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Sandieson L.,Child Health Research Institute | Sandieson L.,Sciencetech Inc. | Hwang J.T.K.,Child Health Research Institute | Kelly G.M.,Child Health Research Institute
Stem Cells and Development | Year: 2014

Retinoic acid (RA) induces mouse F9 cells to form primitive endoderm (PrE) and increased levels of reactive oxygen species (ROS) accompany differentiation. ROS are obligatory for differentiation and while H2O2 alone induces PrE, antioxidants attenuate the response to RA. Evidence shows that ROS can modulate the Wnt/β-catenin pathway and in this study, we show that extraembryonic endoderm formation is dependent on the redox state of nucleoredoxin (NRX). In undifferentiated F9 cells, NRX interacted with dishevelled 2 (Dvl2) and while this association was enhanced under reduced conditions, it decreased following H2O2 treatment. Depleting NRX levels caused morphological changes like those induced by RA, while increasing protein kinase A activity further induced these PrE cells to parietal endoderm. Reduced NRX levels also correlated to an increase in T-cell-factors-lymphoid enhancer factors-mediated transcription, indicative of canonical Wnt signaling. Together these results indicate that a mechanism exists whereby NRX maintains canonical Wnt signaling in the off state in F9 cells, while increased ROS levels lift these constraints. Dvl2 no longer bound to NRX is now positioned to prime the Wnt pathway(s) required for PrE formation. © Copyright 2014, Mary Ann Liebert, Inc. 2014.


When we are in a deep slumber our brain's activity ebbs and flows in big, obvious waves, like watching a tide of human bodies rise up and sit down around a sports stadium. It's hard to miss. Now, Stanford researchers have found, those same cycles exist in wake as in sleep, but with only small sections sitting and standing in unison rather than the entire stadium. It's as if tiny portions of the brain are independently falling asleep and waking back up all the time. What's more, it appears that when the neurons have cycled into the more active, or "on," state they are better at responding to the world. The neurons also spend more time in the on state when paying attention to a task. This finding suggests processes that regulate brain activity in sleep might also play a role in attention. "Selective attention is similar to making small parts of your brain a little bit more awake," said Tatiana Engel, a postdoctoral fellow and co-lead author on the research, which is scheduled to publish Dec. 1 in Science. Former graduate student Nicholas Steinmetz was the other co-lead author, who carried out the neurophysiology experiments in the lab of Tirin Moore, a professor of neurobiology and one of the senior authors. Understanding these newly discovered cycles requires knowing a bit about how the brain is organized. If you were to poke a pin directly into the brain, all the brain cells you'd hit would respond to the same types of things. In one column they might all be responding to objects in a particular part of the visual field - the upper right, for example. The team used what amounts to sets of very sensitive pins that can record activity from a column of neurons in the brain. In the past, people had known that individual neurons go through phases of being more or less active, but with this probe they saw for the first time that all the neurons in a given column cycled together between firing very rapidly then firing at a much slower rate, similar to coordinated cycles in sleep. "During an on state the neurons all start firing rapidly," said Kwabena Boahen, a professor of bioengineering and electrical engineering at Stanford and a senior author on the paper. "Then all of a sudden they just switch to a low firing rate. This on and off switching is happening all the time, as if the neurons are flipping a coin to decide if they are going to be on or off." Those cycles, which occur on the order of seconds or fractions of seconds, weren't as visible when awake because the wave doesn't propagate much beyond that column, unlike in sleep when the wave spreads across almost the entire brain and is easy to detect. The team found that the higher and lower activity states relate to the ability to respond to the world. The group had their probe in a region of the brain in monkeys that specifically detects one part of the visual world. The monkeys had been trained to pay attention to a cue indicating that something in a particular part of the visual field - the upper right, say, or the lower left - was about to change slightly. The monkeys then got a treat if they correctly identified that they'd seen that change. When the team gave a cue to where a change might occur, the neurons within the column that senses that part of the world all began spending more time in the active state. In essence, they all continued flipping between states in unison, but they spent more time in the active state if they were paying attention. If the stimulus change came when the cells were in a more active state, the monkey was also more likely to correctly identify the change. "The monkey is very good at detecting stimulus changes when neurons in that column are in the on state but not in the off state," Engel said. Even when the monkey knew to pay attention to a particular area, if the neurons cycled to a lower activity state the monkey frequently missed stimulus change. Engel said this finding is something that might be familiar to many people. Sometimes you think you are paying attention, she pointed out, but you will still miss things. The scientists said the findings also relate to previous work, which found that more alert animals and humans tend to have pupils that are more dilated. In the current work, when the brain cells were spending more time in an active state the monkey's pupils were also more dilated. The findings demonstrate an interaction between synchronous oscillations in the brain, attention to a task and external signs of alertness. "It seems that the mechanisms underlying attention and arousal are quite interdependent," Moore said. A question that comes out of this work is why the neurons cycle into a lower activity state when we're awake. Why not just stay in the more active state all the time in case that's when the saber tooth tiger attacks? One answer could relate to energy. "There is a metabolic cost associated with neurons firing all the time," Boahen said. The brain uses a lot of energy and maybe giving the cells a chance to do the energetic equivalent of sitting down allows the brain to save energy. Also, when neurons are very active they generate cellular byproducts that can damage the cells. Engel pointed out that the low-activity states could allow time to clear out this neuronal waste. "This paper suggests places to look for these answers," Engel said. Additional co-authors include colleagues from Newcastle University. Kwabena Boahen is also a member of Stanford Bio-X and the Stanford Neurosciences Institute. Tirin Moore is also an HHMI investigator as well as a member of Stanford Bio-X, the Stanford Neurosciences Institute and the Child Health Research Institute. The work was funded by the NIH, Stanford NeuroVentures, the HHMI, the MRC and the Wellcome Trust.


