Hot off the presses! Oct 01 Nat Methods

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

Latest Articles Include:

• In celebration of methods
  - Nat Methods 6(10):687 (2009)
  As evidenced by the cake adorning the cover, Nature Methods is five years old. To celebrate this anniversary, we look at methodological development and its role in scientific inquiry.
• Online image analysis software for photoactivation localization microscopy
  - Nat Methods 6(10):689-690 (2009)
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• My5C: web tools for chromosome conformation capture studies
  - Nat Methods 6(10):690-691 (2009)
  I want to purchase this article Register now Price: US$18 In order to purchase this article you must be a registered user. I want to subscribe to Nature Methods Select this option to purchase a personal subscription to Nature Methods.
• Two photons as exciting as one
  - Nat Methods 6(10):693 (2009)
  Channelrhodopsin-2 can be efficiently activated by infrared two-photon excitation light and stimulates action potentials in cultured neurons.
• 'Blue' lighting cell signaling research
  - Nat Methods 6(10):694-695 (2009)
  By fusing a light-sensitive domain of an oat plant protein to Rac1, researchers created a genetically encoded protein fusion that can be reversibly activated with blue light and control cell movement—an attractive alternative to current caging tools.
• A leader anyone can follow
  - Nat Methods 6(10):694-695 (2009)
  The development of leader sequences that stimulate mRNA translation in a species-independent manner could offer new possibilities for eukaryotic protein production and proteomic research.
• News in brief
  - Nat Methods 6(10):695 (2009)
  
• Illuminating lipids
  - Nat Methods 6(10):696 (2009)
  Visualization of choline-containing phospholipids in cells and in vivo is made possible by the metabolic incorporation of a choline analog with an alkyne handle for click chemistry–based labeling.
• Engineering bacteria in yeast
  - Nat Methods 6(10):698 (2009)
  Bacterial genomes shuttled into yeast can be easily altered before transplantation back into bacteria.
• Technical matters: method, knowledge and infrastructure in twentieth-century life science
  - Nat Methods 6(10):701-705 (2009)
  Conceptual breakthroughs in science tend to garner accolades and attention. But, as the invention of tissue culture and the development of isotopic tracers show, innovative methods open up new fields and enable the solution of longstanding problems.
• Seeing things: from microcinematography to live cell imaging
  - Nat Methods 6(10):707-709 (2009)
  From histology to microcinematography, from cytochemistry to live cell imaging, the history of visualization technology in the life sciences may be understood as a series of cycles of action and reaction between static and dynamic modes of representing life.
• Is sequencing enlightenment ending the dark age of the transcriptome?
  - Nat Methods 6(10):711-713 (2009)
  Sequencing-based technologies for RNA discovery are playing a key role in deciphering the transcriptome and hold the potential to provide us with a census of RNAs and their functions.
• Engineered fluorescent proteins: innovations and applications
  - Nat Methods 6(10):713-717 (2009)
  Despite expansion of the fluorescent protein and optical highlighter palette into the orange to far-red range of the visible spectrum, achieving performance equivalent to that of EGFP has continued to elude protein engineers.
• Comparative analysis to guide quality improvements in proteomics
  - Nat Methods 6(10):717-719 (2009)
  The potential of mass spectrometry–based proteomics to advance biology and biomedicine is nearly unlimited but so is its potential for generating bad data. Apart from the pursuit of technological progress in protocols and instruments, stringent comparative analyses of different approaches are critical for fully developing the discipline.
• From information to knowledge: new technologies for defining gene function
  - Nat Methods 6(10):721-723 (2009)
  A wide range of methodology will be needed to bridge the gap between genome sequence and mechanistic understanding in biology. Recent advances in high-throughput genetic screening address this task.
• Five years of Methods
  - Nat Methods 6(10):724-725 (2009)
  Introduction A fun selection of papers published in Nature Methods since 2004. Fastest citation rate Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L. & Wold, B. Nat. Methods 5, 621–628 (2008). Cloonan, N. et al. Nat. Methods 5, 613–619 (2008). Some methods take a field by storm, and RNA-seq is one of those. The year 2008 saw several high-profile papers describing transcriptome profiling with second-generation sequencing platforms. The analysis of mammalian cell transcripts by Barbara Wold's group was received with instant interest by our readers as was a related paper by another group. Most surprising Fortin, D.L. et al. Nat. Methods 5, 331–338 (2008). Imbuing cellular proteins with new characteristics generally requires the introduction of engineered genes or proteins. But Richard Kramer and colleagues showed that attaching a photoisomerizable small molecule to endogenous K+ channels in neurons allows specific light-mediated control of their activity. Fastest to publication Xu, R.H. et al. Nat. Methods 2, 185–190 (2005). This report from James Thomson's group on proliferation mechanisms in human embryonic stem cells not only made culturing them easier but also sped through the review process to make it online just about a month after submission. Seen by the most economists Choi, W. et al. Nat. Methods 4, 717–719 (2007). It seems impossible to predict where a news article about a paper published in Nature Methods will appear. This was aptly demonstrated when an article by Michael Feld and colleagues on a new microscopy method called tomographic phase microscopy was covered in the 16 August 2007 issue of The Economist. Most 'insulting' Nishimura, N. et al. Nat. Methods 3, 99–108 (2006). Authors and editors try to avoid the appearance of insults in papers. But the January 2006 issue of Nature Methods not only had a paper by David Kleinfeld and colleagues devoted to targeted insults but also featured it on the cover. The laser-based insults were designed to model strokes in mice, though. Leaving the least trace