Hot off the presses! Jan 07 LANCET

The Jan 07 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:

• A new year in medicine
  - LANCET 377(9759):1 (2011)
  
• On synthetic biology
  - LANCET 377(9759):2 (2011)
  
• A breath of fresh indoor air
  - LANCET 377(9759):2 (2011)
  
• Will an aspirin a day help keep fatal cancer away?
  - LANCET 377(9759):3-4 (2011)
  
• Rituximab maintenance in follicular lymphoma: PRIMA
  - LANCET 377(9759):4-6 (2011)
  
• Wormy mothers, healthy babies: case closed or conundrum?
  - LANCET 377(9759):6-8 (2011)
  
• Pathogenic C difficile is here (and everywhere) to stay
  - LANCET 377(9759):8-9 (2011)
  
• The white plague returns to London—with a vengeance
  - LANCET 377(9759):10-11 (2011)
  
• Health and philanthropy—the tobacco connection
  - LANCET 377(9759):11-13 (2011)
  
• Offline: Revising our expectations
  - LANCET 377(9759):14 (2011)
  
• Myths and realities about drug addiction in Mexico
  - LANCET 377(9759):15-16 (2011)
  
• Pulse oximeters breathe life into surgery in poorer nations
  - LANCET 377(9759):17-18 (2011)
  
• Eyewitness accounts from surgeons in Gaza
  - LANCET 377(9759):19 (2011)
  
• Journey through the afterlife
  - LANCET 377(9759):20 (2011)
  
• Clare Gerada: Chair of UK's Royal College of General Practitioners
  - LANCET 377(9759):21 (2011)
  
• Of wandering doctors, cities, and humane hospitals
  - LANCET 377(9759):22-23 (2011)
  
• Frank John Fenner
  - LANCET 377(9759):24 (2011)
  
• Oral sucrose for procedural pain in infants
  - LANCET 377(9759):25 (2011)
  
• Oral sucrose for procedural pain in infants
  - LANCET 377(9759):25 (2011)
  
• Oral sucrose for procedural pain in infants
  - LANCET 377(9759):25-26 (2011)
  
• Oral sucrose for procedural pain in infants
  - LANCET 377(9759):26 (2011)
  
• Oral sucrose for procedural pain in infants
  - LANCET 377(9759):26-27 (2011)
  
• Oral sucrose for procedural pain in infants – Authors' reply
  - LANCET 377(9759):27-28 (2011)
  
• International response to Niger's hunger crisis
  - LANCET 377(9759):28 (2011)
  
• The Greek economic crisis: a primary health-care perspective
  - LANCET 377(9759):28-29 (2011)
  
• When will the sun shine on Cyprus's National Health Service?
  - LANCET 377(9759):29 (2011)
  
• Reporting on the modes of data collection
  - LANCET 377(9759):30 (2011)
  
• All important contributions to papers should be recognised
  - LANCET 377(9759):30 (2011)
  
• Department of Error
  - LANCET 377(9759):30 (2011)
  
• Department of Error
  - LANCET 377(9759):30 (2011)
  
• Effect of daily aspirin on long-term risk of death due to cancer: analysis of individual patient data from randomised trials
  - LANCET 377(9759):31-41 (2011)
  Background Treatment with daily aspirin for 5 years or longer reduces subsequent risk of colorectal cancer. Several lines of evidence suggest that aspirin might also reduce risk of other cancers, particularly of the gastrointestinal tract, but proof in man is lacking. We studied deaths due to cancer during and after randomised trials of daily aspirin versus control done originally for prevention of vascular events. Methods We used individual patient data from all randomised trials of daily aspirin versus no aspirin with mean duration of scheduled trial treatment of 4 years or longer to determine the effect of allocation to aspirin on risk of cancer death in relation to scheduled duration of trial treatment for gastrointestinal and non-gastrointestinal cancers. In three large UK trials, long-term post-trial follow-up of individual patients was obtained from death certificates and cancer registries. Results In eight eligible trials (25 570 patients, 674 cancer deaths), allocation to aspirin reduced death due to cancer (pooled odds ratio [OR] 0·79, 95% CI 0·68–0·92, p=0·003). On analysis of individual patient data, which were available from seven trials (23 535 patients, 657 cancer deaths), benefit was apparent only after 5 years' follow-up (all cancers, hazard ratio [HR] 0·66, 0·50–0·87; gastrointestinal cancers, 0·46, 0·27–0·77; both p=0·003). The 20-year risk of cancer death (1634 deaths in 12 659 patients in three trials) remained lower in the aspirin groups than in the control groups (all solid cancers, HR 0·80, 0·72–0·88, p<0·0001; gastrointestinal cancers, 0·65, 0·54–0·78, p<0·0001), and benefit increased (interaction p=0·01) with scheduled duration of trial treatment (≥7·5 years: all solid cancers, 0·69, 0·54–0·88, p=0·003; gastrointestinal cancers, 0·41, 0·26–0·66, p=0·0001). The latent period before an effect on deaths was a! bout 5 years for oesophageal, pancreatic, brain, and lung cancer, but was more delayed for stomach, colorectal, and prostate cancer. For lung and oesophageal cancer, benefit was confined to adenocarcinomas, and the overall effect on 20-year risk of cancer death was greatest for adenocarcinomas (HR 0·66, 0·56–0·77, p<0·0001). Benefit was unrelated to aspirin dose (75 mg upwards), sex, or smoking, but increased with age—the absolute reduction in 20-year risk of cancer death reaching 7·08% (2·42–11·74) at age 65 years and older. Interpretation Daily aspirin reduced deaths due to several common cancers during and after the trials. Benefit increased with duration of treatment and was consistent across the different study populations. These findings have implications for guidelines on use of aspirin and for understanding of carcinogenesis and its susceptibility to drug intervention. Funding None.
• Rituximab maintenance for 2 years in patients with high tumour burden follicular lymphoma responding to rituximab plus chemotherapy (PRIMA): a phase 3, randomised controlled trial
  - LANCET 377(9759):42-51 (2011)
  Background Patients with follicular lymphoma can have long survival times, but disease progression typically occurs 3–5 years after initial treatment. We assessed the potential benefit of 2 years of rituximab maintenance after first-line treatment in patients with follicular lymphoma receiving a rituximab plus chemotherapy regimen. Methods The randomised, open-label PRIMA study was undertaken in 223 centres in 25 countries. 1217 patients with previously untreated follicular lymphoma needing systemic therapy received one of three non-randomised immunochemotherapy induction regimens used in routine practice. 1019 patients achieving a complete or partial response were then randomly assigned to receive 2 years of rituximab maintenance therapy (375 mg/m2 every 8 weeks) or observation. Treatment was assigned equally by centralised block randomisation, stratified by induction regimen, response, region, and centre. Neither the participants nor those giving the interventions, assessing outcomes, and analysing data were masked to group assignments. The primary endpoint was progression-free survival (PFS). Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00140582. Findings 505 patients were assigned to rituximab maintenance and 513 to observation (one patient died during randomisation). With a median follow-up of 36 months (IQR 30–42), PFS was 74·9% (95% CI 70·9–78·9) in the rituximab maintenance group (130 patients progressed) and 57·6% (53·2–62·0) in the observation group (218 progressed; hazard ratio [HR] 0·55, 95% CI 0·44–0·68, p<0·0001). 2 years after randomisation, 361 patients (71·5%) in the rituximab maintenance group were in complete or unconfirmed complete response versus 268 (52·2%) in the observation group (p=0·0001). Overall survival did not differ significantly between groups (HR 0·87, 95% CI 0·51–1·47). Grade 3 and 4 adverse events were recorded in 121 patients (24%) in the rituximab maintenance group and 84 (17%) in the observation group (risk ratio 1·46, 95% CI 1·14–1·87; p=0·0026). Infections (grades 2–4) were the most common adverse event, occurring in 197 (39%) and 123 (24%) patients, respe! ctively (risk ratio 1·62, 95% CI 1·35–1·96; p<0·0001). Interpretation 2 years of rituximab maintenance therapy after immunochemotherapy as first-line treatment for follicular lymphoma significantly improves PFS. Funding Groupe d'Etude des Lymphomes de l'Adulte (GELA) and F Hoffmann-La Roche.
• Effect of single-dose anthelmintic treatment during pregnancy on an infant's response to immunisation and on susceptibility to infectious diseases in infancy: a randomised, double-blind, placebo-controlled trial
  - LANCET 377(9759):52-62 (2011)
  Background Helminth infections affect the human immune response. We investigated whether prenatal exposure to and treatment of maternal helminth infections affects development of an infant's immune response to immunisations and unrelated infections. Methods In this randomised, double-blind, placebo-controlled trial, we enrolled 2507 women in the second or third trimester of pregnancy who were planning to deliver in Entebbe General Hospital, Entebbe, Uganda. With a computer-generated random number sequence in blocks of 100, we assigned patients to 440 mg albendazole and 40 mg/kg praziquantel (n=628), 440 mg albendazole and a praziquantel-matching placebo (n=625), 40 mg/kg praziquantel and an albendazole-matching placebo (n=626), or an albendazole-matching placebo and praziquantel-matching placebo (n=628). All participants and hospital staff were masked to allocation. Primary outcomes were immune response at age 1 year to BCG, tetanus, and measles immunisation; incidence of infectious diseases during infancy; and vertical HIV transmission. Analysis was by intention-to-treat. This trial is registered, number ISRCTN32849447. Findings Data were available at delivery for 2356 women, with 2345 livebirths; 2115 (90%) of liveborn infants remained in follow-up at 1 year of age. Neither albendazole nor praziquantel treatments affected infant response to BCG, tetanus, or measles immunisation. However, in infants of mothers with hookworm infection, albendazole treatment reduced interleukin-5 (geometric mean ratio 0·50, 95% CI 0·30–0·81, interaction p=0·02) and interleukin-13 (0·52, 0·34–0·82, 0·0005) response to tetanus toxoid. The rate per 100 person-years of malaria was 40·9 (95% CI 38·3–43·7), of diarrhoea was 134·1 (129·2–139·2), and of pneumonia was 22·3 (20·4–24·4). We noted no effect on infectious disease incidence for albendazole treatment (malaria [hazard ratio 0·95, 95% CI 0·79–1.14], diarrhoea [1·06, 0·96–1·16], pneumonia [1·11, 0·90–1·38]) or praziquantel treatment (malaria [1·00, 0·84–1·20], diarrhoea [1·07, 0·98–1·18], pneumonia [1·00, 0·80–1·2! 4]). In HIV-exposed infants, 39 (18%) were infected at 6 weeks; vertical transmission was not associated with albendazole (odds ratio 0·70, 95% CI 0·35–1·42) or praziquantel (0·60, 0·29–1·23) treatment. Interpretation These results do not accord with the recently advocated policy of routine antenatal anthelmintic treatment, and the value of such a policy may need to be reviewed. Funding Wellcome Trust.
• Clostridium difficile infection in Europe: a hospital-based survey
  - LANCET 377(9759):63-73 (2011)
  Background Little is known about the extent of Clostridium difficile infection in Europe. Our aim was to obtain a more complete overview of C difficile infection in Europe and build capacity for diagnosis and surveillance. Methods We set up a network of 106 laboratories in 34 European countries. In November, 2008, one to six hospitals per country, relative to population size, tested stool samples of patients with suspected C difficile infection or diarrhoea that developed 3 or more days after hospital admission. A case was defined when, subsequently, toxins were identified in stool samples. Detailed clinical data and stool isolates were collected for the first ten cases per hospital. After 3 months, clinical data were followed up. Findings The incidence of C difficile infection varied across hospitals (weighted mean 4·1 per 10 000 patient-days per hospital, range 0·0–36·3). Detailed information was obtained for 509 patients. For 389 of these patients, isolates were available for characterisation. 65 different PCR ribotypes were identified, of which 014/020 (61 patients [16%]), 001 (37 [9%]), and 078 (31 [8%]) were the most prevalent. The prevalence of PCR-ribotype 027 was 5%. Most patients had a previously identified risk profile of old age, comorbidity, and recent antibiotic use. At follow up, 101 (22%) of 455 patients had died, and C difficile infection played a part in 40 (40%) of deaths. After adjustment for potential confounders, an age of 65 years or older (adjusted odds ratio 3·26, 95% CI 1·08–9·78; p=0·026), and infection by PCR-ribotypes 018 (6·19, 1·28–29·81; p=0·023) and 056 (13·01; 1·14–148·26; p=0·039) were significantly associated with complicated disease outcome. Interpretation PCR ribotypes other than 027 are prevalent in European hospitals. The data emphasise the importance of multicountry surveillance to detect and control C difficile infection in Europe. Funding European Centre for Disease Prevention and Control.
• Borderline personality disorder
  - LANCET 377(9759):74-84 (2011)
  Recent research findings have contributed to an improved understanding and treatment of borderline personality disorder. This disorder is characterised by severe functional impairments, a high risk of suicide, a negative effect on the course of depressive disorders, extensive use of treatment, and high costs to society. The course of this disorder is less stable than expected for personality disorders. The causes are not yet clear, but genetic factors and adverse life events seem to interact to lead to the disorder. Neurobiological research suggests that abnormalities in the frontolimbic networks are associated with many of the symptoms. Data for the effectiveness of pharmacotherapy vary and evidence is not yet robust. Specific forms of psychotherapy seem to be beneficial for at least some of the problems frequently reported in patients with borderline personality disorder. At present, there is no evidence to suggest that one specific form of psychotherapy is more effe! ctive than another. Further research is needed on the diagnosis, neurobiology, and treatment of borderline personality disorder.
• Measuring impact in the Millennium Development Goal era and beyond: a new approach to large-scale effectiveness evaluations
  - LANCET 377(9759):85-95 (2011)
  Evaluation of large-scale programmes and initiatives aimed at improvement of health in countries of low and middle income needs a new approach. Traditional designs, which compare areas with and without a given programme, are no longer relevant at a time when many programmes are being scaled up in virtually every district in the world. We propose an evolution in evaluation design, a national platform approach that: uses the district as the unit of design and analysis; is based on continuous monitoring of different levels of indicators; gathers additional data before, during, and after the period to be assessed by multiple methods; uses several analytical techniques to deal with various data gaps and biases; and includes interim and summative evaluation analyses. This new approach will promote country ownership, transparency, and donor coordination while providing a rigorous comparison of the cost-effectiveness of different scale-up approaches.
• Killing two birds with one stone
  - LANCET 377(9759):96 (2011)
  

