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Showing papers in "Proceedings of the National Academy of Sciences of the United States of America in 2002"


Journal ArticleDOI
TL;DR: This article proposes a method for detecting communities, built around the idea of using centrality indices to find community boundaries, and tests it on computer-generated and real-world graphs whose community structure is already known and finds that the method detects this known structure with high sensitivity and reliability.
Abstract: A number of recent studies have focused on the statistical properties of networked systems such as social networks and the Worldwide Web. Researchers have concentrated particularly on a few properties that seem to be common to many networks: the small-world property, power-law degree distributions, and network transitivity. In this article, we highlight another property that is found in many networks, the property of community structure, in which network nodes are joined together in tightly knit groups, between which there are only looser connections. We propose a method for detecting such communities, built around the idea of using centrality indices to find community boundaries. We test our method on computer-generated and real-world graphs whose community structure is already known and find that the method detects this known structure with high sensitivity and reliability. We also apply the method to two networks whose community structure is not well known—a collaboration network and a food web—and find that it detects significant and informative community divisions in both cases.

14,429 citations


Journal ArticleDOI
TL;DR: Detailed deletion and expression analysis shows that miR15 and miR16 are located within a 30-kb region of loss in CLL, and that both genes are deleted or down-regulated in the majority (≈68%) of CLL cases.
Abstract: Micro-RNAs (miR genes) are a large family of highly conserved noncoding genes thought to be involved in temporal and tissue-specific gene regulation MiRs are transcribed as short hairpin precursors (≈70 nt) and are processed into active 21- to 22-nt RNAs by Dicer, a ribonuclease that recognizes target mRNAs via base-pairing interactions Here we show that miR15 and miR16 are located at chromosome 13q14, a region deleted in more than half of B cell chronic lymphocytic leukemias (B-CLL) Detailed deletion and expression analysis shows that miR15 and miR16 are located within a 30-kb region of loss in CLL, and that both genes are deleted or down-regulated in the majority (≈68%) of CLL cases

5,113 citations


Journal ArticleDOI
TL;DR: A powerful method for exploring the properties of the multidimensional free energy surfaces of complex many-body systems by means of coarse-grained non-Markovian dynamics in the space defined by a few collective coordinates is introduced.
Abstract: We introduce a powerful method for exploring the properties of the multidimensional free energy surfaces (FESs) of complex many-body systems by means of coarse-grained non-Markovian dynamics in the space defined by a few collective coordinates. A characteristic feature of these dynamics is the presence of a history-dependent potential term that, in time, fills the minima in the FES, allowing the efficient exploration and accurate determination of the FES as a function of the collective coordinates. We demonstrate the usefulness of this approach in the case of the dissociation of a NaCl molecule in water and in the study of the conformational changes of a dialanine in solution.

4,587 citations


Journal ArticleDOI
TL;DR: Agent-based modeling is a powerful simulation modeling technique that has seen a number of applications in the last few years, including applications to real-world business problems, and its four areas of application are discussed by using real- world applications.
Abstract: Agent-based modeling is a powerful simulation modeling technique that has seen a number of applications in the last few years, including applications to real-world business problems. After the basic principles of agent-based simulation are briefly introduced, its four areas of application are discussed by using real-world applications: flow simulation, organizational simulation, market simulation, and diffusion simulation. For each category, one or several business applications are described and analyzed.

3,969 citations


Journal ArticleDOI
TL;DR: The method of “nearest shrunken centroids” identifies subsets of genes that best characterize each class, which was highly efficient in finding genes for classifying small round blue cell tumors and leukemias.
Abstract: We have devised an approach to cancer class prediction from gene expression profiling, based on an enhancement of the simple nearest prototype (centroid) classifier. We shrink the prototypes and hence obtain a classifier that is often more accurate than competing methods. Our method of "nearest shrunken centroids" identifies subsets of genes that best characterize each class. The technique is general and can be used in many other classification problems. To demonstrate its effectiveness, we show that the method was highly efficient in finding genes for classifying small round blue cell tumors and leukemias.

2,954 citations


Journal ArticleDOI
TL;DR: The results suggest that the expression of PD-L1 can serve as a potent mechanism for potentially immunogenic tumors to escape from host immune responses and that blockade of interaction between PD-1 andPD-L may provide a promising strategy for specific tumor immunotherapy.
Abstract: PD-1 is a receptor of the Ig superfamily that negatively regulates T cell antigen receptor signaling by interacting with the specific ligands (PD-L) and is suggested to play a role in the maintenance of self-tolerance. In the present study, we examined possible roles of the PD-1/PD-L system in tumor immunity. Transgenic expression of PD-L1, one of the PD-L, in P815 tumor cells rendered them less susceptible to the specific T cell antigen receptor-mediated lysis by cytotoxic T cells in vitro, and markedly enhanced their tumorigenesis and invasiveness in vivo in the syngeneic hosts as compared with the parental tumor cells that lacked endogenous PD-L. Both effects could be reversed by anti-PD-L1 Ab. Survey of murine tumor lines revealed that all of the myeloma cell lines examined naturally expressed PD-L1. Growth of the myeloma cells in normal syngeneic mice was inhibited significantly albeit transiently by the administration of anti-PD-L1 Ab in vivo and was suppressed completely in the syngeneic PD-1-deficient mice. These results suggest that the expression of PD-L1 can serve as a potent mechanism for potentially immunogenic tumors to escape from host immune responses and that blockade of interaction between PD-1 and PD-L may provide a promising strategy for specific tumor immunotherapy.

