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

    1
    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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    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 11:53 am on July 29, 2017 Permalink | Reply
    Tags: 3D structure of human chromatin, , ChromEMT, DNA, , , , ,   

    From Salk: “Salk scientists solve longstanding biological mystery of DNA organization” 

    Salk Institute bloc

    Salk Institute for Biological Studies

    July 27, 2017

    Stretched out, the DNA from all the cells in our body would reach Pluto. So how does each tiny cell pack a two-meter length of DNA into its nucleus, which is just one-thousandth of a millimeter across?

    The answer to this daunting biological riddle is central to understanding how the three-dimensional organization of DNA in the nucleus influences our biology, from how our genome orchestrates our cellular activity to how genes are passed from parents to children.

    Now, scientists at the Salk Institute and the University of California, San Diego, have for the first time provided an unprecedented view of the 3D structure of human chromatin—the combination of DNA and proteins—in the nucleus of living human cells.

    In the tour de force study, described in Science on July 27, 2017, the Salk researchers identified a novel DNA dye that, when paired with advanced microscopy in a combined technology called ChromEMT, allows highly detailed visualization of chromatin structure in cells in the resting and mitotic (dividing) stages. By revealing nuclear chromatin structure in living cells, the work may help rewrite the textbook model of DNA organization and even change how we approach treatments for disease.

    “One of the most intractable challenges in biology is to discover the higher-order structure of DNA in the nucleus and how is this linked to its functions in the genome,” says Salk Associate Professor Clodagh O’Shea, a Howard Hughes Medical Institute Faculty Scholar and senior author of the paper. “It is of eminent importance, for this is the biologically relevant structure of DNA that determines both gene function and activity.”

    2
    A new technique enables 3D visualization of chromatin (DNA plus associated proteins) structure and organization within a cell nucleus (purple, bottom left) by painting the chromatin with a metal cast and imaging it with electron microscopy (EM). The middle block shows the captured EM image data, the front block illustrates the chromatin organization from the EM data, and the rear block shows the contour lines of chromatin density from sparse (cyan and green) to dense (orange and red). Credit: Salk Institute.

    Ever since Francis Crick and James Watson determined the primary structure of DNA to be a double helix, scientists have wondered how DNA is further organized to allow its entire length to pack into the nucleus such that the cell’s copying machinery can access it at different points in the cell’s cycle of activity. X-rays and microscopy showed that the primary level of chromatin organization involves 147 bases of DNA spooling around proteins to form particles approximately 11 nanometers (nm) in diameter called nucleosomes. These nucleosome “beads on a string” are then thought to fold into discrete fibers of increasing diameter (30, 120, 320 nm etc.), until they form chromosomes. The problem is, no one has seen chromatin in these discrete intermediate sizes in cells that have not been broken apart and had their DNA harshly processed, so the textbook model of chromatin’s hierarchical higher-order organization in intact cells has remained unverified.

    To overcome the problem of visualizing chromatin in an intact nucleus, O’Shea’s team screened a number of candidate dyes, eventually finding one that could be precisely manipulated with light to undergo a complex series of chemical reactions that would essentially “paint” the surface of DNA with a metal so that its local structure and 3D polymer organization could be imaged in a living cell. The team partnered with UC San Diego professor and microscopy expert Mark Ellisman, one of the paper’s coauthors, to exploit an advanced form of electron microscopy that tilts samples in an electron beam enabling their 3D structure to be reconstructed. By combining their chromatin dye with electron-microscope tomography, they created ChromEMT.

    The team used ChromEMT to image and measure chromatin in resting human cells and during cell division when DNA is compacted into its most dense form—the 23 pairs of mitotic chromosomes that are the iconic image of the human genome. Surprisingly, they did not see any of the higher-order structures of the textbook model anywhere.

    3
    From left: Horng Ou and Clodagh O’Shea. Credit: Salk Institute.

    “The textbook model is a cartoon illustration for a reason,” says Horng Ou, a Salk research associate and the paper’s first author. “Chromatin that has been extracted from the nucleus and subjected to processing in vitro—in test tubes—may not look like chromatin in an intact cell, so it is tremendously important to be able to see it in vivo.”

    What O’Shea’s team saw, in both resting and dividing cells, was chromatin whose “beads on a string” did not form any higher-order structure like the theorized 30 or 120 or 320 nanometers. Instead, it formed a semi-flexible chain, which they painstakingly measured as varying continuously along its length between just 5 and 24 nanometers, bending and flexing to achieve different levels of compaction. This suggests that it is chromatin’s packing density, and not some higher-order structure, that determines which areas of the genome are active and which are suppressed.

    With their 3D microscopy reconstructions, the team was able to move through a 250 nm x 1000 nm x 1000 nm volume of chromatin’s twists and turns, and envision how a large molecule like RNA polymerase, which transcribes (copies) DNA, might be directed by chromatin’s variable packing density, like a video game aircraft flying through a series of canyons, to a particular spot in the genome. Besides potentially upending the textbook model of DNA organization, the team’s results suggest that controlling access to chromatin could be a useful approach to preventing, diagnosing and treating diseases such as cancer.

    “We show that chromatin does not need to form discrete higher-order structures to fit in the nucleus,” adds O’Shea. “It’s the packing density that could change and limit the accessibility of chromatin, providing a local and global structural basis through which different combinations of DNA sequences, nucleosome variations and modifications could be integrated in the nucleus to exquisitely fine-tune the functional activity and accessibility of our genomes.”

    Future work will examine whether chromatin’s structure is universal among cell types or even among organisms.

    Other authors included Sébastien Phan, Thomas Deerinck and Andrea Thor of the UC San Diego.

    The work was largely funded by the W. M. Keck Foundation, the NIH 4D Nucleome Roadmap Initiative and the Howard Hughes Medical Institute, with additional support from the William Scandling Trust, the Price Family Foundation and the Leona M. and Harry B. Helmsley Charitable Trust.

    See the full article here .

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    Salk Institute Campus

    Every cure has a starting point. Like Dr. Jonas Salk when he conquered polio, Salk scientists are dedicated to innovative biological research. Exploring the molecular basis of diseases makes curing them more likely. In an outstanding and unique environment we gather the foremost scientific minds in the world and give them the freedom to work collaboratively and think creatively. For over 50 years this wide-ranging scientific inquiry has yielded life-changing discoveries impacting human health. We are home to Nobel Laureates and members of the National Academy of Sciences who train and mentor the next generation of international scientists. We lead biological research. We prize discovery. Salk is where cures begin.

     
  • richardmitnick 3:16 pm on July 17, 2017 Permalink | Reply
    Tags: A key building block for controlling microbiomes, Controlling gene expression across bacterial colonies, DNA, , Master clock, Programming the clock, ,   

    From UCSD Jacobs School of Engineering: “Scientists at the UC San Diego Center for Microbiome Innovation invent new tool for the Synthetic Biologist’s toolbox” 

    UC San Diego bloc

    UC San Diego


    Jacobs School of Engineering

    July 10, 2017
    Mario Aguilera
    Scripps Institute of Oceanography
    Phone: 858-534-3624
    scrippsnews@ucsd.edu

    Researchers at the University of California San Diego have invented a new method for controlling gene expression across bacterial colonies. The method involves engineering dynamic DNA copy number changes in a synchronized fashion. The results were published in the July 10, 2017 online edition of Nature Genetics.