Yusa, K., Rad, R., Takeda, J. & Bradley, A. Nat. Methods 6, 363–369 (2009). Joining the race to make induced pluripotent stem cells without permanent genome modification, this paper reports on the use of the piggyBac transposon to transiently express the four reprogramming factors and then remove them from the genome without a trace. Top article Irizarry, R.A. et al. Nat. Methods 2, 345–350 (2005). Frustration with irreproducible DNA microarray results begged the question: is this an inherent problem of the technology? In 2005 a consortium said no, showing that data collected on three platforms in ten labs agreed well. Our citation numbers indicate that the community eagerly received this message. Most aptly named Rust, M.J., Bates, M. & Zhuang, X. Nat. Methods 3, 793–795 (2006). Nature Methods published a paper describing a super-resolution imaging method on 9 August 2006, one day before a competing paper was published in Science. The name of the method, 'STORM', seemed to embody the level of interest these two papers generated in the community. Simplest fishing tool Vigneault, F., Sismour, A.M. & Church, G.M. Nat. Methods 5, 777–779 (2008). To fish out microRNAs from a sample of interest, you need good tools, and the less complicated the better. This paper reported a method to prepare preadenylated barcoded oligonucleotides, which could be used for efficient capture and multiplex analysis of microRNA from biological samples. Smallest tools Ebert, M.S., Neilson, J.R. & Sharp, P.A. Nat. Methods 4, 721–726 (2007). Loss-of-function phenotypes for microRNAs were still rare in 2007, explaining the appeal of microRNA sponges. These 'sponge' transcripts, containing multiple binding sites for a microRNA, soak it up and derepress its target, thus creating a miRNA knockdown phenotype. Among the most deadly Herm-Göz, A. et al. Nat. Methods 4, 1003–1005 (2007). Armstrong, C.M. & Goldberg, D.E. Nat. Methods 4, 1007–1009 (2007). Quod licet Homo non licet Apicomplexa—or maybe it is. Two groups showed that an inducible protein destabilization strategy developed for mammalian cells could be adapted for Apicomplexa to study proteins in these parasites that cause malaria and encephalitis. Most expensive instrument Hura, G.L. et al. Nat. Methods 6, 606–612 (2009). Structural biology is relying more and more on that most expensive of instruments, the synchrotron. John Tainer and colleagues used synchrotron radiation to rapidly carry out small-angle X-ray scattering (SAXS)-based analyses of folds and oligomeric states for large numbers of proteins. Most chemical Hennig, A., Bakirci, H. & Nau, W.M. Nat. Methods 4, 629–632 (2007). Examples of how chemistry provides useful tools for biological applications can be found in many of our issues. One such paper describes a new principle for label-free enzymatic assays, taking advantage of macrocycles that reversibly form complexes with fluorescent dyes and enzymatic products. Fastest amplification Atarashi, R. et al. Nat. Methods 4, 645–650 (2007). Atarashi, R. et al. Nat. Methods 5, 211–212 (2008). The protein misfolding cyclic amplification (PMCA) assay is widely used to detect the protease-resistant prion protein responsible for 'mad cow' and other prion diseases. With several improvements to the protocol, Byron Caughey and colleagues made this assay faster and more sensitive. Among the sweetest Structurally speaking, sugars are complex molecules, but they are also predictable in their protein binding preferences. With an evanescent-field method to detect glycans bound to arrayed lectins, Jun Hirabayashi and colleagues showed that they could not only profile glycans but do so quantitatively. Kuno, A. et al. Nat. Methods 2, 851–856 (2005).
• Staging worms for next-generation analysis
  - Nat Methods 6(10):727-728 (2009)
  Automated stage-specific sorting of Caenorhabditis elegans embryos enables next-generation molecular and biochemical analysis of development.
• Nanoscale 3D cellular imaging by axial scanning transmission electron tomography
  - Nat Methods 6(10):729-731 (2009)
  Electron tomography provides three-dimensional structural information about supramolecular assemblies and organelles in a cellular context, but image degradation, caused by scattering of transmitted electrons, limits applicability in specimens thicker than 300 nm. We found that scanning transmission electron tomography of 1,000-nm-thick samples using axial detection provided resolution comparable to that of conventional electron tomography. We demonstrated the method by reconstructing a human erythrocyte infected with the malaria parasite Plasmodium falciparum.
• Somatic cell nuclear transfer in zebrafish
  Siripattarapravat K Pinmee B Venta PJ Chang CC Cibelli JB - Nat Methods 6(10):733-735 (2009)
  We developed a method for somatic cell nuclear transfer in zebrafish using laser-ablated metaphase II eggs as recipients, the micropyle for transfer of the nucleus and an egg activation protocol after nuclear reconstruction. We produced clones from cells of both embryonic and adult origins, although the latter did not give rise to live adult clones.
• Genetically encoded FRET sensors to monitor intracellular Zn2+ homeostasis
  Vinkenborg JL Nicolson TJ Bellomo EA Koay MS Rutter GA Merkx M - Nat Methods 6(10):737-740 (2009)
  We developed genetically encoded fluorescence resonance energy transfer (FRET)-based sensors that display a large ratiometric change upon Zn2+ binding, have affinities that span the pico- to nanomolar range and can readily be targeted to subcellular organelles. Using this sensor toolbox we found that cytosolic Zn2+ was buffered at 0.4 nM in pancreatic cells, and we found substantially higher Zn2+ concentrations in insulin-containing secretory vesicles.
• Proteomics strategy for quantitative protein interaction profiling in cell extracts
  - Nat Methods 6(10):741-744 (2009)
  We report a proteomics strategy to both identify and quantify cellular target protein interactions with externally introduced ligands. We determined dissociation constants for target proteins interacting with the ligand of interest by combining quantitative mass spectrometry with a defined set of affinity purification experiments. We demonstrate the general utility of this methodology in interaction studies involving small-molecule kinase inhibitors, a tyrosine-phosphorylated peptide and an antibody as affinity ligands.
