Friday, October 29, 2010

Hot off the presses! Nov 01 trends cell biol

The Nov 01 issue of the trends cell biol is now up on Pubget (About trends cell biol): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

  • Contents page + Editorial Board
    - trends cell biol 20(11):i (2010)
  • Corrigendum: Acidic calcium stores open for business: expanding the potential for intracellular Ca2+ signaling: [Trends in Cell Biology 20 (2010), 277–286]
    - trends cell biol 20(11):627 (2010)
  • Dicing with dogma: de-branching the lamellipodium
    - trends cell biol 20(11):628-633 (2010)
    The primary event in the movement of a migrating eukaryotic cell is the extension of cytoplasmic sheets termed lamellipodia composed of networks of actin filaments. Lamellipodia networks are thought to arise through the branching of new filaments from the sides of old filaments, producing a dendritic array. Recent studies by electron tomography have revealed the three dimensional organization of lamellipodia and show, contrary to previous evidence, that actin filaments do not form dendritic arrays in vivo. These findings signal a reconsideration of the structural basis of protrusion and about the roles of the different actin nucleating and elongating complexes involved in the process.
  • Timing control in regulatory networks by multisite protein modifications
    - trends cell biol 20(11):634-641 (2010)
    Computational and experimental studies have yielded quantitative insights into the role for multisite phosphorylation, and other protein modifications, in cell function. This work has emphasized the creation of thresholds and switches for cellular decisions. To date, the dynamics of phosphorylation events have been disregarded yet could be equally relevant for cell function. Here, we discuss theoretical predictions about the kinetic functions of multisite phosphorylation in regulatory networks and how these predictions relate to experimental findings. Using DNA replication as an example, we demonstrate that multisite phosphorylations can support coherent origin firing and robustness against rereplication. We suggest that multisite protein modifications provide a molecular mechanism to robustly time cellular events in the cell cycle, the circadian clock and signal transduction.
  • Divide and ProsPer: The emerging role of PtdIns3P in cytokinesis
    - trends cell biol 20(11):642-649 (2010)
    Cytokinesis is the final step of cell division whereby the dividing cells separate physically. Failure of this process has been proposed to cause tumourigenesis. Several specific lipids are essential for cytokinesis, and recent evidence has revealed that phosphatidylinositol 3-phosphate (PtdIns3P) — a well-known regulator of endosomal trafficking, receptor signaling, nutrient sensing and autophagy — plays an evolutionarily conserved role during cytokinesis. The emerging picture is that PtdIns3P and its regulators and effectors constitute a novel regulatory mechanism for cytokinesis. Elucidating the role of PtdIns3P in cytokinesis might contribute to insight into mechanisms of tumour development and suppression.
  • WASH, WHAMM and JMY: regulation of Arp2/3 complex and beyond
    - trends cell biol 20(11):650-661 (2010)
    Arp2/3 complex mediates the nucleation of actin filaments in multiple subcellular processes, and is activated by nucleation-promoting factors (NPFs) from the Wiskott–Aldrich Syndrome family. In exciting new developments, this family has grown by three members: WASH, WHAMM and JMY, which extend the repertoire of dynamic membrane structures that are remodeled following Arp2/3 activation in vivo. These novel NPFs share an actin- and Arp2/3-interacting WCA module, and combine Arp2/3 activation with additional biochemical functions, including capping protein inhibition, microtubule engagement or Arp2/3-independent actin nucleation, none of which had been previously associated with canonical WCA-harboring proteins. Uncovering the physiological relevance of these unique activities will require concerted efforts from multiple disciplines, and is sure to impact our understanding of how the cytoskeleton controls so many dynamic subcellular events.
  • Histone demethylases in development and disease
    - trends cell biol 20(11):662-671 (2010)
    Histone modifications serve as regulatory marks that are instrumental for the control of transcription and chromatin architecture. Strict regulation of gene expression patterns is crucial during development and differentiation, where diverse cell types evolve from common predecessors. Since the first histone lysine demethylase was discovered in 2004, a number of demethylases have been identified and implicated in the control of gene expression programmes and cell fate decisions. Histone demethylases are now emerging as important players in developmental processes and have been linked to human diseases such as neurological disorders and cancer.
  • In control at the ER: PTP1B and the down-regulation of RTKs by dephosphorylation and endocytosis
    - trends cell biol 20(11):672-679 (2010)
    Receptor tyrosine kinases (RTKs) control the cellular response to a range of stimuli by binding extracellular factors and transmitting appropriate signals to intracellular sites. Protein tyrosine phosphatase 1B (PTP1B) modulates the activity of several RTKs by directly targeting the phosphorylated tyrosine residues that dictate their signaling output. Interestingly, the phenotypes of PTP1B deficiency in different contexts point to a more complex role in regulating RTK signaling. Exciting recent results indicate that the endocytic down-regulation of RTKs could be directly controlled by PTP1B. Microscopy studies have demonstrated an effect of PTP1B on post-endocytic internalization of RTKs into multivesicular bodies, and specific substrates that could influence their endosomal trafficking have been identified. These findings reveal a novel link between two important mechanisms of RTK signal attenuation and highlight the multifaceted impact of PTP1B on cell signaling.
  • Cell cholesterol homeostasis: Mediation by active cholesterol
    - trends cell biol 20(11):680-687 (2010)
    Recent evidence suggests that the major pathways mediating cell cholesterol homeostasis respond to a common signal: active membrane cholesterol. Active cholesterol is the fraction that exceeds the complexing capacity of the polar bilayer lipids. Increments in plasma membrane cholesterol exceeding this threshold have an elevated chemical activity (escape tendency) and redistribute via diverse transport proteins to both circulating plasma lipoproteins and intracellular organelles. Active cholesterol thereby prompts several feedback responses. It is the substrate for its own esterification and for the synthesis of regulatory side-chain oxysterols. It also stimulates manifold pathways that down-regulate the biosynthesis, curtail the ingestion and increase the export of cholesterol. Thus, the abundance of cell cholesterol is tightly coupled to that of its polar lipid partners through active cholesterol.
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Thursday, October 28, 2010

Hot off the presses! Nov 05 LANCET

The Nov 05 issue of the LANCET is now up on Pubget (About LANCET): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

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Hot off the presses! Oct 29 Immunity

The Oct 29 issue of the Immunity is now up on Pubget (About Immunity): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

  • A Decade of Vaccines: Integrating Immunology and Vaccinology for Rational Vaccine Design
    - Immunity 33(4):437-440 (2010)
    Vaccination stands as one of the most successful public health measures of the last century. New approaches will be needed, however, to develop highly effective vaccines to prevent tuberculosis, HIV-AIDS, and malaria and to eradicate polio. Current advances in immunology and technology have set the stage for rational vaccine design to begin a "Decade of Vaccines."
  • Vaccines and the Future of Human Immunology
    - Immunity 33(4):441-450 (2010)
    In this issue of Immunity, a collection of detailed reviews summarizes needs, opportunities, and roadblocks to the development of new vaccines, all in the context of our current knowledge and understanding of key aspects of immune function and microbial interactions with the host. This Perspective is designed to provide a broad overview that discusses our present limitations in designing effective novel vaccines for diseases that do not typically induce robust resistance in infected individuals and how the addition of a systems-level, multiplexed approach to the analysis of the human immune system can complement traditional highly focused research efforts to accelerate our progress toward this goal and the improvement of human health.
  • From Vaccines to Memory and Back
    - Immunity 33(4):451-463 (2010)
    Vaccines work by eliciting an immune response and consequent immunological memory that mediates protection from infection or disease. Recently, new methods have been developed to dissect the immune response in experimental animals and humans, which have led to increased understanding of the molecular mechanisms that control differentiation and maintenance of memory T and B cells. In this review we will provide an overview of the cellular organization of immune memory and underline some of the outstanding questions on immunological memory and how they pertain to vaccination strategies. Finally we will discuss how we can learn about antigen design from the interrogation of our memory T and B cells—a journey from vaccines to memory and back.
  • Designing Vaccines Based on Biology of Human Dendritic Cell Subsets
    - Immunity 33(4):464-478 (2010)
    The effective vaccines developed against a variety of infectious agents, including polio, measles, and hepatitis B, represent major achievements in medicine. These vaccines, usually composed of microbial antigens, are often associated with an adjuvant that activates dendritic cells (DCs). Many infectious diseases are still in need of an effective vaccine including HIV, malaria, hepatitis C, and tuberculosis. In some cases, the induction of cellular rather than humoral responses may be more important because the goal is to control and eliminate the existing infection rather than to prevent it. Our increased understanding of the mechanisms of antigen presentation, particularly with the description of DC subsets with distinct functions, as well as their plasticity in responding to extrinsic signals, represent opportunities to develop novel vaccines. In addition, we foresee that this increased knowledge will permit us to design vaccines that will reprogram the immune syste! m to intervene therapeutically in cancer, allergy, and autoimmunity.
  • Vaccination Strategies to Promote Mucosal Antibody Responses
    - Immunity 33(4):479-491 (2010)
    There are great interest and demand for the development of vaccines to prevent and treat diverse microbial infections. Mucosal vaccines elicit immune protection by stimulating the production of antibodies at mucosal surfaces and systemic districts. Being positioned in close proximity to a large community of commensal microbes, the mucosal immune system deploys a heterogeneous population of cells and a complex regulatory network to maintain the balance between surveillance and tolerance. A successful mucosal vaccine relies on leveraging the functions of these immune cells and regulatory components. We review the important cellular interactions and molecular pathways underlying the induction and regulation of mucosal antibody responses and discuss their implications on mucosal vaccination.
  • Vaccine Adjuvants: Putting Innate Immunity to Work
    - Immunity 33(4):492-503 (2010)
    Adjuvants enhance immunity to vaccines and experimental antigens by a variety of mechanisms. In the past decade, many receptors and signaling pathways in the innate immune system have been defined and these innate responses strongly influence the adaptive immune response. The focus of this review is to delineate the innate mechanisms by which adjuvants mediate their effects. We highlight how adjuvants can be used to influence the magnitude and alter the quality of the adaptive response in order to provide maximum protection against specific pathogens. Despite the impressive success of currently approved adjuvants for generating immunity to viral and bacterial infections, there remains a need for improved adjuvants that enhance protective antibody responses, especially in populations that respond poorly to current vaccines. However, the larger challenge is to develop vaccines that generate strong T cell immunity with purified or recombinant vaccine antigens.
  • Immunologic Basis of Vaccine Vectors
    - Immunity 33(4):504-515 (2010)
    Efforts to make vaccines against infectious diseases as well as immunotherapies for cancer, autoimmune diseases and allergy have utilized a variety of heterologous expression systems, including viral and bacterial vectors, as well as DNA and RNA constructs. This review explores the immunologic rationale and provides an update of insights obtained from preclinical and clinical studies of such vaccines.
  • Systems Vaccinology
    - Immunity 33(4):516-529 (2010)
    Vaccination is one of the greatest triumphs of modern medicine, yet we remain largely ignorant of the mechanisms by which successful vaccines stimulate protective immunity. Two recent advances are beginning to illuminate such mechanisms: realization of the pivotal role of the innate immune system in sensing microbes and stimulating adaptive immunity, and advances in systems biology. Recent studies have used systems biology approaches to obtain a global picture of the immune responses to vaccination in humans. This has enabled the identification of early innate signatures that predict the immunogenicity of vaccines, and identification of potentially novel mechanisms of immune regulation. Here, we review these advances and critically examine the potential opportunities and challenges posed by systems biology in vaccine development.
  • Reverse Vaccinology: Developing Vaccines in the Era of Genomics
    - Immunity 33(4):530-541 (2010)
    The sequence of microbial genomes made all potential antigens of each pathogen available for vaccine development. This increased by orders of magnitude potential vaccine targets in bacteria, parasites, and large viruses and revealed virtually all their CD4+ and CD8+ T cell epitopes. The genomic information was first used for the development of a vaccine against serogroup B meningococcus, and it is now being used for several other bacterial vaccines. In this review, we will first summarize the impact that genome sequencing has had on vaccine development, and then we will analyze how the genomic information can help further our understanding of immunity to infection or vaccination and lead to the design of better vaccines by diving into the world of T cell immunity.
  • Induction of Immunity to Human Immunodeficiency Virus Type-1 by Vaccination
    - Immunity 33(4):542-554 (2010)
    Recent findings have brought optimism that development of a successful human immunodeficiency virus type-1 (HIV-1) vaccine lies within reach. Studies of early events in HIV-1 infection have revealed when and where HIV-1 is potentially vulnerable to vaccine-targeted immune responses. With technical advances in human antibody production, clues about how antibodies recognize HIV-1 envelope proteins have uncovered new targets for immunogen design. A recent vaccine regimen has shown modest efficacy against HIV-1 acquisition. However, inducing long-term T and B cell memory and coping with HIV-1 diversity remain high priorities. Mediators of innate immunity may play pivotal roles in blocking infection and shaping immunity; vaccine strategies to capture these activities are under investigation. Challenges remain in integrating basic, preclinical and clinical research to improve predictions of types of immunity associated with vaccine efficacy, to apply these insights to immuno! gen design, and to accelerate evaluation of vaccine efficacy in persons at-risk for infection.
  • Malaria Vaccine Design: Immunological Considerations
    - Immunity 33(4):555-566 (2010)
    The concept of a malaria vaccine has sparked great interest for decades; however, the challenge is proving to be a difficult one. Immune dysregulation by Plasmodium and the ability of the parasite to mutate critical epitopes in surface antigens have proved to be strong defense weapons. This has led to reconsideration of polyvalent and whole parasite strategies and ways to enhance cellular immunity to malaria that may be more likely to target conserved antigens and an expanded repertoire of antigens. These and other concepts will be discussed in this review.