    Until now, methods for controlling or programming bacterial cells involved transcriptional and post-transcriptional regulation. UC San Diego researchers led by Jeff Hasty, a professor of bioengineering and biology and member of the UC San Diego Center for Microbiome Innovation, describe a new method, which involves cutting circular pieces of bacterial DNA called plasmids, effectively destroying the DNA and turning off regulation.

    The study also demonstrates how DNA concentration can be increased to turn on a synthetic gene circuit. By controlling DNA copy number, researchers can effectively regulate gene expression.

    Synthetic Biology – which can involve altering biological systems for some purpose – is emerging as an engineering discipline. The field was firmly established in 2000, with the description of synthetic biological circuits in which parts of a cell are designed to perform functions, similar to the way an electronic circuit works. Also similar to an electronic circuit, the task performed by a biological circuit can be turned on and off. At the same time, researchers described the making of a “genetic clock”, which involves placing genes in a particular order so that they’ll be turned on at a specific time. This approach has also helped researchers understand natural “oscillators”, such as our sleep-wake cycle.

    Since these early inventions, Hasty and his team have shown how engineered cellular oscillations can be synchronized within a bacterial colony using plasmids, synthetically designed by the researchers themselves. Now, the team is adding a new tool to the Synthetic Biologist’s toolbox – a “master clock” of sorts that will allow researchers to coordinate subprocesses in bacterial cells.

    “This remarkable achievement is a key building block for controlling microbiomes”, said Rob Knight, professor of pediatrics at UC San Diego with a joint appointment in computer science and engineering. Knight leads the Center for Microbiome Innovation. “By controlling different strains with the same master clock, or by giving different strains their own clocks, we can start to engineer population-level dynamics to control specific microbiome functions.”

    Examples of these functions might include interaction with host cells at particular times of day, such as timed release of neurotransmitters produced by the bacteria, or interactions with other bacteria such as antifungal production triggered by a meal rich in sugar.

    Programming the clock

    The researchers used an endonuclease from Saccharomyces cerevisiae, a species of yeast, expressed alongside a plasmid containing the nuclease recognition sequence to temporarily reduce the plasmid’s copy number below natural levels.

    “We found that plasmid replication is so strong that we couldn’t cut them all,” said Hasty. “This was good news, because it meant we could down-regulate gene expression, but not eliminate it.”

    The researchers reasoned that the method could be used to regulate an entire suite of genes and promoters, and tested their idea using a previously constructed circuit to produce sustained cycling of DNA plasmid concentration across a colony of E. coli cells.

    The circuit works by using a small molecule, known as AHL, to coordinate gene expression across a colony of bacterial cells. Once on, the genes driven by the promoter are also activated, including the AHL-producing gene itself. Thanks to this positive feedback loop, the more AHL accumulates, the more it is produced. Because AHL is small enough to diffuse between cells and turn on the promoter in neighboring cells, the genes activated by it would also be produced in high amounts, leading to a phenomenon known as quorum sensing.

    Hasty and his team employed the endonuclease to reduce the number of these plasmids present in the colony and used this mechanism as negative feedback to driving the oscillations in gene expression. Using quorum sensing, the feedback system was coupled across the colony of cells.

    “We observed regular oscillations of gene expression in microfluidic chambers at different colony length scales and over extended time periods,” said Hasty. “By incorporating elements for both positive and negative copy number regulation, we were able to improve the robustness of the circuit.”

    See the full article here .

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    UC San Diego Campus

    About the Jacobs School
    Innovation Happens Here

    The UC San Diego Jacobs School of Engineering is a premier research school set apart by our entrepreneurial culture and integrative engineering approach.

    The Jacobs School’s Mission:

    Educate Tomorrow’s Technology Leaders
    Conduct Leading Edge Research and Drive Innovation
    Transfer Discoveries for the Benefit of Society

    The Jacobs School’s Values:

    Engineering for the global good
    Exponential impact through entrepreneurism
    Collaboration to enrich relevance
    Our education models focus on deep and broad engineering fundamentals, enhanced by real-world design and research, often in partnership with industry. Through our Team Internship Program and GlobalTeams in Engineering Service program, for example, we encourage students to develop their communications and leadership skills while working in the kind of multi-disciplinary team environment experienced by real-world engineers.

    We are home to exciting research centers, such as the San Diego Supercomputer Center, a national resource for data-intensive computing; our Powell Structural Research Laboratories, the largest and most active in the world for full-scale structural testing; and the Qualcomm Institute, which is the UC San Diego division of the California Institute for Telecommunications and Information Technology (Calit2), which is forging new ground in multi-disciplinary applications for information technology.

    Located at the hub of San Diego’s thriving information technology, biotechnology, clean technology, and nanotechnology sectors, the Jacobs School proactively seeks corporate partners to collaborate with us in research, education and innovation.

    The University of California, San Diego (also referred to as UC San Diego or UCSD), is a public research university located in the La Jolla area of San Diego, California, in the United States.[12] The university occupies 2,141 acres (866 ha) near the coast of the Pacific Ocean with the main campus resting on approximately 1,152 acres (466 ha).[13] Established in 1960 near the pre-existing Scripps Institution of Oceanography, UC San Diego is the seventh oldest of the 10 University of California campuses and offers over 200 undergraduate and graduate degree programs, enrolling about 22,700 undergraduate and 6,300 graduate students. UC San Diego is one of America’s Public Ivy universities, which recognizes top public research universities in the United States. UC San Diego was ranked 8th among public universities and 37th among all universities in the United States, and rated the 18th Top World University by U.S. News & World Report ‘s 2015 rankings.

     
  • richardmitnick 8:47 am on July 17, 2017 Permalink | Reply
    Tags: , CRISPR-Cas3, DNA, , , ,   

    From HMS: “Bringing CRISPR into Focus” 

    Harvard University

    Harvard University

    Harvard Medical School

    Harvard Medical School

    June 29, 2017
    KEVIN JIANG

    CRISPR-Cas3 is a subtype of the CRISPR-Cas system, a widely adopted molecular tool for precision gene editing in biomedical research. Aspects of its mechanism of action, however, particularly how it searches for its DNA targets, were unclear, and concerns about unintended off-target effects have raised questions about the safety of CRISPR-Cas for treating human diseases.

    Harvard Medical School and Cornell University scientists have now generated near-atomic resolution snapshots of CRISPR that reveal key steps in its mechanism of action. The findings, published in Cell on June 29, provide the structural data necessary for efforts to improve the efficiency and accuracy of CRISPR for biomedical applications.

    Through cryo-electron microscopy, the researchers describe for the first time the exact chain of events as the CRISPR complex loads target DNA and prepares it for cutting by the Cas3 enzyme. These structures reveal a process with multiple layers of error detection—a molecular redundancy that prevents unintended genomic damage, the researchers say.

    High-resolution details of these structures shed light on ways to ensure accuracy and avert off-target effects when using CRISPR for gene editing.

    “To solve problems of specificity, we need to understand every step of CRISPR complex formation,” said Maofu Liao, assistant professor of cell biology at Harvard Medical School and co-senior author of the study. “Our study now shows the precise mechanism for how invading DNA is captured by CRISPR, from initial recognition of target DNA and through a process of conformational changes that make DNA accessible for final cleavage by Cas3.”

    Target search

    Discovered less than a decade ago, CRISPR-Cas is an adaptive defense mechanism that bacteria use to fend off viral invaders. This process involves bacteria capturing snippets of viral DNA, which are then integrated into its genome and which produce short RNA sequences known as crRNA (CRISPR RNA). These crRNA snippets are used to spot “enemy” presence.

