Hot off the presses! Oct 01 Nat Med

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Latest Articles Include:

• Critical comparisons
  - Nat Med 16(10):1049 (2010)
  Nature Medicine | Editorial Critical comparisons Journal name:Nature MedicineVolume: 16 ,Page:1049Year published:(2010)DOI:doi:10.1038/nm1010-1049 The US Food and Drug Administration (FDA) is increasingly conservative in its assessment of data in new drug applications derived from noninferiority trials. Such caution might be similarly warranted in approving generic biologics. View full text Additional data
• Pooled studies can raise misleading alarm bells, regulators say
  - Nat Med 16(10):1051 (2010)
  Two years ago, drug safety researchers released a critical report linking certain inhaled lung medications, including the blockbuster drug Spiriva, to elevated risks of heart attack, stroke and death from heart disease by more than 50% compared with other inhaled lung drugs or placebo treatment. Notably, the scientists reached this conclusion by pooling the results of 17 clinical trials, all of which tested the inhaled drugs for at least 30 days.
• Sugar treatment doesn't pacify those concerned about preemies
  - Nat Med 16(10):1052 (2010)
  MONTREAL—Preterm and critically ill newborns admitted to neonatal intensive care units often receive pacifiers coated in sucrose to ease their pain. Research has shown that sucrose reduces babies' crying time and improves other behavioral measures of pain.
• 'Pay-for-delay' decision may be left to lawmakers
  - Nat Med 16(10):1052 (2010)
  In the US, the question of whether or not pharmaceutical companies should be able to pay off competitors challenging their patent exclusivity may now be left to Congress."The effort has been fought to a stalemate in the courts," says Lauren Fuller, director of legislative affairs for the Academy of Managed Care Pharmacy, a managed-care advocacy group that supports outlawing such 'pay-for-delay' deals.
• Fluctuating baseline pain implicated in failure of clinical trials
  - Nat Med 16(10):1053 (2010)
  MONTREAL—In the study of pain, the gold standard for assessing discomfort and suffering in human clinical trials is simply to ask participants how much pain they are feeling. Most experts agree that this metric is flawed, but they also acknowledge the lack of suitable biomarkers or other objective alternatives to replace self-reported measurements.
• Talkin' 'bout my (third) generation
  - Nat Med 16(10):1053 (2010)
  In 2004, officials at the US National Human Genome Research Institute (NHGRI) set an ambitious goal: to reduce the cost of sequencing an entire human genome by four orders of magnitude within a decade. At the time, shortly after the publication of the Human Genome Project, a new three-billion-base-pair genome sequence still cost more than $10 million.
• Sequencing of superbugs seen as key to combating their spread
  - Nat Med 16(10):1054 (2010)
  HINXTON, UK—Although the toll from hospital-acquired infections in the UK has declined in recent years from a high of close to 10,000 deaths in 2007 to around half that number today, the recurrent outbreaks are still troubling enough to merit alarm bells around the world. Part of the problem faced by hospitals is that current diagnostic methods have not kept pace with the need for rapid public health interventions, such as moving patients to isolation wards or emptying and deep-cleaning contaminated wards.
• Genomics uncovers microbe resistance
  - Nat Med 16(10):1054-1055 (2010)
  HINXTON, UK—In May 2004, a 64-year-old woman with a hemodialysis catheter entered a community hospital in Indiana with traces of the bacterium Enterococcus faecalis in her blood. Testing revealed the strain to be ampicillin susceptible but vancomycin resistant.
• Targeting hotspots of transmission promises to reduce malaria
  - Nat Med 16(10):1055 (2010)
  HINXTON, UK—Over the past decade, public health officials have beefed up their efforts to eradicate malaria, rolling out insecticide-treated bed nets, increasing indoor residual spraying and boosting access to artemisinin-combination therapy. But, according to a new mathematical model, the existing interventions will not be enough to eliminate malaria in areas hardest hit by the disease (PLoS Med 7, e1000324, 2010).
• As pediatric trials go global, some worry who really benefits
  - Nat Med 16(10):1056 (2010)
  Although children suffer from many of the same diseases as adults and are often treated with the same drugs, only a small fraction of approved medicines is ever tested in pediatric clinical trials. To encourage more safety and efficacy studies of drugs in children, in 1997 the US Food and Drug Administration (FDA) created a special provision that grants six-month patent extensions to medicines screened in children.
• Companies hope to bring DNA storage in from the cold
  - Nat Med 16(10):1056-1057 (2010)
  Would scientists willingly junk their fridges, given the chance? Some companies hope their new methods of storing dried genetic material at room temperature will convince researchers to do exactly that.
• Following Europe's lead, Congress moves to ban ape research
  - Nat Med 16(10):1057 (2010)
  For all the monkey business in Washington, DC, US lawmakers have decided to get serious about protecting chimpanzees. But doing so creates a conundrum: although the apes are intelligent and caring creatures, they are also considered by many to be the best animal model for developing a vaccine for hepatitis C, a human liver disease that leads to nearly 350,000 deaths each year worldwide.
• Correction: 'Personalized investigation'
  - Nat Med 16(10):1057 (2010)
  In the September 2010 issue of Nature Medicine, the article entitled 'Personalized investigation' (Nat. Med. 16, 953, 2010
• In season, atmospheric conditions can drive disease
  - Nat Med 16(10):1058 (2010)
  When winter comes, it usually brings more than just snow. The frigid temperatures coincide with a predictable uptick in colds and influenza.
• Call for renewed focus on rare mutations grows more common
  - Nat Med 16(10):1058 (2010)
  In the past five years, scientists have identified more than 3,000 common genetic mutations associated with diseases including cancer, Alzheimer's and diabetes, thanks to insights gleaned from genome-wide association studies (GWASs). But the inherent value of these studies has come under scrutiny, in part because they largely ignore rare mutations.
• A disease—or gene—by any other name would cause a stink
  - Nat Med 16(10):1059 (2010)
  Biomedically speaking, what's in a name? A lot, as it turns out.
• News in brief
  - Nat Med 16(10):1060-1061 (2010)
  Aug 23A US federal judge in Washington, DC issued an injunction prohibiting government funds from being used for stem cell research, throwing dozens of ongoing studies into jeopardy. An appeals court had decided to temporarily lift the injunction as Nature Medicine went to press.
• Straight talk with...Jules Hirsch
  - Nat Med 16(10):1062 (2010)
  Nature Medicine | News Straight talk with...Jules Hirsch * Roxanne Palmer Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Page:1062Year published:(2010)DOI:doi:10.1038/nm1010-1062 Abstract Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg On 26 October 1910, nine years after the oil baron John D. Rockefeller opened the first research facility in the US devoted exclusively to experimental medicine, the Rockefeller Institute for Medical Research in New York (now Rockefeller University) admitted the country's first research participant in a hospital dedicated completely to clinical studies. Conceived as a place where patient care and laboratory investigations of disease would complement each other, the hospital was home to the development of methadone treatment of heroin addiction and the discovery of autoantibodies in rheumatoid arthritis and lupus, among other findings. The hospital also served as a model for dozens of other clinical research centers, including the US National Institutes of Health's own Clinical Center, which opened its doors in 1953. Emeritus professor Jules Hirsch, a metabolism researcher who joined Rockefeller in 1954, has had a front-row seat for more than half of Rockefeller University Hospital's 100-year history, including four years as the hospital's physician-in-chief and 12 years heading up its institutional review board. As the hospital gears up to celebrate its centennial anniversary later this month, sat down with Hirsch in his office at the Upper East Side hospital to discuss the clinical center's impact on biomedical research and education. View full text Additional data
• Nothing to sneeze at
  - Nat Med 16(10):1063-1065 (2010)
  Nature Medicine | News Nothing to sneeze at * Erica Westly1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Pages:1063–1065Year published:(2010)DOI:doi:10.1038/nm1010-1063 A hundred years after scientists first linked histamine molecules to allergies, the study of histamine pathways is in the midst of a revival, thanks in part to the discovery of a new type of receptor. This fourth known histamine receptor now provides an attractive drug target for seasonal allergies, asthma and possibly even cancer. traces the histamine reaction. View full text Additional data Affiliations * Erica Westly is a freelance science writer based in Brooklyn, New York.
• A timeline of histamine and its receptors
  - Nat Med 16(10):1064 (2010)
  1907— Adolf Windaus and his associate W. Vogt produce histamine synthetically by removing the carboxyl group from the amino acid histidine.
• Sunshine biomedicine
  - Nat Med 16(10):1066-1069 (2010)
  Nature Medicine | News Sunshine biomedicine * Christopher Mims1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Pages:1066–1069Year published:(2010)DOI:doi:10.1038/nm1010-1066 Over the past eight years, the state of Florida has invested close to a billion dollars to attract renowned research organizations to open up shop within its borders. But even with the arrival of several heavyweights, including the Scripps Research Institute and the Max Planck Institute, some continue to question whether the investment was worthwhile. reports. View full text Additional data Affiliations * Christopher Mims is a science writer based in Gainesville, Florida.
• Advocates deserve room at the decision-making table
  - Nat Med 16(10):1070 (2010)
  Nature Medicine | News Advocates deserve room at the decision-making table * Jeff Sheehy1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Page:1070Year published:(2010)DOI:doi:10.1038/nm1010-1070 Patient advocates are often dismissed by the scientific establishment for focusing too much on cures and treatments at the expense of basic research. But advocates help create a biomedical research enterprise that is more attuned to the needs and preferences of the public—the very people who ultimately support and are meant to benefit from the enterprise. As such, scientists and government officials would be wise to heed patient advocates' advice. View full text Additional data Affiliations * Jeff Sheehy is director of communications at the University of California–San Francisco's AIDS Research Institute and chair of the CIRM Governing Board's science subcommittee.
• Living forever
  - Nat Med 16(10):1071 (2010)
  Immortal cell lines, such as HeLa cells, are the backbone of many experiments conducted by today's cell and molecular biologists, but most of them give little thought to the origins of the 'standard' human cell lines they use. In many cases these cells originated from tissue discarded from medical procedures, with the patient, as a person, being dissociated from the scientist.
• Stem cells on the move
  - Nat Med 16(10):1073-1074 (2010)
  Mobilization of hematopoietic stem and progenitor cells (HSPCs) from bone marrow into peripheral blood by the cytokine granulocyte colony–stimulating factor (G-CSF) has become the preferred source of HSPCs for stem cell transplants1, 2, 3, 4, 5, 6, 7, 8, 9. However, G-CSF fails to mobilize sufficient numbers of stem cells in up to 10% of donors, precluding autologous transplantation in those donors or substantially delaying transplant recovery time2. Consequently, new regimens are needed to increase the number of stem cells in peripheral blood upon mobilization. Using a forward genetic approach in mice, we mapped the gene encoding the epidermal growth factor receptor (Egfr) to a genetic region modifying G-CSF–mediated HSPC mobilization. Amounts of EGFR in HSPCs inversely correlated with the cells' ability to be mobilized by G-CSF, implying a negative role for EGFR signaling in mobilization. In combination with G-CSF treatment, genetic reduction of EGFR activity in ! HSPCs (in waved-2 mutant mice) or treatment with the EGFR inhibitor erlotinib increased mobilization. Increased mobilization due to suppression of EGFR activity correlated with reduced activity of cell division control protein-42 (Cdc42), and genetic Cdc42 deficiency in vivo also enhanced G-CSF–induced mobilization. Our findings reveal a previously unknown signaling pathway regulating stem cell mobilization and provide a new pharmacological approach for improving HSPC mobilization and thereby transplantation outcomes.
• High glucose, no cry
  - Nat Med 16(10):1074-1076 (2010)
  During fasting, mammals maintain normal glucose homeostasis by stimulating hepatic gluconeogenesis1. Elevations in circulating glucagon and epinephrine, two hormones that activate hepatic gluconeogenesis, trigger the cAMP-mediated phosphorylation of cAMP response element–binding protein (Creb) and dephosphorylation of the Creb-regulated transcription coactivator-2 (Crtc2)—two key transcriptional regulators of this process2. Although the underlying mechanism is unclear, hepatic gluconeogenesis is also regulated by the circadian clock, which coordinates glucose metabolism with changes in the external environment3, 4, 5, 6. Circadian control of gene expression is achieved by two transcriptional activators, Clock and Bmal1, which stimulate cryptochrome (Cry1 and Cry2) and Period (Per1, Per2 and Per3) repressors that feed back on Clock-Bmal1 activity. Here we show that Creb activity during fasting is modulated by Cry1 and Cry2, which are rhythmically expressed in the li! ver. Cry1 expression was elevated during the night-day transition, when it reduced fasting gluconeogenic gene expression by blocking glucagon-mediated increases in intracellular cAMP concentrations and in the protein kinase A–mediated phosphorylation of Creb. In biochemical reconstitution studies, we found that Cry1 inhibited accumulation of cAMP in response to G protein–coupled receptor (GPCR) activation but not to forskolin, a direct activator of adenyl cyclase. Cry proteins seemed to modulate GPCR activity directly through interaction with Gsα. As hepatic overexpression of Cry1 lowered blood glucose concentrations and improved insulin sensitivity in insulin-resistant db/db mice, our results suggest that compounds that enhance cryptochrome activity may provide therapeutic benefit to individuals with type 2 diabetes.
• A sweet path toward tolerance in the gut
  - Nat Med 16(10):1076-1077 (2010)
  We propose that a C-type lectin receptor, SIGNR-1 (also called Cd209b), helps to condition dendritic cells (DCs) in the gastrointestinal lamina propria (LPDCs) for the induction of oral tolerance in a model of food-induced anaphylaxis. Oral delivery of BSA bearing 51 molecules of mannoside (Man51-BSA) substantially reduced the BSA-induced anaphylactic response. Man51-BSA selectively targeted LPDCs that expressed SIGNR1 and induced the expression of interleukin-10 (IL-10), but not IL-6 or IL-12 p70. We found the same effects in IL-10–GFP knock-in (tiger) mice treated with Man51-BSA. The Man51-BSA–SIGNR1 axis in LPDCs, both in vitro and in vivo, promoted the generation of CD4+ type 1 regulatory T (Tr1)-like cells that expressed IL-10 and interferon-γ (IFN-γ), in a SIGNR-1– and IL-10–dependent manner, but not of CD4+CD25+Foxp3+ regulatory T cells. The Tr1-like cells could transfer tolerance. These results suggest that sugar-modified antigens might be used to ind! uce oral tolerance by targeting SIGNR1 and LPDCs.