  • Future Vaccination Strategies against Tuberculosis: Thinking outside the Box
    - Immunity 33(4):567-577 (2010)
    With almost a dozen vaccine candidates in clinical trials, tuberculosis (TB) research and development is finally reaping the first fruits of its labors. Vaccine candidates in clinical trials may prevent TB disease reactivation by efficiently containing the pathogen Mycobacterium tuberculosis (Mtb). Future research should target vaccines that achieve sterile eradication of Mtb or even prevent stable infection. These are ambitious goals that can be reached only by highly cooperative engagement of basic immunologists, vaccinologists, and clinical researchers—or in other words, by translation from basic immunology to vaccine research and development, as well as reverse translation of insights from clinical trials back to hypothesis-driven research in the basic laboratory. Here, we review current and future strategies toward the rational design of novel vaccines against TB, as well as the progress made thus far, and the hurdles that need to be overcome in the near and dis! tant future.
  • Recycling Endosomes and TLR Signaling— The Rab11 GTPase Leads the Way
    - Immunity 33(4):578-580 (2010)
    The ability of Toll-like receptors (TLRs) to activate innate immunity depends on their transport to pathogen-containing organelles. In this issue of Immunity, Husebye et al. (2010) report that delivery of TLR4 to phagosomes occurs via a recycling endosome intermediate, which is controlled by the GTPase Rab11a.
  • A Flt3L Encounter: mTOR Signaling in Dendritic Cells
    - Immunity 33(4):580-582 (2010)
    The signaling pathway of the cytokine Flt3L in dendritic cells (DCs) is poorly defined. In this issue of Immunity, Sathaliyawala et al. (2010) report that the kinase mTOR functions as a mediator of Flt3L signaling in the development and homeostasis of DCs, particularly of the CD8+ and CD103+ DCs.
  • The Rab11a GTPase Controls Toll-like Receptor 4-Induced Activation of Interferon Regulatory Factor-3 on Phagosomes
    - Immunity 33(4):583-596 (2010)
    Toll-like receptor 4 (TLR4) is indispensable for recognition of Gram-negative bacteria. We described a trafficking pathway for TLR4 from the endocytic recycling compartment (ERC) to E. coli phagosomes. We found a prominent colocalization between TLR4 and the small GTPase Rab11a in the ERC, and Rab11a was involved in the recruitment of TLR4 to phagosomes in a process requiring TLR4 signaling. Also, Toll-receptor-associated molecule (TRAM) and interferon regulatory factor-3 (IRF3) localized to E. coli phagosomes and internalization of E. coli was required for a robust interferon-β induction. Suppression of Rab11a reduced TLR4 in the ERC and on phagosomes leading to inhibition of the IRF3 signaling pathway induced by E. coli, whereas activation of the transcription factor NF-κB was unaffected. Moreover, Rab11a silencing reduced the amount of TRAM on phagosomes. Thus, Rab11a is an important regulator of TLR4 and TRAM transport to E. coli phagosomes thereby controlling IR! F3 activation from this compartment.
  • Mammalian Target of Rapamycin Controls Dendritic Cell Development Downstream of Flt3 Ligand Signaling
    - Immunity 33(4):597-606 (2010)
    Dendritic cells (DCs) comprise distinct functional subsets including CD8− and CD8+ classical DCs (cDCs) and interferon-secreting plasmacytoid DCs (pDCs). The cytokine Flt3 ligand (Flt3L) controls the development of DCs and is particularly important for the pDC and CD8+ cDC and their CD103+ tissue counterparts. We report that mammalian target of rapamycin (mTOR) inhibitor rapamycin impaired Flt3L-driven DC development in vitro, with the pDCs and CD8+-like cDCs most profoundly affected. Conversely, deletion of the phosphoinositide 3-kinase (PI3K)-mTOR negative regulator Pten facilitated Flt3L-driven DC development in culture. DC-specific Pten targeting in vivo caused the expansion of CD8+ and CD103+ cDC numbers, which was reversible by rapamycin. The increased CD8+ cDC numbers caused by Pten deletion correlated with increased susceptibility to the intracellular pathogen Listeria. Thus, PI3K-mTOR signaling downstream of Flt3L controls DC development, and its restriction! by Pten ensures optimal DC pool size and subset composition.
  • MicroRNA-155 Promotes Autoimmune Inflammation by Enhancing Inflammatory T Cell Development
    - Immunity 33(4):607-619 (2010)
    Mammalian noncoding microRNAs (miRNAs) are a class of gene regulators that have been linked to immune system function. Here, we have investigated the role of miR-155 during an autoimmune inflammatory disease. Consistent with a positive role for miR-155 in mediating inflammatory responses, Mir155−/− mice were highly resistant to experimental autoimmune encephalomyelitis (EAE). miR-155 functions in the hematopoietic compartment to promote the development of inflammatory T cells including the T helper 17 (Th17) cell and Th1 cell subsets. Furthermore, the major contribution of miR-155 to EAE was CD4+ T cell intrinsic, whereas miR-155 was also required for optimum dendritic cell production of cytokines that promoted Th17 cell formation. Our study shows that one aspect of miR-155 function is the promotion of T cell-dependent tissue inflammation, suggesting that miR-155 might be a promising therapeutic target for the treatment of autoimmune disorders.
  • Carcinoembryonic Antigen-Related Cell Adhesion Molecule-1 Regulates Granulopoiesis by Inhibition of Granulocyte Colony-Stimulating Factor Receptor
    - Immunity 33(4):620-631 (2010)
    Although carcinoembryonic antigen-related cell adhesion moclecule-1 (CEACAM1) is an activation marker for neutrophils and delays neutrophil apoptosis, the role of CEACAM1 in granulopoiesis and neutrophil-dependent host immune responses has not been investigated. CEACAM1 expression correlated with granulocytic differentiation, and Ceacam1−/− mice developed neutrophilia because of loss of the Src-homology-phosphatase-1 (SHP-1)-dependent inhibition of granulocyte colony-stimulating factor receptor (G-CSFR) signal transducer and activator of transcription (Stat3) pathway provided by CEACAM1. Moreover, Ceacam1−/− mice were hypersensitive to Listeria Monocytogenes (LM) infection with an accelerated mortality. Reintroduction of CEACAM1 into Ceacam1−/− bone marrow restored normal granulopoiesis and host sensitivity to LM infection, while mutation of its immunoreceptor tyrosine-based inhibitory motifs (ITIMs) abrogated this restoration. shRNA-mediated reduction of S! tat3 amounts rescued normal granulopoiesis, attenuating host sensitivity to LM infection in Ceacam1−/− mice. Thus, CEACAM1 acted as a coinhibitory receptor for G-CSFR regulating granulopoiesis and host innate immune response to bacterial infections.
  • Intravital Imaging Reveals Distinct Dynamics for Natural Killer and CD8+ T Cells during Tumor Regression
    - Immunity 33(4):632-644 (2010)
    Recognition of NKG2D ligands by natural killer (NK) cells plays an important role during antitumoral responses. To address how NKG2D engagement affects intratumoral NK cell dynamics, we performed intravital microscopy in a Rae-1β-expressing solid tumor. This NKG2D ligand drove NK cell accumulation, activation, and motility within the tumor. NK cells established mainly dynamic contacts with their targets during tumor regression. In sharp contrast, cytotoxic T lymphocytes (CTLs) formed stable contacts in tumors expressing their cognate antigen. Similar behaviors were observed during effector functions in lymph nodes. In vitro, contacts between NK cells and their targets were cytotoxic but did not elicit sustained calcium influx nor adhesion, whereas CTL contact stability was critically dependent on extracellular calcium entry. Altogether, our results offer mechanistic insight into how NK cells and CTLs can exert cytotoxic activity with remarkably different contact dynam! ics.
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Hot off the presses! Oct 29 Cell

The Oct 29 issue of the Cell is now up on Pubget (About Cell): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

  • In This Issue
    - cell 143(3):327, 329 (2010)
  • Select: Gut Microbes
    - cell 143(3):331, 333 (2010)
    Our intestines host trillions of bacteria, most of which are beneficial to our health most of the time. Occasionally, however, a change in conditions, or the entry of a pathogenic strain, leads to disease. Recent papers shed new light onto the complex interactions that determine intestinal health and disease.
  • ATRX: Put Me on Repeat
    - cell 143(3):335-336 (2010)
    Mutations in the chromatin-remodeling protein ATRX cause alpha thalassaemia and mental retardation, but the severity of the disorder is independent of the specific mutation. In this issue of Cell, Law et al. (2010) demonstrate that ATRX alters gene expression by binding to G-rich tandem repeats, and the degree of transcriptional silencing caused by ATRX mutations correlates with the number of repeats.
  • Egg's ZP3 Structure Speaks Volumes
    - cell 143(3):337-338 (2010)
    Binding of mammalian sperm to eggs depends in part on ZP3, a glycoprotein in the egg's extracellular coat, the zona pellucida. In this issue, Han et al. (2010) describe the structure of an avian ZP3 homolog, providing insights into ZP3 processing and polymerization and the roles of the ZP3 polypeptide and its carbohydrate in sperm binding.
  • Monocytes Join the Dendritic Cell Family
    - cell 143(3):339-340 (2010)
    Dendritic cells are professional antigen-presenting cells that mediate immunity and tolerance. Cheong et al. (2010) uncover a new route for dendritic cell production in vivo. They show that in response to infection by gram-negative bacteria, monocytes are recruited to the lymph node where they rapidly differentiate into dendritic cells that present antigens to T cells.
  • Ephecting Excitatory Synapse Development
    - cell 143(3):341-342 (2010)
    Alterations in synapse number and morphology are associated with devastating psychiatric and neurologic disorders. In this issue of Cell, Margolis et al. (2010) show that the RhoA-guanine exchange factor (GEF) Ephexin5 limits the numbers of excitatory synapses that neurons receive, thus identifying a new mechanism controlling synaptogenesis.
  • Chemoaffinity Revisited: Dscams, Protocadherins, and Neural Circuit Assembly
    - cell 143(3):343-353 (2010)
    The chemoaffinity hypothesis for neural circuit assembly posits that axons and their targets bear matching molecular labels that endow neurons with unique identities and specify synapses between appropriate partners. Here, we focus on two intriguing candidates for fulfilling this role, Drosophila Dscams and vertebrate clustered protocadherins (Pcdhs). In each, a complex genomic locus encodes large numbers of neuronal transmembrane proteins with homophilic binding specificity, individual members of which are expressed combinatorially. Although these properties suggest that Dscams and Pcdhs could act as specificity molecules, they may do so in ways that challenge traditional views of how neural circuits assemble.
  • DNA Damage-Mediated Induction of a Chemoresistant Niche
    - cell 143(3):355-366 (2010)
    While numerous cell-intrinsic processes are known to play decisive roles in chemotherapeutic response, relatively little is known about the impact of the tumor microenvironment on therapeutic outcome. Here, we use a well-established mouse model of Burkitt's lymphoma to show that paracrine factors in the tumor microenvironment modulate lymphoma cell survival following the administration of genotoxic chemotherapy. Specifically, IL-6 and Timp-1 are released in the thymus in response to DNA damage, creating a "chemo-resistant niche" that promotes the survival of a minimal residual tumor burden and serves as a reservoir for eventual tumor relapse. Notably, IL-6 is released acutely from thymic endothelial cells in a p38-dependent manner following genotoxic stress, and this acute secretory response precedes the gradual induction of senescence in tumor-associated stromal cells. Thus, conventional chemotherapies can induce tumor regression while simultaneously eliciting str! ess responses that protect subsets of tumor cells in select anatomical locations from drug action. PaperFlick To view the video inline, enable JavaScript on your browser. However, you can download and view the video by clicking on the icon below Download this Video (17520 K)
  • ATR-X Syndrome Protein Targets Tandem Repeats and Influences Allele-Specific Expression in a Size-Dependent Manner
    - cell 143(3):367-378 (2010)
    ATRX is an X-linked gene of the SWI/SNF family, mutations in which cause syndromal mental retardation and downregulation of α-globin expression. Here we show that ATRX binds to tandem repeat (TR) sequences in both telomeres and euchromatin. Genes associated with these TRs can be dysregulated when ATRX is mutated, and the change in expression is determined by the size of the TR, producing skewed allelic expression. This reveals the characteristics of the affected genes, explains the variable phenotypes seen with identical ATRX mutations, and illustrates a new mechanism underlying variable penetrance. Many of the TRs are G rich and predicted to form non-B DNA structures (including G-quadruplex) in vivo. We show that ATRX binds G-quadruplex structures in vitro, suggesting a mechanism by which ATRX may play a role in various nuclear processes and how this is perturbed when ATRX is mutated. PaperClip To listen to this audio, enable JavaScript on your browser. However, you can download and play the audio by clicking on the icon below Download this Audio (3918 K)
  • Upf1 Senses 3′UTR Length to Potentiate mRNA Decay
    - cell 143(3):379-389 (2010)
    The selective degradation of mRNAs by the nonsense-mediated decay pathway is a quality control process with important consequences for human disease. From initial studies using RNA hairpin-tagged mRNAs for purification of messenger ribonucleoproteins assembled on transcripts with HIV-1 3′ untranslated region (3′UTR) sequences, we uncover a two-step mechanism for Upf1-dependent degradation of mRNAs with long 3′UTRs. We demonstrate that Upf1 associates with mRNAs in a 3′UTR length-dependent manner and is highly enriched on transcripts containing 3′UTRs known to elicit NMD. Surprisingly, Upf1 recruitment and subsequent RNA decay can be antagonized by retroviral RNA elements that promote translational readthrough. By modulating the efficiency of translation termination, recognition of long 3′UTRs by Upf1 is uncoupled from the initiation of decay. We propose a model for 3′UTR length surveillance in which equilibrium binding of Upf1 to mRNAs precedes a kinetica! lly distinct commitment to RNA decay.
