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  • richardmitnick 9:43 pm on September 21, 2017 Permalink | Reply
    Tags: , Biology, Green algae could hold clues for engineering faster-growing crops,   

    From Princeton: “Green algae could hold clues for engineering faster-growing crops” 

    Princeton University
    Princeton University

    Sept. 21, 2017
    Yasemin Saplakoglu

    Two new Princeton-led studies provide a detailed look at an essential part of algae’s growth machinery, with the eventual goal of applying this knowledge to improving the growth of crops. In this image, the researchers used a technique called cryo-electron tomography to image an algal structure called the pyrenoid, which concentrates carbon dioxide to make it more readily available for photosynthetic enzymes (purple). The yellow tubules inside the green tubes are thought to bring carbon and other materials into the pyrenoid.
    Still from video courtesy of Benjamin Engel, Max Planck Institute of Biochemistry

    Two new studies of green algae — the scourge of swimming pool owners and freshwater ponds — have revealed new insights into how these organisms siphon carbon dioxide from the air for use in photosynthesis, a key factor in their ability to grow so quickly. Understanding this process may someday help researchers improve the growth rate of crops such as wheat and rice.

    In the studies published this week in the journal Cell, the Princeton-led team reported the first detailed inventory of the cellular machinery — located in an organelle known as the pyrenoid — that algae use to collect and concentrate carbon dioxide. The researchers also found that the pyrenoid, long thought to be a solid structure, actually behaves like a liquid droplet that can dissolve into the surrounding cellular medium when the algal cells divide.

    “Understanding how algae can concentrate carbon dioxide is a key step toward the goal of improving photosynthesis in other plants,” said Martin Jonikas, an assistant professor of molecular biology at Princeton and leader of the studies, which included collaborators at the Max Planck Institute of Biochemistry in Germany and the Carnegie Institution for Science on the Stanford University campus. “If we could engineer other crops to concentrate carbon, we could address the growing world demand for food,” Jonikas said.

    Aquatic algae and a handful of other plants have developed carbon-concentrating mechanisms that boost the rate of photosynthesis, the process by which plants turn carbon dioxide and sunlight into sugars for growth. All plants use an enzyme called Rubisco to “fix” carbon dioxide into sugar that can be used or stored by the plant.

    Algae have an advantage over many land plants because they cluster the Rubisco enzymes inside the pyrenoid, where the enzymes encounter high concentrations of carbon dioxide pumped in from the air. Having more carbon dioxide around allows the Rubisco enzymes to work faster.

    In the first of the two studies reported this week, the researchers conducted a sweeping search for proteins involved in the carbon-concentrating mechanism of an algae species known as Chlamydomonas reinhardtii. Using techniques the researchers developed for rapidly labeling and evaluating algal proteins, the researchers identified the locations and functions of each protein, detailing the physical interactions between the proteins to create a pyrenoid “interactome.”

    The search revealed 89 new pyrenoid proteins, including ones that the researchers think usher carbon into the pyrenoid and others that are required for formation of the pyrenoid. They also identified three previously unknown layers of the pyrenoid that surround the organelle like the layers of an onion. “The information represents the best assessment yet of how this essential carbon-concentrating machinery is organized and suggests new avenues for exploring how it works,” said Luke Mackinder, the study’s first author and a former postdoctoral researcher at the Carnegie Institution who now leads a team of researchers at the University of York, U.K.

    In the second study [Cell], the researchers report that the pyrenoid, long thought to be a solid structure, is actually liquid-like. Techniques used in previous studies required the researchers to kill and chemically preserve the algae before imaging them. In this new study, the researchers imaged the algae while the organisms were living by using a yellow fluorescent protein to label Rubisco.

    The researchers conducted a large-scale search for proteins associated with the pyrenoid. The resulting data allowed researchers to propose the most detailed model yet of the spatial organization of the pyrenoid.
    Image courtesy of Luke Mackinder, et al. Cell 2017

    While observing the algae, Elizabeth Freeman Rosenzweig, then a Carnegie Institution graduate student, and Mackinder used a high-powered laser to destroy the fluorescent label on Rubisco in half of the pyrenoid, while leaving the label in the other half of the pyrenoid intact. Within minutes, the fluorescence redistributed to the entire pyrenoid, showing that the enzymes easily moved around as they would in a liquid.

    Benjamin Engel, a postdoctoral researcher and project leader at the Max Planck Institute of Biochemistry, further explored this finding using another imaging technique called cryo-electron tomography. He froze and prepared whole algae cells and then imaged them with an electron microscope, which is so sensitive that it can resolve the structures of individual molecules.

    The technique enabled Engel to visualize the pyrenoid in three dimensions and at nanometer-resolution. By comparing these images with those of liquid systems, the researchers confirmed that the pyrenoid was organized like a liquid. “This is one of the rare examples where classical genetics, cell biology and high-resolution imaging approaches were all brought together in one investigation,” Engel said.

    The study enabled the team to ask how a pyrenoid is passed down to the next generation when the single-celled algae divide into two daughter cells. Freeman Rosenzweig noted that the pyrenoid sometimes fails to divide, leaving one of the daughter cells with no pyrenoid.

    Using the fluorescent proteins, the team observed that the cell that failed to receive half the pyrenoid in fact could still form one spontaneously. They found that each daughter cell receives some amount of the pyrenoid in its dissolved form and that these nearly undetectable components can condense into a full-fledged pyrenoid.

    “We think the pyrenoid dissolution before cell division and condensation after division may be a redundant mechanism to ensure that both daughter cells get pyrenoids,” Jonikas said. “That way, both daughter cells will have this key organelle that’s critical for assimilating carbon.”

    To further explore how this might happen, Jonikas collaborated with Ned Wingreen, Princeton’s Howard A. Prior Professor in the Life Sciences and of Molecular Biology. Wingreen and his team created a computer simulation of the interactions between Rubisco and another protein called EPYC1 [PNAS] — discovered to be crucial to the pyrenoid by Mackinder and others on Jonikas’ team — which acts like glue to stick together multiple Rubiscos.

    The computer simulation suggested that the state of the pyrenoid — whether a condensed liquid droplet or dissolved into the surrounding compartment — depended on the number of binding sites on EPYC1. In the simulation, Rubisco has eight binding sites, or eight places where EPYC1 can dock to a Rubisco. If EPYC1 has four binding sites, then two EPYC1s exactly fill all of the docking sites on one Rubisco, and vice versa. Because these fully bonded Rubisco-EPYC1 complexes are small, they form a dissolved state. But if EPYC1 has three or five binding sites, it cannot fill all of the Rubisco sites, and there are open sites on the Rubiscos for binding by additional EPYC1s, which also have free sites that can attract other Rubiscos. The result is a clump of Rubiscos and EPYC1s that form a liquid-like droplet.

    The change in the system’s phase depending on the ratio of EPYC1 to Rubisco binding sites can be considered a “magic number” effect, a term typically used in physics to describe conditions where a specific number of particles form an unusually stable state. “These magic numbers, besides being relevant for pyrenoid systems, may have some currency in the field of polymer physics and potentially in synthetic biology,” Wingreen said.

    Wingreen and Jonikas are continuing their collaboration and hope to develop the project both theoretically — by exploring different flexibilities and configurations of Rubisco and EPYC1 — and experimentally, by combining the two proteins in a test tube and manipulating the number of binding sites.

    “The previous thinking was that the more binding sites they have, the more the proteins tend to cluster,” Jonikas said. “The discovery that there is a magic number effect is important not only for pyrenoids, but perhaps for many other liquid-like organelles found throughout nature.”

    With additional studies, these findings may yield important insights into ensuring the availability of fast-growing crops for an expanding world population.

    See the full article here .

