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  • richardmitnick 10:42 am on July 31, 2017 Permalink | Reply
    Tags: , , , HMS- Harvard Medical School, , It wouldn’t have been obvious that PTPN2 is a good drug target for the immunotherapy of cancer, , PD-1 checkpoint inhibitors have transformed the treatment of many cancers   

    From HMS: “Attack Mode “ 

    Harvard University

    Harvard University

    Harvard Medical School

    Harvard Medical School

    7.31.17
    KAT MCALPINE

    1
    Genetic screening for cancer immunotherapy targets. Cancer cells (colored shapes), each with a different CRISPR-Cas9-mediated gene knocked out. T cells (red) destroy the cancer cells that have had essential immune evasion genes knocked out. Image: Haining Lab, Dana-Farber/Boston Children’s.

    A novel screening method developed by a team at Harvard Medical School and Dana-Farber/Boston Children’s Cancer and Blood Disorders Center—using CRISPR-Cas9 genome editing technology to test the function of thousands of tumor genes in mice—has revealed new drug targets that could potentially enhance the effectiveness of PD-1 checkpoint inhibitors, a promising new class of cancer immunotherapy.

    In findings published online today by Nature http://www.nature.com/nature/journal/v547/n7664/full/nature23270.html the Dana-Farber/Boston Children’s team, led by pediatric oncologist W. Nick Haining, reports that deletion of the PTPN2 gene in tumor cells made them more susceptible to PD-1 checkpoint inhibitors. PD-1 blockade is a drug that “releases the brakes” on immune cells, enabling them to locate and destroy cancer cells.

    “PD-1 checkpoint inhibitors have transformed the treatment of many cancers,” said Haining, HMS associate professor of pediatrics at Dana-Farber/Boston Children’s, associate member of the Broad Institute of MIT and Harvard, and senior author on the new paper. “Yet despite the clinical success of this new class of cancer immunotherapy, the majority of patients don’t reap a clinical benefit from PD-1 blockade.”

    That, Haining said, has triggered a rush of additional trials to investigate whether other drugs, when used in combination with PD-1 inhibitors, can increase the number of patients whose cancer responds to the treatment.

    “The challenge so far has been finding the most effective immunotherapy targets and prioritizing those that work best when combined with PD-1 inhibitors,” Haining said. “So, we set out to develop a better system for identifying new drug targets that might aid the body’s own immune system in its attack against cancer.

    “Our work suggests that there’s a wide array of biological pathways that could be targeted to make immunotherapy more successful,” Haining continued. “Many of these are surprising pathways that we couldn’t have predicted. For instance, without this screening approach, it wouldn’t have been obvious that PTPN2 is a good drug target for the immunotherapy of cancer.”

    Sifting through thousands of potential targets

    To cast a wide net, the paper’s first author Robert Manguso, a graduate student in Haining’s lab, designed a genetic screening system to identify genes used by cancer cells to evade immune attack. He used CRISPR-Cas9, a genome editing technology that works like a pair of molecular scissors to cleave DNA at precise locations in the genetic code, to systematically knock out 2,368 genes expressed by melanoma skin cancer cells. Manguso was then able to identify which genes, when deleted, made the cancer cells more susceptible to PD-1 blockade.

    Manguso started by engineering the melanoma skin cancer cells so that they all contained Cas9, the cutting enzyme that is part of the CRISPR editing system. Then, using a virus as a delivery vehicle, he programmed each cell with a different single-guide-RNA (sgRNA) sequence of genetic code. In combination with the Cas9 enzyme, the sgRNA codes—about 20 amino acids in length—enabled 2,368 different genes to be eliminated.

    By injecting the tumor cells into mice and treating them with PD-1 checkpoint inhibitors, Manguso was then able to tally up which modified tumor cells survived. Those that perished had been sensitized to PD-1 blockade as a result of their missing gene.

    Using this approach, Manguso and Haining first confirmed the role of two genes already known to be immune evaders—PD-L1 and CD47, drug inhibitors that are already in clinical trials. They then discovered a variety of new immune evaders that, if inhibited therapeutically, could enhance PD-1 cancer immunotherapy. One such newly found gene of particular interest is PTPN2.

    “PTPN2 usually puts the brakes on the immune signaling pathways that would otherwise smother cancer cells,” Haining said. “Deleting PTPN2 ramps up those immune signaling pathways, making tumor cells grow slower and die more easily under immune attack.”

    Gaining more ground

    With the new screening approach in hand, Haining’s team is quickly scaling up their efforts to search for additional novel drug targets that could boost immunotherapy.