  • The Long Noncoding RNA, Jpx, Is a Molecular Switch for X Chromosome Inactivation
    - cell 143(3):390-403 (2010)
    Once protein-coding, the X-inactivation center (Xic) is now dominated by large noncoding RNAs (ncRNA). X chromosome inactivation (XCI) equalizes gene expression between mammalian males and females by inactivating one X in female cells. XCI requires Xist, an ncRNA that coats the X and recruits Polycomb proteins. How Xist is controlled remains unclear but likely involves negative and positive regulators. For the active X, the antisense Tsix RNA is an established Xist repressor. For the inactive X, here, we identify Xic-encoded Jpx as an Xist activator. Jpx is developmentally regulated and accumulates during XCI. Deleting Jpx blocks XCI and is female lethal. Posttranscriptional Jpx knockdown recapitulates the knockout, and supplying Jpx in trans rescues lethality. Thus, Jpx is trans-acting and functions as ncRNA. Furthermore, ΔJpx is rescued by truncating Tsix, indicating an antagonistic relationship between the ncRNAs. We conclude that Xist is controlled by two RNA-base! d switches: Tsix for Xa and Jpx for Xi.
  • Insights into Egg Coat Assembly and Egg-Sperm Interaction from the X-Ray Structure of Full-Length ZP3
    - cell 143(3):404-415 (2010)
    ZP3, a major component of the zona pellucida (ZP) matrix coating mammalian eggs, is essential for fertilization by acting as sperm receptor. By retaining a propeptide that contains a polymerization-blocking external hydrophobic patch (EHP), we determined the crystal structure of an avian homolog of ZP3 at 2.0 Å resolution. The structure unveils the fold of a complete ZP domain module in a homodimeric arrangement required for secretion and reveals how EHP prevents premature incorporation of ZP3 into the ZP. This suggests mechanisms underlying polymerization and how local structural differences, reflected by alternative disulfide patterns, control the specificity of ZP subunit interaction. Close relative positioning of a conserved O-glycan important for sperm binding and the hypervariable, positively selected C-terminal region of ZP3 suggests a concerted role in the regulation of species-restricted gamete recognition. Alternative conformations of the area around the O-g! lycan indicate how sperm binding could trigger downstream events via intramolecular signaling.
  • Microbial Stimulation Fully Differentiates Monocytes to DC-SIGN/CD209+ Dendritic Cells for Immune T Cell Areas
    - cell 143(3):416-429 (2010)
    Dendritic cells (DCs), critical antigen-presenting cells for immune control, normally derive from bone marrow precursors distinct from monocytes. It is not yet established if the large reservoir of monocytes can develop into cells with critical features of DCs in vivo. We now show that fully differentiated monocyte-derived DCs (Mo-DCs) develop in mice and DC-SIGN/CD209a marks the cells. Mo-DCs are recruited from blood monocytes into lymph nodes by lipopolysaccharide and live or dead gram-negative bacteria. Mobilization requires TLR4 and its CD14 coreceptor and Trif. When tested for antigen-presenting function, Mo-DCs are as active as classical DCs, including cross-presentation of proteins and live gram-negative bacteria on MHC I in vivo. Fully differentiated Mo-DCs acquire DC morphology and localize to T cell areas via L-selectin and CCR7. Thus the blood monocyte reservoir becomes the dominant presenting cell in response to select microbes, yielding DC-SIGN+ cells with! critical functions of DCs.
  • Endophilin Functions as a Membrane-Bending Molecule and Is Delivered to Endocytic Zones by Exocytosis
    - cell 143(3):430-441 (2010)
    Two models have been proposed for endophilin function in synaptic vesicle (SV) endocytosis. The scaffolding model proposes that endophilin's SH3 domain recruits essential endocytic proteins, whereas the membrane-bending model proposes that the BAR domain induces positively curved membranes. We show that mutations disrupting the scaffolding function do not impair endocytosis, whereas those disrupting membrane bending cause significant defects. By anchoring endophilin to the plasma membrane, we show that endophilin acts prior to scission to promote endocytosis. Despite acting at the plasma membrane, the majority of endophilin is targeted to the SV pool. Photoactivation studies suggest that the soluble pool of endophilin at synapses is provided by unbinding from the adjacent SV pool and that the unbinding rate is regulated by exocytosis. Thus, endophilin participates in an association-dissociation cycle with SVs that parallels the cycle of exo- and endocytosis. This endop! hilin cycle may provide a mechanism for functionally coupling endocytosis and exocytosis.
  • EphB-Mediated Degradation of the RhoA GEF Ephexin5 Relieves a Developmental Brake on Excitatory Synapse Formation
    - cell 143(3):442-455 (2010)
    The mechanisms that promote excitatory synapse formation and maturation have been extensively studied. However, the molecular events that limit excitatory synapse development so that synapses form at the right time and place and in the correct numbers are less well understood. We have identified a RhoA guanine nucleotide exchange factor, Ephexin5, which negatively regulates excitatory synapse development until EphrinB binding to the EphB receptor tyrosine kinase triggers Ephexin5 phosphorylation, ubiquitination, and degradation. The degradation of Ephexin5 promotes EphB-dependent excitatory synapse development and is mediated by Ube3A, a ubiquitin ligase that is mutated in the human cognitive disorder Angelman syndrome and duplicated in some forms of Autism Spectrum Disorders (ASDs). These findings suggest that aberrant EphB/Ephexin5 signaling during the development of synapses may contribute to the abnormal cognitive function that occurs in Angelman syndrome and, poss! ibly, ASDs.
  • Imaging Activity-Dependent Regulation of Neurexin-Neuroligin Interactions Using trans-Synaptic Enzymatic Biotinylation
    - cell 143(3):456-469 (2010)
    The functions of trans-synaptic adhesion molecules, such as neurexin and neuroligin, have been difficult to study due to the lack of methods to directly detect their binding in living neurons. Here, we use biotin labeling of intercellular contacts (BLINC), a method for imaging protein interactions based on interaction-dependent biotinylation of a peptide by E. coli biotin ligase, to visualize neurexin-neuroligin trans-interactions at synapses and study their role in synapse development. We found that both developmental maturation and acute synaptic activity stimulate the growth of neurexin-neuroligin adhesion complexes via a combination of neurexin and neuroligin surface insertion and internalization arrest. Both mechanisms require NMDA receptor activity. We also discovered that disruption of activity-induced neurexin-neuroligin complex growth prevents recruitment of the AMPA receptor, a hallmark of mature synapses. Our results provide support for neurexin-neuroligin f! unction in synapse maturation and introduce a general method to study intercellular protein-protein interactions.
  • Nucleosome-Interacting Proteins Regulated by DNA and Histone Methylation
    - cell 143(3):470-484 (2010)
    Modifications on histones or on DNA recruit proteins that regulate chromatin function. Here, we use nucleosomes methylated on DNA and on histone H3 in an affinity assay, in conjunction with a SILAC-based proteomic analysis, to identify "crosstalk" between these two distinct classes of modification. Our analysis reveals proteins whose binding to nucleosomes is regulated by methylation of CpGs, H3K4, H3K9, and H3K27 or a combination thereof. We identify the origin recognition complex (ORC), including LRWD1 as a subunit, to be a methylation-sensitive nucleosome interactor that is recruited cooperatively by DNA and histone methylation. Other interactors, such as the lysine demethylase Fbxl11/KDM2A, recognize nucleosomes methylated on histones, but their recruitment is disrupted by DNA methylation. These data establish SILAC nucleosome affinity purifications (SNAP) as a tool for studying the dynamics between different chromatin modifications and provide a modification b! inding "profile" for proteins regulated by DNA and histone methylation.
  • Retraction Notice to: Assembly of Endogenous oskar mRNA Particles for Motor-Dependent Transport in the Drosophila Oocyte
    - cell 143(3):485 (2010)
  • SnapShot: Neural Crest
    - cell 143(3):486-486.e1 (2010)
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Hot off the presses! Nov 01 Nat Meth

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Latest Articles Include:

  • Nobel thoughts
    - Nat Meth 7(11):859 (2010)
    ARTICLE NAVIGATION - ISSUE Previous November 2010, Volume 7 No 11 pp859-935 * In This Issue * Editorial * This Month * Correspondence * Research Highlights * Technology Feature * News and Views * Brief Communications * ArticlesAbout the cover In This Issue PDF - In This Issue Editorial Nobel thoughts - p859 doi:10.1038/nmeth1110-859 The community of scientists should celebrate the Nobel Prize, even if awards bestowed on one discipline are associated with another discipline. A new prize might help. Abstract - Nobel thoughts | Full Text - Nobel thoughts | PDF (65 KB) - Nobel thoughts This Month The author file: Rolf Zeller and Javier Lopez-Rios - p861 Monya Baker doi:10.1038/nmeth1110-861 Gene cutting and pasting just got a whole lot faster. Abstract - The author file: Rolf Zeller and Javier Lopez-Rios | Full Text - The author fileRolf Zeller and Javier Lopez-Rios | PDF (152 KB) - The author fileRolf Zeller and Javier Lopez-Rios Points of View: Gestalt principles (Part 1) - p863 Bang Wong doi:10.1038/nmeth1110-863 Full Text - Points of ViewGestalt principles (Part 1) | PDF (119 KB) - Points of ViewGestalt principles (Part 1) ADVERTISEMENT Correspondence Membrane molecules mobile even after chemical fixation - pp865 - 866 Kenji A K Tanaka, Kenichi G N Suzuki, Yuki M Shirai, Shusaku T Shibutani, Manami S H Miyahara, Hisae Tsuboi, Miyako Yahara, Akihiko Yoshimura, Satyajit Mayor, Takahiro K Fujiwara & Akihiro Kusumi doi:10.1038/nmeth.f.314 Full Text - Membrane molecules mobile even after chemical fixation | PDF (642 KB) - Membrane molecules mobile even after chemical fixation | Supplementary information Federal policy and the use of pluripotent stem cells - pp866 - 867 Christopher Thomas Scott, Jennifer B McCormick, Mindy C DeRouen & Jason Owen-Smith doi:10.1038/nmeth1110-866 Full Text - Federal policy and the use of pluripotent stem cells | PDF (153 KB) - Federal policy and the use of pluripotent stem cells Research Highlights Finding the trees in the forest - p869 Nicole Rusk doi:10.1038/nmeth1110-869 The integration of quantitative proteomics and analysis by machine learning yields a refined list of proteins involved in chromosome function. Abstract - Finding the trees in the forest | Full Text - Finding the trees in the forest | PDF (211 KB) - Finding the trees in the forest Protein structure gets exciting - pp870 - 871 Allison Doerr doi:10.1038/nmeth1110-870a Researchers determined the excited-state structure of a small protein using nuclear magnetic resonance spectroscopy. Abstract - Protein structure gets exciting | Full Text - Protein structure gets exciting | PDF (168 KB) - Protein structure gets exciting Many mini mind promoters - pp870 - 871 Natalie de Souza doi:10.1038/nmeth1110-870b Tools to drive restricted gene expression in the brain. Abstract - Many mini mind promoters | Full Text - Many mini mind promoters | PDF (168 KB) - Many mini mind promoters News in brief - p871 doi:10.1038/nmeth1110-871 Full Text - News in brief | PDF (143 KB) - News in brief Species collage - p872 Erika Pastrana doi:10.1038/nmeth1110-872 A new study reports the first viable rat-mouse chimeras and uses rat induced pluripotent stem cells to rescue organ deficiency in mice. Abstract - Species collage | Full Text - Species collage | PDF (108 KB) - Species collage Hidden code in the protein code - p874 Monya Baker doi:10.1038/nmeth1110-874 Apparently redundant codons may not be redundant after all. Abstract - Hidden code in the protein code | Full Text - Hidden code in the protein code | PDF (83 KB) - Hidden code in the protein code Self-healing light beams - p876 Daniel Evanko doi:10.1038/nmeth1110-876 The self-reconstructing properties of Bessel beams provide healing benefits in highly scattering media. Abstract - Self-healing light beams | Full Text - Self-healing light beams | PDF (108 KB) - Self-healing light beams Technology Feature From promising to practical: tools to study networks of neurons - pp877 - 883 Monya Baker doi:10.1038/nmeth1110-877 Combinations of electrophysiology, two-photon microscopy and new tools for detecting neural activity show how neurons function in circuits. Abstract - From promising to practical: tools to study networks of neurons | Full Text - From promising to practical: tools to study networks of neurons | PDF (733 KB) - From promising to practical: tools to study networks of neurons News and Views Defining pluripotency - pp885 - 887 Martin F Pera doi:10.1038/nmeth1110-885 Retroviral marking of single human embryonic stem cells shows that cultures of these cells contain subpopulations with distinct functional properties. Full Text - Defining pluripotency | PDF (857 KB) - Defining pluripotency See also:Article by Stewart et al. DNA construction: homemade or ordered out? - pp887 - 889 Peter A Carr doi:10.1038/nmeth1110-887 Automation and optimization of DNA construction results in the efficient production of large target sequences. Full Text - DNA construction: homemade or ordered