    Acting like a barcode, crRNA is loaded onto members of the CRISPR family of enzymes, which perform the function of sentries that roam the bacteria and monitor for foreign code. If these riboprotein complexes encounter genetic material that matches its crRNA, they chop up that DNA to render it harmless. CRISPR-Cas subtypes, notably Cas9, can be programmed with synthetic RNA in order to cut genomes at precise locations, allowing researchers to edit genes with unprecedented ease.

    To better understand how CRISPR-Cas functions, Liao partnered with Ailong Ke of Cornell University. Their teams focused on type 1 CRISPR, the most common subtype in bacteria, which utilizes a riboprotein complex known as CRISPR Cascade for DNA capture and the enzyme Cas3 for cutting foreign DNA.

    Through a combination of biochemical techniques and cryo-electron microscopy, they reconstituted stable Cascade in different functional states, and further generated snapshots of Cascade as it captured and processed DNA at a resolution of up to 3.3 angstroms—or roughly three times the diameter of a carbon atom.

    1
    A sample cryo-electron microscope image of CRISPR molecules(left). The research team combined hundreds of thousands of particles into 2D averages (right), before turning them into 3D projections. Image: Xiao et al.

    Seeing is believing

    In CRISPR-Cas3, crRNA is loaded onto CRISPR Cascade, which searches for a very short DNA sequence known as PAM that indicates the presence of foreign viral DNA.

    Liao, Ke and their colleagues discovered that as Cascade detects PAM, it bends DNA at a sharp angle, forcing a small portion of the DNA to unwind. This allows an 11-nucleotide stretch of crRNA to bind with one strand of target DNA, forming a “seed bubble.”

    The seed bubble acts as a fail-safe mechanism to check whether the target DNA matches the crRNA. If they match correctly, the bubble is enlarged and the remainder of the crRNA binds with its corresponding target DNA, forming what is known as an “R-loop” structure.

    Once the R-loop is completely formed, the CRISPR Cascade complex undergoes a conformational change that locks the DNA into place. It also creates a bulge in the second, non-target strand of DNA, which is run through a separate location on the Cascade complex.

    Only when a full R-loop state is formed does the Cas3 enzyme bind and cut the DNA at the bulge created in the non-target DNA strand.

    The findings reveal an elaborate redundancy to ensure precision and avoid mistakenly chopping up the bacteria’s own DNA.

    2
    CRISPR forms a “seed bubble” state, which acts as an initial fail-safe mechanism to ensure that CRISPR RNA matches its target DNA. Image: Liao Lab/HMS.

    “To apply CRISPR in human medicine, we must be sure the system is accurate and that it does not target the wrong genes,” said Ke, who is co-senior author of the study. “Our argument is that the CRISPR-Cas3 subtype has evolved to be a precise system that carries the potential to be a more accurate system to use for gene editing. If there is mistargeting, we know how to manipulate the system because we know the steps involved and where we might need to intervene.”

    Setting the sights

    Structures of CRISPR Cascade without target DNA and in its post-R-loop conformational states have been described, but this study is the first to reveal the full sequence of events from seed bubble formation to R-loop formation at high resolution.

    In contrast to the scalpel-like Cas9, CRISPR-Cas3 acts like a shredder that chews DNA up beyond repair. While CRISPR-Cas3 has, thus far, limited utility for precision gene editing, it is being developed as a tool to combat antibiotic-resistant strains of bacteria. A better understanding of its mechanisms may broaden the range of potential applications for CRISPR-Cas3.

    In addition, all CRISPR-Cas subtypes utilize some version of an R-loop formation to detect and prepare target DNA for cleavage. The improved structural understanding of this process can now enable researchers to work toward modifying multiple types of CRISPR-Cas systems to improve their accuracy and reduce the chance of off-target effects in biomedical applications.

    “Scientists hypothesized that these states existed but they were lacking the visual proof of their existence,” said co-first author Min Luo, postdoctoral fellow in the Liao lab at HMS. “The main obstacles came from stable biochemical reconstitution of these states and high-resolution structural visualization. Now, seeing really is believing.”

    “We’ve found that these steps must occur in a precise order,” Luo said. “Evolutionarily, this mechanism is very stringent and has triple redundancy, to ensure that this complex degrades only invading DNA.”

    Additional authors on the study include Yibei Xiao, Robert P. Hayes, Jonathan Kim, Sherwin Ng, and Fang Ding.

    This work is supported by National Institutes of Health grants GM 118174 and GM102543.

    See the full article here .

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    HMS campus

    Established in 1782, Harvard Medical School began with a handful of students and a faculty of three. The first classes were held in Harvard Hall in Cambridge, long before the school’s iconic quadrangle was built in Boston. With each passing decade, the school’s faculty and trainees amassed knowledge and influence, shaping medicine in the United States and beyond. Some community members—and their accomplishments—have assumed the status of legend. We invite you to access the following resources to explore Harvard Medical School’s rich history.

    Harvard University campus

    Harvard is the oldest institution of higher education in the United States, established in 1636 by vote of the Great and General Court of the Massachusetts Bay Colony. It was named after the College’s first benefactor, the young minister John Harvard of Charlestown, who upon his death in 1638 left his library and half his estate to the institution. A statue of John Harvard stands today in front of University Hall in Harvard Yard, and is perhaps the University’s best known landmark.

    Harvard University has 12 degree-granting Schools in addition to the Radcliffe Institute for Advanced Study. The University has grown from nine students with a single master to an enrollment of more than 20,000 degree candidates including undergraduate, graduate, and professional students. There are more than 360,000 living alumni in the U.S. and over 190 other countries.

     
  • richardmitnick 1:21 pm on July 8, 2017 Permalink | Reply
    Tags: , , , , DNA, , , , , UCSD Comet supercomputer   

    From Science Node: “Cracking the CRISPR clock” 

    Science Node bloc
    Science Node

    05 Jul, 2017
    Jan Zverina

    SDSC Dell Comet supercomputer

    Capturing the motion of gyrating proteins at time intervals up to one thousand times greater than previous efforts, a team led by University of California, San Diego (UCSD) researchers has identified the myriad structural changes that activate and drive CRISPR-Cas9, the innovative gene-splicing technology that’s transforming the field of genetic engineering.

    By shedding light on the biophysical details governing the mechanics of CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats) activity, the study provides a fundamental framework for designing a more efficient and accurate genome-splicing technology that doesn’t yield ‘off-target’ DNA breaks currently frustrating the potential of the CRISPR-Cas9- system, particularly for clinical uses.


    Shake and bake. Gaussian accelerated molecular dynamics simulations and state-of-the-art supercomputing resources reveal the conformational change of the HNH domain (green) from its inactive to active state. Courtesy Giulia Palermo, McCammon Lab, UC San Diego.

    “Although the CRISPR-Cas9 system is rapidly revolutionizing life sciences toward a facile genome editing technology, structural and mechanistic details underlying its function have remained unknown,” says Giulia Palermo, a postdoctoral scholar with the UC San Diego Department of Pharmacology and lead author of the study [PNAS].

    See the full article here
    .

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    Science Node is an international weekly online publication that covers distributed computing and the research it enables.

    “We report on all aspects of distributed computing technology, such as grids and clouds. We also regularly feature articles on distributed computing-enabled research in a large variety of disciplines, including physics, biology, sociology, earth sciences, archaeology, medicine, disaster management, crime, and art. (Note that we do not cover stories that are purely about commercial technology.)

    In its current incarnation, Science Node is also an online destination where you can host a profile and blog, and find and disseminate announcements and information about events, deadlines, and jobs. In the near future it will also be a place where you can network with colleagues.