• Good and bad lipids in the lung
  - Nat Med 16(10):1078-1079 (2010)
  Pneumonia remains the leading cause of death from infection in the US, yet fundamentally new conceptual models underlying its pathogenesis have not emerged. We show that humans and mice with bacterial pneumonia have markedly elevated amounts of cardiolipin, a rare, mitochondrial-specific phospholipid, in lung fluid and find that it potently disrupts surfactant function. Intratracheal cardiolipin administration in mice recapitulates the clinical phenotype of pneumonia, including impaired lung mechanics, modulation of cell survival and cytokine networks and lung consolidation. We have identified and characterized the activity of a unique cardiolipin transporter, the P-type ATPase transmembrane lipid pump Atp8b1, a mutant version of which is associated with severe pneumonia in humans and mice. Atp8b1 bound and internalized cardiolipin from extracellular fluid via a basic residue–enriched motif. Administration of a peptide encompassing the cardiolipin binding motif or At! p8b1 gene transfer in mice lessened bacteria-induced lung injury and improved survival. The results unveil a new paradigm whereby Atp8b1 is a cardiolipin importer whose capacity to remove cardiolipin from lung fluid is exceeded during inflammation or when Atp8b1 is defective. This discovery opens the door for new therapeutic strategies directed at modulating the abundance or molecular interactions of cardiolipin in pneumonia.
• A small kiss of death for cancer
  - Nat Med 16(10):1079-1081 (2010)
  Inactivation of the p53 tumor suppressor pathway allows cell survival in times of stress and occurs in many human cancers; however, normal embryonic stem cells and some cancers such as neuroblastoma maintain wild-type human TP53 and mouse Trp53 (referred to collectively as p53 herein). Here we describe a miRNA, miR-380-5p, that represses p53 expression via a conserved sequence in the p53 3′ untranslated region (UTR). miR-380-5p is highly expressed in mouse embryonic stem cells and neuroblastomas, and high expression correlates with poor outcome in neuroblastomas with neuroblastoma derived v-myc myelocytomatosis viral-related oncogene (MYCN) amplification. miR-380 overexpression cooperates with activated HRAS oncoprotein to transform primary cells, block oncogene-induced senescence and form tumors in mice. Conversely, inhibition of endogenous miR-380-5p in embryonic stem or neuroblastoma cells results in induction of p53, and extensive apoptotic cell death. In vivo de! livery of a miR-380-5p antagonist decreases tumor size in an orthotopic mouse model of neuroblastoma. We demonstrate a new mechanism of p53 regulation in cancer and stem cells and uncover a potential therapeutic target for neuroblastoma.
• The persistence of memory
  - Nat Med 16(10):1082-1083 (2010)
  Cell therapy with pluripotent stem cells brought hope to people in need of regenerative medicine. The generation of induced pluripotent stem cells (iPSCs) from adult cells is a promising alternative to stem cells derived from embryos.
• Peering into the aftermath: The inhospitable host?
  - Nat Med 16(10):1084-1085 (2010)
  Nature Medicine | Between Bedside and Bench Peering into the aftermath: The inhospitable host? * Robert S Kerbel1robert.kerbel@sri.utoronto.ca Search for this author in: * NPG journals * PubMed * Google Scholar * John M L Ebos1jebos@sri.utoronto.ca Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorsJournal name:Nature MedicineVolume: 16 ,Pages:1084–1085Year published:(2010)DOI:doi:10.1038/nm1010-1084 Much of how the human host responds to a tumor and to anticancer therapy is still not fully fleshed out. The cytokines and other mediators involved in this response may be both allies and enemies in the quest for more effective treatments or even a cancer cure. In 'Bedside to Bench', Robert Kerbel and John Ebos discuss recent human studies in healthy individuals and people with minimal residue or no disease showing release of host-derived cytokines after antiangiogenic therapy. The increase in proangiogenic factors such as VEGF and PIGF and other cytokines involved in metastasis and tumor repopulation in a host may threaten therapeutic success but may also suggest new prognostic markers and other treatment strategies. In 'Bench to Bedside', Michael Karin and Florian Greten peruse how using JAK2 inhibitors to block STAT3 in tumors could halt cancer progression. JAK2 inhibitors, already being tested in clinical trials to treat myeloproliferative diseases, may also prove valid ! as anticancer drugs. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Robert S. Kerbel and John M.L. Ebos are at the Sunnybrook Research Institute in the Department of Molecular and Cellular Biology at Sunnybrook Health Sciences Centre, Toronto, Canada and the Department of Medical Biophysics at the University of Toronto, Toronto, Canada. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Robert S Kerbel (robert.kerbel@sri.utoronto.ca) or * John M L Ebos (jebos@sri.utoronto.ca) Additional data
• Peering into the aftermath: JAKi rips STAT3 in cancer
  - Nat Med 16(10):1085-1087 (2010)
  Persistent activation of the transcription factor signal transducer and activator of transcription-3 (STAT3) occurs in a large number of solid malignancies, and a plethora of genetic studies suggest that inhibition of this oncogenic transcriptional regulator, which supports the proliferation and survival of malignant cells, could be an effective treatment modality. A recent study by Hedvat et al.1 described the development of a specific inhibitor of the tyrosine kinase Janus kinase-2 (JAK2), which has a central role in STAT3 activation in various human cancer cells1.
• Research Highlights
  - Nat Med 16(10):1088-1089 (2010)
  
• How to win a Lasker? Take a close look at Bathers and Bulls
  - Nat Med 16(10):1091-1096 (2010)
  Some of our most influential scientists have spent their spare time moonlighting as amateur philosophers. Famous figures like the physiologist Claude Bernard, the biochemist Hans Krebs, the immunologist Peter Medawar, the physicist Richard Feynman and the molecular biologist James Watson have offered all sorts of guidance on how to become more creative and make groundbreaking discoveries.
• A historical perspective on leptin
  - Nat Med 16(10):1097-1099 (2010)
  As the only child of a self-employed and largely self-taught Canadian radio-and-refrigeration repairman, I spent much of my childhood investigating how things worked by taking them apart. As I grew older, I developed a keen interest in science, and, while I was an undergraduate at McMaster University, my scientific interests converged on biology, geology and chemistry.
• A tale of two hormones
  - Nat Med 16(10):1100-1106 (2010)
  "Der Mensch denkt, Gott lenkt." ("Man proposes, God disposes.") —German proverb
• Vascular endothelial growth factor and age-related macular degeneration: from basic science to therapy
  - Nat Med 16(10):1107-1111 (2010)
  It is almost intuitive that blood vessels have a central role in biology and medicine. Indeed, even though the concept of blood circulation was not established until a few centuries ago, mankind had known for millennia that blood vessels are indispensable for bringing nourishment to organs and limbs.
• Thalassemia: the long road from the bedside through the laboratory to the community
  - Nat Med 16(10):1112-1115 (2010)
  I owe my long interest in inherited blood diseases and tropical medicine to a series of characteristically bizarre decisions by the British Army. In 1958, 2 years after qualifying in medicine from Liverpool University, I was drafted for 2 years of compulsory National Service.
• Passive neutralizing antibody controls SHIV viremia and enhances B cell responses in infant macaques
  Ng CT Jaworski JP Jayaraman P Sutton WF Delio P Kuller L Anderson D Landucci G Richardson BA Burton DR Forthal DN Haigwood NL - Nat Med 16(10):1117-1119 (2010)
  Nature Medicine | Brief Communication Passive neutralizing antibody controls SHIV viremia and enhances B cell responses in infant macaques * Cherie T Ng1, 2, 9 Search for this author in: * NPG journals * PubMed * Google Scholar * J Pablo Jaworski3 Search for this author in: * NPG journals * PubMed * Google Scholar * Pushpa Jayaraman1, 2, 9 Search for this author in: * NPG journals * PubMed * Google Scholar * William F Sutton3 Search for this author in: * NPG journals * PubMed * Google Scholar * Patrick Delio4 Search for this author in: * NPG journals * PubMed * Google Scholar * LaRene Kuller4 Search for this author in: * NPG journals * PubMed * Google Scholar * David Anderson4 Search for this author in: * NPG journals * PubMed * Google Scholar * Gary Landucci5 Search for this author in: * NPG journals * PubMed * Google Scholar * Barbra A Richardson6 Search for this author in: * NPG journals * PubMed * Google Scholar * Dennis R Burton7, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Donald N Forthal5 Search for this author in: * NPG journals * PubMed * Google Scholar * Nancy L Haigwood1, 2, 3haigwoon@ohsu.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 16 ,Pages:1117–1119Year published:(2010)DOI:doi:10.1038/nm.2233Received27 April 2007Accepted07 September 2010Published online03 October 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Maternal HIV-1–specific antibodies are efficiently transferred to newborns, but their role in disease control is unknown. We administered neutralizing IgG, including the human neutralizing monoclonal IgG1b12, at levels insufficient to block infection, to six newborn macaques before oral challenge with simian-HIV strain SF162P3 (SHIVSF162P3). All of the macaques rapidly developed neutralizing antibodies and had significantly reduced plasma viremia for six months. These studies support the use of neutralizing antibodies in enhancing B cell responses and viral control in perinatal settings. View full text Figures at a glance * Figure 1: Matched IgG, but not mismatched IgG, affects plasma viral load in SHIVSF162P3- infected infant macaques. (,) Neutralizing activity of matched IgG, with IgG1b12 or without IgG1b12, and mismatched IgG against the challenge virus SHIVSF162P3 () and heterologous virus SHIV89.6P (). Vertical line at 2 × 103 denotes the estimated in vivo passive IgG concentration of 2 mg ml−1. () Mean peripheral blood mononuclear cell (PBMC) -proviral loads during the 24 weeks after initiation of infection (w.p.i.) as quantified by real-time PCR. () Mean plasma viral loads as quantified by real-time PCR during the 24 weeks after initiation of infection. () Differences in AUC comparing normal IgG, mismatched IgG and matched IgG for the entire 24 weeks. () Differences in AUC calculations for post-acute viremia between all treatment groups (weeks 8–24). Horizontal bar indicates the median value for the group. P values are indicated. Error bars indicate s.d. * Figure 2: Matched IgG, but not mismatched IgG, improves humoral responses up to 24 weeks after infection. () Left, mean HIV-1SF162 gp120–specific IgG concentration (± s.d.) as determined by kinetic ELISA for macaques treated with normal IgG, matched IgG or mismatched IgG. Right, analysis of the longitudinal effect of the SHIVIG pretreatments on Env-specific IgG development (weeks 8–24) using AUC analysis (Mann-Whitney U test). () Left, neutralizing activity in the plasma of each group against SHIVSF162P3 clone MC17, reported as the mean titers (± s.d.) for each group. ID50 is the plasma dilution necessary to inhibit infection by 50%. Right, AUC from 8 weeks after infection to 24 weeks after infection. P values indicate differences between groups (Mann-Whitney U test). () Mean ADCVI levels (± s.d.) evaluated to examine sources of virus inhibition other than neutralization. () CD4+ T cell counts per μl blood at 24 weeks after infection; dotted line indicates 250 cells per μl blood. Author information * Author information * Supplementary information Affiliations * Pathobiology Program, Department of Global Health, University of Washington, Seattle, Washington, USA. * Cherie T Ng, * Pushpa Jayaraman & * Nancy L Haigwood * Seattle Biomedical Research Institute, Seattle, Washington, USA. * Cherie T Ng, * Pushpa Jayaraman & * Nancy L Haigwood * Oregon National Primate Research Center and Vaccine & Gene Therapy Institute, Oregon Health & Science University, Beaverton, Oregon, USA. * J Pablo Jaworski, * William F Sutton & * Nancy L Haigwood * Washington National Primate Research Center, University of Washington, Seattle, Washington, USA. * Patrick Delio, * LaRene Kuller & * David Anderson * Division of Infectious Diseases, Department of Medicine, University of California–Irvine, Irvine, California, USA. * Gary Landucci & * Donald N Forthal * Department of Biostatistics, University of Washington, Seattle, Washington, USA. * Barbra A Richardson * Department of Immunology and Microbiology and International AIDS Vaccine Initiative Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, California, USA. * Dennis R Burton * Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Boston, Massachusetts, USA. * Dennis R Burton * Present addresses: The Scripps Research Institute, La Jolla, California, USA (C.T.N.) and Department of Medicine, Harvard University, Boston, Massachusetts, USA (P.J.). * Cherie T Ng & * Pushpa Jayaraman Contributions C.T.N., J.P.J., W.F.S. and G.L. were responsible for experimental work, analyses and preparation of figures; P.D. and L.K. provided macaque care and clinical and laboratory assessments; D.A. oversaw the study at the Washington National Primate Research Center; C.T.N., D.N.F., D.R.B., D.A. and P.J. contributed to the writing of the manuscript; B.A.R. performed the statistical analyses; D.R.B. provided IgG1b12; N.L.H. was responsible for study design and coordination and writing the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Nancy L Haigwood (haigwoon@ohsu.edu) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (372K) Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Methods Additional data
• Dynamic regulation of cardiolipin by the lipid pump Atp8b1 determines the severity of lung injury in experimental pneumonia
  Ray NB Durairaj L Chen BB McVerry BJ Ryan AJ Donahoe M Waltenbaugh AK O'Donnell CP Henderson FC Etscheidt CA McCoy DM Agassandian M Hayes-Rowan EC Coon TA Butler PL Gakhar L Mathur SN Sieren JC Tyurina YY Kagan VE McLennan G Mallampalli RK - Nat Med 16(10):1120-1127 (2010)