out? | PDF (214 KB) - DNA construction: homemade or ordered out? See also:Brief Communication by Gibson et al. Pacing lightly: optogenetics gets to the heart - pp889 - 891 Björn C Knollmann doi:10.1038/nmeth1110-889 Transfer of the light-activated cation channel channelrhodopsin-2 gene enables optical control of heart muscle membrane potential. Full Text - Pacing lightly: optogenetics gets to the heart | PDF (400 KB) - Pacing lightly: optogenetics gets to the heart See also:Brief Communication by Bruegmann et al. Brief Communications Dual RMCE for efficient re-engineering of mouse mutant alleles - pp893 - 895 Marco Osterwalder, Antonella Galli, Barry Rosen, William C Skarnes, Rolf Zeller & Javier Lopez-Rios doi:10.1038/nmeth.1521 An efficient one-step method for re-engineering mouse mutant alleles harboring loxP and FRT sites is reported. It may be applied to the large collection of targeted alleles from the International Knockout Mouse Consortium. Abstract - Dual RMCE for efficient re-engineering of mouse mutant alleles | Full Text - Dual RMCE for efficient re-engineering of mouse mutant alleles | PDF (565 KB) - Dual RMCE for efficient re-engineering of mouse mutant alleles | Supplementary information Optogenetic control of heart muscle in vitro and in vivo- pp897 - 900 Tobias Bruegmann, Daniela Malan, Michael Hesse, Thomas Beiert, Christopher J Fuegemann, Bernd K Fleischmann & Philipp Sasse doi:10.1038/nmeth.1512 Stimulation of the light-activated cation channel channelrhodopsin-2 can depolarize heart muscle in vitro and in vivo, resulting in precise localized stimulation and constant prolonged depolarization of genetically targeted cardiomyocytes and cardiac tissue. Abstract - Optogenetic control of heart muscle in vitro and in vivo | Full Text - Optogenetic control of heart muscle in vitro and in vivo | PDF (1,355 KB) - Optogenetic control of heart muscle in vitro and in vivo | Supplementary information See also:News and Views by Knollmann Chemical synthesis of the mouse mitochondrial genome - pp901 - 903 Daniel G Gibson, Hamilton O Smith, Clyde A Hutchison III, J Craig Venter & Chuck Merryman doi:10.1038/nmeth.1515 Using 600 oligonucleotides with 60 bases each and three enzymes, the authors assemble the entire mouse mitochondrial genome in four isothermal reactions. Abstract - Chemical synthesis of the mouse mitochondrial genome | Full Text - Chemical synthesis of the mouse mitochondrial genome | PDF (488 KB) - Chemical synthesis of the mouse mitochondrial genome | Supplementary information See also:News and Views by Carr Efficient CNS gene delivery by intravenous injection - pp905 - 907 Jean-Pierre Louboutin, Alena A Chekmasova, Elena Marusich, J Roy Chowdhury & David S Strayer doi:10.1038/nmeth.1518 Recombinant SV40 viral vectors intravenously injected into mice pretreated with mannitol effectively deliver transgenes to adult neurons in several regions of the central nervous system. Abstract - Efficient CNS gene delivery by intravenous injection | Full Text - Efficient CNS gene delivery by intravenous injection | PDF (1,239 KB) - Efficient CNS gene delivery by intravenous injection | Supplementary information De novo assembly and analysis of RNA-seq data - pp909 - 912 Gordon Robertson, Jacqueline Schein, Readman Chiu, Richard Corbett, Matthew Field, Shaun D Jackman, Karen Mungall, Sam Lee, Hisanaga Mark Okada, Jenny Q Qian, Malachi Griffith, Anthony Raymond, Nina Thiessen, Timothee Cezard, Yaron S Butterfield, Richard Newsome, Simon K Chan, Rong She, Richard Varhol, Baljit Kamoh, Anna-Liisa Prabhu, Angela Tam, YongJun Zhao, Richard A Moore, Martin Hirst, Marco A Marra, Steven J M Jones, Pamela A Hoodless & Inanc Birol doi:10.1038/nmeth.1517 The Trans-ABySS pipeline is an integrated approach for transcript assembly and analysis to identify new mRNA isoforms and structures. Abstract - De novo assembly and analysis of RNA-seq data | Full Text - De novo assembly and analysis of RNA-seq data | PDF (515 KB) - De novo assembly and analysis of RNA-seq data | Supplementary information Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples - pp913 - 915 Isaäc J Nijman, Michal Mokry, Ruben van Boxtel, Pim Toonen, Ewart de Bruijn & Edwin Cuppen doi:10.1038/nmeth.1516 By pooling barcoded genomes of thirty rats before enrichment of a 1.4-megabase target sequence, mutation discovery in 770 genes is achieved with high accuracy. Abstract - Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples | Full Text - Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples | PDF (372 KB) - Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples | Supplementary information Articles Clonal tracking of hESCs reveals differential contribution to functional assays - pp917 - 922 Morag H Stewart, Sean C Bendall, Marilyne Levadoux-Martin & Mickie Bhatia doi:10.1038/nmeth.1519 Retroviral integration is used to mark clones in human embryonic stem cell cultures and clonal distribution is assessed after functionally testing the cells with different methods. Distinct subsets of clones are detected after in vitro differentiation versus teratoma formation in vivo. Abstract - Clonal tracking of hESCs reveals differential contribution to functional assays | Full Text - Clonal tracking of hESCs reveals differential contribution to functional assays | PDF (1,421 KB) - Clonal tracking of hESCs reveals differential contribution to functional assays | Supplementary information See also:News and Views by Pera Trans-SILAC: sorting out the non-cell-autonomous proteome - pp923 - 927 Oded Rechavi, Matan Kalman, Yuan Fang, Helly Vernitsky, Jasmine Jacob-Hirsch, Leonard J Foster, Yoel Kloog & Itamar Goldstein doi:10.1038/nmeth.1513 Proteins can be transferred between cells in contact, such as via trogocytosis in lymphocytes, or acquired via bacteria-host interactions during infection. A quantitative proteomics approach to identify such non-cell-autonomous proteins is described. Abstract - Trans-SILAC: sorting out the non-cell-autonomous proteome | Full Text - Trans-SILAC: sorting out the non-cell-autonomous proteome | PDF (957 KB) - Trans-SILAC: sorting out the non-cell-autonomous proteome | Supplementary information Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors - pp929 - 935 Brigitte Anliker, Tobias Abel, Sabrina Kneissl, Juraj Hlavaty, Antonio Caputi, Julia Brynza, Irene C Schneider, Robert C Münch, Helga Petznek, Roland E Kontermann, Ulrike Koehl, Ian C D Johnston, Kari Keinänen, Ulrike C Müller, Christine Hohenadl, Hannah Monyer, Klaus Cichutek & Christian J Buchholz doi:10.1038/nmeth.1514 A targeting method for lentiviral vectors relying on the use of single-chain antibodies recognizing cell-surface antigens is applied to generate lentiviral vectors specific for endothelial cells, hematopoietic progenitors and neurons. Abstract - Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors | Full Text - Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors | PDF (2,053 KB) - Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors | Supplementary information ADVERTISEMENT
  • The author file: Rolf Zeller and Javier Lopez-Rios
    - Nat Meth 7(11):861 (2010)
    ARTICLE NAVIGATION - ISSUE Previous November 2010, Volume 7 No 11 pp859-935 * In This Issue * Editorial * This Month * Correspondence * Research Highlights * Technology Feature * News and Views * Brief Communications * ArticlesAbout the cover In This Issue PDF - In This Issue Editorial Nobel thoughts - p859 doi:10.1038/nmeth1110-859 The community of scientists should celebrate the Nobel Prize, even if awards bestowed on one discipline are associated with another discipline. A new prize might help. Abstract - Nobel thoughts | Full Text - Nobel thoughts | PDF (65 KB) - Nobel thoughts This Month The author file: Rolf Zeller and Javier Lopez-Rios - p861 Monya Baker doi:10.1038/nmeth1110-861 Gene cutting and pasting just got a whole lot faster. Abstract - The author file: Rolf Zeller and Javier Lopez-Rios | Full Text - The author fileRolf Zeller and Javier Lopez-Rios | PDF (152 KB) - The author fileRolf Zeller and Javier Lopez-Rios Points of View: Gestalt principles (Part 1) - p863 Bang Wong doi:10.1038/nmeth1110-863 Full Text - Points of ViewGestalt principles (Part 1) | PDF (119 KB) - Points of ViewGestalt principles (Part 1) ADVERTISEMENT Correspondence Membrane molecules mobile even after chemical fixation - pp865 - 866 Kenji A K Tanaka, Kenichi G N Suzuki, Yuki M Shirai, Shusaku T Shibutani, Manami S H Miyahara, Hisae Tsuboi, Miyako Yahara, Akihiko Yoshimura, Satyajit Mayor, Takahiro K Fujiwara & Akihiro Kusumi doi:10.1038/nmeth.f.314 Full Text - Membrane molecules mobile even after chemical fixation | PDF (642 KB) - Membrane molecules mobile even after chemical fixation | Supplementary information Federal policy and the use of pluripotent stem cells - pp866 - 867 Christopher Thomas Scott, Jennifer B McCormick, Mindy C DeRouen & Jason Owen-Smith doi:10.1038/nmeth1110-866 Full Text - Federal policy and the use of pluripotent stem cells | PDF (153 KB) - Federal policy and the use of pluripotent stem cells Research Highlights Finding the trees in the forest - p869 Nicole Rusk doi:10.1038/nmeth1110-869 The integration of quantitative proteomics and analysis by machine learning yields a refined list of proteins involved in chromosome function. Abstract - Finding the trees in the forest | Full Text - Finding the trees in the forest | PDF (211 KB) - Finding the trees in the forest Protein structure gets exciting - pp870 - 871 Allison Doerr doi:10.1038/nmeth1110-870a Researchers determined the excited-state structure of a small protein using nuclear magnetic resonance spectroscopy. Abstract - Protein structure gets exciting | Full Text - Protein structure gets exciting | PDF (168 KB) - Protein structure gets exciting Many mini mind promoters - pp870 - 871 Natalie de Souza doi:10.1038/nmeth1110-870b Tools to drive restricted gene expression in the brain. Abstract - Many mini mind promoters | Full Text - Many mini mind promoters | PDF (168 KB) - Many mini mind promoters News in brief - p871 doi:10.1038/nmeth1110-871 Full Text - News in brief | PDF (143 KB) - News in brief Species collage - p872 Erika Pastrana doi:10.1038/nmeth1110-872 A new study reports the first viable rat-mouse chimeras and uses rat induced pluripotent stem cells to rescue organ deficiency in mice. Abstract - Species collage | Full Text - Species collage | PDF (108 KB) - Species collage Hidden code in the protein code - p874 Monya Baker doi:10.1038/nmeth1110-874 Apparently redundant codons may not be redundant after all. Abstract - Hidden code in the protein code | Full Text - Hidden code in the protein code | PDF (83 KB) - Hidden code in the protein code Self-healing light beams - p876 Daniel Evanko doi:10.1038/nmeth1110-876 The self-reconstructing properties of Bessel beams provide healing benefits in highly scattering media. Abstract - Self-healing light beams | Full Text - Self-healing light beams | PDF (108 KB) - Self-healing light beams Technology Feature From promising to practical: tools to study networks of neurons - pp877 - 883 Monya Baker doi:10.1038/nmeth1110-877 Combinations of electrophysiology, two-photon microscopy and new tools for detecting neural activity show how neurons function in circuits. Abstract - From promising to practical: tools to study networks of neurons | Full Text - From promising to practical: tools to study networks of neurons | PDF (733 KB) - From promising to practical: tools to study networks of neurons News and Views Defining pluripotency - pp885 - 887 Martin F Pera doi:10.1038/nmeth1110-885 Retroviral marking of single human embryonic stem cells shows that cultures of these cells contain subpopulations with distinct functional properties. Full Text - Defining pluripotency | PDF (857 KB) - Defining pluripotency See also:Article by Stewart et al. DNA construction: homemade or ordered out? - pp887 - 889 Peter A Carr doi:10.1038/nmeth1110-887 Automation and optimization of DNA construction results in the efficient production of large target sequences. Full Text - DNA construction: homemade or ordered out? | PDF (214 KB) - DNA construction: homemade or ordered out? See also:Brief Communication by Gibson et al. Pacing lightly: optogenetics gets to the heart - pp889 - 891 Björn C Knollmann doi:10.1038/nmeth1110-889 Transfer of the light-activated cation channel channelrhodopsin-2 gene enables optical control of heart muscle membrane potential. Full Text - Pacing lightly: optogenetics gets to the heart | PDF (400 KB) - Pacing lightly: optogenetics gets to the heart See also:Brief Communication by Bruegmann et al. Brief Communications Dual RMCE for efficient re-engineering of mouse mutant alleles - pp893 - 895 Marco Osterwalder, Antonella Galli, Barry Rosen, William C Skarnes, Rolf Zeller & Javier Lopez-Rios doi:10.1038/nmeth.1521 An efficient one-step method for re-engineering mouse mutant alleles harboring loxP and FRT sites is reported. It may be applied to the large collection of targeted alleles from the International Knockout Mouse Consortium. Abstract - Dual RMCE for efficient re-engineering of mouse mutant alleles | Full Text - Dual RMCE for efficient re-engineering of mouse mutant alleles | PDF (565 KB) - Dual RMCE for efficient re-engineering of mouse mutant alleles | Supplementary information Optogenetic control of heart muscle in vitro and in vivo- pp897 - 900 Tobias Bruegmann, Daniela Malan, Michael Hesse, Thomas Beiert, Christopher J Fuegemann, Bernd K Fleischmann & Philipp Sasse doi:10.1038/nmeth.1512 Stimulation of the light-activated cation channel channelrhodopsin-2 can depolarize heart muscle in vitro and in vivo, resulting in precise localized stimulation and