Hot off the presses! Dec 21 PLoS Biol

The Dec 21 issue of the PLoS Biol is now up on Pubget (About PLoS 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:

• Cancer Courts Immune Response to Aid Growth
  - PLoS Biol 8(12):e1001004 (2010)
  
• Stress Brings Memories to the Fore
  - PLoS Biol 8(12):e1001007 (2010)
  
• Melanopsin Ganglion Cells: A Different Way of Seeing Things
  - PLoS Biol 8(12):e1001003 (2010)
  
• Protein Keeps Adjacent Tissues Growing in Synchrony
  - PLoS Biol 8(12):e1001006 (2010)
  
• How Can Vaccines Against Influenza and Other Viral Diseases Be Made More Effective?
  - PLoS Biol 8(12):e1000571 (2010)
  
• Neuro Nonsense
  - PLoS Biol 8(12):e1001005 (2010)
  
• Genomic DNA Sequences from Mastodon and Woolly Mammoth Reveal Deep Speciation of Forest and Savanna Elephants
  - PLoS Biol 8(12):e1000564 (2010)
  To elucidate the history of living and extinct elephantids, we generated 39,763 bp of aligned nuclear DNA sequence across 375 loci for African savanna elephant, African forest elephant, Asian elephant, the extinct American mastodon, and the woolly mammoth. Our data establish that the Asian elephant is the closest living relative of the extinct mammoth in the nuclear genome, extending previous findings from mitochondrial DNA analyses. We also find that savanna and forest elephants, which some have argued are the same species, are as or more divergent in the nuclear genome as mammoths and Asian elephants, which are considered to be distinct genera, thus resolving a long-standing debate about the appropriate taxonomic classification of the African elephants. Finally, we document a much larger effective population size in forest elephants compared with the other elephantid taxa, likely reflecting species differences in ancient geographic structure and range and differences! in life history traits such as variance in male reproductive success.
• A Mitochondrial Superoxide Signal Triggers Increased Longevity in Caenorhabditis elegans
  - PLoS Biol 8(12):e1000556 (2010)
  The nuo-6 and isp-1 genes of C. elegans encode, respectively, subunits of complex I and III of the mitochondrial respiratory chain. Partial loss-of-function mutations in these genes decrease electron transport and greatly increase the longevity of C. elegans by a mechanism that is distinct from that induced by reducing their level of expression by RNAi. Electron transport is a major source of the superoxide anion (O⋅–), which in turn generates several types of toxic reactive oxygen species (ROS), and aging is accompanied by increased oxidative stress, which is an imbalance between the generation and detoxification of ROS. These observations have suggested that the longevity of such mitochondrial mutants might result from a reduction in ROS generation, which would be consistent with the mitochondrial oxidative stress theory of aging. It is difficult to measure ROS directly in living animals, and this has held back progress in determining their function in aging. Her! e we have adapted a technique of flow cytometry to directly measure ROS levels in isolated mitochondria to show that the generation of superoxide is elevated in the nuo-6 and isp-1 mitochondrial mutants, although overall ROS levels are not, and oxidative stress is low. Furthermore, we show that this elevation is necessary and sufficient to increase longevity, as it is abolished by the antioxidants NAC and vitamin C, and phenocopied by mild treatment with the prooxidant paraquat. Furthermore, the absence of effect of NAC and the additivity of the effect of paraquat on a variety of long- and short-lived mutants suggest that the pathway triggered by mitochondrial superoxide is distinct from previously studied mechanisms, including insulin signaling, dietary restriction, ubiquinone deficiency, the hypoxic response, and hormesis. These findings are not consistent with the mitochondrial oxidative stress theory of aging. Instead they show that increased superoxide generation acts ! as a signal in young mutant animals to trigger changes of gene! expression that prevent or attenuate the effects of subsequent aging. We propose that superoxide is generated as a protective signal in response to molecular damage sustained during wild-type aging as well. This model provides a new explanation for the well-documented correlation between ROS and the aged phenotype as a gradual increase of molecular damage during aging would trigger a gradually stronger ROS response.
• Live Imaging of Innate Immune Cell Sensing of Transformed Cells in Zebrafish Larvae: Parallels between Tumor Initiation and Wound Inflammation
  - PLoS Biol 8(12):e1000562 (2010)
  It has not previously been possible to live image the earliest interactions between the host environment and oncogene-transformed cells as they initiate formation of cancers within an organism. Here we take advantage of the translucency of zebrafish larvae to observe the host innate immune cell response as oncogene-transformed melanoblasts and goblet cells multiply within the larval skin. Our studies indicate activation of leukocytes at very early stages in larvae carrying a transformed cell burden. Locally, we see recruitment of neutrophils and macrophages by 48 h post-fertilization, when transformed cells are still only singletons or doublets, and soon after this we see intimate associations between immune and transformed cells and frequent examples of cytoplasmic tethers linking the two cell types, as well as engulfment of transformed cells by both neutrophils and macrophages. We show that a major component of the signal drawing inflammatory cells to oncogenic HRASG! 12V-transformed cells is H2O2, which is also a key damage cue responsible for recruiting neutrophils to a wound. Our short-term blocking experiments show that preventing recruitment of immune cells at these early stages results in reduced growth of transformed cell clones and suggests that immune cells may provide a source of trophic support to the transformed cells just as they do at a site of tissue repair. These parallels between the inflammatory responses to transformed cells and to wounds reinforce the suggestion by others that cancers resemble non-healing wounds.
• High-Throughput Chemical Screen Identifies a Novel Potent Modulator of Cellular Circadian Rhythms and Reveals CKIα as a Clock Regulatory Kinase
  - PLoS Biol 8(12):e1000559 (2010)
  The circadian clock underlies daily rhythms of diverse physiological processes, and alterations in clock function have been linked to numerous pathologies. To apply chemical biology methods to modulate and dissect the clock mechanism with new chemical probes, we performed a circadian screen of ∼120,000 uncharacterized compounds on human cells containing a circadian reporter. The analysis identified a small molecule that potently lengthens the circadian period in a dose-dependent manner. Subsequent analysis showed that the compound also lengthened the period in a variety of cells from different tissues including the mouse suprachiasmatic nucleus, the central clock controlling behavioral rhythms. Based on the prominent period lengthening effect, we named the compound longdaysin. Longdaysin was amenable for chemical modification to perform affinity chromatography coupled with mass spectrometry analysis to identify target proteins. Combined with siRNA-mediated gene knock! down, we identified the protein kinases CKIδ, CKIα, and ERK2 as targets of longdaysin responsible for the observed effect on circadian period. Although individual knockdown of CKIδ, CKIα, and ERK2 had small period effects, their combinatorial knockdown dramatically lengthened the period similar to longdaysin treatment. We characterized the role of CKIα in the clock mechanism and found that CKIα-mediated phosphorylation stimulated degradation of a clock protein PER1, similar to the function of CKIδ. Longdaysin treatment inhibited PER1 degradation, providing insight into the mechanism of longdaysin-dependent period lengthening. Using larval zebrafish, we further demonstrated that longdaysin drastically lengthened circadian period in vivo. Taken together, the chemical biology approach not only revealed CKIα as a clock regulatory kinase but also identified a multiple kinase network conferring robustness to the clock. Longdaysin provides novel possibilities in manipulati! ng clock function due to its ability to simultaneously inhibit! several key components of this conserved network across species.
• Stress-Induced Out-of-Context Activation of Memory
  - PLoS Biol 8(12):e1000570 (2010)
  Inappropriate recollections and responses in stressful conditions are hallmarks of post-traumatic stress disorder and other anxiety and mood disorders, but how stress contributes to the disorders is unclear. Here we show that stress itself reactivates memories even if the memory is unrelated to the stressful experience. Forced-swim stress one day after learning enhanced memory recall. One-day post-learning amnestic treatments were ineffective unless administered soon after the swim, indicating that a stressful experience itself can reactivate unrelated consolidated memories. The swim also triggered inter-hemispheric transfer of a lateralized memory, confirming stress reactivates stable memories. These novel effects of stress on memory required the hippocampus although the memories themselves did not, indicating hippocampus-dependent modulation of extrahippocampal memories. These findings that a stressful experience itself can activate memory suggest the novel hypothesi! s that traumatic stress reactivates pre-trauma memories, linking them to memory for the trauma and pathological facilitation of post-traumatic recall.
• Melanopsin Contributions to Irradiance Coding in the Thalamo-Cortical Visual System
  - PLoS Biol 8(12):e1000558 (2010)
  Photoreception in the mammalian retina is not restricted to rods and cones but extends to a subset of retinal ganglion cells expressing the photopigment melanopsin (mRGCs). These mRGCs are known to drive such reflex light responses as circadian photoentrainment and pupillomotor movements. By contrast, until now there has been no direct assessment of their contribution to conventional visual pathways. Here, we address this deficit. Using new reporter lines, we show that mRGC projections are much more extensive than previously thought and extend across the dorsal lateral geniculate nucleus (dLGN), origin of thalamo-cortical projection neurons. We continue to show that this input supports extensive physiological light responses in the dLGN and visual cortex in mice lacking rods+cones (a model of advanced retinal degeneration). Moreover, using chromatic stimuli to isolate melanopsin-derived responses in mice with an intact visual system, we reveal strong melanopsin input t! o the ∼40% of neurons in the LGN that show sustained activation to a light step. We demonstrate that this melanopsin input supports irradiance-dependent increases in the firing rate of these neurons. The implication that melanopsin is required to accurately encode stimulus irradiance is confirmed using melanopsin knockout mice. Our data establish melanopsin-based photoreception as a significant source of sensory input to the thalamo-cortical visual system, providing unique irradiance information and allowing visual responses to be retained even in the absence of rods+cones. These findings identify mRGCs as a potential origin for aspects of visual perception and indicate that they may support vision in people suffering retinal degeneration.
• A Novel Neural Substrate for the Transformation of Olfactory Inputs into Motor Output
  - PLoS Biol 8(12):e1000567 (2010)
  It is widely recognized that animals respond to odors by generating or modulating specific motor behaviors. These reactions are important for daily activities, reproduction, and survival. In the sea lamprey, mating occurs after ovulated females are attracted to spawning sites by male sex pheromones. The ubiquity and reliability of olfactory-motor behavioral responses in vertebrates suggest tight coupling between the olfactory system and brain areas controlling movements. However, the circuitry and the underlying cellular neural mechanisms remain largely unknown. Using lamprey brain preparations, and electrophysiology, calcium imaging, and tract tracing experiments, we describe the neural substrate responsible for transforming an olfactory input into a locomotor output. We found that olfactory stimulation with naturally occurring odors and pheromones induced large excitatory responses in reticulospinal cells, the command neurons for locomotion. We have also identified t! he anatomy and physiology of this circuit. The olfactory input was relayed in the medial part of the olfactory bulb, in the posterior tuberculum, in the mesencephalic locomotor region, to finally reach reticulospinal cells in the hindbrain. Activation of this olfactory-motor pathway generated rhythmic ventral root discharges and swimming movements. Our study bridges the gap between behavior and cellular neural mechanisms in vertebrates, identifying a specific subsystem within the CNS, dedicated to producing motor responses to olfactory inputs.
• A Genetic and Functional Relationship between T Cells and Cellular Proliferation in the Adult Hippocampus
  - PLoS Biol 8(12):e1000561 (2010)
  Neurogenesis continues through the adult life of mice in the subgranular zone of the dentate gyrus in the hippocampus, but its function remains unclear. Measuring cellular proliferation in the hippocampus of 719 outbred heterogeneous stock mice revealed a highly significant correlation with the proportions of CD8+ versus CD4+ T lymphocyte subsets. This correlation reflected shared genetic loci, with the exception of the H-2Ea locus that had a dominant influence on T cell subsets but no impact on neurogenesis. Analysis of knockouts and repopulation of TCRα-deficient mice by subsets of T cells confirmed the influence of T cells on adult neurogenesis, indicating that CD4+ T cells or subpopulations thereof mediate the effect. Our results reveal an organismal impact, broader than hitherto suspected, of the natural genetic variation that controls T cell development and homeostasis.
• Guidance Receptor Degradation Is Required for Neuronal Connectivity in the Drosophila Nervous System
  - PLoS Biol 8(12):e1000553 (2010)