    You can read Science Node via our homepage, RSS, or email. For the complete iSGTW experience, sign up for an account or log in with OpenID and manage your email subscription from your account preferences. If you do not wish to access the website’s features, you can just subscribe to the weekly email.”

     
  • richardmitnick 10:21 am on July 3, 2017 Permalink | Reply
    Tags: , , DNA, , ,   

    From HMS: “Bringing CRISPR into Focus” 

    Harvard University

    Harvard University

    Harvard Medical School

    Harvard Medical School

    June 29, 2017
    KEVIN JIANG

    CRISPR-Cas3 is a subtype of the CRISPR-Cas system, a widely adopted molecular tool for precision gene editing in biomedical research. Aspects of its mechanism of action, however, particularly how it searches for its DNA targets, were unclear, and concerns about unintended off-target effects have raised questions about the safety of CRISPR-Cas for treating human diseases.

    Harvard Medical School and Cornell University scientists have now generated near-atomic resolution snapshots of CRISPR that reveal key steps in its mechanism of action. The findings, published in Cell on June 29, provide the structural data necessary for efforts to improve the efficiency and accuracy of CRISPR for biomedical applications.

    Through cryo-electron microscopy, the researchers describe for the first time the exact chain of events as the CRISPR complex loads target DNA and prepares it for cutting by the Cas3 enzyme. These structures reveal a process with multiple layers of error detection—a molecular redundancy that prevents unintended genomic damage, the researchers say.

    High-resolution details of these structures shed light on ways to ensure accuracy and avert off-target effects when using CRISPR for gene editing.

    “To solve problems of specificity, we need to understand every step of CRISPR complex formation,” said Maofu Liao, assistant professor of cell biology at Harvard Medical School and co-senior author of the study. “Our study now shows the precise mechanism for how invading DNA is captured by CRISPR, from initial recognition of target DNA and through a process of conformational changes that make DNA accessible for final cleavage by Cas3.”

    Target search

    Discovered less than a decade ago, CRISPR-Cas is an adaptive defense mechanism that bacteria use to fend off viral invaders. This process involves bacteria capturing snippets of viral DNA, which are then integrated into its genome and which produce short RNA sequences known as crRNA (CRISPR RNA). These crRNA snippets are used to spot “enemy” presence.

    Acting like a barcode, crRNA is loaded onto members of the CRISPR family of enzymes, which perform the function of sentries that roam the bacteria and monitor for foreign code. If these riboprotein complexes encounter genetic material that matches its crRNA, they chop up that DNA to render it harmless. CRISPR-Cas subtypes, notably Cas9, can be programmed with synthetic RNA in order to cut genomes at precise locations, allowing researchers to edit genes with unprecedented ease.

    To better understand how CRISPR-Cas functions, Liao partnered with Ailong Ke of Cornell University. Their teams focused on type 1 CRISPR, the most common subtype in bacteria, which utilizes a riboprotein complex known as CRISPR Cascade for DNA capture and the enzyme Cas3 for cutting foreign DNA.

    Through a combination of biochemical techniques and cryo-electron microscopy, they reconstituted stable Cascade in different functional states, and further generated snapshots of Cascade as it captured and processed DNA at a resolution of up to 3.3 angstroms—or roughly three times the diameter of a carbon atom.

    1
    A sample cryo-electron microscope image of CRISPR molecules(left). The research team combined hundreds of thousands of particles into 2D averages (right), before turning them into 3D projections. Image: Xiao et al.

    Seeing is believing

    In CRISPR-Cas3, crRNA is loaded onto CRISPR Cascade, which searches for a very short DNA sequence known as PAM that indicates the presence of foreign viral DNA.

    Liao, Ke and their colleagues discovered that as Cascade detects PAM, it bends DNA at a sharp angle, forcing a small portion of the DNA to unwind. This allows an 11-nucleotide stretch of crRNA to bind with one strand of target DNA, forming a “seed bubble.”

    The seed bubble acts as a fail-safe mechanism to check whether the target DNA matches the crRNA. If they match correctly, the bubble is enlarged and the remainder of the crRNA binds with its corresponding target DNA, forming what is known as an “R-loop” structure.

    Once the R-loop is completely formed, the CRISPR Cascade complex undergoes a conformational change that locks the DNA into place. It also creates a bulge in the second, non-target strand of DNA, which is run through a separate location on the Cascade complex.

    Only when a full R-loop state is formed does the Cas3 enzyme bind and cut the DNA at the bulge created in the non-target DNA strand.

    The findings reveal an elaborate redundancy to ensure precision and avoid mistakenly chopping up the bacteria’s own DNA.

    2
    CRISPR forms a “seed bubble” state, which acts as an initial fail-safe mechanism to ensure that CRISPR RNA matches its target DNA. Image: Liao Lab/HMS

    “To apply CRISPR in human medicine, we must be sure the system is accurate and that it does not target the wrong genes,” said Ke, who is co-senior author of the study. “Our argument is that the CRISPR-Cas3 subtype has evolved to be a precise system that carries the potential to be a more accurate system to use for gene editing. If there is mistargeting, we know how to manipulate the system because we know the steps involved and where we might need to intervene.”

    Setting the sights

    Structures of CRISPR Cascade without target DNA and in its post-R-loop conformational states have been described, but this study is the first to reveal the full sequence of events from seed bubble formation to R-loop formation at high resolution.

    In contrast to the scalpel-like Cas9, CRISPR-Cas3 acts like a shredder that chews DNA up beyond repair. While CRISPR-Cas3 has, thus far, limited utility for precision gene editing, it is being developed as a tool to combat antibiotic-resistant strains of bacteria. A better understanding of its mechanisms may broaden the range of potential applications for CRISPR-Cas3.

    In addition, all CRISPR-Cas subtypes utilize some version of an R-loop formation to detect and prepare target DNA for cleavage. The improved structural understanding of this process can now enable researchers to work toward modifying multiple types of CRISPR-Cas systems to improve their accuracy and reduce the chance of off-target effects in biomedical applications.

    “Scientists hypothesized that these states existed but they were lacking the visual proof of their existence,” said co-first author Min Luo, postdoctoral fellow in the Liao lab at HMS. “The main obstacles came from stable biochemical reconstitution of these states and high-resolution structural visualization. Now, seeing really is believing.”

    “We’ve found that these steps must occur in a precise order,” Luo said. “Evolutionarily, this mechanism is very stringent and has triple redundancy, to ensure that this complex degrades only invading DNA.”

    Additional authors on the study include Yibei Xiao, Robert P. Hayes, Jonathan Kim, Sherwin Ng, and Fang Ding.

    This work is supported by National Institutes of Health

    See the full article here .

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    Established in 1782, Harvard Medical School began with a handful of students and a faculty of three. The first classes were held in Harvard Hall in Cambridge, long before the school’s iconic quadrangle was built in Boston. With each passing decade, the school’s faculty and trainees amassed knowledge and influence, shaping medicine in the United States and beyond. Some community members—and their accomplishments—have assumed the status of legend. We invite you to access the following resources to explore Harvard Medical School’s rich history.

    Harvard University campus

    Harvard is the oldest institution of higher education in the United States, established in 1636 by vote of the Great and General Court of the Massachusetts Bay Colony. It was named after the College’s first benefactor, the young minister John Harvard of Charlestown, who upon his death in 1638 left his library and half his estate to the institution. A statue of John Harvard stands today in front of University Hall in Harvard Yard, and is perhaps the University’s best known landmark.