    Haining says the team is expanding their approach to move from screening thousands of genes at a time to eventually being able to screen the whole genome and to move beyond melanoma to colon, lung, renal carcinoma and more. He’s assembled a large team of scientists spanning Dana-Farber/Boston Children’s and the Broad to tackle the technical challenges that accompany screening efforts on such a large scale.

    In the meantime, while more new potential drug targets are likely around the corner, Haining’s team is taking action based on their findings about PTPN2.

    “We’re thinking hard about what a PTPN2 inhibitor would look like,” said Haining. “It’s easy to imagine making a small molecule drug that turns off PTPN2.”

    This work was supported by the Broad Institute of Harvard and MIT (BroadIgnite and Broadnext10 awards) and the National Institute of General Medical Sciences (T32GM007753).

    See the full article here .

    Please help promote STEM in your local schools.

    STEM Icon

    Stem Education Coalition

    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 10:24 am on July 31, 2017 Permalink | Reply
    Tags: , , HMS- Harvard Medical School, , ,   

    From HMS: “Making the Makers” 

    Harvard University

    Harvard University

    Harvard Medical School

    Harvard Medical School

    July 21, 2017
    KEVIN JIANG

    1
    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

    2
    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 .

    Please help promote STEM in your local schools.

    STEM Icon

    Stem Education Coalition

    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 8:47 am on July 17, 2017 Permalink | Reply
    Tags: , CRISPR-Cas3, , , , HMS- Harvard Medical School,   

    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 .

    Please help promote STEM in your local schools.

    STEM Icon

    Stem Education Coalition

    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 10:21 am on July 3, 2017 Permalink | Reply
    Tags: , , , , HMS- Harvard Medical School,   

    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 .

    Please help promote STEM in your local schools.

    STEM Icon

    Stem Education Coalition

    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 9:51 pm on July 1, 2017 Permalink | Reply
    Tags: Cloning Thousands of Genes for Massive Protein Libraries, HMS- Harvard Medical School, , LASSO (long-adapter single-strand oligonucleotide) probes, New DNA-based LASSO molecule probe can bind target genome regions for functional cloning and analysis, , University of Trento   

    From Rutgers: “Cloning Thousands of Genes for Massive Protein Libraries” 

    Rutgers University
    Rutgers University

    June 26, 2017
    Todd B. Bates

    1
    New DNA-based LASSO molecule probe can bind target genome regions for functional cloning and analysis. Photo: Jennifer E. Fairman/JHU

    Discovering the function of a gene requires cloning a DNA sequence and expressing it. Until now, this was performed on a one-gene-at-a-time basis, causing a bottleneck. Scientists at Rutgers University-New Brunswick in collaboration with Johns Hopkins University and Harvard Medical School have invented a technology to clone thousands of genes simultaneously and create massive libraries of proteins from DNA samples, potentially ushering in a new era of functional genomics.

    “We think that the rapid, affordable, and high-throughput cloning of proteins and other genetic elements will greatly accelerate biological research to discover functions of molecules encoded by genomes and match the pace at which new genome sequencing data is coming out,” said Biju Parekkadan, an associate professor in the Department of Biomedical Engineering at Rutgers University-New Brunswick.

    In a study published online today in the journal Nature Biomedical Engineering, the researchers showed that their technology – LASSO (long-adapter single-strand oligonucleotide) probes – can capture and clone thousands of long DNA fragments at once.

    As a proof-of-concept, the researchers cloned more than 3,000 DNA fragments from E. coli bacteria, commonly used as a model organism with a catalogued genome sequence available.

    “We captured about 95 percent of the gene targets we set out to capture, many of which were very large in DNA length, which has been challenging in the past,” Parekkadan said. “I think there will certainly be more improvements over time.”

    They can now take a genome sequence (or many of them) and make a protein library for screening with unprecedented speed, cost-effectiveness and precision, allowing rapid discovery of potentially beneficial biomolecules from a genome.

    In conducting their research, they coincidentally solved a longstanding problem in the genome sequencing field. When it comes to genetic sequencing of individual genomes, today’s gold standard is to sequence small pieces of DNA one by one and overlay them to map out the full genome code. But short reads can be hard to interpret during the overlaying process and there hasn’t been a way to sequence long fragments of DNA in a targeted and more efficient way. LASSO probes can do just this, capturing DNA targets of more than 1,000 base pairs in length where the current format captures about 100 base pairs.

