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News Article | May 11, 2017
Site: www.eurekalert.org

Adult stem cells have the ability to transform into many types of cells, but tracing the path individual stem cells follow as they mature and identifying the molecules that trigger these fateful decisions are difficult in a living animal. University of California, Berkeley, neuroscientists have now combined new techniques for sequencing the RNA in single cells with detailed statistical analysis to more easily track individual stem cells in the nose, uncovering clues that someday could help restore smell to those who have lost it. The results are published this week in the journal Cell Stem Cell. "A stem cell's job is twofold: to replace or recreate mature cells that are lost over time, both through normal aging and after injury, and to replace themselves so that the process can continue over the life of the animal," said senior author John Ngai, the Coates Family Professor of Neuroscience and a member of UC Berkeley's Helen Wills Neuroscience Institute and the Berkeley Stem Cell Center. "We are getting closer to understanding how mature sensory neurons are generated from olfactory stem cells, an understanding that's key for an eventual stem cell therapy to restore function." Ngai noted that perhaps one-quarter of all people over the age of 50 have some loss of smell, yet doctors have little understanding why, and no treatments for most cases. There's not even a standardized test for loss of smell, as there is for vision or hearing loss, in spite of widespread reports of suffering by patients who have lost their sense of smell. "Some cases of anosmia -- the loss of the sense of smell -- are due to traumatic injury, and there is generally not a whole lot you can do about that," he said. "But some are age-related, or occur for reasons we don't quite know. In the case of age-related anosmias, it could be because the stem cells are just not doing their job replacing the cells that are naturally lost over time. One idea is that if we could harness the very stem cells that are in the noses of people who are losing smell, maybe we can figure out a way to restore function, by getting them to regenerate the cells that are lost." Ngai, who directs the Functional Genomics Laboratory in UC Berkeley's California Institute for Quantitative Biosciences, focuses on the cells and regulatory molecules involved in our sense of smell. Olfactory cells in the nose are unusual in that they are part of the body's outer layer, or epithelium, but also part of the nervous system, incorporating neurons that connect directly with the smell centers in the brain. His group has been working with adult olfactory stem cells that give rise to the neurons that sense odors and other cells, such as sustentacular cells, that support the neurons. A new technique for sequencing the RNA in a single cell has been revolutionary, Ngai said, allowing researchers to trace which stem cells in a densely packed tissue become specialized, based on the mRNA present in the cell, which indicates which genes are being expressed. Nevertheless, it is difficult to follow stem cells that can potentially differentiate into different types of cells. Ngai's group teamed up with UC Berkeley statisticians and computer scientists - led by Sandrine Dudoit, a professor of biotstatistics and statistics, Elizabeth Purdom, a professor of statistics, and Nir Yosef, a professor of electrical engineering and computer sciences - to develop a way to analyze the experimental data and identify cells with similar RNA profiles, indicative of specific cell types and developmental states. As a result, the team was able to trace the paths that cells take as they turn into sustentacular cells -- which seems to be the default fate for olfactory stem cells -- and into neurons and other types of cells. They also were able to identify a signaling pathway known as "Wnt" that triggers the olfactory stem cell to become a sensory neuron. Ngai cautions that the immediate implications of the work are limited to animal models, which provide the necessary foundation for eventually addressing human anosmias. "But with this information, we now have a window into what controls the process and therefore a window into manipulating or coopting that process to stimulate regeneration" he said. "There has been a lot of work on Wnt signaling pathways, for example, so there are a lot of small-molecule drugs that could be tested to trigger a stem cell to mature into a neuron." The sequencing and statistical techniques the team developed can also be used by others studying regulation of stem cells in other tissues, organ systems or organisms, he said. The work was spearheaded by senior postdoctoral researcher Russell Fletcher and graduate student Diya Das, and was funded by the National Institute on Deafness and Other Communications Disorders, the National Institute of Mental health, the National Institute on Aging, National Human Genome Research Institute, the National Center for Research Resources, the National Institute of General Medical Sciences, the California Institute of Regenerative Medicine and the Berkeley Siebel Stem Cell Center. Other co-authors include Levi Gadye, Kelly Street, Ariane Baudhuin, Allon Wagner, Michael Cole, Quetzal Flores, Yoon Gi Choi and Davide Risso.