  Axon pathfinding and synapse formation rely on precise spatiotemporal localization of guidance receptors. However, little is known about the neuron-specific intracellular trafficking mechanisms that underlie the sorting and activity of these receptors. Here we show that loss of the neuron-specific v-ATPase subunit a1 leads to progressive endosomal guidance receptor accumulations after neuronal differentiation. In the embryo and in adult photoreceptors, these accumulations occur after axon pathfinding and synapse formation is complete. In contrast, receptor missorting occurs sufficiently early in neurons of the adult central nervous system to cause connectivity defects. An increase of guidance receptors, but not of membrane proteins without signaling function, causes specific gain-of-function phenotypes. A point mutant that promotes sorting but prevents degradation reveals spatiotemporally specific guidance receptor turnover and accelerates developmental defects in phot! oreceptors and embryonic motor neurons. Our findings indicate that a neuron-specific endolysosomal degradation mechanism is part of the cell biological machinery that regulates guidance receptor turnover and signaling.
• Prox1 Regulates the Notch1-Mediated Inhibition of Neurogenesis
  - PLoS Biol 8(12):e1000565 (2010)
  Activation of Notch1 signaling in neural progenitor cells (NPCs) induces self-renewal and inhibits neurogenesis. Upon neuronal differentiation, NPCs overcome this inhibition, express proneural genes to induce Notch ligands, and activate Notch1 in neighboring NPCs. The molecular mechanism that coordinates Notch1 inactivation with initiation of neurogenesis remains elusive. Here, we provide evidence that Prox1, a transcription repressor and downstream target of proneural genes, counteracts Notch1 signaling via direct suppression of Notch1 gene expression. By expression studies in the developing spinal cord of chick and mouse embryo, we showed that Prox1 is limited to neuronal precursors residing between the Notch1+ NPCs and post-mitotic neurons. Physiological levels of Prox1 in this tissue are sufficient to allow binding at Notch1 promoter and they are critical for proper Notch1 transcriptional regulation in vivo. Gain-of-function studies in the chick neural tube and mou! se NPCs suggest that Prox1-mediated suppression of Notch1 relieves its inhibition on neurogenesis and allows NPCs to exit the cell cycle and differentiate. Moreover, loss-of-function in the chick neural tube shows that Prox1 is necessary for suppression of Notch1 outside the ventricular zone, inhibition of active Notch signaling, down-regulation of NPC markers, and completion of neuronal differentiation program. Together these data suggest that Prox1 inhibits Notch1 gene expression to control the balance between NPC self-renewal and neuronal differentiation.
• A dp53-Dependent Mechanism Involved in Coordinating Tissue Growth in Drosophila
  - PLoS Biol 8(12):e1000566 (2010)
  Coordination of growth between and within organs contributes to the generation of well-proportioned organs and functionally integrated adults. The mechanisms that help to coordinate the growth between different organs start to be unravelled. However, whether an organ is able to respond in a coordinated manner to local variations in growth caused by developmental or environmental stress and the nature of the underlying molecular mechanisms that contribute to generating well-proportioned adult organs under these circumstances remain largely unknown. By reducing the growth rates of defined territories in the developing wing primordium of Drosophila, we present evidence that the tissue responds as a whole and the adjacent cell populations decrease their growth and proliferation rates. This non-autonomous response occurs independently of where growth is affected, and it is functional all throughout development and contributes to generate well-proportioned adult structures. ! Strikingly, we underscore a central role of Drosophila p53 (dp53) and the apoptotic machinery in these processes. While activation of dp53 in the growth-depleted territory mediates the non-autonomous regulation of growth and proliferation rates, effector caspases have a unique role, downstream of dp53, in reducing proliferation rates in adjacent cell populations. These new findings indicate the existence of a stress response mechanism involved in the coordination of tissue growth between adjacent cell populations and that tissue size and cell cycle proliferation can be uncoupled and are independently and non-autonomously regulated by dp53.
• Phosphorylation of Mouse Immunity-Related GTPase (IRG) Resistance Proteins Is an Evasion Strategy for Virulent Toxoplasma gondii
  - PLoS Biol 8(12):e1000576 (2010)
  Virulence of complex pathogens in mammals is generally determined by multiple components of the pathogen interacting with the functional complexity and multiple layering of the mammalian immune system. It is most unusual for the resistance of a mammalian host to be overcome by the defeat of a single defence mechanism. In this study we uncover and analyse just such a case at the molecular level, involving the widespread intracellular protozoan pathogen Toxoplasma gondii and one of its most important natural hosts, the house mouse (Mus musculus). Natural polymorphism in virulence of Eurasian T. gondii strains for mice has been correlated in genetic screens with the expression of polymorphic rhoptry kinases (ROP kinases) secreted into the host cell during infection. We show that the molecular targets of the virulent allelic form of ROP18 kinase are members of a family of cellular GTPases, the interferon-inducible IRG (immunity-related GTPase) proteins, known from earlier ! work to be essential resistance factors in mice against avirulent strains of T. gondii. Virulent T. gondii strain ROP18 kinase phosphorylates several mouse IRG proteins. We show that the parasite kinase phosphorylates host Irga6 at two threonines in the nucleotide-binding domain, biochemically inactivating the GTPase and inhibiting its accumulation and action at the T. gondii parasitophorous vacuole membrane. Our analysis identifies the conformationally active switch I region of the GTP-binding site as an Achilles' heel of the IRG protein pathogen-resistance mechanism. The polymorphism of ROP18 in natural T. gondii populations indicates the existence of a dynamic, rapidly evolving ecological relationship between parasite virulence factors and host resistance factors. This system should be unusually fruitful for analysis at both ecological and molecular levels since both T. gondii and the mouse are widespread and abundant in the wild and are well-established model species wi! th excellent analytical tools available.
• HER2 Phosphorylation Is Maintained by a PKB Negative Feedback Loop in Response to Anti-HER2 Herceptin in Breast Cancer
  - PLoS Biol 8(12):e1000563 (2010)
  Herceptin (trastuzumab) is used in patients with breast cancer who have HER2 (ErbB2)–positive tumours. However, its mechanisms of action and how acquired resistance to Herceptin occurs are still poorly understood. It was previously thought that the anti-HER2 monoclonal antibody Herceptin inhibits HER2 signalling, but recent studies have shown that Herceptin does not decrease HER2 phosphorylation. Its failure to abolish HER2 phosphorylation may be a key to why acquired resistance inevitably occurs for all responders if Herceptin is given as monotherapy. To date, no studies have explained why Herceptin does not abolish HER2 phosphorylation. The objective of this study was to investigate why Herceptin did not decrease HER2 phosphorylation despite being an anti-HER2 monoclonal antibody. We also investigated the effects of acute and chronic Herceptin treatment on HER3 and PKB phosphorylation in HER2-positive breast cancer cells. Using both Förster resonance energy transf! er (FRET) methodology and conventional Western blot, we have found the molecular mechanisms whereby Herceptin fails to abolish HER2 phosphorylation. HER2 phosphorylation is maintained by ligand-mediated activation of EGFR, HER3, and HER4 receptors, resulting in their dimerisation with HER2. The release of HER ligands was mediated by ADAM17 through a PKB negative feedback loop. The feedback loop was activated because of the inhibition of PKB by Herceptin treatment since up-regulation of HER ligands and ADAM17 also occurred when PKB phosphorylation was inhibited by a PKB inhibitor (Akt inhibitor VIII, Akti-1/2). The combination of Herceptin with ADAM17 inhibitors or the panHER inhibitor JNJ-26483327 was able to abrogate the feedback loop and decrease HER2 phosphorylation. Furthermore, the combination of Herceptin with JNJ-26483327 was synergistic in tumour inhibition in a BT474 xenograft model. We have determined that a PKB negative feedback loop links ADAM17 and HER ligands ! in maintaining HER2 phosphorylation during Herceptin treatment! . The activation of other HER receptors via ADAM17 may mediate acquired resistance to Herceptin in HER2-overexpressing breast cancer. This finding offers treatment opportunities for overcoming resistance in these patients. We propose that Herceptin should be combined with a panHER inhibitor or an ADAM inhibitor to overcome the acquired drug resistance for patients with HER2-positive breast cancer. Our results may also have implications for resistance to other therapies targeting HER receptors.
• Connecting Variability in Global Transcription Rate to Mitochondrial Variability
  - PLoS Biol 8(12):e1000560 (2010)
  Populations of genetically identical eukaryotic cells show significant cell-to-cell variability in gene expression. However, we lack a good understanding of the origins of this variation. We have found marked cell-to-cell variability in average cellular rates of transcription. We also found marked cell-to-cell variability in the amount of cellular mitochondrial mass. We undertook fusion studies that suggested that variability in transcription rate depends on small diffusible factors. Following this, in vitro studies showed that transcription rate has a sensitive dependence on [ATP] but not on the concentration of other nucleotide triphosphates (NTPs). Further experiments that perturbed populations by changing nutrient levels and available [ATP] suggested this connection holds in vivo. We found evidence that cells with higher mitochondrial mass, or higher total membrane potential, have a faster rate of transcription per unit volume of nuclear material. We also found evi! dence that transcription rate variability is substantially modulated by the presence of anti- or prooxidants. Daughter studies showed that a cause of variability in mitochondrial content is apparently stochastic segregation of mitochondria at division. We conclude by noting that daughters that stochastically inherit a lower mitochondrial mass than their sisters have relatively longer cell cycles. Our findings reveal a link between variability in energy metabolism and variability in transcription rate.
• Domain Swapping in Allosteric Modulation of DNA Specificity
  - PLoS Biol 8(12):e1000554 (2010)
  SgrAI is a type IIF restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-modulation of cleavage activity and sequence specificity. Previous studies have shown that DNA bound dimers of SgrAI oligomerize into an activated form with higher DNA cleavage rates, although previously determined crystal structures of SgrAI bound to DNA show only the DNA bound dimer. A new crystal structure of the type II restriction endonuclease SgrAI bound to DNA and Ca2+ is now presented, which shows the close association of two DNA bound SgrAI dimers. This tetrameric form is unlike those of the homologous enzymes Cfr10I and NgoMIV and is formed by the swapping of the amino-terminal 24 amino acid residues. Two mutations predicted to destabilize the swapped form of SgrAI, P27W and P27G, have been made and shown to eliminate both the oligomerization of the DNA bound SgrAI dimers as well as the allosteric stimulation of DNA cleavage by SgrAI. A m! echanism involving domain swapping is proposed to explain the unusual allosteric properties of SgrAI via association of the domain swapped tetramer of SgrAI bound to DNA into higher order oligomers.
• S. aureus MscL Is a Pentamer In Vivo but of Variable Stoichiometries In Vitro: Implications for Detergent-Solubilized Membrane Proteins
  - PLoS Biol 8(12):e1000555 (2010)
  While the bacterial mechanosensitive channel of large conductance (MscL) is the best studied biological mechanosensor and serves as a paradigm for how a protein can sense and respond to membrane tension, the simple matter of its oligomeric state has led to debate, with models ranging from tetramers to hexamers. Indeed, two different oligomeric states of the bacterial mechanosensitive channel MscL have been resolved by X-ray crystallography: The M. tuberculosis channel (MtMscL) is a pentamer, while the S. aureus protein (SaMscL) forms a tetramer. Because several studies suggest that, like MtMscL, the E. coli MscL (EcoMscL) is a pentamer, we re-investigated the oligomeric state of SaMscL. To determine the structural organization of MscL in the cell membrane we developed a disulfide-trapping approach. Surprisingly, we found that virtually all SaMscL channels in vivo are pentameric, indicating this as the physiologically relevant and functional oligomeric state. Complement! ing our in vivo results, we purified SaMscL and assessed its oligomeric state using three independent approaches (sedimentation equilibrium centrifugation, crosslinking, and light scattering) and established that SaMscL is a pentamer when solubilized in Triton X-100 and C8E5 detergents. However, performing similar experiments on SaMscL solubilized in LDAO, the detergent used in the crystallographic study, confirmed the tetrameric oligomerization resolved by X-ray crystallography. We further demonstrate that this stoichiometric shift is reversible by conventional detergent exchange experiments. Our results firmly establish the pentameric organization of SaMscL in vivo. Furthermore they demonstrate that detergents can alter the subunit stoichiometry of membrane protein complexes in vitro; thus, in vivo assays are necessary to firmly establish a membrane protein's true functionally relevant oligomeric state.
• Structure of a Classical MHC Class I Molecule That Binds "Non-Classical" Ligands
  - PLoS Biol 8(12):e1000557 (2010)
  Chicken YF1 genes share a close sequence relationship with classical MHC class I loci but map outside of the core MHC region. To obtain insights into their function, we determined the structure of the YF1*7.1/β2-microgloblin complex by X-ray crystallography at 1.3 Å resolution. It exhibits the architecture typical of classical MHC class I molecules but possesses a hydrophobic binding groove that contains a non-peptidic ligand. This finding prompted us to reconstitute YF1*7.1 also with various self-lipids. Seven additional YF1*7.1 structures were solved, but only polyethyleneglycol molecules could be modeled into the electron density within the binding groove. However, an assessment of YF1*7.1 by native isoelectric focusing indicated that the molecules were also able to bind nonself-lipids. The ability of YF1*7.1 to interact with hydrophobic ligands is unprecedented among classical MHC class I proteins and might aid the chicken immune system to recognize a diverse lig! and repertoire with a minimal number of MHC class I molecules.