    The team also reported the capture and cloning of the first protein library, or suite of proteins, from a human microbiome sample. Shedding light on the human microbiome at a molecular level is a first step toward improving precision medicine efforts that affect the microbial communities that colonize our gut, skin and lungs, Parekkadan added. Precision medicine requires a deep and functional understanding, at a molecular level, of the drivers of healthy and disease-forming microbiota.

    Today, the pharmaceutical industry screens synthetic chemical libraries of thousands of molecules to find one that may have a medicinal effect, said Parekkadan, who joined Rutgers’ School of Engineering in January.

    “Our vision is to apply the same approach but rapidly screen non-synthetic, biological or ‘natural’ molecules cloned from human or other genomes, including those of plants, animals and microbes,” he said. “This could transform pharmaceutical drug discovery into biopharmaceutical drug discovery with much more effort.”

    The next phase, which is underway, is to improve the cloning process, build libraries and discover therapeutic proteins found in our genomes, Parekkadan said.

    Other authors include Lorenzo Tosi, Viswanadham Sridhara, Yunlong Yang, Dongli Guan and Polina Shpilker of Harvard Medical School; Nicola Segata of the University of Trento in Trento, Italy; and H. Benjamin Larman of Johns Hopkins University.

    See the full article here .

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    Rutgers, The State University of New Jersey, is a leading national research university and the state’s preeminent, comprehensive public institution of higher education. Rutgers is dedicated to teaching that meets the highest standards of excellence; to conducting research that breaks new ground; and to providing services, solutions, and clinical care that help individuals and the local, national, and global communities where they live.

    Founded in 1766, Rutgers teaches across the full educational spectrum: preschool to precollege; undergraduate to graduate; postdoctoral fellowships to residencies; and continuing education for professional and personal advancement.

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  • richardmitnick 12:13 pm on June 17, 2017 Permalink | Reply
    Tags: , , HMS- Harvard Medical School, ,   

    From HMS: “Staving Off Stem Cell Cancer Risk” 

    Harvard University

    Harvard University

    Harvard Medical School

    Harvard Medical School

    April 26, 2017 [Never saw this one.]
    HANNAH ROBBINS

    1
    Image: BlackJack3D/Getty Images

    Regenerative medicine using human pluripotent stem cells to grow transplantable tissue outside the body carries the promise to treat a range of intractable disorders, such as diabetes and Parkinson’s disease.

    As stem cell lines grow in a lab dish, however, they often acquire mutations in the TP53 (p53) gene, an important tumor suppressor responsible for controlling cell growth and division, according to new research from a team at Harvard Medical School, the Harvard Stem Cell Institute and the Stanley Center for Psychiatric Research at the Broad Institute of MIT and Harvard.

    The findings suggest that genetic sequencing technologies should be used to screen stem cell cultures so that those with mutated cells can be excluded from scientific experiments and clinical therapies. If such methods are not employed, the researchers said, it could lead to an elevated cancer risk in patients receiving transplants.

    The paper, published online in the journal Nature on April 26, comes at just the right time, the researchers said, as experimental treatments using human pluripotent stem cells are ramping up across the country.

    “Our results underscore the need for the field of regenerative medicine to proceed with care,” said the study’s co-corresponding author, Kevin Eggan, a principal faculty member at HSCI and director of stem cell biology at the Stanley Center.

    The team said that the new research should not discourage the pursuit of experimental treatments, but instead should be heeded as a call to rigorously screen all cell lines for mutations at various stages of development as well as immediately before transplantation.

    “Fortunately,” said Eggan, this additional series of genetic quality-control checks “can be readily performed with precise, sensitive and increasingly inexpensive sequencing methods.”

    Hidden mutations

    Researchers can use human stem cells to recreate human tissue in the lab. Eggan’s lab in Harvard University’s Department of Stem Cell and Regenerative Biology uses human stem cells to study the mechanisms of brain disorders, including amyotrophic lateral sclerosis, intellectual disability and schizophrenia.

    Eggan has also been working with Steve McCarroll, associate professor of genetics at HMS and director of genetics at the Stanley Center, to study how genes shape the biology of neurons, which can be derived from human stem cells.

    McCarroll’s lab recently discovered a common precancerous condition in which a blood stem cell in the body acquires a so-called pro-growth mutation and then outcompetes a person’s normal stem cells, becoming the dominant generator of that person’s blood cells. People with this condition are 12 times more likely to develop blood cancer later in life.

    The current study’s lead authors, Florian Merkle and Sulagna Ghosh, collaborated with Eggan and McCarroll to test whether laboratory-grown stem cells might be vulnerable to an analogous process.