"Since opening its doors a year ago, JLABS @ Toronto has successfully attracted a multitude of promising companies from our province's life sciences community, led by academic hospitals, world-class research institutes, top scientists and a strong health start-up network," said the Honourable Reza Moridi, Minister of Research, Innovation and Science. "They have helped our province continue to build up Ontario's vibrant innovation ecosystem, create good jobs, and strengthen our position in the global knowledge economy while also providing access to incredible resources for our life science entrepreneurs." Johnson & Johnson Innovation seeks to fuel the best science and technology, no matter where it is located, to solve the greatest unmet medical and healthcare needs of our time. In addition to offering emerging life science companies modular lab units, office space, shared core laboratory equipment, business facilities, third-party services and educational events, JLABS links the entrepreneurs of Toronto with the full breadth of Johnson & Johnson Innovation, including opportunities for funding, access to research and development experts from medical technology, consumer healthcare product and the pharmaceutical teams at Janssen Inc. JLABS @ Toronto provides access to scientific, industry and capital funding experts from across the industry, and operates under the JLABS no-strings attached model, which means resident companies can completely retain their intellectual property and there are no first rights of refusal. "Our goal is to support early stage innovators with the resources and network needed to grow, and as evident by the 40 companies that reside within JLABS @ Toronto, we are already accomplishing what we set out to do in just one year of operation," said Melinda Richter, head of JLABS, Johnson & Johnson Innovation. "The no-strings attached model has been very important to our success in attracting so many quality companies, as it allows entrepreneurs the freedom to operate and do what is best for their company. We are hopeful that providing JLABS to the life sciences ecosystem in Toronto will support continued economic growth and development in the region." Since opening last year, JLABS @ Toronto has helped boost the Canadian life sciences ecosystem by hosting more than 5,300 attendees at over 50 events. More than 90 experts from a multitude of industries have spoken at these events in the last year. JLABS @ Toronto resident companies are receiving access to expertise from across the Johnson & Johnson Family of Companies. "Through their world-class incubator, Johnson & Johnson Innovation is providing much needed infrastructure and access to funding sources for early-stage innovators to help drive their ideas forward," said the Honourable Brad Duguid, Minister of Economic Development & Growth. "Ontario welcomes JLABS @ Toronto's innovative and flexible platform, providing a total of 40 companies with access to incredible business resources while allowing researchers to keep the freedom and flexibility they need to be successful." JLABS @ Toronto has facilitated over 150 meetings of startups with visiting funding experts from throughout North America. Through a unique collaboration, JLABS @ Toronto companies will be considered for funding and mentoring from Spectrum 28, a Silicon Valley venture capital firm. The following are the new companies* accepted into JLABS @ Toronto since May 11th, 2016: Following the leadership of the government of Ontario's investment in JLABS @ Toronto, the Johnson & Johnson Alberta Health Innovation Partnership (JAHIP), a collaboration that includes Alberta Economic Development and Trade, Janssen Inc. and the University Hospital Foundation in Edmonton recently opened the doors to JLABS POD @ Alberta, a secure video conference system at the University of Alberta. This is the first- of- its- kind in Canada and will provide an access point to the Johnson & Johnson Innovation family, offering Alberta health researchers and entrepreneurs access to: the Johnson & Johnson Innovation broader network of therapeutic area experts in pharmaceutical, medical devices and consumer and digital health. "Since its launch just one year ago, JLABS @ Toronto has attracted some of the region's brightest and freshest talent," said Chris Halyk, President, Janssen Inc. "The JLABS model in Ontario and now the JLABS POD in Alberta, enables us to boost the Canadian innovation and life sciences ecosystem and provide meaningful resources to help drive exciting new science forward. Janssen is proud to be part of such a strong collaboration of partners that helped make JLABS @ Toronto and JLABS POD @ Alberta a reality." JLABS facilities have incubated more than 207 companies to date and are currently home to over 150 companies advancing biotech, pharmaceutical, medical device, consumer and digital health programs. A total of 35 collaborations have been formed between companies residing at JLABS and the Johnson & Johnson Family of Companies. JLABS @ Toronto is located at MaRS Discovery District and is a collaboration between Johnson & Johnson Innovation LLC, the University of Toronto, MaRS, Janssen Inc., MaRS Innovation and the Government of Ontario. It is also supported by the following hospital partners: Centre for Addiction and Mental Health, the Hospital for Sick Children, Sinai Health System, St. Michael's Hospital, Sunnybrook Health Sciences Centre and University Health Network. All JLABS locations are accepting applications from biotech, pharmaceutical, medical device, consumer and digital health companies. To apply, visit www.jnjinnovation.com/jlabs. About Johnson & Johnson Innovation Johnson & Johnson Innovation LLC focuses on accelerating all stages of innovation worldwide and forming collaborations between entrepreneurs and Johnson & Johnson's global healthcare businesses. Johnson & Johnson Innovation provides scientists, entrepreneurs and emerging companies with one-stop access to science and technology experts who can facilitate collaborations across the pharmaceutical, medical device and consumer companies of Johnson & Johnson. Under the Johnson & Johnson Innovation umbrella of businesses, we connect with innovators through our regional Innovation Centers, JLABS, Johnson & Johnson Innovation – JJDC, Inc. and our Business Development teams to create customized deals and novel collaborations that speed development of innovations to solve unmet needs in patients. For more information please visit: www.jnjinnovation.com. About Johnson & Johnson Innovation, JLABS Johnson & Johnson Innovation, JLABS (JLABS) is a global network of open innovation ecosystems, enabling and empowering innovators to create and accelerate the delivery of life-saving, life-enhancing health and wellness solutions to patients around the world. JLABS achieves this by providing the optimal environment for emerging companies to catalyze growth and optimize their research and development by opening them to vital industry connections, delivering entrepreneurial programs and providing a capital-efficient, flexible platform where they can transform the scientific discoveries of today into the breakthrough healthcare solutions of tomorrow.  At JLABS we value great ideas and are passionate about removing obstacles to success to help innovators unleash the potential of their early scientific discoveries.  JLABS is a no-strings-attached model, which means entrepreneurs are free to develop their science while holding on to their intellectual property.  JLABS is open to entrepreneurs across a broad healthcare spectrum including pharmaceutical, medical device, consumer and digital health sectors. JLABS currently has nine locations in innovation hot spots across North America and produces entrepreneurial programs and campaigns to seek out the best science, like the QuickFire Challenges around the globe. The JLABS flagship opened in 2012 in San Diego at Janssen's West Coast Research Center, and since then, has established two locations in San Francisco - one through a collaboration with the California Institute for Quantitative Biosciences (QB3) and a second standalone facility in South San Francisco.  JLABS is also located in Boston through a collaboration with LabCentral, in Lowell, Massachusetts through a collaboration with UMass, in Houston through a collaboration with the Texas Medical Center (TMC), in Toronto through a collaboration with the Ontario Government and the University of Toronto and a new JLABS @ NYC (in collaboration with the New York Genome Center (opening in 2018)).  For more information about JLABS, please visit www.jlabs.jnjinnovation.com. To view the original version on PR Newswire, visit:http://www.prnewswire.com/news-releases/johnson--johnson-innovation-announces-40-resident-companies-now-at-jlabs--toronto-300455763.html