constant prolonged depolarization of genetically targeted cardiomyocytes and cardiac tissue. Abstract - Optogenetic control of heart muscle in vitro and in vivo | Full Text - Optogenetic control of heart muscle in vitro and in vivo | PDF (1,355 KB) - Optogenetic control of heart muscle in vitro and in vivo | Supplementary information See also:News and Views by Knollmann Chemical synthesis of the mouse mitochondrial genome - pp901 - 903 Daniel G Gibson, Hamilton O Smith, Clyde A Hutchison III, J Craig Venter & Chuck Merryman doi:10.1038/nmeth.1515 Using 600 oligonucleotides with 60 bases each and three enzymes, the authors assemble the entire mouse mitochondrial genome in four isothermal reactions. Abstract - Chemical synthesis of the mouse mitochondrial genome | Full Text - Chemical synthesis of the mouse mitochondrial genome | PDF (488 KB) - Chemical synthesis of the mouse mitochondrial genome | Supplementary information See also:News and Views by Carr Efficient CNS gene delivery by intravenous injection - pp905 - 907 Jean-Pierre Louboutin, Alena A Chekmasova, Elena Marusich, J Roy Chowdhury & David S Strayer doi:10.1038/nmeth.1518 Recombinant SV40 viral vectors intravenously injected into mice pretreated with mannitol effectively deliver transgenes to adult neurons in several regions of the central nervous system. Abstract - Efficient CNS gene delivery by intravenous injection | Full Text - Efficient CNS gene delivery by intravenous injection | PDF (1,239 KB) - Efficient CNS gene delivery by intravenous injection | Supplementary information De novo assembly and analysis of RNA-seq data - pp909 - 912 Gordon Robertson, Jacqueline Schein, Readman Chiu, Richard Corbett, Matthew Field, Shaun D Jackman, Karen Mungall, Sam Lee, Hisanaga Mark Okada, Jenny Q Qian, Malachi Griffith, Anthony Raymond, Nina Thiessen, Timothee Cezard, Yaron S Butterfield, Richard Newsome, Simon K Chan, Rong She, Richard Varhol, Baljit Kamoh, Anna-Liisa Prabhu, Angela Tam, YongJun Zhao, Richard A Moore, Martin Hirst, Marco A Marra, Steven J M Jones, Pamela A Hoodless & Inanc Birol doi:10.1038/nmeth.1517 The Trans-ABySS pipeline is an integrated approach for transcript assembly and analysis to identify new mRNA isoforms and structures. Abstract - De novo assembly and analysis of RNA-seq data | Full Text - De novo assembly and analysis of RNA-seq data | PDF (515 KB) - De novo assembly and analysis of RNA-seq data | Supplementary information Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples - pp913 - 915 Isaäc J Nijman, Michal Mokry, Ruben van Boxtel, Pim Toonen, Ewart de Bruijn & Edwin Cuppen doi:10.1038/nmeth.1516 By pooling barcoded genomes of thirty rats before enrichment of a 1.4-megabase target sequence, mutation discovery in 770 genes is achieved with high accuracy. Abstract - Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples | Full Text - Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples | PDF (372 KB) - Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples | Supplementary information Articles Clonal tracking of hESCs reveals differential contribution to functional assays - pp917 - 922 Morag H Stewart, Sean C Bendall, Marilyne Levadoux-Martin & Mickie Bhatia doi:10.1038/nmeth.1519 Retroviral integration is used to mark clones in human embryonic stem cell cultures and clonal distribution is assessed after functionally testing the cells with different methods. Distinct subsets of clones are detected after in vitro differentiation versus teratoma formation in vivo. Abstract - Clonal tracking of hESCs reveals differential contribution to functional assays | Full Text - Clonal tracking of hESCs reveals differential contribution to functional assays | PDF (1,421 KB) - Clonal tracking of hESCs reveals differential contribution to functional assays | Supplementary information See also:News and Views by Pera Trans-SILAC: sorting out the non-cell-autonomous proteome - pp923 - 927 Oded Rechavi, Matan Kalman, Yuan Fang, Helly Vernitsky, Jasmine Jacob-Hirsch, Leonard J Foster, Yoel Kloog & Itamar Goldstein doi:10.1038/nmeth.1513 Proteins can be transferred between cells in contact, such as via trogocytosis in lymphocytes, or acquired via bacteria-host interactions during infection. A quantitative proteomics approach to identify such non-cell-autonomous proteins is described. Abstract - Trans-SILAC: sorting out the non-cell-autonomous proteome | Full Text - Trans-SILAC: sorting out the non-cell-autonomous proteome | PDF (957 KB) - Trans-SILAC: sorting out the non-cell-autonomous proteome | Supplementary information Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors - pp929 - 935 Brigitte Anliker, Tobias Abel, Sabrina Kneissl, Juraj Hlavaty, Antonio Caputi, Julia Brynza, Irene C Schneider, Robert C Münch, Helga Petznek, Roland E Kontermann, Ulrike Koehl, Ian C D Johnston, Kari Keinänen, Ulrike C Müller, Christine Hohenadl, Hannah Monyer, Klaus Cichutek & Christian J Buchholz doi:10.1038/nmeth.1514 A targeting method for lentiviral vectors relying on the use of single-chain antibodies recognizing cell-surface antigens is applied to generate lentiviral vectors specific for endothelial cells, hematopoietic progenitors and neurons. Abstract - Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors | Full Text - Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors | PDF (2,053 KB) - Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors | Supplementary information ADVERTISEMENT
  • Points of View: Gestalt principles (Part 1)
    - Nat Meth 7(11):863 (2010)
    ARTICLE NAVIGATION - ISSUE Previous November 2010, Volume 7 No 11 pp859-935 * In This Issue * Editorial * This Month * Correspondence * Research Highlights * Technology Feature * News and Views * Brief Communications * ArticlesAbout the cover In This Issue PDF - In This Issue Editorial Nobel thoughts - p859 doi:10.1038/nmeth1110-859 The community of scientists should celebrate the Nobel Prize, even if awards bestowed on one discipline are associated with another discipline. A new prize might help. Abstract - Nobel thoughts | Full Text - Nobel thoughts | PDF (65 KB) - Nobel thoughts This Month The author file: Rolf Zeller and Javier Lopez-Rios - p861 Monya Baker doi:10.1038/nmeth1110-861 Gene cutting and pasting just got a whole lot faster. Abstract - The author file: Rolf Zeller and Javier Lopez-Rios | Full Text - The author fileRolf Zeller and Javier Lopez-Rios | PDF (152 KB) - The author fileRolf Zeller and Javier Lopez-Rios Points of View: Gestalt principles (Part 1) - p863 Bang Wong doi:10.1038/nmeth1110-863 Full Text - Points of ViewGestalt principles (Part 1) | PDF (119 KB) - Points of ViewGestalt principles (Part 1) ADVERTISEMENT Correspondence Membrane molecules mobile even after chemical fixation - pp865 - 866 Kenji A K Tanaka, Kenichi G N Suzuki, Yuki M Shirai, Shusaku T Shibutani, Manami S H Miyahara, Hisae Tsuboi, Miyako Yahara, Akihiko Yoshimura, Satyajit Mayor, Takahiro K Fujiwara & Akihiro Kusumi doi:10.1038/nmeth.f.314 Full Text - Membrane molecules mobile even after chemical fixation | PDF (642 KB) - Membrane molecules mobile even after chemical fixation | Supplementary information Federal policy and the use of pluripotent stem cells - pp866 - 867 Christopher Thomas Scott, Jennifer B McCormick, Mindy C DeRouen & Jason Owen-Smith doi:10.1038/nmeth1110-866 Full Text - Federal policy and the use of pluripotent stem cells | PDF (153 KB) - Federal policy and the use of pluripotent stem cells Research Highlights Finding the trees in the forest - p869 Nicole Rusk doi:10.1038/nmeth1110-869 The integration of quantitative proteomics and analysis by machine learning yields a refined list of proteins involved in chromosome function. Abstract - Finding the trees in the forest | Full Text - Finding the trees in the forest | PDF (211 KB) - Finding the trees in the forest Protein structure gets exciting - pp870 - 871 Allison Doerr doi:10.1038/nmeth1110-870a Researchers determined the excited-state structure of a small protein using nuclear magnetic resonance spectroscopy. Abstract - Protein structure gets exciting | Full Text - Protein structure gets exciting | PDF (168 KB) - Protein structure gets exciting Many mini mind promoters - pp870 - 871 Natalie de Souza doi:10.1038/nmeth1110-870b Tools to drive restricted gene expression in the brain. Abstract - Many mini mind promoters | Full Text - Many mini mind promoters | PDF (168 KB) - Many mini mind promoters News in brief - p871 doi:10.1038/nmeth1110-871 Full Text - News in brief | PDF (143 KB) - News in brief Species collage - p872 Erika Pastrana doi:10.1038/nmeth1110-872 A new study reports the first viable rat-mouse chimeras and uses rat induced pluripotent stem cells to rescue organ deficiency in mice. Abstract - Species collage | Full Text - Species collage | PDF (108 KB) - Species collage Hidden code in the protein code - p874 Monya Baker doi:10.1038/nmeth1110-874 Apparently redundant codons may not be redundant after all. Abstract - Hidden code in the protein code | Full Text - Hidden code in the protein code | PDF (83 KB) - Hidden code in the protein code Self-healing light beams - p876 Daniel Evanko doi:10.1038/nmeth1110-876 The self-reconstructing properties of Bessel beams provide healing benefits in highly scattering media. Abstract - Self-healing light beams | Full Text - Self-healing light beams | PDF (108 KB) - Self-healing light beams Technology Feature From promising to practical: tools to study networks of neurons - pp877 - 883 Monya Baker doi:10.1038/nmeth1110-877 Combinations of electrophysiology, two-photon microscopy and new tools for detecting neural activity show how neurons function in circuits. Abstract - From promising to practical: tools to study networks of neurons | Full Text - From promising to practical: tools to study networks of neurons | PDF (733 KB) - From promising to practical: tools to study networks of neurons News and Views Defining pluripotency - pp885 - 887 Martin F Pera doi:10.1038/nmeth1110-885 Retroviral marking of single human embryonic stem cells shows that cultures of these cells contain subpopulations with distinct functional properties. Full Text - Defining pluripotency | PDF (857 KB) - Defining pluripotency See also:Article by Stewart et al. DNA construction: homemade or ordered out? - pp887 - 889 Peter A Carr doi:10.1038/nmeth1110-887 Automation and optimization of DNA construction results in the efficient production of large target sequences. Full Text - DNA construction: homemade or ordered out? | PDF (214 KB) - DNA construction: homemade or ordered out? See also:Brief Communication by Gibson et al. Pacing lightly: optogenetics gets to the heart - pp889 - 891 Björn C Knollmann doi:10.1038/nmeth1110-889 Transfer of the light-activated cation channel channelrhodopsin-2 gene enables optical control of heart muscle membrane potential. Full Text - Pacing lightly: optogenetics gets to the heart | PDF (400 KB) - Pacing lightly: optogenetics gets to the heart See also:Brief Communication by Bruegmann et al. Brief Communications Dual RMCE for efficient re-engineering of mouse mutant alleles - pp893 - 895 Marco Osterwalder, Antonella Galli, Barry Rosen, William C Skarnes, Rolf Zeller & Javier Lopez-Rios doi:10.1038/nmeth.1521 An efficient one-step method for re-engineering mouse mutant alleles harboring loxP and FRT sites is reported. It may be applied to the large collection of targeted alleles from the International Knockout Mouse Consortium. Abstract - Dual RMCE for efficient re-engineering of mouse mutant alleles | Full Text - Dual RMCE for efficient re-engineering of mouse mutant alleles | PDF (565 KB) - Dual RMCE for efficient re-engineering of mouse mutant alleles | Supplementary information Optogenetic control of heart muscle in vitro and in vivo- pp897 - 900 Tobias Bruegmann, Daniela Malan, Michael Hesse, Thomas Beiert, Christopher J Fuegemann, Bernd K Fleischmann & Philipp Sasse doi:10.1038/nmeth.1512 Stimulation of the light-activated cation channel channelrhodopsin-2 can depolarize heart muscle in vitro and in vivo, resulting in precise localized stimulation and constant prolonged depolarization of genetically targeted cardiomyocytes and cardiac tissue. Abstract - Optogenetic control of heart muscle in vitro and in vivo | Full Text - Optogenetic control of heart muscle in vitro and in vivo | PDF (1,355 KB) - Optogenetic control of heart muscle in vitro and in vivo | Supplementary information See also:News and Views by Knollmann Chemical synthesis of the mouse mitochondrial genome - pp901 - 903 Daniel G Gibson, Hamilton O Smith, Clyde A Hutchison III, J Craig Venter & Chuck Merryman doi:10.1038/nmeth.1515 Using 600 oligonucleotides with 60 bases each and three enzymes, the authors assemble the entire mouse mitochondrial genome in four isothermal reactions. Abstract - Chemical synthesis of the mouse mitochondrial genome | Full Text - Chemical synthesis of the mouse mitochondrial genome | PDF (488 KB) - Chemical synthesis of the mouse mitochondrial genome | Supplementary information See also:News and Views by Carr Efficient CNS gene delivery by intravenous injection - pp905 - 907 Jean-Pierre Louboutin, Alena A Chekmasova, Elena Marusich, J Roy Chowdhury & David S Strayer doi:10.1038/nmeth.1518 Recombinant SV40 viral vectors intravenously injected into mice pretreated with mannitol effectively deliver transgenes to adult neurons in several regions of the central nervous system. Abstract - Efficient CNS gene delivery by intravenous injection | Full Text - Efficient CNS gene delivery by intravenous injection | PDF (1,239 KB) - Efficient CNS gene delivery by intravenous injection | Supplementary information De novo assembly and analysis of RNA-seq data - pp909 - 912 Gordon Robertson, Jacqueline Schein, Readman Chiu, Richard Corbett, Matthew Field, Shaun D Jackman, Karen Mungall, Sam Lee, Hisanaga Mark Okada, Jenny Q Qian, Malachi Griffith, Anthony Raymond, Nina Thiessen, Timothee Cezard, Yaron S Butterfield, Richard Newsome, Simon K Chan, Rong She, Richard Varhol, Baljit Kamoh, Anna-Liisa Prabhu, Angela Tam, YongJun Zhao, Richard A Moore, Martin Hirst, Marco A Marra, Steven J M Jones, Pamela A Hoodless & Inanc Birol doi:10.1038/nmeth.1517 The Trans-ABySS pipeline is an integrated approach for transcript assembly and analysis to identify new mRNA isoforms and structures. Abstract - De novo assembly and analysis of RNA-seq data | Full Text - De novo assembly and analysis of RNA-seq data | PDF (515 KB) - De novo assembly and analysis of RNA-seq data | Supplementary information Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples - pp913 - 915 Isaäc J Nijman, Michal Mokry, Ruben van Boxtel, Pim Toonen, Ewart de Bruijn & Edwin Cuppen doi:10.1038/nmeth.1516 By pooling barcoded genomes of thirty rats before enrichment of a 1.4-megabase target sequence, mutation discovery in 770 genes is achieved with high accuracy. Abstract - Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples | Full Text - Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples | PDF (372 KB) - Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples | Supplementary information Articles Clonal tracking of hESCs reveals differential contribution to functional assays - pp917 - 922 Morag H Stewart, Sean C Bendall, Marilyne Levadoux-Martin & Mickie Bhatia doi:10.1038/nmeth.1519 Retroviral integration is used to mark clones in human embryonic stem cell cultures and clonal distribution is assessed after functionally testing the cells with different methods. Distinct subsets of clones are detected after in vitro differentiation versus teratoma formation in vivo. Abstract - Clonal tracking of hESCs reveals differential contribution to functional assays | Full Text - Clonal tracking of hESCs reveals differential contribution to functional assays | PDF (1,421 KB) - Clonal tracking of hESCs reveals differential contribution to functional assays | Supplementary information See also:News and Views by Pera Trans-SILAC: sorting out the non-cell-autonomous proteome - pp923 - 927 Oded Rechavi, Matan Kalman, Yuan Fang, Helly Vernitsky, Jasmine Jacob-Hirsch, Leonard J Foster, Yoel Kloog & Itamar Goldstein doi:10.1038/nmeth.1513 Proteins can be transferred between cells in contact, such as via trogocytosis in lymphocytes, or acquired via bacteria-host interactions during infection. A quantitative proteomics approach to identify such non-cell-autonomous proteins is described. Abstract - Trans-SILAC: sorting out the non-cell-autonomous proteome | Full Text - Trans-SILAC: sorting out the non-cell-autonomous proteome | PDF (957 KB) - Trans-SILAC: sorting out the non-cell-autonomous proteome | Supplementary information Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors - pp929 - 935 Brigitte Anliker, Tobias Abel, Sabrina Kneissl, Juraj Hlavaty, Antonio Caputi, Julia Brynza, Irene C Schneider, Robert C Münch, Helga Petznek, Roland E Kontermann, Ulrike Koehl, Ian C D Johnston, Kari Keinänen, Ulrike C Müller, Christine Hohenadl, Hannah Monyer, Klaus Cichutek & Christian J Buchholz doi:10.1038/nmeth.1514 A targeting method for lentiviral vectors relying on the use of single-chain antibodies recognizing cell-surface antigens is applied to generate lentiviral vectors specific for endothelial cells, hematopoietic progenitors and neurons. Abstract - Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors | Full Text - Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors | PDF (2,053 KB) - Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors | Supplementary information ADVERTISEMENT
  • Membrane molecules mobile even after chemical fixation
    - Nat Meth 7(11):865-866 (2010)
    Nature Methods | Editorial Nobel thoughts Journal name:Nature MethodsVolume: 7 ,Page:859Year published:(2010)DOI:doi:10.1038/nmeth1110-859Published online28 October 2010 The community of scientists should celebrate the Nobel Prize, even if awards bestowed on one discipline are associated with another discipline. A new prize might help. View full text Additional data
  • Federal policy and the use of pluripotent stem cells
    - Nat Meth 7(11):866-867 (2010)
    Nature Methods | This Month The author file: Rolf Zeller and Javier Lopez-Rios * Monya Baker Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 7 ,Page:861Year published:(2010)DOI:doi:10.1038/nmeth1110-861Published online28 October 2010 Gene cutting and pasting just got a whole lot faster. View full text Additional data
  • Finding the trees in the forest
    - Nat Meth 7(11):869 (2010)
    Gestalt principles of perception are theories proposed by German psychologists in the 1920s to explain how people organize visual information1. Gestalt is a German word meaning shape or form.
  • Protein structure gets exciting
    - Nat Meth 7(11):870-871 (2010)
    Nature Methods | Correspondence Membrane molecules mobile even after chemical fixation * Kenji A K Tanaka1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Kenichi G N Suzuki2, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Yuki M Shirai1 Search for this author in: * NPG journals * PubMed * Google Scholar * Shusaku T Shibutani1 Search for this author in: * NPG journals * PubMed * Google Scholar * Manami S H Miyahara1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hisae Tsuboi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Miyako Yahara1 Search for this author in: * NPG journals * PubMed * Google Scholar * Akihiko Yoshimura3 Search for this author in: * NPG journals * PubMed * Google Scholar * Satyajit Mayor4 Search for this author in: * NPG journals * PubMed * Google Scholar * Takahiro K Fujiwara5 Search for this author in: * NPG journals * PubMed * Google Scholar * Akihiro Kusumi1, 5akusumi@frontier.kyoto-u.ac.jp Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature MethodsVolume: 7 ,Pages:865–866Year published:(2010)DOI:doi:10.1038/nmeth.f.314Published online03 October 2010 To the Editor: Fixation of cells and tissues is the critical first step for histochemical or cytochemical investigations1, 2. Recent efforts to visualize lipid rafts by immunofluorescence and immunoelectron microscopy3, particularly in nonstimulated cells, have yielded varied results, raising a question about the efficacy of chemical cross-linking fixation protocols for blocking the lateral diffusion of membrane molecules and thus their antibody-induced clustering3, 4. In a literature search we did not find any systematic investigation of this issue. Here we investigated whether the lateral diffusion of both raft-associated and non–raft-associated molecules is blocked upon chemical cross-linking and treatment with cold methanol in human T24 cells. We used single fluorescent molecule tracking, which allowed us to directly observe the variation in molecular immobilization after fixation (Fig. 1a, Supplementary Figs. 1 and 2, Supplementary Videos 1 and 2, and Supplementary Methods). View full text Author information * 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 Primary authors * These authors contributed equally to this work. * Kenji A K Tanaka & * Kenichi G N Suzuki Affiliations * Membrane Mechanisms Project, International Cooperative Research Project, Japan Science and Technology Agency (JST), Institute for Integrated Cell-Material Sciences (iCeMS) and Research Center for Nano Medical Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan. * Kenji A K Tanaka, * Yuki M Shirai, * Shusaku T Shibutani, * Manami S H Miyahara, * Hisae Tsuboi, * Miyako Yahara & * Akihiro Kusumi * Precursory Research for Embryonic Science and Technology, JST, iCeMS, Kyoto University, Kyoto, Japan. * Kenichi G N Suzuki * Department of Microbiology and Immunology, School of Medicine, Keio University and Core Research for Evolutional Science and Technology, JST, Tokyo, Japan. * Akihiko Yoshimura * National Centre for Biological Sciences, Bangalore, India. * Satyajit Mayor * Center for Meso-Bio Single-Molecule Imaging, iCeMS, Kyoto University, Kyoto, Japan. * Takahiro K Fujiwara & * Akihiro Kusumi Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Akihiro Kusumi (akusumi@frontier.kyoto-u.ac.jp) Supplementary information * Author information * Supplementary information Movies * Supplementary Video 1 (3M) A typical video clip showing the diffusion of single Halo-GPI molecules on the apical plasma membrane at 37 °C. Before chemical cross-linking (untreated, left), and after treatment with 4% PFA for 30 min (center) or with 4% PFA + 0.1% GA for 30 min (right). * Supplementary Video 2 (2M) A typical video clip showing the diffusion of single Halo–H-Ras molecules on the apical plasma membrane at 37 °C. Before chemical cross-linking (untreated, left), and after treatment with 4% PFA for 30 min (center) or with 4% PFA + 0.1% GA for 30 min (right). PDF files * Supplementary Text and Figures (6M) Supplementary Figures 1–6, Supplementary Methods Additional data
  • Many mini mind promoters
    - Nat Meth 7(11):870-871 (2010)
    In March 2009, US President Barack H. Obama ended eight years of restrictions on federal funding for human embryonic stem cell (hESC) research1.
  • News in brief
    - Nat Meth 7(11):871 (2010)
    Nature Methods | Research Highlights Finding the trees in the forest * Nicole Rusk Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 7 ,Page:869Year published:(2010)DOI:doi:10.1038/nmeth1110-869Published online28 October 2010 The integration of quantitative proteomics and analysis by machine learning yields a refined list of proteins involved in chromosome function. View full text Subject terms: * Cell Biology Additional data
  • Species collage
    - Nat Meth 7(11):872 (2010)
    Nature Methods | Research Highlights Protein structure gets exciting * Allison Doerr Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 7 ,Pages:870–871Year published:(2010)DOI:doi:10.1038/nmeth1110-870aPublished online28 October 2010 Researchers determined the excited-state structure of a small protein using nuclear magnetic resonance spectroscopy. View full text Subject terms: * Structural biology Additional data
  • Hidden code in the protein code
    - Nat Meth 7(11):874 (2010)
    Nature Methods | Research Highlights Many mini mind promoters * Natalie de Souza Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 7 ,Pages:870–871Year published:(2010)DOI:doi:10.1038/nmeth1110-870bPublished online28 October 2010 Tools to drive restricted gene expression in the brain. View full text Subject terms: * Gene expression Additional data
  • Self-healing light beams
    - Nat Meth 7(11):876 (2010)
    Molecular engineering Imaging Epigenetics Bioinformatics Biophysics
  • From promising to practical: tools to study networks of neurons
    - Nat Meth 7(11):877-883 (2010)
    Nature Methods | Research Highlights Species collage * Erika Pastrana Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 7 ,Page:872Year published:(2010)DOI:doi:10.1038/nmeth1110-872Published online28 October 2010 A new study reports the first viable rat-mouse chimeras and uses rat induced pluripotent stem cells to rescue organ deficiency in mice. View full text Subject terms: * Stem Cells Additional data
  • Defining pluripotency
    - Nat Meth 7(11):885-887 (2010)
    Nature Methods | Research Highlights Hidden code in the protein code * Monya Baker Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 7 ,Page:874Year published:(2010)DOI:doi:10.1038/nmeth1110-874Published online28 October 2010 Apparently redundant codons may not be redundant after all. View full text Subject terms: * Biochemistry Additional data
  • DNA construction: homemade or ordered out?
    - Nat Meth 7(11):887-889 (2010)
    Nature Methods | Research Highlights Self-healing light beams * Daniel Evanko Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 7 ,Page:876Year published:(2010)DOI:doi:10.1038/nmeth1110-876Published online28 October 2010 The self-reconstructing properties of Bessel beams provide healing benefits in highly scattering media. View full text Subject terms: * Microscopy Additional data
  • Pacing lightly: optogenetics gets to the heart
    - Nat Meth 7(11):889-891 (2010)
    Nature Methods | Technology Feature From promising to practical: tools to study networks of neurons * Monya Baker1techfeatures@nature.com Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 7 ,Pages:877–883Year published:(2010)DOI:doi:10.1038/nmeth1110-877Published online28 October 2010 Combinations of electrophysiology, two-photon microscopy and new tools for detecting neural activity show how neurons function in circuits. 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 * Monya Baker is technology editor for Nature and Nature Methods Corresponding author Correspondence to: * Monya Baker (techfeatures@nature.com) Additional data
  • Dual RMCE for efficient re-engineering of mouse mutant alleles
    - Nat Meth 7(11):893-895 (2010)
    Retroviral marking of single human embryonic stem cells shows that cultures of these cells contain subpopulations with distinct functional properties.
  • Optogenetic control of heart muscle in vitro and in vivo
    - Nat Meth 7(11):897-900 (2010)
    Automation and optimization of DNA construction results in the efficient production of large target sequences.
  • Chemical synthesis of the mouse mitochondrial genome
    - Nat Meth 7(11):901-903 (2010)
    Transfer of the light-activated cation channel channelrhodopsin-2 gene enables optical control of heart muscle membrane potential.