2,631 citations


Journal ArticleDOI
TL;DR: This work presents the stepwise evolution of DsRed to a dimer and then either to a genetic fusion of two copies of the protein, i.e., a tandem dimer, or to a true monomer designated mRFP1 (monomeric red fluorescent protein).
Abstract: All coelenterate fluorescent proteins cloned to date display some form of quaternary structure, including the weak tendency of Aequorea green fluorescent protein (GFP) to dimerize, the obligate dimerization of Renilla GFP, and the obligate tetramerization of the red fluorescent protein from Discosoma (DsRed). Although the weak dimerization of Aequorea GFP has not impeded its acceptance as an indispensable tool of cell biology, the obligate tetramerization of DsRed has greatly hindered its use as a genetically encoded fusion tag. We present here the stepwise evolution of DsRed to a dimer and then either to a genetic fusion of two copies of the protein, i.e., a tandem dimer, or to a true monomer designated mRFP1 (monomeric red fluorescent protein). Each subunit interface was disrupted by insertion of arginines, which initially crippled the resulting protein, but red fluorescence could be rescued by random and directed mutagenesis totaling 17 substitutions in the dimer and 33 in mRFP1. Fusions of the gap junction protein connexin43 to mRFP1 formed fully functional junctions, whereas analogous fusions to the tetramer and dimer failed. Although mRFP1 has somewhat lower extinction coefficient, quantum yield, and photostability than DsRed, mRFP1 matures >10 times faster, so that it shows similar brightness in living cells. In addition, the excitation and emission peaks of mRFP1, 584 and 607 nm, are ≈25 nm red-shifted from DsRed, which should confer greater tissue penetration and spectral separation from autofluorescence and other fluorescent proteins.

2,451 citations


Journal ArticleDOI
TL;DR: It is shown that heterogeneity plays an ambiguous role in determining a system's stability: increasingly heterogeneous thresholds make the system more vulnerable to global cascades; but anincreasingly heterogeneous degree distribution makes it less vulnerable.
Abstract: The origin of large but rare cascades that are triggered by small initial shocks is a phenomenon that manifests itself as diversely as cultural fads, collective action, the diffusion of norms and innovations, and cascading failures in infrastructure and organizational networks. This paper presents a possible explanation of this phenomenon in terms of a sparse, random network of interacting agents whose decisions are determined by the actions of their neighbors according to a simple threshold rule. Two regimes are identified in which the network is susceptible to very large cascades—herein called global cascades—that occur very rarely. When cascade propagation is limited by the connectivity of the network, a power law distribution of cascade sizes is observed, analogous to the cluster size distribution in standard percolation theory and avalanches in self-organized criticality. But when the network is highly connected, cascade propagation is limited instead by the local stability of the nodes themselves, and the size distribution of cascades is bimodal, implying a more extreme kind of instability that is correspondingly harder to anticipate. In the first regime, where the distribution of network neighbors is highly skewed, it is found that the most connected nodes are far more likely than average nodes to trigger cascades, but not in the second regime. Finally, it is shown that heterogeneity plays an ambiguous role in determining a system's stability: increasingly heterogeneous thresholds make the system more vulnerable to global cascades; but an increasingly heterogeneous degree distribution makes it less vulnerable.

2,450 citations


Journal ArticleDOI
Robert L. Strausberg, Elise A. Feingold1, Lynette H. Grouse1, Jeffery G. Derge2, Richard D. Klausner1, Francis S. Collins1, Lukas Wagner1, Carolyn M. Shenmen1, Gregory D. Schuler1, Stephen F. Altschul1, Barry R. Zeeberg1, Kenneth H. Buetow1, Carl F. Schaefer1, Narayan K. Bhat1, Ralph F. Hopkins1, Heather Jordan1, Troy Moore3, Steve I Max3, Jun Wang3, Florence Hsieh, Luda Diatchenko, Kate Marusina, Andrew A Farmer, Gerald M. Rubin4, Ling Hong4, Mark Stapleton4, M. Bento Soares5, Maria de Fatima Bonaldo5, Thomas L. Casavant5, Todd E. Scheetz5, Michael J. Brownstein1, Ted B. Usdin1, Shiraki Toshiyuki, Piero Carninci, Christa Prange6, Sam S Raha7, Naomi A Loquellano7, Garrick J Peters7, Rick D Abramson7, Sara J Mullahy7, Stephanie Bosak, Paul J. McEwan, Kevin McKernan, Joel A. Malek, Preethi H. Gunaratne8, Stephen Richards8, Kim C. Worley8, Sarah Hale8, Angela M. Garcia8, Stephen W. Hulyk8, Debbie K Villalon8, Donna M. Muzny8, Erica Sodergren8, Xiuhua Lu8, Richard A. Gibbs8, Jessica Fahey9, Erin Helton9, Mark Ketteman9, Anuradha Madan9, Stephanie Rodrigues9, Amy Sanchez9, Michelle Whiting9, Anup Madan9, Alice C. Young1, Yuriy O. Shevchenko1, Gerard G. Bouffard1, Robert W. Blakesley1, Jeffrey W. Touchman1, Eric D. Green1, Mark Dickson10, Alex Rodriguez10, Jane Grimwood10, Jeremy Schmutz10, Richard M. Myers10, Yaron S.N. Butterfield11, Martin Krzywinski11, Ursula Skalska11, Duane E. Smailus11, Angelique Schnerch11, Jacqueline E. Schein11, Steven J.M. Jones11, Marco A. Marra11 
TL;DR: The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene.
Abstract: The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http:mgc.nci.nih.gov).

