Precise control of protein turnover is essential for cellular homeostasis. The ubiquitin-proteasome system is well established as a major regulator of protein degradation, but an understanding of how inherent structural features influence the lifetimes of proteins is lacking. We report that yeast, mouse, and human proteins with terminal or internal intrinsically disordered segments have significantly shorter half-lives than proteins without these features. The lengths of the disordered segments that affect protein half-life are compatible with the structure of the proteasome. Divergence in terminal and internal disordered segments in yeast proteins originating from gene duplication leads to significantly altered half-life. Many paralogs that are affected by such changes participate in signaling, where altered protein half-life will directly impact cellular processes and function. Thus, natural variation in the length and position of disordered segments may affect protein half-life and could serve as an underappreciated source of genetic variation with important phenotypic consequences.  •Genomic principles describing how disordered segments influence protein half-life•Proteins with terminal or internal disordered segments have a short half-life•The relationship is evolutionarily conserved between yeast, mouse, and human•Divergence in disordered segments after gene duplication impacts protein half-life Cellular function requires precise adjustment of protein concentrations. Protein degradation is the endpoint of gene expression and thus a primary determinant of protein abundance. Van der Lee et al. report that the length and number of disordered regions in substrates are two fundamental genetic and evolvable parameters that affect protein half-life and hence protein abundance in cells. They suggest that natural variation in disordered segments of proteins may serve as an underappreciated source of genetic variation with important phenotypic consequences. Protein degradation is the endpoint of gene expression, and correct turnover of proteins is essential for cellular function. Indeed, protein half-life impacts virtually all cellular processes including the cell cycle , DNA repThe proteasome mediates controlled and selective degradation of most proteins in eukaryotic cells, and access to the proteasome is key to controlling the half-life of substrates . SubstraAn important feature implicated in affecting protein half-life is the presence of polypeptide regions that do not adopt a defined 3D structure, typically called intrinsically disordered, or unstructured regions . DisordeIn this work, we develop a theory of how disordered segments influence protein half-life, through a systematic analysis of multiple data sets describing sequence, structure, expression, evolutionary relationships, and experimental half-life measurements from both unicellular and multicellular organisms. We present evidence that proteins with a long terminal or internal disordered segment have a significantly shorter in vivo half-life in yeast on a genomic scale. The same relationship is found in mouse and human. Upon gene duplication, divergence in terminal and internal disordered segments leads to altered half-life of paralogous proteins. Many affected paralogs participate in signaling pathways, where altered half-life will influence signaling outcomes. We suggest specific biochemical mechanisms by which disordered segments may influence degradation rates, how these changes might modulate cellular function and phenotype, and how natural variation in the length and position of intrinsically disordered protein regions may contribute to the evolution of protein half-life.To investigate the relationship between the structural architecture of proteins and their cellular stability, we inferred the disorder status of every residue in the proteomes of yeast, mouse, and human using the DISOPRED2 , IUPRED We first classified yeast proteins into two groups depending on the length of the disordered termini, treating the N and C termini separately: those with short (≤30 residues) and those with long (>30 residues) disordered tails A. The le−6, Mann-Whitney U test, a nonparametric test for assessing whether two samples come from the same underlying distribution [H0]; U test; Proteins with a long disordered N terminus have a significantly shorter half-life compared to proteins with a short disordered N terminus  and found that they display a longer half-life compared to proteins with a long disordered N terminus  on protein turnover. However, the fraction of disordered residues , which is an estimate of the packing, folding, and structural stability of a protein, also correlates with half-life, although previous studies disagree on the extent of the effect . ProteinComparison of the half-life distributions of proteins from different classes with similar overall disorder degrees C revealsU