University of California, Berkeley, neuroscientists have now combined new techniques for sequencing the RNA in single cells with detailed statistical analysis to more easily track individual stem cells in the nose, uncovering clues that someday could help restore smell to those who have lost it. The results are published this week in the journal Cell Stem Cell. "A stem cell's job is twofold: to replace or recreate mature cells that are lost over time, both through normal aging and after injury, and to replace themselves so that the process can continue over the life of the animal," said senior author John Ngai, the Coates Family Professor of Neuroscience and a member of UC Berkeley's Helen Wills Neuroscience Institute and the Berkeley Stem Cell Center. "We are getting closer to understanding how mature sensory neurons are generated from olfactory stem cells, an understanding that's key for an eventual stem cell therapy to restore function." Ngai noted that perhaps one-quarter of all people over the age of 50 have some loss of smell, yet doctors have little understanding why, and no treatments for most cases. There's not even a standardized test for loss of smell, as there is for vision or hearing loss, in spite of widespread reports of suffering by patients who have lost their sense of smell. "Some cases of anosmia—the loss of the sense of smell—are due to traumatic injury, and there is generally not a whole lot you can do about that," he said. "But some are age-related, or occur for reasons we don't quite know. In the case of age-related anosmias, it could be because the stem cells are just not doing their job replacing the cells that are naturally lost over time. One idea is that if we could harness the very stem cells that are in the noses of people who are losing smell, maybe we can figure out a way to restore function, by getting them to regenerate the cells that are lost." Ngai, who directs the Functional Genomics Laboratory in UC Berkeley's California Institute for Quantitative Biosciences, focuses on the cells and regulatory molecules involved in our sense of smell. Olfactory cells in the nose are unusual in that they are part of the body's outer layer, or epithelium, but also part of the nervous system, incorporating neurons that connect directly with the smell centers in the brain. His group has been working with adult olfactory stem cells that give rise to the neurons that sense odors and other cells, such as sustentacular cells, that support the neurons. A new technique for sequencing the RNA in a single cell has been revolutionary, Ngai said, allowing researchers to trace which stem cells in a densely packed tissue become specialized, based on the mRNA present in the cell, which indicates which genes are being expressed. Nevertheless, it is difficult to follow stem cells that can potentially differentiate into different types of cells. Ngai's group teamed up with UC Berkeley statisticians and computer scientists - led by Sandrine Dudoit, a professor of biotstatistics and statistics, Elizabeth Purdom, a professor of statistics, and Nir Yosef, a professor of electrical engineering and computer sciences - to develop a way to analyze the experimental data and identify cells with similar RNA profiles, indicative of specific cell types and developmental states. As a result, the team was able to trace the paths that cells take as they turn into sustentacular cells—which seems to be the default fate for olfactory stem cells—and into neurons and other types of cells. They also were able to identify a signaling pathway known as "Wnt" that triggers the olfactory stem cell to become a sensory neuron. Ngai cautions that the immediate implications of the work are limited to animal models, which provide the necessary foundation for eventually addressing human anosmias. "But with this information, we now have a window into what controls the process and therefore a window into manipulating or coopting that process to stimulate regeneration" he said. "There has been a lot of work on Wnt signaling pathways, for example, so there are a lot of small-molecule drugs that could be tested to trigger a stem cell to mature into a neuron." The sequencing and statistical techniques the team developed can also be used by others studying regulation of stem cells in other tissues, organ systems or organisms, he said. Explore further: Neuroscientists find genetic trigger that makes stem cells differentiate in nose epithelia