2,184 citations


Journal ArticleDOI
TL;DR: The principle that c-MYC transcription can be controlled by ligand-mediated G-quadruplex stabilization is established, establishing the principle that the purine-rich strand of the DNA in this region can form two different intramolecular G- quadruplex structures.
Abstract: The nuclease hypersensitivity element III1 upstream of the P1 promoter of c-MYC controls 85–90% of the transcriptional activation of this gene. We have demonstrated that the purine-rich strand of the DNA in this region can form two different intramolecular G-quadruplex structures, only one of which seems to be biologically relevant. This biologically relevant structure is the kinetically favored chair-form G-quadruplex, which is destabilized when mutated with a single G → A transition, resulting in a 3-fold increase in basal transcriptional activity of the c-MYC promoter. The cationic porphyrin TMPyP4, which has been shown to stabilize this G-quadruplex structure, is able to suppress further c-MYC transcriptional activation. These results provide compelling evidence that a specific G-quadruplex structure formed in the c-MYC promoter region functions as a transcriptional repressor element. Furthermore, we establish the principle that c-MYC transcription can be controlled by ligand-mediated G-quadruplex stabilization.

1,969 citations


Journal ArticleDOI
TL;DR: Application of fractal analysis may provide new approaches to assessing cardiac risk and forecasting sudden cardiac death, as well as to monitoring the aging process, and similar approaches show promise in assessing other regulatory systems, such as human gait control in health and disease.
Abstract: According to classical concepts of physiologic control, healthy systems are self-regulated to reduce variability and maintain physiologic constancy. Contrary to the predictions of homeostasis, however, the output of a wide variety of systems, such as the normal human heartbeat, fluctuates in a complex manner, even under resting conditions. Scaling techniques adapted from statistical physics reveal the presence of long-range, power-law correlations, as part of multifractal cascades operating over a wide range of time scales. These scaling properties suggest that the nonlinear regulatory systems are operating far from equilibrium, and that maintaining constancy is not the goal of physiologic control. In contrast, for subjects at high risk of sudden death (including those with heart failure), fractal organization, along with certain nonlinear interactions, breaks down. Application of fractal analysis may provide new approaches to assessing cardiac risk and forecasting sudden cardiac death, as well as to monitoring the aging process. Similar approaches show promise in assessing other regulatory systems, such as human gait control in health and disease. Elucidating the fractal and nonlinear mechanisms involved in physiologic control and complex signaling networks is emerging as a major challenge in the postgenomic era.

Journal ArticleDOI
TL;DR: Comparison of canineCOX-3 activity with murine COX-1 and -2 demonstrates that this enzyme is selectively inhibited by analgesic/antipyretic drugs such as acetaminophen, phenacetin, antipyrine, and dipyrone, and is potently inhibited by some nonsteroidal antiinflammatory drugs.
Abstract: Two cyclooxygenase isozymes, COX-1 and -2, are known to catalyze the rate-limiting step of prostaglandin synthesis and are the targets of nonsteroidal antiinflammatory drugs. Here we describe a third distinct COX isozyme, COX-3, as well as two smaller COX-1-derived proteins (partial COX-1 or PCOX-1 proteins). COX-3 and one of the PCOX-1 proteins (PCOX-1a) are made from the COX-1 gene but retain intron 1 in their mRNAs. PCOX-1 proteins additionally contain an in-frame deletion of exons 5–8 of the COX-1 mRNA. COX-3 and PCOX mRNAs are expressed in canine cerebral cortex and in lesser amounts in other tissues analyzed. In human, COX-3 mRNA is expressed as an ≈5.2-kb transcript and is most abundant in cerebral cortex and heart. Intron 1 is conserved in length and in sequence in mammalian COX-1 genes. This intron contains an ORF that introduces an insertion of 30–34 aa, depending on the mammalian species, into the hydrophobic signal peptide that directs COX-1 into the lumen of the endoplasmic reticulum and nuclear envelope. COX-3 and PCOX-1a are expressed efficiently in insect cells as membrane-bound proteins. The signal peptide is not cleaved from either protein and both proteins are glycosylated. COX-3, but not PCOX-1a, possesses glycosylation-dependent cyclooxygenase activity. Comparison of canine COX-3 activity with murine COX-1 and -2 demonstrates that this enzyme is selectively inhibited by analgesic/antipyretic drugs such as acetaminophen, phenacetin, antipyrine, and dipyrone, and is potently inhibited by some nonsteroidal antiinflammatory drugs. Thus, inhibition of COX-3 could represent a primary central mechanism by which these drugs decrease pain and possibly fever.