    “Cells in the lab, like cells in the body, acquire mutations all the time,” said McCarroll, co-corresponding author of the study. “Mutations in most genes have little impact on the larger tissue or cell line. But cells with a pro-growth mutation can outcompete other cells, become very numerous and ‘take over’ a tissue.”

    “We found that this process of clonal selection—the basis of cancer formation in the body—is also routinely happening in laboratories.”

    A p53 problem

    To find acquired mutations, the researchers performed genetic analyses on 140 stem cell lines. Twenty-six lines had been developed for therapeutic purposes using Good Manufacturing Practices, a quality control standard set by regulatory agencies in multiple countries. The remaining 114 were listed on the NIH registry of human pluripotent stem cells.

    “While we expected to find some mutations, we were surprised to find that about 5 percent of the stem cell lines we analyzed had acquired mutations in a tumor-suppressing gene called p53,” said Merkle.

    Nicknamed the “guardian of the genome,” p53 controls cell growth and cell death. People who inherit p53 mutations develop a rare disorder called Li-Fraumeni syndrome, which confers a near 100 percent risk of developing cancer in a wide range of tissue types.

    The specific mutations that the researchers observed are dominant negative mutations, meaning that when present on even one copy of p53, they compromise the function of the normal protein. The same dominant negative mutations are among the most commonly observed mutations in human cancers.

    “They are among the worst p53 mutations to have,” said co-lead author Ghosh.

    The researchers performed a sophisticated set of DNA analyses to rule out the possibility that these mutations had been inherited rather than acquired as the cells grew in the lab.

    Ensuring safety

    In subsequent experiments, the scientists found that p53 mutant cells outperformed and outcompeted nonmutant cells in the lab dish. In other words, a culture with a million healthy cells and one p53 mutant cell, said Eggan, could quickly become a culture of only mutant cells.

    “The spectrum of tissues at risk for transformation when harboring a p53 mutation includes many of those that we would like to target for repair with regenerative medicine using human pluripotent stem cells,” said Eggan.

    Those organs include the pancreas, brain, blood, bone, skin, liver and lungs.

    However, Eggan and McCarroll emphasized that now that this phenomenon has been found, inexpensive gene-sequencing tests will allow researchers to identify and remove from the production line cell cultures with concerning mutations that might prove dangerous after transplantation.

    The researchers point out in their paper that screening approaches already exist to identify these p53 mutations and others that confer cancer risk. Such techniques are being used in cancer diagnostics.

    In fact, an ongoing clinical trial that is transplanting cells derived from induced pluripotent stem cells (iPSCs) is using gene sequencing to ensure the transplanted cell products are free of dangerous mutations.

    This work was supported by the Harvard Stem Cell Institute, the Stanley Center for Psychiatric Research, the Rosetrees Trust, the Azrieli Foundation, the Howard Hughes Medical Institute, the Wellcome Trust, the Medical Research Council, the Academy of Medical Sciences and grants from the National Institutes of Health (HL109525, 5P01GM099117, 5K99NS08371, HG006855, MH105641).

    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 1:13 pm on May 30, 2017 Permalink | Reply
    Tags: Amping Up Antibodies, , HMS- Harvard Medical School   

    From HMS: “Amping Up Antibodies” 

    Harvard University

    Harvard University

    Harvard Medical School

    Harvard Medical School

    May 26, 2017
    NANCY FLIESLER

    1
    Artist’s rendition of antibodies. Image: urfinguss/Getty Images

    It began with the proteins.

    Before Watson and Crick unraveled DNA’s double helix in the 1950s, biochemists snipped, ground and pulverized animal tissues to extract and study proteins, the workhorses of the body.

    Then, in 1990, the Human Genome Project launched. It promised to uncover the underpinnings of all human biology and the keys to treating disease. Funding for DNA and RNA tools and studies skyrocketed as protein science fell behind.

    While genomics unveiled a wealth of information, including the identity of genes that lead to disease when mutated, researchers still do not fully understand what all the genes really do and how mutations change their function and cause disease.

    Now proteins are promising to provide the missing link.

    Biological chemist, immunologist and structural biologist Timothy Springer is reinventing protein science with new technologies. Now Springer has launched the Institute for Protein Innovation (IPI). Currently housed at Harvard Medical School, the nonprofit aims to create a massive, openly available resource of protein technologies to accelerate drug discovery and development.

    “Proteins lag behind DNA and RNA in institutional research support and funding,” said Springer, Latham Family Professor at HMS and Boston Children’s Hospital. “Yet proteins serve as the targets of almost all drugs and in many cases as therapeutic drugs themselves.”

