communications by researchers from the University of Chicago Medicine Section of Hematology/Oncology cautions that microRNAs are not straightforward weapons against cancer.

One promising anti-cancer target is a microRNA called miR-196b, which is associated with certain types of leukemia associated with translocations of the mixed lineage leukemia (MLL) gene. In a translocation, two chromosomes are accidentally broken and the pieces are put back together incorrectly, leaving two unnatural hybrid genes. In this Frankenstein manner, the MLL gene can be abnormally combined with 60 different partner genes, and this “fusion protein” boosts the transcription of a handful of genes, such as HOXA9 and MEIS1, that cause white blood cells to grow and proliferate uncontrollably.

In 2009, a team of researchers including assistant professor of medicine Jianjun Chen and University of Chicago legend Janet Rowley discovered that miR-196b expression is also boosted by MLL fusions, and that the microRNA is necessary for the immortality and proliferation of the leukemic cells. The finding suggested that reducing levels of miR-196b could be an effective therapeutic strategy in fighting leukemia, while raising the levels of the microRNA would accelerate the disease.

But when Chen’s laboratory tested the second part of that hypothesis by experimentally boosting the levels of miR-196b in mice, they found the exact opposite effect: higher levels of the supposedly cancer-promoting microRNA actually delayed the onset of leukemia.

“It was a surprise result for us, because people already reported that by knocking down expression you delay leukemogenesis, so we expected overexpression would promote leukemogenesis,” said first author Zejuan Li, research associate assistant professor at the University of Chicago Medicine. “We didn’t believe the result, we thought something had gone wrong during our experiment. So we repeated and repeated and repeated several times and we got the same result. Finally, we found this mechanism.”

The miR-196b mystery boiled down to looking at the full set of gene transcripts that the microRNA targets and represses. Surprisingly, the researchers discovered that miR-196b reduces the expression of HOXA9 and MEIS1, two oncogenes also upregulated in MLL-related forms of leukemia. But this anti-cancer action is opposed by another target of the microRNA — a tumor suppressor gene called Fas. Since like many microRNAs, miR-196b has many different targets in the genome (41 were detected by this paper alone), it’s a fallacy to consider the factor as either one-dimensionally pro-cancer or anti-cancer.

“I think this is a very common phenomenon, and this should cause caution for the basic research scientists,” Chen said. “When doing research [on one microRNA function], you could be ignoring half of the potential targets.”

So why in the world would a cell need a regulatory factor with seemingly contradictory effects?

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A cell is full of language. There’s the four-letter code of DNA, the slightly different four-letter dialect of RNA, and the three-letter words that direct the construction of proteins, which are built out of an alphabet of 20 amino acids. In recent years, scientists have slowly revealed another vocabulary superimposed on top of this language, comprised of chemical groups attached to genes and proteins. When groups such as methyl or phosphate are stuck to various places on a protein or gene, they can dramatically change its function, switching it on or off or marking it for transport or destruction. On a disease level, these changes can contribute to cancer, aging, and other conditions, making them an enticing target for drug design.

One area where protein modification is making a big splash is the relatively new field of epigenetics, which looks at how changes to DNA and DNA-related proteins can affect gene expression. The methylation of DNA is known to turn genes off, and a number of modifications to histones - proteins that package and organize DNA - can also have functional consequences. Scientists suspected that they hadn’t found all of the modifications possible on histones, but discovering each new modification and proving its role was considered a painstaking process requiring years of experiments. Finding a new modification, such as Yingming Zhao’s 2010 discovery of lysine succinylation, is an achievement worthy of publication in a high-ranking journal.

So what happened when Zhao’s laboratory discovered sixty-seven new modifications to histones in one paper? The research not only was published in the esteemed journal Cell, but was featured by the journal as one of its top five 2011 highlights.

Zhao, a professor in the Ben May Department of Cancer Research at the University of Chicago Medicine, said he believes the extra accolades reflect the volume of his laboratory’s latest breakthrough, and the paper’s expected influence on how scientists will understand the language of protein modification and epigenetic mechanism.

“If we are going to understand epigenetics and its role in disease we need to identify the full vocabulary of the histone modifications, a major group of epigenetic marks,” Zhao said. ” We thought we had a comprehensive histone vocabulary because of extensive studies by the whole research community in the past 3-4 decades, but in our study, in a single paper, we increased it by 70 percent.”