  Nature Medicine | Article Dynamic regulation of cardiolipin by the lipid pump Atp8b1 determines the severity of lung injury in experimental pneumonia * Nancy B Ray1, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Lakshmi Durairaj1, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Bill B Chen2 Search for this author in: * NPG journals * PubMed * Google Scholar * Bryan J McVerry2 Search for this author in: * NPG journals * PubMed * Google Scholar * Alan J Ryan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Donahoe2 Search for this author in: * NPG journals * PubMed * Google Scholar * Alisa K Waltenbaugh2 Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher P O'Donnell2 Search for this author in: * NPG journals * PubMed * Google Scholar * Florita C Henderson1 Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher A Etscheidt1 Search for this author in: * NPG journals * PubMed * Google Scholar * Diann M McCoy1 Search for this author in: * NPG journals * PubMed * Google Scholar * Marianna Agassandian1 Search for this author in: * NPG journals * PubMed * Google Scholar * Emily C Hayes-Rowan2 Search for this author in: * NPG journals * PubMed * Google Scholar * Tiffany A Coon2 Search for this author in: * NPG journals * PubMed * Google Scholar * Phillip L Butler3 Search for this author in: * NPG journals * PubMed * Google Scholar * Lokesh Gakhar3 Search for this author in: * NPG journals * PubMed * Google Scholar * Satya N Mathur1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jessica C Sieren1 Search for this author in: * NPG journals * PubMed * Google Scholar * Yulia Y Tyurina4 Search for this author in: * NPG journals * PubMed * Google Scholar * Valerian E Kagan4 Search for this author in: * NPG journals * PubMed * Google Scholar * Geoffrey McLennan1, 5, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Rama K Mallampalli2, 7mallampallirk@upmc.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 16 ,Pages:1120–1127Year published:(2010)DOI:doi:10.1038/nm.2213Received11 June 2010Accepted17 August 2010Published online19 September 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Pneumonia remains the leading cause of death from infection in the US, yet fundamentally new conceptual models underlying its pathogenesis have not emerged. We show that humans and mice with bacterial pneumonia have markedly elevated amounts of cardiolipin, a rare, mitochondrial-specific phospholipid, in lung fluid and find that it potently disrupts surfactant function. Intratracheal cardiolipin administration in mice recapitulates the clinical phenotype of pneumonia, including impaired lung mechanics, modulation of cell survival and cytokine networks and lung consolidation. We have identified and characterized the activity of a unique cardiolipin transporter, the P-type ATPase transmembrane lipid pump Atp8b1, a mutant version of which is associated with severe pneumonia in humans and mice. Atp8b1 bound and internalized cardiolipin from extracellular fluid via a basic residue–enriched motif. Administration of a peptide encompassing the cardiolipin binding motif or Atp8b1 g! ene transfer in mice lessened bacteria-induced lung injury and improved survival. The results unveil a new paradigm whereby Atp8b1 is a cardiolipin importer whose capacity to remove cardiolipin from lung fluid is exceeded during inflammation or when Atp8b1 is defective. This discovery opens the door for new therapeutic strategies directed at modulating the abundance or molecular interactions of cardiolipin in pneumonia. View full text Figures at a glance * Figure 1: Quantification of cardiolipin in subjects with pneumonia. () Median (gray line) and distribution (black circles) of cardiolipin abundance in tracheal aspirates from subjects with nonpulmonary critical illness (NPCI, n = 5), pneumonia (PNA, n = 17) and CHF (n = 6). () Quantification of cardiolipin in mice with pneumonia. C57BL/6 mice were infected i.t. with E. coli (1 × 106 CFU per mouse, n = 4), H. influenzae (2 × 108 CFU per mouse, n = 5) or left uninfected (n = 3). Mice were killed 48 h (E. coli) or 72 h (H. influenzae) later and their lungs were lavaged and processed for cardiolipin determination. Inset, cardiolipin concentrations after acid instillation. Mice (six per group) were given HCl (pH 1.5, 2 ml per kg body weight i.t.) before being killed 30 min later for analysis of BAL cardiolipin abundance. () Cellular cardiolipin uptake in primary type II lung epithelia. Cells (from n = 10 mice) were cultured with [3H]cardiolipin for 2 h at 37 °C in the presence or absence of E. coli (multiplicity of infection = 100) or H. influ! enzae (multiplicity of infection = 10). Data are quantified as disintegrations per minute (d.p.m.). In and , **P < 0.01 and ***P < 0.001 versus control (means ± s.d.), as determined by one-way analysis of variance (ANOVA). * Figure 2: Biophysical effects of cardiolipin. (,) Surface tension. Cardiolipin (CL), lysophosphatidylcholine (LPC) and phosphatidylserine (PS) were reconstituted with Infasurf (liposomes in CaCl2 (5 mM)) with apoprotein () or without apoprotein (), and dynamic surface tension (γmin) was measured with a pulsating bubble surfactometer. Effect of amount of lipid on surface tension is magnified in inset in . (–) Compliance (), quasistatic pressure-volume curves (), elastance () and resistance () in mice (three to five per group) that were anesthetized, paralyzed and mechanically ventilated with positive-end expiratory pressure of 3, as determined with a FlexiVent system after i.t. application of phosphatidylglycerol (PG) or cardiolipin (x axis in , and represents amount of lipid). Significance was determined by one-way ANOVA. *P < 0.05, **P < 0.01 and ***P < 0.001 versus control. * Figure 3: Effect of cardiolipin on lung structure and epithelial cell viability. () MicroCT scan images were obtained on live mice (in vivo) 1 h after i.t. administration of cardiolipin (50 nmol, (low), 100 nmol (high)) versus control mice (top images). In separate studies, mice were given cardiolipin as above, and their lungs were fixed and processed for microCT scanning (ex vivo). Images are representative of two mice per group. Fixed tissue was also processed for H & E staining, and the bottom images show a high magnification (100×) image of foamy cells. (,) Lung edema. Mice given cardiolipin (15 mM in 50 μl saline, i.t.; n = 3) or vehicle (50 μl saline; n = 3) were assessed for Evans Blue dye extravasation in lung fluid. Wet-dry weights of lungs were also determined (). ***P < 0.01 versus control. () Apoptosis. Left, poly(ADP-ribose) polymerase (PARP) cleavage (arrows) as a marker of apoptosis in primary mouse type II cells exposed to Infasurf (120 nmol ml−1) or cardiolipin (5–15 mol%). Right, analysis of lung DNA fragmentation in mice given i! .t. cardiolipin (50 nmol). (,) Cell viability () or lactate dehydrogenase (LDH) release (, primary type II cells) after cardiolipin exposure at 2 h, 2.5 h, 4 h and 16 h of lipid exposure. NP-40 was used as a positive control for LDH release. Data represent three separate experiments, where *P < 0.05, **P < 0.01 and ***P < 0.001 versus control, as determined by one-way ANOVA. * Figure 4: Bacteria-induced lung injury after Atp8b1 overexpression. () Fluorescence uptake images from time-lapse microscopy in a lentivirus (LV)-transduced V5-Atp8b1 stable cell line labeled with NBD-cardiolipin or NBD-phosphatidylserine. () Fluorescence uptake of the cells in . **P < 0.05 and ***P < 0.001 by an unpaired Student's t test. (,) Effect of Atp8b1 gene transfer on lung mechanics. Mice (eight per group) received an empty vector (Ad5) or Ad5-Atp8b1 i.t. and 24 h later were given E. coli for 48 h before analysis of lung compliance (, bottom) and elastance () determined as in Figure 2. () A representative immunoblot showing amounts of V5-immunoreactive Atp8b1 in lung tissue from two mice receiving Ad5 or Ad5-Atp8b1 (top). The inset in shows cardiolipin concentrations in BAL fluid after Ad5-Atp8b1 or Ad5 infection. Significance was determined by a one-way ANOVA in and , where *P < 0.05 for empty + E. coli versus other groups (means ± s.d.). () Lung DNA fragmentation in mice (n = 6) treated as in . () Kaplan-Meier survival curve for ! mice infected with Ad5 empty or Ad5-Atp8b1 and infected with E. coli (5 × 106 CFU per mouse, n = 14 mice per group, P < 0.001, log-rank test). * Figure 5: Bacteria-induced lung injury in Atp8b1-mutant mice. () NBD-labeled cardiolipin uptake (left) is quantified in human A549 alveolar type II (ATII)-like cells transfected with Atp8b1-specific siRNA or control RNA. Immunoblotting (right) with 25 μg of protein loaded per lane using cell lysates from siRNA studies. Blots were probed with antibodies specific for Atp8b1, lysophosphatidylcholine acyltransferase-1 (LPCAT1), extracellular signal–regulated kinase-1 (Erk1), importin-α and β-actin (used as specificity controls). **P < 0.01. () HaeIII restriction digest patterns of genomic DNA used for genotyping wild-type (WT), heterozygous (Het) and mutant (Mut) mice. Atp8b1G308V/G308V mutant genomic DNA has a HaeIII restriction site (GGCC) in which the second G is mutated to T (glycine to valine) and is not recognized in restriction digests. () Top, Atp8b1 immunoblotting in mouse tissues. Bottom, densitometric analysis from six mice per group. () Cardiolipin concentrations in lung lavage from Atp8b1-mutant and wild-type littermates ! (three per group). **P < 0.01. () NBD-cardiolipin uptake in primary type II epithelia isolated from mutants or wild-type littermates (five mice per group). Shown is NBD-cardiolipin cellular uptake initially after labeling (t = 0) and after 1 min. *P < 0.05 versus other groups. (,) Lung mechanics in Atp8b1-mutant mice. Mutants and wild-type littermates uninfected or infected (seven per group) with E. coli were analyzed for determination of quasistatic volume-pressure curves (, (uninfected)) and elastance (). Statistical significance was determined by a one-way ANOVA in . **P < 0.01 WT versus Mut + E. coli, and *P < 0.05 WT + E. coli versus Mut + E. coli. () Lung DNA fragmentation in wild-type and mutant mice (three per group) treated as in . () Kaplan-Meier survival curve for wild-type and Atp8b1-mutant mice infected with E. coli (5 × 106 CFU per mouse, seven mice per group, P = 0.11, log-rank test). Data shown as means ± s.d. * Figure 6: Bacterial lung injury after CBD peptide administration. () [3H]cardiolipin uptake is shown after culturing MLE cells with Atp8b1 CBD-GST fusion peptide (CBD) or GST (control) (10 μg of GST peptide or GST-CBD peptide per dish). **P < 0.01. (–) Effect of CBD peptide administration on lung mechanics. C57/BL6 mice (six mice per group) were uninfected (control) or infected with E. coli, and after 48 h, mice were given vehicle or CBD peptide i.t. into lungs before biophysical analysis. Shown are quasistatic volume-pressure curves (), compliance () and elastance (). Significance was determined by a one-way ANOVA where in ***P < 0.001 for vehicle versus all other groups and in *P < 0.05 for 25 μg versus either control or vehicle and ***P < 0.001 for vehicle versus control, 100 μg or 250 μg. () Cytokine concentrations in BAL from mice (n = 4 per group) infected with E. coli or uninfected before i.t. administration of CBD. All P values are <0.05 for vehicle buffer + E. coli. (0+) versus other groups. () Kaplan-Meier survival curve fo! r mice given i.t. CBD peptide (100 μg) and infected with E. coli (5 × 106 CFU per mouse, n = 14 mice per group, P = 0.001, log-rank test). Data shown is means ± s.d. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Nancy B Ray & * Lakshmi Durairaj Affiliations * Department of Internal Medicine, University of Iowa, Iowa City, Iowa, USA. * Nancy B Ray, * Lakshmi Durairaj, * Alan J Ryan, * Florita C Henderson, * Christopher A Etscheidt, * Diann M McCoy, * Marianna Agassandian, * Satya N Mathur, * Jessica C Sieren & * Geoffrey McLennan * Department of Internal Medicine, Acute Lung Injury Center of Excellence, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. * Bill B Chen, * Bryan J McVerry, * Michael Donahoe, * Alisa K Waltenbaugh, * Christopher P O'Donnell, * Emily C Hayes-Rowan, * Tiffany A Coon & * Rama K Mallampalli * Department of Biochemistry, University of Iowa, Iowa City, Iowa, USA. * Phillip L Butler & * Lokesh Gakhar * Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. * Yulia Y Tyurina & * Valerian E Kagan * Department of Radiology, University of Iowa, Iowa City, Iowa, USA. * Geoffrey McLennan * Department of Biomedical Engineering, University of Iowa, Iowa City, Iowa, USA. * Geoffrey McLennan * Medical Specialty Service Line, Veteran's Affairs Pittsburgh Healthcare System, Pittsburgh, Pennsylvania, USA. * Rama K Mallampalli Contributions N.B.R. designed and executed cardiolipin-ATP8b1 binding, in vitro imaging and immunological studies and wrote the manuscript. L.D. edited the manuscript and conducted the human studies. B.B.C. performed in vitro (cardiolipin uptake, biochemical and molecular) experiments and all mouse studies. B.J.M. and M.D. contributed to human studies and statistical analyses. A.K.W., T.A.C., M.A., P.L.B., F.C.H. and S.N.M. assisted with in vitro studies. A.J.R. and C.P.O. assisted with mouse studies. D.M.M., E.C.H.-R. and C.A.E. conducted cardiolipin analysis. L.G. conducted surfactant studies. J.C.S. and G.M. designed and conducted in vivo imaging. V.E.K. designed and executed mass spectrometry of cardiolipin, with assistance from Y.Y.T., and provided editorial suggestions. R.K.M. revised the manuscript and directed the study. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Rama K Mallampalli (mallampallirk@upmc.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (728K) Supplementary Table 1, Supplementary Figures 1–13 and Supplementary Methods Additional data
• Oral tolerance to food-induced systemic anaphylaxis mediated by the C-type lectin SIGNR1
  Zhou Y Kawasaki H Hsu SC Lee RT Yao X Plunkett B Fu J Yang K Lee YC Huang SK - Nat Med 16(10):1128-1133 (2010)