  • Efficient CNS gene delivery by intravenous injection
    - Nat Meth 7(11):905-907 (2010)
    Nature Methods | Brief Communication Dual RMCE for efficient re-engineering of mouse mutant alleles * Marco Osterwalder1 Search for this author in: * NPG journals * PubMed * Google Scholar * Antonella Galli1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Barry Rosen2 Search for this author in: * NPG journals * PubMed * Google Scholar * William C Skarnes2 Search for this author in: * NPG journals * PubMed * Google Scholar * Rolf Zeller1rolf.zeller@unibas.ch Search for this author in: * NPG journals * PubMed * Google Scholar * Javier Lopez-Rios1javier.lopez-rios@unibas.ch Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MethodsVolume: 7 ,Pages:893–895Year published:(2010)DOI:doi:10.1038/nmeth.1521Received13 April 2010Accepted20 September 2010Published online17 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 We have developed dual recombinase-mediated cassette exchange (dRMCE) to efficiently re-engineer the thousands of available conditional alleles in mouse embryonic stem cells. dRMCE takes advantage of the wild-type loxP and FRT sites present in these conditional alleles and in many gene-trap lines. dRMCE is a scalable, flexible tool to introduce tags, reporters and mutant coding regions into an endogenous locus of interest in an easy and highly efficient manner. View full text Figures at a glance * Figure 1: The principle of dRMCE to re-engineer mouse conditional alleles. () Schematic of the target locus shows the configuration of a conditional mouse allele with a genomic region flanked by two loxP sites and an outside selection cassette flanked by two FRT sites. Upon transfection, the combination of iCre- and Flpo-mediated recombination in cis results in a deleted allele flanked by single loxP and FRT sites, which serves as a 'docking site' for insertion of the replacement vector. ex, exon. () Schematic representation of replacement in the Smad4 locus by dRMCE. The target locus is a Smad4 conditional allele (Smad4f) with a promoterless selection cassette. Co-transfection of the pDIRE and pDREV-1 plasmids induces replacement, probably through production of the Smad4− deleted allele as intermediate. Correct trans insertion of the replacement vector results in the Smad4YFP allele. F1–F4 and R1–R3 denote primers used for PCR screening of colonies (see Supplementary Table 2 for sequences). H2B-Venus, YFP fusion protein with histone 2B; lacZ! , β-galactosidase coding region; neo, neomycin resistance coding region; puro, puromycin resistance cassette; rox, Dre recombinase target sites11; SA, splice acceptor; T, autocleavable T2A peptide coding region14. () PCR screening reveals a large number of clones with correct 3′ and 5′ replacement (69%). Col, colony; 3′ recombination, 5′ recombination, correct replacement at the 3′ and 5′ ends, respectively. () The parental Smad4f cells are β-galactosidase positive. (,) Clones with correct replacement (Smad4YFP) lack β-galactosidase activity but show YFP fluorescence. () Micrograph shows single cells expressing the H2B-Venus fusion protein engineered by dRMCE. Scale bars: 100 μm (–), 5 μm (). * Figure 2: dRMCE for efficient modification of difficult-to-target loci. () Schematic shows the conditional Hand2 allele (Hand2f) used as a target locus for insertion of a Flag epitope tag into the Hand2 coding region. After co-transfection of the replacement vector (pRVH2) and pDIRE plasmid into heterozygous Hand2f mouse embryonic stem cells, dRMCE-mediated replacement results in the Hand2Flag allele. The PGK-hygro (hygromycin resistance gene) selection cassette is flanked by the attB and attP target sites for excision by the φC31 recombinase. F5–F7 and R5–R7 denote primers used for PCR screening and genotyping. E, EcoRV site required to detect correct 5′ replacement by combining PCR amplification with an EcoRV restriction digestion. () PCR screening at both ends of the locus identified Hand2 colonies with correct replacement (13%). Scheme at right shows PCR fragment patterns indicative of particular genomic configurations. 3′ recombination, 5′ recombination, correct replacement at the 3′ and 5′ ends, respectively. A, B, primers t! o amplify a region serving as positive control (see Supplementary Table 2 for sequences). () Gels show germline transmission of the Hand2Flag allele (lanes 1, 5, 6). Above, PCR analysis to detect Hand2Flag allele. Below, PCR detection of wild-type allele. () In situ detection of Hand2 transcripts in a wild-type (Hand2+/+; left) and Hand2Flag/Flag (right) mouse embryo at embryonic day 10.5. Note expression in the posterior limb bud mesenchyme (arrow), branchial arches (black arrowhead) and heart (white arrowhead). Scale bar, 500 μm. Author information * Author information * Supplementary information Affiliations * Developmental Genetics, Department of Biomedicine, University of Basel, Basel, Switzerland. * Marco Osterwalder, * Antonella Galli, * Rolf Zeller & * Javier Lopez-Rios * The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK. * Barry Rosen & * William C Skarnes * Present address: Department of Medicine and Genetics & Development, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, New York, USA. * Antonella Galli Contributions M.O., J.L.-R. and R.Z. conceived and designed the experiments. M.O., J.L.-R. and B.R. designed and constructed the dRMCE tool-kit vectors. B.R. and W.C.S. provided the IKMC mouse embryonic stem cell lines. M.O., A.G., J.L.-R. and B.R. performed the experiments. J.L.-R., W.C.S. and R.Z. wrote the paper. Competing financial interests A dRMCE patent is pending. Corresponding authors Correspondence to: * Javier Lopez-Rios (javier.lopez-rios@unibas.ch) or * Rolf Zeller (rolf.zeller@unibas.ch) Supplementary information * Author information * Supplementary information Zip files * Supplementary Data (32K) Vector sequences in GenBank format. PDF files * Supplementary Text and Figures (8M) Supplementary Figures 1–5, Supplementary Tables 1 and 2 Additional data
  • De novo assembly and analysis of RNA-seq data
    - Nat Meth 7(11):909-912 (2010)
    Nature Methods | Brief Communication Optogenetic control of heart muscle in vitro and in vivo * Tobias Bruegmann1 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniela Malan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Hesse1 Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Beiert1 Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher J Fuegemann1 Search for this author in: * NPG journals * PubMed * Google Scholar * Bernd K Fleischmann1 Search for this author in: * NPG journals * PubMed * Google Scholar * Philipp Sasse1philipp.sasse@uni-bonn.de Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 7 ,Pages:897–900Year published:(2010)DOI:doi:10.1038/nmeth.1512Received09 June 2010Accepted30 August 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 Electrical stimulation is the standard technique for exploring electrical behavior of heart muscle, but this approach has considerable technical limitations. Here we report expression of the light-activated cation channel channelrhodopsin-2 for light-induced stimulation of heart muscle in vitro and in mice. This method enabled precise localized stimulation and constant prolonged depolarization of cardiomyocytes and cardiac tissue resulting in alterations of pacemaking, Ca2+ homeostasis, electrical coupling and arrhythmogenic spontaneous extrabeats. View full text Figures at a glance * Figure 1: Generation and characterization of ChR2-expressing cardiomyocytes in vitro. (,) Immunostainings showing ESCs expressing the classical stem cell marker Oct4 in the nucleus (, red) as well as cardiomyocytes in embryoid bodies expressing α-actinin (, red) overlaid with fluorescence images of the native EYFP signal (green; membrane bound). Nuclei are shown in blue. Scale bar, 20 μm. () Frequency analysis of spontaneously beating embryoid bodies upon pulsed light stimulation at 100 beats per minute (bpm) (blue dashed line; 20 ms, 0.6 mW mm−2; ) and continuous light stimulation (blue bar; 30 s, 0.6 mW mm−2; ). Shown are representative examples of six experiments. () Membrane potential recording of a ChR2-EYFP–expressing cardiomyocyte upon stimulation with blue light (20 mW mm−2) for durations indicated by blue bars. A representative example of five experiments is shown. () Cytosolic Ca2+ imaging traces directly after termination of ChR2 stimulation (47 mW mm−2) for 20 ms (black), 200 ms, 500 ms or 800 ms (red) (durations are indicated by bars ! below the traces). Shown is a representative example of nine experiments. * Figure 2: Expression and function of ChR2 in ventricular cardiomyocytes from CAG-ChR2 mice. () Fluorescence image of the native membrane-bound ChR2-EYFP signal (green) overlaid with α-actinin immunostaining (red) in cardiomyocytes of the ventricle and colocalization with the t-tubulus system (inset). Nuclei are shown in blue. Scale bars, 20 μm. () Inward currents evoked at a holding potential of −40 mV by light stimulation at 0.09, 0.18, 0.45 and 1.75 mW mm−2 (from top to bottom). Monoexponential fit to measure the time constant of decay is shown in red. pA, picoampere; pF, picofarad. () Relationship between light intensity and peak or steady-state currents (holding potential was −40 mV). Error bars, s.d. (n = 7 cells). () Current (I) and voltage (membrane potenial) relationship of light-induced steady-state currents. Error bars, s.d. (n = 7 cells). () Repetitive action potential generation by 1-ms light pulses (blue bars) of 0.91 mW mm−2. () Stimulation-response diagram with percentages of cardiomyocytes showing a 1:1 light pulse to action potential coup! ling depending on the light intensity and duration of light pulses (data for a minimum of 13 cells were used to generate each data point). () Action potential generation by light pulses (10 ms; light blue line) of different intensities in a representative single cell (), and analysis of the delay to action potential threshold and peak (; error bars, s.d.; n = 5 cells). * Figure 3: Light-induced stimulation of ChR2-expressing hearts in vivo. () Pulsed light stimulation (illumination area, 38 mm2; 10 ms, 2.8 mW mm−2, blue) of the right atrium () or three indicated ventricular areas () and parallel recordings of the electrocardiogram (black). () Strength-duration curve for threshold of 1:1 coupling in atria (n = 5) and ventricles (n = 4) at 450 beats per min (illumination area, 2.0 mm2). () Electrocardiogram recording (black traces) during pulsed (20 ms) light stimulations of the left ventricle in an area of 0.8 mm2 (1.1 mW mm−2, top) or 0.05 mm2 (7.2 mW mm−2, bottom). () Continuous light stimulation (blue bar, 3.9 mW mm−2) of a left ventricular area (0.2 mm2) and recording of the electrocardiogram (black). Author information * Author information * Supplementary information Affiliations * Institute of Physiology I, Life and Brain Center, University of Bonn, Bonn, Germany. * Tobias Bruegmann, * Daniela Malan, * Michael Hesse, * Thomas Beiert, * Christopher J Fuegemann, * Bernd K Fleischmann & * Philipp Sasse Contributions T. Bruegmann, B.K.F. and P.S. designed the study and prepared the manuscript. T. Bruegmann, D.M., T. Beiert and P.S. performed experiments and analyzed data. C.J.F. and M.H. generated the transgenic mice. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Philipp Sasse (philipp.sasse@uni-bonn.de) Supplementary information * Author information * Supplementary information Movies * Supplementary Video 1 (3M) Video of a spontaneously beating embryoid body with ChR2-expressing cardiomyocytes. Light stimulation (100 ms, 7.1 mW mm−2) is indicated by a blue box in right upper corner. Recording and display frame rate is 20 frames s−1. PDF files * Supplementary Text and Figures (972K) Supplementary Figures 1–6, Supplementary Note 1 Additional data
  • Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples
    - Nat Meth 7(11):913-915 (2010)
    Nature Methods | Brief Communication Chemical synthesis of the mouse mitochondrial genome * Daniel G Gibson1dgibson@jcvi.org Search for this author in: * NPG journals * PubMed * Google Scholar * Hamilton O Smith2 Search for this author in: * NPG journals * PubMed * Google Scholar * Clyde A Hutchison III2 Search for this author in: * NPG journals * PubMed * Google Scholar * J Craig Venter1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Chuck Merryman1cmerryman@jcvi.org Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MethodsVolume: 7 ,Pages:901–903Year published:(2010)DOI:doi:10.1038/nmeth.1515Received26 May 2010Accepted08 September 2010Published online10 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 We describe a one-step, isothermal assembly method for synthesizing DNA molecules from overlapping oligonucleotides. The method cycles between in vitro recombination and amplification until the desired length is reached. As a demonstration of its simplicity and robustness, we synthesized the entire 16.3-kilobase mouse mitochondrial genome from 600 overlapping 60-mers. View full text Figures at a glance * Figure 1: Schematic demonstrating assembly of the synthetic mouse mitochondrial genome. The 60-base oligos (red lines) were assembled in groups of eight into seventy-five 284-bp cassettes (red arrows). These were joined in sets of five to produce fifteen 1.2-kb assemblies (blue arrows) and then again in sets of five to produce three 5.6-kb assemblies (green arrows). These three fragments were recombined into a complete 16.5-kb genome (orange arrow), which includes a 221-bp repeat. NotI restriction sites (N, black lines) were designed to release the 284-bp cassettes from the pUC19 vector (gray lines). * Figure 2: Summary of results for obtaining the 75 sequence-verified first-stage assemblies. (,) The 75 reactions of eight oligos each were pooled and transformed into E. coli () or individually transformed (). The number of correct clones obtained for each segment (1–75) is shown. Accession codes * Accession codes * Author information * Supplementary information Referenced accessions GenBank * NC_005089 Author information * Accession codes * Author information * Supplementary information Affiliations * The J. Craig Venter Institute, Rockville, Maryland, USA. * Daniel G Gibson & * Chuck Merryman * The J. Craig Venter Institute, San Diego, California, USA. * Hamilton O Smith & * Clyde A Hutchison III Contributions D.G.G. and C.M. designed research, performed research, analyzed data and wrote the paper. H.O.S., C.A.H. III and J.C.V. designed research and analyzed data. Competing financial interests J.C.V. is chief executive officer and co-chief scientific officer of Synthetic Genomics, Inc (SGI). H.O.S. is co-chief scientific officer and a member of the board of directors of SGI. C.A.H. III is chairman of the SGI Scientific Advisory Board. J.C.V., H.O.S. and C.A.H. III hold SGI stock. Corresponding authors Correspondence to: * Daniel G Gibson (dgibson@jcvi.org) or * Chuck Merryman (cmerryman@jcvi.org) Supplementary information * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–6, Supplementary Tables 1–15, Supplementary Note 1 Additional data
  • Clonal tracking of hESCs reveals differential contribution to functional assays
    - Nat Meth 7(11):917-922 (2010)