• Large-scale sorting of C. elegans embryos reveals the dynamics of small RNA expression
  - Nat Methods 6(10):745-751 (2009)
  Caenorhabditis elegans is one of the most prominent model systems for embryogenesis, but collecting many precisely staged embryos has been impractical. Thus, early C. elegans embryogenesis has not been amenable to most high-throughput genomics or biochemistry assays. To overcome this problem, we devised a method to collect staged C. elegans embryos by fluorescence-activated cell sorting (eFACS). In a proof-of-principle experiment, we found that a single eFACS run routinely yielded tens of thousands of almost perfectly staged 1-cell stage embryos. As the earliest embryonic events are driven by posttranscriptional regulation, we combined eFACS with second-generation sequencing to profile the embryonic expression of small, noncoding RNAs. We discovered complex and orchestrated changes in the expression between and within almost all classes of small RNAs, including microRNAs and 26G-RNAs, during embryogenesis.
• A Flp-nick system to study repair of a single protein-bound nick in vivo
  - Nat Methods 6(10):753-757 (2009)
  We present the Flp-nick system, which allows introduction of a protein-bound nick at a single genomic site in Saccharomyces cerevisiae and thus mimics a stabilized topoisomerase I–DNA cleavage complex. We took advantage of a mutant Flp recombinase that can introduce a nick at a specific Flp recombinase recognition target site that has been integrated in the yeast genome. The genetic requirement for cells to cope with this insult is the same as for cells treated with camptothecin, which traps topoisomerase I–DNA cleavage complexes genome-wide. Hence, a single protein-bound nick is enough to kill cells if functional repair pathways are lacking. The Flp-nick system can be used to dissect repair, checkpoint and replication fork management pathways activated by a single genomic insult, and it allows the study of events at the damage site, which so far has been impossible to address.
• An approach for extensibly profiling the molecular states of cellular subpopulations
  - Nat Methods 6(10):759-765 (2009)
  Microscopy often reveals the existence of phenotypically distinct cellular subpopulations. However, additional characterization of observed subpopulations can be limited by the number of biomolecular markers that can be simultaneously monitored. Here we present a computational approach for extensibly profiling cellular subpopulations by freeing one or more imaging channels to monitor additional probes. In our approach, we trained classifiers to re-identify subpopulations accurately based on an enhanced collection of phenotypic features extracted from only a subset of the original markers. Then we constructed subpopulation profiles step-wise from replicate experiments, in which cells were labeled with different but overlapping marker sets. We applied our approach to identify molecular differences among subpopulations and to identify functional groupings of markers, in populations of differentiating mouse preadipocytes, polarizing human neutrophil-like cells and dividing! human cancer cells.
• Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms
  - Nat Methods 6(10):767-772 (2009)
  Biological pathways are structured in complex networks of interacting genes. Solving the architecture of such networks may provide valuable information, such as how microorganisms cause disease. Here we present a method (Tn-seq) for accurately determining quantitative genetic interactions on a genome-wide scale in microorganisms. Tn-seq is based on the assembly of a saturated Mariner transposon insertion library. After library selection, changes in frequency of each insertion mutant are determined by sequencing the flanking regions en masse. These changes are used to calculate each mutant's fitness. Using this approach, we determined fitness for each gene of Streptococcus pneumoniae, a causative agent of pneumonia and meningitis. A genome-wide screen for genetic interactions of five query genes identified both alleviating and aggravating interactions that could be divided into seven distinct categories. Owing to the wide activity of the Mariner transposon, Tn-seq has t! he potential to contribute to the exploration of complex pathways across many different species.
• Getting inside their minds
  - Nat Methods 6(10):773-781 (2009)
  Neuroscientists are taking advantage of powerful new tools for fluorescence imaging that enable detailed visualization of the structure and activity of neuronal circuits within the living brain.

Hot off the presses! Sep 01 Nat Med

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

Latest Articles Include:

• In the land of the monolingual
  - Nat Med 15(9):975 (2009)
  Translating a basic finding into a new therapy requires us to speak many languages—scientific, clinical, legal and financial. Yet most of us are hopelessly 'monolingual', a limitation that substantially slows translational research. Steps have been taken to address this problem, but a lot remains to be done.
• A change is in the wind as 'adaptive' clinical trials catch on
  Hutson S - Nat Med 15(9):977 (2009)
  In 2003, researchers stopped the ASTIN study, a clinical trial of a drug suspected to help stroke victims, because the drug was found to be ineffective. Of course, the fact that it was stopped isn't wholly remarkable: more than 80% of drugs that enter clinical trials fail, according to estimates by the Center for Drug Evaluation and Research of the US Food and Drug Administration (FDA).
• Pandemic vaccine enters clinical trials
  Willyard C - Nat Med 15(9):978 (2009)
  The majority of the seasonal flu vaccine has already been shipped ahead of flu season in the Northern Hemisphere, but candidate vaccines against pandemic H1N1—which had killed nearly 1,800 people as of August—are just entering clinical trials. Health officials predict that the first doses should be available, at the earliest, in September.
• New plan seeks to accelerate African diagnostic capacity
  Wenner M - Nat Med 15(9):978 (2009)
  Africa is home to 60% of the world's HIV/AIDS burden, 90% of its malaria cases and nearly a quarter of the globe's tuberculosis sufferers, but only a handful of clinical laboratories can properly identify and treat patients. That could soon change, however, thanks to an ambitious accreditation initiative unveiled on 27 July that seeks to double Africa's diagnostic laboratory capacity over the next two years.