The laboratory put the pedal to the floor on histone modification discovery by developing new technologies and improving the resolution of pre-existing methods. By using the staple lab method of mass spectometry, which measures the mass and charge of particles, and an algorithm of their own design called PTMap, the researchers could scan the entire histone at once and detect more protein modifications than ever before. Their scan yielded a total of 130 different modification sites — 63 that had previously been observed and reported, and 67 that were new to science.

So large was the yield of new modifications that the current paper wasn’t big enough to fully explore the dynamics and function of all of them. The research team chose to more thoroughly study the novel modification that appeared the most: lysine crotonylation, where a crotonyl group is added to the amino acid lysine. Experiments determined that histone lysine crotonylation is evolutionarily conserved (found in yeast, flies, and humans), is often found in promoter or enhancer regions upstream of genes, suggesting a role in transcriptional control. Furthermore, histone lysine crotonylation was found to be enriched on sex chromosomes, specifically marking testis-specific genes, which implies a role in spermatogenesis.

As for what the other never-before-seen modifications are doing in cells, that will have to wait for future papers, Zhao said.

“This is still a very preliminary study,” Zhao said. “Hopefully, we and the research community will figure out if these modifications have a role in cancer and other diseases. Given the fact that lysine methylation and acetylation pathways are already popular drug targets, I assume these new modifications and the enzymes that regulate these modifications and pathways are highly likely to be drug targets for diseases.”

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In books and movies, time travel is typically fraught with negative consequences. Any attempt to change the past — say, stopping the JFK assassination, or taking your mom to the Enchantment Under the Sea dance — is bound to produce ripples of change that alter the future. But what if you could safely contain a trip back in time within the boundaries of a test tube? In a new paper published in Nature, a University of Chicago geneticist used a form of “molecular time travel” to observe a crucial event in the evolutionary history of life on Earth…and extinguish a favorite argument of intelligent design advocates.

The concept of “irreducible complexity” is a favorite talking point of the forces against evolution, both today and historically. As the argument goes, the complex structures found within modern organisms — from the eye to the microscopic protein machines that conduct business in cells — are far too complicated to be the result of the random genetic mutations and selective forces at the core of Darwin’s grand theory. The argument is so old that Darwin himself addressed it in On the Origin of Species, speculating on how an accumulation of small changes could lead from a simple photoreceptor to the wondrous eye shared by many organisms today.

The best way to demonstrate how the minute changes of evolution could produce great complexity is to capture that process in action. But to happen upon such a leap live would be a biological needle in an enormous haystack. A better strategy would be to pick a historic leap in complexity from the evolutionary past, and then go back and observe how it happened. Easy, right?

To accomplish this task, Joe Thornton, a new faculty member in the Departments of Human Genetics and Ecology & Evolution, developed the method of “molecular time travel.” Instead of a Delorean, Thornton’s method uses a computational analysis of the genes from modern-day species to resurrect the genes of ancestral species that lived hundreds of millions of years ago. For the new paper, Thornton and colleagues at the University of Oregon decided to “travel” back to look at a complex molecular machine found in various species of fungus.

“Our strategy was to use ‘molecular time travel’ to reconstruct and experimentally characterize all the proteins in this molecular machine just before and after it increased in complexity,” said Thornton, professor of human genetics and evolution & ecology at the University of Chicago, professor of biology at the University of Oregon, and an Early Career Scientist of the Howard Hughes Medical Institute. “By reconstructing the machine’s components as they existed in the deep past,” Thornton said, “we were able to establish exactly how each protein’s function changed over time and identify the specific genetic mutations that caused the machine to become more elaborate.”

Their target was a molecular machine called the V-ATPase proton pump, which helps maintain the proper acidity of compartments within cells. In modern Fungi, this pump contains a six-part ring made up of three separate proteins, but that wasn’t always the case. Some 800 million years ago, that same ring was made from only two proteins, meaning some kind of event occurred around then to increase the complexity of this machine.

Thornton’s group calculated the genetic sequence of the ring proteins from that ancient ancestor using the sequences of 139 modern Fungi family members, computationally tracing their common elements back up the Tree of Life to their ancient predecessor. The researchers could then reproduce the protein before the split (called Anc.3-11) and the two proteins that came after the split (Anc.3 and Anc.11), and see how they functioned in the proton pump’s ring.

Surprisingly, the “newer” proteins were less versatile than the ancestral Anc.3-11, which could substitute for either of its descendants when transplanted into modern Fungi. The result suggests that the pump’s increase in complexity resulted not from the evolution of a new, “better-designed” function, but from an initial loss of versatility.

“It’s counter-intuitive but simple: complexity increased because protein functions were lost, not gained,” Thornton said. “Just as in society, complexity increases when individuals and institutions forget how to be generalists and come to depend on specialists with increasingly narrow capacities.”

