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— Tissue banks are biorepositories for the preservation of human and animal tissues used for disease diagnosis, biodiversity studies, and research. These banks help in the storage of various types of tissue samples such as skin, bone, cornea, heart valves, umbilical cord, and human soft tissues. Tissue banks vary significantly in size, with tissue banks in medical institutions storing a moderate number of samples while national tissue banks store a large number of samples. These samples are used to support research, particularly studies in genetic disorders, personalized medicine, and stem cell research for treating genetic conditions and maintaining and updating age demographic databases. Publisher's analysts forecast the global tissue banking market for emerging applications to grow at a CAGR of 5.24% during the period 2016-2020. Covered in this report The report covers the present scenario and the growth prospects of the global tissue banking market for emerging applications for 2016-2020. To calculate the market size, we consider revenue generated from the sales of products used for: Therapeutics, medical research, and cosmetics. The market is divided into the following segments based on geography: -EMEA -Americas -APAC Publisher's report, Global Tissue Banking Market 2016-2020, has been prepared based on an in-depth market analysis with inputs from industry experts. The report covers the market landscape and its growth prospects over the coming years. The report also includes a discussion of the key vendors operating in this market. Key vendors -Beckman Coulter -BioCision -Tecan Group -Thermo Fisher Scientific Other prominent vendors -BioKryo -Brooks Automation -Chernobyl Tissue Bank -Cureline -Eppendorf -IMA Pharma -LifeLink Tissue Bank -Tata Memorial Hospital Tissue Bank -The PXE International Blood and Tissue Bank -Tissue Banks International -TuBaFrost Group -Wisconsin Tissue Bank Get Sample of the Report at: http://www.reportsweb.com/inquiry&RW0001332779/sample . Table of Contents PART 01: Executive summary PART 02: Scope of the report PART 03: Market research methodology PART 04: Introduction PART 05: Market landscape PART 06: Market segmentation by application PART 07: Market segmentation by product PART 08: Market segmentation by tissue PART 09: Market segmentation by end-user PART 10: Geographical segmentation PART 11: Market drivers PART 12: Impact of drivers PART 13: Market challenges PART 14: Impact of drivers and challenges PART 15: Market trends PART 16: Vendor landscape PART 17: Major vendors PART 18: Appendix PART 19: Explore Publisher For more information, please visit http://www.reportsweb.com/Global-Tissue-Banking-Market-2016-2020


News Article | October 26, 2016
Site: www.eurekalert.org

Personalized therapies can potentially improve the outcomes of patients with lung cancer, but how to best design such an approach is not always clear. A team of scientists from Baylor College of Medicine and the University of Texas MD Anderson Cancer Center analyzed vast amounts of molecular data from a set of more than 1,000 non-small cell lung cancers that allowed them to break down the cancers into distinct subtypes, each with its own molecular profile and potentially different response to therapy. The results appear in Oncogene. "People may think of lung cancer as one disease, but lung cancer is a collection of diverse subtypes of cells and each subtype may respond differently to the same therapy," said senior author Dr. Chad Creighton, associate professor of medicine and member of the Dan L Duncan Comprehensive Cancer Center Division of Biostatistics at Baylor College of Medicine. By using molecular data - which would inform scientists, for instance, about which genes and proteins the cancer expresses and changes in its DNA- the researchers were able to tease out the different subtypes and have a more refined system of classification for non-small cell lung cancer. "Any two given lung cancers may have very different molecular profiles," said Creighton. "One would have certain genes turned on and produce certain proteins while the other cancer would have different genes turned on and express different proteins. This would imply that one cancer subtype might be more vulnerable to specific therapies while the other might be more susceptible to other therapies." The researchers found that the tumors are complex in more than one way. They are complex because of the diversity of alterations involving tumor cells, but also in the variety of non-tumor cells that are part of the tumor microenvironment. "One big part of the study was to try to tease out the cells in the tumor that actually are not cancer," said Creighton. "Those cells include lymphocytes - a type of immune cell that tries to attack the cancer. Lymphocytes showed up in the tumor profile and we found out that only specific subsets of lung cancer show this really strong pattern for lymphocyte infiltration. These cancer subtypes also show evidence for immune check point pathway, which is a pathway by which cancer cells learn to evade the immune system which normally would actively attack and kill cancer cells. Many cancers have learned some 'tricks' so they can turn on or off specific genes or proteins that allow them to evade the immune response." With these types of analyses the researchers identified specific subsets of cancer cells that they think might be more responsive to immunotherapy - a therapy to boost the body's natural defenses to fight the cancer. Immunotherapy has had a lot of success in a subset of human cancers. "The Cancer Genome Atlas, the cancer initiative that generated these data that we analyzed, involves data for over 10,000 cancers," said Creighton. "That information is now in the public domain and we are currently going through all of the data, not just for lung cancer, but for all the cancer types. We call this pan cancer analysis. We are trying to find features that not just apply to one type of cancer, but across multiple types. Much of our work in the next year is going to be focusing on pan cancer work that might point at ways of treating multiple types of cancer." Other contributors to this work include Fengju Chen, Yiqun Zhang, Edwin Parra, Jaime Rodríguez, Carmen Behrens, Rehan Akbani, Yiling Lu, Jon Kurie, Don Gibbons, Gordon Mills and Ignacio Wistuba, who are affiliated with Baylor and/or the University of Texas MD Anderson Cancer Center. Financial support for this project was provided by the National Institutes of Health (NIH) grant 2R01CA125123-09, MD Anderson's Institutional Tissue Bank Award 2P30CA016672, The University of Texas Lung Specialized Programs of Research Excellence grant P50CA70907, Cancer Prevention and Research Institute of Texas (CPRIT) grant RP120713 C2, P2 and RP150405 and Department of Defense PROSPECT grant W81XWH-07-1-0306.