• Sealants get specific
  Siva N - Nat Med 15(9):978 (2009)
  Researchers from the Massachusetts Institute of Techology say they have new insights into how a glue-like material might be tailored to work with specific tissues and organs. The authors of the paper, published online this summer, think that the current surgical sealants used to repair wounds have not reached their true potential.
• New technologies promise safer sex for women
  May M - Nat Med 15(9):979 (2009)
  With nearly 7,000 people being infected with HIV every day, societies around the world need new techniques to keep sex safe. Currently, condoms provide the highest protection against HIV infection during intercourse.
• Growing pains plague children's health study
  Wenner M - Nat Med 15(9):979 (2009)
  The congressionally mandated National Children's Study (NCS), the largest long-term study of environmental and genetic effects on child health in the US to date, experienced some growing pains this month. A US Senate committee released a report on 4 August stating that it will reevaluate NCS funding after its 18-month pilot phase finishes in 2010, raising questions about whether the full study will move forward as planned.
• News in brief
  - Nat Med 15(9):980-981 (2009)
  Jul 22A report from the US National Academies, including the Institute of Medicine, urged researchers to do a better job of divulging the data behind their study results, as well as the computer programs used to analyze the raw data. Some experts, however, criticized the report for failing to outline detailed rules about such data sharing.
• Straight talk with...Ian Lipkin
  Westly E - Nat Med 15(9):982-983 (2009)
  How do you sequence a virus that no one has identified before? With the right technology, it's not as difficult a task as it might seem, says Ian Lipkin, director of the Center for Infection and Immunity at Columbia University's Mailman School of Public Health. Lipkin has been working on the technology involved with viral discovery since the late 1980s, when he became the first researcher to identify a microbe using only molecular tools. He and the Center's team of about 50 researchers have identified close to 200 new viruses so far. Most recently, Lipkin and his colleague Thomas Briese identified a new hemorrhagic fever virus that killed several patients in southern Africa last year. Now, in addition to processing samples from around the world, Lipkin has been working to export his sequencing technology—and the expertise needed to use it—to the developing world. Erica Westly spoke with Lipkin about how the viral discovery techniques he uses could help prevent futur e viral disease outbreaks, from swine flu to the unknown.
• Boosting our best shot
  Schubert C - Nat Med 15(9):984-988 (2009)
  Vaccines work by training the immune system to target pathogens, but many types of shots need added substances called adjuvants to elicit a robust response. Despite the power of adjuvants, only one, called alum, is approved in the US. Charlotte Schubert looks at recent discoveries that could translate into a wider range of adjuvants and perhaps help provide future protection against diseases ranging from malaria to H1N1 'swine' flu.
• Illuminating alum
  Schubert C - Nat Med 15(9):985 (2009)
  Alum, the world's most widely used adjuvant, got its start in the 1920s when vaccinologists found that mixing it into their preparations gave a boost to the diphtheria vaccine. Researchers then proposed that it worked by glomming onto vaccine components, causing them to be released slowly into the bloodstream.
• Swine flu agitates the adjuvant debate
  Schubert C - Nat Med 15(9):986-987 (2009)
  US regulatory agencies will face a big decision, as early as this fall, as to whether to approve the use of adjuvants for pandemic H1N1 flu vaccine on an emergency basis.Adjuvants used in some seasonal flu vaccines in Europe have the potential to boost the effectiveness of pandemic H1N1 vaccine and also substantially reduce dosage—thereby stretching supplies.
• Mouse fertility is not dependent on the CREB coactivator Crtc1
  Breuillaud L Halfon O Magistretti PJ Pralong FP Cardinaux JR - Nat Med 15(9):989-990 (2009)
  Cyclic AMP response element–binding protein (CREB)-regulated transcription coactivators (Crtcs), also known as transducers of regulated CREB activity (TORCs), are coactivators that function as calcium- and cyclic AMP–sensitive coincidence detectors1, 2. Ubiquitously expressed Crtc2 is a key regulator of energy metabolism modulating gluconeogenesis and insulin signaling3, 4, 5.
• Reply to: "Mouse fertility is not dependent on the CREB coactivator Crtc1"
  Altarejos J Goebel N Conkright M Inoue H Xie J Arias C Sawchenko P Montminy M - Nat Med 15(9):991 (2009)
  We find Cardinaux's correspondence1 quite interesting. Both laboratories used a practically identical approach to knock out the Crtc1 gene in similar genetic strains.
• Innovating for impact: The Affordable Medicines Facility-malaria (AMFm)
  Adeyi O Atun R - Nat Med 15(9):991 (2009)
  Your story1 on AMFm does not provide a balanced picture of the evidence pertaining to the proposed approach adopted by AMFm and the prevailing development approaches used to combat malaria.AMFm is an innovative financing mechanism to expand access to affordable artemisinin-based combination therapies (ACTs) for malaria, thereby saving lives and reducing the use of inappropriate treatments.
• The vaccine-autism controversy
  Geschwind D - Nat Med 15(9):992 (2009)
  Autism is a devastating developmental neuropsychiatric disorder whose symptoms emerge during the first three years of life. Although recent genetic studies have provided major advances in our understanding of autism's etiologies in up to 10% of cases, the causes of most cases are not known.
• Blocking the path of lymphatic vessels
  Skobe M Dana R - Nat Med 15(9):993-994 (2009)
  Identification of an endogenous inhibitor of lymphatic vessel formation provides a glimpse at how lymphatic vessel growth is restrained (pages 1023–1030). The findings might be exploited to lower transplant rejection rates.
• Two sides to cilia in cancer
  Toftgård R - Nat Med 15(9):994-996 (2009)
  The primary cilium can keep cancer at bay, or it can instigate tumor development, according to studies in mice (pages 1055–1061 and 1062–1065). The outcome depends on the nature of the initiating event, which involves signaling through the Hedgehog pathway.