  Nature Medicine | Article Oral tolerance to food-induced systemic anaphylaxis mediated by the C-type lectin SIGNR1 * Yufeng Zhou1, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Hirokazu Kawasaki1, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Shih-Chang Hsu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Reiko T Lee2 Search for this author in: * NPG journals * PubMed * Google Scholar * Xu Yao1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Beverly Plunkett1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jinrong Fu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kuender Yang4 Search for this author in: * NPG journals * PubMed * Google Scholar * Yuan C Lee2 Search for this author in: * NPG journals * PubMed * Google Scholar * Shau-Ku Huang1, 5, 6skhuang@jhmi.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 16 ,Pages:1128–1133Year published:(2010)DOI:doi:10.1038/nm.2201Received09 September 2009Accepted27 July 2010Published online12 September 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We propose that a C-type lectin receptor, SIGNR-1 (also called Cd209b), helps to condition dendritic cells (DCs) in the gastrointestinal lamina propria (LPDCs) for the induction of oral tolerance in a model of food-induced anaphylaxis. Oral delivery of BSA bearing 51 molecules of mannoside (Man51-BSA) substantially reduced the BSA-induced anaphylactic response. Man51-BSA selectively targeted LPDCs that expressed SIGNR1 and induced the expression of interleukin-10 (IL-10), but not IL-6 or IL-12 p70. We found the same effects in IL-10–GFP knock-in (tiger) mice treated with Man51-BSA. The Man51-BSA–SIGNR1 axis in LPDCs, both in vitro and in vivo, promoted the generation of CD4+ type 1 regulatory T (Tr1)-like cells that expressed IL-10 and interferon-γ (IFN-γ), in a SIGNR-1– and IL-10–dependent manner, but not of CD4+CD25+Foxp3+ regulatory T cells. The Tr1-like cells could transfer tolerance. These results suggest that sugar-modified antigens might be used to induce or! al tolerance by targeting SIGNR1 and LPDCs. View full text Figures at a glance * Figure 1: Antigen-induced anaphylaxis in C3H/Hej mice. () The severity of anaphylactic responses, scored 30 min after antigen challenge by a scoring system24 from 0 (no sign of shock) to 5 (death), in mice sensitized with PBS, BSA (200 μg per mouse) or Man51-BSA (Man, 200 μg per mouse) plus cholera toxin (10 μg per mouse) and challenged with BSA (1 mg per mouse). () Mice were pretreated daily on 3 consecutive days with BSA or Man51-BSA at 200 μg per mouse, followed by oral sensitization and challenge with BSA. *P < 0.05. () Plasma histamine concentrations in the same groups of mice as in . *P < 0.05. () Relative amounts of specific IgE, IgG1 and IgG2a antibodies in naive mice and those after treatment (before BSA sensitization) with BSA or Man51-BSA or after BSA sensitization. *P < 0.05 versus BSA; n = 8–14 mice per group. (,) Symptom scores () and plasma histamine concentration () in sensitized and challenged mice that received spleen cell transfer from mice treated with BSA or Man51-BSA. *P < 0.05; n = 6–8 mice per gro! up. () Representative flow cytometry analysis of splenic Foxp3+ Treg cells in mice treated with BSA or Man51-BSA. The percentage of CD25+Foxp3+ cells gated on CD4+ cells and the relative intensity (MFI) of Foxp3 are shown. n = 3–5 mice per group. Results are representative of two independent experiments. Data are expressed as means ± s.e.m. * Figure 2: Man51-BSA targets the DC subset in the lamina propria. () A representative flow cytometry analysis of three main LPDC or macrophage subsets in mice that received oral administration of FITC-BSA or FITC-Man51-BSA. () Immunocytochemical analysis of small intestine samples from the groups of mice in . Two-color merge pictures; Ab, antibody; scale bar, 50 μm. () Flow cytometry analysis of SIGNR1 expression in three cell subsets from lamina propria and spleen. () The relative levels of SIGNR1 gene expression in LPDCs and splenic CD11c+ DCs (SPDCs) measured by quantitative RT-PCR. Data are expressed as means ± s.e.m. of two experiments. * Figure 3: Binding analysis of neoglyco-antigens. () Solid-phase binding analyses. The relative binding activity was expressed as absorbance at 490 nm after subtracting the background. () Representative competition analysis. The MFI of SIGNR1-transfectants stained with FITC-labeled dextran (10 μg ml−1; dextran-FITC) in the presence or absence of varying concentrations of neoglyco-antigens as competitors as indicated, as determined by flow cytometry. (,) Concentrations of IL-10 () and IL-6 and IL-12p70 () in CD11c+ DCs from lamina propria. (,) Concentrations of IL-10 in CD11c−CD11b+ cells from lamina propria () and CD11c+ DCs from spleen (). Cells in – were stimulated with medium alone (M), BSA (20 μg ml−1), Man51-BSA (Man, 20 μg ml−1) or CpG (1 μM) for 24 h.*P < 0.05. Data are expressed as means ± s.e.m of three to five experiments in each panel. * Figure 4: Inhibition analyses of IL-10 induction by Man51-BSA in LPDCs. (–) The concentrations of IL-10 in CD11c+ LPDCs (2 × 105 per condition). () Cells were stimulated with medium alone, BSA (20 μg ml−1) or Man51-BSA (20 μg ml−1) with or without a blocking SIGNR1-specific antibody (50 μg ml−1), isotype control (Ig Ctr) or, in separate assays, mannan (20 μg ml−1). () Cells were stimulated as in with or without an antibody specific to mannose receptor (CD206; 50 μg ml−1) or isotype control. () Cells were stimulated as in and pretreated with a MyD88 peptide inhibitor or a control peptide (200 μM). DCs were stimulated with 1 μM CpG in separate cultures for comparison. The concentrations of IL-10 in LPDCs after 24 h simulation were measured by ELISA. *P < 0.05. () Flow cytometry analysis of CD11c+GFP+ (IL-10+) cells in lamina propria of IL-10-GFP tiger mice. Data are expressed as means ± s.e.m of three experiments in each panel. * Figure 5: Analysis of cytokine expression in T cells. (,) Expression of IL-10 and IFN-γ (as measured by ELISA) in T cells in vitro when cocultured with Man51-BSA–pulsed LPDCs (CD11c+), in the presence or absence of SIGNR1-specific or isotype control (Ig Ctr) antibody () or neutralizing IL-10–specific (5 μg ml−1) or isotype control (Ig Ctr) antibody (). () Expression of IL-10 and IFN-γ in T cells from mice receiving oral administration of PBS, BSA or Man51-BSA (200 μg per mouse). *P < 0.05 versus PBS or BSA. (,) Symptom scores () and plasma histamine concentration () in sensitized and challenged mice receiving transfers of CD4+ T cells or CD11c+ DCs from mice treated with BSA or Man51-BSA. *P < 0.05; n = 6–8 mice per group. Data are expressed as means ± s.e.m. * Figure 6: Reversal of Man51-BSA–mediated tolerance in SIGNR1- and IL-10–deficient mice. (,) IL-10 () and IFN-γ () abundance in splenic CD4+ T cells from wild-type (WT) or SIGNR1-deficient (SIGNR1 KO) mice that were treated with oral administration of BSA or Man51-BSA and stimulated. *P < 0.05. (,) Symptom scores () and plasma histamine concentrations () in sensitized and challenged wild-type and SIGNR1-deficient mice. *P < 0.05; n = 6–8 mice per group. (,) Symptom scores () and plasma histamine () in C3H/HeJ mice treated with BSA or Man51-BSA that received IL-10R–specific or isotype control (Ig Ctr) antibodies. *P < 0.05; n = 4–6 mice per group. (,) Antigen-induced symptom scores () and specific IgG1 response () in wild-type mice adoptively transferred with splenic CD4+ T cells from IL-10–deficient (IL-10 KO) or wild-type (WT) mice treated with BSA or Man51-BSA. *P < 0.05; n = 4–6 mice per group. Data are expressed as means ± s.e.m. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Yufeng Zhou & * Hirokazu Kawasaki Affiliations * Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland, USA. * Yufeng Zhou, * Hirokazu Kawasaki, * Shih-Chang Hsu, * Xu Yao, * Beverly Plunkett, * Jinrong Fu & * Shau-Ku Huang * Department of Biology, Johns Hopkins University, Baltimore, Maryland, USA. * Reiko T Lee & * Yuan C Lee * Institute of Dermatology, Chinese Academy of Medical Sciences, Nanjing, China. * Xu Yao * Chang Gung Memorial Hospital at Kaohsiung and Chang Gung University, Taiwan. * Kuender Yang * Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan. * Shau-Ku Huang * Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan. * Shau-Ku Huang Contributions Y.Z. conducted experiments involving characterizing mucosal DC subsets and their regulatory role, analyzed data and wrote the manuscript; H.K. performed in vivo experiments on antigen-induced anaphylactic responses, analyzed data and wrote the manuscript; S.-C.H. conducted experiments involving binding analyses of neoglyco-antigens; R.T.L. synthesized neoglyco-antigens and wrote the manuscript; X.Y. conducted in vitro experiments characterizing T cell responses; B.P. performed flow cytometric experiments and edited the manuscript; J.F. conducted in vivo cell transfer experiments; K.Y. contributed to the design and preparation of neoglyco-antigens; Y.C.L. designed and prepared neoglyco-antigens and supervised their synthesis; S.-K.H. planned, designed, supervised and coordinated the overall research efforts. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Shau-Ku Huang (skhuang@jhmi.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (600K) Supplementary Figures 1–6 and Supplementary Methods Additional data
• miR-380-5p represses p53 to control cellular survival and is associated with poor outcome in MYCN-amplified neuroblastoma
  Swarbrick A Woods SL Shaw A Balakrishnan A Phua Y Nguyen A Chanthery Y Lim L Ashton LJ Judson RL Huskey N Blelloch R Haber M Norris MD Lengyel P Hackett CS Preiss T Chetcuti A Sullivan CS Marcusson EG Weiss W L'etoile N Goga A - Nat Med 16(10):1134-1140 (2010)
  Nature Medicine | Article miR-380-5p represses p53 to control cellular survival and is associated with poor outcome in MYCN-amplified neuroblastoma * Alexander Swarbrick1, 2, 20a.swarbrick@garvan.org.au Search for this author in: * NPG journals * PubMed * Google Scholar * Susan L Woods3, 4, 20 Search for this author in: * NPG journals * PubMed * Google Scholar * Alexander Shaw1, 5, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Asha Balakrishnan7 Search for this author in: * NPG journals * PubMed * Google Scholar * Yuwei Phua1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Akira Nguyen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Yvan Chanthery8 Search for this author in: * NPG journals * PubMed * Google Scholar * Lionel Lim8 Search for this author in: * NPG journals * PubMed * Google Scholar * Lesley J Ashton9 Search for this author in: * NPG journals * PubMed * Google Scholar * Robert L Judson8 Search for this author in: * NPG journals * PubMed * Google Scholar * Noelle Huskey8 Search for this author in: * NPG journals * PubMed * Google Scholar * Robert Blelloch10, 11 Search for this author in: * NPG journals * PubMed * Google Scholar * Michelle Haber9 Search for this author in: * NPG journals * PubMed * Google Scholar * Murray D Norris9 Search for this author in: * NPG journals * PubMed * Google Scholar * Peter Lengyel7 Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher S Hackett8 Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Preiss2, 5, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Albert Chetcuti12 Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher S Sullivan13 Search for this author in: * NPG journals * PubMed * Google Scholar * Eric G Marcusson14 Search for this author in: * NPG journals * PubMed * Google Scholar * William Weiss15, 16 Search for this author in: * NPG journals * PubMed * Google Scholar * Noelle L'Etoile17 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrei Goga7, 18, 19andrei.goga@ucsf.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 16 ,Pages:1134–1140Year published:(2010)DOI:doi:10.1038/nm.2227Received14 December 2009Accepted27 August 2010Published online26 September 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Inactivation of the p53 tumor suppressor pathway allows cell survival in times of stress and occurs in many human cancers; however, normal embryonic stem cells and some cancers such as neuroblastoma maintain wild-type human TP53 and mouse Trp53 (referred to collectively as p53 herein). Here we describe a miRNA, miR-380-5p, that represses p53 expression via a conserved sequence in the p53 3′ untranslated region (UTR). miR-380-5p is highly expressed in mouse embryonic stem cells and neuroblastomas, and high expression correlates with poor outcome in neuroblastomas with neuroblastoma derived v-myc myelocytomatosis viral-related oncogene (MYCN) amplification. miR-380 overexpression cooperates with activated HRAS oncoprotein to transform primary cells, block oncogene-induced senescence and form tumors in mice. Conversely, inhibition of endogenous miR-380-5p in embryonic stem or neuroblastoma cells results in induction of p53, and extensive apoptotic cell death. In vivo delivery! of a miR-380-5p antagonist decreases tumor size in an orthotopic mouse model of neuroblastoma. We demonstrate a new mechanism of p53 regulation in cancer and stem cells and uncover a potential therapeutic target for neuroblastoma. View full text Figures at a glance * Figure 1: The p53 3′ UTR contains binding sites for miR-380-5p, a developmentally restricted miRNA. () Alignment of human, mouse, rat and hamster p53 3′ UTRs, identifying a highly conserved 104-bp region. The predicted miR-380-5p binding sites are indicated in red. () Northern blot of miR-380 using total RNA from mouse embryonic, human fetal and adult tissues. () Quantitative RT-PCR (qRT-PCR) analysis of miR-380-5p expression in normal brain, embryonic carcinoma (P19) and mouse ES cells. () qRT-PCR analysis showing miR-380-5p expression in ES cells differentiated to the neuronal lineage. () Immunofluorescent staining for Sox1 (green) in cultures of neural progenitor cells. Scale bar, 65 μm. (,) Immunofluorescent staining for an early neuronal marker, Tuj1 (red), and a marker of astrocytes, GFAP (green), in differentiated cultures of neurons () and astrocytes (). Scale bars: 150 μm (); 100 μm (). In and , error bars depict s.d.; in , independent biological replicates indicated by separate bars. *P < 0.0002. * Figure 2: miR-380-5p is required for ES cell survival. () Activity of a miR-380 reporter either alone or after transfection with a control LNA (LNA-Ctrl) or an LNA directed against miR-380-5p (LNA-380). () Left, amount of ES cell death 24h after transfection of wild-type (WT) or Trp53−/− ES cells with LNA-Ctrl or LNA-380. Right, cell images 24 h post transfection with LNA-Ctrl or LNA-380. () qRT-PCR analysis of relative miR-380-5p expression in WT, Trp53−/− and Dgcr8−/− ES cells. () Western blots showing p53 induction and PARP cleavage (indicated by the arrow) after knockdown of miR-380-5p by LNA-380 compared to LNA-ctrl–transfected wild-type, Trp53−/− and Dgcr8−/− ES cells that were ultraviolet irradiated (UV). In , and , results are from at least three independent experiments (performed in triplicate in ). In –, error bars depict s.d. *P < 0.00002; **P < 0.00007; ***P < 0.0004; NS, not significant. Scale bars, 200 μm. * Figure 3: miR-380-5p targets p53 and decreases cell death after genotoxic stress. () Western blot showing p53 expression in MCF10a cells transfected with either a scrambled (SC) miRNA or miR-380-5p. () Quantification of p53 protein in MCF10a cells transfected with SC miRNA or miR-380-5p with or without UV treatment, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). () qRT-PCR analysis of TP53 mRNA in MCF10a cells after expression of miR-380, normalized to GAPDH levels. () Luciferase activity of TP53 3′UTR reporters after expression of miR-380. (,) Relative cell death of MCF10a cell populations stably expressing miR-380 or SC 24 h after treatment with ultraviolet light () or cisplatin (). Error bars depict s.e.m. (,) or s.d. (–). , and – are results from at least three independent repeats; in experiments were performed in triplicate. *P < 0.0009, **P < 0.03, ***P < 0.002, ****P < 0.05, *****P < 0.03, ******P < 0.001. * Figure 4: miR-380 prevents oncogene-induced senescence and increases tumor incidence in a mouse mammary cancer model. () The incidence of palpable mammary tumors (1 tumor per mouse) arising from cells infected with the indicated miRNA-encoding retrovirus plus HRASV12 after 6 weeks, control group with empty vector or expressing scrambled control (vector/SC). n = 15; miR-125b, n = 10; miR-380, n = 15; p53 shRNA, n = 17. () qRT-PCR of mature miR-380-5p in tumors arising from cells infected with HRASV12 and miR-380 or p53 shRNA virus. MCF10a cells that stably express miR-380 or a scrambled miRNA are shown as controls. () Immunohistochemical staining of mammary tumors. SA-β-gal, senescence-associated β-galactosidase. () qRT-PCR for p21waf1 expression normalized to GAPDH in tumors arising