News Article | December 19, 2016
Site: www.eurekalert.org

CAMBRIDGE, Mass. (Dec. 19, 2016) -- Investigators from Whitehead Institute, the Ragon Institute of MGH, MIT and Harvard and the Broad Institute of MIT and Harvard have used CRISPR-Cas9 gene-editing technology to identify three promising new targets for treatment of HIV infection. In their report receiving advance online publication in Nature Genetics, the research team describes how screening with CRISPR for human genes that are essential for HIV infection but not for cellular survival identified five genes -- three of which had not been identified in earlier studies using RNA interference. Their method can also be used to identify therapeutic targets for other viral pathogens. "We were surprised to find that there are so few host factors required for HIV infection given some of the previous literature," observes David M. Sabatini, Whitehead Institute Faculty Member and co-corresponding author of the Nature Genetics paper. "The beauty of the CRISPR-based genetic screens is the clear and robust results they yield," notes Sabatini, who is also member of the Broad Institute and Professor of Biology at Massachusetts Institute of Technology. "Current anti-HIV medications overwhelmingly target viral proteins," says Ryan J. Park of the Ragon Institute and the Broad Institute, co-lead author of the report. "Because HIV mutates so rapidly, drug-resistant strains frequently emerge, particularly when patients miss doses of their medication. Developing new drugs to target human genes required for HIV infection is a promising approach to HIV therapy, with potentially fewer opportunities for the development of resistance." Bruce Walker, director of the Ragon Institute and co-corresponding author of the Nature Genetics paper, explains, "Viruses are very small and have very few genes - HIV has only 9, while humans have more than 19,000 - so viruses commandeer human genes to make essential building blocks for their replication. Our goal was to identify human genes, also called host genes, that are essential for HIV to replicate but could be eliminated without harming a human patient." Tim Wang, a doctoral student conducting research at Whitehead Institute and the Broad Institute, and co-lead author of the report, explains, "CRISPR makes it possible to completely knock out genes at the DNA level; and our genome-wide, CRISPR-Cas9-based approach targets more than 18,500 genes, the vast majority of human protein-coding genes. Our study demonstrates how CRISPR-based screens can be applied to identify host factors critical to the survival of other viral pathogens but dispensable for host cell viability. Broad application of this method should pinpoint a novel class of potential therapeutic targets that have previously been underexplored for the treatment of infectious disease." Co-corresponding author Nir Hacohen, an institute member at the Broad Institute and director of Cancer Immunology at Massachusetts General Hospital (MGH), adds, "An important aspect of our study was to focus on human T cells, the primary targets of HIV, and to identify host genes with the most dramatic role in viral infection of T cells." Previous research has identified several host dependency factors, including two proteins required for HIV to enter CD4 T cells, the primary target of the virus: the CD4 molecule itself, to which the virus binds, and CCR5, which facilitates the binding of common HIV strains. Individuals with a particular CCR5 mutation are immune to those viral strains - indeed the only individual considered cured of HIV infection received a bone marrow transplant from a donor with that CCR5 mutation - but while therapeutic CCR5 inhibitors have been developed and are in clinical use, they can cause serious side effects. Three 2008 studies that used RNA interference (RNAi) to identify potential host dependency factors identified more than 800 possible targets; but the little overlap among the results of the studies suggested a high rate of false positive results. In addition, none of those studies was performed using the immune cells targeted by HIV, which also reduces the likelihood that the identified genes actually participate in HIV's infection of CD4 T cells. Whitehead Institute's Tim Wang explains that, "RNAi suppresses but does not completely block gene