• Connecting obesity, aging and diabetes
  Ahima RS - Nat Med 15(9):996-997 (2009)
  Obesity accelerates the aging of adipose tissue, a process only now beginning to come to light at the molecular level. Experiments in mice suggest that obesity increases the formation of reactive oxygen species in fat cells, shortens telomeres—and ultimately results in activation of the p53 tumor suppressor, inflammation and the promotion of insulin resistance (pages 1082–1087).
• Cardiovascular biomarker questioned
  - Nat Med 15(9):998 (2009)
  A study in the Journal of the American Medical Association addresses a long-standing controversy in cardiovascular medicine: does a protein in the bloodstream associated with cardiovascular disease, C-reactive protein (CRP), actually cause disease? The analysis, an amalgamation of data from 120,000 individuals, suggests that the answer is no1.
• A safer stem cell: on guard against cancer
  Jandial R Snyder EY - Nat Med 15(9):999-1001 (2009)
  Before stem cell therapies become mainstream, several hurdles must be overcome. One challenge is developing air-tight approaches to assure that stem cell transplantation does not give rise to tumors. Another is finding safe ways to induce pluripotency in adult stem cells, which can then be used for transplantation. In Bedside to Bench, Evan Snyder and Rahul Jandial discuss the risks of tumorigenesis in stem cell therapies, and, in Bench to Bedside, Laura Clarke and Derek van der Kooy examine new ways to induce pluripotency.
• A safer stem cell: inducing pluripotency
  Clarke L van der Kooy D - Nat Med 15(9):1001-1002 (2009)
  The isolation of human embryonic stem cells1 (hESCs) theoretically gave scientists the starting material to produce any cell type in the body for use in regenerative medicine. One major obstacle to this approach has been the inevitable host immunological response to transplanted foreign tissue.
• Research Highlights
  - Nat Med 15(9):1004-1005 (2009)
  
• The advancement of translational medicine—from regional challenges to global solutions
  Albani S Prakken B - Nat Med 15(9):1006-1009 (2009)
  Translating the progress in molecular medicine into new therapies has met with limited success; the route from idea to drug has many hurdles and is a very fragmented process. This fragmentation is evident at all levels—academia, industry and governments—and across geographic boundaries.
• Cancer stem cells: mirage or reality?
  Gupta PB Chaffer CL Weinberg RA - Nat Med 15(9):1010-1012 (2009)
  The similarities and differences between normal tissue stem cells and cancer stem cells (CSCs) have been the source of much contention, with some recent studies calling into question the very existence of CSCs. An examination of the literature indicates, however, that the CSC model rests on firm experimental foundations and that differences in the observed frequencies of CSCs within tumors reflect the various cancer types and hosts used to assay these cells. Studies of stem cells and the differentiation program termed the epithelial-mesenchymal transition (EMT) point to the possible existence of plasticity between stem cells and their more differentiated derivatives. If present, such plasticity would have major implications for the CSC model and for future therapeutic approaches.
• Involvement of interleukin-21 in the epidermal hyperplasia of psoriasis
  Caruso R Botti E Sarra M Esposito M Stolfi C Diluvio L Giustizieri ML Pacciani V Mazzotta A Campione E Macdonald TT Chimenti S Pallone F Costanzo A Monteleone G - Nat Med 15(9):1013-1015 (2009)
  T cells are crucial mediators of the skin damage in psoriasis. We here show that interleukin-21 (IL-21), a T cell–derived cytokine, is highly expressed in the skin of individuals with psoriasis, stimulates human keratinocytes to proliferate and causes epidermal hyperplasia when injected intradermally into mice. In the human psoriasis xenograft mouse model, blockade of IL-21 activity resolves inflammation and reduces keratinocyte proliferation. Blocking IL-21 may represent a new therapeutic strategy in psoriasis.
• A human colonic commensal promotes colon tumorigenesis via activation of T helper type 17 T cell responses
  Wu S Rhee KJ Albesiano E Rabizadeh S Wu X Yen HR Huso DL Brancati FL Wick E McAllister F Housseau F Pardoll DM Sears CL - Nat Med 15(9):1016-1022 (2009)
  The intestinal flora may promote colon tumor formation. Here we explore immunologic mechanisms of colonic carcinogenesis by a human colonic bacterium, enterotoxigenic Bacteroides fragilis (ETBF). ETBF that secretes B. fragilis toxin (BFT) causes human inflammatory diarrhea but also asymptomatically colonizes a proportion of the human population. Our results indicate that whereas both ETBF and nontoxigenic B. fragilis (NTBF) chronically colonize mice, only ETBF triggers colitis and strongly induces colonic tumors in multiple intestinal neoplasia (Min) mice. ETBF induces robust, selective colonic signal transducer and activator of transcription-3 (Stat3) activation with colitis characterized by a selective T helper type 17 (TH17) response distributed between CD4+ T cell receptor- (TCR)+ and CD4–8–TCR+ T cells. Antibody-mediated blockade of interleukin-17 (IL-17) as well as the receptor for IL-23, a key cytokine amplifying TH17 responses, inhibits ETBF-induced colitis, colonic hyperplasia and tumor formation. These results show a Stat3- and TH17-dependent pathway for inflammation-induced cancer by a common human commensal bacterium, providing new mechanistic insight into human colon carcinogenesis.