News Article | March 1, 2017
Site: www.eurekalert.org

It's what's missing in the tumor genome, not what's mutated, that thwarts treatment of metastatic melanoma with immune checkpoint blockade drugs, researchers at The University of Texas MD Anderson Cancer Center report in Science Translational Medicine. Whole exome sequencing of tumor biopsies taken before, during and after treatment of 56 patients showed that outright loss of a variety of tumor-suppressing genes with influence on immune response leads to resistance of treatment with both CTLA4 and PD1 inhibitors. The team's research focuses on why these treatments help 20-30 percent of patients -- with some complete responses that last for years - but don't work for others. Their findings indicate that analyzing loss of blocks of the genome could provide a new predictive indicator. "Is there a trivial or simple (genomic) explanation? There doesn't seem to be one," said co-senior author Andrew Futreal, Ph.D., professor and chair of Genomic Medicine and co-leader of MD Anderson's Moon Shots Program™. "There's no obvious correlation between mutations in cancer genes or other genes and immune response in these patients." "There are, however, pretty strong genomic copy loss correlates of resistance to sequential checkpoint blockade that also pan out for single-agent treatment," Futreal said. Doctoral candidate Whijae Roh, co-lead author, Futreal, and co-senior author Jennifer Wargo, M.D., associate professor of Surgical Oncology and Genomic Medicine, and colleagues analyzed the genomic data for non-mutational effects. "We found a higher burden of copy number loss correlated to response to immune checkpoint blockade and to lower immune scores, a measure of immune activation in the tumor's microenvironment," said Roh, a graduate student in the University of Texas MD Anderson UTHealth Graduate School of Biomedical Sciences. "We also found copy loss has an effect that is independent of mutational load in the tumors." Melanoma tumors with larger volumes of genetic alterations, called mutational load, provide more targets for the immune system to detect and are more susceptible to checkpoint blockade, although that measure is not conclusive alone. "Combining mutational load and copy number loss could improve prediction of patient response," Wargo said. When the team stratified patients in another data set of patients by whether they had high or low copy loss or high or low mutational load, they found that 11 of 26 patients with high mutational load and low copy loss had a clinical benefit, while only 4 or 26 with low mutational load and high copy loss benefited from treatment. In the trial, patients were treated first with the immune checkpoint inhibitor ipilimumab, which blocks a brake called CTLA4 on T cells, the immune system's specialized warriors, freeing them to attack. Patients whose melanoma did not react then went on to anti-PD1 treatment (nivolumab), which blocks a second checkpoint on T cells. Biopsies were taken, when feasible, before, during and after treatment for molecular analysis to understand response and resistance. To better understand the mechanisms at work, the team analyzed tumor genomes for recurrent copy loss among 9 tumor biopsies from patients who did not respond to either drug and had high burden of copy number loss. They found repeated loss of blocks of chromosomes 6, 10 and 11, which harbor 13 known tumor-suppressing genes. Analysis of a second cohort of patients confirmed the findings, with no recurrent tumor-suppressor loss found among any of the patients who had a clinical benefit or long-term survival after treatment. Ipilimumab sometimes wins when it fails The researchers also found a hint that treatment with ipilimumab, even if it fails, might prime the patient's immune system for successful anti-PD1 treatment. The team analyzed the genetic variability of a region of the T cell receptors, a feature of T cells that allows them to identify, attack and remember an antigen target found on an abnormal cell or an invading microbe. They looked for evidence of T cell "clonality," an indicator of active T cell response. Among eight patients with longitudinal samples taken before treatment with both checkpoint types, all three who responded to anti-PD1 therapy had shown signs of T cell activation after anti-CTLA treatment. Only one of the five non-responders had similar indicators of T cell clonality. "That's evidence that anti-CTLA4 in some cases primes T cells for the next step, anti-PD1 immunotherapy. It's well known that if you don't have T cells in the tumor, anti-PD1 won't do anything, it doesn't bring T cells into the tumor," Futreal says. Overall, they found that T cell clonality predicts response to PD1 blockade but not to CTLA-4 blockade. "Developing an assay to predict response will take an integrated analysis, thinking about genomic signatures and pathways, to understand the patient when you start therapy and what happens as they begin to receive therapy," Wargo said. "Changes from pretreatment to on-therapy activity will be important as well." The Science Translational Medicine paper is the third set of findings either published or presented at scientific meetings by the team, which is led by Futreal and Wargo, who also is co-leader of the Melanoma Moon Shot™. Immune-monitoring analysis showed that presence of immune infiltrates in a tumor after anti-PD1 treatment begins is a strong predictor of success. They also presented evidence that the diversity and composition of a patient's gut bacteria also affects response to anti-PD1 therapy. The serial biopsy approach is a hallmark of the Adaptive Patient-Oriented Longitudinal Learning and Optimization™ (APOLLO) platform of the Moon Shots Program™, co-led by Futreal that systematically gathers samples and data to understand tumor response and resistance to treatment over time. The Moon Shots Program™ is designed to reduce cancer deaths by accelerating development of therapies, prevention and early detection from scientific discoveries. Futreal holds the The Robert A. Welch Distinguished University Chair in Chemistry at MD Anderson. Co-authors with Roh, Futreal and Wargo are co-first authors Pei-Ling Chen, M.D., of Genomic Medicine and Pathology, and Alexandre Reuben, Ph.D., of Surgical Oncology; also Christine Spencer, Feng Wang, Ph.D., Zachary Cooper, Ph.D., Curtis Gumbs, Latasha Little, Qing Chang, Wei-Shen Chen, M.D., and Jason Roszik, Ph.D., of Genomic Medicine; Michael Tetzlaff, Ph.D., M.D., and Victor Prieto, M.D., Ph.D., of Pathology; Peter Prieto, M.D., Vancheswaran Gopalakrishnan, Jacob L. Austin-Breneman, Hong Jiang, Ph.D., and Jeffrey Gershenwald, M.D., of Surgical Oncology; John Miller, Ph.D., Oncology Research for Biologics and Immunotherapy Translation (ORBIT); Sangeetha Reddy, M.D., Division of Cancer Medicine; Khalida Wani, Ph.D., Mariana Petaccia De Macedo, M.D., Ph.D., Eveline Chen, and Alexander Lazar, M.D., Ph.D., of Translational Molecular Pathology; Michael Davies, M.D., Ph.D., Hussein Tawbi, M.D., Ph.D., Patrick Hwu, M.D., Wen-Jen Hwu, M.D., Ph.D., Adi Diab, M.D., Isabella Glitza, M.D., Ph.D., Sapna Patel, M.D., Scott Woodman, M.D., Ph.D., and Rodabe Amaria, M.D., of Melanoma Medical Oncology; Jianhua Hu, Ph.D., of Biostatistics; Padmanee Sharma, M.D., Ph.D., and James Allison, Ph.D., of Immunology; Lynda Chin, M.D., University of Texas System; and Jianhua Zhang Ph.D., of the Institute for Applied Cancer Science. Wargo, Sharma and Allison are all members of the Parker Institute for Cancer Immunotherapy. The research was funded by MD Anderson's Melanoma Moon Shot™, the Melanoma Research Alliance Team Science Award, the John G. and Marie Stella Kenedy Memorial Foundation, the University of Texas System STARS program; the Cancer Prevention and Research Institute of Texas; the American Society of Clinical Oncology; Conquer Cancer Foundation; the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation; and grants from the National Cancer Institute of the National Institutes of Health (U54CA163125, 1K08CA160692-01A1, T32CA009599, NIH T32 CA009666, R01 CA187076-02) and MD Anderson's Institutional Tissue Bank (2P30CA016672) Spencer and Gopalakrishnan are graduate students in The University of Texas Health Science Center at Houston School of Public Health.