Journal ArticleDOI
TL;DR: A structural model for amyloid fibrils formed by the 40-residue β-amyloid peptide associated with Alzheimer's disease (Aβ1–40) is presented, based on a set of experimental constraints from solid state NMR spectroscopy and incorporates the cross-β structural motif established by x-ray fiber diffraction.
Abstract: We present a structural model for amyloid fibrils formed by the 40-residue beta-amyloid peptide associated with Alzheimer's disease (Abeta(1-40)), based on a set of experimental constraints from solid state NMR spectroscopy. The model additionally incorporates the cross-beta structural motif established by x-ray fiber diffraction and satisfies constraints on Abeta(1-40) fibril dimensions and mass-per-length determined from electron microscopy. Approximately the first 10 residues of Abeta(1-40) are structurally disordered in the fibrils. Residues 12-24 and 30-40 adopt beta-strand conformations and form parallel beta-sheets through intermolecular hydrogen bonding. Residues 25-29 contain a bend of the peptide backbone that brings the two beta-sheets in contact through sidechain-sidechain interactions. A single cross-beta unit is then a double-layered beta-sheet structure with a hydrophobic core and one hydrophobic face. The only charged sidechains in the core are those of D23 and K28, which form salt bridges. Fibrils with minimum mass-per-length and diameter consist of two cross-beta units with their hydrophobic faces juxtaposed.

Journal ArticleDOI
TL;DR: This finding suggests that reaction of cysteine thiols is followed by rapid formation of protein disulfide linkages, which are the direct sensors of inducers of the phase 2 system.
Abstract: Coordinate induction of phase 2 proteins and elevation of glutathione protect cells against the toxic and carcinogenic effects of electrophiles and oxidants. All inducers react covalently with thiols at rates that are closely related to their potencies. Inducers disrupt the cytoplasmic complex between the actin-bound protein Keap1 and the transcription factor Nrf2, thereby releasing Nrf2 to migrate to the nucleus where it activates the antioxidant response element (ARE) of phase 2 genes and accelerates their transcription. We cloned, overexpressed, and purified murine Keap1 and demonstrated on native gels the formation of complexes of Keap1 with the Neh2 domain of Nrf2 and their concentration-dependent disruption by inducers such as sulforaphane and bis(2-hydroxybenzylidene)acetone. The kinetics, stoichiometry, and order of reactivities of the most reactive of the 25 cysteine thiol groups of Keap1 have been determined by tritium incorporation from [3H]dexamethasone mesylate (an inducer and irreversible modifier of thiols) and by UV spectroscopy with sulforaphane, 2,2′-dipyridyl disulfide and 4,4′-dipyridyl disulfide (titrants of thiol groups), and two closely related Michael reaction acceptors [bis(2- and 4-hydroxybenzylidene)acetones] that differ 100-fold in inducer potency and the UV spectra of which are bleached by thiol addition. With large excesses of these reagents nearly all thiols of Keap1 react, but sequential reaction with three successive single equivalents (per cysteine residue) of dipyridyl disulfides revealed excellent agreement with pseudo-first order kinetics, rapid successive declines in reaction velocity, and the stoichiometric formation of two equivalents of thiopyridone per reacted cysteine. This finding suggests that reaction of cysteine thiols is followed by rapid formation of protein disulfide linkages. The most reactive residues of Keap1 (C257, C273, C288, and C297) were identified by mapping the dexamethasone-modified cysteines by mass spectrometry of tryptic peptides. These residues are located in the intervening region between BTB and Kelch repeat domains of Keap1 and probably are the direct sensors of inducers of the phase 2 system.

Journal ArticleDOI
TL;DR: This work provides the first direct experimental evidence for dry adhesion of gecko setae by van der Waals forces, and suggests a possible design principle underlying the repeated, convergent evolution of dry adhesive microstructures in gecko, anoles, skinks, and insects.
Abstract: Geckos have evolved one of the most versatile and effective adhesives known. The mechanism of dry adhesion in the millions of setae on the toes of geckos has been the focus of scientific study for over a century. We provide the first direct experimental evidence for dry adhesion of gecko setae by van der Waals forces, and reject the use of mechanisms relying on high surface polarity, including capillary adhesion. The toes of live Tokay geckos were highly hydrophobic, and adhered equally well to strongly hydrophobic and strongly hydrophilic, polarizable surfaces. Adhesion of a single isolated gecko seta was equally effective on the hydrophobic and hydrophilic surfaces of a microelectro-mechanical systems force sensor. A van der Waals mechanism implies that the remarkable adhesive properties of gecko setae are merely a result of the size and shape of the tips, and are not strongly affected by surface chemistry. Theory predicts greater adhesive forces simply from subdividing setae to increase surface density, and suggests a possible design principle underlying the repeated, convergent evolution of dry adhesive microstructures in gecko, anoles, skinks, and insects. Estimates using a standard adhesion model and our measured forces come remarkably close to predicting the tip size of Tokay gecko seta. We verified the dependence on size and not surface type by using physical models of setal tips nanofabricated from two different materials. Both artificial setal tips stuck as predicted and provide a path to manufacturing the first dry, adhesive microstructures.