from cells infected with HRASV12 and p53 shRNA or miR-380 retrovirus compared to the primary MMECs. In and , error bars depict s.d.; each column represents a separate tumor. Scale bars, 100 μm. * Figure 5: miR-380-5p is expressed in mouse and human neuroblastoma and is associated with poor outcome in subjects with MYCN amplification. () qRT-PCR for miR-380-5p expression in tumors and neuroendocrine ganglion (SCG) tissue from wild type and transgenic mice. () miR-380-5p expression detected by qRT-PCR in primary human neuroblastoma samples taken before chemotherapy. miR-380-5p expression was normalized to U6 small nuclear 2 RNA, normal human brain expression (indicated by red dashed line); 'low' and 'high' designate the lowest quartile of miR-380-5p expression and the remainder, respectively. () Kaplan-Meier survival curves of event-free survival (EFS) in subgroups of subjects with neuroblastoma according to relative expression level of miR-380-5p, all with MYCN amplification (n = 22). Subjects were dichotomized around the lower quartile of miR-380-5p expression. High: n = 6, mean miR-380-5p expression = 3.19, s.e.m. = 0.83. Low: n = 16, mean miR-380-5p expression = 0.49, s.e.m. = 0.13. P = 0.004. In , error bars depict s.d., *P < 0.02, **P < 0.02. * Figure 6: Treatment with miR-380-5p antagonist induces p53-dependent cell death in neuroblastoma cells and decreases tumor growth in vivo. () Western blots showing p53 and p21waf1 induction and PARP cleavage after knockdown of miR-380-5p by LNA-380 compared to control LNA in NBL-WS cells (left) but not TP53-mutant BE(2)C cells (right). Arrow indicates cleaved PARP. () Images of NBL-WS cells 24 h after mock transfection (mock) or treatment with the indicated LNAs; an LNA directed against let7e (LNA-let7e) was included as an additional control. Scale bars, 200 μm. () MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay showing that knockdown of miR-380-5p by LNA-380 induces rapid loss of cell viability in NBL-WS cells (left) but not BE(2)C cells (right). Dox, doxorubicin treatment. () Tumor size after systemic treatment with miR-380 antagonist (anti-miR380) for 3 weeks; mass depicted is the weight of the kidney (indicated by dashed red line) plus associated tumor (n = 5 mice for each treatment). () Representative images of kidneys and associated neuroblastoma tumor! mass from two different mice for each treatment group. Scale bars, 1 cm. In –, results are representative of at least three independent experiments, error bars depict s.e.m. *P < 0.01. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Alexander Swarbrick & * Susan L Woods Affiliations * Cancer Research Program, Garvan Institute of Medical Research, Sydney, New South Wales, Australia. * Alexander Swarbrick, * Alexander Shaw, * Yuwei Phua & * Akira Nguyen * St. Vincent's Clinical School, University of New South Wales, Sydney, New South Wales, Australia. * Alexander Swarbrick, * Yuwei Phua & * Thomas Preiss * G.W. Hooper Research Foundation, University of California–San Francisco, San Francisco, California, USA. * Susan L Woods * Division of Genetics & Population Health, Queensland Institute of Medical Research, Brisbane, Queensland, Australia. * Susan L Woods * Victor Chang Cardiac Research Institute, Sydney, New South Wales, Australia. * Alexander Shaw & * Thomas Preiss * School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, Australia. * Alexander Shaw & * Thomas Preiss * Department of Medicine, University of California–San Francisco, San Francisco, California, USA. * Asha Balakrishnan, * Peter Lengyel & * Andrei Goga * Biomedical Sciences Program, University of California–San Francisco, San Francisco, California, USA. * Yvan Chanthery, * Lionel Lim, * Robert L Judson, * Noelle Huskey & * Christopher S Hackett * Children's Cancer Institute Australia for Medical Research, Sydney, New South Wales, Australia. * Lesley J Ashton, * Michelle Haber & * Murray D Norris * Department of Urology, University of California–San Francisco, San Francisco, California, USA. * Robert Blelloch * Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research and Center for Reproductive Sciences, University of California–San Francisco, San Francisco, California, USA. * Robert Blelloch * Children's Cancer Research Unit, The Children's Hospital at Westmead, Westmead, New South Wales, Australia. * Albert Chetcuti * Section of Molecular Genetics and Microbiology, University of Texas, Austin, Texas, USA. * Christopher S Sullivan * Regulus Therapeutics, San Diego, California, USA. * Eric G Marcusson * Department of Neurology, University of California–San Francisco, San Francisco, California, USA. * William Weiss * Department of Pediatrics, University of California–San Francisco, San Francisco, California, USA. * William Weiss * Center for Neuroscience, University of California–Davis, Davis, California, USA. * Noelle L'Etoile * Helen Diller Cancer Center, University of California–San Francisco, San Francisco, California, USA. * Andrei Goga * Liver Center, University of California–San Francisco, San Francisco, California, USA. * Andrei Goga Contributions S.L.W., A. Swarbrick and A.G. conceived and designed the experiments, discussed the results and wrote the manuscript. A. Swarbrick, S.L.W., A. Shaw, Y.P., A.N., A.G., R.L.J., C.S.S., C.S.H., P.L., A.B., N.H., Y.C. and L.L. performed experiments. L.J.A. and M.D.N. performed statistical analysis of the human neuroblastoma data set. A.C. provided human samples and clinical data, and E.G.M. provided anti-miRs for in vivo studies. M.H., T.P., W.W., N.L. and C.S.S. supervised experiments or experimental design. Competing financial interests E.G.M. is an employee and shareholder of Regulus Therapeutics. Corresponding authors Correspondence to: * Andrei Goga (andrei.goga@ucsf.edu) or * Alexander Swarbrick (a.swarbrick@garvan.org.au) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Methods Additional data
• Pharmacological inhibition of EGFR signaling enhances G-CSF–induced hematopoietic stem cell mobilization
  Ryan MA Nattamai KJ Xing E Schleimer D Daria D Sengupta A Köhler A Liu W Gunzer M Jansen M Ratner N Le Cras TD Waterstrat A Van Zant G Cancelas JA Zheng Y Geiger H - Nat Med 16(10):1141-1146 (2010)
  Nature Medicine | Letter Pharmacological inhibition of EGFR signaling enhances G-CSF–induced hematopoietic stem cell mobilization * Marnie A Ryan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kalpana J Nattamai1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ellen Xing1 Search for this author in: * NPG journals * PubMed * Google Scholar * David Schleimer1 Search for this author in: * NPG journals * PubMed * Google Scholar * Deidre Daria1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Amitava Sengupta1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anja Köhler3 Search for this author in: * NPG journals * PubMed * Google Scholar * Wei Liu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Matthias Gunzer3 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Jansen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nancy Ratner1 Search for this author in: * NPG journals * PubMed * Google Scholar * Timothy D Le Cras4 Search for this author in: * NPG journals * PubMed * Google Scholar * Amanda Waterstrat5 Search for this author in: * NPG journals * PubMed * Google Scholar * Gary Van Zant6 Search for this author in: * NPG journals * PubMed * Google Scholar * Jose A Cancelas1, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Yi Zheng1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hartmut Geiger1, 2hartmut.geiger@cchmc.org Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 16 ,Pages:1141–1146Year published:(2010)DOI:doi:10.1038/nm.2217Received10 March 2010Accepted24 August 2010Published online26 September 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Mobilization of hematopoietic stem and progenitor cells (HSPCs) from bone marrow into peripheral blood by the cytokine granulocyte colony–stimulating factor (G-CSF) has become the preferred source of HSPCs for stem cell transplants1, 2, 3, 4, 5, 6, 7, 8, 9. However, G-CSF fails to mobilize sufficient numbers of stem cells in up to 10% of donors, precluding autologous transplantation in those donors or substantially delaying transplant recovery time2. Consequently, new regimens are needed to increase the number of stem cells in peripheral blood upon mobilization. Using a forward genetic approach in mice, we mapped the gene encoding the epidermal growth factor receptor (Egfr) to a genetic region modifying G-CSF–mediated HSPC mobilization. Amounts of EGFR in HSPCs inversely correlated with the cells' ability to be mobilized by G-CSF, implying a negative role for EGFR signaling in mobilization. In combination with G-CSF treatment, genetic reduction of EGFR activity in HSPCs ! (in waved-2 mutant mice) or treatment with the EGFR inhibitor erlotinib increased mobilization. Increased mobilization due to suppression of EGFR activity correlated with reduced activity of cell division control protein-42 (Cdc42), and genetic Cdc42 deficiency in vivo also enhanced G-CSF–induced mobilization. Our findings reveal a previously unknown signaling pathway regulating stem cell mobilization and provide a new pharmacological approach for improving HSPC mobilization and thereby transplantation outcomes. View full text Figures at a glance * Figure 1: Regulation of G-CSF–mediated mobilization is linked to a 5-Mbp interval on mouse chromosome 11 containing the Egfr locus. () Frequency of CFCs after G-CSF induced mobilization in C57BL/6 (B6, n = 10) and line G (n = 10) (0–36 Mbp on chromosome 11) mice. *P < 0.05 versus C57BL/6. PB, peripheral blood. () Genetic constitution of C57BL/6.D2 chromosome 11 (line G) and the new subcongenic lines generated from line G (B, C57BL/6 allele, D, DBA/2 allele). The column headings indicate the PCRmarkers that define the underlying SNPs. The square represents the 5-Mbp interval between 14.7 and 19.5 Mbp. () G-CSF induced mobilization in subcongenic lines 106 (D2 interval 8.9–36.7 Mbp) (n = 4), 338 (D2 interval 26.1–36.7 Mbp) (n = 7), 1023 (D2 interval 8.9–26.1 Mbp) (n = 4) and 1804 (D2 interval 14.7–19.5 Mbp) (n = 8) relative to C57BL/6 and line G. *P < 0.05 versus C57BL/6, #P < 0.05 versus line G. () Relative differences in expression in HPCs (Lin−c-Kit+ cells) from the bone marrow of C57BL/6 and line G mice of the indicated genes in the 5-Mbp interval represented on the MOE430 chip. The level o! f expression for C57BL/6 set to 1. Data are based on three independent hybridizations per genotype. *P < 0.05. Plek, plekstrin; Pno1 partner of NOB1 homolog; Wdr92, WD repeat domain 92; Etaa1, Ewing tumor-associated antigen-1; Meis1, myeloid ectropic viral integration-1. () Egfr expression by quantitative RT-PCR in bone marrow–derived HPCs (Lin−c-Kit+ cells) from C57BL/6, line G and line 1804 mice; for C57BL/6 and line G, n = 3 repeats per experimental group (four mice per group); for line 1804, n = 2 repeats per experimental group (four mice per group). Steady state refers to expression in HPCs from nonmobilized mice; mobilized refers to expression in HPCs from G-CSF–mobilized mice. *P < 0.05 versus C57BL/6 at steady state, #P < 0.05 versus C57BL/6 mobilized. Error bars represent the mean ± s.e.m., except for expression data in line 1804 in , where they represent s.d. * Figure 2: EGF reduces G-CSF–induced mobilization of HSPCs. () Mobilization efficiency of C57BL/6 mice after a single dose of EGF on day 5 of the standard G-CSF regimen (n = 6, at least three mice per group), *P < 0.05 versus G-CSF only. () Schematic of the setup for competitive transplant experiments in to measure repopulating units in peripheral blood using identical volumes of peripheral blood as donor tissue from mice treated with G-CSF (n = 3) or G-CSF and EGF (n = 4) in competition with identical numbers of C57BL/6 CD45.1+ bone marrow cells. BM, bone marrow. () Repopulating unit values based on donor chimerism measured by flow cytometry in peripheral blood 3 months after transplant. *P < 0.05. () Mobilization of line 1804 compared to C57BL/6 mice after G-CSF and EGF treatment. #P < 0.05 versus G-CSF alone, *P < 0.05 C57BL/6 versus line 1804 at the same dose of EGF. () Expression of known EGFR ligands in total bone marrow. RT-PCR was performed with specific primers for the genes encoding epidermal growth factor (EGF), TGF-α, HB! -EGF and betacellulin (BTC) using cDNA isolated from total bone marrow (TBM) and lung (positive control). Error bars represent the mean ± s.e.m. * Figure 3: Genetic and pharmacological inhibition of EGFR activity enhances G-CSF–mediated mobilization. () Schematic of the experimental setup of the transplants in to determine mobilization of wa2/+ bone marrow cells. () CFCs in peripheral blood from WT and wa2/+ recipient mice (n = 6 per group) mobilized by G-CSF. *P < 0.05 versus WT. () Schematic of the experimental setup for competitive transplant experiments in and . () Donor chimerism in hematopoietic cells in peripheral blood of recipients before mobilization. () Percentage of Ly5.2+ donor colonies after mobilization (determined by flow cytometry of at least 30 individual CFCs per mouse), n = 6 mice per group. *P < 0.05 versus before mobilization and versus WT. () Mobilization in response to G-CSF or G-CSF and erlotinib treatment (2.5–10.0 μg per g body weight, administered on days 3, 4 and 5 of the G-CSF regimen). *P < 0.05 versus G-CSF. () Schematic of the setup for competitive transplant experiments in to measure hematopoietic stem cell frequency in peripheral blood after mobilization by G-CSF or G-CSF plus erloti! nib (5 μg per g body weight) in competition with identical numbers of C57BL/6 CD45.1+ bone marrow cells, n = 3 repeats per experimental group (three recipient mice per group). () Repopulating units based on donor chimerism determined by flow cytometry in peripheral blood 3 months after transplant. *P < 0.05 versus G-CSF. Error bars represent the mean ± s.e.m. * Figure 4: Cdc42 regulates G-CSF–mediated mobilization in response to EGFR signaling. () Representative immunoblot showing increased amounts of activated Cdc42 in LDBM cells from G-CSF–mobilized C57BL/6 mice after EGF treatment (n = 3 independent experiments, at least three mice per group). The ratio of activated Cdc42 to actin in response to EGF treatment was normalized relative to the ratio of activated Cdc42 to actin in mice treated with only G-CSF. () Representative immunoblot showing decreased amounts of activated Cdc42 in LDBM cells from G-CSF mobilized C57BL/6 mice in response to erlotinib (n = 3 independent experiments, at least three mice per group). The ratio of activated Cdc42 to actin in response to erlotinib treatment was normalized relative to the ratio of activated Cdc42 to actin in mice treated with only G-CSF. () Quantification of the amount of the activated (GTP-bound) form of Cdc42 in LDBM cells upon G-CSF– or G-CSF plus erlotinib (5.0 μg per g body weight)-induced mobilization. Statistical analyses are based on three independent weste! rn blots from three independent experiments with three mice in each group. *P < 