News Article | February 27, 2017
Site: www.rdmag.com

In movies and TV shows, dolphins are often portrayed as heroes who save humans through remarkable feats of strength and tenacity. Now dolphins could save the day for humans in real life, too - with the help of emerging technology that can measure thousands of proteins and an improved database full of genetic data. "Dolphins and humans are very, very similar creatures," said NIST's Ben Neely, a member of the Marine Biochemical Sciences Group and the lead on a new project at the Hollings Marine Laboratory, a research facility in Charleston, South Carolina that includes the National Institute of Standards and Technology (NIST) as one of its partner institutions. "As mammals, we share a number of proteins and our bodies function in many similar ways, even though we are terrestrial and dolphins live in the water all their lives." Neely and his colleagues have just finished creating a detailed, searchable index of all the proteins found in the bottlenose dolphin genome. A genome is the complete set of genetic material present in an organism. Neely's project is built on years of marine mammal research and aims to provide a new level of bioanalytical measurements. The results of this work will aid wildlife biologists, veterinary professionals and biomedical researchers. Protein Maps Could Help Dolphins and Humans Although a detailed map of the bottlenose dolphin (Tursiops truncatus) genome was first compiled in 2008, recent technological breakthroughs enabled the creation of a new, more exhaustive map of all of the proteins produced by the dolphins' DNA. Neely led the process to generate the new genome with the help of colleagues at the Hollings Marine Laboratory. For this project, the initial genomic sequencing and assembly were completed by Dovetail Genomics, a private U.S.-based company. Next, the genome was annotated by the National Center for Biotechnology Information at the National Library of Medicine (NCBI) using previously deposited data generated in large part by the National Oceanic and Atmospheric Administration's National Centers for Coastal Ocean Science Marine Genomics Core. "Once you can identify all of the proteins and know their amounts as expressed by the genome," Neely explained, "you can figure out what's going on in the bottlenose dolphin's biological systems in this really detailed manner." Neely's study is part of an emerging field called proteomics. In the case of dolphins, proteomic work has a wide variety of potential applications. The zoo and aquarium industry, which generates revenues of approximately $16 billion a year, could use it to improve the care of bottlenose dolphins. In addition, improved dolphin proteomics could improve assessments of wild dolphin populations, and provide an immense amount of data on environmental contaminants and the safety and health of the world's oceanic food web. Comparing the proteins of humans and these other mammals is already providing researchers with a wealth of new information about how the human body works. Those findings could eventually be used to develop new, more precise treatment methods for common medical problems. As marine mammals descend, they shut off the blood flow to many of their organs, which has long puzzled and intrigued biologists. In contrast, if blood stops flowing to the organs of a human's body for even a few seconds, the result can be a stroke, kidney failure, or even death. Studies have recently revealed that lesser-known proteins in the blood of marine mammals may be playing a big role in the dives by protecting bottlenose dolphins' kidneys and hearts from damage when blood flow and oxygen flow start and stop repeatedly during those underwater forays. One of these proteins is known as vanin-1. Humans produce vanin-1, but in much smaller amounts. Researchers would like to gather more information on whether or not elevating levels of vanin-1 may offer protection to kidneys. "There's this gap in the knowledge about genes and the proteins they make. We are missing a huge piece of the puzzle in how these animals do what they do," said Mike Janech from the Medical University of South Carolina. His group has been researching vanin-1 (link is external) and has identified numerous other potential biomedical applications for the dolphin genome just created by NIST. "Genes carry the information of life," Janech said. "But proteins execute the functions." Vanin-1 is just one example of how genomic information about this mammalian cousin might prove useful. There may be hundreds of other similar applications, including some related to the treatment of high blood pressure and diabetes. This represents another avenue for biomimicry, which seeks solutions to human problems by examining and imitating nature's patterns and strategies. In the past, biomimicry was solely focused on the structural aspects of animal body parts such as arms and legs or functional patterns of things like noses and sniffing. But as the study of DNA has evolved, so too has our ability to examine the things happening at the most minute levels within another mammal's body. "We are now entering what could be called the post-model-organism era," Neely said. Instead of looking only for a structure to model, imitate or learn from, scientists are looking at the complete molecular landscape of genes and proteins of these creatures for model processes, too. "With abundant genomic resources it is now possible to study non-model organisms with similar molecular machinery in order to tackle difficult biomedical problems." To gather the needed protein information, Neely and his team used a specimen provided by the National Marine Mammal Tissue Bank (link is external) (NMMTB), the longest running project of NIST's Marine Environmental Specimen Bank. Half of the approximately 4,000 marine mammal specimens in the NMMTB are collected as a part of the Marine Mammal Health and Stranding Response Program (link is external). The specimen provided for Neely's study was known to originate very close to the Hollings Marine Lab. The new, state-of-the-art genome immediately began providing new biochemical insights. Studies at NIST are ongoing to validate the updated protein maps using an ultra-high-resolution tribrid mass spectrometer, which is the most powerful tool available to identify and quantify proteins. Other Mammal Proteins Seem Promising, Too Neely said the results demonstrate the utility of re-mapping genomes with the improved bioanalytical capabilities provided by new genomic sequencing technology coupled to high-resolution mass spectrometers. The data from this project will also be available in the public domain so that the results will be easy for others to access and use for diverse applications and research. This is the first of many such projects to be undertaken by the Charleston group whereby new analytical techniques could be applied to marine animals. Studying other diving marine mammals can improve our understanding of the molecular mechanisms involved in diving. Also, sea lion proteins may have much to tell us about metastatic cancer, which especially intrigues Neely and his colleagues. As a research chemist, Neely says he has not really spent much time before now observing marine mammals as a part of his work hours. He does encounter dolphins when he goes out surfing along the Carolina coastline, though. "It's amazing to think that we are at a point where cutting-edge research in marine mammals can directly advance human biomedical discoveries," he said.


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

In movies and TV shows, dolphins are often portrayed as heroes who save humans through remarkable feats of strength and tenacity. Now dolphins could save the day for humans in real life, too - with the help of emerging technology that can measure thousands of proteins and an improved database full of genetic data. "Dolphins and humans are very, very similar creatures," said NIST's Ben Neely, a member of the Marine Biochemical Sciences Group and the lead on a new project at the Hollings Marine Laboratory, a research facility in Charleston, South Carolina that includes the National Institute of Standards and Technology (NIST) as one of its partner institutions. "As mammals, we share a number of proteins and our bodies function in many similar ways, even though we are terrestrial and dolphins live in the water all their lives." Neely and his colleagues have just finished creating a detailed, searchable index of all the proteins found in the bottlenose dolphin genome. A genome is the complete set of genetic material present in an organism. Neely's project is built on years of marine mammal research and aims to provide a new level of bioanalytical measurements. The results of this work will aid wildlife biologists, veterinary professionals and biomedical researchers. Although a detailed map of the bottlenose dolphin (Tursiops truncatus) genome was first compiled in 2008, recent technological breakthroughs enabled the creation of a new, more exhaustive map of all of the proteins produced by the dolphins' DNA. Neely led the process to generate the new genome with the help of colleagues at the Hollings Marine Laboratory. For this project, the initial genomic sequencing and assembly were completed by Dovetail Genomics, a private U.S.-based company. Next, the genome was annotated by the National Center for Biotechnology Information at the National Library of Medicine (NCBI) using previously deposited data generated in large part by the National Oceanic and Atmospheric Administration's National Centers for Coastal Ocean Science Marine Genomics Core. "Once you can identify all of the proteins and know their amounts as expressed by the genome," Neely explained, "you can figure out what's going on in the bottlenose dolphin's biological systems in this really detailed manner." Neely's study is part of an emerging field called proteomics. In the case of dolphins, proteomic work has a wide variety of potential applications. The zoo and aquarium industry, which generates revenues of approximately $16 billion a year, could use it to improve the care of bottlenose dolphins. In addition, improved dolphin proteomics could improve assessments of wild dolphin populations, and provide an immense amount of data on environmental contaminants and the safety and health of the world's oceanic food web. Comparing the proteins of humans and these other mammals is already providing researchers with a wealth of new information about how the human body works. Those findings could eventually be used to develop new, more precise treatment methods for common medical problems. As marine mammals descend, they shut off the blood flow to many of their organs, which has long puzzled and intrigued biologists. In contrast, if blood stops flowing to the organs of a human's body for even a few seconds, the result can be a stroke, kidney failure, or even death. Studies have recently revealed that lesser-known proteins in the blood of marine mammals may be playing a big role in the dives by protecting bottlenose dolphins' kidneys and hearts from damage when blood flow and oxygen flow start and stop repeatedly during those underwater forays. One of these proteins is known as vanin-1. Humans produce vanin-1, but in much smaller amounts. Researchers would like to gather more information on whether or not elevating levels of vanin-1 may offer protection to kidneys. "There's this gap in the knowledge about genes and the proteins they make. We are missing a huge piece of the puzzle in how these animals do what they do," said Mike Janech from the Medical University of South Carolina. His group has been researching vanin-1 (link is external) and has identified numerous other potential biomedical applications for the dolphin genome just created by NIST. "Genes carry the information of life," Janech said. "But proteins execute the functions." Vanin-1 is just one example of how genomic information about this mammalian cousin might prove useful. There may be hundreds of other similar applications, including some related to the treatment of high blood pressure and diabetes. This represents another avenue for biomimicry, which seeks solutions to human problems by examining and imitating nature's patterns and strategies. In the past, biomimicry was solely focused on the structural aspects of animal body parts such as arms and legs or functional patterns of things like noses and sniffing. But as the study of DNA has evolved, so too has our ability to examine the things happening at the most minute levels within another mammal's body. "We are now entering what could be called the post-model-organism era," Neely said. Instead of looking only for a structure to model, imitate or learn from, scientists are looking at the complete molecular landscape of genes and proteins of these creatures for model processes, too. "With abundant genomic resources it is now possible to study non-model organisms with similar molecular machinery in order to tackle difficult biomedical problems." To gather the needed protein information, Neely and his team used a specimen provided by the National Marine Mammal Tissue Bank (link is external) (NMMTB), the longest running project of NIST's Marine Environmental Specimen Bank. Half of the approximately 4,000 marine mammal specimens in the NMMTB are collected as a part of the Marine Mammal Health and Stranding Response Program (link is external). The specimen provided for Neely's study was known to originate very close to the Hollings Marine Lab. The new, state-of-the-art genome immediately began providing new biochemical insights. Studies at NIST are ongoing to validate the updated protein maps using an ultra-high-resolution tribrid mass spectrometer, which is the most powerful tool available to identify and quantify proteins. Neely said the results demonstrate the utility of re-mapping genomes with the improved bioanalytical capabilities provided by new genomic sequencing technology coupled to high-resolution mass spectrometers. The data from this project will also be available in the public domain so that the results will be easy for others to access and use for diverse applications and research. This is the first of many such projects to be undertaken by the Charleston group whereby new analytical techniques could be applied to marine animals. Studying other diving marine mammals can improve our understanding of the molecular mechanisms involved in diving. Also, sea lion proteins may have much to tell us about metastatic cancer, which especially intrigues Neely and his colleagues. As a research chemist, Neely says he has not really spent much time before now observing marine mammals as a part of his work hours. He does encounter dolphins when he goes out surfing along the Carolina coastline, though. "It's amazing to think that we are at a point where cutting-edge research in marine mammals can directly advance human biomedical discoveries," he said.