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Latest Articles Include:

• Method of the Year 2010
  - Nat Meth 8(1):1 (2011)
  Nature Methods | Editorial Method of the Year 2010 Journal name:Nature MethodsVolume: 8,Page:1Year published:(2011)DOI:doi:10.1038/nmeth.f.321Published online20 December 2010 With the capacity to control cellular behaviors using light and genetically encoded light-sensitive proteins, optogenetics has opened new doors for experimentation across biological fields. View full text Additional data
• The Author file: Erik Jorgensen
  - Nat Meth 8(1):3 (2011)
  Nature Methods | This Month The author file: Erik Jorgensen * Monya Baker Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:3Year published:(2011)DOI:doi:10.1038/nmeth0111-3Published online29 December 2010 Fluorescent proteins can be located in electron micrographs. View full text Additional data
• Negative space
  - Nat Meth 8(1):5 (2011)
  Nature Methods | This Month Negative space * Bang Wong1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:5Year published:(2011)DOI:doi:10.1038/nmeth0111-5Published online29 December 2010 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Negative space, also known as whitespace, refers to the unmarked areas of the page. Collectively, it is the margins and the gaps between text blocks and images. Whitespace is as much a part of a composition as the titles, words and pictures. The Swiss typographer Jan Tschichold calls whitespace 'the lungs of a good design'1. In addition to giving elements breathing room, judicious use of whitespace can dramatically improve the visual appeal and effectiveness of figures, posters and slides. The term whitespace stems from the printing practice in which white paper is generally used. Margins and gaps that separate blocks of text make it easier to access written material because they provide a visual structure. Well-planned negative space balances the positive (nonwhite) space and is key to aesthetic. Asian art makes wide use of negative space to create harmony and to add dimension to flat silkscreen prints. View full text Figures at a glance * Figure 1: Empty space defines the shape of an object. (,) Ribbon diagram of a protein () and with the negative space masked in black (). * Figure 2: Whitespace can be used to structure content. () An example of a scientific poster. () A space study reveals that contents in sections 1–6 are scattered and whitespace is fragmented. () An example of consolidated whitespace organizing contents. 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 * Bang Wong is the creative director of the Broad Institute of the Massachusetts Institute of Technology and Harvard and an adjunct assistant professor in the Department of Art as Applied to Medicine at The Johns Hopkins University School of Medicine. Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data
• Preassembled zinc-finger arrays for rapid construction of ZFNs
  - Nat Meth 8(1):7 (2011)
  Nature Methods | Correspondence Preassembled zinc-finger arrays for rapid construction of ZFNs * Seokjoong Kim1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Mi Jung Lee2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Hyojin Kim1 Search for this author in: * NPG journals * PubMed * Google Scholar * Mijin Kang2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jin-Soo Kim1 Contact Jin-Soo Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature MethodsVolume: 8,Page:7Year published:(2011)DOI:doi:10.1038/nmeth0111-7aPublished online29 December 2010 To the Editor: Since the publication of our Correspondence1 and the reply of Joung et al.2, we improved zinc-finger nuclease (ZFN) modular assembly. ZFNs are artificial restriction enzymes3 composed of tailor-made zinc-finger DNA-binding arrays and the FokI nuclease domain, which can induce site-specific mutations4 and large chromosomal deletions5 in higher eukaryotic cells and organisms. 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. * Seokjoong Kim & * Mi Jung Lee Affiliations * Department of Chemistry, Seoul National University, Seoul, South Korea. * Seokjoong Kim, * Hyojin Kim & * Jin-Soo Kim * ToolGen, Inc., Biotechnology Incubating Center, Seoul National University, Seoul, South Korea. * Mi Jung Lee & * Mijin Kang Competing financial interests M.J.L. and M.K. are employees of ToolGen, Inc. J.-S.K. holds stock in ToolGen, Inc. Corresponding author Correspondence to: * Jin-Soo Kim Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (268K) Supplementary Figures 1–2, Supplementary Tables 1–2, Supplementary Methods Additional data
• Live-cell dSTORM with SNAP-tag fusion proteins
  - Nat Meth 8(1):7-9 (2011)
  Nature Methods | Correspondence Live-cell dSTORM with SNAP-tag fusion proteins * Teresa Klein1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anna Löschberger1 Search for this author in: * NPG journals * PubMed * Google Scholar * Sven Proppert1 Search for this author in: * NPG journals * PubMed * Google Scholar * Steve Wolter1 Search for this author in: * NPG journals * PubMed * Google Scholar * Sebastian van de Linde1 Search for this author in: * NPG journals * PubMed * Google Scholar * Markus Sauer1 Contact Markus Sauer Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:7–9Year published:(2011)DOI:doi:10.1038/nmeth0111-7bPublished online29 December 2010 To the Editor: In the September 2010 issue of Nature Methods we demonstrated live-cell direct stochastic optical reconstruction microscopy (dSTORM) of histone H2B proteins using a trimethoprim chemical tag (TMP tag) for genetic encoding with photostable standard fluorophores1. The method takes advantage of the fact that cells contain the reducing thiol glutathione—a cysteine-containing tripeptide—at millimolar concentrations, which enables reversible photoswitching of synthetic organic fluorophores2, 3. The generality of the method can be easily understood considering that most Alexa Fluors (Invitrogen) and Atto fluorophores (ATTO-TEC) belong to the class of rhodamine and oxazine derivatives that have similar redox properties, that is, the triplet state of rhodamine and oxazine fluorophores is reduced by thiols such as glutathione3. View full text Subject terms: * Biophysics 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 Affiliations * Department of Biotechnology and Biophysics, Julius Maximilians University Wuerzburg, Wuerzburg, Germany. * Teresa Klein, * Anna Löschberger, * Sven Proppert, * Steve Wolter, * Sebastian van de Linde & * Markus Sauer Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Markus Sauer Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (652K) Supplementary Figures 1–4, Supplementary Methods Additional data
• Programming molecular instruments
  - Nat Meth 8(1):11 (2011)
  Nature Methods | Research Highlights Programming molecular instruments * Nicole Rusk Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:11Year published:(2011)DOI:doi:10.1038/nmeth0111-11Published online29 December 2010 Small conditional RNAs prove their mettle in multiplexed mRNA imaging and show promise as potential cancer therapeutics. View full text Subject terms: * Small RNAs Additional data
• The birth of a ribosome
  - Nat Meth 8(1):12-13 (2011)
  Nature Methods | Research Highlights The birth of a ribosome * Allison Doerr Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Pages:12–13Year published:(2011)DOI:doi:10.1038/nmeth0111-12aPublished online29 December 2010 A team of researchers applied a 'discovery single-particle profiling' experimental strategy to visualize the assembly of the ribosome via time-resolved electron microscopy. View full text Subject terms: * Imaging Additional data
• A model brain
  - Nat Meth 8(1):12-13 (2011)
  Nature Methods | Research Highlights A model brain * Natalie de Souza Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Pages:12–13Year published:(2011)DOI:doi:10.1038/nmeth0111-12bPublished online29 December 2010 A predictive model of intercellular metabolic interactions in the human brain is reported. View full text Subject terms: * Systems Biology Additional data
• News in brief
  - Nat Meth 8(1):13 (2011)
  Nature Methods | Research Highlights News in brief Journal name:Nature MethodsVolume: 8,Page:13Year published:(2011)DOI:doi:10.1038/nmeth0111-13Published online29 December 2010 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Video-rate stimulated Raman scattering Stimulated Raman scattering (SRS) microscopy is a label-free optical imaging technique based on the detection of signature molecular bond vibrations. Saar et al. now report technical developments that greatly increase the SRS imaging speed, allowing video-rate SRS microscopy. These advances facilitated SRS imaging of the skin and of small molecule drug penetration into the skin of living mice and humans, highlighting the potential utility of SRS for clinical imaging. Saar, B.G.et al. Science330, 1368–1370 (2010). View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data
• The dyes that came in from the cold
  - Nat Meth 8(1):14 (2011)
  Nature Methods | Research Highlights The dyes that came in from the cold * Michael Eisenstein Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:14Year published:(2011)DOI:doi:10.1038/nmeth0111-14Published online29 December 2010 Three groups achieve room-temperature, single-molecule detection of nonfluorescent, photon-absorbing compounds. View full text Subject terms: * Single molecule Additional data
• Unraveling synapse diversity
  - Nat Meth 8(1):16 (2011)
  Nature Methods | Research Highlights Unraveling synapse diversity * Erika Pastrana Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:16Year published:(2011)DOI:doi:10.1038/nmeth0111-16Published online29 December 2010 Array tomography opens the door to the large-scale exploration of molecular diversity of individual brain synapses. View full text Subject terms: * Neuroscience Additional data
• RNA-based reprogramming
  - Nat Meth 8(1):18 (2011)
  Nature Methods | Research Highlights RNA-based reprogramming * Monya Baker Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:18Year published:(2011)DOI:doi:10.1038/nmeth0111-18Published online29 December 2010 RNA molecules can both induce pluripotency and direct differentiation. View full text Subject terms: * Stem Cells Additional data
• Light tools
  - Nat Meth 8(1):19-22 (2011)
  Nature Methods | News Feature Light tools * Monya Baker1 Contact Monya Baker Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Pages:19–22Year published:(2011)DOI:doi:10.1038/nmeth.f.322Published online20 December 2010 Optogenetics grows from an idea into a discipline. Monya Baker reports. View full text Additional data Affiliations * Monya Baker is technology editor for Nature and Nature Methods Corresponding author Correspondence to: * Monya Baker
• Optogenetics: controlling cell function with light
  - Nat Meth 8(1):24-25 (2011)
  Nature Methods | Primer Optogenetics: controlling cell function with light * Erika Pastrana Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Pages:24–25Year published:(2011)DOI:doi:10.1038/nmeth.f.323Published online20 December 2010 A brief description of the basic steps required to control cellular function with optogenetics is presented. View full text Additional data
• Optogenetics
  - Nat Meth 8(1):26-29 (2011)
  Nature Methods | Commentary Optogenetics * Karl Deisseroth1 Contact Karl Deisseroth Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Pages:26–29Year published:(2011)DOI:doi:10.1038/nmeth.f.324Published online20 December 2010 Optogenetics is a technology that allows targeted, fast control of precisely defined events in biological systems as complex as freely moving mammals. By delivering optical control at the speed (millisecond-scale) and with the precision (cell type–specific) required for biological processing, optogenetic approaches have opened new landscapes for the study of biology, both in health and disease. View full text Figures at a glance * Figure 1: Graphical illustration of 'optogenetics' emerging in the scientific literature. Demonstration of single-component optogenetic control of neurons with microbial opsins4 was followed by corresponding optogenetic terminology2 in October 2006, and corresponding optogenetic control of freely moving mammals using microbial opsins and the fiberoptic neural interface9, 10. Also marked are identifications of bacteriorhodopsin3, halorhodopsin5 and channelrhodopsin6, all of which were much later (2005–2010) shown to function as fast, single-component optogenetic tools in neurons. Numbers indicate only publications searchable by 'optogenetics' or derivatives thereof on 1 December 2010. * Figure 2: Principle of optogenetics in neuroscience. Targeted excitation (as with a blue light–activated channelrhodopsin) or inhibition (as with a yellow light–activated halorhodopsin), conferring cellular specificity and even projection specificity not feasible with electrodes while maintaining high temporal (action-potential scale) precision. 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 * Karl Deisseroth is at the Howard Hughes Medical Institute, Department of Bioengineering and Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, California, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Karl Deisseroth Additional data
• From cudgel to scalpel: toward precise neural control with optogenetics
  - Nat Meth 8(1):30-34 (2011)