    Partnering with co-founder Andrew Kruse, assistant professor of biological chemistry and molecular pharmacology at HMS, Springer formally launched the IPI on May 10 with $15 million in grants and philanthropy.

    Accelerating antibodies

    Nearly half of all drugs on the market today are proteins, mostly monoclonal antibodies that home in on specific targets in the body, just as our own antibodies target proteins made by disease-causing organisms. Researchers spent decades producing antibodies in the lab that blocked or activated proteins’ function. This helped investigators figure out what genes and proteins do in the body.

    Seeing their success, drug developers began seeking to mass-produce antibodies that would target protein culprits in diseases such as cancer. However, the development of antibodies for use as therapeutics faced—and still faces—major hurdles.

    First, antibodies are traditionally produced by immunizing animals like mice with a target human protein. That process is often slow and not always effective: If the mouse and human proteins are too similar, the mouse immune system won’t recognize the human protein as foreign and won’t make an antibody. This leaves many extremely important molecules without targeting antibodies.

    Second, many antibodies used in published research studies have not been properly validated through extra testing. This makes it difficult to reproduce the results of pivotal antibody-based studies, slowing scientific progress.

    Probing for proteins

    To circumvent these problems, researchers including British biochemist Greg Winter invented robust “molecular display” technologies. They involve creating immense libraries of unique antibodies or antibody fragments.

    Researchers insert the genes for antibodies into one-celled organisms such as yeast, modified so that the antibodies made by the yeast are displayed on their surface. Billions of yeast cells, each bearing a unique antibody, are then mixed with the protein of interest. The final step is fishing out the yeast that bind to the target proteins most strongly, through a two-step process.

    Because the yeast make the antibodies as instructed by inserted genes, the organisms can be used to create antibodies to molecules that a mouse immune system wouldn’t respond to. In addition, the technique can be scaled up for the high-throughput approaches of genomics and proteomics.

    But there are other problems yet to solve. Many proteins that would be used as targets in the antibody selection process are still extremely difficult to produce. Expertise in antibody manufacture is also lacking.

    Springer believes the solution is an infrastructure to make, develop and validate synthetic antibodies and their target proteins at a scale never before attempted. The IPI’s first major effort will be to develop open-source libraries of well-validated antibodies targeting every human protein found outside cells.

    Academic entrepreneurship

    Unlike Springer’s other commercial start-ups, the IPI is an academic-entrepreneurial hybrid. By straddling the traditional divide, Springer hopes to create alliances between leaders in academic research, biotechnology, the pharmaceutical industry and biomedical investing, while providing open-source resources to the scientific community at little or no charge. The institute will publish its technical processes and share its know-how freely, equipping the next generation of protein scientists.

    Springer intends for the IPI to stay afloat through a mix of public and philanthropic investment, including $5 million from the Massachusetts Life Sciences Center and a $10 million gift from Springer himself. With the 2018 NIH budget poised to be cut 18 percent, the effort could not have come at a better time.

    Springer conducted his postdoctoral studies with Nobel laureate César Milstein, who co-invented monoclonal antibody technology in 1975. This invention has led to far more drugs than have come from genomics. Springer sees IPI as the embodiment of Milstein’s vision of using antibodies to target disease.

    “César would love this,” said Springer. “He really wanted to have the promise of antibodies fulfilled.”

    See the full article here .

    Please help promote STEM in your local schools.

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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 9:23 am on April 21, 2017 Permalink | Reply
    Tags: , Autism studies, HMS- Harvard Medical School,   

    From HMS: “A strengths-based approach to autism” 

    Harvard University

    Harvard University

    Harvard Medical School

    Harvard Medical School

    April 20, 2017
    Monique Tello, MD, MPH

    1
    No image caption. No image credit.

    At our son’s 18-month checkup five years ago, our pediatrician expressed concern. Gio wasn’t using any words, and would become so frustrated he would bang his head on the ground. Still, my husband and I were in denial. We dragged our feet. Meanwhile, our son grunted and screamed; people said things. Finally we started therapy with early intervention services.

    A few months later, after hundreds of pages of behavior questionnaires for us and hours of testing for Gio, we heard the words: “Your son meets criteria for a diagnosis of autism spectrum disorder…”

    Our journey has taken us through several behavioral approaches with many different providers. Today, Gio is doing very well, in an integrated first grade in public school. He can speak, read, write, and play. His speech and syntax can be hard to understand, but we are thrilled that we can communicate with him.