expression - which could allow a targeted gene to produce enough protein to permit HIV infection - and it also can suppress expression of additional genes besides the intended target, leading to a false positive result." Using CRISPR to screen a cell line derived from HIV-susceptible CD4 T cells identified five genes that, when inactivated, protected cells from HIV infection without affecting cellular survival. In addition to CD4 and CCR5, the screen identified genes for two enzymes -- TPST2 and SLC35B2 -- that modify the CCR5 molecule in a way that is required for the binding of HIV. An additional gene identified through the screen was ALCAM, which is involved in cell-to-cell adhesion. When CD4 T cells are exposed to low amounts of virus, as might be seen in natural transmission, loss of ALCAM was associated with striking protection from HIV infection. Park explains, "ALCAM is necessary for cell-to-cell adhesion in our cell line, allowing more efficient viral transfer from one cell to the next. In fact, we found that artificially inducing the aggregation of cells lacking ALCAM restored the cell-to-cell transmission of HIV. Further studies are needed to investigate whether targeting these genes would be toxic to humans. However, even if systemic inhibition has toxic effects, gene therapy approaches that selectively target these genes only in CD4 T cells or their precursors may avoid these toxicities, although it's important to note that gene therapy remains a challenging and potentially costly therapeutic approach." Eric S. Lander, of the Broad Institute, is a co-corresponding author of the Nature Genetics paper, along with Sabatini, Hacohen, and Walker - who is also the Phillip and Susan Ragon Professor of Medicine at Harvard Medical School, a clinician in the MGH Division of Infectious Diseases and an associate member of the Broad Institute. Additional co-authors are Dylan Koundakjian, Pedro Lamothe-Molina, Blandine Monel, Wilfredo Garcia-Beltran and Alicja Piechocka-Trocha, Ragon Institute; Judd F. Hultquist, Kathrin Schumann, Alexander Marson and Nevan J. Krogan University of California, San Francisco; Haiyan Yu, Broad Institute; and Kevin M. Krupczak, a member of the Sabatini lab at Whitehead Institute when the study was conducted. The study was supported by funds from the Ragon Institute and the Howard Hughes Medical Institute. The study, formally titled A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors, will appear via Advance Online Publication on the Nature Genetics website on 19 December 2016. 1Ragon Institute of Massachusetts General Hospital (MGH), Massachusetts Institute of Technology (MIT), and Harvard University, Cambridge, Massachusetts, USA 7Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biosciences, QB3, University of California at San Francisco (UCSF), San Francisco, California, USA 11Department of Microbiology and Immunology, University of California at San Francisco, San Francisco, California, USA 13Department of Medicine, University of California at San Francisco, San Francisco, California, USA 19Institute of Medical Engineering and Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA The following funding acknowledgements from the authors appear at the end of the paper: This work was supported by the Howard Hughes Medical Institute (D.M.S. and B.D.W.), the National Institutes of Health (grants CA103866 (D.M.S.), F31 CA189437 (T.W.), P50 GM082250 (A.M. and N.J.K.), U19 AI106754 (J.F.H. and N.J.K.), and P01 AI090935 (N.J.K.)), the National Human Genome Research Institute (grant 2U54HG003067-10; E.S.L.), the National Science Foundation (T.W.), the MIT Whitaker Health Sciences Fund (T.W.), the UCSF Sandler Fellowship (A.M.), a gift from J. Aronov (A.M.), the UCSF MPHD T32 Training Grant (J.F.H.), and the Deutsche Forschungsgemeinschaft (grant SCHU3020/2-1; K.S.). Support was also provided by NIH-funded Centers for AIDS Research (grant P30 AI027763, UCSF Center for AIDS Research (N.J.K.) and grant P30 AI060354, Harvard University Center for AIDS Research (B.D.W.)), which are supported by the following NIH co-funding and participating Institutes and Centers: NIAID, NCI, NICHD, NHLBI, NIDA, NIMH, NIA, FIC, and OAR. D.M.S. and B.D.W. are investigators of the Howard Hughes Medical Institute. R.J.P. is a Howard Hughes Medical Institute Research Fellow.