News Article | February 23, 2017
Site: phys.org

"Dolphins and humans are very, very similar creatures," said NIST's Ben Neely, a member of the Marine Biochemical Sciences Group and the lead on a new project at the Hollings Marine Laboratory, a research facility in Charleston, South Carolina that includes the National Institute of Standards and Technology (NIST) as one of its partner institutions. "As mammals, we share a number of proteins and our bodies function in many similar ways, even though we are terrestrial and dolphins live in the water all their lives." Neely and his colleagues have just finished creating a detailed, searchable index of all the proteins found in the bottlenose dolphin genome. A genome is the complete set of genetic material present in an organism. Neely's project is built on years of marine mammal research and aims to provide a new level of bioanalytical measurements. The results of this work will aid wildlife biologists, veterinary professionals and biomedical researchers. Protein Maps Could Help Dolphins and Humans Although a detailed map of the bottlenose dolphin (Tursiops truncatus) genome was first compiled in 2008, recent technological breakthroughs enabled the creation of a new, more exhaustive map of all of the proteins produced by the dolphins' DNA. Neely led the process to generate the new genome with the help of colleagues at the Hollings Marine Laboratory. For this project, the initial genomic sequencing and assembly were completed by Dovetail Genomics , a private U.S.-based company. Next, the genome was annotated by the National Center for Biotechnology Information at the National Library of Medicine (NCBI) using previously deposited data generated in large part by the National Oceanic and Atmospheric Administration's National Centers for Coastal Ocean Science Marine Genomics Core. "Once you can identify all of the proteins and know their amounts as expressed by the genome," Neely explained, "you can figure out what's going on in the bottlenose dolphin's biological systems in this really detailed manner." Neely's study is part of an emerging field called proteomics. In the case of dolphins, proteomic work has a wide variety of potential applications. The zoo and aquarium industry, which generates revenues of approximately $16 billion a year, could use it to improve the care of bottlenose dolphins. In addition, improved dolphin proteomics could improve assessments of wild dolphin populations, and provide an immense amount of data on environmental contaminants and the safety and health of the world's oceanic food web. Comparing the proteins of humans and these other mammals is already providing researchers with a wealth of new information about how the human body works. Those findings could eventually be used to develop new, more precise treatment methods for common medical problems. As marine mammals descend, they shut off the blood flow to many of their organs, which has long puzzled and intrigued biologists. In contrast, if blood stops flowing to the organs of a human's body for even a few seconds, the result can be a stroke, kidney failure, or even death. Studies have recently revealed that lesser-known proteins in the blood of marine mammals may be playing a big role in the dives by protecting bottlenose dolphins' kidneys and hearts from damage when blood flow and oxygen flow start and stop repeatedly during those underwater forays. One of these proteins is known as vanin-1. Humans produce vanin-1, but in much smaller amounts. Researchers would like to gather more information on whether or not elevating levels of vanin-1 may offer protection to kidneys. "There's this gap in the knowledge about genes and the proteins they make. We are missing a huge piece of the puzzle in how these animals do what they do," said Mike Janech from the Medical University of South Carolina. His group has been researching vanin-1 and has identified numerous other potential biomedical applications for the dolphin genome just created by NIST. "Genes carry the information of life," Janech said. "But proteins execute the functions." Vanin-1 is just one example of how genomic information about this mammalian cousin might prove useful. There may be hundreds of other similar applications, including some related to the treatment of high blood pressure and diabetes. This represents another avenue for biomimicry, which seeks solutions to human problems by examining and imitating nature's patterns and strategies. In the past, biomimicry was solely focused on the structural aspects of animal body parts such as arms and legs or functional patterns of things like noses and sniffing. But as the study of DNA has evolved, so too has our ability to examine the things happening at the most minute levels within another mammal's body. "We are now entering what could be called the post-model-organism era," Neely said. Instead of looking only for a structure to model, imitate or learn from, scientists are looking at the complete molecular landscape of genes and proteins of these creatures for model processes, too. "With abundant genomic resources it is now possible to study non-model organisms with similar molecular machinery in order to tackle difficult biomedical problems." To gather the needed protein information, Neely and his team used a specimen provided by the National Marine Mammal Tissue Bank (NMMTB), the longest running project of NIST's Marine Environmental Specimen Bank. Half of the approximately 4,000 marine mammal specimens in the NMMTB are collected as a part of the Marine Mammal Health and Stranding Response Program . The specimen provided for Neely's study was known to originate very close to the Hollings Marine Lab. The new, state-of-the-art genome immediately began providing new biochemical insights. Studies at NIST are ongoing to validate the updated protein maps using an ultra-high-resolution tribrid mass spectrometer, which is the most powerful tool available to identify and quantify proteins. Other Mammal Proteins Seem Promising, Too Neely said the results demonstrate the utility of re-mapping genomes with the improved bioanalytical capabilities provided by new genomic sequencing technology coupled to high-resolution mass spectrometers. The data from this project will also be available in the public domain so that the results will be easy for others to access and use for diverse applications and research. This is the first of many such projects to be undertaken by the Charleston group whereby new analytical techniques could be applied to marine animals. Studying other diving marine mammals can improve our understanding of the molecular mechanisms involved in diving. Also, sea lion proteins may have much to tell us about metastatic cancer, which especially intrigues Neely and his colleagues. As a research chemist, Neely says he has not really spent much time before now observing marine mammals as a part of his work hours. He does encounter dolphins when he goes out surfing along the Carolina coastline, though. "It's amazing to think that we are at a point where cutting-edge research in marine mammals can directly advance human biomedical discoveries," he said. Explore further: Researchers probing the beneficial secrets in dolphins' proteins