    The difference between typical and functional

    Longtime autism researcher Laurent Mottron wrote a recent scientific editorial in which he points out that the current approach to treating a child with autism is based on changing them, making them conform, suppressing repetitive behaviors, intervening with any “obsessive” interests. Our family experienced this firsthand. Some of our early behavioral therapists would see Gio lie on the ground to play, his face level with the cars and trucks he was rolling into long rows, and they would tell us, “Make him sit up. No lying down. Let’s rearrange the cars. Tell him, they don’t always have to be in a straight line, Gio!”

    To me, this approach seemed rigid. We don’t all have to act in the exact same way. These kids need to function, not robotically imitate “normal.”

    Why not leverage difference rather than extinguish it?

    We naturally gravitated towards Stanley Greenspan’s “DIR/Floortime” approach, in which therapists and parents follow the child’s lead, using the child’s interests to engage them, and then helping the child to progress and develop.

    Mottron’s research supports Greenspan’s approach: study the child to identify his or her areas of interest. The more intense the interest the better, because that’s what the child will find stimulating. Let them fully explore that object or theme (shiny things? purple things? wheels?) because these interests help the developing brain to figure out the world.

    Then, use that interest as a means to engage with the child, and help them make more connections. Mottron suggests that parents and teachers get on the same level with the child and engage in a similar activity — be it rolling cars and trucks, or lining them up. When the child is comfortable, add in something more. Maybe, make the cars and trucks talk to each other.

    But, don’t pressure the child to join the conversation. Let them be exposed to words, conversations, and songs, without forced social interaction. This is how early language skills can be taught in a non-stressful way, acknowledging and aligning with the autistic brain. The ongoing relationship and engagement will foster communication.

    Basically, what both Greenspan and Mottron are advocating are methods of teaching autistic children to relate, adapt, and function in the world, without “forcing the autism out of them.”

    The concept of accepting autistic kids as they are, and incorporating the natural ways they think into educational and therapeutic techniques, feels right to me. Gio is different from most kids, and really, he’s not interested in most kids. Our attempts to push him to participate in “fun” group activities like soccer, Easter egg hunts, and birthday parties have all been spectacular failures. Maybe the real failure was ours: by pushing him to “fit in,” we deny his true nature. Yes, the way he thinks is sometimes mysterious to us, but he clearly has great strengths: a remarkable ability to focus and persevere, to experiment with his ideas, and to follow his vision.

    World-renowned autism expert and animal rights activist Temple Grandin (who is herself autistic, and very open about her preference for animal rather than human companionship!) sums up Mottron’s approach perfectly: “The focus should be on teaching people with autism to adapt to the social world around them while still retaining the essence of who they are, including their autism.”

    Sources

    generallymedicine blog post: Screaming Frustration: Our Two-Year-Old Won’t Talk

    generallymedicine blog post: They Dropped the A-Bomb On Us

    generallymedicine blog post: So Our Son Is Autistic… and It’s Going to Be Okay

    generallymedicine blog post: Should we let our kid hang out by himself most of the time?

    generallymedicine blog post: Autism Awareness… and Coolness

    Greenspan, Stanley (2006). Engaging Autism. Da Capo Press. http://www.stanleygreenspan.com

    Mottron, L. Should we change targets and methods of early intervention in autism, in favor of a strengths-based education? European Child & Adolescent Psychiatry, February 2017, e-pub ahead of print.

    Related Information: The Autism Revolution

    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 2:40 pm on April 3, 2017 Permalink | Reply
    Tags: , HMS- Harvard Medical School, , Taking Their Best Shot   

    From HMS: “Taking Their Best Shot’ 

    Harvard University

    Harvard University

    Harvard Medical School

    Harvard Medical School

    March 24, 2017
    NANCY FLIESLER

    1
    Image: Esben_H/Getty Images

    In many parts of the world, babies have just one chance to be vaccinated: when they’re born. Unfortunately, newborns’ immune systems don’t respond well to most vaccines. That’s why, in the U.S., most immunizations start at two months of age.

    Currently, only BCG (for tuberculosis), polio and hepatitis B vaccines work in newborns, and the last two require multiple doses. But new research raises the possibility of one-shot vaccinations at birth—with huge implications for reducing infant mortality.

    The trick? Specially designed additives—adjuvants—tailored to stimulate newborns’ unique immune systems.

    Two papers published today demonstrate strong vaccine responses in newborn mice, and, more importantly, in newborn monkeys, the ultimate preclinical test. Both studies used formulations designed to maximize safety.