test; Disordered segments could influence half-life either indirectly, by embedding short peptide motifs that serve as destruction signals such as ubiquitination sites or docking sites for ubiquitinating enzymes , or direGiven that the ubiquitin-proteasome system and the architecture of the proteasome itself are conserved from yeast to mammals , we hypoOur observations suggest that protein turnover rates could be tuned by divergence in terminal or internal disordered segments during evolution A. To tes−6, Mann-Whitney U test; We classified the pairs of paralogs into (1) those that during evolution maintained N-terminal disorder of roughly equal length  and (2) pairs with disordered N termini of different length . Paralogous protein pairs that diverged in the length of N-terminal disorder show significantly larger differences in half-lives than pairs that maintained roughly equal N-terminal disorder  Sequence motifs and the presence of regulatory proteins such as ubiquitin ligases contribute to the overall half-life of a protein by determining when substrates will be ubiquitinated and hence targeted to the proteasome. (2) Upon recognition of the ubiquitinated substrate by the proteasome, the presence of disordered segments of sufficient size either at the terminus or internally may facilitate efficient degradation initiation, thereby leading to lower half-life. (3) The overall degree of disorder may contribute to a general trend in lowering half-life by increasing the processivity of degradation upon engagement (after recognition and initiation) by the proteasome. Thus, our observations suggest that disordered segments influence protein half-life as an underlying factor that can be modulated by other cellular mechanisms, sequence determinants and the structural stability of the substrate.MKK1, MKK2, HOG1, and STE7), serine/threonine kinases , and cyclin-dependent kinases (PHO85 and CAK1). Paralogs that have diverged in terminal or internal disordered segments are generally enriched in nucleotide binding, kinase regulatory activity, and phosphoproteins. Alterations in the degradation rate of kinases, for instance, can have significant implications for the dynamics of signaling networks  molecules . In lineWe observed that divergence in disordered regions might influence protein half-life among paralogs. An outstanding question is whether such changes in half-life through divergence of disordered segments are under selective pressure. Natural variation leading to alteration of disordered regions may provide a simple means for regulatory subfunctionalization of paralogous proteins upon gene duplication. It also suggests a mechanism for divergence of half-life among orthologous proteins between species. Thus, while it is clear that the emergence of destruction signals such as ubiquitination sites and dedicated ubiquitin ligases affect targeting of a protein for degradation, variation in disordered segments may provide a simple evolutionary mechanism for fine-tuning protein turnover rates.Several genetic and molecular mechanisms may generate diversity in terminal or internal disordered segments. These include repeat expansion, alternative splicing, and alternative transcription start sites, all of which can influence the length of terminal and/or internal disorder of protein products, thereby potentially influencing the half-life. This idea is supported by the observation that protein disorder is common in insertions and deletions . FurtherSaccharomyces cerevisiae), mouse, and human. Intrinsic disorder was predicted for all reviewed protein sequences of these organisms  using three complementary methods: DISOPRED2, IUPRED long, and PONDR VLS1. The presence and length of N-terminal, C-terminal, and internal disordered segments were then calculated using different algorithms and integrated with the half-life data. Proteomes were classified into groups according to the length of disordered segments: (1) proteins with short and long disordered termini  and (2) proteins with and without internal disordered segments (at least 40 disordered residues). The overall degree of disorder of a protein was calculated as the fraction of disordered residues (number of disordered residues divided by sequence length). The distributions of half-life values and protein disorder were analyzed using appropriate statistical tests.Protein half-life data and other data A were coSee the Yeast paralog pairs were obtained from an all-against-all sequence comparison using BLASTClust . More diSee the R.v.d.L. and M.M.B. designed the project and analyzed and interpreted the results. R.v.d.L. performed the majority of the calculations. B.L. performed additional calculations, and K.K., J.G., N.S.d.G., and M.F. carried out specific analyses. B.L., K.K., M.A.H., M.F., and A.M. assisted in interpretation. R.v.d.L. and M.M.B. wrote the manuscript with help from B.L., M.A.H., M.F., and A.M.