  Nature Methods | Commentary From cudgel to scalpel: toward precise neural control with optogenetics * Simon Peron1 Search for this author in: * NPG journals * PubMed * Google Scholar * Karel Svoboda1 Contact Karel Svoboda Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:30–34Year published:(2011)DOI:doi:10.1038/nmeth.f.325Published online20 December 2010 Optogenetics is routinely used to activate and inactivate genetically defined neuronal populations in vivo. A second optogenetic revolution will occur when spatially distributed and sparse neural assemblies can be precisely manipulated in behaving animals. View full text Figures at a glance * Figure 1: Manipulating neural assemblies with light. () Mapping neural assemblies, for example, using calcium imaging and two-photon laser scanning microscopy, in populations of neurons. () Silencing neurons based on their response type. () Activating neurons to elicit specific activity patterns. Stippled border indicates expression of both excitatory (cyan) and inhibitory (yellow) transducers. * Figure 2: Methods for two-photon photostimulation with ChR2. () Photostimulation with a stationary diffraction limited excitation volume in a two-photon microscope produces subthreshold excitation. () Photostimulation with a diffraction-limited excitation volume scanned rapidly over the cell of interest, here with a spiral pattern. () Photostimulation with extended excitation volumes, defined by temporal focusing () or temporal focusing combined with generalized phase contrast (), to excite one () or multiple () neurons. Transducer expression is indicated with a cyan border; cells attaining desired response are indicated in yellow. * Figure 3: Effects of dense packing of neural element on the precision of photostimulation. () Stimulation of the targeted cell (target) and undesired stimulation (undesired response). () Volume electron microscopy reconstruction showing 600 axons in a 9.1 μm × 9.0 μm × 4.1 μm volume of cortical tissue (hippocampal CA1 stratum radiatum; reconstruction provided by D.B. Chklovskii; see ref. 18). * Figure 4: Hypothetical scheme to manipulate distributed, sparse assemblies. () Neurons that are active during a well-defined behavioral epoch are selected for expression by illuminating with diffuse light. () Neurons expressing the optogenetic transducer. () Activation of the transducer in specific assemblies using diffuse light. 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 * Simon Peron and Karel Svoboda are at the Howard Hughes Medical Institute Janelia Farm Research Campus, Ashburn, Virginia, USA. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Karel Svoboda Additional data
• The promise of optogenetics in cell biology: interrogating molecular circuits in space and time
  - Nat Meth 8(1):35-38 (2011)
  Nature Methods | Commentary The promise of optogenetics in cell biology: interrogating molecular circuits in space and time * Jared E Toettcher1 Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher A Voigt2 Search for this author in: * NPG journals * PubMed * Google Scholar * Orion D Weiner3 Search for this author in: * NPG journals * PubMed * Google Scholar * Wendell A Lim4 Contact Wendell A Lim Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:35–38Year published:(2011)DOI:doi:10.1038/nmeth.f.326Published online20 December 2010 Optogenetic modules offer cell biologists unprecedented new ways to poke and prod cells. The combination of these precision perturbative tools with observational tools, such as fluorescent proteins, may dramatically accelerate our ability to understand the inner workings of the cell. View full text Figures at a glance * Figure 1: Modes of light-regulated biochemistry. () Protein activity can be put directly under light control by fusion to light-responsive domains or residues (green). Upon stimulation with light (gold arrow), allosteric inhibition is removed, leading to activation. (,) Protein activity can be indirectly controlled using light-dependent anchoring to a subcellular compartment () or scaffolding (). * Figure 2: In vivo biochemistry: from component lists to signal processing. () Cell regulatory networks are comprised of cascades of interacting proteins as well as feedback and feed-forward loops. Typically, they are stimulated by extracellular ligands or pharmacological agents and observed by following protein levels or pathway activity (such as a transcriptional response). () Classical chemical and genetic perturbations block or enhance individual nodes to identify phenotype changes. (,) Light-gated inputs can be used to specifically and precisely perturb activation at distinct nodes in a pathway (inputs 1–4, labeled I1 to I4) (), using a rich set of temporal inputs including fixed levels of activation and frequency-modulated signals (). * Figure 3: Reversibility and spatial precision. () Optogenetics allows the investigator to apply spatially restricted light inputs (orange). However, diffusion of protein activity (red) from the site of activation destroys the applied spatial pattern. () Faithful spatial patterns can be maintained by coupling local activation with global inactivation (green), either by implementing an inactivating light wavelength or a short-lived active state. 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 * Jared E. Toettcher is at the Department of Cellular and Molecular Pharmacology and the Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, USA. * Christopher A. Voigt is at the Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, USA. * Orion D. Weiner is at the Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, USA. * Wendell A. Lim is at the Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, USA. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Wendell A Lim Additional data
• Channelrhodopsin engineering and exploration of new optogenetic tools
  - Nat Meth 8(1):39-42 (2011)
  Nature Methods | Commentary Channelrhodopsin engineering and exploration of new optogenetic tools * Peter Hegemann1 Contact Peter Hegemann Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas Möglich1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:39–42Year published:(2011)DOI:doi:10.1038/nmeth.f.327Published online20 December 2010 Rhodopsins from microalgae and eubacteria are powerful tools for manipulating the function of neurons and other cells, but these tools still have limitations. We discuss engineering approaches that can help advance optogenetics. View full text Figures at a glance * Figure 1: A simplified kinetic model of channelrhodopsin function. The model includes two closed states; one prevails after dark adaptation (DA state), whereas the other (LA state) is only occupied after several hundred milliseconds in the light. Light absorption and subsequent isomerization of the retinal chromophore (red) converts both dark states into open conducting states, O1 and O2. * Figure 2: Structural model of a channelrhodopsin. () Three-dimensional computer model of ChR2. Mutations of the amino acid residues shown in stick representation are known to substantially influence absorption, conductance (without selectivity change), kinetics and ion selectivity, as indicated for each residue. The retinal moiety is shown in yellow, residues conserved in all four known channelrhodopsins are colored blue, and residues that differ in various channelrhodopsins are colored gray. Oxygen, nitrogen and sulfur atoms are colored red, blue and dark yellow, respectively. Graphics are based on the coordinates of H. salinarum bacteriorhodopsin28 and were drawn with Pymol (Schrödinger). () Structures of the two possible chromophore dark state isomers: the all-trans, 15-anti form and the 13-cis, 15-syn form. 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 * Peter Hegemann and Andreas Möglich are at Humboldt Universität zu Berlin, Department of Biology, Biophysics, Berlin, Germany. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Peter Hegemann Additional data
• Zinc-finger nucleases
  - Nat Meth 8(1):43 (2011)
  Nature Methods | Methods to Watch Zinc-finger nucleases * Natalie de Souza Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:43Year published:(2011)DOI:doi:10.1038/nmeth.f.328Published online20 December 2010 Genome-engineering tools with improved design and efficiency will become widely used. View full text Additional data
• Targeted proteomics
  - Nat Meth 8(1):43 (2011)
  Nature Methods | Methods to Watch Targeted proteomics * Allison Doerr Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:43Year published:(2011)DOI:doi:10.1038/nmeth.f.329Published online20 December 2010 Targeted analysis of proteins on a broad scale with mass spectrometry is becoming a reality. View full text Additional data
• Torrents of sequence
  - Nat Meth 8(1):44 (2011)
  Nature Methods | Methods to Watch Torrents of sequence * Nicole Rusk Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:44Year published:(2011)DOI:doi:10.1038/nmeth.f.330Published online20 December 2010 In 2011, we will see the arrival of new and improved sequencing technologies. View full text Additional data
• Seamless delivery
  - Nat Meth 8(1):44 (2011)
  Nature Methods | Methods to Watch Seamless delivery * Nicole Rusk Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:44Year published:(2011)DOI:doi:10.1038/nmeth.f.331Published online20 December 2010 The payoffs for efficient cargo delivery into living cells make the development of better methods worthwhile. View full text Additional data
• Single-molecule structure determination
  - Nat Meth 8(1):45 (2011)
  Nature Methods | Methods to Watch Single-molecule structure determination * Allison Doerr Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:45Year published:(2011)DOI:doi:10.1038/nmeth.f.332Published online20 December 2010 X-ray free-electron lasers may enable single-molecule structure determination. View full text Additional data
• Adaptive optics for biological imaging
  - Nat Meth 8(1):45 (2011)
  Nature Methods | Methods to Watch Adaptive optics for biological imaging * Erika Pastrana Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:45Year published:(2011)DOI:doi:10.1038/nmeth.f.333Published online20 December 2010 The use of adaptive optics to correct light distortions promises to greatly improve the imaging quality of thick biological tissues. View full text Additional data
• Networking to understand disease
  - Nat Meth 8(1):46 (2011)
  Nature Methods | Methods to Watch Networking to understand disease * Natalie de Souza Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:46Year published:(2011)DOI:doi:10.1038/nmeth.f.334Published online20 December 2010 The application of systems approaches to human disease will continue to expand. View full text Additional data
• Fast 3D super-resolution fluorescence microscopy
  - Nat Meth 8(1):46 (2011)
  Nature Methods | Methods to Watch Fast 3D super-resolution fluorescence microscopy * Erika Pastrana Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:46Year published:(2011)DOI:doi:10.1038/nmeth.f.335Published online20 December 2010 High-speed fluorescence imaging in all three dimensions at nanometer resolution will resolve, in finer detail, the workings of the living cell. View full text Additional data
• Screening: the age of fishes
  - Nat Meth 8(1):47-51 (2011)
  Nature Methods | Technology Feature Screening: the age of fishes * Monya Baker1 Contact Monya Baker Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Pages:47–51Year published:(2011)DOI:doi:10.1038/nmeth0111-47Published online29 December 2010 Advances in microfluidics and imaging, combined with some high-profile studies, are increasing interest in whole-organism screening. 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 Additional data
• Zinc-finger nucleases transition to the CoDA
  - Nat Meth 8(1):53-55 (2011)
  Nature Methods | News and Views Zinc-finger nucleases transition to the CoDA * David J Segal1 Contact David J Segal Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Pages:53–55Year published:(2011)DOI:doi:10.1038/nmeth0111-53Published online29 December 2010 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Potent and easily produced custom zinc-finger nucleases will be game-changers for the field, at a time when the field itself is changing. 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 * David J. Segal is at the Genome Center and Department of Biochemistry and Molecular Medicine, University of California–Davis, Davis, California, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * David J Segal Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data
• At last, functional glycomics
  - Nat Meth 8(1):55-57 (2011)
  Nature Methods | News and Views At last, functional glycomics * Joseph Zaia1 Contact Joseph Zaia Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Pages:55–57Year published:(2011)DOI:doi:10.1038/nmeth0111-55Published online29 December 2010 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Microarrays made from naturally expressed glycolipids help winnow function from heterogeneous glycan structures. 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 * Joseph Zaia is at Boston University, Boston, Massachusetts, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Joseph Zaia Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data
• Seeing is believing
  - Nat Meth 8(1):57-58 (2011)
  Nature Methods | News and Views Seeing is believing * Jahar Bhattacharya1 Contact Jahar Bhattacharya Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Pages:57–58Year published:(2011)DOI:doi:10.1038/nmeth0111-57Published online29 December 2010 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Stabilization with a suction window permits in vivo imaging of the mouse lung vasculature with video-rate two-photon microscopy. 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 * Jahar Bhattacharya is at the Lung Biology Lab, Department of Medicine, Columbia University Medical Center, New York, New York, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Jahar Bhattacharya Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data
• Assemblies: the good, the bad, the ugly
  - Nat Meth 8(1):59-60 (2011)
  Nature Methods | Commentary Assemblies: the good, the bad, the ugly * Ewan Birney1 Contact Ewan Birney Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Pages:59–60Year published:(2011)DOI:doi:10.1038/nmeth0111-59Published online29 December 2010 The low cost of short-read sequencing has motivated the development of de novo assemblies from only short-read data; impressively, assemblies for large mammalian genomes are now available. However, this is still a developing field, and these de novo assemblies have many artifacts, as do all de novo assemblies. 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 * Ewan Birney is at the European Molecular Biology Laboratory–European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, UK. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Ewan Birney Additional data
• Limitations of next-generation genome sequence assembly
  - Nat Meth 8(1):61-65 (2011)