News Article | February 15, 2017
Site: www.eurekalert.org

Researchers at the Stanford University School of Medicine used heart muscle cells made from stem cells to rank commonly used chemotherapy drugs based on their likelihood of causing lasting heart damage in patients. Drugs known as tyrosine kinase inhibitors can be an effective treatment for many types of cancers, but they also have severe and sometimes fatal side effects. Using lab-grown heart cells, Stanford researchers were able to assess the drugs' various effects on heart muscle cells, including whether the cells survived, were able to beat rhythmically and effectively, responded appropriately to electrophysiological signals and communicated with one another. The researchers found that their assay can accurately identify those tyrosine kinase inhibitors already known to be the most dangerous in patients. In the future, they believe their system may prove useful in the early stages of drug development to screen new compounds for cardiotoxicity. "This type of study represents a critical step forward from the usual process running from initial drug discovery and clinical trials in human patients," said Joseph Wu, MD, PhD, director of the Stanford Cardiovascular Institute and a professor of cardiovascular medicine and of radiology. "It will help pharmaceutical companies better focus their efforts on developing safer drugs, and it will provide patients more effective drugs with fewer side effects." A paper describing the research will be published Feb. 15 in Science Translational Medicine. Wu, who holds the Simon H. Stertzer Professorship, is the senior author. Former graduate student Arun Sharma, PhD, is the lead author. "We used multiple measurements to accurately predict which of the tyrosine kinase inhibitors were the most cardiotoxic," said Sharma. "The drugs with the lowest safety indices in our study were also those identified by the Food and Drug Administration as the most cardiotoxic to patients. Other drugs that are not as cardiotoxic performed much better in our assays." Validating the researchers' cardiac-safety test on drugs with extensive clinical track records is necessary before the assay can be used to predict with confidence the likely clinical outcomes of drugs still under development. Sharma, Wu and their colleagues created heart muscle cells called cardiomyocytes from induced pluripotent stem cells, or iPS cells, from 11 healthy people and two people with kidney cancer. They grew the lab-made cardiomyocytes in a dish and tested the effects of 21 commonly used tyrosine kinase inhibitors on the cells. They found that treatment with drug levels equivalent to those taken by patients often caused the cells to beat irregularly and begin to die. The cells also displayed differences in the electrophysiological signaling that controls their contraction. The researchers used these and other measurements to develop a cardiac safety index for each drug. They found that those drugs known to be particularly dangerous to heart function, such as nilotinib, which is approved for the treatment of chronic myelogenous leukemia, and vandetanib, which is approved for the treatment of some types of thyroid cancer, also had the lowest safety indices based on the assay; conversely, those known to be better tolerated by patients ranked higher on their safety index. Prescribing information for both nilotinib and vandetanib contains warnings from the FDA about the drugs' potential cardiotoxicity. An activity increase in an insulin responsive pathway Six of the 21 tyrosine kinase inhibitors tested were assigned cardiac safety indices at or below 0.1 -- the threshold limit at which the researchers designated a drug highly cardiotoxic. Three of these six are known to inhibit the same two signaling pathways: VEGFR2 and PDGFR. The researchers noticed that cells treated with these three drugs ramped up the activity of a cellular signaling pathway that responds to insulin or IGF1, an insulinlike growth factor. This discovery, coupled with the fact that treatment with insulin or IGF1 is known to enhance heart function during adverse cardiac events such as heart attacks, led the researchers to experiment further. They found that exposing the cells to insulin or IGF1 made it less likely they would die due to tyrosine kinase inhibitors blocking the VEGFR2 and PDGFR pathways. Although more research is needed, these findings suggest it may be possible to alleviate some of the heart damage in patients receiving these chemotherapies. The current study mirrors another by Wu's lab that was published in April 2016 in Nature Medicine. That research focused on the toxic effect of a chemotherapy drug called doxorubicin on iPS cell-derived cardiomyocytes. Doxorubicin, which indiscriminately kills any replicating cells, is increasingly being replaced by more targeted, cancer-specific therapies such as the tyrosine kinase inhibitors tested in the current study. "The switch from doxorubicin is a result of the paradigm shift in cancer treatment to personalized, precise treatment as emphasized by President Obama's 2015 Precision Medicine Initiative," said Wu. "Moving even further, we're discovering that many tyrosine kinase inhibitors are themselves significantly cardiotoxic, and some have been withdrawn from the market. There is a critical need for a way to 'safety test' all drugs earlier in development before they are administered to patients. Our drug safety index is a step in that direction." Other Stanford co-authors are Paul Burridge, PhD, a former instructor at the Cardiovascular Institute; graduate student Wesley McKeithan; postdoctoral scholars Praveen Shukla, PhD, Tomoya Kitani, MD, Haodi Wu, PhD, and Alexandra Holmström, PhD; instructors Nazish Sayed, MD, PhD, Elena Matsa, PhD, and Jared Churko, PhD; medical student Anusha Kumar; undergraduate student Yuan Zhang; assistant professor of medicine Alice Fan, MD; associate professor of medicine Sean Wu, MD, PhD; and professor of medicine Mark Mercola, PhD. The research was funded by the American Heart Association, the National Science Foundation, the National Institutes of Health (a Director's Pioneer Award and grants R01HL113006, R01HL130020, R01HL128170, R01HL123968 and R24HL117756), the Lucile Packard Foundation for Children's Health, the Stanford Child Health Research Institute and the Burroughs Wellcome Foundation. Wu is a member of the Stanford Cancer Institute, Bio-X and the Child Health Research Institute. Mercola is on the scientific advisory board for Vala Sciences, a company offering high-content screening services, and Wu is on the scientific advisory board of Stem Cell Theranostics, a company using patient-specific iPS cells for drug discovery. The Stanford University School of Medicine consistently ranks among the nation's top medical schools, integrating research, medical education, patient care and community service. For more news about the school, please visit http://med. . The medical school is part of Stanford Medicine, which includes Stanford Health Care and Stanford Children's Health. For information about all three, please visit http://med. .