    “Our efforts have led to adjuvant approaches that may enable earlier protection of newborns and young infants from life-threatening infectious diseases, such as pneumococcus, pertussis or even respiratory syncytial virus,” said Ofer Levy, HMS associate professor of pediatrics at Boston Children’s Hospital and a senior author of both papers.

    Dramatic response

    The first of the new studies, led by David Dowling , an HMS research fellow in pediatrics in the Division of Infectious Diseases at Boston Children’s, tested an adjuvant called 3M-052. The adjuvant was manufactured by 3M Drug Delivery Systems, which partially funded the study. It works by stimulating two specific toll-like receptors (pathogen-sensing proteins), TLR7 and TLR8.

    Newborn rhesus monkeys received a series of three shots with the existing Prevnar 13 pneumococcal vaccine—“the same as my children got,” said Levy, who directs the Precision Vaccines Program at Boston Children’s.

    The team chose pneumococcal vaccine as a test case because Streptococcus pnumoniae can cause potentially fatal pneumonia, meningitis and sepsis in infants.

    Prevnar 13 already comes with an adjuvant, Alum, but half the monkeys were randomized to also receive 3M-052. All were monitored at the Tulane National Primate Research Center.

    As reported in JCI Insight, the immune response was dramatic.

    At day 28, even before their second dose, the monkeys receiving 3M-052 were producing robust quantities of antibodies. In fact, their antibody levels were 10 to 100 times higher than those with Prevnar 13 alone—high enough to ensure protection against infection. They also showed dramatically enhanced production of CD4+ T cells and B cells specific to Streptococcus pneumoniae.

    “The protective antibody response we saw was so strong that it’s conceivable that you could get protection with one shot,” said Levy. “This is critical because in many parts of the world, birth is the most reliable point of healthcare contact. After birth, it becomes challenging to bring children in for repeated clinic visits.”

    The 3M-052 adjuvant was chemically modified to minimize side effects. An added lipid “tail,” which doesn’t mix well with water, keeps the adjuvant from getting into the bloodstream, where it could cause side effects.

    “Rather than floating all over the place causing fever and chills, it stays put in the muscle and enhances the immune response to the vaccine,” said Levy.

    Alternate approach

    The second study, published in the Journal of Allergy and Clinical Immunology, was led by Dowling and Evan Scott of Northwestern University. It tested a different adjuvant, CL075, and a different type of formulation.

    This time, vaccine and adjuvant were encapsulated in nanoparticles designed to maximize the immune response while avoiding side effects. Specifically, the particles were engineered to be taken up by antigen-presenting cells, which instruct lymphocytes to make antibodies.

    When the team added the particles to human cells in a dish or injected them into mice expressing the human TLR8 gene, immune responses were as good as or better than those induced by the BCG vaccine (one of the few vaccines that works in newborns).

    Next steps

    The team now plans to develop highly stable vaccine formulations, obtain more safety data and further characterize responses in newborns versus older infants.

    Levy intends to work with collaborators from around the world, via the Precision Vaccines Program, to work towards launching human trials.

    “There’s not a long list of vaccines that can be given at birth, and we need better vaccine formulations against a range of early-life infectious pathogens,” said Levy. “We hope to meet these challenges.”

    Jeffrey Hubbell of École Polytechnique Fédérale de Lausanne in Switzerland, now at the University of Chicago, was co-senior author on the second paper with Levy.

    The research was funded by the Bill & Melinda Gates Foundation, the National Institutes of Health/NIAID, the European Research Council and Boston Children’s Hospital. The Levy Laboratory also received sponsored research support from 3M Drug Delivery Systems.

    See the full article here .

    Please help promote STEM in your local schools.

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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 10:15 am on March 20, 2017 Permalink | Reply
    Tags: Angelman’s syndrome, , , Cerebellin 1 (CBLN1), Chemogenetics, Circuit Breaker, HMS- Harvard Medical School, Isodicentric chromosome 15q, , The gene UBE3A   

    From HMS: “Circuit Breaker” Autism Studies 

    Harvard University

    Harvard University

    Harvard Medical School

    Harvard Medical School

    March 16, 2017
    JACQUELINE MITCHELL

    1
    ktsimage/Getty Images

    Harvard Medical School researchers at Beth Israel Deaconess Medical Center have gained new insight into the genetic and neuronal circuit mechanisms that may contribute to impaired sociability in some forms of autism spectrum disorder.

    Led by Matthew Anderson, HMS associate professor of pathology and director of neuropathology at Beth Israel Deaconess, the scientists determined how a gene linked to one common form of autism works in a specific population of brain cells to impair sociability.