0.05. () Quantification of progenitor cell adhesion to a layer of FBMD-1 stromal cells after G-CSF or G-CSF and EGF (200 ng ml−1) treatment, n = 4 experiments. *P < 0.05 versus PBS, #P < 0.05 versus G-CSF. () Quantification of progenitor cell adhesion to a layer of FBMD-1 stromal cells after G-CSF or G-CSF plus erlotinib (10 μM) treatment (data represent at least three separate experiments). *P < 0.05 versus PBS, #P < 0.05 versus G-CSF. () Frequency of CFCs in peripheral blood in WT mice (littermates) mobilized with G-CSF and treated with EGF (0.8 μg per g body weight) on day 5. n = 3 experiments, at least three mice per group, *P < 0.05. () Frequency of CFCs in peripheral blood of wa2/+ mice mobilized with G-CSF and treated with EGF (0.8 μg per g body weight) on day 5. n = 3 experiments, at least three mice per group. () Frequency of CFCs in peripheral blood of WT-reconstituted C57BL/6.SJL! (BoyJ) mice in response to G-CSF or G-CSF plus EGF after treat! ment with polyI:C. n = 3 experiments, at least three mice per group, *P < 0.05 versus G-CSF. () Frequency of CFCs in peripheral blood of mice reconstituted with Cdc42−/− hematopoietic cells and treated with polyI:C (n = 12 mice per group). P = 0.4715 G-CSF versus G-CSF plus EGF. () Representative immunoblots showing the amount of activated Cdc42 in LDBM cells in response to G-CSF in 'poor mobilizer' C57BL/6 mice and the 'better mobilizer' line 1804 (representative of two individual experiments with three mice per group). The ratio of activated Cdc42 to actin was normalized relative to the ratio of activated Cdc42 to actin in PBS (control)-treated C57BL/6 mice. Error bars represent the mean ± s.e.m. Accession codes * Accession codes * Author information * Supplementary information Referenced accessions ArrayExpress * E-MEXP-2911 Author information * Accession codes * Author information * Supplementary information Affiliations * Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center (CCHMC), Cincinnati, Ohio, USA. * Marnie A Ryan, * Kalpana J Nattamai, * Ellen Xing, * David Schleimer, * Deidre Daria, * Amitava Sengupta, * Wei Liu, * Michael Jansen, * Nancy Ratner, * Jose A Cancelas, * Yi Zheng & * Hartmut Geiger * Department of Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany. * Deidre Daria & * Hartmut Geiger * Institute of Molecular and Clinical Immunology, Otto von Guericke University, Magdeburg, Germany. * Anja Köhler & * Matthias Gunzer * Division of Pulmonary Biology, CCHMC, Cincinnati, Ohio, USA. * Timothy D Le Cras * Department of Biological Sciences, Eastern Kentucky University, Richmond, Kentucky, USA. * Amanda Waterstrat * Department of Internal Medicine, Markey Cancer Center, Division of Hematology/Oncology, University of Kentucky, Lexington, Kentucky, USA. * Gary Van Zant * Hoxworth Blood Center, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA. * Jose A Cancelas Contributions M.A.R. performed most of the experiments with help from K.J.N., A.S., D.S., D.D., W.L. and J.A.C. M.J. and A.W. performed microarray expression analyses. E.X. performed initial experiments on the inhibition of mobilization of EGF and generated the new congenic strains. J.A.C., N.R., T.D.L.C., G.V.Z., M.G., A.K. and Y.Z. consulted on most of the experiments and provided reagents or data for some experiments. H.G. performed some experiments and planned and supervised all experiments. Competing financial interests M.A.R. and H.G. are listed on a patent application [AU: About what aspect of this work?] to the US Patent Office filed by the Cincinnati Children's Hospital Medical Center. Corresponding author Correspondence to: * Hartmut Geiger (hartmut.geiger@cchmc.org) Supplementary information * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (584K) Supplementary Figures 1–7, Supplementary Tables 1 and 2 and Supplementary Methods Additional data
• Transcriptional analysis of HIV-specific CD8+ T cells shows that PD-1 inhibits T cell function by upregulating BATF
  Quigley M Pereyra F Nilsson B Porichis F Fonseca C Eichbaum Q Julg B Jesneck JL Brosnahan K Imam S Russell K Toth I Piechocka-Trocha A Dolfi D Angelosanto J Crawford A Shin H Kwon DS Zupkosky J Francisco L Freeman GJ Wherry EJ Kaufmann DE Walker BD Ebert B Haining WN - Nat Med 16(10):1147-1151 (2010)
  Nature Medicine | Letter Transcriptional analysis of HIV-specific CD8+ T cells shows that PD-1 inhibits T cell function by upregulating BATF * Michael Quigley1 Search for this author in: * NPG journals * PubMed * Google Scholar * Florencia Pereyra2, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Björn Nilsson3, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Filippos Porichis2, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Catia Fonseca1 Search for this author in: * NPG journals * PubMed * Google Scholar * Quentin Eichbaum2 Search for this author in: * NPG journals * PubMed * Google Scholar * Boris Julg2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jonathan L Jesneck1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Kathleen Brosnahan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Sabrina Imam1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kate Russell1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ildiko Toth2 Search for this author in: * NPG journals * PubMed * Google Scholar * Alicja Piechocka-Trocha2 Search for this author in: * NPG journals * PubMed * Google Scholar * Douglas Dolfi4 Search for this author in: * NPG journals * PubMed * Google Scholar * Jill Angelosanto4 Search for this author in: * NPG journals * PubMed * Google Scholar * Alison Crawford4 Search for this author in: * NPG journals * PubMed * Google Scholar * Haina Shin4 Search for this author in: * NPG journals * PubMed * Google Scholar * Douglas S Kwon2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jennifer Zupkosky2 Search for this author in: * NPG journals * PubMed * Google Scholar * Loise Francisco5 Search for this author in: * NPG journals * PubMed * Google Scholar * Gordon J Freeman6 Search for this author in: * NPG journals * PubMed * Google Scholar * E John Wherry4 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel E Kaufmann2, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Bruce D Walker2bwalker@partners.org Search for this author in: * NPG journals * PubMed * Google Scholar * Benjamin Ebert3, 6, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * W Nicholas Haining1, 9nicholas_haining@dfci.harvard.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 16 ,Pages:1147–1151Year published:(2010)DOI:doi:10.1038/nm.2232Received13 July 2010Accepted07 September 2010Published online03 October 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg CD8+ T cells in chronic viral infections such as HIV develop functional defects including loss of interleukin-2 (IL-2) secretion and decreased proliferative potential that are collectively termed 'exhaustion'1. Exhausted T cells express increased amounts of multiple inhibitory receptors, such as programmed death-1 (PD-1)2, 3, that contribute to impaired virus-specific T cell function. Although reversing PD-1 inhibition is therefore an attractive therapeutic strategy, the cellular mechanisms by which PD-1 ligation results in T cell inhibition are not fully understood. PD-1 is thought to limit T cell activation by attenuating T cell receptor (TCR) signaling4, 5. It is not known whether PD-1 also acts by upregulating genes in exhausted T cells that impair their function. Here we analyzed gene expression profiles from HIV-specific CD8+ T cells in individuals with HIV and show that PD-1 coordinately upregulates a program of genes in exhausted CD8+ T cells from humans and mice. Th! is program includes upregulation of basic leucine transcription factor, ATF-like (BATF), a transcription factor in the AP-1 family. Enforced expression of BATF was sufficient to impair T cell proliferation and cytokine secretion, whereas BATF knockdown reduced PD-1 inhibition. Silencing BATF in T cells from individuals with chronic viremia rescued HIV-specific T cell function. Thus, inhibitory receptors can cause T cell exhaustion by upregulating genes—such as BATF—that inhibit T cell function. Such genes may provide new therapeutic opportunities to improve T cell immunity to HIV. View full text Figures at a glance * Figure 1: Transcriptional profiles of HIV-specific CD8+ T cells show coordinate upregulation of genes induced by PD-1 signaling. () HIV viral load in controllers and progressors. Horizontal lines indicate median viral load in each cohort. () Genes differentially expressed in Gag-specific CD8+ T cells from controllers or progressors ranked by moderated t statistic. Each column represents an individual sample and each row an individual gene, colored to indicate normalized expression. The top 200 genes (Supplementary Table 2) in either direction are shown. () IL-2 secretion from PD-1–expressing Jurkat cells cultured with inhibitory PD-1–CD3-CD28 beads or control CD3-CD28 beads, as measured by ELISA (**P = 0.007). () Differentially expressed genes in PD-1 Jurkat cells cultured as in . The top 100 differentially expressed genes (Supplementary Table 5) from either condition are shown. () Enrichment analysis of PD-1 signature in HIV-specific CD8+ T cell profiles. The top 200 genes in the PD-1-specific signature were tested for enrichment in the rank-ordered list of genes differentially expressed in progr! essor versus controller HIV-specific CD8+ T cells. The x axis indicates the t statistic measured for each of the ~20,000 genes assayed in HIV-specific T cells, ranked in order of their differential expression in progressor versus controller classes. The y axis indicates the cumulative distribution of all genes (black) or of a set of 200 PD-1 signature genes (red). Gene sets that are related to the class distinction on the x axis would be expected to deviate from the dotted line (that is, shifted toward the left if enriched in profiles of CD8+ T cells from progressors). () Enrichment of PD-1 signature in tetramer-sorted CD8+ T cells specific for various human viral pathogens. PD-1 signature genes were tested for enrichment by single-sample enrichment analysis in gene expression profiles from sorted tetramer+CD8+ T cells specific for the indicated pathogens or in naive CD8+ T cells. Each point represents the relative enrichment of PD-1 signature genes in an individual sample.! The y axis indicates normalized enrichment score (*P < 0.05; ! **P < 0.01; ***P < 0.001 by Wilcoxon ranked-sum test). * Figure 2: Expression of BATF is upregulated by PD-1 and increased in exhausted T cells. () A Venn diagram representation of three transcription factors upregulated in Gag-specific T cells from HIV progressors and Jurkat cells after PD-1 ligation (t > 2.0). () BATF expression measured by real-time quantitative PCR in primary human CD4+ and CD8+ T cells cultured with CD3-CD28 beads or PD-L1–Ig–CD3-CD28 beads for 4 d. Data represent independent experiments with four to ten normal donors and are shown as expression relative to the CD3-CD28 bead condition (**P = 0.001; *P = 0.02; paired t test). () Relative BATF expression in arbitrary expression units from Affymetrix analysis of sorted naive (CD62L+CD45RA+) or HIV Gag-specific CD8+ T cell populations from controllers and progressors. () Batf expression measured by real-time quantitative PCR in LCMV-specific CD8+ T cells from mice infected with LCMV Armstrong or LCMV clone 13 relative to naïve mice (*P < 0.05; **P < 0.01). () PD-1 expression on LCMV-specific CD8+ T cells measured by flow cytometry after infecti! on with the indicated viruses (*P < 0.05; **P < 0.01; ***P < 0.001). Error bars represent means ± s.e.m. * Figure 3: BATF inhibits T cell function. () FACS analysis of carboxyfluorescein succinimidyl ester (CFSE)-labeled primary human CD4+ or CD8+ T cells from healthy volunteers transduced with a lentivirus expressing BATF (bottom) or with control vector (top) and cultured for 4 d with CD3-CD28 beads. () Summary data of proliferation (percentage CFSEdimannexin V−, top) and cell death (percentage annexin V+, bottom) in primary human CD4+ or CD8+ T cells (n = 14) transduced as in and cultured for 4 d with CD3-CD28 beads. () IL-2 and IFN-γ secretion by primary human CD4+ T cells (n = 10) transduced as in and cultured with CD3-CD28 beads. Data are shown normalized to the empty vector condition. NS, not significant. () BATF expression in PD-1–expressing Jurkat cells lentivirally transduced with shRFP (control) or two separate shBATF sequences measured by western blotting (top) or quantitative PCR (bottom). () IL2 expression by PD-1 Jurkat cells transduced with shRFP or shBATF cultured with no beads or either PD-1–CD3-! CD28 or CD3-CD28 beads for 18 h. Data show IL2 expression (± s.e.m.) measured by quantitative PCR (***P < 0.001; **P = 0.01). Data are normalized to the gene encoding β-actin and are presented as fold change with respect to unstimulated conditions. () Correlation between BATF silencing and IL-2 secretion in PD-1–expressing Jurkat cells transfected with five sequence-independent shBATF constructs or a control hairpin and cultured with PD-1–CD3-CD28 beads. BATF expression was measured by quantitative PCR and presented as fold change relative to control hairpin. Error bars represent means ± s.e.m. * Figure 4: BATF silencing improves HIV-specific T cell function. () Efficacy of siRNA uptake in CD3+ T cells cultured with a mixture of siRNA pool and fluorescent oligonucleotides (to monitor transduction) either with (transfected) or without (untransfected) electroporation. () Silencing of BATF by siRNA sequences targeting BATF in CD3+ T cells from a representative chronic progressor measured by quantitative PCR. Expression (mean ± s.e.m.) normalized to a housekeeping gene is presented as fold change relative to control siRNA, (–) BATF silencing enhances HIV-specific cytokine secretion in CD8+ () and CD4+ (,) T cells from chronic progressors. Peripheral blood mononuclear cells (PBMCs) depleted of CD4+ () or CD8+ (,) T cells were electroporated with the indicated siRNA pools and cultured with or without HIV Gag peptides for 4 d, and IFN-γ (,) or IL-2 () amounts were measured by a highly sensitive cytokine bead assay. In each panel, the left graph shows results from a representative subject, and the right graph shows summary data (CD8+! responses, 26 HIV epitope responses in four subjects; CD4+ responses, HIV Gag peptide pool in seven subjects). Cytokine amounts shown are adjusted for background secretion, and statistical significance was evaluated with the paired t test. () Proliferation of CFSE-labeled CD8+ T cells, as measured by the fraction of CFSEdimCD25+ cells 6 d after transfection and peptide stimulation of PBMCs. Data represent nine HIV epitope-specific responses in four subjects. Accession codes * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE24082 Author information * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Florencia Pereyra, * Björn Nilsson & * Filippos Porichis Affiliations * Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA. * Michael Quigley, * Catia Fonseca, * Jonathan L Jesneck, * Kathleen Brosnahan, * Sabrina Imam, * Kate Russell & * W Nicholas Haining * Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Charlestown, Massachusetts, USA. * Florencia Pereyra, * Filippos Porichis, * Quentin Eichbaum, * Boris Julg, * Ildiko Toth, * Alicja Piechocka-Trocha, * Douglas S Kwon, * Jennifer Zupkosky, * Daniel E Kaufmann & * Bruce D Walker * Cancer Program, Broad Institute, Cambridge, Massachusetts, USA. * Björn Nilsson, * Jonathan L Jesneck & * Benjamin Ebert * Department of Microbiology and Institute for Immunology, University of Pennsylvania, Philadelphia, Pennsylvania, USA. * Douglas Dolfi, * Jill Angelosanto, * Alison Crawford, * Haina Shin & * E John Wherry * Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA. * Loise Francisco * Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA. * Gordon J Freeman & * Benjamin Ebert * Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts, USA. * Daniel E Kaufmann * Division of Hematology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Benjamin Ebert * Division of Hematology/Oncology, Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA. * W Nicholas Haining Contributions M.Q. designed and performed experiments, analyzed data and helped write the paper. F. Pereyra designed the clinical components of the study. B.N. and J.L.J. designed and performed computational experiments. F. Porichis, D.S.K., J.Z. and D.E.K. designed and performed siRNA experiments in samples from subjects with HIV. C.F., Q.E., B.J., K.B., S.I., K.R., I.T., A.P.-T., D.D. and L.F. all performed experiments. G.J.F. designed experiments and developed PD-L1–Ig. J.A., A.C., H.S. and E.J.W. designed and performed mouse experiments and analyzed data. W.N.H., B.E. and B.D.W. conceived of the study and designed the experiments. W.N.H. analyzed data and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Bruce D Walker (bwalker@partners.org) or * W Nicholas Haining (nicholas_haining@dfci.harvard.edu) Supplementary information * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–7, Supplementary Tables 1–6 and Supplementary Methods Additional data