News Article | February 27, 2017
Site: www.eurekalert.org

A combination of two cancer drugs inhibited both dengue and Ebola virus infections in mice in a study led by Stanford University School of Medicine researchers, despite the fact that these two viruses are vastly different from each other. In laboratory-dish experiments, the drug combination, which has previously shown efficacy against the hepatitis C virus, also was effective against West Nile and Zika viruses, both of which are relatives of the hepatitis C virus, and multiple other unrelated viruses. The multi-institution study, to be published online Feb. 27 in the Journal of Clinical Investigation, also pinpointed the specific molecular mechanism by which these drugs derail a variety of RNA viruses, whose genetic material consists not of DNA but of its close relative, RNA. "We've shown that a single combination of drugs can be effective across a broad range of viruses -- even when those viruses hail from widely separated branches of the evolutionary tree," said the study's senior author, Shirit Einav, MD, assistant professor of infectious diseases and of microbiology and immunology. The study's lead authors are former Stanford postdoctoral scholars Elena Bekerman, PhD, now at Gilead Sciences Inc., and Gregory Neveu, PhD, now at the University of Lyon and French National Institute of Health and Medical Research. The reason the drugs used in the study are able to combat infections by such different viruses is that their disabling action is directed not at the virus but at proteins of the host cell it's trying to infect, Einav said. Einav and her team are investigating strategies for combatting RNA viruses, such as dengue and Ebola. These viruses have a faulty replication process that results in frequent errors as their genetic material is copied, rendering them especially prone to mutations. Consequently, they swiftly acquire resistance to a typical antiviral drug that targets a specific viral enzyme, Einav said. "The 'one drug, one bug' approach can be quite successful, as in the case of hepatitis C virus," for which a concerted effort has generated several approved antiviral treatments, she said. But it took more than 10 years of research, she noted, and drug development costs typically exceed $2 billion. Making matters worse, Einav added, is the impossibility of predicting what the next emerging viral threat will look like. "We're always getting blindsided," she said. The deadly Ebola epidemic of a few years ago has subsided but could return at any time. Dengue infects an estimated 390 million people annually in over 100 countries. Four distinct strains of the dengue virus exist, hampering the development of a vaccine and boosting the chances of a once-infected person's re-infection by a different strain against which that person hasn't achieved sufficient immunity. Secondary infections can become life-threatening. While an Ebola vaccine has shown promise, it's not yet approved. A recently approved dengue vaccine has only limited efficacy. No viable antiviral drugs are currently available for either virus. Viruses are cut-rate brigands: They produce nothing on their own, but rather hijack the machinery of our cells. Hepatitis C, dengue, Ebola and other viruses hop onto molecular "buses" that whisk cargo between cell compartments. These buses shuttle the viruses around inside of cells. The buses' routes and fares are regulated by numerous cellular enzymes. Two such enzymes, which go by the acronyms AAK1 and GAK, essentially lower the fares charged by the molecular buses by tweaking them so they bind more strongly to their cargo. The standard antiviral approach aims to disable a specific viral enzyme. Einav and her associates' alternative approach took advantage of viruses' total dependence on infected cells' molecular machinery. The two-drug drug combination Einav's team put to work against dengue and Ebola impedes AAK1's and GAK's activity, effectively pricing bus fares beyond the viral budget. Erlotinib and sunitinib, each approved by the Food and Drug Administration more than a decade ago, are prescribed for various cancer indications. Neither AAK1 nor GAK are the primary targets of these drugs in their cancer-fighting roles. But Einav's group discovered, by accessing publicly available databases, that the two drugs impair AAK1 and GAK activity, too. Einav and her colleagues previously demonstrated that erlotinib and sunitinib inhibit hepatitis C virus infection in cells. In the new study, the investigators conducted experiments in lab dishes to show that both drugs inhibit viral infection by impeding the activity of AAK1 and GAK. Next, they tested the combination in lab dishes against the dengue and Ebola viruses, and observed that viral activity was strongly inhibited in both. While the dengue virus is a relatively close cousin of hepatitis C, it is quite different from the Ebola virus. The same drug combination also showed efficacy against a variety of other RNA viruses related to hepatitis C, including the Zika and West Nile viruses, and even against several unrelated viruses. In a prevention experiment in mice, the investigators administered the erlotinib-sunitinib combination once daily starting on the day of dengue-virus infection, employing the two drugs for five days at doses comparable to those approved for use against cancer in humans. All the control mice died between days four and eight. But of those treated with the drug combination, 65 to 100 percent, depending on the individual experiment, survived and regained their pre-infection weight and mobility. Given individually, the drugs provided substantially less protection, Einav said. In another experiment designed to test the drugs as a therapy, the combination retained substantial antiviral efficacy as long as it was given less than 48 hours after infection. In a similar prevention experiment with the Ebola virus, the scientists administered the drug daily for 10 days starting at six hours before infection. Some 90 percent of the control mice died within a week or two. But half the mice receiving the drug combination survived. Again, the drugs were substantially less effective when given individually. Additional lab experiments showed that the combination profoundly inhibited the dengue virus's ability to develop drug resistance. There's no possible way for viral mutations to alter the proteins of the cells it infects, Einav said, and no easy way for the virus to mutate around its dependence on those proteins. Stanford's Office of Technology Licensing has filed for patents on intellectual property associated with the findings. Other Stanford study co-authors are Claude Nagamine, DVM, PhD, assistant professor of comparative medicine; and research scientist Robert Mateo, PhD. The study was carried out in collaboration with researchers from the University of Chicago, the U.S. Army Medical Research Institute of Infectious Diseases in Maryland, the Washington University School of Medicine in St. Louis and the University of Leuven in Belgium. The study was funded by the National Institute of Health (grants IU19AI10966201 and U19A1083019); the American Cancer Society; the Doris Duke Charitable Foundation; the Department of Defense; Stanford Bio-X; the Stanford Spark program; the Stanford Translational Research and Applied Medicine program; Spectrum, which administers Stanford's Clinical and Translational Science Award (grant UL1TR001085) from the NIH; the Stanford Child Health Research Institute; and the Taiwan Ministry of Science and Technology. Stanford's departments of Medicine and of Microbiology and Immunology also supported the work. The Stanford University School of Medicine consistently ranks among the nation's top medical schools, integrating research, medical education, patient care and community service. For more news about the school, please visit http://med. . The medical school is part of Stanford Medicine, which includes Stanford Health Care and Lucile Packard Children's Hospital Stanford. For information about all three, please visit http://med. .