News Article | January 29, 2016
Site: phys.org

"I intentionally heat up an object a little bit, and it wants to expand because it's heating up, so it generates an outgoing pressure pulse. We measure those pressure pulses with ultrasound receivers," Patch explained. "A few years ago, we landed a grant through UWM's instrumentation award program to purchase a research-friendly ultrasound system. Now I can use a transducer just like those in hospitals to 'listen' for the signal." Patch uses that existing technology to create 3D images of prostates with the goal of crafting a diagnostic tool that spots cancer without the risks associated with biopsies. She started by imaging cancerous prostates immediately after they were removed from patients at Froedtert & the Medical College of Wisconsin. "Lots of folks at MCW have been very supportive of this project, from Dr. (William) See in urology, to the MCW Tissue Bank and MCW's Clinical & Translational Science Institute," Patch said. To create thermoacoustic images, Patch needed a way to heat prostates uniformly. In the basement of the UWM Physics building, a souped-up FM radio transmitter propagates high-power VHF (very high frequency) pulses through her bench-top imaging system. The signal is driven by electrical conductivity. Healthy prostate glands produce fluid that is about three times more conductive than blood or plasma. Unhealthy prostate glands produce less conductive fluid. Patch and collaborator Dr. David Hull compare the thermoacoustic images to the corresponding prostate samples to determine whether the images could be used for cancer diagnosis. If thermoacoustic imaging proves as effective as more costly techniques, Patch would look to image prostates still inside patients. The process would be similar to the current transrectal biopsy now used for diagnosing tumors. "We are looking for surgeons in town to help us move to the next level," she said. "To drum up funds to build a prototype, we'll need to have physicians and patients on board who will allow us to perform thermoacoustic imaging the biopsy procedure." Longer term, she hopes to image other abdominal organs, like the liver and pancreas. It's also possible that thermoacoustic imaging could do more than just detect cancer; it could be used to treat it. Proton therapy is a method of cancer treatment in which doctors direct a beam of charged particles at a tumor. Unlike regular radiation treatments, in which X-rays can affect a wide area of the body, particle beams deposit most of their energy at a certain point known as the Bragg peak, and then die away almost completely. In principle, treatment can be focused on the tumor, leaving the healthy tissue beyond the Bragg peak untouched. Positioning errors, however, result in treating healthy tissue and under-treating the tumor. Patch and scientists working on the Lawrence Berkeley National Lab's (LBNL) 88-inch cyclotron worked together to detect thermoacoustic emissions from the Bragg peak. Patch tested her newest ultrasound equipment at Berkeley last summer with an upgraded cyclotron that accelerates protons to approximately one-third the speed of light. "LBNL donated a day's worth of time of on the cyclotron. We did some experiments and it worked better than I thought it would," Patch said. LBNL also provided staff support, including technicians who modified electronics, operators who controlled the beam and a scientist coaxed out of retirement. The experiment involved pulsing a proton beam at a "phantom," a model of human tissue used for ultrasound. The team designed a phantom with a cavity that mimicked a portion of the intestine, because gas pockets wreak havoc with treatment plans. Scientists George Noid and Allen Li at MCW took CT scans of the phantom, which Patch used to estimate the Bragg peak when the cavity was empty and when it was filled with olive oil. "The beam could penetrate 2 centimeters in the oil. But when the cavity is empty, the beam flies right through and doesn't slow down until it enters phantom material. With my ultrasound transducers, we can see that difference pretty accurately," Patch said. Her results are preliminary, but Patch thinks that if the team continues to see positive results, thermoacoustics could improve the accuracy of proton therapy.


