Latest Articles Include:
- An empiric victory
- Nat Biotech 28(6):529 (2010)
Nature Biotechnology | Editorial An empiric victory Journal name:Nature BiotechnologyVolume:28,Page:529Year published:(2010)DOI:doi:10.1038/nbt0610-529 Provenge already looks like the product of a bygone era. View full text Additional data - Landmark approval for Dendreon's cancer vaccine
- Nat Biotech 28(6):531-532 (2010)
The April 29 approval of Seattle-based Dendreon's prostate cancer vaccine, Provenge (sipuleucel-T), is being hailed as a victory for cancer immunotherapy. For Dendreon, the US Food and Drug Administration's (FDA) go-ahead marks the end of a tortuous regulatory path, marked not only by missteps by the company but also by controversy at the FDA, not least the decision in 2007 by the Center for Biologics Evaluation and Research (CBER) to act against its advisory panel's positive recommendations. - Firms chase diabetic inflammation with anti-IL-1β antibodies
- Nat Biotech 28(6):533-534 (2010)
A recent US patent award to Xoma for XOMA 052, an anti-interleukin-1β(IL-1β) IgG2 humanized monoclonal antibody (mAb), has spiced up what was already an intriguing contest to uncover disease-modifying therapies for type 2 diabetes. The Berkeley, California–based antibody developer is aggressively staking out territory in a rapidly emerging—and potentially lucrative—field, which has also captured the attention of Novartis and Eli Lilly. - African GM safety drill
- Nat Biotech 28(6):534 (2010)
The African Union has set up a school to educate and train future regulators in genetically modified (GM) crop biosafety. The African Biosafety Network of Expertise (ABNE) was officially launched in April in Ouagadougou, Burkina Faso, with a five-year, $10. - Burgeoning stem cell product market lures major suppliers
- Nat Biotech 28(6):535-536 (2010)
Life sciences supplier Lonza has struck a deal with Axiogenesis of Cologne, Germany, to offer mouse embryonic stem cell–derived cardiomyocytes in its product catalog. The agreement, signed in March, is the latest move of several large reagent and material suppliers to grab a slice of the rapidly expanding market for stem cell products for use in in vitro assays and testing kits for predictive toxicology. - GMP cell lines to order
- Nat Biotech 28(6):536 (2010)
Eden Biodesign and Millipore have struck a deal to offer a service of mammalian cell lines on demand for companies developing antibodies and protein therapeutics. The collaboration marries Millipore's Ubiquitous Chromatin Opening Element (UCOE) expression technology with Eden's cGMP production. - Open-access fermenter
- Nat Biotech 28(6):536 (2010)
The UK's first open-access facility will soon be available for firms wanting to ramp up biotech processes. The UK's Centre for Process Innovation (CPI) is expanding the capacity of its National Industrial Biotechnology Facility (NIBF) in Wilton from 1 to 10 tons to provide startups and established businesses with equipment and expertise for proof-of-concept development. - Glyphosate resistance threatens Roundup hegemony
- Nat Biotech 28(6):537-538 (2010)
Weeds are becoming increasingly resistant to glyphosate, a report from the US National Academy of Sciences (NAS) released in April has found. The driving force, according to the report, is farmers' dependence on the weed killer accompanied by the widespread adoption of genetically modified (GM) herbicide-tolerant crops. - SBIR grants wax
- Nat Biotech 28(6):538 (2010)
Awards under the Small Business Innovation Research (SBIR) program have just been given a boost. As of March 30, the cap for SBIR phase I awards has risen from $100,000 to $150,000, and for phase II awards from $750,000 to $1,000,000. - Relief over stem cell lines
- Nat Biotech 28(6):538 (2010)
The US National Institutes of Health (NIH) announced the addition of 13 lines to its Stem Cell Registry. The news was cheered by the research community, as the two most widely studied lines— H7 (WA07) and H9 (WA09) owned by the WiCell Research Institute of Madison—were included in the batch approved by NIH director Francis Collins. - Obama appoints bioethics panel to offer practical advice
- Nat Biotech 28(6):539 (2010)
In April, President Barack Obama named 11 more members to the Presidential Commission for the Study of Bioethical Issues. They will join commission chair Amy Gutmann, president of the University of Pennsylvania, and commission vice-chair James Wagner, Emory University president, who were appointed last year. - GSK's RNA splash
- Nat Biotech 28(6):539 (2010)
Antisense-drug developer Isis Pharmaceuticals and GlaxoSmithKline (GSK) have forged a collaboration to develop drugs for rare diseases that could earn the Carlsbad, California–based biotech up to $1.5 billion dollars in licensing fees and milestones. - Germany caps drug prices
- Nat Biotech 28(6):539 (2010)
The German coalition government is putting into place new rules that will allow health insurers to influence the pricing of new medications. The changes are intended to save the healthcare system around |[euro]|2 ($2. - Biotech breeding goes bovine
- Nat Biotech 28(6):540-543 (2010)
Nature Biotechnology | News | News Feature Biotech breeding goes bovine * Stephen Strauss1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature BiotechnologyVolume:28,Pages:540–543Year published:(2010)DOI:doi:10.1038/nbt0610-540 Dairy farmers are rapidly adopting molecular profiling to accelerate the process of siring cows. But this seismic shift in breeding practices is raising new questions and translating more slowly to the beef industry. Stephen Strauss reports. View full text Additional data Affiliations * Toronto * Stephen Strauss - Up for grabs
- Nat Biotech 28(6):544-546 (2010)
Nature Biotechnology | News | News Feature Up for grabs * Michael Eisenstein1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature BiotechnologyVolume:28,Pages:544–546Year published:(2010)DOI:doi:10.1038/nbt0610-544 As issued patents on induced pluripotent stem (iPS) cells stack up, the specter of a patent thicket looms. Michael Eisenstein investigates. View full text 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 PDF files * Supplementary Text and Figures (76K) Supplementary Table 1 Additional data Affiliations * Philadelphia * Michael Eisenstein - Beyond venture capital
- Nat Biotech 28(6):547-549 (2010)
- 1 out of 27—European politicians score poorly in agbiotech
- Nat Biotech 28(6):551-552 (2010)
To the Editor: We wish to express our concern and dismay at the apparent lack of intergovernmental engagement by European governments regarding the proven positive roles of modern biotechnologies as key tools supporting efforts to address the issue of food security, especially in developing countries. This was shown clearly by the failure of 26 of the 27 members states of the European Union to send any official government delegations to participate and engage in the recent United Nations Food and Agriculture Organization (FAO; Rome) intergovernmental conference (ABDC-10) on 'Agricultural biotechnologies in developing countries' (http://www. - Split approvals and hot potatoes
- Nat Biotech 28(6):552-553 (2010)
To the Editor: The letter by Gerhart Ryffel in the April issue outlines some of the public perception concerns surrounding the European Union's (EU; Brussels) recent sanctioning of the cultivation of a genetically modified (GM) potato—the first for any GM plant in 12 years. But readers should be far more concerned about the form of approval granted by EU authorities. - Why drought tolerance is not the new Bt
- Nat Biotech 28(6):553-554 (2010)
To the Editor: Given rapid uptake of Bacillus thuringiensis toxin (Bt) cotton by farmers in several developing countries, it is often assumed that poor farmers will clamor for drought-tolerant varieties in an era of tightening water resources and climate change. There are, however, important differences between Bt-mediated insect resistance and drought tolerance. - Health impact in China of folate-biofortified rice
- Nat Biotech 28(6):554-556 (2010)
Nature Biotechnology | Opinion and Comment | Correspondence Health impact in China of folate-biofortified rice * Hans De Steur1 Search for this author in: * NPG journals * PubMed * Google Scholar * Xavier Gellynck1 Search for this author in: * NPG journals * PubMed * Google Scholar * Sergei Storozhenko2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ge Liqun3 Search for this author in: * NPG journals * PubMed * Google Scholar * Willy Lambert4 Search for this author in: * NPG journals * PubMed * Google Scholar * Dominique Van Der Straeten2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jacques Viaene1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature BiotechnologyVolume:28,Pages:554–556Year published:(2010)DOI:doi:10.1038/nbt0610-554 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg To the Editor: Despite efforts to reduce the burden of malnutrition, large numbers of people still consume insufficient micronutrients, including folate1. Folate deficiency, characterized by a suboptimal daily intake of folate (<400 μg) may lead to the onset of diseases and disorders, such as neural-tube defects (NTD), megaloblastic anemia and aggravation of iron-deficiency anemia2. In line with the main micronutrient deficiencies (zinc, iron and vitamin A), folate deficiency is more prevalent in less developed, non-Western countries. In China, for instance, ~20% of the population is considered to be folate deficient3 (for an overview on folate deficiency and NTDs, see Supplementary Discussion, sections 2 and 3, respectively). View full text Author information * Author information * Supplementary information Affiliations * Department of Agricultural Economics, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium. * Hans De Steur, * Xavier Gellynck & * Jacques Viaene * Unit Plant Hormone Signalling and Bio-imaging, Department of Physiology, Ghent University, Ghent, Belgium. * Sergei Storozhenko & * Dominique Van Der Straeten * Rural Economy Research Institute, Liaoning Academy of Agricultural Sciences, Shenyang, Liaoning, P.R. China. * Ge Liqun * Laboratory of Toxicology, Department of Bioanalysis, Ghent University, Ghent, Belgium. * Willy Lambert Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Hans De Steur (hans.desteur@ugent.be) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Discussion Additional data - Alive and kicking
- Nat Biotech 28(6):556 (2010)
To the Editor: As CEO of the companies involved, I would like to bring to the attention of your readers several inaccuracies in a News article in the March issue entitled 'Resuscitated deCODE refocuses on diagnostics'. The article erroneously reports that deCODE (Reykjavik, Iceland) ". - Pluripotent patents make prime time: an analysis of the emerging landscape
- Nat Biotech 28(6):557-559 (2010)
Nature Biotechnology | Feature | Patents Pluripotent patents make prime time: an analysis of the emerging landscape * Brenda M Simon1 Search for this author in: * NPG journals * PubMed * Google Scholar * Charles E Murdoch1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher T Scott1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorsJournal name:Nature BiotechnologyVolume:28,Pages:557–559Year published:(2010)DOI:doi:10.1038/nbt0610-557 An examination of three patents in the fast-moving iPS space may help determine their ultimate value. 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 * Brenda M. Simon and Charles E. Murdoch are at the Stanford University Center for Law and the Biosciences Stanford, California, USA. * Christopher T Scott * Charles E. Murdoch and Christopher T. Scott are at the Stanford University Program on Stem Cells in Society, Stanford, California, USA. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Brenda M Simon (bmsimon@stanford.edu or cscott@stanford.edu) or * Christopher T Scott (bmsimon@stanford.edu or cscott@stanford.edu) Additional data - Recent patent applications in epigenetics
- Nat Biotech 28(6):560 (2010)
Recent patent applications in epigenetics Table 1 - Raising the bar for cancer therapy models
- Nat Biotech 28(6):561-562 (2010)
The failure rate of double-blind, often placebocontrolled randomized phase 3 trials is higher in oncology than in any other therapeutic area. In non-small cell lung cancer, for example, with the exception of a bevacizumab (Avastin) trial, every one of over a dozen phase 3 trials combining a 'targeted' biologic agent with standard chemotherapy used for first-line treatment has failed to provide an overall survival benefit. - Scalable pluripotent stem cell culture
- Nat Biotech 28(6):562-563 (2010)