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    Princeton University Campus

    About Princeton: Overview

    Princeton University is a vibrant community of scholarship and learning that stands in the nation’s service and in the service of all nations. Chartered in 1746, Princeton is the fourth-oldest college in the United States. Princeton is an independent, coeducational, nondenominational institution that provides undergraduate and graduate instruction in the humanities, social sciences, natural sciences and engineering.

    As a world-renowned research university, Princeton seeks to achieve the highest levels of distinction in the discovery and transmission of knowledge and understanding. At the same time, Princeton is distinctive among research universities in its commitment to undergraduate teaching.

    Today, more than 1,100 faculty members instruct approximately 5,200 undergraduate students and 2,600 graduate students. The University’s generous financial aid program ensures that talented students from all economic backgrounds can afford a Princeton education.

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  • richardmitnick 10:24 pm on September 20, 2017 Permalink | Reply
    Tags: , Biology, , ,   

    From SLAC: “High-Speed Movie Aids Scientists Who Design Glowing Molecules” 

    SLAC Lab

    September 20, 2017
    Amanda Solliday

    Aequorea victoria, also called the crystal jelly, is a bioluminescent jellyfish that lives near the Pacific coast of North America. (Gary Kavanagh/iStockphoto.com)

    The Coherent X-Ray Imaging (CXI) instrument makes use of the brilliant hard X-ray pulses from the Linac Coherent Light Source. The equipment is tailored for X-ray crystallography experiments. (SLAC National Accelerator Laboratory)


    With SLAC’s X-ray laser, a research team captured ultrafast changes in fluorescent proteins between “dark” and “light” states. The insights allowed the scientists to design improved markers for biological imaging.

    The crystal jellyfish swims off the coast of the Pacific Northwest and can illuminate the waters when disturbed. That glow comes from proteins that absorb energy and then release it as bright flashes.

    To track many of life’s activities, biologists took a cue from this same jellyfish.

    Scientists collected one of the proteins found in the sea creatures, green fluorescent protein (GFP), and engineered a molecular light switch that would glow or remain dark depending on specific experimental conditions. The glowing labels are attached to molecules in living cells so researchers can highlight them during imaging experiments. They use these fluorescent markers to understand how a cell responds to changes in its environment, identify which molecules interact within a cell and track the effects of genetic mutations.

    Researchers have studied GFP and other fluorescent proteins for decades to better understand their glowing action and improve their function in scientific studies, but they have never been able to observe the ultrafast changes that occur between “off” and “on” states until now.

    In a recent experiment conducted at the Department of Energy’s SLAC National Accelerator Laboratory, a research team used bright, ultrafast X-ray pulses from SLAC’s X-ray free-electron laser to create a high-speed movie of a fluorescent protein in action. With that information, the scientists began to design a marker that switches more easily, a quality that can improve resolution during biological imaging.

    “We think that this approach will open a world of possibilities to tailor fluorescent proteins,” says Martin Weik, scientist at the Institute of Structural Biology in Grenoble, France and one of the authors on the publication. “We not only have the structure of the fluorescent protein, but now we can see what is happening between one static state and the other.”

    Nature Chemistry published the study on Sept. 11.

    Filming a Molecular Light Switch

    To observe these intermediate states, the scientists initiated a photochemical reaction in the fluorescent protein with an optical laser at the Coherent X-ray Imaging instrument at the Linac Coherent Light Source, followed by X-ray snapshots at distinct time delays. The optical laser provides energy in the form of photons, mimicking what happens in nature.

    “Atoms move around in the photoactive site of the molecule as a result of absorption of a photon,” says Sebastien Boutet, SLAC scientist and a co-author of the paper. “This structural change turns the protein from a dark state to a light-emitting (fluorescent) state.”

    There’s a vast body of literature calculating what might happen between the two states, but no one studying the protein was able to see the structural changes in the switch as the photon is absorbed. The molecular switch was just too fast for traditional X-ray imaging techniques.

    In this study, the femtosecond X-ray pulses generated by LCLS—arriving in just millionths of a billionth of a second—allowed the team to create stop-action images of the process at an extremely close interval after the proteins were activated by the optical laser.

    A Door Half Open

    The high-speed snapshots were used to generate a movie starting from the dark state, and gave the researchers insights that they used to design more efficient switchable light-emitting proteins. They found a clue in the time the molecules spent between fluorescent and non-fluorescent states.

    “After a picosecond, and for a very short time, this molecular switch is stuck between on and off,” says Ilme Schlichting, scientist at the Max-Planck Institute in Heidelberg, Germany and one of the authors on the publication. “People have predicted this, but to actually visualize its structure is extremely exciting.”

    “It’s as if there’s a door and it’s neither closed nor completely open; it’s half open,” she says. “And now we are learning what can go through the door, what might be blocking it and how it works in real time.”

    In this study, the scientists found that an amino acid blocked the door and prevented the switch from flipping as easily as possible.

    The researchers shortened the amino acid in a mutated version of the fluorescent protein. This engineered version switched more easily and gave better contrast. These traits will allow scientists to observe cellular activity with greater precision.

    “Contrast is essential in imaging. It’s like on a TV screen, where to see the best picture, you want the dark to be extremely dark and the color to be super bright and colorful,” says Jacques-Philippe Colletier, a scientist at the Institute of Structural Biology who contributed to the research.

    This new molecular movie featuring the jellyfish-inspired proteins lights the way to capture more of life’s microscopic details. The team will continue to fine-tune the protein for other desired characteristics that make it ideal for “super-resolution microscopy,” a type of light microscopy where scientists are able to see illuminated details of cells not distinguishable with conventional light microscopy methods.

    The research collaboration included several French institutions, including the Institute of Structural Biology, University of Lille, University of Paris-Sud and the University of Rennes, as well as Max Planck Institutes in Germany and SLAC.

    LCLS is a DOE Office of Science User Facility.

    See the full article here .

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    SLAC Campus
    SLAC is a multi-program laboratory exploring frontier questions in photon science, astrophysics, particle physics and accelerator research. Located in Menlo Park, California, SLAC is operated by Stanford University for the DOE’s Office of Science.

  • richardmitnick 8:57 am on September 20, 2017 Permalink | Reply
    Tags: , , Biology, , , First user groups, ,   

    From XFEL: “First users at European XFEL” 

    XFEL bloc

    European XFEL

    No writer credit

    DESY’s Anton Barty (left) and Henry Chapman (right), seen at the SPB/SFX instrument, were in one of the first two user groups. (Photo: DESY, Lars Berg)

    The first users have now started experiments at the new international research facility in Schenefeld.

    “This is a very important event, and we are very happy that the first users have now arrived at European XFEL so we can do a full scale test of the facility” said European XFEL Managing Director Prof. Dr. Robert Feidenhans’l. ”The instruments and the supporting teams have made great progress in the recent weeks and months. Together with our first users, we will now do the first real commissioning experiments and collect valuable scientific data. At the same time, we will continue to further advance our facility and concentrate on further improving the integration and stability of the instrumentation” he added.

    The first two instruments available for users in the underground experiment hall are the FXE (Femtosecond X-Ray Experiments) instrument, and the SPB/SFX (Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography) instrument.

    The FXE instrument will enable the research of extremely fast processes. It will be possible to create “molecular movies” showing the progression of chemical reactions which, for example, will help improve our understanding of how catalysts work, or how plants convert light into usable chemical energy. The first seven experiments conducted at FXE highlight the range of methods available at the instrument and the diversity of topics of study possible. Experiments will include using different spectroscopy methods to track ultrafast reactions and electron movement in model molecules, probe organic light emitting diodes, or investigate the recombination of nitrogen and oxygen in the muscle tissue protein myoglobin.