News Article | January 6, 2016
Site: www.nature.com

No statistical methods were used to predetermine sample size. Clinical samples GBM1w, GBM2w, GBM3w, GBM4w, GBM5w, GBM6w, GBM7w, AA15 m, AA16 m, AA17 m, OD18 m and AA19 m were obtained as frozen specimens from the Massachusetts General Hospital Pathology Tissue Bank, or received directly after surgical resection and flash frozen (Extended Data Table 1). All samples were acquired with Institutional Review Board approval, and were de-identified before receipt. GBM1w was obtained at autopsy; the remaining samples were surgical resections. IDH status was determined for all clinical samples by SNaPshot multiplex PCR31. PDGFRA status was confirmed by FISH analysis. Tissue (200–500 μg) was mechanically minced with a sterile razor blade before further processing. Gliomaspheres were maintained in culture as described32, 33. In brief, neurosphere cultures contain Neurobasal media supplemented with 20 ng ml−1 recombinant EGF (R and D Systems), 20 ng ml−1 FGF2 (R and D Systems), 1× B27 supplement (Invitrogen), 0.5× N2 supplement (Invitrogen), 3 mM L-glutamine, and penicillin/streptomycin. Cultures were confirmed to be mycoplasma-free via PCR methods. GSC4 and GSC6 gliomasphere lines were derived from IDH wild-type tumours resected at Massachusetts General Hospital, and have been previously described and characterized32, 33, 34. BT142 gliomasphere line (IDH1 mutant)35 was obtained from ATCC, and cultured as described above except 25% conditioned media was carried over each passage. BT142 G-CIMP status was confirmed by evaluating LINE methylation with the Global DNA Methylation Assay – LINE-1 kit (Active Motif), as described36, and by methylation-sensitive restriction digests. GSC119 was derived from an IDH1 mutant tumour (confirmed by SNaPshot) resected at Massachusetts General Hospital. We confirmed IDH1 mutant status of GSC119 by RNA-seq (82 out of 148 reads overlapping the relevant position in the transcript correspond the mutant allele). The gliomasphere models were derived from tumours of the following types: GSC4 and GSC6: primary glioblastoma; BT142: grade III oligoastrocytoma; GSC119: secondary glioblastoma, G-CIMP. Clinical specimens and models used in this study are detailed in Extended Data Table 1. ChIP-seq was performed as described previously32. In brief, cultured cells or minced tissue was fixed in 1% formaldehyde and snap frozen in liquid nitrogen and stored at −80 °C at least overnight. Sonication of tumour specimens and gliomaspheres was calibrated such that DNA was sheared to between 400 and 2,000 bp. CTCF was immunoprecipitated with a monoclonal rabbit CTCF antibody, clone D31H2 (Cell Signaling 3418). H3K27ac was immunoprecipitated with an antibody from Active Motif (39133). ChIP DNA was used to generate sequencing libraries by end repair (End-It DNA repair kit, Epicentre), 3′ A base overhang addition via Klenow fragment (NEB), and ligation of barcoded sequencing adapters. Barcoded fragments were amplified via PCR. Libraries were sequenced as 38-base paired-end reads on an Illumina NextSeq500 instrument or as 50-base single-end reads on a MiSeq instrument. Sequencing libraries are detailed in Extended Data Table 2. H3K27ac maps for GSC6 were previously deposited to the GEO under accession GSM1306340. Genomic data has been deposited into GEO as GSE70991. For sequence analysis, identical reads were collapsed to a single paired-end read to avoid PCR duplicates. To avoid possible saturation, reads were downsampled to 5% reads collapsed as PCR duplicates, or 5 million fragments. Reads were aligned to hg19 using BWA, and peaks were called using HOMER. ChIP-seq tracks were visualized using Integrative Genomics Viewer (IGV, http://www.broadinstitute.org/igv/). To detect peaks lost in IDH mutants, we called signal over all peaks in a 100-bp window centred on the peaks. To control for copy number changes, we first called copy number profiles from input sequencing data using CNVnator37. We then removed all regions where at least one sample had a strong deletion (<0.25), and normalized by copy number. To account for batch effects and difference in ChIP efficiency, we quantile normalized each data set. Peaks were scored as lost or gained if the difference in signal between a given tumour and the average of the five wild-type tumours was at least twofold lower or higher, with a signal of at least 1 in all wild-type or IDH mutant tumours. Fisher exact test confirmed that the overlap between peaks lost in the IDH mutant tumours is highly significant (P < 10−100). GC content over CTCF peaks lost (or retained) in the IDH mutant glioma specimens was averaged over 200-bp windows centred on each peak lost in IDH mutant tumours. Methylation levels were quantified over these same regions for 3 IDH mutant and 3 IDH wild-type tumours, using TCGA data generated by whole genome bisulfite sequencing10. In brief, methylation levels (percentage) based on proportion of reads with protected CpG were averaged over all CpG di-nucleotides in these regions, treating each tumour separately. Occupancy of the CTCF site in the boundary element adjacent to the PDGFRA locus was quantified by ChIP qPCR, using the following primers: PDGFRActcfF: 5′-GTCACAGTAGAACCACAGAT-3′; PDGFRActcfR: 5′-TAAGTATACTGGTCCTCCTC-3′. Equal masses of ChIP or input (WCE) DNA were used as input for PCR, and CTCF occupancy was quantified as a ratio between ChIP and WCE, determined by 2−ΔCt. CTCF peak intensity was further normalized as ratio to two invariant peaks, at PSMB1 and SPG11, using the following primers: PSMB1ctcfF: 5′-CCTTCCTAGTCACTCAGTAA-3′; PSMB1ctcfR: 5′-CAGTGTTGACTCATCCAG-3′; SPG11ctcfF: 5′-CAGTACCAGCCTCTCTAG-3′; SPG11ctcfR: 5′-CTAAGCTAGGCCTTCAAG-3′. RNA-seq data for 357 normal brain samples was downloaded from GTEx20. RNA-seq data and copy number profiles for lower grade gliomas were downloaded from TCGA23, 24. Contact domains of IMR90, GM12878, K562 and NHEK cells were obtained from published HiC data15. Genes were assigned to the inner-most domain in which their transcription start site fell within. Gene pairs were considered to be in the same domain if they were assigned to the same domain in both GM12878 and IMR90. Gene pairs were considered to span a boundary if they were assigned to different domains in both GM12878 and IMR90, and separated by a CTCF-binding site in IDH wild-type tumours. Gene pairs that did not fit either criterion were excluded from this analysis. The plot of correlation vs distance for brain GTEx samples is based on Pearson correlations for all relevant pairs, smoothed by locally weighted scatterplot smoothing with weighted linear least squares (LOESS). To assess the bias in correlation differences, we computed the difference of Pearson correlations between wild-type and IDH mutant gliomas for all gene pairs separated by <180 kb. In Fig. 1e, this difference in correlations is plotted against the significance of this difference (estimated by Fisher z-transformation). For each gene pair, we omitted samples with a deletion or amplification of one of the genes at or above threshold of the minimal arm level deletion or amplification (to avoid copy number bias). To ensure robustness, we also repeated the analysis using boundaries defined from HiC data for K562 and NHEK. This yielded similar results: 84% pairs gaining correlation cross boundary versus 71% expected (P < 8 × 10−3), 54% pairs losing correlation are within the same domain versus 29% expected (P < 3 × 10−8). Repeating the analysis with only the 14,055 genes that have expressed over 1 transcripts per million (TPM) in at least half the samples also yielded similar results (Extended Data Fig. 7): 92% pairs gaining correlation cross boundary versus 69% expected (P < 2 × 10−3), 73% pairs losing correlation are within the same domain versus 31% expected (P < 8 × 10−4). To detect boundaries deregulated in IDH mutant gliomas, we scanned for gene pairs, separated by <1 Mb, with a significant difference in correlation between wild-type and IDH mutant tumours (Fisher z-transformation, FDR <1%). We omitted amplified or deleted samples as described above. To ensure robustness to noise from lowly expressed genes, we first filtered out 6,476 genes expressed <1 TPM in more than half of the samples (keeping 14,055 genes). We considered all domains and boundaries scored in IMR90 HiC data13. Gene pairs crossing a CTCF peak and an IMR90 boundary (that is, can be assigned to different domains) that were significantly more correlated in IDH mutant tumours were considered to support the loss of that boundary. Gene pairs not crossing a boundary (that is, can be assigned to the same domain) that were significantly less correlated in IDH mutant tumours were considered to support the loss of a flanking boundary. We collated a set of deregulated boundaries, supported by at least one cross-boundary pair gaining correlation and at least one intra-domain pair losing correlation. Each was assigned a P value equal to the product of both supporting pairs (best P value was chosen if there were more supporting pairs). If both boundaries of a domain were deregulated, or if the same pair of gene pairs (one losing and one gaining correlations) were supporting more than one boundary due to overlapping domains, the entries were merged (Supplementary Table 