    The research, published today in the journal Nature, reveals the neurobiological control of sociability and could represent important first steps toward interventions for patients with autism.

    Anderson and colleagues focused on the gene UBE3A, multiple copies of which cause a form of autism in humans (called isodicentric chromosome 15q). Conversely, the lack of this same gene in humans leads to a developmental disorder called Angelman’s syndrome, characterized by increased sociability.

    In previous work, Anderson’s team demonstrated that mice engineered with extra copies of the UBE3A gene show impaired sociability, as well as heightened repetitive self grooming and reduced vocalizations with other mice.

    “In this study, we wanted to determine where in the brain this social behavior deficit arises and where and how increases of the UBE3A gene repress it,” said Anderson, who is also director of the Autism BrainNET, Boston Node.

    “We had tools in hand that we built ourselves. We not only introduced the gene into specific brain regions of the mouse, but we could also direct it to specific cell types to test which ones played a role in regulating sociability,” Anderson said.

    When Anderson and colleagues compared the brains of the mice engineered to model autism to those of normal—or wild type—mice, they observed that the increased UBE3A gene copies interacted with nearly 600 other genes.

    After analyzing and comparing protein interactions between the UBE3A regulated gene and genes altered in human autism, the researchers noticed that increased doses of UBE3A repressed Cerebellin genes.

    Cerebellin is a family of genes that physically interact with other autism genes to form glutamatergic synapses, the junctions where neurons communicate with each other via the neurotransmitter glutamate.

    The researchers chose to focus on one of them, Cerebellin 1 (CBLN1), as the potential mediator of UBE3A’s effects. When they deleted CBLN1 in glutamate neurons, they recreated the same impaired sociability produced by increased UBE3A.

    “Selecting Cerebellin 1 out of hundreds of other potential targets was something of a leap of faith,” Anderson said. “When we deleted the gene and were able to reconstitute the social deficits, that was the moment we realized we’d hit the right target. Cerebellin 1 was the gene repressed by UBE3A that seemed to mediate its effects,” he said.

    In another series of experiments, Anderson and colleagues demonstrated an even more definitive link between UBE3A and CBLN1. Seizures are a common symptom among people with autism including this genetic form.

    Seizures themselves, when sufficiently severe, also impaired sociability.

    Anderson’s team suspected this seizure-induced impairment of sociability was the result of repressing the Cerebellin genes. Indeed, the researchers found that deleting UBE3A, upstream from Cerebellin genes, prevented the seizure-induced social impairments and blocked seizures ability to repress CBLN1.

    “If you take away UBE3A, seizures can’t repress sociability or Cerebellin,” said Anderson. “The flip side is, if you have just a little extra UBE3A—as a subset of people with autism do—and you combine that with less severe seizures, you can get a full-blown loss of social interactions.”

    The researchers next conducted a variety of brain-mapping experiments to locate where in the brain these crucial seizure-gene interactions take place.

    “We mapped this seat of sociability to a surprising location,“ Anderson explained. Most scientists would have thought they take place in the cortex—the area of the brain where sensory processing and motor commands take place—but, in fact, these interactions take place in the brain stem, in the reward system.”

    Then the researchers used their engineered mouse model to confirm the precise location as the ventral tegmental area, part of the midbrain that plays a role in the reward system and addiction.

    Anderson and colleagues used chemogenetics—an approach that makes use of modified receptors introduced into neurons that respond to drugs but not to naturally occurring neurotransmitters—to switch this specific group of neurons on or off.

    Turning these neurons on could magnify sociability and rescue seizure and UBE3A-induced sociability deficits.

    “We were able to abolish sociability by inhibiting these neurons, and we could magnify and prolong sociability by turning them on,” said Anderson. “So we have a toggle switch for sociability. It has a therapeutic flavor; someday, we might be able to translate this into a treatment that will helps patients.”

    The researchers thank Oriana DiStefano, Greg Salimando and Rebecca Broadhurst for colony work and the HMS Neurobiology Imaging Facility (NINDS P30 Core Center Grant #NS07203).

    This work was supported an American Academy of Neurology Research Training Fellowship, the National Institutes of Health (grants 1R25NS070682, 1R01NS08916, 1R21MH100868 and 1R21HD079249), the Nancy Lurie Marks Family Foundation, the Landreth Family Foundation, the Simons Foundation, Autism Speaks/National Alliance for Autism Research and the Klarman Family Foundation.

    See the full article here .

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