    The first user group at the FXE instrument. No image credit

    The SPB/SFX instrument will be used to gain a better understanding of the shape and function of biomolecules, such as proteins, that are otherwise difficult to study. Several of the seven first experiments at this instrument will focus on method development for these new research opportunities at European XFEL or ways to reduce the amount of precious sample used for the examination of biological processes. Other groups will be studying biological structures and processes such as the Melbourne virus and the water splitting process in photosynthesis.

    The first user group at the SPB/SFX instrument. No image credit

    In this first round of beamtime a total of 14 groups, of up to 80 users each and travelling to Schenefeld from across the globe, will conduct experiments at European XFEL until March 2018. Each group will have about five days of 12 hours of beamtime.

    See the full article here .

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    XFEL Campus

    The Hamburg area will soon boast a research facility of superlatives: The European XFEL will generate ultrashort X-ray flashes—27 000 times per second and with a brilliance that is a billion times higher than that of the best conventional X-ray radiation sources.

    The outstanding characteristics of the facility are unique worldwide. Starting in 2017, it will open up completely new research opportunities for scientists and industrial users.

  • richardmitnick 1:51 pm on September 14, 2017 Permalink | Reply
    Tags: , , Biology, , , ,   

    From EPFL: “Unexpected facets of Antarctica emerge from the labs” 

    EPFL bloc

    École Polytechnique Fédérale de Lausanne EPFL

    Sarah Perrin

    the Akademik Treshnikov Russian icebreaker

    Six months after the Antarctic Circumnavigation Expedition ended, the teams that ran the 22 scientific projects are hard at work sorting through the many samples they collected. Some preliminary findings were announced during a conference in Crans Montana organized by the Swiss Polar Institute, who just appointed Konrad Steffen as new scientific director (see the interview below).

    Nearly 30,000 samples were taken during the Antarctic Circumnavigation Expedition (ACE). And now, barely six months after the voyage ended, the research teams tasked with analyzing the samples have already produced some initial figures and findings. These were presented in Crans Montana during a conference put together earlier this week by the Swiss Polar Institute (SPI), the EPFL-based entity that ran the expedition. The event, called “High altitudes meet high latitudes,” brought together world-renowned experts in polar and alpine research in an exercise aimed at highlighting the many similarities between these two fields of study.

    Over the course of three months – from December 2016 to March 2017 – 160 researchers from 23 different countries sailed around the Great White Continent on board a Russian icebreaker. They ran 22 research projects in an effort to learn more about the impact of climate change on these fragile and little-known regions. The valuable samples, taken from the Southern Ocean, the atmosphere and a handful of remote islands, are now back at the labs of the 73 scientific institutions involved in the expedition.

    The route of the ACE expedition.

    Most of the teams that ran the 22 projects are still carrying out the preliminary task of sorting through and identifying the samples, which means the initial results are necessarily incomplete and provisional. It is only later that the samples will be analyzed. Some important observations can nevertheless be made at this stage.

    A solid database

    The sum total of the samples collected represents an impressive and valuable database. The SPI must now come up with ways to organize, group and present the data so that researchers can readily access and make use of it. What’s more, “the large number of potential collaborations and exchanges between projects is becoming clear,” says David Walton, the chief scientist on the expedition. “Some research projects have been found to have links with as much as nine others.” And some startling figures have already been released – here is a look at just a few of them.

    For the SubIce project, around 100 meters of ice cores were taken on five subantarctic islands and the Mertz Glacier, which sits on the edge of the Antarctic continent. The chemical composition of the cores will be analyzed in an attempt to trace climate change over recent decades. In some places, like Balleny, Peter 1st or Bouvet Islands, it was the first time an ice sample had ever been taken. “Of all the islands where we were able to take samples, that last one was the farthest from the continent,” says Liz Thomas, from British Antarctic Survey. “It’s also the island where the ice in the samples is the most granular. Our findings confirm significant seasonal variations at this location.”

    The air on the continent is so pure that even the hottest cup of tea does not produce any steam. “No particles, no clouds,” explains Julia Schmale, a researcher with the Paul-Scherrer-Institute who measured for aerosols – tiny chemical particles like grains of sand, dust, pollen, soot, sulfuric acid, and so on – throughout the expedition. These particles attach to water molecules and aggregate to form clouds. On Mertz Glacier, her measurements revealed aerosol levels below 100 particles per cm3, which is less than the level found in a cleanroom.

    Christel Hassler and her team, from the University of Geneva, studied bacteria and virus populations in the Southern Ocean. The team took some 170 samples from all around the continent. For the time being, their work consists in isolating and culturing the numerous cells found in the samples. “We will then analyze their DNA in order to identify them,” says Marion Fourquez, a marine biologist. “That will show us whether we have come across any new bacterial strains that have yet never been observed in this region.”

    Bacteria collected on the sedimental floor beneath Mertz glacier, on the Antarctic continent, as part of Christel Hassler’s project (University of Geneva). ©M.Fourquez.

    One of the subsequent lines of research will be to determine their geographical distribution. The researchers will be able to tell if there’s a link between the presence of a given bacterium and that of other microorganisms by comparing their data with data from other projects, like Nicolas Cassar’s. Cassar, from Duke University in the United States, measured concentrations of phytoplankton, which sit at the very bottom of the region’s food chain. “This approach worked out well, and we have nearly continuous samples from along the entire route,” says Walton.

    More than 3,000 whales

    Brian Miller, from the Australian Antarctic Division, was interested in somewhat larger animals. For his project, he used a piece of sophisticated acoustic equipment to listen for and count the number of whales in the Southern Ocean. Walton notes: “In around 500 hours of recordings, the researchers counted for example over 3,000 individual blue whales, although we actually saw only three or so at the surface.” These cetaceans appear to be particularly plentiful in the depths of the Ross Sea.

    Peter Ryan, from the University of Cape Town in South Africa, observed and counted bird populations. He discovered that one of the largest colonies of king penguins, on Pig Island in the Crozet archipelago, had declined drastically – he estimates the numerical loss to be around 75%. “That’s around half a million animals,” says Walton. “We don’t know if they’ve died or migrated to other colonies, like the one in St. Andrews Bay, in South Georgia, which is actually in a growth phase.”

    More complete and detailed results will be published in the coming months.

    Detailed information on SPI and ACE can be found on http://spi-ace-expedition.ch


    “We urgently need to coordinate our efforts.”

    Konrad Steffen, a glaciologist and the new scientific director of the Swiss Polar Institute (SPI), has been involved in polar research for the past 40 years. His work has focused primarily on the Arctic, particularly the changes taking place within Greenland’s ice sheet. He is also a professor at ETH Zurich and director of the Swiss Federal Institute for Forest, Snow and Landscape Research WSL.

    Professor Steffen, why is the Swiss Polar Institute so necessary today?

    Research in this field tended to be conducted by small groups that organized their own expeditions and ran their own projects. In Switzerland, there had never been any kind of initiative aimed at coordinating all this work. The effects of climate change on polar and alpine regions are now so evident that we urgently need to coordinate our efforts and conduct cross-disciplinary research. This is what we did with the ACE project, where researchers from fields like oceanography, glaciology and biology came together in an attempt to improve our understanding of the climate-change process in a region.

    What for you is the top priority when it comes to the polar regions?

    At the SPI, one of our aims is to devise a strategic plan within the scientific community. More personally, I think that we urgently need to assess the mass balance of ice sheets across the globe. That’s what will have the greatest and swiftest impact in terms of rising sea levels and changes to our coastlines. Instead of studying individual glaciers in the Alps, we need to look at the bigger picture and observe in detail how the atmosphere interacts with large ice sheets, such as those in Greenland and the Antarctic. We need to connect the dots to see how the system as a whole is affected.

    What made the ACE such an innovative expedition?

    There have been many scientific expeditions to the Antarctic, but they usually only cover part of the continent. This was the first time that an expedition went all the way around the continent in one three-month period, studying all the oceans during the same season. That provides a fuller picture of the issues, such as microplastics – during the trip, we really saw that they were everywhere! The expedition also served up attractive career opportunities for budding young scientists and enabled several research groups to establish long-term partnerships.