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As another year comes to a close we’d like to look back at the fascinating research breakthroughs and inspiring patient stories from 2011. ScienceLife ran 168 posts this year, and while we wish we could highlight all of them, here are a handful of our favorites from each month.

January

Patrick Wilson found out that the H1N1 virus could end up helping us fight all types of flu. Stephen Pruett-Jones studied how some male birds mimic the sounds of predators to pick up the ladies (with an audio clip). We interviewed David Gozal about his study on the link between childhood obesity and lack of sleep, and took a look at NCAA regulations mandating sickle cell testing for athletes.

February

Harold Pollack gave a lecture on why violent crime in urban, minority communities should be considered a public health epidemic. Siri Atma Greeley studied the actual medical benefit of widespread genetic testing. Stacy Lindau wanted to know why so few women get help for sexual problems after surviving cancer. We talked to Bana Jabri about the causes of celiac disease, and Sliman Bensmaïa showed us how the brain processes the basic elements of touch very much like it handles visual information.

March

Sola Olopade educated women in Nigeria about using clean-burning stoves to prevent indoor pollution. Stefano Allesina and Jonathan Levine looked at how rock-paper-scissors helps explain evolution. Joshua Miller went to Yellowstone Park to see what stories the ghostly bones of animals can tell, and Scott Eggener questioned the wisdom of indiscriminate prostate cancer screening.

Photo by Gerald Waddell

Andrea King studied the wide range of responses to drinking alcohol, and why it can be fun for some people and a bummer for others. Cheryl Reed took a ride in a helicopter with our UCAN nurses. Kamal Sharma looked at the genes that control animals’ gait, and Ningqi Hou studied how urban environments can dictate how much exercise people get.

May

Daniel McGehee looked at the long-term effects of nicotine on the brain. Habibul Ahsan went to Bangladesh to study the health impacts of accidental exposure to arsenic in drinking water. The brain’s overlooked supporting cells got their due at a conference on neuroscience, and we remembered a landmark discovery about a once popular drug taken during pregnancy that we now know can cause cancer.

June

As we headed into summer, Diana Lauderdale used Google to track MRSA. We learned about an extraordinary transplant where a man received a new heart, liver AND kidney. Daniel Geynisman gave us the rundown on whether or not cell phones are killing us (they’re not, as long as you don’t use them in the car), and some UChicago undergrads studied what happens to gorillas on the birth control pill.

July

We spoke to Donald Jensen and Andrew Aronsohn about the new outlook for patients with hepatitis C. Igor Schneider made a time machine to find the genetic switch for limb development. Farr Curlin led a study about the benefits of addressing spiritual needs alongside medical care, and Adam Cifu looked at the phenomenon of scientific study reversals.

August

Stefano Allesina dug into the long, shady history of nepotism in academia in Italy. John Schneider talked about his work addressing sexual health and stigma in India. Michael Becker discovered a new treatment for the Royal Disease, and we had the rare chance to name check a Spiderman villain in a post.

September

Martha McClintock and Suzanne Conzen studied the connection between social isolation, stress and breast cancer. Gallego Romero traveled to India to search for the origins of lactose intolerance. Stephanie Dulawa developed a mouse model for OCD, and Paul Vezina looked at a different kind of obsession, compulsive gambling.

October

Arshiya Baig started a pilot project to help people learn about life with diabetes through pictures. Manyuan Long found that some of the youngest genes are in the brain. Jens Ludwig and Stacy Lindau published a landmark study about the connection between neighborhood poverty and health, and Issam Awad studied a rare brain disease that soon could be treated with a drug instead of surgery.

November

Cathy Pfister and Tim Wootton figured out how to use seashells to track climate change over the years. Lianne Kurina found a link between loneliness and sleep quality. Shantanu Nundy, Monica Peek and Marshall Chin developed a program to send text message reminders to people with diabetes, and Pan Chen looked at the links between childhood abuse and aggressive behavior in adults.

December

Inbal Ben-Ami Bartal, Jean Decety and Peggy Mason discovered that rats can show empathy for their fellow rats in distress. Maciej Lesniak performed a scary but amazing brain surgery on a patient who was awake. Cathryn Nagler searched for the source of food allergies within our bodies, while Stafano Guandalini uncovered the challenges in educating doctors about one of those allergies, celiac disease.

Whew. Hope you were able to click through at least a few of those. We look forward to another great year of research in 2012. We’re taking a break next week, but we’ll be back on January 5. Happy holidays!