Plant molecular farming (PMF), defined as the practice of using plants to produce human therapeutic proteins, has received worldwide interest. PMF has grown and advanced considerably over the past two decades. A number of therapeutic proteins have been produced in plants, some of which have been through pre-clinical or clinical trials and are close to commercialization. Plants have the potential to mass-produce pharmaceutical products with less cost than traditional methods. Tobacco-derived antibodies have been tested and used to combat the Ebola outbreak in Africa. Genetically engineered immunoadhesin (DPP4-Fc) produced in green plants has been shown to be able to bind to MERS-CoV (Middle East Respiratory Syndrome), preventing the virus from infecting lung cells. Biosafety concerns (such as pollen contamination and immunogenicity of plant-specific glycans) and costly downstream extraction and purification requirements, however, have hampered PMF production from moving from the laboratory to industrial application. In this review, the challenges and opportunities of PMF are discussed. Topics addressed include; transformation and expression systems, plant bioreactors, safety concerns, and various opportunities to produce topical applications and health supplements. The production of plant-derived pharmaceuticals has attracted great interest. Mapp Biopharmaceutical Inc., a company located in San Diego, CA, USA has produced a drug in tobacco leaves called ZMapp, which has been used to combat the 2014 Ebola virus outbreak in Africa . Currentet al. [The concept of using plants to produce recombinant pharmaceutical proteins, referred to as plant molecular farming (PMF) or pharming (PMP), is not new. Human growth hormone, initially produced in tobacco and sunflower in 1986, was the first-plant-derived recombinant therapeutic protein . Mason eet al. . Severalet al. ,8,9,10, et al. ,12,13, pet al. ,15,16, hIn May 2012, the first PMF-derived enzyme, ELELYSO™  , was approved for human use by the FDA . ELELYSOIdentifying potential genes suitable for PMF and general approaches is becoming more simple and straight forward. Facilitated by the rapid progress in genomics, proteomics, and bioinformatics, a greater number of useful genes are being identified and characterized. Additionally, relatively routine molecular methods have become available for placing the genes of interest into plant expression vectors and transforming them into plants see .An example of an early proof of concept for PMF is the production of plant-derived edible human vaccines, using leafy plants or fruits ,33,34,35E. coli, yeast, or mammalian cell expression systems . Levels of expression in subsequent generations may also vary. Given this scenario, it is very difficult to accurately quantify the appropriate dosage of edible vaccines for children and adult patients. Edible vaccines can also trigger immune tolerance after oral administration. Lastly, most of the ingested protein will be degraded by digestive processes. Collectively, these disadvantages greatly restrict the clinical use of edible vaccines [Current methods in plant biotechnology cannot precisely control the expression level of transgenes in plants in a consistent manner and not every plant species is readily transformed. This means that the amount of pharmaceutical produced may vary in each plant species, or even in different plant parts  launched the large-scale production of transgenic maize that produces trypsin. However, the USDA discovered that plant remnants from a previous ProdiGene trial had contaminated a nearby conventional field. ProdiGene was fined $250,000 and charged $3 million to cover the cleanup operation of mishandling the field test. The punitive action forced ProdiGene into bankruptcy. This event represented a significant setback to the commercial use of PMF . In ordeStreptococcal colonization for at least four months in humans [et al. (2014) [Salvia miltiorrhiza. The product combined the medicinal function of both FGF-1 and bioactive compounds within the medicinal plant. Topical application of extracts obtained from the transgenic medicinal plant significantly stimulated fibroblast cells, promoted blood vessel formation, and accelerated the healing process of burn wounds in mice. This is an example of how PMF can be used to combine the therapeutic function of a recombinant protein and the inherent properties of a medicinal plant. Topical application of a plant extract would significantly reduce the cost of purification and downstream processing. In general, topical application is safer than oral consumption or injection, which would help to address concerns about public safety.Some reports have indicated that subcutaneous injections of plant-derived proteins could induce an immunogenic response to plant-specific glycans ,51,52,53n humans . Topicaln humans . Tan et . (2014)  expresseet al. (2014) [A primary objective of PMF is to reduce the cost of producing novel therapeutic proteins. Using PMF to create a vegetable, seed or fruit health supplement could be a practical alternative to using PMF to develop a processed pharmaceutical drug. Guan . (2014) ,58 expre. (2014) .It often takes six