    Harvard University has 12 degree-granting Schools in addition to the Radcliffe Institute for Advanced Study. The University has grown from nine students with a single master to an enrollment of more than 20,000 degree candidates including undergraduate, graduate, and professional students. There are more than 360,000 living alumni in the U.S. and over 190 other countries.

     
  • richardmitnick 8:06 am on June 23, 2017 Permalink | Reply
    Tags: A Single Electron’s Tiny Leap Sets Off ‘Molecular Sunscreen’ Response, , , DNA, , , ,   

    From SLAC: “A Single Electron’s Tiny Leap Sets Off ‘Molecular Sunscreen’ Response” 


    SLAC Lab

    June 22, 2017
    Glennda Chui

    1
    Thymine – the molecule illustrated in the foreground – is one of the four basic building blocks that make up the double helix of DNA. It’s such a strong absorber of ultraviolet light that the UV in sunlight should deactivate it, yet this does not happen. Researchers used an X-ray laser at SLAC National Accelerator Laboratory to observe the infinitesimal leap of a single electron that sets off a protective response in thymine molecules, allowing them to shake off UV damage. (Greg Stewart/SLAC National Accelerator Laboratory)

    In experiments at the Department of Energy’s SLAC National Accelerator Laboratory, scientists were able to see the first step of a process that protects a DNA building block called thymine from sun damage: When it’s hit with ultraviolet light, a single electron jumps into a slightly higher orbit around the nucleus of a single oxygen atom.

    This infinitesimal leap sets off a response that stretches one of thymine’s chemical bonds and snaps it back into place, creating vibrations that harmlessly dissipate the energy of incoming ultraviolet light so it doesn’t cause mutations.

    The technique used to observe this tiny switch-flip at SLAC’s Linac Coherent Light Source (LCLS) X-ray free-electron laser can be applied to almost any organic molecule that responds to light – whether that light is a good thing, as in photosynthesis or human vision, or a bad thing, as in skin cancer, the scientists said. They described the study in Nature Communications today.

    SLAC/LCLS

    “All of these light-sensitive organic molecules tend to absorb light in the ultraviolet. That’s not only why you get sunburn, but it’s also why your plastic eyeglass lenses offer some UV protection,” said Phil Bucksbaum, a professor at SLAC and Stanford University and director of the Stanford PULSE Institute at SLAC. “You can even see these effects in plastic lawn furniture – after a couple of seasons it can become brittle and discolored simply due to the fact that the plastic was absorbing ultraviolet light all the time, and the way it absorbs sun results in damage to its chemical bonds.”

    Catching Electrons in Action

    Thymine and the other three DNA building blocks also strongly absorb ultraviolet light, which can trigger mutations and skin cancer, yet these molecules seem to get by with minimal damage. In 2014, a team led by Markus Guehr ­– then a SLAC senior staff scientist and now on the faculty of the University of Potsdam in Germany – reported that they had found the answer: The stretch-snap of a single bond and resulting energy-dissipating vibrations, which took place within 200 femtoseconds, or millionths of a billionth of a second after UV light exposure.

    But what made the bond stretch? The team knew the answer had to involve electrons, which are responsible for forming, changing and breaking bonds between atoms. So they devised an ingenious way to catch the specific electron movements that trigger the protective response.

    It relied on the fact that electrons don’t orbit an atom’s nucleus in neat concentric circles, like planets orbiting a sun, but rather in fuzzy clouds that take a different shape depending on how far they are from the nucleus. Some of these orbitals are in fact like a fuzzy sphere; others look a little like barbells or the start of a balloon animal. You can see examples here.

    2
    No image caption or credit, but there is a comment,
    “I see the distribution in different orbitals. So if for example I take the S orbitals, they are all just a sphere. So wont the 2S orbital overlap with the 1S overlap, making the electrons in each orbital “meet” at some point? Or have I misunderstood something?”

    Strong Signal Could Solve Long-Standing Debate

    For this new experiment, the scientists hit thymine molecules with a pulse of UV laser light and tuned the energy of the LCLS X-ray laser pulses so they would home in on the response of the oxygen atom that’s at one end of the stretching, snapping bond.

    The energy from the UV light excited one of the atom’s electrons to jump into a higher orbital. This left the atom in a sort of tippy state where just a little more energy would boost a second electron into a higher orbital; and that second jump is what sets off the protective response, changing the shape of the molecule just enough to stretch the bond.

    The first jump, which was previously known to happen, is difficult to detect because the electron winds up in a rather diffuse orbital cloud, Guehr said. But the second, which had never been observed before, was much easier to spot because that electron ended up in an orbital with a distinctive shape that gave off a big signal.

    “Although this was a very tiny electron movement, the signal kind of jumped out at us in the experiment,” Guehr said. “I always had a feeling this would be a strong transition, just intuitively, but when we saw this come in it was a special moment, one of the best moments an experimentalist can have.”

    Settling a Longstanding Debate

    Study lead author Thomas Wolf, an associate staff scientist at SLAC, said the results should settle a longstanding debate about how long after UV exposure the protective response kicks in: It happens 60 femtoseconds after UV light hits. This time span is important, he said, because the longer the atom spends in the tippy state between the first jump and the second, the more likely it is to undergo some sort of reaction that could damage the molecule.

    Henrik Koch, a theorist at NTNU in Norway who was a guest professor at Stanford at the time, led the study with Guehr. He led the effort to model, understand and interpret what happened in the experiment, and he participated in it to an unusual extent, Guehr said.

    “He is extremely experienced in applying theory to methodology development, and he had this curiosity to bring this to our experiment,” Guehr said. “He was so fascinated by this research that he did something completely untypical of a theorist – he came to LCLS, into the control room, and he wanted to see the data coming in. I found that completely amazing and very motivating. It turned out that some of my previous thinking was completely right but other aspects were completely wrong, and Henrik did the right theory at the right level so we could learn from it.”

    See the full article here .

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    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.
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  • richardmitnick 7:31 pm on June 21, 2017 Permalink | Reply
    Tags: , DNA, Gene Therapy, , heterochromatin protein 1a (HP1a),   

    From LBNL: “Researchers Find New Mechanism for Genome Regulation” 

    Berkeley Logo

    Berkeley Lab

    June 21, 2017
    Sarah Yang
    scyang@lbl.gov
    (510) 486-4575

    Berkeley Lab study could have implications for improving gene therapy.

    The same mechanisms that quickly separate mixtures of oil and water are at play when controlling the organization in an unusual part of our DNA called heterochromatin, according to a new study by researchers at the Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab).

    Researchers studying genome and cell biology provide evidence that heterochromatin organizes large parts of the genome into specific regions of the nucleus using liquid-liquid phase separation, a mechanism well known in physics but whose importance for biology has only recently been revealed.

    2
    Liquid-like fusion of heterochromatin protein 1a droplets is shown in the embryo of a fruit fly. (Credit: Amy Strom/Berkeley Lab)

    They present their findings June 21 in the journal Nature, addressing a long-standing question about how DNA functions are organized in space and time, including how genes are regulated to be silenced or expressed.

    “The importance of DNA sequences in health and disease has been clear for decades, but we only recently have come to realize that the organization of sections of DNA into different physical domains or compartments inside the nucleus is critical to promote distinct genome functions,” said study corresponding author, Gary Karpen, senior scientist at Berkeley Lab’s Biological Systems and Engineering Division.

    The long stretches of DNA in heterochromatin contain sequences that, for the most part, need to be silenced for cells to work properly. Scientists once thought that compaction of the DNA was the primary mechanism for controlling which enzymes and molecules gain access to the sequences. It was reasoned that the more tightly wound the strands, the harder it would be to get to the genetic material inside.