    Nature Methods | Brief Communication Efficient CNS gene delivery by intravenous injection * Jean-Pierre Louboutin1jplouboutin@hotmail.com Search for this author in: * NPG journals * PubMed * Google Scholar * Alena A Chekmasova1 Search for this author in: * NPG journals * PubMed * Google Scholar * Elena Marusich1 Search for this author in: * NPG journals * PubMed * Google Scholar * J Roy Chowdhury2 Search for this author in: * NPG journals * PubMed * Google Scholar * David S Strayer1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 7 ,Pages:905–907Year published:(2010)DOI:doi:10.1038/nmeth.1518Received19 May 2010Accepted13 September 2010Published online17 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 We administered recombinant SV40-derived viral vectors (rSV40s) intravenously to mice with or without prior intraperitoneal injection of mannitol to deliver transgenes to the central nervous system (CNS). We detected transgene-expressing cells (mainly neurons) most prominently in the cortex and spinal cord; prior intraperitoneal mannitol injection increased CNS gene delivery tenfold. Intravenous injection of rSV40s, particularly with mannitol pretreatment, resulted in extensive expression of multiple transgenes throughout the CNS. View full text Figures at a glance * Figure 1: AU1 expression from a transgene after intravenous injection of recombinant SV40 virus. () AU1 immunostaining in coronal cryostat sections of brains of mice injected intravenously with SV(RevM10.AU1) or control SV(BUGT), with or without prior intraperitoneal administration of mannitol. The motor cortex is shown. Scale bar corresponds to 60 μm in the top three images and 30 μm in the bottom image. (–) Percentages of AU1-positive cells in the indicated brain regions after indicated treatments (), with different doses of intravenously injected vector with prior mannitol treatment () and after different numbers of injections of intravenous vector with prior mannitol treatment of mice (). Error bars, ± s.e.m. (n = 5 mice for each treatment group). **P < 0.001. In and , unless indicated on the plots, P < 0.05 between the different doses () and between the numbers of injections (). * Figure 2: Transgene-expressing cells were mostly neurons. () Cryostat sections of mice brains (motor cortex is shown) immunostained for AU1 and stained with NeuroTrace one month after intravenous injection with the indicated vectors with or without prior intraperitoneal mannitol injection. Arrows point to neurons expressing AU1. Insets show higher-magnification images. Scale bar corresponds to 60 μm in large images and 15 μm in insets. () Higher magnification of another field of the cryostat section shown in the bottom image in . (–) Representative images of sections from mice injected with SV(RevM10.AU1) and prior intraperitoneal mannitol administration, immunostained for NeuN and AU1 (), Iba1 and AU1 (), and GFAP and AU1 (). Five mice were examined in each treatment group. Data are representative of three experiments. Scale bars, 10 μm (), 20 μm (), 25 μm () and 40 μm (). * Figure 3: Distribution of AU1 expression in the brain. () Image of a cryostat section through the cingulate cortex of mice one month after injection of viral vectors with prior mannitol treatment. Sections were labeled with DAPI (left) and immunostained for AU1 (right). M1, primary motor cortex; M2, secondary motor cortex; CG, cingulate cortex; PrL, prelimbic cortex; IL, infralimbic cortex; Pir, piriform cortex; LO, lateral orbital cortex; and AI, agranular insular cortex. () Images showing transgene expression (AU1, green) in the indicated brain areas overlaid with NeuroTrace staining (red). DG, dentate gyrus; Mol, molecular layer of the dentate gyrus; ML, molecular layer of the cerebellum; PCL, Purkinje cell layer; and GCL, granule cell layer. () Percentages of total cells (DAPI-stained) and of neurons (NeuroTrace-stained) expressing AU1 in the indicated areas of the brain. Error bars, s.e.m. (n = 5 mice in each treatment group). Scale bars, 240 μm () and 30 μm (). * Figure 4: Expression of transgene-encoded AU1 in the spinal cord. () Cryostat section through upper thoracic spinal cord of mice one month after injection of viral vectors with prior mannitol treatment. Section was labeled with DAPI (left) and immunostained for AU1 (right). DH, dorsal horn; VH, ventral horn; and CC, central canal. Scale bar corresponds to 240 μm in large images and 120 μm in insets. () Images of AU1-expressing cells in the upper thoracic cord under the indicated conditions. Scale bar, 60 μm. () AU1-immunolabeled and NeuroTrace-stained sections through the thoracic and lumbar spinal cord. The shown scale bar corresponds to 60 μm for the top images and 45 μm for the middle and bottom images. Some AU1-expressing neurons had the morphology and size of sensory or motor neurons (arrowheads), others of interneurons (arrows). Data are representative of three experiments with five mice in each group. (–) Percentages of total cells (DAPI-stained) and of neurons (NeuroTrace (NT)-stained) expressing the transgenes SOD1 (), GPx1! () and Flag () in different areas of the brain. Error bars, s.e.m. (n = 5 mice in each group). Author information * Author information * Supplementary information Affiliations * Department of Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA. * Jean-Pierre Louboutin, * Alena A Chekmasova, * Elena Marusich & * David S Strayer * Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA. * J Roy Chowdhury Contributions J.-P.L. designed and performed experiments, processed and analyzed data, and wrote the paper. A.A.C. performed experiments and designed vectors. E.M. designed vectors. J.R.C. analyzed data and provided collaboration and grant support. D.S.S. coordinated the project and helped to write the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jean-Pierre Louboutin (jplouboutin@hotmail.com) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (18M) Supplementary Figures 1–4 Additional data
  • Trans-SILAC: sorting out the non-cell-autonomous proteome
    - Nat Meth 7(11):923-927 (2010)
    Nature Methods | Brief Communication De novo assembly and analysis of RNA-seq data * Gordon Robertson1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jacqueline Schein1 Search for this author in: * NPG journals * PubMed * Google Scholar * Readman Chiu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard Corbett1 Search for this author in: * NPG journals * PubMed * Google Scholar * Matthew Field1 Search for this author in: * NPG journals * PubMed * Google Scholar * Shaun D Jackman1 Search for this author in: * NPG journals * PubMed * Google Scholar * Karen Mungall1 Search for this author in: * NPG journals * PubMed * Google Scholar * Sam Lee2 Search for this author in: * NPG journals * PubMed * Google Scholar * Hisanaga Mark Okada1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jenny Q Qian1 Search for this author in: * NPG journals * PubMed * Google Scholar * Malachi Griffith1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anthony Raymond1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nina Thiessen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Timothee Cezard1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Yaron S Butterfield1 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard Newsome1 Search for this author in: * NPG journals * PubMed * Google Scholar * Simon K Chan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Rong She1 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard Varhol1 Search for this author in: * NPG journals * PubMed * Google Scholar * Baljit Kamoh1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anna-Liisa Prabhu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Angela Tam1 Search for this author in: * NPG journals * PubMed * Google Scholar * YongJun Zhao1 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard A Moore1 Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Hirst1 Search for this author in: * NPG journals * PubMed * Google Scholar * Marco A Marra1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Steven J M Jones1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Pamela A Hoodless2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Inanc Birol1ibirol@bcgsc.ca Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 7 ,Pages:909–912Year published:(2010)DOI:doi:10.1038/nmeth.1517Received18 June 2010Accepted13 September 2010Published online10 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 We describe Trans-ABySS, a de novo short-read transcriptome assembly and analysis pipeline that addresses variation in local read densities by assembling read substrings with varying stringencies and then merging the resulting contigs before analysis. Analyzing 7.4 gigabases of 50-base-pair paired-end Illumina reads from an adult mouse liver poly(A) RNA library, we identified known, new and alternative structures in expressed transcripts, and achieved high sensitivity and specificity relative to reference-based assembly methods. View full text Figures at a glance * Figure 1: Representation of transcripts and contigs across assemblies. () Distributions of normalized mean transcript coverage from read-to-genome alignments and assembly k-mer length, for unmerged contigs from assemblies for every other k value between 26 and 50 bp (left to right, with the curve for each k value in a different color). Results are shown for all Ensembl v54 mouse transcripts (gray), and for contigs that cover at least 80% of the transcript's total exon length. Inset, distribution of transcripts for each each k value. () Result of contig merging for main contigs from assemblies with k values of 26–50 bp. 'Buried' contigs are those with an exact sequence match within a longer 'parent' contig from another assembly. 'Untouched' contigs have no sequence match in another assembly. * Figure 2: Performance comparisons between ABySS and reference-based transcriptome analysis tools. () Number of Ensembl v54 transcripts reconstructed to 80% by ABySS, Cufflinks and Scripture by a single contig as a function of mean read coverage, C. () Intron-level sensitivity and specificity of Trans-ABySS, Cufflinks, Scripture and TopHat, relative to all 298,893 nonredundant introns from UCSC genome browser, RefSeq, Ensembl and AceView transcript models. Tophat split-read alignments are shown as a curve for intron support levels ranging from 1 to 100 reads. Alignments of Trans-ABySS de novo contigs, and reference-based Tophat Cufflinks and Scripture generated contigs, are represented as points, each of which represents a set of contigs. Two points are shown for ABySS: non-reference-based filtering with (light blue) or without (dark blue) contig-level splice-site filtering. Author information * Author information * Supplementary information Affiliations * Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, Canada. * Gordon Robertson, * Jacqueline Schein, * Readman Chiu, * Richard Corbett, * Matthew Field, * Shaun D Jackman, * Karen Mungall, * Hisanaga Mark Okada, * Jenny Q Qian, * Malachi Griffith, * Anthony Raymond, * Nina Thiessen, * Timothee Cezard, * Yaron S Butterfield, * Richard Newsome, * Simon K Chan, * Rong She, * Richard Varhol, * Baljit Kamoh, * Anna-Liisa Prabhu, * Angela Tam, * YongJun Zhao, * Richard A Moore, * Martin Hirst, * Marco A Marra, * Steven J M Jones & * Inanc Birol * Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada. * Sam Lee & * Pamela A Hoodless * Department of Medical Genetics, University of British Columbia, Vancouver, Canada. * Marco A Marra, * Steven J M Jones & * Pamela A Hoodless * Present address: University of Edinburgh, Edinburgh, UK. * Timothee Cezard Contributions G.R. and J.S. wrote the paper. J.S., G.R. and K.M. reviewed predictions and recommended analysis methods. G.R. coordinated analysis and validation. B.K., A.-L.P. and A.T. constructed libraries under the supervision of YJ.Z. S.L. generated biological material and performed RT-PCR validation. R.A.M. supervised sequencing activities. Y.S.B., T.C., R. Corbett, R. Chiu, M.F., M.G., J.Q.Q., R.N., H.M.O., N.T., R.V., S.K.C. and R.S. developed analysis methods and code and performed analyses. R. Corbett and R. Chiu performed comparisons with reference-based methods. S.D.J. develops and maintains ABySS and generated the ABySS assemblies. A.R. contributed algorithms and code for ABySS. M.A.M., S.J.M.J. and P.A.H. directed research. S.J.M.J. suggested analysis methods. YJ.Z. and M.H. developed the WTSS protocol. J.S. supervised activities. P.A.H. supervised validation. I.B. developed ABySS and Trans-ABySS and directed bioinformatics work. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Inanc Birol (ibirol@bcgsc.ca) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–21, Supplementary Tables 1–4, Supplementary Note Additional data
  • Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors
    - Nat Meth 7(11):929-935 (2010)
    Nature Methods | Brief Communication Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples * Isaäc J Nijman1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Michal Mokry1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Ruben van Boxtel1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Pim Toonen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ewart de Bruijn1 Search for this author in: * NPG journals * PubMed * Google Scholar * Edwin Cuppen1, 2e.cuppen@hubrecht.eu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 7 ,Pages:913–915Year published:(2010)DOI:doi:10.1038/nmeth.1516Received02 June 2010Accepted06 September 2010Published online17 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 Targeted genomic enrichment followed by next-generation DNA sequencing has dramatically increased efficiency of mutation-discovery efforts. We describe a protocol for genomic enrichment of pooled barcoded samples in a single assay that increases experimental flexibility and efficiency. We screened 770 genes (1.4 megabases) in thirty N-ethyl-N-nitrosourea (ENU)-mutagenized rats and identified known variants at >96% sensitivity as well as new mutations at a false positive rate < 1 in 8 megabases. View full text Accession codes * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE22024 Author information * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Isaäc J Nijman, * Michal Mokry & * Ruben van Boxtel Affiliations * Hubrecht Institute, Developmental Biology and Stem Cell Research, Royal Netherlands Academy of Arts and Sciences and the University Medical Center Utrecht, Utrecht, The Netherlands. * Isaäc J Nijman, * Michal Mokry, * Ruben van Boxtel, * Pim Toonen, * Ewart de Bruijn & * Edwin Cuppen * Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands. * Edwin Cuppen Contributions R.v.B., I.J.N., M.M. and E.C. designed the experiments. R.v.B. and P.T. performed rat ENU mutagenesis. M.M. performed library preparation and genomic enrichments. E.d.B. performed SOLiD sequencing. I.J.N. analyzed the data. R.v.B. performed capillary sequencing reconfirmation experiments. I.J.N. and E.C. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Edwin Cuppen (e.cuppen@hubrecht.eu) Supplementary information * Accession codes * Author information * Supplementary information Excel files * Supplementary Table 1 (2M) List of rat genes targeted in the next-generation reverse genetic. * Supplementary Table 6 (116K) Complete list of polymorphic positions identified in the next-generation reverse genetics screen. Zip files * Supplementary Software (4k) Custom SNP filtering PERL script. PDF files * Supplementary Text and Figures (564K) Supplementary Figures 1–2 and Supplementary Tables 2–5 Additional data
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