months to a year or more to produce transgenic plants. Several generations are required to generate plants that are homozygous for the transgene. Most transformation technologies also result in the gene being inserted randomly into the plant genome. The factor of random insertion, along with the need to identify regulatory elements (promoters) to drive a high-level of foreign gene expression, often results in a low yield of the recombinant protein even in plants that have been stably transformed. The time needed to utilize a standard PMF approach is unsuitable for addressing sudden viral epidemics, such as an outbreak of Ebola, severe acute respiratory syndrome (SARS), or MERS-CoV. Transient expression systems can be used as an alternative approach to produce recombinant protein within three to five days ,61,62,63et al. (2010) [Agrobacterium infiltration of leaves for transient expression is very lab intensive and is the primary reason that sufficient supplies of the Ebola vaccine are not available. In September 2014, the U.S Department of Health and Human Service (HHS) provided a grant of $42.2 million to Mapp Biopharmaceutical Inc. for developing a method for large-scale production of ZMapp. With further optimization of the transient expression system, large-scale production in a short time period (one week) may become feasible [Various viral vectors have been developed for small- or medium-scale PMF production. For example, Mason . (2010)  develope. (2010) . Agrobacfeasible .et al. [Plant-cell-culture based bioreactors currently show more promise than traditional PMF using whole plants to produce pharmaceuticals ,68,69. Set al. .etc. have all been produced in moss cultures. Cultures of the moss, Physcomitrella patens (P. patens), are the most commonly used plant material within bioreactors to enable protein production. A German biopharmaceutical company, Greenovation Biotech GmbH, has developed a P. patens-based platform (Bryo-Technology) for large-scale, high quality, recombinant protein production. Examples include Moss-GAA for Pompe Disease, Moss-GBA for Gaucher’s Disease, and Moss-AGAL for Fabry Disease. Moss-AGAL has completed preclinical trials and is entering Phase I testing  in Arabidopsis resulted in high yield of recombinant protein  [Seed-based systems for PMF have been developed in various plant species, including bidopsis ,92, tobabidopsis , rice [9bidopsis , and corbidopsis . Recombiprotein) . The abuprotein) . The levprotein) .Different subcellular compartments have been targeted for recombinant protein in order to increase protein stability in seeds. The ER (endoplasmic reticulum) compartment in plant cells has been demonstrated to contain few proteases and therefore represents a relatively protective environment. Retention of proteins in the ER therefore increases protein stability and yield due to the lack of protein degradation. KDEL, an ER signal peptide, was used to target the deposition of a recombinant protein to the ER ,97. RecoTargeting of PMF-based products to seeds allows for long-term protein stability, as well as easy harvesting, storage, and transportation. Relative to leaves, seeds contain fewer native proteins and less phenolic compounds and secondary metabolites. Seeds are easy to surface wash and sterilize which also facilitates commercial production. Collectively, the advantages of producing recombinant proteins in seeds make this platform a cost-effective platform for PMF-based products ,98.N-glycan reaches the Golgi apparatus. This pathway produces β--linked galactose and sialic acid in animals and N-glycan  xylose and core α-) fucose moieties in plant. Glycosylation of native plant proteins is essential for their proper function during plant growth and development [Greater than 50% of human proteins are glycosylated. One third of all approved pharmaceuticals are glycoproteins . Plants elopment ,100,101.N-glycan [Plant-specific, hyperglycosylated proteins, however, may be immunogenic in humans. Immunization of mice and rats with horseradish peroxidase elicited the production of antibodies (Abs) specific for plant glycans . ImmunizN-glycan . Even thN-glycan ,102.N-Glycans are produced [et al. (2004) [Arabidopsis, resulting in the absence of plant-specific glycans, in order to produce recombinant protein with humanized glycan structures. A recombinant human antibody fragment and an active enzyme were successfully produced with a controlled glycosylation pattern using this triple knockout Arabidopsis mutant [Lemna [Researchers have addressed concerns about plant-specific glycosylation by altering the pathway that plant cells use to process the recombinant protein. Recombinant proteins have been targeted to the endoplasmic reticulum (ER) where non-immunogenic, high-Man-type produced . Other sproduced  or by siproduced ,105,106.. (2004)  produceds mutant . RNAi lit [Lemna , alfalfaTM) to treat the rare genetic disorder, Gaucher’s disease. The enzyme is targeted to the vacuole of carrot cells. Unlike the equivalent product produced in CHO cells, which express a recombinant protein with a terminal sialic residue that needs to be removed by an exoglycosidase to expose the terminal mannose moiety, the