Salt M.B.,University of California at San Francisco | Bandyopadhyay S.,University of California at San Francisco | Bandyopadhyay S.,California Institute for Quantitative Biosciences | McCormick F.,University of California at San Francisco
Cancer Discovery | Year: 2014

Tumors showing evidence of epithelial-to-mesenchymal transition (EMT) have been associated with metastasis, drug resistance, and poor prognosis. Heterogeneity along the EMT spectrum is observed between and within tumors. To develop effective therapeutics, a mechanistic understanding of how EMT affects the molecular requirements for proliferation is needed. We found that although cells use phosphoinositide 3-kinase (PI3K) for proliferation in both the epithelial and mesenchymal states, EMT rewires the mechanism of PI3K pathway activation. In epithelial cells, autocrine ERBB3 activation maintains PI3K signaling, whereas after EMT, downregulation of ERBB3 disrupts autocrine signaling to PI3K. Loss of ERBB3 leads to reduced serum-independent proliferation after EMT that can be rescued through reactivation of PI3K by enhanced signaling from p110α, ERBB3 reexpression, or growth factor stimulation. In vivo, we demonstrate that PIK3CA expression is upregulated in mesenchymal tumors with low levels of ERBB3. This study defines how ERBB3 downregulation after EMT affects PI3K-dependent proliferation. © 2014 American Association for Cancer Research.


Ideker T.,University of California at San Diego | Krogan N.J.,University of California at San Francisco | Krogan N.J.,California Institute for Quantitative Biosciences | Krogan N.J.,Gladstone
Molecular Systems Biology | Year: 2012

Protein and genetic interaction maps can reveal the overall physical and functional landscape of a biological system. To date, these interaction maps have typically been generated under a single condition, even though biological systems undergo differential change that is dependent on environment, tissue type, disease state, development or speciation. Several recent interaction mapping studies have demonstrated the power of differential analysis for elucidating fundamental biological responses, revealing that the architecture of an interactome can be massively re-wired during a cellular or adaptive response. Here, we review the technological developments and experimental designs that have enabled differential network mapping at very large scales and highlight biological insight that has been derived from this type of analysis. We argue that differential network mapping, which allows for the interrogation of previously unexplored interaction spaces, will become a standard mode of network analysis in the future, just as differential gene expression and protein phosphorylation studies are already pervasive in genomic and proteomic analysis. © 2012 EMBO and Macmillan Publishers Limited All rights reserved.


Heredia J.E.,University of California at San Francisco | Mukundan L.,University of California at San Francisco | Chen F.M.,University of California at San Francisco | Mueller A.A.,Stanford University | And 7 more authors.
Cell | Year: 2013

In vertebrates, activation of innate immunity is an early response to injury, implicating it in the regenerative process. However, the mechanisms by which innate signals might regulate stem cell functionality are unknown. Here, we demonstrate that type 2 innate immunity is required for regeneration of skeletal muscle after injury. Muscle damage results in rapid recruitment of eosinophils, which secrete IL-4 to activate the regenerative actions of muscle resident fibro/adipocyte progenitors (FAPs). In FAPs, IL-4/IL-13 signaling serves as a key switch to control their fate and functions. Activation of IL-4/IL-13 signaling promotes proliferation of FAPs to support myogenesis while inhibiting their differentiation into adipocytes. Surprisingly, type 2 cytokine signaling is also required in FAPs, but not in myeloid cells, for rapid clearance of necrotic debris, a process that is necessary for timely and complete regeneration of tissues. © 2013 Elsevier Inc.


Smith C.A.,University of California at San Francisco | Kortemme T.,University of California at San Francisco | Kortemme T.,California Institute for Quantitative Biosciences
Journal of Molecular Biology | Year: 2010