    Are any other expeditions in the pipeline?

    Yes, the next one is planned for 2019. The aim is to sail around Greenland. We are in the process of looking for a vessel and determining what sort of research will be undertaken during the trip.


    See the full article here .

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    EPFL campus

    EPFL is Europe’s most cosmopolitan technical university with students, professors and staff from over 120 nations. A dynamic environment, open to Switzerland and the world, EPFL is centered on its three missions: teaching, research and technology transfer. EPFL works together with an extensive network of partners including other universities and institutes of technology, developing and emerging countries, secondary schools and colleges, industry and economy, political circles and the general public, to bring about real impact for society.

  • richardmitnick 7:26 am on September 8, 2017 Permalink | Reply
    Tags: , Biology, Human Cell Atlas hopes to unravel mysteries hidden in our genes, , Microsatellites   

    From Horizon: “Human Cell Atlas hopes to unravel mysteries hidden in our genes” 



    06 September 2017
    Richard Gray

    A process called droplet microfluidics isolates thousands of cells in microscopic water droplets allowing up-close analysis of genetic material. Image credit – Dr Linas Mazutis

    A major international project is attempting to create the first comprehensive three-dimensional map of all human cells which could end up revealing secrets about our health and how our bodies function.

    It is nearly 350 years since scientists first discovered that our bodies are made up of tiny building blocks known as cells. Today we still know very little about their nature, but if we did, we could better understand how our bodies work, how diseases afflict us and how we age.

    A global project called Human Cell Atlas is now attempting to create the first comprehensive three-dimensional map of the human body in order to unravel some of these mysteries.

    ‘It will hopefully have the same impact as when the human genome was sequenced,’ said Dr Linas Mazutis, a biochemist at Vilnius University in Lithuania. ‘A human cell atlas could set the stage to develop new technologies and provide new answers about the human body.’

    The project could also lead to better diagnosis and treatment of diseases, but taking a census of all human cells is no simple task – there are an estimated 40 trillion in each adult body.

    Dr Mazutis coordinates an EU-funded project called Cells-in-drops, which is aimed at developing some of the techniques needed to create this enormous map of human cells.

    Their technology allows thousands of individual cells to be rapidly isolated into microscopic water droplets around 100 micrometres across – about the same width as a human hair.

    Known as droplet microfluidics, this approach can be loaded with biochemical reagents that break open the cells, spilling their contents into the water they are encased in.

    This essentially turns each droplet into a microscopic test tube where the genetic material, or its other contents, can be analysed.

    ‘We are particularly looking at gene expression programs of single cells,’ said Dr Mazutis.

    This is important as not all genes, or DNA, in every cell in the human body are switched on – in some tissues certain genes are deactivated while others are amplified.

    DNA can also be read in different ways depending on the cell it is in, meaning different proteins can be produced from the same genetic code.

    To help unravel this complexity, Dr Mazutis co-developed a technique with colleagues at Harvard University that analyses another type of genetic material called ribonucleic acid (RNA).


    RNA plays an important role in cells by helping to translate the DNA code into proteins the cell needs to grow or replicate. Analysing this can then reveal details about a cell’s activity and functions.

    The technique used by Dr Mazutis converts the RNA from a cell in one of the microdroplets back into DNA, but adds a unique barcode into the sequence of genes. The genetic material from all of the droplets are then pooled together and analysed in bulk.

    As the genetic material from each droplet has been labelled with a barcode, it means the RNA sequences from each cell can be individually identified.

    Compared to previous techniques, which required separating cells into 96 individual wells on a plastic plate, it is orders of magnitude faster and cheaper, something that will be essential for building the Human Cell Atlas.

    Doing this for 40 trillion cells, however, will generate staggering volumes of data, requiring expertise from around the world in many different disciplines, but scientists are already seeing what may come out of it.

    Professor Ehud Shapiro, a computer scientist and biologist at the Weizmann Institute of Science in Rehovot, Israel, said: ‘The Human Cell Atlas 1.0 is trying to map all cell types in the human body.

    ‘Once that is achieved, the thought is to look at questions of cell lineage to produce a sort of 4D atlas of human cell development over time.’

    Cellular history

    The first multi-cellular organism to have its cellular history mapped was a tiny 1 millimetre-long nematode worm called Caenorhabditis elegans. Composed of just 1 000 cells when fully grown, the worm was filmed as it matured from a cell into an adult, allowing scientists to follow its development.

    This feat has not been repeated with any larger organism due to the difficulties in tracking cells in this way. But Prof. Shapiro and his team are developing techniques that allow them to reconstruct the lineage of cells in the human body, using tiny errors that occur in parts of the genome known as microsatellites.

    These are composed of long repeats of the same code, which suffer errors as the DNA is copied when cells divide and replicate.

    Prof. Shapiro and his colleagues calculated that they could reconstruct the family tree of a cell if they could track one million of these errors in its DNA. But so far it has only been possible to examine just a few hundred microsatellite errors in each cell.

    However, Prof. Shapiro has being developing new techniques to look at thousands and even tens of thousands at a time.

    In a project funded by the EU’s European Research Council, called LineageDiscovery, he is now working on an advanced technology called padlock, or molecular inversion, which uses open loop-shaped genetic probes to target potential sites of errors.

    ‘So far we can use 12 000 padlock probes at once and we are now working on using 50 000 probes,’ explained Prof. Shapiro. ‘It makes a million probes seem not so far away.’

    If successful, unlocking the human cell lineage tree in this way could answer some of the fundamental questions bothering biologists today. For example, it could help uncover why some cancers spread to new areas in the body.

    See the full article here .

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  • richardmitnick 2:28 pm on September 1, 2017 Permalink | Reply
    Tags: , Biology, Going with the Ion Flow, Ion channels, , Northwestern University   

    From Northwestern: “Going with the Ion Flow” 

    Northwestern U bloc
    Northwestern University



    Northwestern Medicine scientists are diving deep into the structure and function of ion channels to inform new therapies.

    A growing cohort of talented Northwestern Medicine scientists is working to unlock the secrets of ion channels and discover how these tiny molecular machines contribute to an array of diseases, from brain tumors and epilepsy to kidney disease and devastating immune deficiencies.

    This group of investigators, including seasoned faculty like Alfred George Jr., MD, Magerstadt Professor and chair of Pharmacology, and newcomers like Paul DeCaen, PhD, assistant professor in the same department, are not only fundamentally altering understanding of disorders, they’re also revealing how existing treatments work and pointing to potential new treatment strategies.

    “All of this expertise provides fertile ground for making new discoveries,” says DeCaen, a former Howard Hughes and Harvard University fellow who joined Northwestern in October 2016. “And a world-class hospital here gives us access to the medical perspective on ion channel-linked diseases.”

    Ion channels are a class of proteins that control the flow of ions such as calcium, sodium or potassium across the membranes of cells, DeCaen explains. Maintaining a proper flow of ions is critical to a multitude of bodily functions, from the transmission of messages between brain cells to the beating of the heart.

    “It seems like a simple job, but it ends up frequently being problematic,” he says.

    Mutations in the genes that encode ion channels have been linked to many medical conditions. To understand how these mutations lead to disease, ion channel investigators try to piece together the three-dimensional molecular structures of ion channels.

    For example, DeCaen and colleagues from the lab of Erhu Cao, PhD, at the University of Utah took this approach to better understand a gene called polycystic kidney disease 2 (PKD2). Mutations in the gene had been found in patients who develop large cysts in their kidneys that cause organ failure. Scientists knew the gene encoded an ion channel that controls the flow of ions, but did not know which ions. Work from DeCaen’s lab pointed to potassium and sodium.