News Article | December 17, 2015
Site: www.biosciencetechnology.com

In his role as a pediatrician, Manish Butte, M.D., Ph.D., will often push and prod a patient’s abdomen, feeling for abnormalities — a swollen spleen, a hardened lymph node or an unusual lump in the intestines or liver. There are still some things that can only be gleaned by touch, and Butte believes this notion applies to individual cells as well. Yet researchers’ ability to probe and measure the features of living cells has been almost nonexistent. Recently, a team of Stanford scientists and engineers set out to right that imbalance with a new technique for rapidly mapping cells. They succeeded by engineering a major advancement in a technology known as atomic force microscopy, or AFM, which itself was invented at Stanford in 1986. A paper describing the work was published online Nov. 11 in ACS Nano. Butte, an assistant professor of pediatric immunology, is the senior author. Lead authorship is shared by Andrew Wang, Ph.D., a former postdoctoral scholar in Butte’s lab, and Karthik Vijayrhagavan, Ph.D., who was a graduate student and member of the microphotonics lab led by Olav Solgaard, Ph.D., a professor of electrical engineering. “What a cell feels like — its mechanical properties that affect how it makes contact with other cells and tissues — is much more important than what it looks like, but the technology just wasn’t there to allow us to examine it,” Butte said. “There is a lot to be learned from studying the mechanics of a cell and its structures just beneath the surface.” The way Butte and his colleagues use AFM to measure the mechanical properties of cells is akin to the way a builder taps her knuckles along a drywall, listening for the change in pitch that will tell her a wooden stud is on the other side. When an AFM probe taps the surface of a cell, it vibrates, and the pattern of these vibrations, like the sound waves reflecting from the stud, gives mechanical information about the structures of the cell being touched. However, existing AFM probes are relatively large and, as a result, insensitive to high frequencies, which communicate much of the key information about a cell’s innards. The Stanford team’s device couples a very small probe with a traditional one. This assembly allows the device to sense faster oscillations than conventional devices and, accordingly, to take more detailed and much faster measurements. “The main difference between this and previous atomic force microscopes is that we are able to measure the impact of the probe on the cell very fast and get specific readings, whereas typical AFMs simply provide an average. This allows us to accurately measure some very soft materials for the first time,” said Solgaard, who also is a co-author of the paper. Current probes measure cellular stiffness by tapping against the cell around one or two times per second — the fastest that the large probes can make measurements. The small probe, however, can make detailed measurements easily at five to 10,000 taps per second because of its sensitivity. He likened the leap in sensitivity to the difference between driving a Cadillac Escalade down the road and pushing a Hot Wheel toy car along the same surface: “The small Hot Wheel will feel every little bump so much more than the large Cadillac.” AFMs measure movement of the probe by bouncing a laser off its tip. As the tip moves up and down, the laser is reflected. The Stanford invention couples the small probe with the large one by means of a fork-shaped structure called an interferometric grating. The grating produces a diffraction pattern based on the movements of the small probe, and allows the AFM to conveniently capture its measurements. “Our tip actually produces a second signal, and that is what allows us to get much greater detail. From an engineering standpoint, it’s an extremely simple, beautiful solution,” Solgaard said, referring to the diffracted signals from the grating. Best of all, the team’s device can be directly attached to existing AFMs, potentially saving millions of dollars on new equipment that could otherwise be spent on research. A new AFM can run as much as $500,000, according to Solgaard. The objective is the cellular equivalent of Butte pressing a child’s abdomen. “We want to study cell stiffness to understand what is beneath the surface and how cells are structured,” Wang said. As a demonstration, the team measured a section of a red blood cell, making approximately 4 million total measurements in about 10 minutes — all without damaging the delicate cellular exterior. “The same measurements would have taken more than a month to complete using conventional atomic force microscopes,” said Vijayraghavan. The technology is so fast that the team was able to create a series of time-lapse images of a living cell, each taken just seven minutes apart, a previously unimaginable pace. The practical applications of the device range from basic scientific understanding of cellular structure to immunology and oncology. Scientific understanding of the mechanical forces at play in cells is so lacking that the field — now being called mechanobiology — is really in its infancy, according to Butte. The mechanical forces in the body can come from tissues, which range in stiffness from softest brain matter to stiffest bones, from gravity, and even from the pushing and pulling movements of other cells. Cancer cells make their environment mechanically rigid by secreting chemicals that stiffen up the extracellular matrix. Cancer cells likewise interpret the mechanical forces of a tissue to make decisions about growth and metastasis. Surprising feedback loops like this also appear to occur for stem cells in the bone marrow and during embryonic development. How immune cells interpret mechanical forces is still totally unknown. “The lowest-hanging fruit is cancer. Cancers are often stiffer than normal, healthy tissues and we can use that knowledge to diagnose disease. But first, you have to have good data, which our device provides,” Wang said. He has already used an early form of the new Stanford probe in pilot work on breast cancer specimens taken from mastectomies. For his part, Butte plans to use fast AFM to study the immune system. He hopes to explore why otherwise disease-fighting T cells often remain dormant once inside a tumor. He theorizes that the mechanical stiffness of the tumorous tissue may be preventing T cells from freely making contact with cancer cells and from triggering their cancer-fighting functions. In essence, the tumor may be too crowded for the T cells to work. On the other end of the stiffness gamut, he believes that the soft mechanical properties of chronically inflamed or infected tissues provoke the immune system into over-activity, like autoimmunity. It is a theory no one has yet explored due to technical barriers, which the fast AFM could overcome. Butte’s lab has begun a broad effort to link mechanical forces with immune responses at the molecular, cellular and tissue scales. “There is so much we don’t know about the mechanical properties of various cell types and diseased tissues. Almost nothing, in fact,” Butte said. “The first step is to probe. Now, we can do that.” The work was funded by the National Institutes of Health, the National Science Foundation, the Stanford Center for Probing the Nanoscale, the Stanford Child Health Research Institute and Stanford Bio-X.