  Nature Methods | Perspective Limitations of next-generation genome sequence assembly * Can Alkan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Saba Sajjadian1 Search for this author in: * NPG journals * PubMed * Google Scholar * Evan E Eichler1 Contact Evan E Eichler Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:61–65Year published:(2011)DOI:doi:10.1038/nmeth.1527Published online21 November 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg High-throughput sequencing technologies promise to transform the fields of genetics and comparative biology by delivering tens of thousands of genomes in the near future. Although it is feasible to construct de novo genome assemblies in a few months, there has been relatively little attention to what is lost by sole application of short sequence reads. We compared the recent de novo assemblies using the short oligonucleotide analysis package (SOAP), generated from the genomes of a Han Chinese individual and a Yoruban individual, to experimentally validated genomic features. We found that de novo assemblies were 16.2% shorter than the reference genome and that 420.2 megabase pairs of common repeats and 99.1% of validated duplicated sequences were missing from the genome. Consequently, over 2,377 coding exons were completely missing. We conclude that high-quality sequencing approaches must be considered in conjunction with high-throughput sequencing for comparative genomics an! alyses and studies of genome evolution. View full text Subject terms: * Genetics and genomics Author information * Abstract * Author information * Supplementary information Affiliations * Department of Genome Sciences, University of Washington School of Medicine and Howard Hughes Medical Institute, Seattle, Washington, USA. * Can Alkan, * Saba Sajjadian & * Evan E Eichler Contributions C.A. and E.E.E. conceived the study and wrote the manuscript. C.A. and S.S. analyzed the data. Competing financial interests E.E.E. is a scientific advisory board member of Pacific Biosciences. Corresponding author Correspondence to: * Evan E Eichler Supplementary information * Abstract * Author information * Supplementary information Excel files * Supplementary Table 1 (408K) Contamination found in reported human new sequence insertions from the genomes of two individuals. * Supplementary Table 3 (5M) Analysis of nonredundant autosomal genes in the YH genome assembly. * Supplementary Table 5 (2M) Assigned positions of duplicated sequences (YH) to the NCBI build 36 assembly. Text files * Supplementary Table 4 (12M) Analysis of nonredundant autosomal coding exons in the YH genome. NOTE: This is a tab-delimited text file with 171,751 rows of data. Confirm that all data will load into your application before proceeding. PDF files * Supplementary Text and Figures (404K) Supplementary Figures 1–2, Supplementary Table 2, Supplementary Note Additional data
• Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA)
  - Nat Meth 8(1):67-69 (2011)
  Nature Methods | Brief Communication Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA) * Jeffry D Sander1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Elizabeth J Dahlborg1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Mathew J Goodwin1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Lindsay Cade4 Search for this author in: * NPG journals * PubMed * Google Scholar * Feng Zhang5 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel Cifuentes6 Search for this author in: * NPG journals * PubMed * Google Scholar * Shaun J Curtin7 Search for this author in: * NPG journals * PubMed * Google Scholar * Jessica S Blackburn1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Stacey Thibodeau-Beganny1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Yiping Qi5 Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher J Pierick5 Search for this author in: * NPG journals * PubMed * Google Scholar * Ellen Hoffman6 Search for this author in: * NPG journals * PubMed * Google Scholar * Morgan L Maeder1, 2, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Cyd Khayter1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Deepak Reyon9 Search for this author in: * NPG journals * PubMed * Google Scholar * Drena Dobbs9 Search for this author in: * NPG journals * PubMed * Google Scholar * David M Langenau1, 3, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Robert M Stupar7 Search for this author in: * NPG journals * PubMed * Google Scholar * Antonio J Giraldez6 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel F Voytas5 Search for this author in: * NPG journals * PubMed * Google Scholar * Randall T Peterson4, 10, 11 Search for this author in: * NPG journals * PubMed * Google Scholar * Jing-Ruey J Yeh4, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * J Keith Joung1, 2, 3, 8 Contact J Keith Joung Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:67–69Year published:(2011)DOI:doi:10.1038/nmeth.1542Received17 June 2010Accepted16 November 2010Published online12 December 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe context-dependent assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis. Using CoDA-generated ZFNs, we rapidly altered 20 genes in Danio rerio, Arabidopsis thaliana and Glycine max. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multigene pathways or genome-wide alterations. View full text Subject terms: * Molecular biology Author information * Author information * Supplementary information Affiliations * Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA. * Jeffry D Sander, * Elizabeth J Dahlborg, * Mathew J Goodwin, * Jessica S Blackburn, * Stacey Thibodeau-Beganny, * Morgan L Maeder, * Cyd Khayter, * David M Langenau & * J Keith Joung * Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA. * Jeffry D Sander, * Elizabeth J Dahlborg, * Mathew J Goodwin, * Stacey Thibodeau-Beganny, * Morgan L Maeder, * Cyd Khayter & * J Keith Joung * Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA. * Jeffry D Sander, * Jessica S Blackburn, * David M Langenau & * J Keith Joung * Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts, USA. * Lindsay Cade, * Randall T Peterson & * Jing-Ruey J Yeh * Department of Genetics, Cell Biology and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA. * Feng Zhang, * Yiping Qi, * Christopher J Pierick & * Daniel F Voytas * Genetics Department, Yale University School of Medicine, New Haven, Connecticut, USA. * Daniel Cifuentes, * Ellen Hoffman & * Antonio J Giraldez * Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota, USA. * Shaun J Curtin & * Robert M Stupar * Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts, USA. * Morgan L Maeder, * David M Langenau & * J Keith Joung * Department of Genetics, Development and Cell Biology and Bioinformatics and Computational Biology Program, Iowa State University, Ames, Iowa, USA. * Deepak Reyon & * Drena Dobbs * Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA. * Randall T Peterson & * Jing-Ruey J Yeh * Broad Institute, Cambridge, Massachusetts, USA. * Randall T Peterson Contributions J.D.S. and J.K.J. conceived of the CoDA engineering method; J.D.S., S.J.C., D.M.L., R.M.S., A.J.G., D.F.V., R.T.P., J.-R.J.Y. and J.K.J. designed research; J.D.S., E.J.D., M.J.G., L.C., F.Z., D.C., S.J.C., J.S.B., S.T.-B., Y.Q., C.J.P., E.H., M.L.M. and C.K. performed experiments; J.D.S., D.R. and D.D. identified potential genomic CoDA target sites; and J.D.S., R.M.S., D.F.V., R.T.P., J.-R.J.Y. and J.K.J. wrote the paper. Competing financial interests F.Z. and D.F.V. are paid for work performed for Cellectis Plant Sciences. Corresponding author Correspondence to: * J Keith Joung Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–5, Supplementary Tables 1–5 and Supplementary Discussion Additional data
• SAINT: probabilistic scoring of affinity purification–mass spectrometry data
  - Nat Meth 8(1):70-73 (2011)
  Nature Methods | Brief Communication SAINT: probabilistic scoring of affinity purification–mass spectrometry data * Hyungwon Choi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Brett Larsen2 Search for this author in: * NPG journals * PubMed * Google Scholar * Zhen-Yuan Lin2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ashton Breitkreutz2 Search for this author in: * NPG journals * PubMed * Google Scholar * Dattatreya Mellacheruvu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Damian Fermin1 Search for this author in: * NPG journals * PubMed * Google Scholar * Zhaohui S Qin3, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Mike Tyers2, 4, 5, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Anne-Claude Gingras2, 4 Contact Anne-Claude Gingras Search for this author in: * NPG journals * PubMed * Google Scholar * Alexey I Nesvizhskii1, 7 Contact Alexey I Nesvizhskii Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MethodsVolume: 8,Pages:70–73Year published:(2011)DOI:doi:10.1038/nmeth.1541Received28 June 2010Accepted09 November 2010Published online05 December 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 present 'significance analysis of interactome' (SAINT), a computational tool that assigns confidence scores to protein-protein interaction data generated using affinity purification–mass spectrometry (AP-MS). The method uses label-free quantitative data and constructs separate distributions for true and false interactions to derive the probability of a bona fide protein-protein interaction. We show that SAINT is applicable to data of different scales and protein connectivity and allows transparent analysis of AP-MS data. View full text Figures at a glance * Figure 1: Probability model in SAINT. (,) Interaction data in the presence () and absence () of control purifications. Schematic of the experimental AP-MS procedure is shown at the top and a spectral count interaction table is illustrated at the bottom. Ctrl, control; rep, replicate; freq, frequency. () Modeling spectral count distributions for true and false interactions. For the interaction between prey i and bait j, SAINT uses all relevant data for the two proteins, as shown in the column of the bait (green) and the data in the row of the prey (orange) in and . () Probability is calculated for each replicate by application of Bayes rule, and a summary probability is calculated for the interaction pair (i,j). * Figure 2: Analysis of TIP49 and DUB datasets. () Benchmarking of filtered interactions in the TIP49 dataset by the overlap with interactions previously reported in BioGRID and iRefWeb databases. () Co-annotation of interaction partners to common GO terms in 'biological processes' in the TIP49 dataset. () Benchmarking against BioGRID and iRefWeb in the DUB dataset. () Co-annotation to GO terms in the DUB dataset. Author information * Author information * Supplementary information Affiliations * Department of Pathology, University of Michigan, Ann Arbor, Michigan, USA. * Hyungwon Choi, * Dattatreya Mellacheruvu, * Damian Fermin & * Alexey I Nesvizhskii * Centre for Systems Biology, Samuel Lunenfeld Research Institute, Toronto, Ontario, Canada. * Brett Larsen, * Zhen-Yuan Lin, * Ashton Breitkreutz, * Mike Tyers & * Anne-Claude Gingras * Department of Biostatistics and Center for Statistical Genetics, University of Michigan, Ann Arbor, Michigan, USA. * Zhaohui S Qin * Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. * Mike Tyers & * Anne-Claude Gingras * Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK. * Mike Tyers * Centre for Systems Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK. * Mike Tyers * Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, USA. * Alexey I Nesvizhskii * Present address: Department of Biostatistics and Bioinformatics, Emory University, Atlanta, Georgia, USA. * Zhaohui S Qin Contributions H.C. and A.I.N. developed, implemented and tested the SAINT method; H.C. wrote the software; B.L., A.B., Z.-Y.L., A.-C.G. and M.T. generated data for the initial SAINT modeling and provided feedback on the model performance; D.M. and D.F. assisted with data analysis and processing; Z.S.Q. contributed to statistical model development; H.C., A.-C.G. and A.I.N. wrote the manuscript; A.I.N. and A.-C.G. conceived the study; A.I.N. directed the project with input from A.-C.G. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Anne-Claude Gingras or * Alexey I Nesvizhskii Supplementary information * Author information * Supplementary information Excel files * Supplementary Table 1 (1008K) Data for the TIP49 dataset. () List of all detected interactions and scores from PP-NSAF, CompPASS and SAINT. () All interactions in control purifications were included in a separate table after merging of 35 technical replicate purifications into 9 purifications. () Table of technical replicates of control purifications. () GO terms enrichment in top scoring interactions for each scoring method. * Supplementary Table 2 (3M) Data for the DUB dataset. () List of all detected interactions and scores from CompPASS and SAINT. (–) GO terms enrichment in top scoring interactions for each scoring method. * Supplementary Table 3 (100K) Data for the CDC23 dataset. List of all detected interactions with SAINT scores and results reported by t-test. Zip files * Supplementary Software (2M) PDF files * Supplementary Text and Figures (240K) Supplementary Figure 1 Additional data
• Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures
  - Nat Meth 8(1):74-79 (2011)
  Nature Methods | Article Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures * Yannick Doyon1 Contact Yannick Doyon Search for this author in: * NPG journals * PubMed * Google Scholar * Thuy D Vo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Matthew C Mendel1 Search for this author in: * NPG journals * PubMed * Google Scholar * Shon G Greenberg1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jianbin Wang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Danny F Xia1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeffrey C Miller1 Search for this author in: * NPG journals * PubMed * Google Scholar * Fyodor D Urnov1 Search for this author in: * NPG journals * PubMed * Google Scholar * Philip D Gregory1 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael C Holmes1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:74–79Year published:(2011)DOI:doi:10.1038/nmeth.1539Received04 June 2010Accepted05 November 2010Published online05 December 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Zinc-finger nucleases (ZFNs) drive efficient genome editing by introducing a double-strand break into the targeted gene. Cleavage is induced when two custom-designed ZFNs heterodimerize upon binding DNA to form a catalytically active nuclease complex. The importance of this dimerization event for subsequent cleavage activity has stimulated efforts to engineer the nuclease interface to prevent undesired homodimerization. Here we report the development and application of a yeast-based selection system designed to functionally interrogate the ZFN dimer interface. We identified critical residues involved in dimerization through the isolation of cold-sensitive nuclease domains. We used these residues to engineer ZFNs that have superior cleavage activity while suppressing homodimerization. The improvements were portable to orthogonal domains, allowing the concomitant and independent cleavage of two loci using two different ZFN pairs. These ZFN architectures provide a general means! for obtaining highly efficient and specific genome modification. View full text Subject terms: * Molecular biology * Genetics and genomics Figures at a glance * Figure 1: Isolation of FokI cold-sensitive mutants. () Schematic of two independent single-strand