    “We now know what ions move through the channel, but no one had any idea of what it looked like in three-dimensional space,” DeCaen says. “Since function follows form, we figured that this is an important knowledge gap to fill.”

    So, the team chilled the protein to a very low temperature and then used a powerful electron microscope to get the first glimpse of the protein’s configuration. The results were published in the journal Cell last year.

    “Now that we know what the ion channel looks like, we can see how mutations that cause alterations in its structure may cause it to malfunction in the disease state,” he says. “We can start to do some pie-in-the-sky thinking about developing small molecules that can affect the ion channel’s function.”

    For example, in polycystic kidney disease it is not clear whether mutations cause the PKD2 channel to be continually open, allowing an unending flow of ions, or if the mutation closes the channel. There might even be a mix of on/off effects depending on the specific mutation. So, DeCaen and colleagues are using electrophysiological techniques to find out. Their results could inform the design of drugs to combat the disease.

    DeCaen has also been consulting Northwestern clinicians about complications beyond cysts in patients with polycystic kidney disease. These clinical insights might provide clues on the function of these ion channels throughout the body and potentially suggest treatment strategies.

    “In ion channel research, you need a broad range of expertise in medicine,” DeCaen explained. “You need a neurologist, a cardiac arrhythmias expert and kidney disease experts. We have that large pool of scientists and clinicians here at Northwestern.”

    Working with George, and Jennifer Kearney, PhD, associate professor of Pharmacology, DeCaen is also probing the role of ion channels in epilepsy. His lab is recreating the structure of a bacterial version of an epilepsy-linked sodium channel as a first step toward recreating the mammalian version. So far, the work has yielded unexpected clinical benefits.

    “This gave us our first glimpse into how anti-epileptic drugs work,” DeCaen says. It has also suggested potential antibacterial treatments that would target the channel.

    The applications of this line of research go even further: This summer, George and colleagues showed how mutations in a sodium channel called Nav1.9 can lead to a disorder where people are unable to feel pain. The findings, published in The Journal of Clinical Investigation, might have implications for the development of novel therapies for pain.

    “Ion channels represent an under-appreciated class of druggable protein targets,” says George. “A goal for the Department of Pharmacology has been to place ion channels at the center stage of research efforts to find new drug targets.”


    Meanwhile, Murali Prakriya, PhD, associate professor of Pharmacology, focuses on the Ca2+ release-activated Ca2+ (CRAC) channel. Originally described in immune cells, CRAC channels are found in the plasma membranes of most, if not all, human cells. When the channel opens, it allows calcium ions to flow into the cell, signaling functions such as gene expression and cell proliferation. A growing number of diseases are associated with abnormalities in CRAC channel function including immunodeficiencies, muscular dystrophy and neurological diseases such as Alzheimer’s disease.

    “CRAC calcium channels are widespread and important for many biological processes, from the birth of cells to the death of cells,” Prakriya says. “Therefore, dissecting how CRAC channel activity is controlled and regulated in different contexts is of great interest.”

    His lab is working to understand how CRAC channels operate and contribute to immune host defense mechanisms, the detection of allergens in the lung airways, and brain function.

    “If you lose CRAC channel function through mutations, human patients develop devastating immune deficiencies and muscle weakness,” he explains. “Children born with these symptoms often die in the first six months of life. The simplest infections are quite dangerous to these children.”

    In a paper published in Nature Communications early this year, Prakriya worked with Megumi Yamashita, PhD, DDS, research assistant professor of Pharmacology, and Priscilla Yeung, a student in Feinberg’s Medical Scientist Training Program, to reveal how the CRAC channel opens and closes. This research identified the molecular structure in the channel that functions as the gate, as well as the movements in the channel pore that open the gate.

    First, the scientists used electrophysiology and microscopy techniques to systematically probe the contributions of different regions of the CRAC channel protein to pore opening, identifying an oily amino acid as the channel gate in the process. Then, computer simulations developed by University of Toronto collaborators helped reveal how this amino acid impedes ion conduction.

    “In ion channels, the pore is usually filled with water, so one way to close the pore is to present an oily, hydrophobic chemical group in the pore to prevent water and ions from going through — similar to the way that oil and water don’t mix. To open the pore, the hydrophobic group swings out of the way allowing the pore to fill with water and ions,” Prakriya explains. “The presence of the oily amino acid in the pore creates a closed channel state.”

    These conclusions have important clinical implications. Some human mutations in the gene encoding the CRAC channel leave the gate open and cause uncontrolled bleeding, neurological problems and muscle weakness because the cells in these individuals have excessive levels of calcium all the time.

    “We showed that one of these mutations affected the oiliness of the gate region, thereby chronically filling the pore with water and ions,” Prakriya says. “As a consequence, ions were going through when they shouldn’t.”

    Prakriya’s lab is currently working to understand the molecular signals that open the hydrophobic gate and to identify small molecules that can interact with the gate to alter the channel’s activity. These could correct defects in cell signaling and ameliorate symptoms associated with aberrant CRAC channel activity seen in immune, muscular and neurodegenerative diseases.

    Anatomy of an Ion Channel
    Ion channels are a class of proteins that control the flow of ions such as calcium, sodium or potassium across the membranes of cells.


    While investigators like DeCaen and Prakriya focus on molecular-level details, Rintaro Hashizume, MD, PhD, assistant professor of Neurological Surgery and of Biochemistry and Molecular Genetics, is using mouse models of brain tumors to begin to translate basic ion channel discoveries into experimental therapeutics.

    Before he joined Northwestern in 2014, Hashizume collaborated with a team of ion channel investigators at the University of California, San Francisco, who figured out that medulloblastoma, a cancerous pediatric brain tumor, was enriched with Ether-a-go-go 2 (EAG2) potassium ion channels.

    The EAG2 channel helps regulate the cell cycle and volume of cells, so the investigators searched for a drug that could inhibit it. They found that thioridazine, used to treat schizophrenia, did the trick. Hashizume gave the drug to mice with human medulloblastoma and showed that it stopped tumor growth and, more importantly, prevented metastasis, which occurs when the tumor spreads to other parts of the body, decreasing patient survival rates. The findings were published in Nature Neuroscience.

    “That’s an important therapeutic advantage of the potassium ion channel blocker — if the tumor doesn’t metastasize you can focus on the management of the original tumor,” he says.

    Hashizume has since launched a pediatric tumor research collaboration with George. Using cells derived from a Northwestern pediatric patient with a brain tumor, Hashizume created a mouse model that will allow the team to probe how the mutation affects ion channel function and test treatments that might correct the problem.

    “That’s an important therapeutic advantage of the potassium ion channel blocker — if the tumor doesn’t metastasize you can focus on the management of the original tumor,” he says.

    Hashizume has since launched a pediatric tumor research collaboration with George. Using cells derived from a Northwestern pediatric patient with a brain tumor, Hashizume created a mouse model that will allow the team to probe how the mutation affects ion channel function and test treatments that might correct the problem.

    See the full article here .

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    South Campus

    On May 31, 1850, nine men gathered to begin planning a university that would serve the Northwest Territory.

    Given that they had little money, no land and limited higher education experience, their vision was ambitious. But through a combination of creative financing, shrewd politicking, religious inspiration and an abundance of hard work, the founders of Northwestern University were able to make that dream a reality.

    In 1853, the founders purchased a 379-acre tract of land on the shore of Lake Michigan 12 miles north of Chicago. They established a campus and developed the land near it, naming the surrounding town Evanston in honor of one of the University’s founders, John Evans. After completing its first building in 1855, Northwestern began classes that fall with two faculty members and 10 students.
    Twenty-one presidents have presided over Northwestern in the years since. The University has grown to include 12 schools and colleges, with additional campuses in Chicago and Doha, Qatar.

    Northwestern is recognized nationally and internationally for its educational programs.