    That mechanism has been questioned in recent years by the discovery that some large protein complexes could get inside the heterochromatin domain, while smaller proteins can remain shut out.

    3
    Shown is purified heterochromatin protein 1a forming liquid droplets in an aqueous solution. On the right side, two drops fuse together over time. (Credit: Amy Strom/Berkeley Lab)

    In this new study of early Drosophila embryos, the researchers observed two non-mixing liquids in the cell nucleus: one that contained expressed genes, and one that contained silenced heterochromatin. They found that heterochromatic droplets fused together just like two drops of oil surrounded by water.

    In lab experiments, researchers purified heterochromatin protein 1a (HP1a), a main component of heterochromatin, and saw that this single component was able to recreate what they saw in the nucleus by forming liquid droplets.

    “We are excited about these findings because they explain a mystery that’s existed in the field for a decade,” said study lead author Amy Strom, a graduate student in Karpen’s lab. “That is, if compaction controls access to silenced sequences, how are other large proteins still able to get in? Chromatin organization by phase separation means that proteins are targeted to one liquid or the other based not on size, but on other physical traits, like charge, flexibility, and interaction partners.”

    The Berkeley Lab study, which used fruit fly and mouse cells, will be published alongside a companion paper in Nature led by UC San Francisco researchers, who showed that the human version of the HP1a protein has the same liquid droplet properties, suggesting that similar principles hold for human heterochromatin.

    4
    Mouse fibroblast cells expressing HP1alpha, the human version of heterochromatin protein 1a. A technique that highlights edges between two liquid phases reveals the liquid droplets in the nucleus. (Credit: Amy Strom/Berkeley Lab)

    Interestingly, this type of liquid-liquid phase separation is very sensitive to changes in temperature, protein concentration, and pH levels.

    “It’s an elegant way for the cell to be able to manipulate gene expression of many sequences at once,” said Strom.

    Other cellular structures, including some involved in disease, are also organized by phase separation.

    “Problems with phase separation have been linked to diseases such as dementia and certain neurodegenerative disorders,” said Karpen.

    He noted that as we age, biological molecules lose their liquid state and become more solid, accumulating damage along the way. Karpen pointed to diseases like Alzheimer’s and Huntington’s, in which proteins misfold and aggregate, becoming less liquid and more solid over time.

    “If we can better understand what causes aggregation, and how to keep things more liquid, we might have a chance to combat these types of disease,” Strom added.

    The work is a big step forward for understanding how DNA functions, but could also help researchers improve their ability to manipulate genes.

    “Gene therapy, or any treatment that relies on tight regulation of gene expression, could be improved by precisely targeting molecules to the right place in the nucleus,” says Karpen. “It is very difficult to target genes located in heterochromatin, but this understanding of the properties linked to phase separation and liquid behaviors could help change that and open up a third of the genome that we couldn’t get to before.”

    This includes targeting gene-editing technologies like CRISPR, which has recently opened up new doors for precise genome manipulation and gene therapy.

    Karpen and Strom have joint appointments at UC Berkeley’s Department of Molecular and Cell Biology. Other study co-authors include Mustafa Mir and Xavier Darzacq at UC Berkeley, and Alexander Emelyanov and Dmitry Fyodorov at the Albert Einstein College of Medicine in New York.

    The National Institutes of Health and the California Institute for Regenerative Medicine helped support this work.

    See the full article here .

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  • richardmitnick 7:46 am on June 15, 2017 Permalink | Reply
    Tags: , DNA, , , When Neurology Becomes Theology, Wilder Penfield   

    From Nautilus: “When Neurology Becomes Theology” 

    Nautilus

    Nautilus

    June 15, 2017
    Robert A. Burton

    A neurologist’s perspective on research into consciousness.

    Early in my neurology residency, a 50-year-old woman insisted on being hospitalized for protection from the FBI spying on her via the TV set in her bedroom. The woman’s physical examination, lab tests, EEGs, scans, and formal neuropsychological testing revealed nothing unusual. Other than being visibly terrified of the TV monitor in the ward solarium, she had no other psychiatric symptoms or past psychiatric history. Neither did anyone else in her family, though she had no recollection of her mother, who had died when the patient was only 2.

    The psychiatry consultant favored the early childhood loss of her mother as a potential cause of a mid-life major depressive reaction. The attending neurologist was suspicious of an as yet undetectable degenerative brain disease, though he couldn’t be more specific. We residents were equally divided between the two possibilities.

    Fortunately an intern, a super-sleuth more interested in data than speculation, was able to locate her parents’ death certificates. The patient’s mother had died in a state hospital of Huntington’s disease—a genetic degenerative brain disease. (At that time such illnesses were often kept secret from the rest of the family.) Case solved. The patient was a textbook example of psychotic behavior preceding the cognitive decline and movement disorders characteristic of Huntington’s disease.

    1
    WHERE’S THE MIND?: Wilder Penfield spent decades studying how brains produce the experience of consciousness, but concluded “There is no good evidence, in spite of new methods, that the brain alone can carry out the work that the mind does.” Montreal Neurological Institute

    As a fledgling neurologist, I’d already seen a wide variety of strange mental states arising out of physical diseases. But on this particular day, I couldn’t wrap my mind around a gene mutation generating an isolated feeling of being spied on by the FBI. How could a localized excess of amino acids in a segment of DNA be transformed into paranoia?

    Though I didn’t know it at the time, I had run headlong into the “hard problem of consciousness,” the enigma of how physical brain mechanisms create purely subjective mental states. In the subsequent 50 years, what was once fodder for neurologists’ late night speculations has mushroomed into the pre-eminent question in the philosophy of mind. As an intellectual challenge, there is no equal to wondering how subatomic particles, mindless cells, synapses, and neurotransmitters create the experience of red, the beauty of a sunset, the euphoria of lust, the transcendence of music, or in this case, intractable paranoia.

    Neuroscientists have long known which general areas of the brain and their connections are necessary for the state of consciousness. By observing both the effects of localized and generalized brain insults such as anoxia and anesthesia, none of us seriously doubt that consciousness arises from discrete brain mechanisms. Because these mechanisms are consistent with general biological principles, it’s likely that, with further technical advances, we will uncover how the brain generates consciousness.

    However, such knowledge doesn’t translate into an explanation for the what of consciousness—that state of awareness of one’s surroundings and self, the experience of one’s feelings and thoughts. Imagine a hypothetical where you could mix nine parts oxytocin, 17 parts serotonin, and 11 parts dopamine into a solution that would make 100 percent of people feel a sense of infatuation 100 percent of the time. Knowing the precise chemical trigger for the sensation of infatuation (the how) tells you little about the nature of the resulting feeling (the what).

    Over my career, I’ve gathered a neurologist’s working knowledge of the physiology of sensations. I realize neuroscientists have identified neural correlates for emotional responses. Yet I remain ignorant of what sensations and responses are at the level of experience. I know the brain creates a sense of self, but that tells me little about the nature of the sensation of “I-ness.” If the self is a brain-generated construct, I’m still left wondering who or what is experiencing the illusion of being me. Similarly, if the feeling of agency is an illusion, as some philosophers of mind insist, that doesn’t help me understand the essence of my experience of willfully typing this sentence.