Journal ArticleDOI
TL;DR: It is shown how the total variation in the level of expression of a given gene can be decomposed into its intrinsic and extrinsic components and theoretically that simultaneous measurement of two identical genes per cell enables discrimination of these two types of noise.
Abstract: Gene expression is a stochastic, or “noisy,” process. This noise comes about in two ways. The inherent stochasticity of biochemical processes such as transcription and translation generates “intrinsic” noise. In addition, fluctuations in the amounts or states of other cellular components lead indirectly to variation in the expression of a particular gene and thus represent “extrinsic” noise. Here, we show how the total variation in the level of expression of a given gene can be decomposed into its intrinsic and extrinsic components. We demonstrate theoretically that simultaneous measurement of two identical genes per cell enables discrimination of these two types of noise. Analytic expressions for intrinsic noise are given for a model that involves all the major steps in transcription and translation. These expressions give the sensitivity to various parameters, quantify the deviation from Poisson statistics, and provide a way of fitting experiment. Transcription dominates the intrinsic noise when the average number of proteins made per mRNA transcript is greater than ≃2. Below this number, translational effects also become important. Gene replication and cell division, included in the model, cause protein numbers to tend to a limit cycle. We calculate a general form for the extrinsic noise and illustrate it with the particular case of a single fluctuating extrinsic variable—a repressor protein, which acts on the gene of interest. All results are confirmed by stochastic simulation using plausible parameters for Escherichia coli.

Journal ArticleDOI
TL;DR: This work has engineer a very potent high-affinity VEGF blocker that has markedly enhanced pharmacokinetic properties and effectively suppresses tumor growth and vascularization in vivo, resulting in stunted and almost completely avascular tumors.
Abstract: Vascular endothelial growth factor (VEGF) plays a critical role during normal embryonic angiogenesis and also in the pathological angiogenesis that occurs in a number of diseases, including cancer. Initial attempts to block VEGF by using a humanized monoclonal antibody are beginning to show promise in human cancer patients, underscoring the importance of optimizing VEGF blockade. Previous studies have found that one of the most effective ways to block the VEGF-signaling pathway is to prevent VEGF from binding to its normal receptors by administering decoy-soluble receptors. The highest-affinity VEGF blocker described to date is a soluble decoy receptor created by fusing the first three Ig domains of VEGF receptor 1 to an Ig constant region; however, this fusion protein has very poor in vivo pharmacokinetic properties. By determining the requirements to maintain high affinity while extending in vivo half life, we were able to engineer a very potent high-affinity VEGF blocker that has markedly enhanced pharmacokinetic properties. This VEGF-Trap effectively suppresses tumor growth and vascularization in vivo, resulting in stunted and almost completely avascular tumors. VEGF-Trap-mediated blockade may be superior to that achieved by other agents, such as monoclonal antibodies targeted against the VEGF receptor.

Journal ArticleDOI
TL;DR: Allogeneic mesenchymal cells offer feasible posttransplantation therapy for osteogenesis imperfecta and likely other disorders originating in mesenchyal precursors, including bone, cartilage, and muscle.
Abstract: Treatment with isolated allogeneic mesenchymal cells has the potential to enhance the therapeutic effects of conventional bone marrow transplantation in patients with genetic disorders affecting mesenchymal tissues, including bone, cartilage, and muscle. To demonstrate the feasibility of mesenchymal cell therapy and to gain insight into the transplant biology of these cells, we used gene-marked, donor marrow-derived mesenchymal cells to treat six children who had undergone standard bone marrow transplantation for severe osteogenesis imperfecta. Each child received two infusions of the allogeneic cells. Five of six patients showed engraftment in one or more sites, including bone, skin, and marrow stroma, and had an acceleration of growth velocity during the first 6 mo postinfusion. This improvement ranged from 60% to 94% (median, 70%) of the predicted median values for age- and sex-matched unaffected children, compared with 0% to 40% (median, 20%) over the 6 mo immediately preceding the infusions. There was no clinically significant toxicity except for an urticarial rash in one patient just after the second infusion. Failure to detect engraftment of cells expressing the neomycin phosphotransferase marker gene suggested the potential for immune attack against therapeutic cells expressing a foreign protein. Thus, allogeneic mesenchymal cells offer feasible posttransplantation therapy for osteogenesis imperfecta and likely other disorders originating in mesenchymal precursors.

Journal ArticleDOI
TL;DR: This work analyzes an international collection of 912 MRSA and methicillin-susceptible S. aureus isolates to establish the likely evolutionary origins of each major MRSA clone, the genotype of the original MRSAclone and its MSSA progenitor, and the extent of acquisition and horizontal movement of the methiillin resistance genes.
Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired infections that are becoming increasingly difficult to combat because of emerging resistance to all current antibiotic classes. The evolutionary origins of MRSA are poorly understood, no rational nomenclature exists, and there is no consensus on the number of major MRSA clones or the relatedness of clones described from different countries. We resolve all of these issues and provide a more thorough and precise analysis of the evolution of MRSA clones than has previously been possible. Using multilocus sequence typing and an algorithm, burst, we analyzed an international collection of 912 MRSA and methicillin-susceptible S. aureus (MSSA) isolates. We identified 11 major MRSA clones within five groups of related genotypes. The putative ancestral genotype of each group and the most parsimonious patterns of descent of isolates from each ancestor were inferred by using burst, which, together with analysis of the methicillin resistance genes, established the likely evolutionary origins of each major MRSA clone, the genotype of the original MRSA clone and its MSSA progenitor, and the extent of acquisition and horizontal movement of the methicillin resistance genes. Major MRSA clones have arisen repeatedly from successful epidemic MSSA strains, and isolates with decreased susceptibility to vancomycin, the antibiotic of last resort, are arising from some of these major MRSA clones, highlighting a depressing progression of increasing drug resistance within a small number of ecologically successful S. aureus genotypes.