  • richardmitnick 8:37 am on August 17, 2017 Permalink | Reply
    Tags: , Biology, , , , SynBio FSP-SynBio Future Science Platform, SynBio-synthetic biology   

    From CSIRO blog: “First steps toward a synthetic biology future” 

    CSIRO bloc

    CSIRO blog

    17 August 2017
    Chris McKay

    The Industrial Revolution set off a wave of technological revolutions. Illustration: D O Hill/Wikimedia Commons

    It was steam power in 18th Century Britain that helped set off the Industrial Revolution, an evolution in technology that would change the course of human history. But that turned out to be only the first in a wave of technological revolutions to follow. From the late 1800s, electricity was being harnessed to allow for mass production, and then in the 1980s, electronics and information technology took the world by storm, heralding the third technological revolution and giving us the digital world we know today.

    Now, we’re in the midst of a fourth technological revolution. Building on the digital revolution that came before it, we’re seeing increasing digital connectedness (think Internet of Things) and a fusing of digital technology with biological systems and technologies. And there has been a step change in the speed at which progress is occurring.

    The trend in the cost of sequencing a human-sized genome since 2001. Image: National Human Genome Research Institute.

    Consider the speed of progress during the IT revolution, which saw computing power doubling roughly every two years in accordance with Moore’s Law. Then contrast that with the rate of progress in the field of biotechnology, which has been exponential: the Human Genome Project, starting in 1990, was a $3 billion USD project that sequenced the human genome for the first time over a period of more than 10 years; then as a result of that work, from 2001 a genome could be sequenced for $100 million USD; and today we can sequence a genome for less than $1000.

    It is in this context that the field of synthetic biology (SynBio) has emerged. SynBio is essentially the application of engineering principles to biology. It involves making things from biological components, such as genetic code, to carry out useful activities. These activities could include sustainable production of fuels, treatment and cure of diseases, controlling invasive pests, or sensing toxins in the environment. Indeed, recent advancements in writing DNA code, printing DNA, and gene editing technology have made SynBio one of the fastest growing areas of modern science. It is a rapidly expanding multi-billion dollar industry with significant potential for generating societal benefits and commercial opportunities.

    That’s why SynBio was among the six new Future Science Platforms we announced last year; a program of investment in areas of science that are set to drive innovation and have the potential to help reinvent and create new industries for Australia. The SynBio Future Science Platform (SynBio FSP) is also growing the capability of a new generation of researchers in partnership with some Australian universities—some of the newest recruits, 11 SynBio Future Science Fellows, will be undertaking work on a suite of innovative projects.

    Future Science Fellow Dr Michele Fabris, based at the University of Technology Sydney’s Climate Change Cluster, will be exploring the potential for photosynthetic microalgae to be modified to carry out new functions, like the production of anti-cancer pharmaceutical compounds. Image: Anna Zhu/UTS

    The research projects cover a broad spectrum of activity. There will be environmental and biocontrol applications, such as the development of cell-tissue structures capable of sensing the environment and eliminating toxins, new tools for targeting antibiotic resistant biofilms, and biosensors providing real-time biological monitoring. Some projects will be exploring the potential to use yeast, microalgae or cyanobacteria cells for the production of valuable pharmaceuticals or fuels, driving innovation in chemical and fibre manufacturing. Other projects will be creating new tools and building blocks that will be fundamental in driving progress in SynBio.

    This work will complement other SynBio FSP research being undertaken at CSIRO that will help us position Australia to play a role in the latest technological revolution. It is research that will allow us to better understand global developments and, where appropriate, contribute responsibly to advances in areas as diverse as healthcare, industrial biotechnology, biosecurity, food and agriculture.

    SynBio FSP’s Future Science Fellowships are co-funded partnerships between CSIRO and the host universities, with each partner contributing matching funding. The host universities are Australian National University, Macquarie University, University of Adelaide, University of Queensland, University of the Sunshine Coast, University of Technology Sydney and University of Western Australia.

    See the full article here .

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    CSIRO, the Commonwealth Scientific and Industrial Research Organisation, is Australia’s national science agency and one of the largest and most diverse research agencies in the world.

    The CSIRO blog is designed to entertain, inform and inspire by generally digging around in the work being done by our terrific scientists, and leaving the techie speak and jargon for the experts.

    We aim to bring you stories from across the vast breadth and depth of our organisation: from the wild sea voyages of our Research Vessel Investigator to the mind-blowing astronomy of our Space teams, right through all the different ways our scientists solve national challenges in areas as diverse as Health, Farming, Tech, Manufacturing, Energy, Oceans, and our Environment.

    If you have any questions about anything you find on our blog, we’d love to hear from you. You can reach us at socialmedia@csiro.au.

    And if you’d like to find out more about us, our science, or how to work with us, head over to CSIRO.au

  • richardmitnick 9:41 am on August 14, 2017 Permalink | Reply
    Tags: Acidic patch, Acidic patch’ regulates access to genetic information, , Biology, , Chromatin remodelers, , , ISWI remodelers,   

    From Princeton: “‘Acidic patch’ regulates access to genetic information” 

    Princeton University
    Princeton University Research Blog

    August 14, 2017
    Pooja Makhijani

    Chromatin remodelers — protein machines that pack and unpack chromatin, the tightly wound DNA-protein complex in cell nuclei — are essential and powerful regulators for critical cellular processes, such as replication, recombination and gene transcription and repression. In a new study published Aug. 2 in the journal Nature, a team led by researchers from Princeton University unravels more details on how a class of ATP-dependent chromatin remodelers, called ISWI, regulate access to genetic information.

    The researchers reported that ISWI remodelers use a structural feature of the nucleosome, known as the “acidic patch,” to remodel chromatin. The nucleosome is the fundamental structural subunit of chromatin, and is often compared to thread wrapped around a spool.

    “The acidic patch is a negatively charged surface, presented on each face of the nucleosome disc, that is formed by amino acids contributed by two different histone proteins, H2A and H2B,” said Geoffrey Dann, a graduate student in the Department of Molecular Biology at Princeton and the study’s lead author. “Histone proteins are overall very positively charged, which makes the negatively charged acidic patch region of the nucleosome very unique. Recognition of the acidic patch has never before been implicated in chromatin remodeling.”

    The research was conducted in the laboratory of Tom Muir, the Van Zandt Williams Jr. Class of 1965 Professor of Chemistry and chair of the Department of Chemistry. Research in the Muir group centers on elucidating the physiochemical basis of protein functions in biomedically relevant systems.

    Because ISWI remodelers are known to interact extensively with nucleosomes, the researchers hypothesized that signals, in the form of chemical modifications on histone proteins embedded within nucleosomes, communicate to the remodelers on which nucleosome to act. Using high throughput screening technology, an assay process often used in drug discovery, allowed the researchers to quickly conduct tens of thousands of biochemical measurements to test their assumptions. “The number of chromatin modifications known to exist in vivo is astronomical,” Dann said.

    Not only did the experiments reveal that ISWI remodelers use the “acidic patch” to remodel chromatin, but also determined that remodeling enzymes outside the family of ISWI remodelers also use this structural feature, “suggesting that this feature may be a general requirement for chromatin remodeling to occur,” Dann said.

    Geoffrey Dann. Photo by Jeffrey Bos.

    Certain chemical modifications that act on histone proteins that are adjacent to the acidic patch also have the ability to enhance or inhibit ISWI remodeling activity, he explained. “A handful of other proteins are known to engage the acidic patch in their interaction with chromatin as well, and we also found that the biochemistry of several of these proteins was affected by such modifications. Interestingly, each protein tested had its own signature response to this collection of modifications.”