1). This definition allows every gene pair to be considered as potential support for a boundary loss. To quantify CTCF occupancy over these deregulated boundaries, we averaged the signal over all CTCF peaks located within a 1-kb window around the boundary, using copy number and quantile normalized CTCF signals. To quantify DNA methylation over the deregulated boundaries, we averaged DNA methylation signals from TCGA data in 200-bp windows as above. Figure 2a depicts significance of disrupted domains and the fold change of genes in them that are upregulated in IDH mutant tumours (compared to median expression in wild type). In addition to PDGFRA, top-ranking genes include CHD4 (P < 10−32), a driver of glioblastoma tumour initiation38, L1CAM (P < 10−8), a regulator of the glioma stem cells and tumour growth39, and other candidate regulators (Supplementary Table 1). To ensure robustness to cell-type-specific boundaries, we repeated the analysis with GM12878-, K562- and NHEK-defined boundaries. This yielded very similar results, and again highlighted PDGFRA as an overexpressed gene adjacent to a disrupted boundary. For the correlation of FIP1L1 and PDGFRA expression, RNA-seq data from the TCGA lower grade glioma (LGG) and glioblastoma (GBM) data sets2, 24 were downloaded and segregated by IDH mutation status and subtype. Patients from the proneural subtype were divided by IDH mutation status, while patients from the mesenchymal, classical or neural subtypes (which had no IDH mutations) were classified as ‘other’. For correlation analysis, patients with copy number variation in either gene were excluded from the analysis to control for effects of co-amplification. For outcome analysis, LGG RNA-seq data and corresponding patient survival data was obtained from TCGA. Patients with sum PDGFRA and FIP1L1 expression of at least one-half of one standard deviation above the mean were classified as ‘high PDGFRA and FIP1L1 expression’ (n = 17), while all other patients were classified as ‘low PDGFRA and FIP1L1 expression’ (n = 201). Data were plotted as Kaplan–Meier curves and statistically analysed via log–rank test. HiC data15 were downloaded from GEO. 5-kb resolution intra-chromosomal contact scores for chromosome 4 for the cell lines IMR90, NHEK, KBM7, K562, HUVEC, HMEC and GM12878 were filtered to the region between 53,700 and 55,400 kb. The average interaction score at each coordinate pair for all cell lines was calculated and used to determine putative insulator elements as local maxima at the interaction point of two domain boundaries. To determine the interactions of the PDGFRA promoter, the interaction scores of all points in the region with the PDGFRA promoter (chr4: 55,090,000) were plotted as a one-dimensional trace. To view the topological domain structure of the region, HiC interaction scores were visualized using Juicebox (http://www.aidenlab.org/juicebox/)15. Data shown is from the IMR90 cell line at 5-kb resolution, normalized to coverage. DNA methylation was analysed in two ways. For gliomaspheres, genomic DNA was isolated via QiaAmp DNA minikit (Qiagen) and subjected to bisulfite conversion (EZ DNA Methylation Gold Kit, Zymo Research). Bisulfite-converted DNA specific to the CTCF-binding site (defined by JASPAR40) in the boundary adjacent to PDGFRA was amplified using the following primers forward: 5′-GAATTATAGATAATGTAGTTAGATGG-3′, reverse: 5′-AAATATACTAATCCTCCTCTCCCAAA-3′. Amplified DNA was used to prepare a sequencing library, which was sequenced as 38-base paired-end reads on a NextSeq500. For tumours, limiting DNA yields required an alternative strategy for methylation analysis. Tumour genomic DNA was isolated from minced frozen sections of tumours by QiaAmp DNA minikit (Qiagen). Genomic DNA was digested using the methylation-sensitive restriction enzyme Hin6I (Thermo) recognizing the restriction site GCGC, or subjected to mock digestion. Protected DNA was quantified by PCR using the following primer set: PDGFRAinsF: 5′-CGTGAGCTGAATTGTGCCTG-3′, PDGFRAinsR: 5′-TGGGAGGACAGTTTAGGGCT-3′, normalizing to mock digestion. 3C analysis was performed using procedures as described previously41, 42. In brief, ~10 million cell equivalents from minced tumour specimens or gliomasphere cultures were fixed in 1% formaldehyde. Fixed samples were lysed in lysis buffer containing 0.2% PMSF using a Dounce pestle. Following lysis, samples were digested with HinDIII (NEB) overnight on a thermomixer at 37 °C rotating at 950 r.p.m. Diluted samples were ligated using T4 DNA ligase (NEB) at 16 °C overnight, followed by RNase and proteinase K treatment. DNA was extracted via phenol/chloroform/isoamyl alcohol (Invitrogen). DNA was analysed via TaqMan PCR using ABI master mix. Primers and probe were synthesized by IDT with the following sequences: common PDGFRA promoter: 5′-GGTCGTGCCTTTGTTTT-3′; FIP1L1 control: 5′-CAGGGAAGAGAGGAAGTTT-3′; FIP1L1 enhancer: 5′-TTAAGTAAGCAGGTAAACTACAT-3′; intragenic enhancer: 5′-AGCCTTTGCCTCCTTTT-3′; intragenic control: 5′-CCACAGGGAGAAGGAAAT-3′; intact promoter: 5′-CAAGGAATTCGTAGGGTTC-3′; probe: 5′-/56-FAM/TTGTATGCG/ZEN/AGATAGAAGCCAGGGCAA/3IABkFQ/-3′. For the reciprocal FIP1L1 enhancer interaction interrogation, the following primer sequences were used: common enhancer primer (as FIP1L1 enhancer primer above): 5′-TTAAGTAAGCAGGTAAACTACAT-3′, PDGFRA promoter (as common PDGFRA promoter above): 5′-GGTCGTGCCTTTGTTTT-3′; SCFD2 promoter: 5′-AATACATGGTCATGATGCTC-3′; FIP1L1 promoter: 5′-AGGCATTGCTTAAACATAAC-3′; FIP1L1 control: 5′-TTATTTGTAGTAGAGGTTACTGG-3′; PDGFRA control: 5′-ATGATAACACCACCATTCAG-3′; FIP1L1 enhancer probe: 5′-/56-FAM/TATCCCAAC/ZEN/CAAATACAGGGCTTGG/3IABkFQ/-3′. To normalize primer signals, bacterial artificial chromosome (BAC) clones CTD-2022B5 and RP11-626H4 were obtained from Invitrogen. BAC DNA was purified via BACMAX DNA Purification kit (Epicentre) and quantified using two primer sets specific to the Chloramphenicol resistance gene: 1F: 5′-TTCGTCTCAGCCAATCCCTG-3′; 1R: 5′-TTTGCCCATGGTGAAAACGG-3′; 2F: GGTTCATCATGCCGTTTGTG-3′; 2R: 5′-CCACTCATCGCAGTACTGTTG-3′. BAC DNA was subjected to a similar 3C protocol, omitting steps related to cell lysis, proteinase or RNase treatment. PCR signal from tumour and gliomasphere 3C was normalized to digestion efficiency and BAC primer signal. BT142 cells were cultured in either 5 μM 5-azacytidine or equivalent DMSO (1:10,000) for 8 days, with drug refreshed every 2 days. The following CRISPR sgRNAs were cloned into the LentiCRISPR vector obtained from the Zhang laboratory43: GFP: 5′-GAGCTGGACGGCGACGTAAA-3′; insulator: 5′-GCCACAGATAATGCAGCTAGA-3′. GSC6 gliomaspheres were mechanically dissociated and plated in 5 μg ml−1 EHS laminin (Sigma) and allowed to adhere overnight, and then infected with lentivirus containing either CRISPR vector for 48 h. Cells were then selected in 1 μg ml−1 puromycin for 4 days, with puromycin-containing media refreshed every 2 days. Genomic DNA was isolated and the region of interest was amplified using the PDGFRAins primer set described above. CRISPR-mediated disruption of this amplified DNA was confirmed via Surveyor Assay (Transgenomic), with amplified uninfected GSC6 genomic DNA being added to each annealing reaction as the unmodified control. To quantify the precise CRISPR alterations, genomic DNA from each construct was amplified using a set of primers closer to the putative deletion site as follows: forward: 5′-TTTGCAATGGGACACGGAGA-3′, reverse: 5′-AGAAATGTGTGGATGTGAGCG-3′. PCR product from these primers was used to prepare a library that was sequenced as 38-base paired-end reads on the Illumina NextSeq500. Total RNA was isolated from CRISPR-infected GSC6 gliomaspheres (insulator or control GFP sgRNA) or BT142 gliomaspheres (5-aza-treated or control condition) using the RNeasy minikit (Qiagen) and used to synthesize cDNA with the SuperScriptIII system (Invitrogen). cDNA was analysed using SYBR mastermix (Applied Biosystems) on a 7500 Fast Real Time System (Applied Biosystems). PDGFRA expression was determined using the following primers: forward: 5′-GCTCAGCCCTGTGAGAAGAC-3′, reverse: 5′-ATTGCGGAATAACATCGGAG-3′, and was normalized to primers for ribosomal protein, large, P0 (RPLP0), as follows: forward: 5′-TCCCACTTGCTGAAAAGGTCA-3′, reverse: 5′-CCGACTCTTCCTTGGCTTCA-3′. Normalization was also verified by β-actin (ACTB), forward: 5′-AGAAAATCTGGCACCACACC-3′, reverse: 5′-AGAGGCGTACAGGGATAGCA-3′. Cells were incubated with PE-conjugated anti-PDGFRa (CD140a) antibody (Biolegend, clone 16A1) for 30 min at room temperature at the dilution specified in the manufacturer’s protocol. Data was analysed and visualized with FlowJo software. Single live cells were selected for analysis via side and forward scatter, and viable cells were selected by lack of an unstained channel (APC) autofluorescence. For the cell growth assay, 2,500 dissociated viable GSC6 cells expressing CRISPR and either GFP or insulator-targeting sgRNA (see above) was plated in 100 μl of media in an opaque-walled tissue culture 96-well plate, in 1 μM dasatinib, 500 nM crenolanib, or equivalent DMSO (1:10,000) as a vehicle control. Cell growth was analysed at days 3, 5 and 7 for dasatinib, or days 3, 7 and 10 for crenolanib, using CellTiter-Glo reagent (Promega) following the manufacturer’s protocol. Data were normalized across days using an ATP standard curve.