All of the animal life on Earth, including human beings, can be traced back to a unicellular ancestor somewhat similar to the modern-day protozoa. In one sense, the hundreds of millions of years of evolution is the story of how organisms became more and more complex, growing from a single cell to trillions of highly specialized cells forming different organs and tissues in a single body. Yet while you could easily tell a protozoa from a human in a police lineup, cells from the two species are made up of many of the same proteins, performing similar jobs. What changed to produce these profound differences in complexity?

One potential area where this complexity may have bloomed is tyrosine phosphorylation, a key cellular signal for pathways that control cell growth, proliferation, and structure. Enzymes called tyrosine kinases add a phosphate group to a wide range of cellular targets, which can act like a light switch, turning their function on or off. The phosphorylated proteins are recognized by another group of proteins with a special “sensor” called the SH2 domain. Because tyrosine kinases will promiscuously phosphorylate many targets in the cell, the very picky SH2 domain proteins are responsible for sorting out the noise.

“Tyrosine kinases tend to be not that selective,” said Piers Nash, assistant professor in the Ben May Department of Cancer Research at the University of Chicago who studies this system. “They’ll phosphorylate a lot of things, and that creates all of these docking sites for SH2-domain-containing proteins. It’s really up to the SH2 domains to interpret those signals and convert them into downstream signaling pathways.”

The more complex the cell, the more unique types of SH2 domains that are needed to perform this important sorting function. In the unicellular cousins of animals, organisms can get by with just a single SH2 domain. But in humans, some 121 SH2 domains are known to exist, managing many different pathways in many different cells. In two recent papers, Nash’s laboratory studied how these SH2 domains manage their impressive selectivity and the evolutionary pathway that they took from simple protozoa to complicated human.

It’s essential that SH2 domains only bind to the right phosphorylated protein — repeatedly screwing up and activating the wrong pathway could lead to diabetes, cancer, or worse. But scientists have struggled to figure out how SH2 domains choose their appropriate target, with some even concluding that they aren’t so selective at all, merely in the right part of the cell at the right time to only bind the correct protein. However, that wasn’t what a research team led Bernard Liu from Nash’s laboratory found when they looked at how SH2 domains bind actual cell targets such as the insulin receptor.

“It turned out that the SH2 domains were exquisitely selective, much more selective than the general motifs for the SH2 domains that had previously been mapped,” Nash said. “So it was clear there was additional information encoded in the peptide that the SH2 domain makes use of.”

The researchers then deduced that the SH2 domains select their target through a kind of language, looking for the exact sequence of amino acids - or “word” - that marks the appropriate match. Because each amino acid (akin to the letters of the word) will either attract a particular SH2 domain or reject its peers, changing only one amino acid can completely change the meaning, like altering the word “light” to “fight.”

“For SH2 domains, that makes all the difference in the world. They can sense incredibly subtle differences,” Nash said. “It’s looking at the entire peptide and seeing both the permissive and the non-permissive residues, integrating that and making this collective decision about what to bind.”

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How does an organ know when to stop growing? It may sound like a riddle, but it’s a serious biological question with the potential for grave consequences. During development, an organism grows from a single cell up to trillions of cells. If that growth process overshoots its goal and doesn’t stop generating new cells, the result can be the unrestrained proliferation of cancer. Scientists have thus looked for the regulators of that growth, a search that led them to a cast of unusual characters: hippos, Yorkies, and warts.

That colorful menagerie is the result of research in fruit flies, where naming conventions steer away from the cold acronyms used by the rest of biology. Researchers of the fruit fly Drosophila melanogaster run screens where individual genes are deleted or suppressed, then name the gene according to the unusual appearance or activity this modified fly displays. So when a genetic deletion created a fly with organs of unusually large size, researchers named that missing gene Hippo. Conversely, the name Yorkie was assigned to a gene that, when deleted, produced a fly that grew abnormally small organs.

In the early 2000s, researchers determined that Hippo and Yorkie - and a handful of other genes found to control organ size - were all part of the same system, dubbed the Hippo-Salvador-Warts (HSW) signaling pathway. These elements were not exclusive to flies, but found in a host of other organisms, suggesting that the system goes far back in evolutionary time as a critical controller of cell function. Early returns also indicate that the HSW pathway is a likely contributor to human cancers, said Rick Fehon, professor and chair of molecular genetics and cell biology at the University of Chicago.

“The basic components are in yeast, worms, flies, and humans, so it’s a really fundamentally conserved pathway,” Fehon said. “It’s a pretty fresh field in general, and I think the mammalian cancer implications are far from having been fully explored.”