Only a dozen years after they were first isolated, human embryonic stem cells (hESCs) are beginning to move from the research laboratory toward the clinic. Several biotech companies have initiated hESC clinical programs; at least two cell therapies based on hESCs have been submitted to the US Food and Drug Administration under Investigational New Drug (IND) applications; and hESC cultures are being retooled for disease modeling and drug screening. - Complex molecular dynamics in the spotlight
- Nat Biotech 28(6):564-565 (2010)
The ability to measure events at the single-molecule level promises to reveal the workings of biological machines in unprecedented detail. Among the various technologies that can achieve single-molecule resolution, the zero-mode waveguide (ZMW) is emerging as a powerful method with unique capabilities. - Detecting methylated bases in real time
- Nat Biotech 28(6):565 (2010)
In many organisms the primary DNA structure is covalently modified to regulate, for example, gene expression and genome structure. In eukaryotes, the dominant modification is methylcytosine, although others, such as hydroxymethylcytosine, have been detected. - Research highlights
- Nat Biotech 28(6):566 (2010)
Taking aim at transcription factors The transcription factor BCL6 facilitates the generation of antibody diversity in B cells by repressing the DNA damage–sensing apparatus, thereby creating genomic instability. But when BCL6 activity goes awry by mutation or translocation, unregulated B-cell growth can ensue and is often associated with diffuse large B-cell lymphomas (DLBCL). - Comparative assessment of methods for aligning multiple genome sequences
- Nat Biotech 28(6):567-572 (2010)
Nature Biotechnology | Computational Biology | Analysis Comparative assessment of methods for aligning multiple genome sequences * Xiaoyu Chen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Tompa1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature BiotechnologyVolume:28,Pages:567–572Year published:(2010)DOI:doi:10.1038/nbt.1637Received21 December 2009Accepted27 April 2010Published online23 May 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 Multiple sequence alignment is a difficult computational problem. There have been compelling pleas for methods to assess whole-genome multiple sequence alignments and compare the alignments produced by different tools. We assess the four ENCODE alignments, each of which aligns 28 vertebrates on 554 Mbp of total input sequence. We measure the level of agreement among the alignments and compare their coverage and accuracy. We find a disturbing lack of agreement among the alignments not only in species distant from human, but even in mouse, a well-studied model organism. Overall, the assessment shows that Pecan produces the most accurate or nearly most accurate alignment in all species and genomic location categories, while still providing coverage comparable to or better than that of the other alignments in the placental mammals. Our assessment reveals that constructing accurate whole-genome multiple sequence alignments remains a significant challenge, particularly for noncodi! ng regions and distantly related species. View full text Figures at a glance * Figure 1: Comparison of coverage of the alignments. The comparison is broken down by species and by location category; also provided is an overall chart that aggregates all four location categories. Species are displayed on the horizontal axis in order of increasing total branch length from human, according to a phylogeny estimated from fourfold-degenerate sites of third codon positions in the ENCODE regions10. The vertical axis represents the number of human residues aligned to each species given on the horizontal axis, in units of kilobase pairs (Kbp). Note that the vertical scales are different in each of the charts. The figure shows that TBA, MLAGAN and Pecan all have comparable coverage in all the placental mammals (chimp through tenrec) across all location categories. For all alignments, note that the coverage decreases approximately as species distance from human increases, particularly in the noncoding location categories. * Figure 2: Comparison percentages agree%, unique% and disagree% for TBA, MAVID and MLAGAN. (See Online Methods for the explanation of why Pecan is excluded.) The comparison is shown for 12 representative species and broken down by location category. Species are displayed on the horizontal axis in order of increasing total branch length from human. Note the trend that agree% decreases and unique% increases as the species distance from human increases and also as one moves from coding to UTR to intronic/intergenic categories. * Figure 3: Comparison of accuracy of the alignments, as measured by suspicious%. The comparison is broken down by species and by location category, plus an overall chart that aggregates all four location categories. Species are displayed on the horizontal axis in order of increasing total branch length from human. For each alignment and each noncoding category, suspicious% generally increases as species distance from human increases, with a noticeable jump between the placental mammals and more distant species. Note that Pecan has the lowest or near lowest suspicious% for every species and location category. * Figure 4: Pairwise alignment scores of suspicious regions versus those for alternative alignments of the same human region. For three representative species S (baboon, mouse and zebrafish) and one representative target alignment (MLAGAN), scatter plots show all points (x′, y′), where x′ is the pairwise human-S alignment score of an MLAGAN alignment region that is suspicious for species S, and y′ is the pairwise human-S alignment score of one of the other three alignments for the same human region that is not suspicious for S. (See Online Methods for the scoring function and Supplementary Fig. 3 for other target alignments.) Alignment scores are normalized by alignment length. The dashed black diagonal line has equation y = x. The solid blue line has equation y – x = μ, where μ is the mean value of y′ – x′ for all points (x′, y′) in the plot. The dotted blue lines have equations y – x = μ ± ó, where ó is the standard deviation of y′ – x′ for all points (x′, y′) in the plot. Note that most points lie above the line y = x, suggesting that most of the suspiciousl! y aligned regions can be improved by one of the alternative alignments. * Figure 5: Summary plot of suspicious% vs. coverage, aggregated over all four location categories. The horizontal axis is on a logarithmic scale. For a given species, points that are lower and farther right represent better performance. Note the comparable performance of Pecan and TBA on placental mammals (orange and green triangles) and the superior accuracy of Pecan on the distant species (orange circles). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Computer Science and Engineering, Department of Genome Sciences, University of Washington, Seattle, Washington, USA. * Xiaoyu Chen & * Martin Tompa Contributions X.C., design, implementation, experimentation, analysis; M.T., design, analysis. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Martin Tompa (tompa@cs.washington.edu) Supplementary information * Abstract * Author information * Supplementary information Excel files * Supplementary Coverage Spreadsheet (24K) * Supplementary Comparison Percentage Spreadsheet (44K) * Supplementary Suspicious Percentage Spreadsheet (28K) PDF files * Supplementary Text and Figures (1M) Supplementary Text and Supplementary Figs. 1–4 Additional data - Rationalizing the development of live attenuated virus vaccines
- Nat Biotech 28(6):573-579 (2010)
Nature Biotechnology | Research | Review Rationalizing the development of live attenuated virus vaccines * Adam S Lauring1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeremy O Jones2, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Raul Andino3 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature BiotechnologyVolume:28,Pages:573–579Year published:(2010)DOI:doi:10.1038/nbt.1635Published online07 June 2010 Abstract * Abstract * 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 The design of vaccines against viral disease has evolved considerably over the past 50 years. Live attenuated viruses (LAVs)—those created by passaging a virus in cultured cells—have proven to be an effective means for preventing many viral diseases, including smallpox, polio, measles, mumps and yellow fever. Even so, empirical attenuation is unreliable in some cases and LAVs pose several safety issues. Although inactivated viruses and subunit vaccines alleviate many of these concerns, they have in general been less efficacious than their LAV counterparts. Advances in molecular virology—creating deleterious gene mutations, altering replication fidelity, deoptimizing codons and exerting control by microRNAs or zinc finger nucleases—are providing new ways of controlling viral replication and virulence and renewing interest in LAV vaccines. Whereas these rationally attenuated viruses may lead to a new generation of safer, more widely applicable LAV vaccines, each approa! ch requires further testing before progression to human testing. View full text Figures at a glance * Figure 1: The microRNA (miRNA)-virus vaccine strategy. Viral replication can be regulated in a tissue-specific manner by incorporating miRNA target sites into the viral genome. In cells that express the miRNA (e.g., brain, top cell), the miRNAs are processed and transported to the cytoplasm, where they mediate cleavage of viral RNA. Viral replication is restricted to cells in which the miRNA is not expressed (e.g., intestine, bottom cell). The engineered virus can therefore trigger a natural immune response in target tissues without the associated risk of dissemination and disease. * Figure 2: Zinc-finger nuclease (ZFN)-virus vaccine strategy. The ZFN is composed of two arrays of three ZF domains fused to a DNA nuclease domain (blue lightning bolt). The nuclease must dimerize to be activated, so each ZFN array is designed to bind the adjacent 9-bp sequences in the virus genome, spaced 5–6 bp apart, allowing the nuclease domains to dimerize and cleave the viral double-stranded DNA. ZFNs can be designed to target multiple, essential viral sequences. By encoding the ZFNs in the viral genome itself and temporally controlling the expression of the ZFNs using viral promoters, the virus can express immunogenic proteins before ZFN cleavage of circular episomal DNA to linear DNA, which is incapable of replication and establishment of latency. Author information * Abstract * Author information Primary authors * These authors contributed equally to this work. * Adam S Lauring & * Jeremy O Jones Affiliations * Department of Medicine, University of California, San Francisco, California, USA. * Adam S Lauring * Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California, USA. * Jeremy O Jones * Department of Microbiology and Immunology, University of California, San Francisco, California, USA. * Raul Andino Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Raul Andino (raul.andino@ucsf.edu.) Additional data - Synthetic polymer coatings for long-term growth of human embryonic stem cells
Villa-Diaz LG Nandivada H Ding J Nogueira-de-Souza NC Krebsbach PH O'Shea KS Lahann J Smith GD - Nat Biotech 28(6):581-583 (2010)