News Article | February 23, 2017
Site: www.futurity.org

Cells within our bodies divide and change over time, with thousands of chemical reactions occurring within each cell daily. This makes it difficult for scientists to understand what’s happening inside. New nanostraws offer a non-disruptive way to find out. A problem with the current method of cell sampling, called lysing, is that it ruptures the cell. Once the cell is destroyed, it can’t be sampled from again. This new sampling system relies on tiny tubes 600 times smaller than a strand of hair that allow researchers to sample a single cell at a time. The nanostraws penetrate a cell’s outer membrane, without damaging it, and draw out proteins and genetic material from the cell’s salty interior. “It’s like a blood draw for the cell,” says Nicholas Melosh, an associate professor of materials science and engineering at Stanford University and senior author of a paper describing the work in the Proceedings of the National Academy of Sciences. The nanostraw sampling technique, according to Melosh, will significantly impact our understanding of cell development and could lead to much safer and effective medical therapies because the technique allows for long term, non-destructive monitoring. “What we hope to do, using this technology, is to watch as these cells change over time and be able to infer how different environmental conditions and ‘chemical cocktails’ influence their development—to help optimize the therapy process,” Melosh says. If researchers can fully understand how a cell works, then they can develop treatments that will address those processes directly. For example, in the case of stem cells, researchers are uncovering ways of growing entire, patient-specific organs. The trick is, scientists don’t really know how stem cells develop. “For stem cells, we know that they can turn into many other cell types, but we do not know the evolution—how do they go from stem cells to, say, cardiac cells? There is always a mystery. This sampling technique will give us a clearer idea of how it’s done,” says Yuhong Cao, a graduate student and first author on the paper. The sampling technique could also inform cancer treatments and answer questions about why some cancer cells are resistant to chemotherapy while others are not. “With chemotherapy, there are always cells that are resistant,” says Cao. “If we can follow the intercellular mechanism of the surviving cells, we can know, genetically, its response to the drug.” The sampling platform on which the nanostraws are grown is tiny—about the size of a gumball. It’s called the Nanostraw Extraction (NEX) sampling system, and it was designed to mimic biology itself. In our bodies, cells are connected by a system of “gates” through which they send each other nutrients and molecules, like rooms in a house connected by doorways. These intercellular gates, called gap junctions, are what inspired Melosh six years ago, when he was trying to determine a non-destructive way of delivering substances, like DNA or medicines, inside cells. The new NEX sampling system is the reverse, observing what’s happening within rather than delivering something new. “It’s a super exciting time for nanotechnology,” Melosh says. “We’re really getting to a scale where what we can make controllably is the same size as biological systems.” Building the NEX sampling system took years to perfect. Not only did Melosh and his team need to ensure cell sampling with this method was possible, they needed to see that the samples were actually a reliable measure of the cell content, and that samples, when taken over time, remained consistent. When the team compared their cell samples from the NEX with cell samples taken by breaking the cells open, they found that 90 percent of the samples were congruous. Melosh’s team also found that when they sampled from a group of cells day after day, certain molecules that should be present at constant levels remained the same, indicating that their sampling accurately reflected the cell’s interior. With help from collaborators Sergiu P. Pasca, assistant professor of psychiatry and behavioral sciences, and Joseph Wu, professor of radiology, Melosh and coworkers tested the NEX sampling method not only with generic cell lines, but also with human heart tissue and brain cells grown from stem cells. In each case, the nanostraw sampling reflected the same cellular contents as lysing the cells. The goal of developing this technology, according to Melosh, was to make an impact in medical biology by providing a platform that any lab could build. Only a few labs across the globe, so far, are employing nanostraws in cellular research, but Melosh expects that number to grow dramatically. “We want as many people to use this technology as possible,” he says. Funding for the work came from the National Institute of Standards and Technology, the Knut and Alice Wallenberg Foundation, the National Institutes of Health, Stanford Bio-X, the Progenitor Cell Biology Consortium, the National Institute of Mental Health, an MQ Fellow award, the Donald E. and Delia B. Baxter Foundation, and the Child Health Research Institute.