• Cryptochrome mediates circadian regulation of cAMP signaling and hepatic gluconeogenesis
  Zhang EE Liu Y Dentin R Pongsawakul PY Liu AC Hirota T Nusinow DA Sun X Landais S Kodama Y Brenner DA Montminy M Kay SA - Nat Med 16(10):1152-1156 (2010)
  Nature Medicine | Letter Cryptochrome mediates circadian regulation of cAMP signaling and hepatic gluconeogenesis * Eric E Zhang1, 2, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Yi Liu3, 4, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Renaud Dentin3 Search for this author in: * NPG journals * PubMed * Google Scholar * Pagkapol Y Pongsawakul1 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew C Liu1, 2, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Tsuyoshi Hirota1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Dmitri A Nusinow1 Search for this author in: * NPG journals * PubMed * Google Scholar * Xiujie Sun4 Search for this author in: * NPG journals * PubMed * Google Scholar * Severine Landais3 Search for this author in: * NPG journals * PubMed * Google Scholar * Yuzo Kodama6 Search for this author in: * NPG journals * PubMed * Google Scholar * David A Brenner6 Search for this author in: * NPG journals * PubMed * Google Scholar * Marc Montminy3montminy@salk.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Steve A Kay1skay@ucsd.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 16 ,Pages:1152–1156Year published:(2010)DOI:doi:10.1038/nm.2214Received26 May 2010Accepted18 August 2010Published online19 September 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg During fasting, mammals maintain normal glucose homeostasis by stimulating hepatic gluconeogenesis1. Elevations in circulating glucagon and epinephrine, two hormones that activate hepatic gluconeogenesis, trigger the cAMP-mediated phosphorylation of cAMP response element–binding protein (Creb) and dephosphorylation of the Creb-regulated transcription coactivator-2 (Crtc2)—two key transcriptional regulators of this process2. Although the underlying mechanism is unclear, hepatic gluconeogenesis is also regulated by the circadian clock, which coordinates glucose metabolism with changes in the external environment3, 4, 5, 6. Circadian control of gene expression is achieved by two transcriptional activators, Clock and Bmal1, which stimulate cryptochrome (Cry1 and Cry2) and Period (Per1, Per2 and Per3) repressors that feed back on Clock-Bmal1 activity. Here we show that Creb activity during fasting is modulated by Cry1 and Cry2, which are rhythmically expressed in the liver. C! ry1 expression was elevated during the night-day transition, when it reduced fasting gluconeogenic gene expression by blocking glucagon-mediated increases in intracellular cAMP concentrations and in the protein kinase A–mediated phosphorylation of Creb. In biochemical reconstitution studies, we found that Cry1 inhibited accumulation of cAMP in response to G protein–coupled receptor (GPCR) activation but not to forskolin, a direct activator of adenyl cyclase. Cry proteins seemed to modulate GPCR activity directly through interaction with Gsα. As hepatic overexpression of Cry1 lowered blood glucose concentrations and improved insulin sensitivity in insulin-resistant db/db mice, our results suggest that compounds that enhance cryptochrome activity may provide therapeutic benefit to individuals with type 2 diabetes. View full text Figures at a glance * Figure 1: Hepatic Creb-Crtc2 activity is modulated by the circadian clock. () Left, CRE-luc reporter activity in mice fasted for 3 h followed by i.p. glucagon administration. The relative effect of fasting at ZT10–13 (ZT13) and ZT22–1 (ZT1) is indicated. Right, bar graph showing CRE-luc activity from hepatic lysates normalized to β-galactosidase activity from co-infected Ad-RSV-β-gal adenovirus. *P < 0.01, n = 5. () Quantitative PCR analysis of gluconeogenic gene expression in mice fasted at ZT10–13 or ZT22–1 and then injected i.p. with glucagon (Glu). *P < 0.01, n = 5. () Immunoblot of Creb and Crtc2 proteins in liver extracts from mice fasted ZT10–13 (ZT13) or ZT22–1 (ZT1) followed by i.p. injection with glucagon. The relative effects ZT13 and ZT1 fasting on hepatic amounts of phospho-Crtc2 (p-Crtc2) and phospho-Creb (Ser133; p-Creb) are also shown. Dephosphorylated Crtc2 (active) runs faster than the p-Crtc2 (inactive) in ZT13 fasting mice. The loading control is heat-shock protein 90 (Hsp90). Data represent means ± s.e.m. * Figure 2: Cry inhibits Creb activity. () CRE-luc reporter activity in Vipr2-expressing mouse fibroblasts synchronized by serum shock and then exposed to VIP at indicated times. Replicate samples (a total of 24) received VIP treatment every 3 h after synchronization from 15–48 h. For clarity, one responsive curve per time point is shown in a different color. Mouse fibroblasts harboring Per2-luc or Bmal1-luc reporters were used to report the two distinct phases of clock gene expression (scale at right); units are the same as at left. Data are representative of three experiments. c.p.s., count per second. () Effect of Cry overexpression on CRE-luc reporter activity. Vipr2–CRE-luc cells were infected with lentiviral particles expressing either GFP, Cry1 or Cry2. Cells were treated with VIP 24 h after medium change (red arrow). (,) CRE-luc activity in transfected cells expressing Vipr2. CRE-luc and Vipr2 expression plasmids were co-transfected into HEK 293T cells along with Per1, Cry1 or Cry2 as indicated. Twenty! -four hours after transfection, cells were treated with VIP to induce CRE-luc activity. NS, not significant. *P < 0.01, Student's t test. Data represent means ± s.e.m. * Figure 3: Cry blocks induction of the gluconeogenic genetic program by Creb and Crtc2. () Left, live imaging analysis of hepatic CRE-luc reporter activity in control (Ad-GFP) and Ad-Cry1 expressing mice. CRE-luc activity in fasted mice (ZT10–13) injected with glucagon is shown. Right, quantitative analysis of CRE-luc activity. Asterisk indicates P < 0.01, n = 5. () Quantitative PCR analysis of G6pc and Pck1 gene expression in control and Cry1-expressing fasted (after i.p. injection of glucagon) and fed mice. *P < 0.05, n = 5. () Effect of Cry1 overexpression on fasting blood glucose concentrations relative to control GFP expressing mice. *P < 0.05, n = 3. () Left, live imaging analysis of CRE-luc reporter activity in mice expressing adenovirally encoded Cry1- and Cry2-specific shRNA (CRYi) or nonspecific shRNA (USi) in liver. Right, quantitative analysis of CRE-luc activity. *P < 0.01, n = 4. () Quantitative PCR analysis of hepatic G6pc and Pck1 mRNA amounts in control (USi) and CRYi-expressing mice. **P < 0.001 for G6pc and *P < 0.01 for Pck1, n = 3. () Glu! cose production assay in primary hepatocytes infected with Ad-CRYi or control. *P < 0.01, n = 3. () Glucose tolerance testing of db/db mice expressing adenovirus encoded Cry1 or GFP. *P < 0.01, n = 5. () Pyruvate tolerance testing of wild-type mice expressing adenovirus-encoded Cry1 or GFP. Fasted mice were injected with pyruvate and blood glucose concentrations were measured at indicated times. *P < 0.05, n = 5. Data represent means ± s.e.m. * Figure 4: Cry inhibits GPCR-dependent increases in adenyl cyclase activity. () Hepatic cAMP concentrations in fed or fasted mice after i.p. injection of glucagon. () Effect of Cry1 overexpression on hepatic cAMP levels at ZT13 in fasted mice after i.p. glucagon administration and in fed mice. () Immunoblot showing the effect of Ad-Cry1 expression on amounts of phospho-Creb (Ser133) in primary hepatocytes treated with glucagon or forskolin (Fsk). () Effect of Adenoviral Cry1 expression on intracellular cAMP concentrations in hepatocytes exposed to forskolin or glucagon. Ctrl, control. () In vitro reconstitution studies showing effect of cytosolic fractions from control (GFP) and Cry1-expressing cells on cAMP accumulation in reactions containing plasma membrane fractions from HEK293T cells. Iso, isoproterenol. () Immunodepletion assays showing effect of antibody presence on cAMP production. Anti-HA, HA-specific antibody. () Immunoblot showing recovery of Gsα and Vipr2 from membrane fractions of transfected HEK293T cells incubated with recombinant GST! -Cry1 or GST-GFP control. Top, input and pull-down signals from 293T cells overexpressing Flag-Gsα, Vipr2-V5, Flag-Per1 and Flag-Luc. Bottom, GST-tagged proteins were purified and stained with Coomassie brilliant blue (CBB). Asterisk indicates truncated GST-Cry1 polypeptide. () Co-immunoprecipitation of Gsα and Cry1. Top, immunoblot showing recovery of HA-Cry1 from immunoprecipitates of Flag-Gsα prepared from HEK293T cells. Asterisks indicate nonspecific signals. IP, immunoprecipitation. Bottom, immunoblot showing recovery of endogenous Gsα from immunoprecipitates of HA-Cry1 prepared from primary mouse hepatocytes. () Schematic diagram indicating that the circadian regulation of cAMP signaling in liver is Cry dependent. Blue oval represents E-box–bound transcription factors CLOCK-BMAL1, and green oval represents Cre-mediated transcription activators or coactivators such as Creb, p300/Cbp and Crtc2. AC, adenyl cyclase. *P < 0.05. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Eric E Zhang & * Yi Liu Affiliations * Section of Cell & Developmental Biology, Division of Biological Sciences, University of California–San Diego, La Jolla, California, USA. * Eric E Zhang, * Pagkapol Y Pongsawakul, * Andrew C Liu, * Tsuyoshi Hirota, * Dmitri A Nusinow & * Steve A Kay * Genomics Institute of the Novartis Research Foundation, San Diego, California, USA. * Eric E Zhang, * Andrew C Liu & * Tsuyoshi Hirota * The Salk Institute for Biological Studies, La Jolla, California, USA. * Yi Liu, * Renaud Dentin, * Severine Landais & * Marc Montminy * Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. * Yi Liu & * Xiujie Sun * Department of Biological Sciences, The University of Memphis, Memphis, Tennessee, USA. * Andrew C Liu * School of Medicine, University of California–San Diego, La Jolla, California, USA. * Yuzo Kodama & * David A Brenner Contributions E.E.Z. and S.A.K. conceived the project. E.E.Z., Y.L., M.M. and S.A.K. designed the research. E.E.Z., Y.L., R.D., P.Y.P., A.C.L., T.H., D.A.N., X.S., S.L. and Y.K. performed the experiments. E.E.Z. and Y.L. analyzed the data. E.E.Z., Y.L., D.A.B., M.M. and S.A.K. wrote the paper. This work is also supported in part by the Clayton Medical Research Foundation (CMRF), and M.M. is a Senior CMRF Investigator. M.M. is also supported by the Kieckhefer Foundation. Competing financial interests S.A.K. is a cofounder of ReSet Therapeutics and is a member of its Scientific Advisory Board. Corresponding authors Correspondence to: * Marc Montminy (montminy@salk.edu) or * Steve A Kay (skay@ucsd.edu) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (564K) Supplementary Figures 1–8 and Methods Additional data
• A dominant-negative mutation in the TRESK potassium channel is linked to familial migraine with aura
  Lafrenière RG Cader MZ Poulin JF Andres-Enguix I Simoneau M Gupta N Boisvert K Lafrenière F McLaughlan S Dubé MP Marcinkiewicz MM Ramagopalan S Ansorge O Brais B Sequeiros J Pereira-Monteiro JM Griffiths LR Tucker SJ Ebers G Rouleau GA - Nat Med 16(10):1157-1160 (2010)