Journal ArticleDOI
TL;DR: It was found that diversity was essential for sustaining the service, because of year-to-year variation in community composition, and conservation and restoration of bee habitat are potentially viable economic alternatives for reducing dependence on managed honey bees.
Abstract: Ecosystem services are critical to human survival; in selected cases, maintaining these services provides a powerful argument for conserving biodiversity. Yet, the ecological and economic underpinnings of most services are poorly understood, impeding their conservation and management. For centuries, farmers have imported colonies of European honey bees (Apis mellifera) to fields and orchards for pollination services. These colonies are becoming increasingly scarce, however, because of diseases, pesticides, and other impacts. Native bee communities also provide pollination services, but the amount they provide and how this varies with land management practices are unknown. Here, we document the individual species and aggregate community contributions of native bees to crop pollination, on farms that varied both in their proximity to natural habitat and management type (organic versus conventional). On organic farms near natural habitat, we found that native bee communities could provide full pollination services even for a crop with heavy pollination requirements (e.g., watermelon, Citrullus lanatus), without the intervention of managed honey bees. All other farms, however, experienced greatly reduced diversity and abundance of native bees, resulting in insufficient pollination services from native bees alone. We found that diversity was essential for sustaining the service, because of year-to-year variation in community composition. Continued degradation of the agro-natural landscape will destroy this “free” service, but conservation and restoration of bee habitat are potentially viable economic alternatives for reducing dependence on managed honey bees.

Journal ArticleDOI
TL;DR: Vectors based on AAV7 and AAV8 should be considered for human gene therapy because of low reactivity to antibodies directed to human AAVs and because gene transfer efficiency in muscle was similar to that obtained with the best known serotype, whereas, in liver, gene transfer was substantially higher than previously described.
Abstract: Tissues from rhesus monkeys were screened by PCR for the presence of sequences homologous to known adeno-associated virus (AAV) serotypes 1-6. DNA spanning entire rep-cap ORFs from two novel AAVs, called AAV7 and AAV8, were isolated. Sequence comparisons among these and previously described AAVs revealed the greatest divergence in capsid proteins. AAV7 and AAV8 were not neutralized by heterologous antisera raised to the other serotypes. Neutralizing antibodies to AAV7 and AAV8 were rare in human serum and, when present, were low in activity. Vectors formed with capsids from AAV7 and AAV8 were generated by using rep and inverted terminal repeats (ITRs) from AAV2 and were compared with similarly constructed vectors made from capsids of AAV1, AAV2, and AAV5. Murine models of skeletal muscle and liver-directed gene transfer were used to evaluate relative vector performance. AAV7 vectors demonstrated efficiencies of transgene expression in skeletal muscle equivalent to that observed with AAV1, the most efficient known serotype for this application. In liver, transgene expression was 10- to 100-fold higher with AAV8 than observed with other serotypes. This improved efficiency correlated with increased persistence of vector DNA and higher number of transduced hepatocytes. The efficiency of AAV8 vector for liver-directed gene transfer of factor IX was not impacted by preimmunization with the other AAV serotypes. Vectors based on these novel, nonhuman primate AAVs should be considered for human gene therapy because of low reactivity to antibodies directed to human AAVs and because gene transfer efficiency in muscle was similar to that obtained with the best known serotype, whereas, in liver, gene transfer was substantially higher than previously described.

Journal ArticleDOI
TL;DR: The complete genomic sequences of human chromosomes 21 and 22 are used to examine the properties of CpG islands in different sequence classes by using a search algorithm that is compatible with the recent detection of 5-methylcytosine in Drosophila, and might suggest that S. cerevisiae has, or once had, C pG methylation.
Abstract: CpG islands are useful markers for genes in organisms containing 5-methylcytosine in their genomes. In addition, CpG islands located in the promoter regions of genes can play important roles in gene silencing during processes such as X-chromosome inactivation, imprinting, and silencing of intragenomic parasites. The generally accepted definition of what constitutes a CpG island was proposed in 1987 by Gardiner-Garden and Frommer [Gardiner-Garden, M. & Frommer, M. (1987) J. Mol. Biol. 196, 261–282] as being a 200-bp stretch of DNA with a C+G content of 50% and an observed CpG/expected CpG in excess of 0.6. Any definition of a CpG island is somewhat arbitrary, and this one, which was derived before the sequencing of mammalian genomes, will include many sequences that are not necessarily associated with controlling regions of genes but rather are associated with intragenomic parasites. We have therefore used the complete genomic sequences of human chromosomes 21 and 22 to examine the properties of CpG islands in different sequence classes by using a search algorithm that we have developed. Regions of DNA of greater than 500 bp with a G+C equal to or greater than 55% and observed CpG/expected CpG of 0.65 were more likely to be associated with the 5′ regions of genes and this definition excluded most Alu-repetitive elements. We also used genome sequences to show strong CpG suppression in the human genome and slight suppression in Drosophila melanogaster and Saccharomyces cerevisiae. This finding is compatible with the recent detection of 5-methylcytosine in Drosophila, and might suggest that S. cerevisiae has, or once had, CpG methylation.