News Article | February 22, 2017
Site: www.eurekalert.org

Cells within our bodies divide and change over time, with thousands of chemical reactions occurring within each cell daily. This makes it difficult for scientists to understand what's happening inside. Now, tiny nanostraws developed by Stanford researchers offer a method of sampling cell contents without disrupting its natural processes. A problem with the current method of cell sampling, called lysing, is that it ruptures the cell. Once the cell is destroyed, it can't be sampled from again. This new sampling system relies on tiny tubes 600 times smaller than a strand of hair that allow researchers to sample a single cell at a time. The nanostraws penetrate a cell's outer membrane, without damaging it, and draw out proteins and genetic material from the cell's salty interior. "It's like a blood draw for the cell," said Nicholas Melosh, an associate professor of materials science and engineering and senior author on a paper describing the work published recently in Proceedings of the National Academy of Sciences. The nanostraw sampling technique, according to Melosh, will significantly impact our understanding of cell development and could lead to much safer and effective medical therapies because the technique allows for long term, non-destructive monitoring. "What we hope to do, using this technology, is to watch as these cells change over time and be able to infer how different environmental conditions and 'chemical cocktails' influence their development - to help optimize the therapy process," Melosh said. If researchers can fully understand how a cell works, then they can develop treatments that will address those processes directly. For example, in the case of stem cells, researchers are uncovering ways of growing entire, patient-specific organs. The trick is, scientists don't really know how stem cells develop. "For stem cells, we know that they can turn into many other cell types, but we do not know the evolution - how do they go from stem cells to, say, cardiac cells? There is always a mystery. This sampling technique will give us a clearer idea of how it's done," said Yuhong Cao, a graduate student and first author on the paper. The sampling technique could also inform cancer treatments and answer questions about why some cancer cells are resistant to chemotherapy while others are not. "With chemotherapy, there are always cells that are resistant," said Cao. "If we can follow the intercellular mechanism of the surviving cells, we can know, genetically, its response to the drug." The sampling platform on which the nanostraws are grown is tiny - about the size of a gumball. It's called the Nanostraw Extraction (NEX) sampling system, and it was designed to mimic biology itself. In our bodies, cells are connected by a system of "gates" through which they send each other nutrients and molecules, like rooms in a house connected by doorways. These intercellular gates, called gap junctions, are what inspired Melosh six years ago, when he was trying to determine a non-destructive way of delivering substances, like DNA or medicines, inside cells. The new NEX sampling system is the reverse, observing what's happening within rather than delivering something new. "It's a super exciting time for nanotechnology," Melosh said. "We're really getting to a scale where what we can make controllably is the same size as biological systems." Building the NEX sampling system took years to perfect. Not only did Melosh and his team need to ensure cell sampling with this method was possible, they needed to see that the samples were actually a reliable measure of the cell content, and that samples, when taken over time, remained consistent. When the team compared their cell samples from the NEX with cell samples taken by breaking the cells open, they found that 90 percent of the samples were congruous. Melosh's team also found that when they sampled from a group of cells day after day, certain molecules that should be present at constant levels remained the same, indicating that their sampling accurately reflected the cell's interior. With help from collaborators Sergiu P. Pasca, assistant professor of psychiatry and behavioral sciences, and Joseph Wu, professor of radiology, Melosh and co-workers tested the NEX sampling method not only with generic cell lines, but also with human heart tissue and brain cells grown from stem cells. In each case, the nanostraw sampling reflected the same cellular contents as lysing the cells. The goal of developing this technology, according to Melosh, was to make an impact in medical biology by providing a platform that any lab could build. Only a few labs across the globe, so far, are employing nanostraws in cellular research, but Melosh expects that number to grow dramatically. "We want as many people to use this technology as possible," he said. "We're trying to help advance science and technology to benefit mankind." Melosh is also a professor in the photon science directorate at SLAC National Accelerator Laboratory, a member of Stanford Bio-X, the Child Health Research Institute, the Stanford Neurosciences Institute, Stanford ChEM-H and the Precourt Institute for Energy. Wu is also the Simon H. Stertzer, MD, Professor; he is director of the Stanford Cardiovascular Institute and a member of Stanford Bio-X, the Child Health Research Institute, Stanford ChEM-H and the Stanford Cancer Institute. Pasca is also a member of Stanford Bio-X, the Child Health Research Institute, the Stanford Neurosciences Institute and Stanford ChEM-H. The work was funded by the National Institute of Standards and Technology, the Knut and Alice Wallenberg Foundation, the National Institutes of Health, Stanford Bio-X, the Progenitor Cell Biology Consortium, the National Institute of Mental Health, an MQ Fellow award, the Donald E. and Delia B. Baxter Foundation and the Child Health Research Institute.


News Article | February 16, 2017
Site: www.eurekalert.org

Alpha cells in the pancreas can be induced in living mice to quickly and efficiently become insulin-producing beta cells when the expression of just two genes is blocked, according to a study led by researchers at the Stanford University School of Medicine. Studies of human pancreases from diabetic cadaver donors suggest that the alpha cells' "career change" also occurs naturally in diabetic humans, but on a much smaller and slower scale. The research suggests that scientists may one day be able to take advantage of this natural flexibility in cell fate to coax alpha cells to convert to beta cells in humans to alleviate the symptoms of diabetes. "It is important to carefully evaluate any and all potential sources of new beta cells for people with diabetes," said Seung Kim, MD, PhD, professor of developmental biology and of medicine. "Now we've discovered what keeps an alpha cell as an alpha cell, and found a way to efficiently convert them in living animals into cells that are nearly indistinguishable from beta cells. It's very exciting." Kim is the senior author of the study, which will be published online Feb. 16 in Cell Metabolism. Postdoctoral scholar Harini Chakravarthy, PhD, is the lead author. "Transdifferentiation of alpha cells into insulin-producing beta cells is a very attractive therapeutic approach for restoring beta cell function in established Type 1 diabetes," said Andrew Rakeman, PhD, the director of discovery research at JDRF, an organization that funds research into Type 1 diabetes. "By identifying the pathways regulating alpha to beta cell conversion and showing that these same mechanisms are active in human islets from patients with Type 1 diabetes, Chakravarthy and her colleagues have made an important step toward realizing the therapeutic potential of alpha cell transdifferentiation." Rakeman was not involved in the study. Cells in the pancreas called beta cells and alpha cells are responsible for modulating the body's response to the rise and fall of blood glucose levels after a meal. When glucose levels rise, beta cells release insulin to cue cells throughout the body to squirrel away the sugar for later use. When levels fall, alpha cells release glucagon to stimulate the release of stored glucose. Although both Type 1 and Type 2 diabetes are primarily linked to reductions in the number of insulin-producing beta cells, there are signs that alpha cells may also be dysfunctional in these disorders. "In some cases, alpha cells may actually be secreting too much glucagon," said Kim. "When there is already not enough insulin, excess glucagon is like adding gas to a fire." Because humans have a large reservoir of alpha cells, and because the alpha cells sometimes secrete too much glucagon, converting some alpha cells to beta cells should be well-tolerated, the researchers believe. The researchers built on a previous study in mice several years ago that was conducted in a Swiss laboratory, which also collaborated on the current study. It showed that when beta cells are destroyed, about 1 percent of alpha cells in the pancreas begin to look and act like beta cells. But this happened very slowly. "What was lacking in that initial index study was any sort of understanding of the mechanism of this conversion," said Kim. "But we had some ideas based on our own work as to what the master regulators might be." Chakravarthy and her colleagues targeted two main candidates: a protein called Arx known to be important during the development of alpha cells and another called DNMT1 that may help alpha cells "remember" how to be alpha cells by maintaining chemical tags on its DNA. The researchers painstakingly generated a strain of laboratory mice unable to make either Arx or DNMT1 in pancreatic alpha cells when the animals were administered a certain chemical compound in their drinking water. They observed a rapid conversion of alpha cells into what appeared to be beta cells in the mice within seven weeks of blocking the production of both these proteins. To confirm the change, the researchers collaborated with colleagues in the laboratory of Stephen Quake, PhD, a co-author and professor of bioengineering and of applied physics at Stanford, to study the gene expression patterns of the former alpha cells. They also shipped the cells to collaborators in Alberta, Canada, and at the University of Illinois to test the electrophysiological characteristics of the cells and whether and how they responded to glucose. "Through these rigorous studies by our colleagues and collaborators, we found that these former alpha cells were -- in every way -- remarkably similar to native beta cells," said Kim. The researchers then turned their attention to human pancreatic tissue from diabetic and nondiabetic cadaver donors. They found that samples of tissue from children with Type 1 diabetes diagnosed within a year or two of their death include a proportion of bi-hormonal cells -- individual cells that produce both glucagon and insulin. Kim and his colleagues believe they may have caught the cells in the act of converting from alpha cells to beta cells in response to the development of diabetes. They also saw that the human alpha cell samples from the diabetic donors had lost the expression of the very genes -- ARX and DNMT1 -- they had blocked in the mice to convert alpha cells into beta cells. "So the same basic changes may be happening in humans with Type 1 diabetes," said Kim. "This indicates that it might be possible to use targeted methods to block these genes or the signals controlling them in the pancreatic islets of people with diabetes to enhance the proportion of alpha cells that convert into beta cells." Kim is a member of Stanford Bio-X, the Stanford Cardiovascular Institute, the Stanford Cancer Institute and the Stanford Child Health Research Institute. Researchers from the University of Alberta, the University of Illinois, the University of Geneva and the University of Bergen are also co-authors of the study. The research was supported by the National Institutes of Health (grants U01HL099999, U01HL099995, UO1DK089532, UO1DK089572 and UC4DK104211), the California Institute for Regenerative Medicine, the Juvenile Diabetes Research Foundation, the Center of Excellence for Stem Cell Genomics, the Wallenberg Foundation, the Swiss National Science Foundation, the NIH Beta-Cell Biology Consortium, the European Union, the Howard Hughes Medical Institute, the H.L. Snyder Foundation, the Elser Trust and the NIH Human Islet Resource Network. Stanford's Department of Developmental Biology also supported the work. The Stanford University School of Medicine consistently ranks among the nation's top medical schools, integrating research, medical education, patient care and community service. For more news about the school, please visit http://med. . The medical school is part of Stanford Medicine, which includes Stanford Health Care and Stanford Children's Health. For information about all three, please visit http://med. .