Protein-protein recognition, frequently mediated by members of large families of interaction domains, is one of the cornerstones of biological function. Here, we present a computational, structure-based method to predict the sequence space of peptides recognized by PDZ domains, one of the largest families of recognition proteins. As a test set, we use a considerable amount of recent phage display data that describe the peptide recognition preferences for 169 naturally occurring and engineered PDZ domains. For both wild-type PDZ domains and single point mutants, we find that 70-80% of the most frequently observed amino acids by phage display are predicted within the top five ranked amino acids. Phage display frequently identified recognition preferences for amino acids different from those present in the original crystal structure. Notably, in about half of these cases, our algorithm correctly captures these preferences, indicating that it can predict mutations that increase binding affinity relative to the starting structure. We also find that we can computationally recapitulate specificity changes upon mutation, a key test for successful forward design of protein-protein interface specificity. Across all evaluated data sets, we find that incorporation backbone sampling improves accuracy substantially, irrespective of using a crystal or NMR structure as the starting conformation. Finally, we report successful prediction of several amino acid specificity changes from blind tests in the DREAM4 peptide recognition domain specificity prediction challenge. Because the foundational methods developed here are structure based, these results suggest that the approach can be more generally applied to specificity prediction and redesign of other protein-protein interfaces that have structural information but lack phage display data. © 2010 Elsevier Ltd.


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

Researchers at UC San Francisco and the academically affiliated Gladstone Institutes have used a newly developed gene-editing system to find gene mutations that make human immune cells resistant to HIV infection. The team built a high-throughput cell-editing platform using a variant of CRISPR/Cas9 technology that allowed them to test how well scores of different genetic tweaks defended immune cells against HIV. The new system allows researchers to quickly modify the genetic code of freshly donated human immune cells and will hopefully accelerate the quest to finally cure HIV+ patients, the researchers said. "This is an ability HIV researchers have wanted for a long time," said postdoctoral researcher Judd F. Hultquist, PhD, one of the new paper's co-lead authors. "I hope this will take what seemed like an insurmountable task a year ago and make it something everyone can do." The research, which was published online October 25, 2016 in Cell Reports, was conducted by the laboratories of co-senior authors Nevan J. Krogan, PhD, a professor of cellular and molecular pharmacology at UCSF, director of the Quantitative Biosciences Institute (QBI) in UCSF's School of Pharmacy, and a senior investigator at the Gladstone Institutes, and Alexander Marson, MD, PhD, an assistant professor of microbiology and immunology in UCSF's School of Medicine. The research was spearheaded by Hultquist, who is in Krogan's lab, and Kathrin Schumann, PhD, a postdoctoral researcher in Marson's lab. Despite great progress made since the 1980s in the ability to treat and control HIV with antiretroviral drugs, there is still no cure for the virus, and millions of people are newly infected every year. Once the virus infiltrates a patient's immune system, it can hide indefinitely within cells' own DNA, impossible to detect or destroy with current technology. As a result, patients must continue on antiretroviral drugs for the rest of their lives. However, not everyone is susceptible to the virus. Scientists have taken inspiration from a group of individuals whose immune cells appear to be naturally resistant to HIV infection, and hope to one day edit HIV patients' immune systems to mimic the biology of these HIV-resistant individuals. "There have been lots of efforts to sequence the genomes of resistant people to discover the mutations that make them immune to the virus," Hultquist said. "But there are many different genes that could be involved: some control the virus's ability to enter immune cells, others control how the virus tricks cells into expressing its genes. Until now, there was no way to test which of these mutations actually confer resistance in primary human T-cells." Despite being the immune system's lead fighters, T cells are delicate - only able to survive outside the body for a couple weeks. They are also resistant to the viruses researchers use in other cell types to deliver DNA instructions about how to build the machinery needed for CRISPR/Cas9 gene editing. Last year, Marson and Schumann successfully used CRISPR to perform precise DNA sequence replacements in primary human T cells for the first time by prefabricating the CRISPR machinery in test tubes, then adding it to the freshly donated immune cells. "It's incredibly fast," Schumann said. "The desired editing occurs rapidly, and then the cell degrades the CRISPR machinery so it can't go on making changes. That's really important: otherwise it's like doing surgery and leaving in the scalpel." In the new paper, Schumann and Hultquist improved the technique by devising an automated system for high-throughput, parallel editing of T cells. The new approach enables the researchers to mutate different candidate genes in hundreds of thousands of T cells from healthy volunteers, expose these mutant cells to the HIV virus, then screen through the cells to find which mutations were able to prevent infection. A key feature of this system is its speed, as donated T cells can only survive outside of the body for two to