annealing (SSA) reporter constructs containing a homodimer site for the CCR5-L ZFN that were integrated in the yeast genome. The PHO5 SSA reporter contains the positive selection cassette natMX conferring dominant resistance to nourseothricin. The MEL1 SSA reporter contains the URA3 gene for negative selection using 5-fluoroorotic acid (5-FOA), in addition to the kanMX cassette conferring dominant resistance to geneticin (G418). A DNA double-stranded break induced by a functional homodimeric ZFN will result in reconstitution of the reporter genes and elimination of both positive and negative selection markers. PHO, 5′ fragment of PHO5; HO5, 3′ fragment of PHO5; MEL, 5′ fragment of MEL1; and EL1, 3′ fragment of MEL1. () Schematic depicting random mutagenesis of the FokI nuclease domain and assembly of the library of mutants. CYC1t, transcription terminator of the CYC1 gene. S. cerevisiae HIS3 gene was included for selectio! n of transformants in yeast. () Relative activity after induction of the reporter strain transformed with the indicated mutant vectors at three different temperatures compared to the wild type. In the leftmost lane (−), the reporter strain was transformed with an expression vector lacking a zinc-finger protein domain. () ZFN expression was monitored by western blot with an antibody to Flag (anti-Flag). Blots with antibody to histone H3 (anti-H3) were used as controls for loading. * Figure 2: Isolated mutations cluster to the FokI dimer interface. () Location of the mutations in the three-dimensional structure of the wild-type FokI dimer19 interface amino acids 470–540. Mutated residues are shown in gold with oxygen atoms in red and nitrogen atoms in blue, and numbers refer to the mutations described in Supplementary Table 1. () Alternative representation of the dimer interface corresponding to an approximate 100° turn around the x axis. () Molecular modeling of the putative salt-bridge interaction between Arg537 and Asp496 in the obligate heterodimeric interface. * Figure 3: Enhanced activity of the ELD:KKK and ELD:KKR architecture. () Autoradiograms showing results of a Cel-1 assay6, 17, 21 to determine the frequency of ZFN-induced indels, conducted on genomic DNA from K562 cells collected 3 and 20 d after transfection with the indicated KDR ZFN constructs (400 ng). () Western blots showing ZFN expression. The NFκB p65 antibody was used as a loading control. () Autoradiograms showing results of a Cel-1 assay to determine the frequency of ZFN-induced indels, conducted on genomic DNA from K562 cells collected 3 d after transfection with the indicated NR3C1 ZFN constructs (80 and 400 ng). () Autoradiograms showing results of a PCR-based assay9 to determine ZFN-driven tag integration frequency, conducted on genomic DNA from K562 cells collected 3 d after transfection with the indicated NR3C1 ZFN constructs (80 and 400 ng). The percentage of BamHI-sensitive DNA resulting from targeted integration (TI) of the donor is indicated below each lane. Arrows denote specific cleavage products. Asterisks indicate a ! nonspecific amplification product present in each lane. WT, wild type. * Figure 4: Improved activity of new ZFN mutants in primary cells. () Autoradiograms showing results of a Cel-1 assay to determine the frequency of ZFN-induced indels, conducted on genomic DNA from PBMCs collected 3 d and 10 d after transfection with the indicated NR3C1 ZFN constructs (100 ng, 200 ng and 400 ng). Arrows denote specific cleavage products. () Mean values (± s.e.m.) of the relative activities of the indicated ZFNs from six independent transfections into PBMCs as determined by Cel-1 assays conducted on genomic DNA collected at day 3 after transfection and using 400 ng of ZFN expression vector. P values were calculated using the two-sample t-test. * Figure 5: Preservation of the obligate heterodimer specificity. () Flow cytometry data for human K562 cells after transfection with the indicated NR3C1 ZFN constructs (80 ng) and stained with antibodies to γ-H2AX. The percentage of positive cells is shown on days 2 and 3 after transfection. Data from two independent transfections are shown. () Autoradiograms showing results of a Cel-1 assay to determine the frequency of ZFN-induced indels at the intended target (NR3C1) and two homodimer off-target sites, found in noncoding regions of RCSD1 and SREBF2 genes, conducted on genomic DNA from K562 cells collected 3 d after transfection. Arrows denote specific cleavage products. *, nonspecific cutting by the Cel-1 nuclease. **, nonspecific amplification product present in each lane. () Western blots showing ZFN expression. The NFκB p65 antibody was used as a loading control. () Autoradiograms showing results of a Cel-1 assay to determine the frequency of ZFN-induced indels, conducted on genomic DNA from K562 cells collected 3 d after transfec! tion with the indicated NR3C1 ZFN constructs (400 ng). Arrows denote specific cleavage products. () ZFN expression monitored as in . Author information * Abstract * Author information * Supplementary information Affiliations * Sangamo BioSciences, Richmond, California, USA. * Yannick Doyon, * Thuy D Vo, * Matthew C Mendel, * Shon G Greenberg, * Jianbin Wang, * Danny F Xia, * Jeffrey C Miller, * Fyodor D Urnov, * Philip D Gregory & * Michael C Holmes Contributions Y.D. and S.G.G. isolated the cold-sensitive mutants. Y.D., T.D.V., M.C.M., J.W. and D.F.X. characterized the engineered domains. Y.D., J.C.M. and M.C.H. designed the experiments. Y.D., F.D.U., P.D.G. and M.C.H. wrote the manuscript. Competing financial interests All authors are employees of Sangamo BioSciences. Corresponding author Correspondence to: * Yannick Doyon Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (5M) Supplementary Figures 1–15, Supplementary Tables 1–2 and Supplementary Notes 1–2 Additional data
• Protein localization in electron micrographs using fluorescence nanoscopy
  - Nat Meth 8(1):80-84 (2011)
  Nature Methods | Article Protein localization in electron micrographs using fluorescence nanoscopy * Shigeki Watanabe1 Search for this author in: * NPG journals * PubMed * Google Scholar * Annedore Punge2 Search for this author in: * NPG journals * PubMed * Google Scholar * Gunther Hollopeter1 Search for this author in: * NPG journals * PubMed * Google Scholar * Katrin I Willig2 Search for this author in: * NPG journals * PubMed * Google Scholar * Robert John Hobson1 Search for this author in: * NPG journals * PubMed * Google Scholar * M Wayne Davis1 Search for this author in: * NPG journals * PubMed * Google Scholar * Stefan W Hell2 Search for this author in: * NPG journals * PubMed * Google Scholar * Erik M Jorgensen1 Contact Erik M Jorgensen Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:80–84Year published:(2011)DOI:doi:10.1038/nmeth.1537Received03 June 2010Accepted20 October 2010Published online21 November 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg A complete portrait of a cell requires a detailed description of its molecular topography: proteins must be linked to particular organelles. Immunocytochemical electron microscopy can reveal locations of proteins with nanometer resolution but is limited by the quality of fixation, the paucity of antibodies and the inaccessibility of antigens. Here we describe correlative fluorescence electron microscopy for the nanoscopic localization of proteins in electron micrographs. We tagged proteins with the fluorescent proteins Citrine or tdEos and expressed them in Caenorhabditis elegans, fixed the worms and embedded them in plastic. We imaged the tagged proteins from ultrathin sections using stimulated emission depletion (STED) microscopy or photoactivated localization microscopy (PALM). Fluorescence correlated with organelles imaged in electron micrographs from the same sections. We used these methods to localize histones, a mitochondrial protein and a presynaptic dense projection! protein in electron micrographs. View full text Subject terms: * Cell biology Figures at a glance * Figure 1: Correlative fluorescence and electron microscopy using histone H2B fusion proteins. (–) Confocal image (), STED image () and electron micrograph () from the same thin GMA section (120 nm) from a worm expressing histone H2B–Citrine. () Correlative STED microscopy and electron micrographs showing histone H2B–Citrine (overlay of the images in and ). The images in – show an intestinal cell nucleus. (–) Sum TIRF image (; represents all the photons detected by the camera during the experiment), PALM image () and electron micrograph () from a thin GMA section (70 nm) from a worm expressing histone H2B–tdEos. () Correlative PALM and electron micrographs showing histone H2B–tdEos (overlay of the images in and ). The images in – show a muscle cell nucleus. Scale bars, 3 μm (–) and 1 μm (–). * Figure 2: Correlative fluorescence and electron microscopy using TOM20 fusion proteins. (–) Confocal image (), STED image () and electron micrograph () from the same GMA thin section (120 nm) of a worm expressing TOM20–Citrine. () Correlative STED and electron micrographs showing TOM20–Citrine (overlay of the images in and ). (–) Sum TIRF image (), PALM image () and electron micrograph () from a thin LR White section (70 nm) of a worm expressing TOM20–tdEos. () Correlative PALM and electron micrographs showing TOM20–tdEos (overlay of the images in and ). PALM images of sections from a worm expressing TOM20–tdEos are from tissue embedded in LR White; all other samples were embedded in GMA. Scale bars, 1 μm (–) and 2 μm (–). * Figure 3: Correlative fluorescence and electron microscopy using α-liprin fusion proteins. (–) Confocal image (), STED image () and electron micrograph () of the same thin GMA section (70 nm) from a worm expressing α-liprin–Citrine. () Correlative STED microscopy and electron micrographs showing α-liprin–Citrine (overlay of the images in and ). (–) Sum TIRF image (), PALM image () and electron micrograph () from a thin section (70 nm) of a section of a worm expressing α-liprin–Dendra. Asterisk in marks a region of predominant background signal, which was discarded by emission time threshold. () Correlative PALM and electron micrographs showing α-liprin–Dendra (overlay of the images in and ). SV, synaptic vesicle. Scale bars, 500 nm. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Biology and Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah, USA. * Shigeki Watanabe, * Gunther Hollopeter, * Robert John Hobson, * M Wayne Davis & * Erik M Jorgensen * Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. * Annedore Punge, * Katrin I Willig & * Stefan W Hell Contributions S.W. and E.M.J. conceived and designed experiments. G.H., R.J.H. and M.W.D. provided strains and advice. S.W. optimized the methods, prepared the samples and performed PALM imaging. A.P. and K.I.W. performed STED imaging. S.W., S.W.H. and E.M.J. wrote the manuscript. S.W.H. and E.M.J. provided funding. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Erik M Jorgensen Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–5, Supplementary Table 1, Supplementary Notes 1–5 Additional data
• Shotgun glycomics: a microarray strategy for functional glycomics
  - Nat Meth 8(1):85-90 (2011)
  Nature Methods | Article Shotgun glycomics: a microarray strategy for functional glycomics * Xuezheng Song1 Search for this author in: * NPG journals * PubMed * Google Scholar * Yi Lasanajak1 Search for this author in: * NPG journals * PubMed * Google Scholar * Baoyun Xia1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jamie Heimburg-Molinaro1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeanne M Rhea2 Search for this author in: * NPG journals * PubMed * Google Scholar * Hong Ju1 Search for this author in: * NPG journals * PubMed * Google Scholar * Chunmei Zhao1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ross J Molinaro2 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard D Cummings1 Contact Richard D Cummings Search for this author in: * NPG journals * PubMed * Google Scholar * David F Smith1 Contact David F Smith Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MethodsVolume: 8,Pages:85–90Year published:(2011)DOI:doi:10.1038/nmeth.1540Received30 August 2010Accepted10 November 2010Published online05 December 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Major challenges of glycomics are to characterize a glycome and identify functional glycans as ligands for glycan-binding proteins (GBPs). To address these issues we developed a general strategy termed shotgun glycomics. We focus on glycosphingolipids (GSLs), a class of glycoconjugates that is challenging to study, recognized by toxins, antibodies and GBPs. We derivatized GSLs extracted from cells with a heterobifunctional fluorescent tag suitable for covalent immobilization. We separated fluorescent GSLs by multidimensional chromatography, quantified them and coupled them to glass slides to create GSL shotgun microarrays. Then we interrogated the microarrays with cholera toxin, antibodies and sera from individuals with Lyme disease to identify biologically relevant GSLs that we subsequently characterized by mass spectrometry. Shotgun glycomics incorporating GSLs and potentially glycoprotein-derived glycans is an approach for accessing the complex glycomes of animal cells an! d is a strategy for focusing structural analyses on functionally important glycans. View full text Subject terms: * Biochemistry Figures at a glance * Figure 1: Schematic for shotgun glycomics. Glycans are released chemically or enzymatically from glycoproteins, and GSLs are modified directly. The fluorescently labeled products (labeled with 'tag') are separated, quantified and printed to create microarrays available for interrogation with GBPs. * Figure 2: Fluorescent derivatization of GSLs for shotgun glycomics. () The derivatization of GSLs with a bifunctional linker. () C18-HPLC profiles of AOAB derivatization of GM1, GD1a, GT1b and BBG mixture detected by fluorescence. () MALDI-TOF spectra of GM1-AOAB, GD1a-AOAB and GT1b-AOAB purified by HPLC as shown in . The spectra were acquired in the reflective negative mode. ([M-H]− represents negative molecular ions generated by loss of a proton). () The normal-phase HPLC profiles of crude ODA treatment of BBG-PNPA conjugates without precipitation, the precipitate and the filtrate of BBG-PNPA mixture after addition of acetonitrile. * Figure 3: Binding assay on the BBG–GSL-AOAB microarray prepared from two-dimensional HPLC separation. (,) Analysis of 0.1 μg ml−1 CTSB () and anti-GD1a at 1:20 dilution of ascites fluid () on the BBG-GSL-AOAB microarray. Error bars, s.d. (n = 4 replicates). (,) Structural characterization of bound fractions 9 () and 24 () by MS and MS/MS. * Figure 4: Binding of sera from individuals with Lyme disease and control sera on the BBG microarray. () Comparison of IgG binding of sera from individuals with Lyme disease and control sera (tested at 1:100 dilution). The average RFUs were normalized in each serum sample by setting the binding of fraction 33 in control serum and serum from individual with Lyme disease to 100. () Distribution of binding by sera from individual with Lyme disease and control sera over six selected GSL-AOAB fractions (fractions 12, 17, 26, 33, 39 and 40). P values were calculated using Student's t-test, *P < 0.05. () Proposed structural characterization of bound fraction 12 by MS and MS/MS. * Figure 5: The GSL microarray from human erythrocytes and its interrogation with lectins and antibodies. () C18-HPLC profiles of O–blood-type erythrocyte GSL-AOAB. () C18-HPLC profiles of A–blood-type erythrocyte GSL-AOAB. Vertical lines denote each fraction collected in and . (–) The binding of plant lectins Aleuria aurantia lectin (AAL) (1 μg ml−1) (), Ulex eurpoaeus agglutinin 1 (UEA-I) (10 μg ml−1) () and Helix pomatia agglutinin (HPA) (10 μg ml−1) () and antibody to blood group A (10 μg ml−1) (), in which GSL-AOAB fractions 1–23 were from human blood group O erythrocytes, fractions 24–48 were from human blood group A erythrocytes and fractions 49–52 were controls, including the AEAB derivatives of lacto-N-neotetraose (LNnT), lacto-N-fucopentaose III (LNFIII), Lewisy-Lewisx (LeYLeX) and biotin. Inset in is a magnified view for fractions 1–48. Error bars, s.d. (n = 4). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA. * Xuezheng Song, * Yi Lasanajak, * Baoyun Xia, * Jamie Heimburg-Molinaro, * Hong Ju, * Chunmei Zhao, * Richard D Cummings & * David F Smith * Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA. * Jeanne M Rhea & * Ross J Molinaro Contributions X.S., R.D.C. and D.F.S. planned the project, and X.S., Y.L., B.X., H.J., C.Z., J.M.R. and R.J.M. carried out the experiments and supplied critical reagents. X.S., Y.L., J.H.-M., R.D.C. and D.F.S. analyzed the data and wrote the manuscript. All authors edited and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Richard D Cummings or * David F Smith Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (5M) Supplementary Figures 1–6 and Supplementary Tables 1–3 Additional data
• Stabilized imaging of immune surveillance in the mouse lung
  - Nat Meth 8(1):91-96 (2011)
  Nature Methods | Article Stabilized imaging of immune surveillance in the mouse lung * Mark R Looney1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Emily E Thornton2, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Debasish Sen2 Search for this author in: * NPG journals * PubMed * Google Scholar * Wayne J Lamm3 Search for this author in: * NPG journals * PubMed * Google Scholar * Robb W Glenny3 Search for this author in: * NPG journals * PubMed * Google Scholar * Matthew F Krummel2 Contact Matthew F Krummel Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:91–96Year published:(2011)DOI:doi:10.1038/nmeth.1543Received22 July 2010Accepted18 November 2010Published online12 December 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Real-time imaging of cellular and subcellular dynamics in vascularized organs requires image resolution and image registration to be simultaneously optimized without perturbing normal physiology. This problem is particularly pronounced in the lung, in which cells may transit at speeds >1 mm s−1 and in which normal respiration results in large-scale tissue movements that prevent image registration. Here we report video-rate, two-photon imaging of a physiologically intact preparation of the mouse lung that is stabilizing and nondisruptive. Using our method, we obtained evidence for differential trapping of T cells and neutrophils in mouse pulmonary capillaries, and observed neutrophil mobilization and dynamic vascular leak in response to stretch and inflammatory models of lung injury in mice. The system permits physiological measurement of motility rates of >1 mm s−1, observation of detailed cellular morphology and could be applied in the future to other organs and tissues! while maintaining intact physiology. View full text Subject terms: * Immunology Figures at a glance * Figure 1: Experimental setup and image stability for intravital imaging of the mouse lung. () Anterior and posterior views of the thoracic suction window fitted with a coverslip. () Side-view rendering of the suction window showing suction chamber, cover slip (green arrows) and vacuum flows (blue arrows near tissue, red arrows toward suction regulator). () Surgical preparation of left thorax with exposed left lung. () Suction window in situ. () Representative images at the indicated depths in a mouse injected with Texas Red–dextran, showing the capillary bed above (left) and below (right) the subpleural alveoli (middle). (,) Still images of CFP fluorescence in an actin-CFP–expressing mouse lung at the indicated times after the start of imaging (color-coded arbitrarily), and a merge of these three images (far right). The plot shows the Pearson's coefficient between time points. Images shown in were captured at 30 fps. In , each frame represents 15 integrated images that are then merged (time points aligned and the Pearson's coefficient from this integration is ! shown). Scale bars, 5 mm (), 10 mm (,) and 50 μm (–). * Figure 2: Perfusion velocities of beads and neutrophils in the lung. () The micrographs show sequential images of individual beads traversing the lung microcirculation (arrowheads), in actin-CFP–expressing mice injected intravenously with red fluorescent microspheres and then imaged at 30 fps. Time elapsed after the first frame is indicated. Scale bar, 50 μm. () Perfusion velocities of individual beads shown as individual dots in small (109 ± 12 μm s−1, mean ± s.e.m. (gray bars), n = 14) and medium-sized blood vessels (280 ± 53 μm s−1, mean ± s.e.m., n = 11; P < 0.001). () The micrograph shows one frame recorded at 30 fps with four representative tracks of neutrophils (colony-stimulating factor 1R (Cfms)-EGFP) and beads inside a vessel of an actin-CFP–expressing mouse injected with fluorescent beads and imaged at 30 fps. Scale bar, 10 μm. () The plots show the average and instantaneous track speeds of neutrophils in small (0.91 ± 0.16 μm s−1, mean ± s.e.m., n = 5) and medium-sized (96.5 ± 37.8 μm s−1, mean ± s.e.m., ! n = 5, *P < 0.05 and **P < 0.001) blood vessels. () Histogram of neutrophil perfusion velocities in a medium-sized blood vessel. Solid line highlights cells that are crawling along the vessel at slow speeds, and dotted line highlights cells with velocities most consistent with flow within vessels. * Figure 3: Perfusion velocities of T cells in the lung. () Average track speeds of naive (2.48 ± 0.49 μm s−1, mean ± s.e.m., n = 4) and activated T cells (0.41 ± 0.07 μm s−1, mean ± s.e.m., n = 4, *P < 0.01) injected into the jugular vein of actin-CFP–expressing mice. () Representative images showing the morphology of naive T cells (CD2 RFP) and T cell blasts (ubiquitin-GFP). Arrows indicate a T cell blast with two leading edges, likely extending into two vascular branches. Scale bar, 10 μm. () Width of the capillary segments containing naive (5.61 ± 0.39 μm, mean ± s.e.m., n = 8) and activated T cells (7.75 ± 0.41 μm, mean ± s.e.m., n = 12, *P < 0.01) are plotted. () Maximal intensity projections of single time points showing the sizes of intravascular naive (left) and activated (right) T cells. Scale bar, 50 μm. Images are from 40 μm z-dimension stack. () Average diameters of naive (7.74 ± 0.23 μm, mean ± s.e.m., n = 12) and activated T cells (11.36 ± 0.40 μm, mean ± s.e.m., n = 12, *P < 0.05, **P < 0! .001 and P < 0.0001). * Figure 4: Imaging inflammation and injury-induced neutrophil dynamics in physiologically intact lungs. () Images of the lung of a LysM-GFP mouse injected with Texas Red–dextran and imaged before (left) and after (right) intratracheal instillation of MIP-2. Scale bar, 50 μm, 40 μm z stack. () Number of GFP+ neutrophils in the imaging field before (16.75 ± 3.06 cells, mean ± s.e.m., n = 4) and after MIP-2 (44.25 ± 4.42 cells, mean ± s.e.m., n = 4, *P < 0.01) intratracheal (IT) instillation. () Number of GFP+ neutrophils in the lung vasculature under continuous suction (n = 4 for each time point). () Representative images of an intravascular GFP+ neutrophil. Scale bar, 10 μm, single z plane at 5:20, 6:40 and 9:20 min:s. () Representative images of a GFP+ neutrophil moving within alveoli. Scale bar, 10 μm, single z plane at 0:40, 5:40 and 9:00 min:s. () Images of the lung of an actin-CFP/c-fms-GFP mouse before and after intratracheal instillation of LPS. Scale bar, 50 μm, 40 μm z stack. () Number of neutrophils per field before (2.75 ± 0.48, mean ± s.e.m., n = 4) an! d after LPS instillation (10.25 ± 1.32, mean ± s.e.m., n = 4, *P < 0.01). () Images of the lung of an actin-CFP mouse injected with Texas Red–dextran and either challenged with intratracheal LPS for 50 min () or subjected to ventilator-induced lung injury for 60 min (). Scale bar, 50 μm, 40 μm z stack. () The plots show the average intensity of fluorescent dextran in the alveolar space at the indicated times after LPS treatment () or ventilator-induced lung injury (). Blue lines are the pretreatment average (n = 3 alveoli), red lines are individual alveoli measured after treatment and black lines are the after treatment average (n = 5 alveoli). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Mark R Looney & * Emily E Thornton Affiliations * Departments of Medicine and Laboratory Medicine, University of California, San Francisco, San Francisco, California, USA. * Mark R Looney * Department of Pathology, University of California, San Francisco, San Francisco, California, USA. * Emily E Thornton, * Debasish Sen & * Matthew F Krummel * Department of Medicine, University of Washington, Seattle, Washington, USA. * Wayne J Lamm & * Robb W Glenny Contributions M.R.L. conceived and designed the experiment, validated and implemented the technique, collected and analyzed data and wrote the manuscript. E.E.T. conceived and designed the experiment, validated and implemented the technique, collected and analyzed data and wrote the manuscript. D.S. implemented the technique and collected and analyzed data. W.J.L. conceived and designed the experiment and validated the technique. R.W.G. conceived and designed the experiment and edited the manuscript. M.F.K. conceived and designed the experiment, provided administrative and financial support and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Matthew F Krummel Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (2M) Ventilation with thoracic suction. Real-time brightfield video of expanding and contracting alveoli during ventilation in the microscope setup at 100A magnification. * Supplementary Movie 2 (5M) Video-rate intravital lung imaging. Video-rate (30 fps, single z plane) two-photon movie of actin-CFP (blue) mouse with unaveraged inspiration and expiration. * Supplementary Movie 3 (192K) Fifteen-frame-averaged intravital lung imaging. Two-photon video of actin-CFP (blue) mouse with averaged (fifteen frames, single z plane) acquisition showing stability and intravascular cellular movement. * Supplementary Movie 4 (244K) Lung circulation with thoracic suction. Two-photon video of wild-type mouse injected with Texas Red dextran (red) marking vasculature. Unlabeled cellular shadows are observed in motion in the labeled vasculature. * Supplementary Movie 5 (2M) Perfusion velocities in the lung. Two-photon video of a single z plane with 1-μm beads (red) flowing through alveolar capillaries of diameter 10–15 μm. Bead tracks are marked with dragon tails. Vasculature marked with actin-CFP (blue). Time is indicated in s:ms. Bead speeds of ~0–400 μm s-1 with an average of ~110 μm s-1 were observed inside capillaries. * Supplementary Movie 6 (9M) Simultaneous imaging of neutrophil and bead velocities in the lung. Video-rate, two-photon movie of a single z plane with i.v. 1-μm beads (red) flowing through vasculature (30 μm diameter) marked with actin-CFP (blue). Neutrophils (c-fms, green) flow through the vessels. Cell (green) and bead (red) tracks are marked with dragon tails. Bead track speed average is 297.6 μm s-1 with a range of 106.8–728.3 μm s-1. * Supplementary Movie 7 (4M) Naive and T-cell blast migration in the lungs. Left-sided movie from an actin-CFP mouse injected with naive CD2-RFP (red) T cells (5 A 107). Right-sided movie from a wild-type mouse injected with T-cell blasts (ubiquitin-GFP, green, 5 A 107). * Supplementary Movie 8 (2M) Neutrophil recruitment into the lung with intratracheal MIP-2. Two-photon video of LysM-GFP marked neutrophils (green) in dextran marked vasculature (red) at baseline (left) and 70 min after intratracheal MIP-2 treatment (right, 5 μg, i.t., 40 μm z stack). * Supplementary Movie 9 (8M) Intravascular neutrophil migratory activity in the lung. Two-photon video of a LysM-GFP neutrophil (green) crawling through dextran marked vasculature (red) at 60 min after MIP-2 treatment (5 μg, i.t.). The center of mass of the intravascular neutrophil is marked with a grey sphere. Flashing yellow arrowheads indicate the alternating leading edge of the intravascular neutrophil. * Supplementary Movie 10 (928K) Extravascular neutrophil migratory activity in the lung. Two-photon video of LysM-GFP neutrophils (green) in dextran (red) marked vasculature in a mouse 60 min after MIP-2 treatment (5 μg, i.t.). The first pass consists of one z plane to show cellular detail with the cell of interest, marked with a white sphere, deforming itself to move between alveolar spaces. The second pass includes 40 μm in z to provide context. * Supplementary Movie 11 (7M) Neutrophil recruitment into the lung after intratracheal LPS. Two-photon video of c-fms–GFP neutrophils (green) in a lung where all cells are marked by actin-CFP (blue) before (left) and 70 min after LPS treatment (5 mg kg-1, i.t., 40 μm z stack). * Supplementary Movie 12 (532K) Lung vascular leak after intratracheal LPS. Two-photon video of lung tissue marked with actin-CFP (blue) showing dextran leak (red) into the extravascular space 50 min after LPS treatment (5 mg kg-1, i.t., 40 μm z stack). * Supplementary Movie 13 (852K) Ventilator-induced lung injury and lung vascular leak. Two-photon video of lung tissue marked with actin-CFP (blue) showing dextran leak (red) into the extravascular space 50 min after induction with high tidal volume lung injury (ventilator-induced lung injury, 40 μm z stack). PDF files * Supplementary Text and Figures (156K) Supplementary Figure 1 Additional data