Nature Biotechnology | Research | Brief Communications Synthetic polymer coatings for long-term growth of human embryonic stem cells * Luis G Villa-Diaz1, 2, 12 Search for this author in: * NPG journals * PubMed * Google Scholar * Himabindu Nandivada3, 12 Search for this author in: * NPG journals * PubMed * Google Scholar * Jun Ding1 Search for this author in: * NPG journals * PubMed * Google Scholar * Naiara C Nogueira-de-Souza1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Paul H Krebsbach2, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * K Sue O'Shea6 Search for this author in: * NPG journals * PubMed * Google Scholar * Joerg Lahann3, 5, 7, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Gary D Smith1, 9, 10, 11 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature BiotechnologyVolume:28,Pages:581–583Year published:(2010)DOI:doi:10.1038/nbt.1631Received14 September 2009Accepted05 April 2010Published online30 May 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 report a fully defined synthetic polymer coating, poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH), which sustains long-term human embryonic stem (hES) cell growth in several different culture media, including commercially available defined media. The development of a standardized, controllable and sustainable culture matrix for hES cells is an essential step in elucidating mechanisms that control hES cell behavior and in optimizing conditions for biomedical applications of hES cells. View full text Figures at a glance * Figure 1: Long-term culture of H9 hES cells on PMEDSAH with MEF-conditioned media. () Schematic diagram showing graft-polymerization used to synthesize the polymer coatings and their chemical structures. Tissue culture polystyrene dishes were first activated by UV ozone and then methacrylate-based monomers were polymerized from the surface. Table lists contact angle, reduced elastic modulus (GPa) (mean ± s.d.), initial hES cell aggregate adhesion (mean ± s.e.m.) and number of passages achieved on each polymer coating. () Fourier transform infrared spectrum of PMEDSAH coating showing distinct bands at 1,732.9 cm−1 and 1,208.4 cm−1, which indicated presence of carbonyl and sulfonate groups, respectively. Table lists elemental composition of PMEDSAH obtained using X-ray photoelectron spectroscopy. Relative composition of these elements was in agreement with the expected chemical composition of PMEDSAH. () Percentage (mean ± s.e.m.) of hES cells expressing OCT3/4 and SOX2 at passages 3 (P03) and 20 (P20). () Relative transcript levels of NANOG, OCT3/4 a! nd SOX2 from hES cells cultured on PMEDSAH and Matrigel. (,) After 25 passages, hES cells cultured on PMEDSAH and Matrigel (Supplementary Fig. 1) () maintained a normal karyotype and () retained pluripotency as demonstrated by teratoma formation in immunosuppressed mice. H&E-stained paraffin sections indicating endoderm (goblet-like cells at arrow), ectoderm (neuroepithelial aggregates at arrow; and cells expressing neuron-restricted protein β-III tubulin in inset) and mesodermal derivatives (cartilage, connective tissue and muscle at arrow). Scale bar, 200 μm. * Figure 2: PMEDSAH supports culture of hES cells in defined medium. () Percentage (mean ± s.e.m.) of cell aggregate adhesion (number of aggregates attached with respect to total aggregates passaged) and population doubling time (twofold increase in colony area) for H9 hES cells cultured on PMEDSAH in MEF-conditioned medium, human cell–conditioned medium and defined medium. P-values calculated using unpaired t-test. () Fluorescence micrographs of colonies of H9 cells cultured on PMEDSAH in StemPro medium showing expression of hES cell markers: OCT3/4, SOX2, SSEA-4, TRA-1-60 and TRA-1-81; and a phase-contrast image. Scale bars, 200 μm. () RT-PCR analysis of RNA from embryoid bodies showing expression of endoderm (GATA4), ectoderm (KRT18) and mesoderm derivatives (VE-cadherin; also known as CDH5). β-Actin (also known as ACTB) was used as positive control, and for each primer set tested, a reaction lacking RNA was assessed in parallel as a negative control. () Micrographs showing immunoreactivity for α-fetoprotein (endoderm), β-III tubuli! n (ectoderm) and smooth muscle actin (mesoderm) demonstrating the pluripotent state of H9 cells cultured on PMEDSAH in StemPro medium. Scale bars, 200 μm. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Luis G Villa-Diaz & * Himabindu Nandivada Affiliations * Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, Michigan, USA. * Luis G Villa-Diaz, * Jun Ding, * Naiara C Nogueira-de-Souza & * Gary D Smith * Department of Biologic and Materials Sciences, University of Michigan, Ann Arbor, Michigan, USA. * Luis G Villa-Diaz & * Paul H Krebsbach * Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan, USA. * Himabindu Nandivada & * Joerg Lahann * Human Biology Research Laboratory, Barretos Cancer Hospital, Sao Paulo, Brazil. * Naiara C Nogueira-de-Souza * Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, USA. * Paul H Krebsbach & * Joerg Lahann * Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA. * K Sue O'Shea * Department of Materials Science and Engineering, University of Michigan, Ann Arbor, Michigan, USA. * Joerg Lahann * Department of Macromolecular Science and Engineering, University of Michigan, Ann Arbor, Michigan, USA. * Joerg Lahann * Department of Urology, University of Michigan, Ann Arbor, Michigan, USA. * Gary D Smith * Department of Molecular and Integrated Physiology, University of Michigan, Ann Arbor, Michigan, USA. * Gary D Smith * Reproductive Sciences Program, University of Michigan, Ann Arbor, Michigan, USA. * Gary D Smith Contributions L.G.V.-D. and H.N. contributed equally to this work and were involved in experimental design, performing hES cell culture experiments, data analysis and manuscript preparation. L.G.V.-D. carried out cell analysis experiments, and H.N. fabricated the polymer coatings and performed surface analysis. J.D. was involved in immunocytochemistry and RT-PCR, while N.C.N.-d.-S. conducted microarray analysis and qPCR. P.H.K. participated in manuscript preparation. K.S.O. participated in manuscript preparation and performed teratoma assays. J.L. and G.D.S. were involved in conceptual and experimental design, as well as in manuscript preparation. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Joerg Lahann (materials: lahann@umich.edu) or * Gary D Smith (cell culture: smithgd@umich.edu) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (356K) Supplementary Tables 1,2, Supplementary Figs. 1–3 and Supplementary Methods Additional data - Assessing therapeutic responses in Kras mutant cancers using genetically engineered mouse models
- Nat Biotech 28(6):585-593 (2010)
Nature Biotechnology | Research | Article Assessing therapeutic responses in Kras mutant cancers using genetically engineered mouse models * Mallika Singh1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anthony Lima1 Search for this author in: * NPG journals * PubMed * Google Scholar * Rafael Molina1 Search for this author in: * NPG journals * PubMed * Google Scholar * Patricia Hamilton1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anne C Clermont1 Search for this author in: * NPG journals * PubMed * Google Scholar * Vidusha Devasthali1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jennifer D Thompson1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jason H Cheng1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hani Bou Reslan2 Search for this author in: * NPG journals * PubMed * Google Scholar * Calvin C K Ho2 Search for this author in: * NPG journals * PubMed * Google Scholar * Timothy C Cao2 Search for this author in: * NPG journals * PubMed * Google Scholar * Chingwei V Lee3 Search for this author in: * NPG journals * PubMed * Google Scholar * Michelle A Nannini4 Search for this author in: * NPG journals * PubMed * Google Scholar * Germaine Fuh3 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard A D Carano2 Search for this author in: * NPG journals * PubMed * Google Scholar * Hartmut Koeppen5 Search for this author in: * NPG journals * PubMed * Google Scholar * Ron X Yu6 Search for this author in: * NPG journals * PubMed * Google Scholar * William F Forrest6 Search for this author in: * NPG journals * PubMed * Google Scholar * Gregory D Plowman7 Search for this author in: * NPG journals * PubMed * Google Scholar * Leisa Johnson1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature BiotechnologyVolume:28,Pages:585–593Year published:(2010)DOI:doi:10.1038/nbt.1640Received28 December 2009Accepted28 April 2010Published online23 May 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 The low rate of approval of novel anti-cancer agents underscores the need for better preclinical models of therapeutic response as neither xenografts nor early-generation genetically engineered mouse models (GEMMs) reliably predict human clinical outcomes. Whereas recent, sporadic GEMMs emulate many aspects of their human disease counterpart more closely, their ability to predict clinical therapeutic responses has never been tested systematically. We evaluated the utility of two state-of-the-art, mutant Kras-driven GEMMs—one of non-small-cell lung carcinoma and another of pancreatic adenocarcinoma—by assessing responses to existing standard-of-care chemotherapeutics, and subsequently in combination with EGFR and VEGF inhibitors. Standard clinical endpoints were modeled to evaluate efficacy, including overall survival and progression-free survival using noninvasive imaging modalities. Comparisons with corresponding clinical trials indicate that these GEMMs model human res! ponses well, and lay the foundation for the use of validated GEMMs in predicting outcome and interrogating mechanisms of therapeutic response and resistance. View full text Figures at a glance * Figure 1: Influence of KRAS mutations in the first-line treatment of NSCLC with chemotherapy versus chemotherapy plus erlotinib. (–) Kaplan-Meier plots from TRIBUTE12 showing the OS () and PFS () data from NSCLC patients with KRAS mutant tumors treated with chemotherapy alone (CP) or chemotherapy plus concurrent erlotinib (CPE). These figures reprinted with permission, ©2008 American Society of Clinical Oncology. All rights reserved. Corresponding OS () and PFS () data in the Kras mutant GEMM for NSCLC; note that the mice were dosed with erlotinib maintenance therapy. Control vehicle-treated cohorts are also shown in the case of the GEMM for comparison. The number of patients or mice in each treatment cohort is indicated in parentheses. Relevant P values (log-rank test) and hazard ratios are depicted for each graph, and the comparator group (denominator) is denoted with a hyphen. *, P values ≤ 0.05. CI95 for each hazard ratio shown are () 1.11–3.79, () 1.05–3.6, () 0.92–8.54 and () 0.67–4.34. () Representative micro-CT images from mice in each of the GEMM cohorts pre-treatment (left) and ! after ~4 weeks on study (right). A visible tumor example is marked with yellow arrows in the control vehicle-treated cohort and mean tumor burden cross-products for each cohort are shown at the bottom left of each image. C, carboplatin; P, paclitaxel; E, erlotinib; HR, hazard ratio; micro-CT, micro-computed tomography. * Figure 2: First-line treatment of PDAC patients and KrasLSL−G12D; p16/p19fl/fl; Pdx1-Cre mice with gemcitabine versus gemcitabine plus erlotinib. (–) Kaplan-Meier plots from NCIC CTG PA.3 (ref. 37) showing the OS () and PFS () data from PDAC patients treated with gemcitabine alone (G) or gemcitabine plus erlotinib (GE). These figures reprinted with permission, © 2008 American Society of Clinical Oncology. All rights reserved. Corresponding OS () and PFS () data in the Kras mutant GEMM for PDAC are shown. Vehicle control-treated cohorts are also shown in the case of the GEMM for comparison. The number of patients or mice in each treatment cohort is indicated in parentheses. Relevant P values (log-rank test) and hazard ratios are depicted for each graph, and the comparator group (denominator) is denoted with a hyphen. CI95 for each hazard ratio shown are () 0.69–0.99, () 0.64–0.92, () 0.35–1.6 and () 0.69–3.2. () Representative high-resolution ultrasound images from mice in each of the GEMM cohorts pre-treatment (left) and after ~11–15 d on study (right). Visible lesions are outlined in yellow and mean tumo! r burden for each cohort are shown at the bottom left of each image. G, gemcitabine; E, erlotinib; HR, hazard ratio. * Figure 3: Anti-VEGF provides significant benefit when combined with chemotherapy as first-line therapy in human patients and KrasLSL−G12D; p53frt/frt mice with late-stage NSCLC. (–) Kaplan-Meier plots from ECOG 4599 (ref. 39) showing the OS () and PFS () data from NSCLC patients treated with chemotherapy alone (CP) or chemotherapy plus bevacizumab (CPA). Corresponding OS () and PFS () data in the Kras mutant GEMM for NSCLC are shown; the control vehicle- and carboplatin-treated cohorts are reproduced from Figure 1 for comparison. The number of patients or mice in each treatment cohort is indicated in parentheses. Relevant P values (log-rank test) and hazard ratios are depicted for each graph, and the comparator group (denominator) is denoted with a hyphen. *, P values ≤ 0.05. CI95 for each hazard ratio shown are () 0.69–0.93, () 0.56–0.76, () 0.032–0.399 and () 0.027–0.341. Panels and reproduced with the kind permission of Alan Sandler. () Representative micro-CT images from mice in each of the GEMM cohorts pre-treatment (left) and after ~4 weeks on study (right). A visible tumor example is marked with yellow arrows in the control-treate! d cohort and mean tumor burden cross-products for each cohort are shown at the bottom left of each image. Control and carboplatin treatment images are reproduced from Figure 1 for comparison. C, carboplatin; P, paclitaxel; A, anti-VEGF; HR, hazard ratio; micro-CT, micro-computed tomography. * Figure 4: First-line treatment of PDAC patients and KrasLSL−G12D; p16/p19fl/fl; Pdx1-Cre mice with gemcitabine versus gemcitabine plus anti-VEGF. (–) Kaplan-Meier plots from CALGB 80303 (ref. 