• Alternatively spliced vascular endothelial growth factor receptor-2 is an essential endogenous inhibitor of lymphatic vessel growth
  Albuquerque RJ Hayashi T Cho WG Kleinman ME Dridi S Takeda A Baffi JZ Yamada K Kaneko H Green MG Chappell J Wilting J Weich HA Yamagami S Amano S Mizuki N Alexander JS Peterson ML Brekken RA Hirashima M Capoor S Usui T Ambati BK Ambati J - Nat Med 15(9):1023-1030 (2009)
  Disruption of the precise balance of positive and negative molecular regulators of blood and lymphatic vessel growth can lead to myriad diseases. Although dozens of natural inhibitors of hemangiogenesis have been identified, an endogenous selective inhibitor of lymphatic vessel growth has not to our knowledge been previously described. We report the existence of a splice variant of the gene encoding vascular endothelial growth factor receptor-2 (Vegfr-2) that encodes a secreted form of the protein, designated soluble Vegfr-2 (sVegfr-2), that inhibits developmental and reparative lymphangiogenesis by blocking Vegf-c function. Tissue-specific loss of sVegfr-2 in mice induced, at birth, spontaneous lymphatic invasion of the normally alymphatic cornea and hyperplasia of skin lymphatics without affecting blood vasculature. Administration of sVegfr-2 inhibited lymphangiogenesis but not hemangiogenesis induced by corneal suture injury or transplantation, enhanced corneal allog raft survival and suppressed lymphangioma cellular proliferation. Naturally occurring sVegfr-2 thus acts as a molecular uncoupler of blood and lymphatic vessels; modulation of sVegfr-2 might have therapeutic effects in treating lymphatic vascular malformations, transplantation rejection and, potentially, tumor lymphangiogenesis and lymphedema (pages 993–994)
• Pericyte contraction induced by oxidative-nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery
  Yemisci M Gursoy-Ozdemir Y Vural A Can A Topalkara K Dalkara T - Nat Med 15(9):1031-1037 (2009)
  Here we show that ischemia induces sustained contraction of pericytes on microvessels in the intact mouse brain. Pericytes remain contracted despite successful reopening of the middle cerebral artery after 2 h of ischemia. Pericyte contraction causes capillary constriction and obstructs erythrocyte flow. Suppression of oxidative-nitrative stress relieves pericyte contraction, reduces erythrocyte entrapment and restores microvascular patency; hence, tissue survival improves. In contrast, peroxynitrite application causes pericyte contraction. We also show that the microvessel wall is the major source of oxygen and nitrogen radicals causing ischemia and reperfusion–induced microvascular dysfunction. These findings point to a major but previously not recognized pathophysiological mechanism; ischemia and reperfusion-induced injury to pericytes may impair microcirculatory reflow and negatively affect survival by limiting substrate and drug delivery to tissue already under m etabolic stress, despite recanalization of an occluded artery. Agents that can restore pericyte dysfunction and microvascular patency may increase the success of thrombolytic and neuroprotective treatments.
• A granulocyte-macrophage colony–stimulating factor and interleukin-15 fusokine induces a regulatory B cell population with immune suppressive properties
  Rafei M Hsieh J Zehntner S Li M Forner K Birman E Boivin MN Young YK Perreault C Galipeau J - Nat Med 15(9):1038-1045 (2009)
  We have previously shown that a granulocyte-macrophage colony–stimulating factor (GM-CSF) and interleukin-15 (IL-15) 'fusokine' (GIFT15) exerts immune suppression via aberrant signaling through the IL-15 receptor on lymphomyeloid cells. We show here that ex vivo GIFT15 treatment of mouse splenocytes generates suppressive regulatory cells of B cell ontogeny (hereafter called GIFT15 Breg cells). Arising from CD19+ B cells, GIFT15 Breg cells express major histocompatibility complex class I (MHCI) and MHCII, surface IgM and IgD, and secrete IL-10, akin to previously described B10 and T2-MZP Breg cells, but lose expression of the transcription factor PAX5, coupled to upregulation of CD138 and reciprocal suppression of CD19. Mice with experimental autoimmune encephalomyelitis went into complete remission after intravenous infusion of GIFT15 Breg cells paralleled by suppressed neuroinflammation. The clinical effect was abolished when GIFT15 Breg cells were derived from mMT ( lacking B cells), MHCII-knockout, signal transducer and activator of transcription-6 (STAT-6)-knockout, IL-10–knockout or allogeneic splenocytes, consistent with a pivotal role for MHCII and IL-10 by sygeneic B cells for the observed therapeutic effect. We propose that autologous GIFT15 Breg cells may serve as a new treatment for autoimmune ailments.
• Impaired Wnt–-catenin signaling disrupts adult renal homeostasis and leads to cystic kidney ciliopathy
  Lancaster MA Louie CM Silhavy JL Sintasath L Decambre M Nigam SK Willert K Gleeson JG - Nat Med 15(9):1046-1054 (2009)
  Cystic kidney disease represents a major cause of end-stage renal disease, yet the molecular mechanisms of pathogenesis remain largely unclear. Recent emphasis has been placed on a potential role for canonical Wnt signaling, but investigation of this pathway in adult renal homeostasis is lacking. Here we provide evidence of a previously unidentified canonical Wnt activity in adult mammalian kidney homeostasis, the loss of which leads to cystic kidney disease. Loss of the Jouberin (Jbn) protein in mouse leads to the cystic kidney disease nephronophthisis, owing to an unexpected decrease in endogenous Wnt activity. Jbn interacts with and facilitates -catenin nuclear accumulation, resulting in positive modulation of downstream transcription. Finally, we show that Jbn is required in vivo for a Wnt response to injury and renal tubule repair, the absence of which triggers cystogenesis.