News Article | February 17, 2017
Site: www.futurity.org

Chemists and gene therapy experts have a new way to insert the code for modified proteins into the cells of mice. They’ve used firefly proteins to make a glow-in-the-dark mouse and, if the technique also works in humans, it could be useful for vaccines or cancer therapies. Not only did the mouse glow, recalls Timothy Blake, a postdoctoral fellow in the Waymouth lab at Stanford University, but it also later woke up and ran around, completely unaware of the complex series of events that had just taken place within its body. Blake says it was the most exciting day of his life. This success, the topic of a recent paper in Proceedings of the National Academy of Sciences, could mark a significant step forward for gene therapy. It’s hard enough getting these protein instructions, called messenger RNA (mRNA), physically into a cell. It’s another hurdle altogether for the cell to actually use them to make a protein. If the technique works in people, it could provide a new way of inserting therapeutic proteins into diseased cells. “It’s almost a childlike enthusiasm we have for this,” says chemistry professor Robert Waymouth. “The code for an insect protein is put into an animal and that protein is not only synthesized in the cells but it’s folded and it becomes fully functional, capable of emitting light.” Although the results are impressive, this technique is remarkably simple and fast. And unlike traditional gene therapy that permanently alters the genetic makeup of the cell, mRNA is short-lived and its effects are temporary. The transient nature of mRNA transmission opens up special opportunities, such as using these compounds for vaccination or cancer immunotherapy. Gene therapy is a decades-old field of research that usually focuses on modifying DNA, the fundamental genetic code. That modified DNA then produces a modified mRNA, which directs the creation of a modified protein. The current work skips the DNA and instead just delivers the protein’s instructions. Previous work has been successful at delivering a different form of RNA—called short interfering RNA, or siRNA—but sending mRNA through a cell membrane is a much bigger problem. While both siRNA and mRNA have many negative charges—so-called polyanions—mRNA is considerably more negatively charged, and therefore more difficult to sneak through the positively charged cell membrane. What the researchers needed was a positively charged delivery method—a polycation—to complex, protect, and shuttle the polyanions. However, this alone would only assure that the mRNA made it through the cell membrane. Once inside, the mRNA needed to detach from the transporter compound in order to make proteins. The researchers addressed this twofold challenge with a novel, deceptively straightforward creation, which they call charge-altering releasable transporters (CARTs). “What distinguishes this polycation approach from the others, which often fail, is the others don’t change from polycations to anything else,” says chemistry professor Paul Wender, coauthor of the paper. “Whereas, the ones that we’re working with will change from polycations to neutral small molecules. That mechanism is really unprecedented.” As part of their change from polycations to polyneutrals, CARTs biodegrade and are eventually excreted from the body. The researchers say CARTs could move the field of gene therapy forward dramatically in several directions. “Gene therapy has been held up as a silver bullet because the idea that you could pick any gene you want is so alluring,” says Jessica Vargas, co-lead author of the study, who was a PhD student in the Wender lab during this research. “With mRNA, there are more limitations because the protein expression is transient, but that opens up other applications where you wouldn’t use other types of gene therapy.” One especially appropriate application of this technology is vaccination. At present, vaccines require introducing part of a virus or an inactive virus into the body in order to elicit an immune response. CARTs could potentially cut out the middleman, directly instructing the body to produce its own antigens. Once the CART dissolves, the immunity remains without any leftover foreign material present. The team is also working on applying their technique to another genetic messenger that would produce permanent effects, making it a complementary option to the temporary mRNA therapies. With the progress already made using mRNA and the potential of their ongoing research, they and others could be closer than ever to making individualized therapeutics using a person’s own cells. “Creating a firefly protein in a mouse is amazing but, more than that, this research is part of a new era in medicine,” says Wender. Funding came from the Department of Energy, the National Science Foundation, the National Institutes of Health, the Chambers Family Foundation for Excellence in Pediatric Research, the Child Health Research Institute, the Stanford Center for Molecular Analysis and Design, and the National Center for Research Resources.