17) showing the OS () and PFS () data from PDAC patients treated with gemcitabine alone (G) or gemcitabine plus bevacizumab (GA). Corresponding OS () and PFS () data in the Kras mutant GEMM for PDAC are shown; the control vehicle- and gemcitabine-treated cohorts are reproduced from Figure 2 for comparison. The number of patients or mice in each treatment cohort is indicated in parentheses. Relevant P values (log-rank test) and hazard ratios are depicted for each graph, and the comparator group (denominator) is denoted with a hyphen. *, P values ≤ 0.05. CI95 for each GEMM hazard ratio shown are () 0.22–0.86 and () 0.28–1.03. Panels and reproduced with the kind permission of Hedy Kindler. () Representative high-resolution ultrasound images from mice in each of the GEMM cohorts pre-treatment (left) and after ~11–15 d on study (right). Visible lesions are outlined in yellow and mean tumor burden for each cohort are shown at! the bottom left of each image. Control and gemcitabine treatment images are reproduced from Figure 2 for comparison. G, gemcitabine; A, anti-VEGF; HR, hazard ratio. * Figure 5: Anti-VEGF is a primary driver of response in the Kras mutant NSCLC GEMM. (,) Anti-VEGF overcomes the negative interaction observed with carboplatin followed by erlotinib maintenance on both OS and PFS. Kaplan-Meier plots showing OS () and PFS () following different dual- and triple-combination regimens using chemotherapy, erlotinib and anti-VEGF are shown. (,) Anti-VEGF confers a significant OS and PFS benefit relative to vehicle control, both as a single agent and in combination with erlotinib. Single-agent effects on OS () and PFS () in comparison with the combination of the two targeted agents, anti-VEGF and erlotinib. Significant P values (log-rank test) and hazard ratios for each graph are as follows: () CA versus CE (P = 0.0003, HR = 0.20, CI95 = 0.08–0.48); EA versus CE (P = 0.0003, HR = 0.15, CI95 = 0.05–0.42); CEA versus CE (P = 0.0012, HR = 0.19, CI95 = 0.07–0.52). () CA versus CE (P = 0.0002, HR = 0.17, CI95 = 0.07–0.43); EA versus CE (P = 0.0011, HR = 0.17, CI95 = 0.06–0.49); CEA versus CE (P = 0.0004, HR = 0.15, CI95 = 0.05! –0.43). () A versus control (P = 0.0001, HR = 0.32, CI95 = 0.18–0.57); A versus E (P = 0.0017, HR = 0.28, CI95 = 0.12–0.62); A versus C (P = 0.024, HR = 0.38, CI95 = 0.16–0.88); EA versus control (P = 0.0052, HR = 0.32, CI95 = 0.15–0.71); EA versus E (P = 0.0084, HR = 0.28, CI95 = 0.11–0.72); EA versus C (P = 0.057, HR = 0.39, CI95 = 0.14–1.03). () A versus control (P = 6.3e–08, HR = 0.17, CI95 = 0.09–0.32); A versus E (P = 3e–06, HR = 0.14, CI95 = 0.06–0.32); A versus C (P = 0.0011, HR = 0.25, CI95 = 0.11–0.58); EA versus control (P = 0.0004, HR = 0.22, CI95 = 0.10–0.51); EA versus E (P = 0.0008, HR = 0.19, CI95 = 0.07–0.50); EA versus C (P = 0.03, HR = 0.34, CI95 = 0.13–0.89). () Representative micro-CT images from mice in each of the cohorts pre-treatment (left) and after ~4 weeks on study (right). Mean tumor burden cross-products for each cohort are shown at the bottom left of each image. The control, C, CE and CA cohorts are reproduced fro! m previous figures. C, carboplatin; A, anti-VEGF; E, erlotinib! ; HR, hazard ratio; CI95, 95% confidence interval; micro-CT, micro-computed tomography. * Figure 6: Gemcitabine is a primary driver of a survival benefit, with incremental benefit conferred by the addition of targeted agents in a Kras mutant PDAC GEMM. (,) Kaplan-Meier plots showing OS () and PFS () following different dual- and triple-combination regimens using chemotherapy, erlotinib and anti-VEGF are shown. (,) Targeted agents do not show single-agent effects, but erlotinib plus anti-VEGF is comparable to conventional chemotherapy with gemcitabine. Single-agent effects on OS () and PFS () in comparison with the combination of the two targeted agents, anti-VEGF and erlotinib. Significant P values (log-rank test) and hazard ratios for each graph are as follows: () GA versus GE (P = 0.20, HR = 0.57, CI95 = 0.24–1.35; note, only the HR is notable in this case), () GA versus GE (P = 0.04, HR = 0.40, CI95 = 0.17–0.94), GEA versus GE (P = 0.17, HR = 0.50, CI95 = 0.19–1.34; note, only the HR is notable in this case), () G versus control (P = 3.4e−07, HR = 0.22, CI95 = 0.12–0.39), G versus E (P = 0.0011, HR = 0.25, CI95 = 0.11–0.57), G versus A (P = 0.0012, HR = 0.28, CI95 = 0.13–0.61), EA versus control (P = 9e−! 05, HR = 0.18, CI95 = 0.07–0.42), EA versus E (P = 0.0028, HR = 0.20, CI95 = 0.07–0.59), EA versus A (P = 0.0037, HR = 0.23, CI95 = 0.08–0.63) and () G versus control (P = 0.018, HR = 0.53, CI95 = 0.32–0.90), G versus E (P = 0.43, HR = 0.72, CI95 = 0.32–1.61), G versus A (P = 0.066, HR = 0.49, CI95 = 0.22–1.05), EA versus control (P = 0.066, HR = 0.47, CI95 = 0.21–1.05), EA versus E (P = 0.39, HR = 0.64, CI95 = 0.23–1.79), EA versus A (P = 0.09, HR = 0.43, CI95 = 0.16–1.15). Unlike the OS data, only the HRs are notable for each of the PFS analyses in except for G versus control. () Representative high-resolution ultrasound images from mice in each of the cohorts pre-treatment (left) and after ~11–15 d on study (right). Visible lesions are outlined in yellow and mean tumor burden for each cohort are shown at the bottom left of each image. The control, G, GE and GA cohorts are reproduced from previous figures. G, gemcitabine; A, anti-VEGF; E, erlotinib; HR! , hazard ratio; CI95, 95% confidence interval. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Molecular Biology, Genentech, Inc., South San Francisco, California, USA. * Mallika Singh, * Anthony Lima, * Rafael Molina, * Patricia Hamilton, * Anne C Clermont, * Vidusha Devasthali, * Jennifer D Thompson, * Jason H Cheng & * Leisa Johnson * Department of Biomedical Imaging, Genentech, Inc., South San Francisco, California, USA. * Hani Bou Reslan, * Calvin C K Ho, * Timothy C Cao & * Richard A D Carano * Department of Antibody Engineering, Genentech, Inc., South San Francisco, California, USA. * Chingwei V Lee & * Germaine Fuh * Department of Cancer Signaling & Translational Oncology, Genentech, Inc., South San Francisco, California, USA. * Michelle A Nannini * Department of Pathology, Genentech, Inc., South San Francisco, California, USA. * Hartmut Koeppen * Department of Biostatistics, Genentech, Inc., South San Francisco, California, USA. * Ron X Yu & * William F Forrest * Department of Tumor Biology & Angiogenesis, Genentech, Inc., South San Francisco, California, USA. * Gregory D Plowman Contributions M.S. and L.J. designed, planned and performed the experiments, analyzed data and wrote the manuscript. A.L., R.M., P.H., A.C.C., V.D., J.D.T., J.H.C., H.B.R., C.C.K.H. and T.C.C. performed experiments and analyzed data. C.V.L. and G.F. developed and provided the B20-4.1.1 anti-VEGF antibody. M.A.N. and R.A.D.C. provided design input and supervised animal dosing and imaging experiments, respectively. G.D.P. provided design input and contributed to manuscript preparation. H.K. carried out histopathological analyses, and R.X.Y. and W.F.F. performed all the statistical analyses and contributed to the writing of the manuscript. Competing financial interests The authors are current or past employees of Genentech, Inc. and/or may have stocks or shares in Roche, Inc. Corresponding authors Correspondence to: * Mallika Singh (msingh@gene.com) or * Leisa Johnson (leisaj@gene.com) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (9M) Supplementary Tables 1,2 and Supplementary Figs. 1–9 Additional data - Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations
Rissin DM Kan CW Campbell TG Howes SC Fournier DR Song L Piech T Patel PP Chang L Rivnak AJ Ferrell EP Randall JD Provuncher GK Walt DR Duffy DC - Nat Biotech 28(6):595-599 (2010)
Nature Biotechnology | Research | Letter Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations * David M Rissin1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Cheuk W Kan1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Todd G Campbell1 Search for this author in: * NPG journals * PubMed * Google Scholar * Stuart C Howes1 Search for this author in: * NPG journals * PubMed * Google Scholar * David R Fournier1 Search for this author in: * NPG journals * PubMed * Google Scholar * Linan Song1 Search for this author in: * NPG journals * PubMed * Google Scholar * Tomasz Piech1 Search for this author in: * NPG journals * PubMed * Google Scholar * Purvish P Patel1 Search for this author in: * NPG journals * PubMed * Google Scholar * Lei Chang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew J Rivnak1 Search for this author in: * NPG journals * PubMed * Google Scholar * Evan P Ferrell1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeffrey D Randall1 Search for this author in: * NPG journals * PubMed * Google Scholar * Gail K Provuncher1 Search for this author in: * NPG journals * PubMed * Google Scholar * David R Walt2 Search for this author in: * NPG journals * PubMed * Google Scholar * David C Duffy1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature BiotechnologyVolume:28,Pages:595–599Year published:(2010)DOI:doi:10.1038/nbt.1641Received01 February 2010Accepted29 April 2010Published online23 May 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The ability to detect single protein molecules1, 2 in blood could accelerate the discovery and use of more sensitive diagnostic biomarkers. To detect low-abundance proteins in blood, we captured them on microscopic beads decorated with specific antibodies and then labeled the immunocomplexes (one or zero labeled target protein molecules per bead) with an enzymatic reporter capable of generating a fluorescent product. After isolating the beads in 50-fl reaction chambers designed to hold only a single bead, we used fluorescence imaging to detect single protein molecules. Our single-molecule enzyme-linked immunosorbent assay (digital ELISA) approach detected as few as ~10–20 enzyme-labeled complexes in 100 μl of sample (~10−19 M) and routinely allowed detection of clinically relevant proteins in serum at concentrations (<10−15 M) much lower than conventional ELISA3, 4, 5. Digital ELISA detected prostate-specific antigen (PSA) in sera from patients who had undergone radic! al prostatectomy at concentrations as low as 14 fg/ml (0.4 fM). View full text Figures at a glance * Figure 1: Digital ELISA based on arrays of femtoliter-sized wells. () Single protein molecules are captured and labeled on beads using standard ELISA reagents (), and beads with or without a labeled immunoconjugate are loaded into femtoliter-volume well arrays for isolation and detection of single molecules by fluorescence imaging (). () Scanning electron micrograph of a small section of a femtoliter-volume well array after bead loading. Beads (2.7 μm diameter) were loaded into an array of wells with diameters of 4.5 μm and depths of 3.25 μm. () Fluorescence image of a small section of the femtoliter-volume well array after signals from single enzymes are generated. Whereas the majority of femtoliter-volume chambers contain a bead from the assay, only a fraction of those beads possess catalytic enzyme activity, indicating a single, bound protein molecule. The concentration of protein in bulk solution is correlated to the percentage of beads that carry a protein molecule. * Figure 2: Digitization of enzyme-linked complexes greatly increases sensitivity compared with bulk, ensemble measurements. () Log-log plot of signal output (% active beads for single-molecule array (SiMoA) or relative fluorescence units (r.f.u.) for plate reader) as a function of the concentration of streptavidin-β-galactosidase (SβG) captured on biotinylated beads. SβG concentrations for the ensemble readout ranged from 3 fM to 300 fM, with a detection limit of 15 × 10−15 M (15 fM; green broken line). For the SiMoA assay, SβG concentrations ranged from 350 zM to 7 fM, demonstrating a linear response of ~10,000-fold, with a calculated detection limit of 220 × 10−21 M (220 zM; red broken line). Error bars are based on the s.d. over three replicates for both technologies. LODs were determined by extrapolating the concentration from the signal equal to background signal plus 3 s.d. of the background signal. () The imprecision from SiMoAs is determined by the Poisson noise of counting single events. The intrinsic variation (Poisson noise) of counting single active beads is given by √n. C! omparing the Poisson noise–associated coefficient of variation (%CV = √n/n) with the SiMoA %CV over three measurements confirmed that the imprecision of the assay is determined by counting error. * Figure 3: Subfemtomolar detection of proteins in serum using digital ELISA. (,) Changes in the percentage of active beads with changes in analyte concentration for human prostate-specific antigen (PSA) spiked into 25% serum () and human tumor necrosis factor-α (TNF-α) spiked into 25% serum (). The concentrations plotted on the x axes refer to the final concentration of spiked protein in the diluted sample. The plots on the left-hand side show the assay response over the concentration range tested in log-log space. The plots on the right-hand side show the assay response in the femtomolar range in linear-linear space to illustrate the limit of detection (LODs) and linearity of response. LODs were determined by extrapolating the concentration from the signal equal to background signal plus 3 s.d. of the background signal. Broken lines, signal at the LOD. Error bars, s.d. over three replicates. * Figure 4: Digital detection of prostate-specific antigen (PSA) in serum samples of patients who had undergone radical prostatectomy. Concentrations of PSA in serum samples from radical prostatectomy patients (), healthy control samples () and Bio-Rad PSA control samples () determined using digital ELISA. Radical prostatectomy patient samples (SeraCare Life Sciences) all had undetectable PSA levels as measured by a leading clinical diagnostic assay (ADVIA Centaur); the green broken line represents the detection limit of the ADVIA Centaur PSA assay (100 pg/ml or 3 pM). All 30 patient samples were above the detection limit of the PSA digital ELISA, shown by the red broken line (0.006 pg/ml or ~200 aM), with the lowest patient PSA concentrations measured at 0.014 pg/ml (~400 aM) using digital ELISA. Patient samples with the lowest PSA levels approached the LOD of the assay, resulting in a large imprecision in the concentration determined (high dose %CV). The digital ELISA was validated for specificity to PSA using control standards (Bio-Rad) and serum from healthy individuals (ProMedDx) that had been assayed ! using the ADVIA Centaur PSA assay (Supplementary Table 3). Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * David M Rissin & * Cheuk W Kan Affiliations * Quanterix Corporation, Cambridge, Massachusetts, USA. * David M Rissin, * Cheuk W Kan, * Todd G Campbell, * Stuart C Howes, * David R Fournier, * Linan Song, * Tomasz Piech, * Purvish P Patel, * Lei Chang, * Andrew J Rivnak, * Evan P Ferrell, * Jeffrey D Randall, * Gail K Provuncher & * David C Duffy * Department of Chemistry, Tufts University, Medford, Massachusetts, USA. * David R Walt Contributions D.M.R., C.W.K., D.R.F., D.R.W. and D.C.D. conceived the approach. D.R.F. built the imaging system. D.M.R., C.W.K., T.G.C., S.C.H., L.S., P.P.P., A.J.R., E.P.F., J.D.R. and G.K.P. conducted the experiments. T.P. wrote the image analysis software. L.C. prepared reagents. D.M.R. and D.C.D. wrote the manuscript. All authors were involved in designing experiments, reviewing and discussing data, and commenting on the manuscript. Competing financial interests All authors are employees or advisors of Quanterix Corporation who have a minority ownership or ownership option position in the company. Corresponding author Correspondence to: * David C Duffy (dduffy@quanterix.com) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (252K) Supplementary Tables 1–3, Supplementary Figs. 1–3 and Supplementary Methods Additional data - Identification of influenza A nucleoprotein as an antiviral target
Kao RY Yang D Lau LS Tsui WH Hu L Dai J Chan MP Chan CM Wang P Zheng BJ Sun J Huang JD Madar J Chen G Chen H Guan Y Yuen KY - Nat Biotech 28(6):600-605 (2010)
Nature Biotechnology | Research | Letter Identification of influenza A nucleoprotein as an antiviral target * Richard Y Kao1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Dan Yang4 Search for this author in: * NPG journals * PubMed * Google Scholar * Lai-Shan Lau1 Search for this author in: * NPG journals * PubMed * Google Scholar * Wayne H W Tsui1 Search for this author in: * NPG journals * PubMed * Google Scholar * Lihong Hu4 Search for this author in: * NPG journals * PubMed * Google Scholar * Jun Dai1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Mei-Po Chan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Che-Man Chan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Pui Wang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Bo-Jian Zheng1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jian Sun4 Search for this author in: * NPG journals * PubMed * Google Scholar * Jian-Dong Huang5 Search for this author in: * NPG journals * PubMed * Google Scholar * Jason Madar6 Search for this author in: * NPG journals * PubMed * Google Scholar * Guanhua Chen4 Search for this author in: * NPG journals * PubMed * Google Scholar * Honglin Chen1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Yi Guan1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Kwok-Yung Yuen1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature BiotechnologyVolume:28,Pages:600–605Year published:(2010)DOI:doi:10.1038/nbt.1638Received23 November 2009Accepted27 April 2010Published online30 May 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Influenza A remains a significant public health challenge because of the emergence of antigenically shifted or highly virulent strains1, 2, 3, 4, 5. Antiviral resistance to available drugs such as adamantanes or neuraminidase inhibitors has appeared rapidly6, 7, 8, 9, creating a need for new antiviral targets and new drugs for influenza virus infections. Using forward chemical genetics, we have identified influenza A nucleoprotein (NP) as a druggable target and found a small-molecule compound, nucleozin, that triggers the aggregation of NP and inhibits its nuclear accumulation. Nucleozin impeded influenza A virus replication in vitro with a nanomolar median effective concentration (EC50) and protected mice challenged with lethal doses of avian influenza A H5N1. Our results demonstrate that viral NP is a valid target for the development of small-molecule therapies. View full text Figures at a glance * Figure 1: Chemical structures and biological activities of nucleozin and related compounds. () Chemical structures of compound FA-1, FA-2, FA-3 and FA-4. () Nucleozin is effective against human H1N1, H3N2 and H5N1 influenza viruses. MDCK cells were infected with different strains of virus and antiviral activities determined by PRA. Oseltamivir (curve in red) was included for comparisons of in vitro efficacies. () Antiviral activity of nucleozin in multicycle growth assays. MDCK cells were infected with A/WSN/33 virus at 0.001 MOI in the presence or absence of nucleozin (0.1 or 1 μM). Zanamivir at 1 μM has stopped viral growth but data were omitted for clarity. Viral titers were determined by plaque assay at the time indicated. () Efficacies of nucleozin added at various time points. MDCK cells were infected at an MOI of 2 and nucleozin (1 μM) was added before infection (−1 h), at the time of infection (0 h) and at 1, 2, 4, 6 and 8 h after infection as indicated. +, controls with no nucleozin added. Viral titers were determined at 12 h after infection by plaque! assay. The experiments were carried out in triplicate and repeated twice for confirmation. The mean value is shown with s.d. () Nucleozin blocked nuclear accumulation of influenza A NP in virus-infected A549 cells. Cells were infected with A/WSN/33 virus (10 MOI) in the presence or absence of 1 μM nucleozin. Influenza A NP accumulated in the nucleus at early-infection stage and was distributed exclusively in the cytoplasm at late-infection stage in the absence of nucleozin. At the indicated time point, cells were fixed and DAPI staining and mouse anti-influenza A NP antibodies were used to define the locations of the nucleus and viral NP, respectively. PFU, plaque forming unit. * Figure 2: Influenza A NP is the molecular target of nucleozin. () Three potential binding sites of nucleozin on the influenza A NP crystal structure as predicted by molecular docking models. Electrostatic surface representation of influenza A NP is color-coded (red, negative; blue, positive; light gray, neutral). Potential binding sites of nucleozin are highlighted by yellow circles. () Escape mutant virus and recombinant virus carrying the Y289H substitution in influenza A NP confer resistance to high concentrations of nucleozin. MDCK cells were infected with A/WSN/33 virus, Y289H escape mutant virus or Y289H variant virus generated by reverse genetics. Antiviral activities determined by PRA. The highest concentration of nucleozin used was limited to 125 μM as fine precipitates appeared at higher concentration that interfered with the determination of plaques. All virus strains were tested in the same experiments for comparisons of in vitro resistance profiles. () MDCK cells were infected with the Y289H escape mutant virus (MOI = 5) i! n the presence or absence of 1 μM nucleozin. The addition of nucleozin did not block the nuclear accumulation of the viral NP. At the indicated time points, cells were fixed and DAPI staining and mouse anti-influenza A NP antibodies were used to define the locations of the nucleus and viral NP, respectively. () Nucleozin inhibits the parental virus NP activity but not theY289H variant virus NP in a luciferase reporter assay. 0, 1, 5 or 25 μM of nucleozin was added to 293T cells transfected with minigenomes containing A/WSN/33 virus NP or Y289H variant NP. Luciferase activities were measured 24 h post-transfection. The experiments were carried out in triplicate and repeated twice. The mean value is shown with s.d. () Nucleozin inhibits the nuclear import of exogenously added A/WSN/33 NP but not the Y289H variant NP. Purified recombinant NP or Y289H variant NP at 25 μM was added to digitonin-treated MDCK cells in the presence or absence of 10 μM nucleozin. Nuclear import ! of proteins was allowed for 30 min and followed by cell fixati! on and immunostaining for the presence of NP in the nucleus. DAPI was used to indicate the location of the nucleus. Images were visualized by confocal microscopy. * Figure 3: Nucleozin interacts with NP and causes NP aggregation. () Binding of nucleozin to NP induced fluorescence quenching. We used 4 μM wild-type WSN NP or Y289H variant NP and 0–25 μM of nucleozin for fluorescence titrations. Internal fluorescence-quenching effect was due to micro-conformational changes upon the binding of nucleozin to the NP. The experiments were carried out in duplicate and repeated three times for confirmation. (–) Nucleozin does not inhibit RNA binding but mediates formation of large NP-RNA complexes not resolvable by gradient gel electrophoresis. Free RNA oligomer and NP-RNA complex were visualized by ethidium bromide staining in and NP was visualized by Coomassie brilliant blue G-250 staining in . RNA incubated with 40 μM of nucleozin in the absence of NP (lane 2) was used as a control. () Visualization and comparison of the effects of nucleozin on recombinant wild-type and Y289H NP (in the absence of RNA); native gradient gel conditions, stained by Coomassie brilliant blue G-250. WT, purified recombinan! t A/WSN/33 NP; Mu, purified recombinant Y289H variant NP; +, in the presence; −, in the absence. Positions of free RNA, NP-RNA complex and NP are indicated. () Visualization of nucleozin-induced NP aggregates in cells. MDCK cells transfected with plasmids expressing wild-type NP or Y289H variant NP were treated with nucleozin for 4 h. DAPI staining and mouse anti-influenza A NP antibodies were used to define the locations of the nucleus and NP respectively. * Figure 4: Efficacies of nucleozin in a mice H5N1 virus infection model. () Nucleozin protected the mice infected with highly pathogenic influenza H5N1 virus. Mice (nine per group) infected with 5 LD50 (median lethal dose) of A/Vietnam/1194/04 H5N1 virus received 100 μl of 20 mg/ml zanamivir, 2.3 mg/ml nucleozin or PBS twice daily intraperitoneally. Treatments stopped at day 7 after infection. Conditions of the mice were monitored for 21 d. () Zanamivir and nucleozin reduced the viral load in the lungs of infected mice when compared to the control (untreated mice). Three mice from each group were euthanized at day 6 and lungs removed for viral load determination by standard plaque assay. Shown are the mean values with s.d. Author information * Author information * Supplementary information Affiliations * Department of Microbiology, The University of Hong Kong, Hong Kong. * Richard Y Kao, * Lai-Shan Lau, * Wayne H W Tsui, * Jun Dai, * Mei-Po Chan, * Che-Man Chan, * Pui Wang, * Bo-Jian Zheng, * Honglin Chen, * Yi Guan & * Kwok-Yung Yuen * Research Center of Infection and Immunology, The University of Hong Kong, Hong Kong. * Richard Y Kao, * Jun Dai, * Bo-Jian Zheng, * Honglin Chen, * Yi Guan & * Kwok-Yung Yuen * State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong. * Richard Y Kao, * Bo-Jian Zheng, * Honglin Chen, * Yi Guan & * Kwok-Yung Yuen * Department of Chemistry, The University of Hong Kong, Hong Kong. * Dan Yang, * Lihong Hu, * Jian Sun & * Guanhua Chen * Department of Biochemistry, The University of Hong Kong, Hong Kong. * Jian-Dong Huang * Department of Computing Sciences, Capilano University, British Columbia, Canada. * Jason Madar Contributions R.Y.K. and K.-Y.Y conceived the study. R.Y.K. designed and performed experiments and analyzed data. D.Y. gave conceptual advice and technical support on chemistry. L.-S.L., W.H.W.T., J.D., M.-P.C., C.-M.C. and P.W. performed experiments. J.S., L.H., and G.C. performed molecular dockings. B.-J.Z. provided animal study data. J.-D.H. gave conceptual advice on protein trafficking. J.M. constructed database and performed HTS data normalization. H.C. and Y.G. provided reverse genetics system. K.-Y.Y. did troubleshooting and provided the grant support. R.Y.K. and K.-Y.Y. supervised the study and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Richard Y Kao (rytkao@hkucc.hku.hk) or * Kwok-Yung Yuen (kyyuen@hkucc.hku.hk) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (660K) Supplementary Tables 1–3 and Supplementary Figs. 1–11 Additional data - Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells
Melkoumian Z Weber JL Weber DM Fadeev AG Zhou Y Dolley-Sonneville P Yang J Qiu L Priest CA Shogbon C Martin AW Nelson J West P Beltzer JP Pal S Brandenberger R - Nat Biotech 28(6):606-610 (2010)
Nature Biotechnology | Research | Letter Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells * Zara Melkoumian1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jennifer L Weber1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * David M Weber1 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrei G Fadeev1 Search for this author in: * NPG journals * PubMed * Google Scholar * Yue Zhou1 Search for this author in: * NPG journals * PubMed * Google Scholar * Paula Dolley-Sonneville1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jiwei Yang2 Search for this author in: * NPG journals * PubMed * Google Scholar * Liqun Qiu2 Search for this author in: * NPG journals * PubMed * Google Scholar * Catherine A Priest2 Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher Shogbon1 Search for this author in: * NPG journals * PubMed * Google Scholar * Arthur W Martin1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jodelle Nelson1 Search for this author in: * NPG journals * PubMed * Google Scholar * Peter West1 Search for this author in: * NPG journals * PubMed * Google Scholar * James P Beltzer1 Search for this author in: * NPG journals * PubMed * Google Scholar * Santona Pal1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ralph Brandenberger2 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature BiotechnologyVolume:28,Pages:606–610Year published:(2010)DOI:doi:10.1038/nbt.1629Received19 October 2009Accepted01 April 2010Published online30 May 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Human embryonic stem cells (hESCs) have two properties of interest for the development of cell therapies: self-renewal and the potential to differentiate into all major lineages of somatic cells in the human body. Widespread clinical application of hESC-derived cells will require culture methods that are low-cost, robust, scalable and use chemically defined raw materials. Here we describe synthetic peptide-acrylate surfaces (PAS) that support self-renewal of hESCs in chemically defined, xeno-free medium. H1 and H7 hESCs were successfully maintained on PAS for over ten passages. Cell morphology and phenotypic marker expression were similar for cells cultured on PAS or Matrigel. Cells on PAS retained normal karyotype and pluripotency and were able to differentiate to functional cardiomyocytes on PAS. Finally, PAS were scaled up to large culture-vessel formats. Synthetic, xeno-free, scalable surfaces that support the self-renewal and differentiation of hESCs will be useful for ! both research purposes and development of cell therapies. View full text Figures at a glance * Figure 1: Development of the PAS surface. () Schematic representation of the peptide-acrylate surface coating process. Vessel surfaces are coated with carboxylic acid containing acrylate, followed by EDC/NHS conjugation of amine-containing peptide. () hESC attachment to PAS in defined medium, X-VIVO 10 + GF in 96-well plates. H1 (white bars) and H7 (black bars) hESCs attach to BSP- or VN-, but not FN- or LM-PAS. AttoPhos relative fluorescent units (RFU) represent alkaline phosphatase activity of attached cells 48 h after seeding. Values are normalized to cell attachment to Matrigel (absolute values from 5,000–55,000). () H7 and H1 hESC growth on PAS in X-VIVO 10 + GF. One million cells were seeded in each well of a 6-well plate and cultured for 4 d. hESC cell number was comparable to Matrigel for BSP-PAS and VN-PAS but not FN-PAS. () H7 hESC colony morphology on day 3 on Matrigel, BSP-PAS and sFN-PAS. Scale bars, 200 μm. () Peptide concentration–dependent H7 hESC attachment and growth. BSP-PAS 6-well plates wer! e prepared with serial dilutions of BSP peptide spiked with 0.25% rhodamine-labeled peptide. 1 × 106 H7 hESCs were seeded per well and cultured for 5 d in X-VIVO + GF. RFU correspond to fluorescent intensity of 0.25% rhodamine-labeled peptide. () ELISA staining with BCIP/NBT using anti-BSP antibodies shows uniform peptide distribution within a well of a 6-well plate. () Peptide surface density–dependent hESC attachment. H7 hESCs were seeded at 1 × 106 cells/well of a BSP-PAS 6-well plate and cultured in X-VIVO 10 + GF for 5 d. Crystal violet staining of cells shows uniform cell distribution and BSP concentration-dependent confluency. * Figure 2: PAS supports long-term culture and pluripotency of H7 hESCs in chemically defined medium X-VIVO 10 + GF. () Doubling time of H7 cells over the course of 12 consecutive passages on BSP-PAS, VN-PAS and Matrigel. Seeding density at each passage was 1 × 106 cells/well of a 6-well plate. Cells were harvested for passaging after 4–5 d. () Phase contrast images of cell colony morphology for H7 cells at passage 2 (top panel) and 10 (bottom panel) on Matrigel and PAS, as indicated. Scale bars, 200 μm. () Indirect immunofluorescence staining of H7 hESCs after ten consecutive passages on BSP-PAS, VN-PAS and Matrigel. Scale bars, 100 μm. () H7 cells retain pluripotency after long-term culture on VN-PAS or BSP-PAS. H7 hESCs were thawed and cultured for eight passages on BSP-PAS, VN-PAS and Matrigel (positive control); cells were harvested and 1 × 107 cells were injected by intramuscular injection into the flank of SCID/bg mice. Tissues from all three germ layers, represented as secretory epithelium (endoderm), cartilage (mesoderm) and neuroepithelium (ectoderm), were identified in ter! atomas formed by H7 hESCs cultured on VN-PAS, BSP-PAS or Matrigel. Scale bar, 50 μm. * Figure 3: Direct differentiation of H7 hESCs into cardiomyocytes on PAS and scalability of PAS production. () Cardiomyocyte differentiation. H7 hESCs were maintained on BSP-PAS in defined X-VIVO 10 + GF medium for ten passages followed by a directed differentiation into cardiomyocytes on the same PAS. Confocal indirect immunofluorescence for cardiomyocyte-specific markers, α-actinin (green) and Nkx2.5 (in red). Scale bar, 50 μm. () Flow cytometry analysis for α-actinin and Nkx2.5 markers. () Uniform peptide distribution in VN-PAS T75 flasks. Surface-bound peptide was visualized by light-purple ELISA BCIP/NBT staining using anti VN-peptide antibodies. () Uniform distribution of alkaline phosphatase–positive H7 hESC colonies after 4 d in culture. H7 hESCs were cultured in X-VIVO 10 + GF in VN-PAS T75 flasks. After 4 d in culture cells were fixed and stained with BCIP/NBT to detect alkaline phosphatase activity. () H7 hESC colonies cultured in X-VIVO 10 + GF in VN-PAS T75 flasks express the nuclear hESC marker OCT4. Scale bar, 200 μm. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Zara Melkoumian & * Jennifer L Weber Affiliations * Corning Life Sciences, Corning Inc., Corning, New York, USA. * Zara Melkoumian, * Jennifer L Weber, * David M Weber, * Andrei G Fadeev, * Yue Zhou, * Paula Dolley-Sonneville, * Christopher Shogbon, * Arthur W Martin, * Jodelle Nelson, * Peter West, * James P Beltzer & * Santona Pal * Geron Corporation, Menlo Park, California, USA. * Jiwei Yang, * Liqun Qiu, * Catherine A Priest & * Ralph Brandenberger Contributions Z.M. conceived, designed, performed and analyzed PAS validation with H7 cells. J.L.W. conceived, designed, performed and analyzed PAS validation with H1 cells, wrote supplementary materials. P.D.-S. and J.Y. designed, performed and analyzed cardiomyocyte differentiation on PAS. J.Y. designed, performed and analyzed EB differentiation. L.Q. assisted J.Y. in performing and analyzing cardiomyocytes and EB differentiation. C.A.P. performed teratoma formation studies. D.M.W., A.G.F. and Y.Z. conceived, designed, developed and fabricated PAS. D.M.W. identified peptides for the study, characterized surface peptide uniformity, contributed to design of peptide conjugation scheme. A.G.F. developed and optimized peptide conjugation and characterized surface peptide density. C.S. assisted Y.Z. in acrylate coating development and fabrication. A.M., J.N. and P.W. developed coating conditions for PAS in T75 format, fabricated PAS-T75. Z.M. and R.B. wrote the manuscript. J.P.B., S.P. and R.! B. provided direction and guidance for the various areas of the project. Competing financial interests Z.M., J.L.W., P.D.-S., J.P.B., S.P., D.M.W., A.G.F., Y.Z., C.S., A.M., J.N. and P.W. are employees of Corning Incorporated. J.Y., L.Q., C.A.P. and R.B. are employees of Geron Corporation. Corresponding authors Correspondence to: * Zara Melkoumian (melkoumiz@corning.com) or * Ralph Brandenberger (RBrandenberger@Geron.com) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Tables 1,2 and Supplementary Figs. 1–6 Additional data - Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511
Rodin S Domogatskaya A Ström S Hansson EM Chien KR Inzunza J Hovatta O Tryggvason K - Nat Biotech 28(6):611-615 (2010)
Nature Biotechnology | Research | Letter Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511 * Sergey Rodin1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anna Domogatskaya1 Search for this author in: * NPG journals * PubMed * Google Scholar * Susanne Ström2 Search for this author in: * NPG journals * PubMed * Google Scholar * Emil M Hansson3 Search for this author in: * NPG journals * PubMed * Google Scholar * Kenneth R Chien3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * José Inzunza5 Search for this author in: * NPG journals * PubMed * Google Scholar * Outi Hovatta2 Search for this author in: * NPG journals * PubMed * Google Scholar * Karl Tryggvason1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature BiotechnologyVolume:28,Pages:611–615Year published:(2010)DOI:doi:10.1038/nbt.1620Received15 December 2009Accepted08 March 2010Published online30 May 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We describe a system for culturing human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells on a recombinant form of human laminin-511, a component of the natural hES cell niche. The system is devoid of animal products and feeder cells and contains only one undefined component, human albumin. The hES cells self-renewed with normal karyotype for at least 4 months (20 passages), after which the cells could produce teratomas containing cell lineages of all three germ layers. When plated on laminin-511 in small clumps, hES cells spread out in a monolayer, maintaining cellular homogeneity with approximately 97% OCT4-positive cells. Adhesion of hES cells was dependent on α6β1 integrin. The use of homogeneous monolayer hES or iPS cell cultures provides more controllable conditions for the design of differentiation methods. This xeno-free and feeder-free system may be useful for the development of cell lineages for therapeutic purposes. View full text Figures at a glance * Figure 1: Adhesion of hES cells to different coatings and expression of laminin chains in hES cells. () Crystal violet staining of hES cells adherent to LN-511 and LN-111. Scale bars, 220 μm (insets, 55 μm). () Contact area of ES cells with different adhesive substrata. Bars represent average relative contact area (compared with the cells plated on poly-D-lysine). Statistical significance (P) was calculated by Student's t-test. Error bars show s.e.m.; number inside each bar indicates number of independent measurements. LN, laminins; MG, Matrigel; PL, poly-D-lysine. () RT-PCR analysis of total RNA isolated from HS420 cells. Primer sets for all known