    The high throughput screening technology method also generated a vast library of data to drive the design of future studies geared toward further understanding ISWI regulation. “This study generated an immense amount of data pointing to many other novel regulatory inputs, in the form of chromatin modifications, into ISWI remodeling activity,” Dann said. “A long-term goal in our lab is to use this data resource as a launch pad for additional studies investigating how chromatin modifications affect ISWI remodeling, and how this plays into the various roles ISWI remodelers assume in the cell.”

    Their findings may also identify a new instrument in cells’ molecular repertoire of chromatin-remodeling tools and spur investigations into potential cancer therapeutic targets. “Mutations in the acidic patch are known to occur in certain types of human cancers, which underscores the emerging importance of the acidic patch in chromatin biology,” Dann said.

    See the full article here .

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    About Princeton: Overview

    Princeton University is a vibrant community of scholarship and learning that stands in the nation’s service and in the service of all nations. Chartered in 1746, Princeton is the fourth-oldest college in the United States. Princeton is an independent, coeducational, nondenominational institution that provides undergraduate and graduate instruction in the humanities, social sciences, natural sciences and engineering.

    As a world-renowned research university, Princeton seeks to achieve the highest levels of distinction in the discovery and transmission of knowledge and understanding. At the same time, Princeton is distinctive among research universities in its commitment to undergraduate teaching.

    Today, more than 1,100 faculty members instruct approximately 5,200 undergraduate students and 2,600 graduate students. The University’s generous financial aid program ensures that talented students from all economic backgrounds can afford a Princeton education.

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  • richardmitnick 3:34 pm on July 31, 2017 Permalink | Reply
    Tags: 3-D microscope gives Johns Hopkins scientists a clearer view, , Biology, , , ,   

    From Hopkins: “3-D microscope gives Johns Hopkins scientists a clearer view” 

    Johns Hopkins
    Johns Hopkins University

    Jill Rosen

    Light-sheet technology allows researchers like Kavli Neuroscience Discovery Institute fellow Audrey Branch to observe how cells, ducts, or veins connect without damaging the cells in the sample Image credit: Will Kirk / Homewood Photography.

    Audrey Branch is trying to learn more about aging by studying old and young brains. Specifically, she’s interested in how cells connect to form memories and what might be going wrong with those connections when older people start to forget things.

    Until recently, getting at that question meant months of tedious specimen preparation. And even then, the very prep that made getting a glimpse of the brain’s core possible—slicing what’s already tiny into thousands of pieces—very likely destroyed the delicate connections the Johns Hopkins neuroscientist needed to see.

    That changed this spring when a new, three-dimensional microscope arrived at the university’s Homewood campus, a cutting-edge tool that not only condenses what had been months of work into just hours, but allows researchers unprecedented views of organs, tissue, and even live specimens.

    Just practicing with it, Branch knew it was a game-changer. She cried when she saw the first pictures of a mouse brain, its individual neurons glowing red, and its spindly dendrites, too—showing quite clearly the links between those cells.

    “It feels so amazing to see the brain in a way that no one has ever seen it before,” she said. “It’s pretty much the greatest thing I’ve ever experienced in science.”

    The selective plane florescence light sheet microscope arrived on campus in April, one of the first in operation on the East Coast and the only one in Maryland. Purchased with a grant from the National Institutes of Health, it cost $360,000.

    Unlike other microscopes, this one illuminates specimens from the side, shooting two perfectly aligned planes of light across an object, illuminating a wafer-thin slice of the whole while the camera captures the image—thousands of times over as the specimen moves through the light. When the images are displayed together, the result is a three-dimensional image or video clip of the full object, sort of like the more familiar CAT scan.

    The technology is very new, but Michael McCaffery, director of the university’s Integrated Imaging Center, expects researchers everywhere will be using it within a few years. Just among the Johns Hopkins community, word of the light sheet is already out and scientists have been lining up to use it—even if that requires the minor inconvenience of bringing specimens over from the medical campus.

    “People really want to use this,” McCaffery said. “It fills a niche that until now was unavailable at Hopkins. Simply, there was no instrument that allowed a researcher to take a whole organ, brain, or cardiac muscle, and image them in three-dimensions, in their entirety.”

    The light sheet is the latest advance in modern microscopy—a world that’s been evolving since fluorescence microscopy became the standard in the 1960s. Now, most researchers use confocal microscopes, which use lasers to illuminate a sample point by point—only extremely tiny samples will work—then create computerized images, pixel by pixel.

    Confocals produce vivid, high-resolution images, but the sample size limitations—nothing thicker than about 70 microns, which is about as wide as a strand of human hair—severely handicapped scientists.

    The new light sheet allows samples up to 12 to 15 millimeters, or about a half an inch. Researchers can study much larger samples, even entire organs. And because the samples don’t have to be cut up, researchers like Branch who are interested in how cells, ducts, or veins connect have a chance to observe them, unspoiled.

    “It’s a very big deal for researchers, particularly those interested in the science of connectomics,” McCaffery said. “Mapping the neuronal connections of the brain is the holy grail of neurology.”

    It’s certainly Branch’s holy grail.

    Branch is a Kavli Neuroscience Discovery Institute fellow working in the Krieger School of Arts and Sciences. She wants to know how newborn neurons, which are key to making memories, connect to other cells in the brain—and how those connections might change as people age.

    Scientists know the number of newborn neurons declines with age, and that likely has something to do with why short-term memory declines with age. What Branch wants to do is audit these newborn cells in a young brain, determining how many there are, where they are, and what other cells they communicate with. She can compare that with an older brain and possibly see which connections have broken when memory loss occurs. If she can target the broken connections, there could be a way to treat the area with a drug and stop or slow cognitive decline.

    Branch has been practicing on the light sheet with mouse brains, and she plans to formally investigate her hypothesis with rat brains, which are bigger and more human-like.

    If she didn’t have the light sheet, Branch would have to slice the brain, which is about the size of an olive pit, into tissue-thin sections—about 250 pieces. Each slice would need to be stained, mounted onto a slide, and then imaged. Each of those images would need to be manually assembled into a composite to approximate the whole.

    All of this work would take about a month. Since Branch’s experiment involves 30 brains, it would take her about two and a half years, “if,” she says, “that’s all I did day in and day out.”

    Worse yet, by slicing the brain, she would lose most of the newborn neurons she needed to find, and probably all of the connections. She figures if she had marked 50 newborn neurons, she’d be lucky to find five.

    “It would be impossible to find the connections,” she says. “And it would be impossible to get an idea of who each of those cells is talking to. Maybe it’s not important, but I’m guessing that’s not the case. Neurons in isolation aren’t interesting; it’s who they’re talking to, it’s how they’re wired.

    “I was just going to have to estimate. I’d have missed a lot of the picture, and that’s all anyone’s been able to do.”

    Guy Bar-Klein, a neuroscientist working in the Hal Dietz Lab at the School of Medicine, has been crossing town to spend time at Homewood’s Dunning Hall with the light sheet to study blood vessels in the heart and brain, hoping to better understand what causes aneurysms.

    Without the light-sheet technology, his view would be limited to a minuscule section of tissue, much too small to get a true sense of its vasculature. Now, he has been looking at samples with intact blood vessels, making it possible to spot and track aneurysms—and possibly pinpoint the underlying issues that caused it to form.

    “It’s very exciting,” Bar-Klein said. “I think it gives us a very substantial advantage in understanding the signaling involved in aneurysm formation.”

    Michael Noë, a pathology resident who studies pancreatic cancer, hopes the light sheet’s three-dimensional perspective will allow him to see relationships between tumors and the surrounding nerves and blood vessels. Tumors often grow around nerves, and Noë expects the new perspective of cancerous ducts and nerves could shed light on why.

    “For almost 200 years, pathologists looked at tissue the same way,” he says. “Three-dimensional is almost a whole new world for us. There is a lot of excitement in the department of pathology to apply this technology for the first time to human samples.”