News Article | February 16, 2017
Site: www.eurekalert.org

Timothy Blake, a postdoctoral fellow in the Waymouth lab, was hard at work on a fantastical interdisciplinary experiment. He and his fellow researchers were refining compounds that would carry instructions for assembling the protein that makes fireflies light up and deliver them into the cells of an anesthetized mouse. If their technique worked, the mouse would glow in the dark. Not only did the mouse glow, but it also later woke up and ran around, completely unaware of the complex series of events that had just taken place within its body. Blake said it was the most exciting day of his life. This success, the topic of a recent paper in Proceedings of the National Academy of Sciences, could mark a significant step forward for gene therapy. It's hard enough getting these protein instructions, called messenger RNA (mRNA), physically into a cell. It's another hurdle altogether for the cell to actually use them to make a protein. If the technique works in people, it could provide a new way of inserting therapeutic proteins into diseased cells. "It's almost a childlike enthusiasm we have for this," said chemistry Professor Robert Waymouth. "The code for an insect protein is put into an animal and that protein is not only synthesized in the cells but it's folded and it becomes fully functional, capable of emitting light." Although the results are impressive, this technique is remarkably simple and fast. And unlike traditional gene therapy that permanently alters the genetic makeup of the cell, mRNA is short-lived and its effects are temporary. The transient nature of mRNA transmission opens up special opportunities, such as using these compounds for vaccination or cancer immunotherapy. Gene therapy is a decades-old field of research that usually focuses on modifying DNA, the fundamental genetic code. That modified DNA then produces a modified mRNA, which directs the creation of a modified protein. The current work skips the DNA and instead just delivers the protein's instructions. Previous work has been successful at delivering a different form of RNA - called short interfering RNA, or siRNA - but sending mRNA through a cell membrane is a much bigger problem. While both siRNA and mRNA have many negative charges - so-called polyanions - mRNA is considerably more negatively charged, and therefore more difficult to sneak through the positively charged cell membrane. What the researchers needed was a positively charged delivery method - a polycation - to complex, protect and shuttle the polyanions. However, this alone would only assure that the mRNA made it through the cell membrane. Once inside, the mRNA needed to detach from the transporter compound in order to make proteins. The researchers addressed this twofold challenge with a novel, deceptively straightforward creation, which they call charge-altering releasable transporters (CARTs). "What distinguishes this polycation approach from the others, which often fail, is the others don't change from polycations to anything else," said chemistry Professor Paul Wender, co-author of the paper. "Whereas, the ones that we're working with will change from polycations to neutral small molecules. That mechanism is really unprecedented." As part of their change from polycations to polyneutrals, CARTs biodegrade and are eventually excreted from the body. This research was made possible through coordination between the chemists and experts in imaging molecules in live animals, who rarely work together directly. With this partnership, the synthesis, characterization and testing of compounds could take as little as a week. "We are so fortunate to engage in this kind of collaborative project between chemistry and our clinical colleagues. It allowed us to see our compounds go from very basic building blocks - all the way from chemicals we buy in a bottle - to putting a firefly gene into a mouse," said Colin McKinlay, a graduate student in the Wender lab and co-lead author of the study. Not only did this enhanced ability to test and re-test new molecules lead to the discovery of their charge-altering behavior, it allowed for quick optimization of their properties and applications. As different challenges arise in the future, the researchers believe they will be able to respond with the same rapid flexibility. After showing that the CARTs could deliver a glowing jellyfish protein to cells in a lab dish, the group wanted to find out if they worked in living mice, which was made possible through the expertise of the Contag lab, run by Christopher Contag, professor of pediatrics and of microbiology and immunology. Together, the multidisciplinary team showed that the CARTs could effectively deliver mRNA that produced glowing proteins in the thigh muscle or in the spleen and liver, depending on where the injection was made. The researchers said CARTs could move the field of gene therapy forward dramatically in several directions. "Gene therapy has been held up as a silver bullet because the idea that you could pick any gene you want is so alluring," said Jessica Vargas, co-lead author of the study, who was a PhD student in the Wender lab during this research. "With mRNA, there are more limitations because the protein expression is transient, but that opens up other applications where you wouldn't use other types of gene therapy." One especially appropriate application of this technology is vaccination. At present, vaccines require introducing part of a virus or an inactive virus into the body in order to elicit an immune response. CARTs could potentially cut out the middleman, directly instructing the body to produce its own antigens. Once the CART dissolves, the immunity remains without any leftover foreign material present. The team is also working on applying their technique to another genetic messenger that would produce permanent effects, making it a complementary option to the temporary mRNA therapies. With the progress already made using mRNA and the potential of their ongoing research, they and others could be closer than ever to making individualized therapeutics using a person's own cells. "Creating a firefly protein in a mouse is amazing but, more than that, this research is part of a new era in medicine," said Wender. Additional co-authors of this study, "Charge-altering releasable transporters (CARTs) for the delivery and release of mRNA in living animals," include Timothy Blake, Jonathan Hardy, Masamitsu Kanada and Christopher Contag. Waymouth is also a professor, by courtesy, of chemical engineering, a member of Stanford Bio-X, a faculty fellow of Stanford ChEM-H and an affiliate of the Stanford Woods Institute for the Environment. Wender is also a professor, by courtesy, of chemical and systems biology, a member of Stanford Bio-X, a member of the Stanford Cancer Institute and a faculty fellow of Stanford ChEM-H. Contag is also a professor, by courtesy, of radiology and of bioengineering, a member of Stanford Bio-X, a member of the Child Health Research Institute and a member of the Stanford Cancer Institute. This work was funded by the Department of Energy, the National Science Foundation, the National Institutes of Health, the Chambers Family Foundation for Excellence in Pediatric Research, the Child Health Research Institute, the Stanford Center for Molecular Analysis and Design and the National Center for Research Resources.

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