• Primary cilia can both mediate and suppress Hedgehog pathway–dependent tumorigenesis
  Wong SY Seol AD So PL Ermilov AN Bichakjian CK Epstein EH Dlugosz AA Reiter JF - Nat Med 15(9):1055-1061 (2009)
  Primary cilia are present on most mammalian cells and are implicated in transducing Hedgehog (Hh) signals during development; however, the prevalence of cilia on human tumors remains unclear, and the role of cilia in cancer has not been examined. Here we show that human basal cell carcinomas (BCCs) are frequently ciliated, and we test the role of cilia in BCC by conditionally deleting Kif3a (encoding kinesin family member 3A) or Ift88 (encoding intraflagellar transport protein 88), genes required for ciliogenesis, in two Hh pathway–dependent mouse tumor models. Ciliary ablation strongly inhibited BCC-like tumors induced by an activated form of Smoothened. In contrast, removal of cilia accelerated tumors induced by activated Gli2, a transcriptional effector of Hh signaling. These seemingly paradoxical effects are consistent with a dual role for cilia in mediating both the activation and the repression of the Hh signaling pathway. Our findings demonstrate that cilia fun ction as unique signaling organelles that can either mediate or suppress tumorigenesis depending on the nature of the oncogenic initiating event.
• Dual and opposing roles of primary cilia in medulloblastoma development
  Han YG Kim HJ Dlugosz AA Ellison DW Gilbertson RJ Alvarez-Buylla A - Nat Med 15(9):1062-1065 (2009)
  Recent work has shown that primary cilia are essential for Hedgehog (Hh) signaling during mammalian development1, 2, 3, 4, 5, 6, 7, 8, 9. It is also known that aberrant Hh signaling can lead to cancer10, but the role of primary cilia in oncogenesis is not known. Cerebellar granule neuron precursors (GNPs) can give rise to medulloblastomas, the most common malignant brain tumor in children11, 12. The primary cilium and Hh signaling are required for GNP proliferation8, 12, 13, 14, 15. We asked whether primary cilia in GNPs have a role in medulloblastoma growth in mice. Genetic ablation of primary cilia blocked medulloblastoma formation when this tumor was driven by a constitutively active Smoothened protein (Smo), an upstream activator of Hh signaling. In contrast, removal of cilia was required for medulloblastoma growth by a constitutively active glioma-associated oncogene family zinc finger-2 (GLI2), a downstream transcription factor. Thus, primary cilia are either requ ired for or inhibit medulloblastoma formation, depending on the initiating oncogenic event. Remarkably, the presence or absence of cilia was associated with specific variants of human medulloblastomas; primary cilia were found in medulloblastomas with activation in HH or WNT signaling but not in most medulloblastomas in other distinct molecular subgroups. Primary cilia could serve as a diagnostic tool and provide new insights into the mechanism of tumorigenesis.
• Interferon regulatory factor-8 regulates bone metabolism by suppressing osteoclastogenesis
  Zhao B Takami M Yamada A Wang X Koga T Hu X Tamura T Ozato K Choi Y Ivashkiv LB Takayanagi H Kamijo R - Nat Med 15(9):1066-1071 (2009)
  Bone metabolism results from a balance between osteoclast-driven bone resorption and osteoblast-mediated bone formation. Diseases such as periodontitis and rheumatoid arthritis are characterized by increased bone destruction due to enhanced osteoclastogenesis1, 2. Here we report that interferon regulatory factor-8 (IRF-8), a transcription factor expressed in immune cells, is a key regulatory molecule for osteoclastogenesis. IRF-8 expression in osteoclast precursors was downregulated during the initial phase of osteoclast differentiation induced by receptor activator of nuclear factor-B ligand (RANKL), which is encoded by the Tnfsf11 gene. Mice deficient in Irf8 showed severe osteoporosis, owing to increased numbers of osteoclasts, and also showed enhanced bone destruction after lipopolysaccharide (LPS) administration. Irf8-/- osteoclast precursors underwent increased osteoclastogenesis in response to RANKL and tumor necrosis factor- (TNF-). IRF-8 suppressed osteoclastog enesis by inhibiting the function and expression of nuclear factor of activated T cells c1 (NFATc1). Our results show that IRF-8 inhibits osteoclast formation under physiological and pathological conditions and suggest a model where downregulation of inhibitory factors such as IRF-8 contributes to RANKL-mediated osteoclastogenesis.
• Syndecan-4 regulates ADAMTS-5 activation and cartilage breakdown in osteoarthritis
  Echtermeyer F Bertrand J Dreier R Meinecke I Neugebauer K Fuerst M Lee YJ Song YW Herzog C Theilmeier G Pap T - Nat Med 15(9):1072-1076 (2009)
  Aggrecan cleavage by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 5 (ADAMTS-5) is crucial for the breakdown of cartilage matrix during osteoarthritis1, 2, a degenerative joint disease that leads to the progressive destruction of articular structures. The mechanisms of ADAMTS-5 activation and their links to the pathogenesis of osteoarthritis remain poorly understood, but syndecans have been shown to be involved in the activation of ADAMTS-4 (ref. 3). Here we show that syndecan-4 is specifically induced in type X collagen–producing chondrocytes both in human osteoarthritis and in murine models of the disease. The loss of syndecan-4 in genetically modified mice and intra-articular injections of syndecan-4–specific antibodies into wild-type mice protect from proteoglycan loss and thereby prevent osteoarthritic cartilage damage in a surgically induced model of osteoarthritis. The occurrence of less severe osteoarthritis-like cartilage de struction in both syndecan-4–deficient mice and syndecan-4–specific antibody–treated wild-type mice results from a marked decrease in ADAMTS-5 activity. Syndecan-4 controls the activation of ADAMTS-5 through direct interaction with the protease and through regulating mitogen-activated protein kinase (MAPK)-dependent synthesis of matrix metalloproteinase-3 (MMP-3). Our data suggest that strategies aimed at the inhibition of syndecan-4 will be of great value for the treatment of cartilage damage in osteoarthritis.