  Nature Medicine | Letter A dominant-negative mutation in the TRESK potassium channel is linked to familial migraine with aura * Ronald G Lafrenière1, 2, 13 Search for this author in: * NPG journals * PubMed * Google Scholar * M Zameel Cader3, 4, 13zameel.cader@dpag.ox.ac.uk Search for this author in: * NPG journals * PubMed * Google Scholar * Jean-François Poulin2 Search for this author in: * NPG journals * PubMed * Google Scholar * Isabelle Andres-Enguix5 Search for this author in: * NPG journals * PubMed * Google Scholar * Maryse Simoneau2 Search for this author in: * NPG journals * PubMed * Google Scholar * Namrata Gupta2 Search for this author in: * NPG journals * PubMed * Google Scholar * Karine Boisvert2 Search for this author in: * NPG journals * PubMed * Google Scholar * François Lafrenière2 Search for this author in: * NPG journals * PubMed * Google Scholar * Shannon McLaughlan2 Search for this author in: * NPG journals * PubMed * Google Scholar * Marie-Pierre Dubé6 Search for this author in: * NPG journals * PubMed * Google Scholar * Martin M Marcinkiewicz7 Search for this author in: * NPG journals * PubMed * Google Scholar * Sreeram Ramagopalan8 Search for this author in: * NPG journals * PubMed * Google Scholar * Olaf Ansorge9 Search for this author in: * NPG journals * PubMed * Google Scholar * Bernard Brais1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jorge Sequeiros10 Search for this author in: * NPG journals * PubMed * Google Scholar * Jose Maria Pereira-Monteiro11 Search for this author in: * NPG journals * PubMed * Google Scholar * Lyn R Griffiths12 Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen J Tucker5 Search for this author in: * NPG journals * PubMed * Google Scholar * George Ebers8 Search for this author in: * NPG journals * PubMed * Google Scholar * Guy A Rouleau1, 2guy.rouleau@umontreal.ca Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 16 ,Pages:1157–1160Year published:(2010)DOI:doi:10.1038/nm.2216Received09 July 2010Accepted23 August 2010Published online26 September 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Migraine with aura is a common, debilitating, recurrent headache disorder associated with transient and reversible focal neurological symptoms1. A role has been suggested for the two-pore domain (K2P) potassium channel, TWIK-related spinal cord potassium channel (TRESK, encoded by KCNK18), in pain pathways and general anaesthesia2. We therefore examined whether TRESK is involved in migraine by screening the KCNK18 gene in subjects diagnosed with migraine. Here we report a frameshift mutation, F139WfsX24, which segregates perfectly with typical migraine with aura in a large pedigree. We also identified prominent TRESK expression in migraine-salient areas such as the trigeminal ganglion. Functional characterization of this mutation demonstrates that it causes a complete loss of TRESK function and that the mutant subunit suppresses wild-type channel function through a dominant-negative effect, thus explaining the dominant penetrance of this allele. These results therefore suppo! rt a role for TRESK in the pathogenesis of typical migraine with aura and further support the role of this channel as a potential therapeutic target. View full text Figures at a glance * Figure 1: Segregation analysis in a large migraine with aura pedigree. () Individuals with clinically confirmed migraine with visual aura are shown as black symbols, unaffected individuals as white symbols and unknown affectation as gray. The proband is indicated with an arrow. The haplotypes on 10q25.2–3, covering an indicative 13 of 141 genotyped SNPs, are shown with different colors to indicate each haplotype; the haplotype segregating with the F139WfsX24 mutation is colored red. Inferred haplotypes are marked with an asterisk. () Linkage region overlying 10q25.2–3, with dots representing the following SNP markers: rs1034178, rs17121613, rs1887984, rs1324288, rs1050755, rs1408817, rs932652, rs151603, rs4751909, rs765173, rs1157117, rs1900500, rs14240 and rs703422. LOD, logarithm of the odds ratio; cM, centiMorgan. () Sequence traces of the c.414–415 CT-deletion mutation predicted to cause a frameshift. MT, mutant allele. * Figure 2: KCNK18 and TRESK expression patterns in mice and humans. () ISH analysis of sagittal whole-body section at E12.5 and E15.5, in a P1 newborn mouse and in an adult mouse head stained with hematoxylin or labeled with an antisense or sense Kcnk18 riboprobe after 4-d exposure, showing mRNA labeling under dark-field illumination. () Horizontal section of a newborn (P1) mouse head showing Kcnk18 expression in the trigeminal ganglion. () Cross section of adult mouse trigeminal ganglion, with hematoxylin staining. Kcnk18 antisense probe revealed strongly labeled large size (>35 μm) neurons (large arrow), lightly labeled medium size neurons (medium arrow) and lightly labeled small size (>20 μm) neurons (small arrows). () Expression pattern of KCNK18 in human tissues, as determined by TaqMan quantitative RT-PCR assay normalized to the expression of the human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Data are given as mean values ± s.e.m. for three independent experiments. () Western blotting of adult mouse brain regions with ! mouse TRESK-specific antibody, showing widespread expression of TRESK in all examined regions of the brain. () TRESK expression in frozen sections of human trigeminal ganglion. A large ganglion cell with surrounding satellite cells is shown. Staining with TRESK-specific antibody reveals dense cytoplasmic staining. B, bone; Br, brain; Cb, cerebellum; H, heart; He, hematoxylin; Hp, hippocampus; Hy, hypothalamus; K, kidney; Li, liver; M, medulla; Mb, midbrain; P, pons; PC, posterior cortex; SC, spinal cord; SG, stellate ganglion; Sk, skin; T, thalamus; TG, trigeminal ganglion; Th, thymus. The asterisks are meant to indicate the particular location of the indicated tissue type. * Figure 3: Electrophysiological characterization of the F139WfsX24 TRESK mutant. () Representative currents in 2 mM K+ from an oocyte expressing WT TRESK. () Representative currents in 2 mM K+ from an oocyte expressing F139WfsX24 mutant TRESK. () Dominant-negative effect of coexpression of mutant F139WfsX24 subunits with WT TRESK. Steady-state current-voltage relationships are plotted for the WT TRESK mRNA coexpressed with varying molar ratios of either WT or F139WfsX24 mutant mRNA. The 1:1 WT to WT ratio represents the wild-type state, whereas 1:1 WT to F139WfsX24 (fs) represents the heterozygous state. No currents are observed in the homozygous fs state, even with fivefold overexpression of the F139WfsX24 mRNA (5:5 fs to fs). () Quantification of the dominant-negative effects. Peak currents measured at 20 mV and normalized to the average of the control group are plotted for each group. In panels c and d, data are given as mean values ± s.e.m. The numbers above the bars represent the number of oocytes measured. () Representative response of the WT TRES! K to 500 nM ionomycin. () Representative response of the F139WfsX24 mutant TRESK to 500 nM ionomycin. As shown above the trace in panels e and f, oocytes were switched from a low K+ to a high K+ solution before the application of the Ca2+ ionophore ionomycin, which activates TRESK through a calcineurin-dependent mechanism. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Ronald G Lafrenière & * M Zameel Cader Affiliations * Centre of Excellence in Neuromics and Department of Medicine, Université de Montréal, Centre Hospitalier de l'Université de Montréal, Research Centre, Notre-Dame Hospital, Montreal, Quebec, Canada. * Ronald G Lafrenière, * Bernard Brais & * Guy A Rouleau * Emerillon Therapeutics, Montreal, Quebec, Canada. * Ronald G Lafrenière, * Jean-François Poulin, * Maryse Simoneau, * Namrata Gupta, * Karine Boisvert, * François Lafrenière, * Shannon McLaughlan & * Guy A Rouleau * Medical Research Council Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK. * M Zameel Cader * Department of Clinical Neurology, John Radcliffe Hospital, Oxford, UK. * M Zameel Cader * Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, UK. * Isabelle Andres-Enguix & * Stephen J Tucker * Faculty of Medicine, Université de Montréal, Montreal Heart Institute, Montreal, Quebec, Canada. * Marie-Pierre Dubé * Cytochem, Montreal, Quebec, Canada. * Martin M Marcinkiewicz * Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK. * Sreeram Ramagopalan & * George Ebers * Department of Neuropathology, University of Oxford, Oxford, UK. * Olaf Ansorge * UnIGENe, Institute for Molecular and Cell Biology, University of Porto, Porto, Portugal. * Jorge Sequeiros * Hospital Santo António, Porto, Portugal. * Jose Maria Pereira-Monteiro * Genomics Research Centre, Griffith Health Institute, Griffith University, Gold Coast, Queensland, Australia. * Lyn R Griffiths Contributions R.G.L. and M.Z.C. planned the experiments and wrote the manuscript. R.G.L. supervised the PCR, dHPLC and sequence analysis, annotated exon and intron structures and verified all data tables and figures. M.Z.C. provided migraine samples, obtained clinical information, performed the linkage analysis and supervised the protein expression studies. M.-P.D. also performed linkage analysis. J.-F.P. supervised PCR, RT-PCR and screening experiments, did DNA sequence analyses and helped write the manuscript. M.S. supervised and performed dHPLC screening experiments and analyses. N.G. supervised PCR and screening experiments. F.L., K.B. and S.M. performed dHPLC and PCR experiments and provided some sequence analysis. M.M.M. conducted the in situ hybridization experiments. S.R. helped obtain clinical information and linkage analysis. O.A. supervised the protein expression studies. I.A.-E. and S.J.T. performed the electrophysiology and helped write the manuscript. L.R.G., G.E., B.B., J.S! . and J.M.P.-M. provided migraine samples and clinical information. G.A.R. supervised all aspects of the project and edited the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Guy A Rouleau (guy.rouleau@umontreal.ca) or * M Zameel Cader (zameel.cader@dpag.ox.ac.uk) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (108K) Supplementary Tables 1 and 2 and Supplementary Methods Additional data
• Orderly recruitment of motor units under optical control in vivo
  Llewellyn ME Thompson KR Deisseroth K Delp SL - Nat Med 16(10):1161-1165 (2010)
  Nature Medicine | Technical Report Orderly recruitment of motor units under optical control in vivo * Michael E Llewellyn1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kimberly R Thompson1 Search for this author in: * NPG journals * PubMed * Google Scholar * Karl Deisseroth1, 2deissero@stanford.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Scott L Delp1, 3delp@stanford.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 16 ,Pages:1161–1165Year published:(2010)DOI:doi:10.1038/nm.2228Received11 January 2010Accepted30 June 2010Published online26 September 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg A drawback of electrical stimulation for muscle control is that large, fatigable motor units are preferentially recruited before smaller motor units by the lowest-intensity electrical cuff stimulation. This phenomenon limits therapeutic applications because it is precisely the opposite of the normal physiological (orderly) recruitment pattern; therefore, a mechanism to achieve orderly recruitment has been a long-sought goal in physiology, medicine and engineering. Here we demonstrate a technology for reliable orderly recruitment in vivo. We find that under optical control with microbial opsins, recruitment of motor units proceeds in the physiological recruitment sequence, as indicated by multiple independent measures of motor unit recruitment including conduction latency, contraction and relaxation times, stimulation threshold and fatigue. As a result, we observed enhanced performance and reduced fatigue in vivo. These findings point to an unanticipated new modality of neura! l control with broad implications for nervous system and neuromuscular physiology, disease research and therapeutic innovation. View full text Figures at a glance * Figure 1: ChR2 in mouse sciatic nerve. () Confocal image of sciatic nerve in cross-section. Red, fluorescently labeled laminin of the basal lamina of the peripheral nerve. Green, YFP fluorescence expressed from ChR2-YFP fusion protein expressed under control of the Thy1 promoter. Scale bar, 50 μm. () Confocal image of sciatic nerve in longitudinal section; staining as in , illustrating several nodes of Ranvier. Scale bar, 50 μm. () YFP fluorescence intensity versus motor axon size in cross-section (n = 4). () Average YFP fluorescence intensity parallel to the long axis of sampled axons, where the origin indicates the center of the node of Ranvier (n = 15, shaded region represents s.d.). * Figure 2: Optogenetic control of peripheral nerve. () An optical or electrical cuff is placed around the sciatic nerve of an anesthetized Thy1::ChR2 mouse. Inset photomicrograph: custom-designed light-emitting diode–based optical cuff. Fine-wire EMG leads are placed in the muscle of interest; EMG plot shows a typical response from optical stimulation. The Achilles tendon is fixed to a force transducer; force traces show typical raw data of contractions at various frequencies of optical stimulation. The sag in force after initial stimulation seen in optical stimulation probably arises from the biophysical properties of the ChR2 channel itself. An example EMG trace is shown for 500 ms at 60 Hz stimulation. () Typical raw EMG and force traces from twitches elicited by optical and electrical stimulations in Thy1::ChR2 mice and control C57BL/6 mice. The colored bars near each trace indicate the duration of stimulation. * Figure 3: Orderly recruitment and fatigue resistance with optical stimulation. Each point represents the mean ± s.e.m. (optical, n = 5 mice, 625 trials; electrical, n = 5 mice, 573 trials; *P < 0.01). Error bars are present at all points and may be smaller than the data-point markers throughout the figure. () Peak force during a single twitch versus rectified iEMG for both electrical and optical stimulation. () Average latency measured from initiation of stimuli to detection of EMG. () Average contraction time measured from 10% of peak force to peak force. () Average relaxation time measured from peak force to 10% of peak force. () Average tetanic tension over 2 min in muscle being stimulated with 250-ms trains at 1 Hz using electrical and optical stimulation (n = 7, shaded region is s.e.m., average body weight (BW) = 0.258 ± 0.01 N, 2 BW is approximately 20% of maximal isometric tension, Supplementary Methods). () Average fatigue index for electrical and optical stimulation, measured as decline in tetanic tension over 2 min (n = 7). () Exemplar teta! nic tension from a single mouse using both optical and electrical stimulation in hindlimbs over 20 min. * Figure 4: Differential recruitment of soleus and lateral gastrocnemius with electrical and optical stimulation. Each point represents mean ± s.e.m. (optical, n = 5 mice, 1,099 trials; electrical, n = 5 mice, 885 trials; *P < 0.01). Error bars are present at all points and may be smaller than the data-point markers throughout the figure. () Rectified iEMG versus estimated optical intensity at surface of the sciatic nerve for soleus (SOL) and lateral gastrocnemius (LG). () Rectified iEMG versus electrical stimulation voltage applied to sciatic nerve. () Optical intensity required to achieve maximum iEMG in soleus and lateral gastrocnemius. () Electrical stimulation to achieve 95% of maximum iEMG in soleus and lateral gastrocnemius. () Distribution of motor axon diameters for soleus and lateral gastrocnemius found in cross-section of the sciatic nerve. () Distribution of soleus and lateral gastrocnemius motor axon depths from the surface of the sciatic nerve. () Example cross-section of the sciatic nerve where retrograde dye was injected into the lateral gastrocnemius only. Scale bar, 1! 00 μm. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Bioengineering, Stanford University, Stanford, California, USA. * Michael E Llewellyn, * Kimberly R Thompson, * Karl Deisseroth & * Scott L Delp * Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, California, USA. * Karl Deisseroth * Department of Mechanical Engineering, Stanford University, Stanford, California, USA. * Scott L Delp Contributions M.E.L. conducted the experiments, performed the analysis and wrote the manuscript. K.R.T. performed the imaging experiments and wrote the manuscript. S.L.D. and K.D. supervised the project and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Scott L Delp (delp@stanford.edu) or * Karl Deisseroth (deissero@stanford.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (436K) Supplementary Figure 1 and Supplementary Methods Additional data