Journal ArticleDOI
TL;DR: MDA-based whole genome amplification by multiple displacement amplification is a simple and reliable method that could have significant implications for genetic studies, forensics, diagnostics, and long-term sample storage.
Abstract: Fundamental to most genetic analysis is availability of genomic DNA of adequate quality and quantity. Because DNA yield from human samples is frequently limiting, much effort has been invested in developing methods for whole genome amplification (WGA) by random or degenerate oligonucleotide-primed PCR. However, existing WGA methods like degenerate oligonucleotide-primed PCR suffer from incomplete coverage and inadequate average DNA size. We describe a method, termed multiple displacement amplification (MDA), which provides a highly uniform representation across the genome. Amplification bias among eight chromosomal loci was less than 3-fold in contrast to 4–6 orders of magnitude for PCR-based WGA methods. Average product length was >10 kb. MDA is an isothermal, strand-displacing amplification yielding about 20–30 μg product from as few as 1–10 copies of human genomic DNA. Amplification can be carried out directly from biological samples including crude whole blood and tissue culture cells. MDA-amplified human DNA is useful for several common methods of genetic analysis, including genotyping of single nucleotide polymorphisms, chromosome painting, Southern blotting and restriction fragment length polymorphism analysis, subcloning, and DNA sequencing. MDA-based WGA is a simple and reliable method that could have significant implications for genetic studies, forensics, diagnostics, and long-term sample storage.

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TL;DR: This work generated and analyzed gene expression from 91 human and mouse samples across a diverse array of tissues, organs, and cell lines to reveal insights into molecular and physiological gene function, mechanisms of transcriptional regulation, disease etiology, and comparative genomics.
Abstract: High-throughput gene expression profiling has become an important tool for investigating transcriptional activity in a variety of biological samples. To date, the vast majority of these experiments have focused on specific biological processes and perturbations. Here, we have generated and analyzed gene expression from a set of samples spanning a broad range of biological conditions. Specifically, we profiled gene expression from 91 human and mouse samples across a diverse array of tissues, organs, and cell lines. Because these samples predominantly come from the normal physiological state in the human and mouse, this dataset represents a preliminary, but substantial, description of the normal mammalian transcriptome. We have used this dataset to illustrate methods of mining these data, and to reveal insights into molecular and physiological gene function, mechanisms of transcriptional regulation, disease etiology, and comparative genomics. Finally, to allow the scientific community to use this resource, we have built a free and publicly accessible website (http://expression.gnf.org) that integrates data visualization and curation of current gene annotations.

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TL;DR: The distribution of 20 variable regions resulting from insertion-deletion events in the genomes of the tubercle bacilli has been evaluated and contradict the often-presented hypothesis that M. tuberculosis, the etiological agent of human tuberculosis evolved from M. bovis, the agent of bovine disease.
Abstract: The distribution of 20 variable regions resulting from insertion-deletion events in the genomes of the tubercle bacilli has been evaluated in a total of 100 strains of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium canettii, Mycobacterium microti, and Mycobacterium bovis. This approach showed that the majority of these polymorphisms did not occur independently in the different strains of the M. tuberculosis complex but, rather, resulted from ancient, irreversible genetic events in common progenitor strains. Based on the presence or absence of an M. tuberculosis specific deletion (TbD1), M. tuberculosis strains can be divided into ancestral and “modern” strains, the latter comprising representatives of major epidemics like the Beijing, Haarlem, and African M. tuberculosis clusters. Furthermore, successive loss of DNA, reflected by region of difference 9 and other subsequent deletions, was identified for an evolutionary lineage represented by M. africanum, M. microti, and M. bovis that diverged from the progenitor of the present M. tuberculosis strains before TbD1 occurred. These findings contradict the often-presented hypothesis that M. tuberculosis, the etiological agent of human tuberculosis evolved from M. bovis, the agent of bovine disease. M. canettii and ancestral M. tuberculosis strains lack none of these deleted regions, and, therefore, seem to be direct descendants of tubercle bacilli that existed before the M. africanum→M. bovis lineage separated from the M. tuberculosis lineage. This observation suggests that the common ancestor of the tubercle bacilli resembled M. tuberculosis or M. canettii and could well have been a human pathogen already.

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TL;DR: In this paper, the authors analyzed lines carrying dSpm insertions in the highly expressed AtrbohD and AtrabohF genes, which are required for full ROI production observed during incompatible interactions with the bacterial pathogen Pseudomonas syringae pv.
Abstract: Reactive oxygen intermediates (ROI) are strongly associated with plant defense responses. The origin of these ROI has been controversial. Arabidopsis respiratory burst oxidase homologues (rboh genes) have been proposed to play a role in ROI generation. We analyzed lines carrying dSpm insertions in the highly expressed AtrbohD and AtrbohF genes. Both are required for full ROI production observed during incompatible interactions with the bacterial pathogen Pseudomonas syringae pv. tomato DC3000(avrRpm1) and the oomycete parasite Peronospora parasitica. We also observed reduced cell death, visualized by trypan blue stain and reduced electrolyte leakage, in the Atrboh mutants after DC3000(avrRpm1) inoculation. However, enhanced cell death is observed after infection of mutant lines with P. parasitica. Paradoxically, although atrbohD mutation eliminated the majority of total ROI production, atrbohF mutation exhibited the strongest effect on cell death.