    Slowly, and with much resistance, it’s dawned on me that the pursuit of the nature of consciousness, no matter how cleverly couched in scientific language, is more like metaphysics and theology. It is driven by the same urges that made us dream up gods and demons, souls and afterlife. The human urge to understand ourselves is eternal, and how we frame our musings always depends upon prevailing cultural mythology. In a scientific era, we should expect philosophical and theological ruminations to be couched in the language of physical processes. We argue by inference and analogy, dragging explanations from other areas of science such as quantum physics, complexity, information theory, and math into a subjective domain. Theories of consciousness are how we wish to see ourselves in the world, and how we wish the world might be.

    My first hint of the interaction between religious feelings and theories of consciousness came from Montreal Neurological Institute neurosurgeon Wilder Penfield’s 1975 book, Mystery of the Mind: A Critical Study of Consciousness and the Human Brain. One of the great men of modern neuroscience, Penfield spent several decades stimulating the brains of conscious, non-anesthetized patients and noting their descriptions of the resulting mental states, including long-lost bits of memory, dreamy states, deju vu, feelings of strangeness, and otherworldliness. What was most startling about Penfield’s work was his demonstration that sensations that normally qualify how we feel about our thoughts can occur in the absence of any conscious thought. For example, he could elicit feelings of familiarity and strangeness without the patient thinking of anything to which the feeling might apply. His ability to spontaneously evoke pure mental states was proof positive that these states arise from basic brain mechanisms.

    And yet, here’s Penfield’s conclusion to his end-of-career magnum opus on the nature of the mind: “There is no good evidence, in spite of new methods, that the brain alone can carry out the work that the mind does.” How is this possible? How could a man who had single-handedly elicited so much of the fabric of subjective states of mind decide that there was something to the mind beyond what the brain did?

    In the last paragraph of his book, Penfield explains, “In ordinary conversation, the ‘mind’ and ‘the spirit of man’ are taken to be the same. I was brought up in a Christian family and I have always believed, since I first considered the matter … that there is a grand design in which all conscious individuals play a role … Since a final conclusion … is not likely to come before the youngest reader of this book dies, it behooves each one of us to adopt for himself a personal assumption (belief, religion), and a way of life without waiting for a final word from science on the nature of man’s mind.”

    Front and center is Penfield’s observation that, in ordinary conversation, the mind is synonymous with the spirit of man. Further, he admits that, in the absence of scientific evidence, all opinions about the mind are in the realm of belief and religion. If Penfield is even partially correct, we shouldn’t be surprised that any theory of the “what” of consciousness would be either intentionally or subliminally infused with one’s metaphysics and religious beliefs.

    To see how this might work, take a page from Penfield’s brain stimulation studies where he demonstrates that the mental sensations of consciousness can occur independently from any thought that they seem to qualify. For instance, conceptualize thought as a mental calculation and a visceral sense of the calculation. If you add 3 + 3, you compute 6, and simultaneously have the feeling that 6 is the correct answer. Thoughts feel right, wrong, strange, beautiful, wondrous, reasonable, far-fetched, brilliant, or stupid. Collectively these widely disparate mental sensations constitute much of the contents of consciousness. But we have no control over the mental sensations that color our thoughts. No one can will a sense of understanding or the joy of an a-ha! moment. We don’t tell ourselves to make an idea feel appealing; it just is. Yet these sensations determine the direction of our thoughts. If a thought feels irrelevant, we ignore it. If it feels promising, we pursue it. Our lines of reasoning are predicated upon how thoughts feel.

    2
    No image caption or credit.

    Shortly after reading Penfield’s book, I had the good fortune to spend a weekend with theoretical physicist David Bohm. Bohm took a great deal of time arguing for a deeper and interconnected hidden reality (his theory of implicate order). Though I had difficulty following his quantum theory-based explanations, I vividly remember him advising me that the present-day scientific approach of studying parts rather than the whole could never lead to any final answers about the nature of consciousness. According to him, all is inseparable and no part can be examined in isolation.

    In an interview in which he was asked to justify his unorthodox view of scientific method, Bohm responded, “My own interest in science is not entirely separate from what is behind an interest in religion or in philosophy—that is to understand the whole of the universe, the whole of matter, and how we originate.” If we were reading Bohm’s argument as a literary text, we would factor in his Jewish upbringing, his tragic mistreatment during the McCarthy era, the lack of general acceptance of his idiosyncratic take on quantum physics, his bouts of depression, and the close relationship between his scientific and religious interests.

    Many of today’s myriad explanations for how consciousness arises are compelling. But once we enter the arena of the nature of consciousness, there are no outright winners.

    Christof Koch, the chief scientific officer of the Allen Institute for Brain Science in Seattle, explains that a “system is conscious if there’s a certain type of complexity. And we live in a universe where certain systems have consciousness. It’s inherent in the design of the universe.”

    According to Daniel Dennett, professor of philosophy at Tufts University and author of Consciousness Explained and many other books on science and philosophy, consciousness is nothing more than a “user-illusion” arising out of underlying brain mechanisms. He argues that believing consciousness plays a major role in our thoughts and actions is the biological equivalent of being duped into believing that the icons of a smartphone app are doing the work of the underlying computer programs represented by the icons. He feels no need to postulate any additional physical component to explain the intrinsic qualities of our subjective experience.

    Meanwhile, Max Tegmark, a theoretical physicist at the Massachusetts Institute of Technology, tells us consciousness “is how information feels when it is being processed in certain very complex ways.” He writes that “external reality is completely described by mathematics. If everything is mathematical, then, in principle, everything is understandable.” Rudolph E. Tanzi, a professor of neurology at Harvard University, admits, “To me the primal basis of existence is awareness and everything including ourselves and our brains are products of awareness.” He adds, “As a responsible scientist, one hypothesis which should be tested is that memory is stored outside the brain in a sea of consciousness.”

    Each argument, taken in isolation, seems logical, internally consistent, yet is at odds with the others. For me, the thread that connects these disparate viewpoints isn’t logic and evidence, but their overall intent. Belief without evidence is Richard Dawkins’ idea of faith. “Faith is belief in spite of, even perhaps because of, the lack of evidence.” These arguments are best read as differing expressions of personal faith.

    For his part, Dennett is an outspoken atheist and fervent critic of the excesses of religion. “I have absolutely no doubt that secular and scientific vision is right and deserves to be endorsed by everybody, and as we have seen over the last few thousand years, superstitious and religious doctrines will just have to give way.” As the basic premise of atheism is to deny that for which there is no objective evidence, he is forced to avoid directly considering the nature of purely subjective phenomena. Instead he settles on describing the contents of consciousness as illusions, resulting in the circularity of using the definition of mental states (illusions) to describe the general nature of these states.

    The problem compounds itself. Dennett is fond of pointing out (correctly) that there is no physical manifestation of “I,” no ghost in the machine or little homunculus that witnesses and experiences the goings on in the brain. If so, we’re still faced with asking what/who, if anything, is experiencing consciousness? All roads lead back to the hard problem of consciousness.

    Though tacitly agreeing with those who contend that we don’t yet understand the nature of consciousness, Dennett argues that we are making progress. “We haven’t yet succeeded in fully conceiving how meaning could exist in a material world … or how consciousness works, but we’ve made progress: The questions we’re posing and addressing now are better than the questions of yesteryear. We’re hot on the trail of the answers.”

    By contrast, Koch is upfront in correlating his religious upbringing with his life-long pursuit of the nature of consciousness. Raised as a Catholic, he describes being torn between two contradictory views of the world—the Sunday view reflected by his family and church, and the weekday view as reflected in his work as a scientist (the sacred and the profane).