• NADPH oxidase-4 mediates myofibroblast activation and fibrogenic responses to lung injury
  Hecker L Vittal R Jones T Jagirdar R Luckhardt TR Horowitz JC Pennathur S Martinez FJ Thannickal VJ - Nat Med 15(9):1077-1081 (2009)
  Members of the NADPH oxidase (NOX) family of enzymes, which catalyze the reduction of O2 to reactive oxygen species, have increased in number during eukaryotic evolution1, 2. Seven isoforms of the NOX gene family have been identified in mammals; however, specific roles of NOX enzymes in mammalian physiology and pathophysiology have not been fully elucidated3, 4. The best established physiological role of NOX enzymes is in host defense against pathogen invasion in diverse species, including plants5, 6. The prototypical member of this family, NOX-2 (gp91phox), is expressed in phagocytic cells and mediates microbicidal activities7, 8. Here we report a role for the NOX4 isoform in tissue repair functions of myofibroblasts and fibrogenesis. Transforming growth factor-1 (TGF-1) induces NOX-4 expression in lung mesenchymal cells via SMAD-3, a receptor-regulated protein that modulates gene transcription. NOX-4–dependent generation of hydrogen peroxide (H2O2) is required for T GF-1–induced myofibroblast differentiation, extracellular matrix (ECM) production and contractility. NOX-4 is upregulated in lungs of mice subjected to noninfectious injury and in cases of human idiopathic pulmonary fibrosis (IPF). Genetic or pharmacologic targeting of NOX-4 abrogates fibrogenesis in two murine models of lung injury. These studies support a function for NOX4 in tissue fibrogenesis and provide proof of concept for therapeutic targeting of NOX-4 in recalcitrant fibrotic disorders.
• A crucial role for adipose tissue p53 in the regulation of insulin resistance
  Minamino T Orimo M Shimizu I Kunieda T Yokoyama M Ito T Nojima A Nabetani A Oike Y Matsubara H Ishikawa F Komuro I - Nat Med 15(9):1082-1087 (2009)
  Various stimuli, such as telomere dysfunction and oxidative stress, can induce irreversible cell growth arrest, which is termed 'cellular senescence'1, 2. This response is controlled by tumor suppressor proteins such as p53 and pRb. There is also evidence that senescent cells promote changes related to aging or age-related diseases3, 4, 5, 6. Here we show that p53 expression in adipose tissue is crucially involved in the development of insulin resistance, which underlies age-related cardiovascular and metabolic disorders. We found that excessive calorie intake led to the accumulation of oxidative stress in the adipose tissue of mice with type 2 diabetes–like disease and promoted senescence-like changes, such as increased activity of senescence-associated -galactosidase, increased expression of p53 and increased production of proinflammatory cytokines. Inhibition of p53 activity in adipose tissue markedly ameliorated these senescence-like changes, decreased the express ion of proinflammatory cytokines and improved insulin resistance in mice with type 2 diabetes–like disease. Conversely, upregulation of p53 in adipose tissue caused an inflammatory response that led to insulin resistance. Adipose tissue from individuals with diabetes also showed senescence-like features. Our results show a previously unappreciated role of adipose tissue p53 expression in the regulation of insulin resistance and suggest that cellular aging signals in adipose tissue could be a new target for the treatment of diabetes (pages 996–967).
• A rapid and efficient single-cell manipulation method for screening antigen-specific antibody–secreting cells from human peripheral blood
  Jin A Ozawa T Tajiri K Obata T Kondo S Kinoshita K Kadowaki S Takahashi K Sugiyama T Kishi H Muraguchi A - Nat Med 15(9):1088-1092 (2009)
  Antigen-specific human monoclonal antibodies (mAbs) are key candidates for therapeutic agents. However, the availability of a suitable screening system for antigen-specific antibody–secreting cells (ASCs) is limited in humans. Here we present a unique method for detecting individual ASCs using microwell array chips, which enables the analysis of live cells on a single-cell basis and offers a rapid, efficient and high-throughput (up to 234,000 individual cells) system for identifying and recovering objective ASCs. We applied the system to detect and retrieve ASCs for hepatitis B virus and influenza viruses from human peripheral blood lymphocytes and produced human mAbs with virus-neutralizing activities within a week. Furthermore, we show that the system is useful for detecting ASCs for multiple antigens as well as for selection of ASCs secreting high-affinity antibodies on a chip. Our method can open the way for the generation of therapeutic antibodies for individual patients.
• Erratum: GOAT links dietary lipids with the endocrine control of energy balance
  Kirchner H Gutierrez JA Solenberg PJ Pfluger PT Czyzyk TA Willency JA Schürmann A Joost HG Jandacek RJ Hale JE Heiman ML Tschöp MH - Nat Med 15(9):1093 (2009)
  Introduction Nat. Med.15, 741–745 (2009); published online 5 June; corrected after print 4 September 2009 In the version of this article initially published, the fourth condition from the top in the key to the bar graphs in Figure 2c was mislabeled as 'mC8'. The correct label is 'hC8'. The error has been corrected in the HTML and PDF versions of the article.
• Corrigendum: Aldosterone impairs vascular reactivity by decreasing glucose-6-phosphate dehydrogenase activity
  Leopold JA Dam A Maron BA Scribner AW Liao R Handy DE Stanton RC Pitt B Loscalzo J - Nat Med 15(9):1093 (2009)
  Introduction Nat. Med.13, 189–197 (2007); published online 4 February 2007; corrected after print 4 September 2009 In the version of this article initially published, the number of one of the grants listed in the Acknowledgments was incorrect; 'HL55993' should have been 'P01 HL81587'. The error has been corrected in the HTML and PDF versions of the article.