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TL;DR: Qdots are small (<10 nm) inorganic nanocrystals that possess unique luminescent properties; their fluorescence emission is stable and tuned by varying the particle size or composition, and these results encourage the construction of more complex nanostructures with capabilities such as disease sensing and drug delivery.
Abstract: Inorganic nanostructures that interface with biological systems have recently attracted widespread interest in biology and medicine. Nanoparticles are thought to have potential as novel intravascular probes for both diagnostic (e.g., imaging) and therapeutic purposes (e.g., drug delivery). Critical issues for successful nanoparticle delivery include the ability to target specific tissues and cell types and escape from the biological particulate filter known as the reticuloendothelial system. We set out to explore the feasibility of in vivo targeting by using semiconductor quantum dots (qdots). Qdots are small (<10 nm) inorganic nanocrystals that possess unique luminescent properties; their fluorescence emission is stable and tuned by varying the particle size or composition. We show that ZnS-capped CdSe qdots coated with a lung-targeting peptide accumulate in the lungs of mice after i.v. injection, whereas two other peptides specifically direct qdots to blood vessels or lymphatic vessels in tumors. We also show that adding polyethylene glycol to the qdot coating prevents nonselective accumulation of qdots in reticuloendothelial tissues. These results encourage the construction of more complex nanostructures with capabilities such as disease sensing and drug delivery.

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TL;DR: The complete genome sequence of uropathogenic Escherichia coli, strain CFT073 is presented and Comparisons indicate that extraintestinal pathogenic E. coli arose independently from multiple clonal lineages.
Abstract: We present the complete genome sequence of uropathogenic Escherichia coli, strain CFT073. A three-way genome comparison of the CFT073, enterohemorrhagic E. coli EDL933, and laboratory strain MG1655 reveals that, amazingly, only 39.2% of their combined (nonredundant) set of proteins actually are common to all three strains. The pathogen genomes are as different from each other as each pathogen is from the benign strain. The difference in disease potential between O157:H7 and CFT073 is reflected in the absence of genes for type III secretion system or phage- and plasmid-encoded toxins found in some classes of diarrheagenic E. coli. The CFT073 genome is particularly rich in genes that encode potential fimbrial adhesins, autotransporters, iron-sequestration systems, and phase-switch recombinases. Striking differences exist between the large pathogenicity islands of CFT073 and two other well-studied uropathogenic E. coli strains, J96 and 536. Comparisons indicate that extraintestinal pathogenic E. coli arose independently from multiple clonal lineages. The different E. coli pathotypes have maintained a remarkable synteny of common, vertically evolved genes, whereas many islands interrupting this common backbone have been acquired by different horizontal transfer events in each strain.

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TL;DR: Findings implicate the angiogenesis factor VEGF in neurogenesis as well in murine cerebral cortical cultures and in adult rat brain in vivo.
Abstract: Vascular endothelial growth factor (VEGF) is an angiogenic protein with neurotrophic and neuroprotective effects. Because VEGF promotes the proliferation of vascular endothelial cells, we examined the possibility that it also stimulates the proliferation of neuronal precursors in murine cerebral cortical cultures and in adult rat brain in vivo. VEGF (>10 ng/ml) stimulated 5-bromo-2'-deoxyuridine (BrdUrd) incorporation into cells that expressed immature neuronal marker proteins and increased cell number in cultures by 20-30%. Cultured cells labeled by BrdUrd expressed VEGFR2/Flk-1, but not VEGFR1/Flt-1 receptors, and the effect of VEGF was blocked by the VEGFR2/Flk-1 receptor tyrosine kinase inhibitor SU1498. Intracerebroventricular administration of VEGF into rat brain increased BrdUrd labeling of cells in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG), where VEGFR2/Flk-1 was colocalized with the immature neuronal marker, doublecortin (Dcx). The increase in BrdUrd labeling after the administration of VEGF was caused by an increase in cell proliferation, rather than a decrease in cell death, because VEGF did not reduce caspase-3 cleavage in SVZ or SGZ. Cells labeled with BrdUrd after VEGF treatment in vivo include immature and mature neurons, astroglia, and endothelial cells. These findings implicate the angiogenesis factor VEGF in neurogenesis as well.

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TL;DR: Although much of the work in self-assembly has focused on molecular components, many of the most interesting applications of self-assembling processes can be found at larger sizes (nanometers to micrometers).
Abstract: Self-assembly is a process in which components, either separate or linked, spontaneously form ordered aggregates. Self-assembly can occur with components having sizes from the molecular to the macroscopic, provided that appropriate conditions are met. Although much of the work in self-assembly has focused on molecular components, many of the most interesting applications of self-assembling processes can be found at larger sizes (nanometers to micrometers). These larger systems also offer a level of control over the characteristics of the components and over the interactions among them that makes fundamental investigations especially tractable.