    In an interview with Nautilus, Koch said, “For reasons I don’t understand and don’t comprehend, I find myself in a universe that had to become conscious, reflecting upon itself.” He added, “The God I now believe in is closer to the God of Spinoza than it is to Michelangelo’s paintings or the God of the Old Testament, a god that resides in this mystical notion of all-nothingness.” Koch admitted, “I’m not a mystic. I’m a scientist, but this is a feeling I have.” In short, Koch exemplifies a truth seldom admitted—that mental states such as a mystical feeling shape how one thinks about and goes about studying the universe, including mental states such as consciousness.

    Both Dennett and Koch have spent a lifetime considering the problem of consciousness; though contradictory, each point of view has a separate appeal. And I appreciate much of Dennett and Koch’s explorations in the same way that I can mull over Aquinas and Spinoza without necessarily agreeing with them. One can enjoy the pursuit without believing in or expecting answers. After all these years without any personal progress, I remain moved by the essential nature of the quest, even if it translates into Sisyphus endlessly pushing his rock up the hill.

    The spectacular advances of modern science have generated a mindset that makes potential limits to scientific inquiry intuitively difficult to grasp. Again and again we are given examples of seemingly insurmountable problems that yield to previously unimaginable answers. Just as some physicists believe we will one day have a Theory of Everything, many cognitive scientists believe that consciousness, like any physical property, can be unraveled. Overlooked in this optimism is the ultimate barrier: The nature of consciousness is in the mind of the beholder, not in the eye of the observer.

    It is likely that science will tell us how consciousness occurs. But that’s it. Although the what of consciousness is beyond direct inquiry, the urge to explain will persist. It is who we are and what we do.

    See the full article here .

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    Welcome to Nautilus. We are delighted you joined us. We are here to tell you about science and its endless connections to our lives. Each month we choose a single topic. And each Thursday we publish a new chapter on that topic online. Each issue combines the sciences, culture and philosophy into a single story told by the world’s leading thinkers and writers. We follow the story wherever it leads us. Read our essays, investigative reports, and blogs. Fiction, too. Take in our games, videos, and graphic stories. Stop in for a minute, or an hour. Nautilus lets science spill over its usual borders. We are science, connected.

     
  • richardmitnick 7:35 am on June 2, 2017 Permalink | Reply
    Tags: Alzheimer’s Parkinson’s and Huntington’s diseases as well as schizophrenia autism and depression., , , DNA, , Microglia–unique,   

    From Salk: “Brain’s immune cells linked to Alzheimer’s, Parkinson’s, schizophrenia” 

    Salk Institute bloc

    Salk Institute for Biological Studies

    Salk and UC San Diego scientists conducted vast microglia survey, revealing links to neurodegenerative diseases and psychiatric illnesses.

    Scientists have, for the first time, characterized the molecular markers that make the brain’s front lines of immune defense–cells called microglia–unique. In the process, they discovered further evidence that microglia may play roles in a variety of neurodegenerative and psychiatric illnesses, including Alzheimer’s, Parkinson’s and Huntington’s diseases as well as schizophrenia, autism and depression.

    “Microglia are the immune cells of the brain, but how they function in the human brain is not well understood,” says Rusty Gage, professor in Salk’s Laboratory of Genetics, the Vi and John Adler Chair for Research on Age-Related Neurodegenerative Disease, and a senior author of the new work. “Our work not only provides links to diseases but offers a jumping off point to better understand the basic biology of these cells.”

    Genes that have previously been linked to neurological diseases are turned on at higher levels in microglia compared to other brain cells, the team reported in Science on May 25, 2017. While the link between microglia and a number of disorders has been explored in the past, the new study offers a molecular basis for this connection.

    “These studies represent the first systematic effort to molecularly decode microglia,” says Christopher Glass, a Professor of Cellular and Molecular Medicine and Professor of Medicine at University of California San Diego, also senior author of the paper. “Our findings provide the foundations for understanding the underlying mechanisms that determine beneficial or pathological functions of these cells.”

    Microglia are a type of macrophage, white blood cells found throughout the body that can destroy pathogens or other foreign materials. They’re known to be highly responsive to their surroundings and respond to changes in the brain by releasing pro-inflammatory or anti-inflammatory signals. They also prune back the connections between neurons when cells are damaged or diseased. But microglia are notoriously hard to study. They can’t be easily grown in a culture dish and quickly die outside of a living brain.

    Nicole Coufal, a pediatric critical care doctor at UC San Diego, who also works in the Gage lab at Salk, wanted to make microglia from stem cells. But she realized there wasn’t any way to identify whether the resulting cells were truly microglia.

    “There was not a unique marker that differentiated microglia from circulating macrophages in the rest of the body,” she says.

    David Gosselin and Dylan Skola in the Glass lab, together with Coufal and their collaborators, set out to characterize the molecular characteristics of microglia. They worked with neurosurgeons at UC San Diego to collect brain tissue from 19 patients, all of who were having brain surgery for epilepsy, a brain tumor or a stroke. They isolated microglia from areas of tissue that were unaffected by disease, as well as from mouse brains, and then set out to study the cells. The work was made possible by a multidisciplinary collaboration between bench scientists, bioinformaticians and clinicians.

    The team used a variety of molecular and biochemical tests–performed within hours of the cells being collected–to characterize which genes are turned on and off in microglia, how the DNA is marked up by regulatory molecules, and how these patterns change when the cells are cultured.

    Microglia, they found, have hundreds of genes that are more highly expressed than other types of macrophages, as well as distinct patterns of gene expression compared to other types of brain cells. After the cells were cultured, however, the gene patterns of the microglia began to change. Within just six hours, more than 2,000 genes had their expression turned down by at least fourfold. The results underscore how dependent microglia are on their surroundings in the brain, and why researchers have struggled to culture them.

    Next, the researchers analyzed whether any of the genes that were upregulated in microglia compared to other cells had been previously implicated in disease. Genes linked to a variety of neurodegenerative and psychiatric diseases, they found, were highly expressed in microglia.

    “A really high proportion of genes linked to multiple sclerosis, Parkinson’s and schizophrenia are much more highly expressed in microglia than the rest of the brain,” says Coufal. “That suggests there’s some kind of link between microglia and the diseases.”

    For Alzheimer’s, more than half of the genes known to affect a person’s risk of developing the disease were expressed more highly in microglia than other brain cells.

    In mice, however, many of the disease genes weren’t as highly expressed in microglia. “That tells us that maybe mice aren’t the best model organisms for some of these diseases,” Coufal says.

    More work is needed to understand exactly how microglia may be altered in people with diseases, but the new molecular profile of microglia offers a way for researchers to begin trying to better culture the cells, or coax stem cells to develop into microglia for future studies.

    Other researchers on the study were Baptiste Jaeger, Carolyn O’Connor, Conor Fitzpatrick, Monique Pena, and Amy Adair of the Salk Institute; Inge Holtman, Johannes Schlachetzki, Eniko Sajti, Martina Pasillas, David Gona, and Michael Levy of the University of California San Diego; and Richard Ransohoff of Biogen.

    The work and the researchers involved were supported by grants from the Larry L. Hillblom Foundation, National Institutes of Health, Canadian Institute of Health Research, Multiple Sclerosis Society of Canada, University of California San Diego, Dutch MS Research Foundation, the Gemmy and Mibeth Tichelaar Foundation, the DFG, the JPB Foundation, Dolby Family Ventures, The Paul G. Allen Family Foundation, the Engman Foundation, the Ben and Wanda Hildyard Chair in Hereditary Diseases.

    See the full article here .

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