    Before researchers can view tissue of any sort with the light-sheet, their samples must be treated to make them translucent, so the microscope’s light can pass through and create an image. Noë has developed a protocol for clearing human tissue and tumors, work he’s hoping to publish.

    Branch expects to have 3-D images of all 30 of her rat brains in three to six months.

    She’ll see every newborn neuron. She’ll see each dendrite. And hopefully, she’ll find answers – she already knows she’ll find more questions.

    “The technology makes it easier to have confidence about our findings,” she says, “It also opens up an opportunity to ask even more questions — things that before, we didn’t even know we could ask.”

    See the full article here .

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    The Johns Hopkins University opened in 1876, with the inauguration of its first president, Daniel Coit Gilman. “What are we aiming at?” Gilman asked in his installation address. “The encouragement of research … and the advancement of individual scholars, who by their excellence will advance the sciences they pursue, and the society where they dwell.”

    The mission laid out by Gilman remains the university’s mission today, summed up in a simple but powerful restatement of Gilman’s own words: “Knowledge for the world.”

    What Gilman created was a research university, dedicated to advancing both students’ knowledge and the state of human knowledge through research and scholarship. Gilman believed that teaching and research are interdependent, that success in one depends on success in the other. A modern university, he believed, must do both well. The realization of Gilman’s philosophy at Johns Hopkins, and at other institutions that later attracted Johns Hopkins-trained scholars, revolutionized higher education in America, leading to the research university system as it exists today.

  • richardmitnick 10:24 am on July 31, 2017 Permalink | Reply
    Tags: , Biology, , , ,   

    From HMS: “Making the Makers” 

    Harvard University

    Harvard University

    Harvard Medical School

    Harvard Medical School

    July 21, 2017

    Rendering of the structure of the eukaryotic ribosome. Ribosomal RNA is represented as a grey tube. Proteins are shown in blue, orange and red. Image: Wikimedia Commons.

    Every living cell, whether a single bacterium or a human neuron, is a biological system as dynamic and complex as any city. Contained within cells are walls, highways, power plants, libraries, recycling centers and much more, all working together in unison to ensure the continuation of life.

    The vast majority of these myriad structures are made of and made by proteins, and those proteins are made by one uniquely important molecular machine, the ribosome.

    In a new study published in Nature on July 20, a team led by Johan Paulsson, professor of systems biology at Harvard Medical School, now reveals the likely origin of several previously mysterious characteristics of the ribosome.

    They mathematically demonstrated that ribosomes are precisely structured to produce additional ribosomes as quickly as possible, in order to support efficient cell growth and division.

    The study’s theoretical predictions accurately reflect observed large-scale features—revealing why are ribosomes made of an unusually large number of small, uniformly sized proteins and a few strands of RNA that vary greatly in size—and provide perspective on the evolution of an exceptional molecular machine.

    “The ribosome is one of the most important molecular complexes in all of life, and it’s been studied across scientific disciplines for decades,” Paulsson said.

    “I was always puzzled by the fact that it seemed like we could explain its finer details, but ribosomes have these bizarre features that have not often been addressed, or if so in an unsatisfying way,” he said.

    Mysterious features

    Atomic structure of a ribosome subunit from an archaea, a type of microorganism. Proteins are shown in blue and RNA chains in orange and yellow. Animation: Wikimedia Commons/David Goodsell.

    Although scientists have unlocked how ribosomes turn genetic information into proteins at atomic resolution, revealing a molecular machine finely tuned for accuracy, speed and control, it hasn’t been clear what advantages lay in its several large-scale features.

    Ribosomes are composed of a puzzlingly large number of different structural proteins—anywhere from 55 to 80, depending on organism type. These proteins are not just more numerous than expected, they are unusually short and uniform in length. Ribosomes are also composed of two to three strands of RNA, which account for up to 70 percent of the total mass of the ribosome.

    “Without understanding why collective features exist, it is a bit like looking at a forest and understanding how chloroplasts and photosynthesis work, and not being able to explain why there are trees instead of grass,” Paulsson said.

    So Paulsson and his collaborators Shlomi Reuveni, an HMS postdoctoral fellow, and Måns Ehrenberg of Uppsala University in Sweden, decided to look at the ribosome in a different light.

    “Our breakthrough came by zooming out from the atomic and looking at the ribosome from a different perspective,” Reuveni said. “We didn’t think of the ribosome as a machine that produces proteins, but rather as the product of the protein production process.”

    Forest for the trees

    For a cell to divide, it must have two full sets of ribosomes to make all the proteins that the daughter cells will need. The speed at which ribosomes can make themselves, therefore, places a hard limit on how fast cell division occurs. Paulsson and his colleagues devised theoretical mathematical models for what the ribosome’s features should look like if speed was the primary selective pressure that drove its evolution.

    The team calculated that distributing the task of making a new ribosome among many ribosomes—each making a small piece of the final product—can increase the rate of production by as much as 30 percent, since each new ribosome helps make more ribosomes as soon as they are created, accelerating the process.

    This represents an enormous advantage for cells that need to divide quickly, such as bacteria. However, the protein production process takes time to initiate, and this overhead cost limits the number of proteins that a ribosome can be made of, according to the math.

    The team’s models predicted that, for maximum self-production efficacy, a ribosome should be made of between 40 and 80 proteins. Each of these proteins should be around three times smaller than an average cellular protein, and they should all be roughly similar in size.

    It turns out that the researchers’ theory, developed completely independently of the laboratory, accurately reflects the observed protein composition of the ribosome.

    “An analogy for our findings would be to think of ribosomes not as a group of carpenters who merely build a lot of houses, but as carpenters who also build other carpenters,” Paulsson said. “There is then an incentive to divide the job into many small pieces that can be done in parallel to more quickly assemble another complete carpenter to help in the process.”

    Theory and reality

    Paulsson and his colleagues also examined ribosomal RNA, which act as a structural component and carry out the ribosome’s enzymatic activity of linking amino acids together into proteins.

    Their analysis showed that, the more RNA a ribosome is made of, the more rapidly it can be produced. This is because cells can make RNA orders of magnitude faster than protein. Thus, while RNA enzymes are thought to be less efficient than protein enzymes, ribosomes have enormous pressure to use as much RNA as possible to maximize the rate at which more ribosomes can be made.

    “Any place the ribosome can get away with using RNA, it should use it because self-production speed can essentially be doubled or tripled,” Paulsson said. “Even if RNA were inferior compared to protein for enzymatic function, there is still a great advantage to using RNA if a cell is trying to produce ribosomes as fast as possible.”

    This observation was predicted to hold primarily for self-producing ribosomes, according to the team. Most other structures in the cell do not self-produce and can sacrifice production speed for the stability and efficacy provided by using protein instead of RNA.

    Taken together, the team’s theory accurately predicts large-scale features of the ribosome that are seen across domains of life. It explains why the fastest growing organisms, such as bacteria, have the shortest ribosomal proteins and the greatest amounts of RNA. At the opposite end of the spectrum are mitochondria—the power plants of eukaryotic cells, which are thought to have once been bacteria that entered a permanent symbiotic state. Mitochondria have their own ribosomes that do not produce themselves. Without this pressure, mitochondrial ribosomes are indeed made of larger proteins and far less RNA than cellular ribosomes.

    “When we started this project, we didn’t have a long list of features that we tried to explain through theory,” Reuveni said. “We started with the theory, and certain features emerged. When we looked at data to compare with what our math predicted, we found in most cases that they matched what is seen in nature.”

    Rather than being mere relics of an evolutionary past, the unusual features of ribosomes thus seem to reflect an additional layer of functional optimization acting on collective properties of its parts, the team writes.

    “While this study is basic science, we are addressing something that is shared by all life,” Paulsson said. “It is important that we understand where the constraints on structure and function come from, because like much of basic science, it is unpredictable what the consequences of new knowledge can unlock in the future.”

    See the full article here .

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