Research and Markets has announced the addition of the "Canadian Market Report for Spinal Bone Graft Substitutes 2016 - MedCore" report to their offering. The vast majority of allograft materials utilized in spinal surgeries in Canada are supplied by large national tissue bank organizations and to a smaller extent, by provincial local tissue banks. In recent years, consolidation of tissue banks has become a growing trend as larger tissue organizations demonstrated abilities to develop more sophisticated and specialized allografts. Major allograft tissue banks in Canada include Mount Sinai Allograft Technologies and the Regional Tissue Bank. Local supply is limited to donor materials received at Canadian Tissue Banks such as Mount Sinai Hospital and the Regional Tissue Bank to a large extent. The majority of the allograft market is supplied domestically as it is simple for a Canadian tissue bank to source bone domestically and cut it up to provide cancellous or corticancellous chips or granules. Therefore, physicians do not feel it necessary to purchase proprietary allograft chips in the Canadian market and instead rely heavily on domestically sourced and distributed allograft. There is however a limited allograft bone graft market for spinal indications that is imported from U.S. sources, mainly from not for profit tissue banks like MTF. For more information about this report visit http://www.researchandmarkets.com/research/2wdvlj/canadian_market


News Article | October 6, 2016
Site: www.chromatographytechniques.com

Researchers at UC San Francisco have discovered a previously unknown mass migration of inhibitory neurons into the brain's frontal cortex during the first few months after birth, revealing a stage of brain development that had previously gone unrecognized. The authors hypothesize that this late-stage migration may play a role in establishing fundamentally human cognitive abilities and that its disruption could underlie a number of neurodevelopmental diseases. Most neurons of the cerebral cortex - the outermost layer of the brain responsible for advanced cognition - migrate outward from their birthplaces deep in the brain to take up their positions within the cortex. Developmental neuroscientists have long thought that most neural migration ends well before an infant is born, but the new paper -- published October 6, 2016 in Science -- suggests for the first time that many neurons continue to migrate and integrate into neural circuits well into infancy. "The dogma among developmental neuroscientists was that after birth all that was left was the fine wiring and pruning," said Mercedes Paredes, MD, PhD, an assistant professor of neurology at UCSF and leader of the new study. "These results suggest there's a whole new phase of human brain development that we had never noticed before." The new study was a collaboration between the labs of co-senior authors Arturo Alvarez-Buylla, PhD, a UCSF professor of neurological surgery who specializes in understanding the migration of immature neurons in the developing brain, and in whose lab Paredes is a postdoctoral researcher, and Eric J. Huang, MD, PhD, a professor of pathology and director of the Pediatric Brain Tissue Bank at the UCSF Newborn Brain Research Institute. Several recent studies - including work by Alvarez-Buylla and Huang - identified small populations of immature neurons deep in the front of the brain that migrate after birth into the orbito-frontal cortex -- a small region of the frontal cortex just above the eyes. Given that the entire frontal cortex continues to expand massively after birth, the researchers sought to discover whether neural migration continues after birth in the rest of the frontal cortex. The team examined brain tissue from the Pediatric Brain Tissue Bank using histological stains for migratory neurons. These studies revealed clusters of immature, migratory neurons widely distributed deep within the frontal lobe of the newborn brain, above the fluid-filled lateral ventricles. MRI imaging of the three-dimensional structure of these clusters revealed a long arc of migratory neurons sitting like a cap in front and on top of the ventricles and stretching from deep behind the eyebrows all the way to the top of the head. "Several labs had observed that there seemed to be many young neurons around birth along the ventricles, but no one knew what they were doing there," said Paredes. "As soon as we looked closely, we were shocked to discover how massive this population was and to find that they were still actively migrating for weeks and weeks after birth." To determine whether these immature neurons - which the researchers dubbed "the Arc" - actively migrate in the newborn brain, researchers used viruses to label immature neurons in tissue samples collected immediately after death and observed that Arc cells move inch-worm style through the brain, much as neurons migrate in the fetal brain. Further histological studies of the cingulate cortex, a portion of the brain's frontal lobe, show that Arc neurons migrate outward from the ventricles into the cortex primarily within the first three months of life, where they differentiate into multiple different subtypes of inhibitory neurons. "It is impressive that these cells can find their way to precise positions within the cortex," said Alvarez-Buylla. "Earlier in fetal development the brain is much smaller and the tissue far less complicated, but at this later stage it is quite a long and treacherous journey." Late migration of inhibitory neurons could play a role in human cognitive abilities, neurological disease Inhibitory neurons, which use the neurotransmitter GABA, make up about 20 percent of the neurons in the cerebral cortex and play a vital role in balancing the brain's need for stability with its ability to learn and change. Imbalanced excitation and inhibition -- particularly in circuits of the frontal lobe of the brain, which are involved in executive control -- have been implicated in many neurological disorders, from autism to schizophrenia. The new research suggests that inhibitory circuits in humans develop significantly later than previously realized. This postnatal migration is much larger than what is seen in mice and other mammals, the authors say, suggesting that it may be an important developmental factor behind the uniqueness of the human brain. The first months of life, when an infant first begins to interact with its environment, is a crucial time for brain development, Huang said. "The timing of this migration corresponds very well with the development of more complex cognitive functions in infants. It suggests that the arrival of these cells could play a role in setting up the basis for complex human cognition." The researchers plan to follow up their study by exploring whether this migration of inhibitory neurons from the Arc to the cortex might be affected in the brains of children with neurological disorders such as autism, which has previously been associated with abnormal inhibitory circuitry in the frontal cortex. "Trying to understand what makes human brain development so unique was what drove me to tackle this research," said Paredes, who works with patients with epilepsy in her clinical practice. "If we don't understand how our brains are built, we won't be able to understand what is going wrong when people suffer from neurological disease."

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