laminin chains were used. bp, base pairs. * Figure 2: Integrin receptors on hES cell surface and their role in hES cell adhesion. () Adhesion-blocking experiment: inhibition of hES cell adhesion to LN-511 by different integrin antibodies. Bars represent inhibition by antibodies to chains indicated below graph (all from Millipore). IgG was used as a control for uninhibited cell adhesion. Error bars show s.e.m. (n = 4). Statistical significance (P) was calculated by Student's t-test. () Adhesion of hES cells to surfaces coated by different integrin antibodies. Bars represent adhesion with antibodies to chains indicated below. Error bars show s.e.m. (n = 4). P calculated by Student's t-test is shown; **P < 0.01. () Immunofluorescence: integrin α6 coexpression with β1 integrin subunit in pluripotent (SOX2-positive) hES cells cultured on LN-511. Scale bars, 37 μm. * Figure 3: Representative immunostaining analysis, RT-PCR, fluorescence-activated cell sorting (FACS) analysis, real-time quantitative RT-PCR and quantitative western blot analysis of HS207 cultured on LN-511, either in O3 medium or in H3 medium free from any animal-derived components. () Growth curves for hES cells cultured in O3 medium on LN-511 and Matrigel. The cells were passaged as described in the Online Methods for the long-term experiment. After each TrypLE Express treatment and subsequent washing, one-third of the cells were plated in clumps on fresh LN-511– or Matrigel-coated dishes. The rest were dissociated into single-cell suspension and counted. Two independent duplicate experiments were performed for each coating. After the fifth passage, a portion of the cells were fixed and analyzed by immunofluorescence staining, confirming that the majority of the cells still expressed Nanog, a marker of pluripotency. () Immunostaining of HS207 cells with antibodies to Nanog, SOX2 and OCT4 after 20 passages (6 months) on LN-511 in O3 medium. Right panels show nuclear 4,6-diamidino-2-phenylindole (DAPI) staining. Scale bars, 0.15 mm. () RT-PCR analysis of total RNA isolated from H207 cells grown on feeder cells (Feeders), on Matrigel after 7 passages i! n O3 medium (MG, p.7 in O3), on LN-511 after 8 passages in H3 medium (LN-511, p.8 in H3) and on LN-511 after 27 passages in O3 medium (LN-511, p.27 in O3). Primer sets were designed for pluripotency markers OCT4 and NANOG, along with differentiation markers Brachyury, α-fetoprotein (AFP), SOX1 and PAX6, and for a housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH). () Real-time quantitative RT-PCR analysis was used to measure numbers of mRNA transcripts of the pluripotency markers OCT4 and NANOG at different time points in HS207 cells cultured on LN-511 and on Matrigel; values shown are normalized to OCT4 and NANOG expression levels in control HS207 cells cultured on feeder layer (Feeders). Number of passages, adhesion surface and medium are denoted as in . Error bars show 95% confidence intervals. () Expression of pluripotency markers OCT4 and SOX2 in HS207 cells cultured on feeder cells, Matrigel and LN-511 at different time points and in differen! t media (denoted as in ) was measured by western blotting and ! quantified by densitometry. Error bars represent range. () FACS analysis of HS207 cells after 25 passages on LN-511 in O3 medium for OCT4, a marker of pluripotency. The percentage of positive cells is listed in parentheses. * Figure 4: Pluripotency of HS207 cells after extensive passaging on LN-511. Teratomas containing components of the three germ layers were formed after HS207 cells that had been cultured for 15 passages on LN-511 were injected subcutaneously into SCID mice. () Cartilage, stained with hematoxylin and eosin (HE). Magnification, ×100. () Developing neural tissue and intestinal endoderm, with HE and periodic acid-Schiff (HE-PAS) staining. Goblet cells are shown in red. Magnification, ×400. () Developing kidney glomerulus, HE staining. Magnification, ×400. () Retinal pigment epithelium, HE staining. Magnification, ×400. () Immunostaining of embryoid bodies formed from HS207 cells after 20 passages on LN-511 revealed expression of markers for the three embryonic cell layers: smooth-muscle (SM) actin, nestin, MAP-2 and AFP. Scale bars, 55 μm. * Figure 5: Immunostaining analysis of different hES and iPS cells grown on LN-511. () H1 and H9 cells after five passages (1 month) on LN-511 in O3 medium expressed pluripotency markers OCT4 (green), Nanog (green) and SOX2 (red). DAPI staining is in blue. Scale bars, 75 μm. () BJ#12 and LDS 1.4 iPS cells after five passages on LN-511 in mTeSR1 medium expressed Nanog (red) and OCT4 (green). Scale bars, 75 μm. Author information * Author information * Supplementary information Affiliations * Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden. * Sergey Rodin, * Anna Domogatskaya & * Karl Tryggvason * Division of Obstetrics and Gynecology, Department of Clinical Sciences, Intervention and Technology, Karolinska Institute and Karolinska University Hospital, Huddinge, Stockholm, Sweden. * Susanne Ström & * Outi Hovatta * Cardiovascular Research Center, Massachusetts General Hospital, Charles River Plaza, Boston, Massachusetts, USA. * Emil M Hansson & * Kenneth R Chien * Department of Stem Cell and Regenerative Biology, Cambridge, Massachusetts, USA. * Kenneth R Chien * Department of Biosciences and Nutrition, Karolinska Institute, Novum, Huddinge, Karolinska Hospital, Huddinge, Stockholm, Sweden. * José Inzunza Contributions S.R. and A.D. contributed to the production and purification of human recombinant laminins, conducted all in vitro experiments with the hES cells and contributed to the planning and design of experiments and to the writing of the manuscript. O.H. established and provided the hES cell lines and contributed to manuscript writing and karyotyping. S.S. contributed to the establishment of the new hES cell lines. E.M.H. and K.R.C. contributed to the iPS cell work. J.I. carried out the teratoma experiments in nude mice. K.T. planned and designed the project and contributed to the writing of the manuscript. Competing financial interests K.T. and S.R. are shareholders in BioLamina. Corresponding author Correspondence to: * Karl Tryggvason (karl.tryggvason@ki.se) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figs. 1–7 and Supplementary Tables 1,2 Additional data - Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe
- Nat Biotech 28(6):617-623 (2010)
Nature Biotechnology | Research | Resources Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe * Dong-Uk Kim1, 14 Search for this author in: * NPG journals * PubMed * Google Scholar * Jacqueline Hayles2, 14 Search for this author in: * NPG journals * PubMed * Google Scholar * Dongsup Kim3, 14 Search for this author in: * NPG journals * PubMed * Google Scholar * Valerie Wood2, 4, 14 Search for this author in: * NPG journals * PubMed * Google Scholar * Han-Oh Park5, 14 Search for this author in: * NPG journals * PubMed * Google Scholar * Misun Won1, 14 Search for this author in: * NPG journals * PubMed * Google Scholar * Hyang-Sook Yoo1, 14 Search for this author in: * NPG journals * PubMed * Google Scholar * Trevor Duhig2 Search for this author in: * NPG journals * PubMed * Google Scholar * Miyoung Nam1 Search for this author in: * NPG journals * PubMed * Google Scholar * Georgia Palmer2 Search for this author in: * NPG journals * PubMed * Google Scholar * Sangjo Han3 Search for this author in: * NPG journals * PubMed * Google Scholar * Linda Jeffery2 Search for this author in: * NPG journals * PubMed * Google Scholar * Seung-Tae Baek1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hyemi Lee1 Search for this author in: * NPG journals * PubMed * Google Scholar * Young Sam Shim1 Search for this author in: * NPG journals * PubMed * Google Scholar * Minho Lee3 Search for this author in: * NPG journals * PubMed * Google Scholar * Lila Kim1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kyung-Sun Heo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Eun Joo Noh1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ah-Reum Lee1 Search for this author in: * NPG journals * PubMed * Google Scholar * Young-Joo Jang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kyung-Sook Chung1 Search for this author in: * NPG journals * PubMed * Google Scholar * Shin-Jung Choi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jo-Young Park1 Search for this author in: * NPG journals * PubMed * Google Scholar * Youngwoo Park1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hwan Mook Kim6 Search for this author in: * NPG journals * PubMed * Google Scholar * Song-Kyu Park6 Search for this author in: * NPG journals * PubMed * Google Scholar * Hae-Joon Park5 Search for this author in: * NPG journals * PubMed * Google Scholar * Eun-Jung Kang5 Search for this author in: * NPG journals * PubMed * Google Scholar * Hyong Bai Kim7 Search for this author in: * NPG journals * PubMed * Google Scholar * Hyun-Sam Kang8 Search for this author in: * NPG journals * PubMed * Google Scholar * Hee-Moon Park9 Search for this author in: * NPG journals * PubMed * Google Scholar * Kyunghoon Kim10 Search for this author in: * NPG journals * PubMed * Google Scholar * Kiwon Song11 Search for this author in: * NPG journals * PubMed * Google Scholar * Kyung Bin Song12 Search for this author in: * NPG journals * PubMed * Google Scholar * Paul Nurse2, 13 Search for this author in: * NPG journals * PubMed * Google Scholar * Kwang-Lae Hoe1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature BiotechnologyVolume:28,Pages:617–623Year published:(2010)DOI:doi:10.1038/nbt.1628Received06 January 2010Accepted30 March 2010Published online16 May 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We report the construction and analysis of 4,836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast genome providing a tool for studying eukaryotic biology. Comprehensive gene dispensability comparisons with budding yeast—the only other eukaryote for which a comprehensive knockout library exists—revealed that 83% of single-copy orthologs in the two yeasts had conserved dispensability. Gene dispensability differed for certain pathways between the two yeasts, including mitochondrial translation and cell cycle checkpoint control. We show that fission yeast has more essential genes than budding yeast and that essential genes are more likely than nonessential genes to be present in a single copy, to be broadly conserved and to contain introns. Growth fitness analyses determined sets of haploinsufficient and haploproficient genes for fission yeast, and comparisons with budding yeast identified specific ribosomal proteins and RNA polymerase subunits, whic! h may act more generally to regulate eukaryotic cell growth. View full text Figures at a glance * Figure 1: Deletion construction and gene dispensability. () Gene deletion cassette containing the KanMX4 gene flanked by unique bar codes (UPTAG/DNTAG) and regions of homology to the gene of interest (RHG). The cassette replaced the ORF of interest by homologous recombination at the RHG regions. () Construction of deletion mutants. All 4,836 protein coding genes were deleted using serial extension PCR (31.3%), block PCR (63.2%) or total gene synthesis (5.4%). The remaining 78 genes could not be confirmed as deleted owing to ambiguous sequencing results, recombination failure or inviability of the heterozygous diploids. () Dispensability of 4,836 protein coding genes. For 3,626 (2,729 + 897) genes the dispensability was previously unknown. ND, not done. * Figure 2: Analysis of gene dispensability. () Chromosome distribution of gene dispensability. Essential genes (tall bars) and nonessential genes (short bars) are distributed randomly throughout the genome except within 100 kb of the telomeres (gray boxes), where nonessential genes are enriched. Upper bars represent genes transcribed left to right and lower bars represent genes transcribed right to left. Filled circles in orange represent centromeres. () Percentage of essential genes versus number of introns. Percentage of essential genes was plotted against the number of introns within genes. In fission yeast, the percentage of essential genes containing introns is significantly (P < 10−14) higher than the percentage of those lacking introns. The dotted line represents the average percentage of essential genes in the total gene set (26.1%). () Percentage of essential genes versus ORFeome localization. The percentage of essential genes was plotted against ten different cellular locations in fission yeast. The dotted! line represents the average percentage of essential genes for the total gene set (26.1%). The number of essential gene products localized to the nucleolus, spindle