While the Hippo to Salvador to Warts to Yorkie pathway has been firmly established, scientists are still looking for how elements upstream turn the pathway on and off. In a new paper published this week in the journal Developmental Cell, Julian Boggiano and Pamela Vanderzalm of Fehon’s laboratory discovered one of these HSW pathway “switches,” and lengthened the cellular chain of how organ size is regulated.

Boggiano and Vanderzalm were looking for proteins that interact with another cell growth regulator called Merlin, a gene responsible for the disease neurofibromatosis in humans. One by one, they depleted a family of proteins called the Sterile 20 kinases, looking for an element that regulates Merlin activity. In the process, they found that suppressing one gene, called Tao-1 (this name originates from studies in mammals, not flies), created a fly that looked similar to Hippo, displaying an abnormal growth of organs called imaginal discs that form the wings and eyes of adult flies (seen above).

“We were looking for one thing, and serendipitously found something else,” said Vanderzalm, a postdoctoral fellow. “Imaginal discs undergo about 1,000 fold growth in four days. During that time they go from about 50 cells to 50,000 cells. You can tell right away that the overall shape is disrupted and wherever we’ve driven Tao-1 RNAi, those cells have a growth advantage, and they’ve overgrown relative to the remaining wild type cells in that tissue. They’re dividing more frequently.”

“That was when we realized it was probably a new component of this pathway,” said Boggiano, a graduate student in the Committee on Development, Regeneration, and Stem Cell Biology.

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Many of the most interesting processes in nature are so fast, they can make “a blink of an eye” look like a millennium. Cellular proteins undergo elaborate transformations in as little as a picosecond - one millionth of one millionth of a second. That astonishing time scale presents an enormous challenge to scientists who would like to study the structure and behavior of those proteins. To catch the extremely fleeting moments of transition between different structural states of one such protein, a University of Chicago laboratory used a strategy straight out of science fiction: freezing time.

Xiaojing Yang, a senior research professional in the laboratory of Keith Moffat, wanted to look at a particular protein called a bacteriophytochrome, a red light photoreceptor from bacteria related to phytochromes in plants. Photoreceptors are found everywhere in nature, from plants to eyes, and are activated by light to change their shape and transform a light signal into a biological signal.  Yang was interested in the contortions a photoreceptor makes when changing from the “dark” state to the “light” state, and chose a particular phytochrome from the bacterial species Pseudomonas aeruginosa.

Yang started with a method called X-ray crystallography, where proteins are maneuvered into crystal formation and then imaged at the atomic level using an X-ray beam. With traditional X-ray crystallography, Yang could determine the structure of the P. aeruginosa phytochrome in the dark state, before activation with the light signal - data she reported in a 2008 PNAS paper. But in order to see the transition between dark and light in finer detail, the researchers needed to develop a new trick.

“We applied an innovative application of crystallography called temperature-scanning cryocrystallography, where we use temperature to mimic time,” Yang said of the work, published this week in Nature. “So this is a new way of doing dynamic crystallography.”

Cryocrystallography, or “cryotrapping,” involves performing the method at a very low temperature to freeze the target molecule in a particular conformation. The temperature-scan aspect that Yang and colleagues added was to hold the crystal at an escalating series of temperatures (from -279° F to -135° F), each one moving the super-fast structural changes the slightest bit forward. The researchers could then do an X-ray scan at each temperature level to capture those normally-fleeting in-between stages.

“We shine the light at different temperatures, higher and higher,” Yang said. “The reaction progresses further as the temperature rises, and then you have a different mixture of reaction intermediates.”

“It’s a cunning way of slowing things down from picoseconds to minutes or even tens of minutes,” said Moffat, Louis Block Professor of Biochemistry & Molecular Biology at UChicago.

The experiments, using ten temperature levels in all, revealed three predominant intermediate states, called L1, L2, and L3. Transitions between states resemble a “molecular earthquake,” Moffat said, with the changes initially appearing at one corner of the light-sensitive chromophore of the phytochrome before spreading outwards until the entire structure is in flux. And as dramatic as the motion is, it’s only the initial steps of the phytochrome’s dance. Moffat uses the analogy of filming a sprint to describe the action they captured - the flash of light that starts the reaction is the starting gun - and says that they are so far only able to observe the first 15 meters of a 100 meter dash.

“We’re not in a position to observe the rest of the race,” Moffat said. “Yes, we would like to see the entire race going all the way to the finish, but we believe that the overall reaction from beginning to end involves quite a large molecular convulsion, which is probably not compatible with the crystals.”

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