three weeks. "If we want to start editing T cells and putting them back into people as a therapy," Krogan said, "I think this will be the gold standard for how to do that quickly, safely, and efficiently." The researchers used the new technique to mutate the genes CXCR4 and CCR5, which encode receptor molecules that different strains of the HIV virus use to sneak in and infect immune cells and which have been targeted in previous cell therapy trials. Inactivating either of these genes successfully blocked HIV infection of the human T cells by the relevant HIV strain. Additional experiments showed the feasibility of creating a two-layer security system for T cells by simultaneously blocking a gene the HIV virus needs to gain entry into cells and a gene the virus needs to survive and reproduce within the cell, resulting in doubly secure resistance. To demonstrate the efficiency and power of the new high-throughput technology, the researchers also developed 146 different CRISPR-based edits, each designed to deactivate one of 45 genes linked to HIV's ability to integrate into host cells. They identified several genes whose absence conferred HIV resistance, some of which had been predicted by previous studies and others that had never been directly tied to HIV infection before. 'Tip of the iceberg' for infectious disease research The researchers plan to use the new platform to identify additional weaknesses in the HIV virus's life cycle that could be exploited either by cell therapy or targeted drugs. They also want to be able to insert more subtle mutations, such as those reported in HIV-resistant individuals, which could alter cell function just enough to confer resistance but without fully deactivating the gene and impeding cell function. However, their greater hope is that the system will have much broader applications than just HIV and eventually be used in labs around the world to study the virus of their choice. "This toolkit has been a huge missing piece in infectious disease research," Marson said. "Now we have the ability to make modifications in human immune cells and right away see the effects. The potential is immense -- this is just the tip of the iceberg." Additional authors on the new paper include Jonathan M. Woo, MS, of UCSF; Michael J. McGregor, MS, of UCSF and the Gladstone Institutes; Lara Manganaro, PhD, and Viviana Simon, MD, PhD, of Mt. Sinai Icahn School of Medicine; and Jennifer Doudna, PhD, of UC Berkeley and the Howard Hughes Medical Institute. Krogan is director of the UCSF site of the California Institute for Quantitative Biosciences (QB3). Marson is a member of the Divisions of Infectious Diseases and Rheumatology in UCSF's Department of Medicine, the UCSF Diabetes Center, and the Helen Diller Family Comprehensive Cancer Center. Marson and Doudna are members of the University of California Innovative Genomics Initiative. This research was supported by UCSF; the National Institutes of Health, National Institute of Allergy and Infectious Disease, and National Institute of General Medical Sciences (R01 AI064001, P50 GM082250, U19 AI106754, P01 AI090935, CFAR, and P30 AI027763); the Deutsche Forschungsgemeinschaft (SCHU3020/2-1); the UCSF Sandler Fellowship; and a gift from Jake Aronov. Marson has filed a patent on the use of Cas9 RNPs to edit the genome of human primary T cells, and serves as an advisor to Juno Therapeutics independently from this work. About UCSF: UC San Francisco (UCSF) is a leading university dedicated to promoting health worldwide through advanced biomedical research, graduate-level education in the life sciences and health professions, and excellence in patient care. It includes top-ranked graduate schools of dentistry, medicine, nursing and pharmacy; a graduate division with nationally renowned programs in basic, biomedical, translational and population sciences; and a preeminent biomedical research enterprise. It also includes UCSF Health, which comprises top-ranked hospitals, UCSF Medical Center and UCSF Benioff Children's Hospitals in San Francisco and Oakland - and other partner and affiliated hospitals and healthcare providers throughout the Bay Area. Please visit http://www. . About the Gladstone Institutes: To ensure our work does the greatest good, the Gladstone Institutes focuses on conditions with profound medical, economic, and social impact--unsolved diseases of the brain, the heart, and the immune system. Affiliated with the University of California, San Francisco, Gladstone is an independent, nonprofit life science research organization that uses visionary science and technology to overcome disease.


Guo H.,Whitehead Institute For Biomedical Research | Guo H.,Howard Hughes Medical Institute | Ingolia N.T.,Howard Hughes Medical Institute | Ingolia N.T.,California Institute for Quantitative Biosciences | And 4 more authors.
Nature | Year: 2010

MicroRNAs (miRNAs) are endogenous g∼1/422-nucleotide RNAs that mediate important gene-regulatory events by pairing to the mRNAs of protein-coding genes to direct their repression. Repression of these regulatory targets leads to decreased translational efficiency and/or decreased mRNA levels, but the relative contributions of these two outcomes have been largely unknown, particularly for endogenous targets expressed at low-to-moderate levels. Here, we use ribosome profiling to measure the overall effects on protein production and compare these to simultaneously measured effects on mRNA levels. For both ectopic and endogenous miRNA regulatory interactions, lowered mRNA levels account for most (≥84%) of the decreased protein production. These results show that changes in mRNA levels closely reflect the impact of miRNAs on gene expression and indicate that destabilization of target mRNAs is the predominant reason for reduced protein output. © 2010 Macmillan Publishers Limited. All rights reserved.

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