pole body and nuclear envelope is higher than average. The number of essential genes compared to the total for each location is: (i) cytoplasm 564/2,113; (ii) nucleus 601/2,068; (iii) mitochondrion 128/450; (iv) ER 98/436; (v) cell periphery 55/326; (vi) nucleolus 89/217; (vii) Golgi 27/224; (viii) spindle pole body 69/181; (ix) nuclear envelope 29/76; and (x) microtubule 20/71. () Comparison of GO analyses of fission yeast and budding yeast genes. Bar chart shows a selection of broad, biologically informative GO terms significantly (P ≤ 0.01) enriched for essential and nonessential genes in fission yeast and budding yeast. For the complete list of processes and for methods used to extract these data, see Supplementary Tables 5 and 6. * Figure 3: Comparative analysis of gene dispensability profiles of fission yeast. Gene dispensability profiles of 4,836 deletion mutants by gene copy number of fission yeast orthologs compared to budding yeast (x axis) and species distribution (y axis). Compared to budding yeast, fission yeast genes consist of 2,841 single-copy genes (n = 1, m ≥1), 855 duplicated genes (n > 1, m ≥1) and 1,140 genes found in fission yeast but not in budding yeast (n ≥ 1, m = 0), where 'n' is the number of genes in fission yeast and 'm' is the number of genes in budding yeast. The term 'eukaryotes' includes human and the term 'variable phyla' includes plants. The area of each circle represents the numbers of genes, where essential and nonessential genes are represented by yellow and blue, respectively. * Figure 4: Dispensability comparison of orthologous pairs from the two yeasts. () Essentiality of nonredundant 2,438 orthologous pairs were compared between the two yeasts. Eighty-three percent of orthologs show conserved dispensability and the remaining 17% show different dispensability. E, essential; NE, nonessential. () Functional distribution of orthologs with different dispensability. The 17% of the orthologous pairs with different dispensability were allocated to one of 31 biological terms, 22 of which are shown here. For the complete list of processes and genes, see Supplementary Table 14. Note that genes annotated to mitochondrial functions, certain amino acid metabolic pathways and protein degradation pathways such as neddylation and sumoylation are mostly essential in one yeast and nonessential in the other yeast, whereas other categories show essential genes (although the specific genes are different) in both yeasts under the conditions used in this study. Because there are some differences in the constituents of the standard rich media used! for each organism, it is possible that in a few cases different dispensability between the two organisms are due to these differences. * Figure 5: A comparison of the relative growth rates for the total set of heterozygous deletion diploids in fission yeast (4,334 genes) and budding yeast (5,921 genes). In fission yeast there are more haploinsufficient genes with a relative growth rate of <0.97 compared to budding yeast (455 versus 356), as shown in the expanded region 0.88–0.97 (Supplementary Table 16). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Dong-Uk Kim, * Jacqueline Hayles, * Dongsup Kim, * Valerie Wood, * Han-Oh Park, * Misun Won & * Hyang-Sook Yoo Affiliations * Integrative Omics Research Centre, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong, Daejeon, Korea. * Dong-Uk Kim, * Misun Won, * Hyang-Sook Yoo, * Miyoung Nam, * Seung-Tae Baek, * Hyemi Lee, * Young Sam Shim, * Lila Kim, * Kyung-Sun Heo, * Eun Joo Noh, * Ah-Reum Lee, * Young-Joo Jang, * Kyung-Sook Chung, * Shin-Jung Choi, * Jo-Young Park, * Youngwoo Park & * Kwang-Lae Hoe * Cancer Research UK, The London Research Institute, London, UK. * Jacqueline Hayles, * Valerie Wood, * Trevor Duhig, * Georgia Palmer, * Linda Jeffery & * Paul Nurse * Department of Bio and Brain Engineering, Korea Advanced Institute of Science & Technology (KAIST), Yuseong, Daejeon, Korea. * Dongsup Kim, * Sangjo Han & * Minho Lee * Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK. * Valerie Wood * Bioneer Corp., Daedeok, Daejeon, Korea. * Han-Oh Park, * Hae-Joon Park & * Eun-Jung Kang * Bioevaluation Centre, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Ochang, Chungcheongbuk-do, Korea. * Hwan Mook Kim, * Song-Kyu Park & * Kwang-Lae Hoe * Department of Bioinformatics & Biotechnology, Korea University, Jochiwon, Chungnam, Korea. * Hyong Bai Kim * School of Biological Sciences, Seoul National University, Seoul, Korea. * Hyun-Sam Kang * Department of Microbiology, Chungnam National University, Yuseong, Daejeon, Korea. * Hee-Moon Park * Division of Life Sciences, Kangwon National University, Chuncheon, Kangwon-do, Korea. * Kyunghoon Kim * Department of Biochemistry, Yonsei University, Seoul, Korea. * Kiwon Song * Department of Food and Nutrition, Chungnam National University, Yuseong, Daejeon, Korea. * Kyung Bin Song * The Rockefeller University, New York, New York, USA. * Paul Nurse Contributions D.-U.K., J.H., H.-O.P., M.W., H.-S.Y., P.N. and K.-L.H. conceived the project; D.-U.K., J.H., D.K., V.W., M.W., T.D., M.N., G.P., S.H., L.J., S.-T.B., H.L., Y.S.S., M.L., L.K., K.-S.H., E.J.N., A.-R.L., Y.-J.J., K.-S.C., S.-J.C., J.-Y.P., Y.P., H.M.K., S.-K.P., H.B.K., H.-S.K., H.-M.P., K.K., K.S. and K.B.S. performed experiments and data analysis; D.K., H.-J.P., E.-J.K. and H.-M.P. performed primer design; D.K. and V.W. performed bioinformatics; D.-U.K., J.H., D.K., V.W., P.N. and K.-L.H. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Kwang-Lae Hoe (kwanghoe@kribb.re.kr) Supplementary information * Abstract * Author information * Supplementary information Excel files * Supplementary Table 1 (2M) The 4,836 deletion set in fission yeast and its genome dataset as a reference (4,914) * Supplementary Table 11 (576K) Spreadsheet of 2,438 'one to one' orthologous pairs in fission yeast and budding yeast (for details, see attached Excel file) * Supplementary Table 15 (1M) Growth fitness data of S. pombe heterozygous deletion mutants in rich YE media * Supplementary Table 16 (264K) List of slow growers from the two yeasts, whose relative fitness is less than 0.97 * Supplementary Table 17 (220K) List of haploinsufficient (lowest 3% ranked by RF) and haploproficient (highest 3% ranked by RF) genes from the two yeasts in rich media * Supplementary Data 1 (3M) All the primer set for the construction of deletion strains * Supplementary Data 4 (2M) Design of Affymertix custom GeneChip Zip files * Supplementary Data 5 (10M) Microarray data set for growth profiling PDF files * Supplementary Text and Figures (5M) Supplementary Tables 2–10,12–14,18 Supplementary Figs. 1–10 and Supplementary Methods * Supplementary Data 2 (19M) Mapping of the deletions * Supplementary Data 3 (232K) Sequence of KanMX4 Additional data - Erratum: The cancer vaccine roller coaster
- Nat Biotech 28(6):624 (2010)
Nature Biotechnology | Erratum Erratum: The cancer vaccine roller coaster * Bruce Goldman Search for this author in: * NPG journals * PubMed * Google Scholar * Laura DeFrancesco Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature BiotechnologyVolume:28,Page:624Year published:(2010)DOI:doi:10.1038/nbt0610-624a Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Biotechnol.27, 129–139 (2009); published online 7 February 2009; corrected after print 7 June 2010 In the version of this article initially published, the Mologen product description in Table 5, page 139, was incomplete and its status incorrectly stated to be compassionate use in India. The product description should have read: Genetically modified allogeneic (human) tumor cells for the expression of IL-7, GM-CSF, CD80 and CD154, in fixed combination with a DNA-based double stem loop immunomodulator (dSLIM). The status should have read: Orphan drug status granted by EMEA in 2006. The error has been corrected in the HTML and PDF versions of the article. Additional data - Erratum: Irish bioethics council axed
- Nat Biotech 28(6):624 (2010)
Nature Biotechnology | Erratum Erratum: Irish bioethics council axed * Cormac Sheridan Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature BiotechnologyVolume:28,Page:624Year published:(2010)DOI:doi:10.1038/nbt0610-624b Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Biotechnol., 112 (2010); published online 5 February 2010; corrected after print 7 June 2010 In the version of this article initially published, a researcher at University College Cork was incorrectly named. His name is Tom (not Barry) Moore. The error has been corrected in the HTML and PDF versions of the article. Additional data - Erratum: Never again
- Nat Biotech 28(6):624 (2010)
Nature Biotechnology | Erratum Erratum: Never again * Chris Scott Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature BiotechnologyVolume:28,Page:624Year published:(2010)DOI:doi:10.1038/nbt0610-624c Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Biotechnol.28, 131 (2010); published online 5 February 2010; corrected after print 7 June 2010 In the version of this article initially published, Art Levinson is incorrectly described as a founder of Genentech, Sandra Horning as senior vice president of global clinical development and Richard Scheller as chief of operations. Their titles should have read: CEO Arthur Levinson moved up to the board of directors.... Sandra Horning...took over as senior vice president, global head, clinical development, hematology/oncology. Executive vice president, research, Richard Scheller.... The errors have been corrected in the HTML and PDF versions of the article. Additional data - Erratum: Resuscitated deCODE refocuses on diagnostics
- Nat Biotech 28(6):624 (2010)
Nature Biotechnology | Erratum Erratum: Resuscitated deCODE refocuses on diagnostics * Mark Ratner Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature BiotechnologyVolume:28,Page:624Year published:(2010)DOI:doi:10.1038/nbt0610-624d Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Biotechnol.28, 192 (2010); published online 8 March 2010; corrected after print 7 June 2010 In the version of this article initially published, it was reported that deCODE had "shuttered its Emerald Biosciences and Emerald Biostructures drug discovery operations"; in fact, the companies were sold to investors. In addition, the correct name of Emerald Biosciences is Emerald BioSystems. The error has been corrected in the HTML and PDF versions of the article. Additional data - Erratum: Biotech in a blink
- Nat Biotech 28(6):624 (2010)
Nature Biotechnology | Erratum Erratum: Biotech in a blink * Ken Garber Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature BiotechnologyVolume:28,Page:624Year published:(2010)DOI:doi:10.1038/nbt0610-624e Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Biotechnol.28, 311–314 (2010); published online 8 April 2010; corrected after print 15 April 2010 In the version of the article originally published, Michael Tolentino was misquoted to the effect that bevasiranib had been shown to persist indefinitely in post-mitotic cells. Tolentino actually stated that the RNA-induced signaling complex persists. The error has been corrected in the HMTL and PDF versions of the article. Additional data - The ABC's of industry: a postdoc program provides a sneak peek into industry careers
- Nat Biotech 28(6):625-626 (2010)
Nature Biotechnology | Careers and Recruitment The ABC's of industry: a postdoc program provides a sneak peek into industry careers * Adnan O Abu-Yousif1 Search for this author in: * NPG journals * PubMed * Google Scholar * Erik C Hett1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ann M Skoczenski1 Search for this author in: * NPG journals * PubMed * Google Scholar * Tayyaba Hasan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature BiotechnologyVolume:28,Pages:625–626Year published:(2010)DOI:doi:10.1038/nbt0610-625 An innovative partnership allows local companies to educate postdocs about careers in industry. 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 * Adnan O. Abu-Yousif, Erik C. Hett, Ann M. Skoczenski and Tayyaba Hasan are at Massachusetts General Hospital, Boston, Massachusetts, USA. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Adnan O Abu-Yousif (aabu-yousif@partners.org) Additional data - People
- Nat Biotech 28(6):628 (2010)
Optimer Pharmaceuticals (San Diego) has announced the appointment of Pedro Lichtinger (right) as president, CEO and member of the board of directors. He joins Optimer with more than 30 years of experience in the pharmaceutical and animal health industries, most recently as president of Pfizer's global primary care business unit, where he oversaw operations in North America, Europe, Korea and Australia.
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