recombinant enzyme produced in a plant-based system already exhibited terminal mannose residues capable of specifically binding to the receptor on a microphage. The plant derived recombinant protein does not need an enzyme to expose mannose residues, which significantly lowers the cost and complexity of downstream processing; thus reducing the costs of production and therapy. Another example is Lemna (duckweed)-derived mAbs . RNAi technology was used to reduce the level of enzymes involved with plant core β- xylose and core α- fucose synthesis The recombinant mAbs produced in the duckweed system contains a single major N-glycan that has a 20-fold better antibody-dependent cell-mediated cytotoxicity and a 10-fold higher cell receptor binding activity than mAbs produced in CHO cells [Efforts to optimize the plant glycosylation pathway have improved the therapeutic safety of PMF-derived recombinant proteins and reduced concerns about plant-specific glycan immunogenicity . ProtaliHO cells .etc. [Nicotiana benthamianai) biomass per week. A transient expression approach is used to produce “biobetter” monoclonal antibodies (mAb) and anti-viral mAb. The downstream processes in this facility include an automated system for the production of various buffers used for separation, high-capacity chromatography, and formulation (cryostorage). Yields of the recombinant protein are in the range of 62%–68% of total protein and the purity of the final product is 95%–98% [Extraction and purification of PMF-derived pharmaceutical proteins can be complex and costly from an industrial perspective. It has been estimated that purification and downstream processing represents 80% to 90% of the cost of producing PMF-derived pharmaceuticals . For eacetc. . Plant setc. . The oveetc. . In 2015etc.  built a  95%–98% . The cos 95%–98% .Plants have the potential to rapidly produce recombinant proteins on a large scale at a relatively low cost compared to other production systems. PMF-based production of pharmaceuticals, topical compounds, and nutritional supplements is feasible, however, concerns about biosafety, human health , and other factors need to be adequately addressed. Downstream processing and purification of PMF products is currently tedious and costly. Systems need to be developed that simplify the purification process in order to make the production of industrial quantities of PMF-based products feasible and cost effective. The right candidate genes, a strong commercial need, and a good production system will build a bridge between basic research on PMF and its commercial application.


Summary: Complex formation and conformational transitions of biological macromolecules in solution can be effectively studied using the information about overall shape and size provided by small angle X-ray scattering (SAXS). Hybrid modeling is often applied to integrate high-resolution models into SAXS data analysis. To facilitate this task, we present SASpy, a PyMOL plugin that provides an easy-to-use graphical interface for SAXS-based hybrid modeling. Through a few mouse clicks in SASpy, low-resolution models can be superimposed to high-resolution structures, theoretical scattering profiles and fits can be calculated and displayed on-the-fly. Mouse-based manual rearrangements of complexes are conveniently applied to rapidly check and interactively refine tentative models. Interfaces to automated rigid-body and flexible refinement of macromolecular models against the experimental SAXS data are provided.Availability and implementation: SASpy is available as open source at: github.com/emblsaxs/saspy/. Working installations of both PyMOL (www.pymol.org) and ATSAS (www.embl-hamburg.de/biosaxs/download.html) are required.Contact:apanjkovich@embl-hamburg.de or svergun@embl-hamburg.de Given the atomic or predicted models of the entire particle or of its domains/subunits, tools are available to compute theoretical scattering profiles, fits and also to perform hybrid modeling and refinement  can be used to obtain size, shape and oligomerization information of biological macromolecules in solution, and the method has become an important part of the structural biology toolbox . The intfinement . Most ofAt present, there is a point-and-click interface to evaluate theoretical SAXS profiles in the Chimera visualization software  and the SAXS profile of the assembly to launch the program SASREF . Using Swww.pymol.org, these are freely available for educational purposes. A local installation of ATSAS version 2.7 or higher , is easy to install as a PyMOL plugin. SASpy requires a working installation of PyMOL and the source code can be downloaded and compiled from sourceforge.net/projects/pymol/. Pre-compiled binaries can be obtained from Schrödinger at


Epigenome-wide association scans (EWAS) are an increasingly powerful and widely-used approach to assess the role of epigenetic variation in human complex traits. However, this rapidly emerging field lacks dedicated visualisation tools that can display features specific to epigenetic datasets.http://epigen.kcl.ac.uk/cometand as an R Bioconductor package. Source code, examples, and full documentation are also available from GitHub.We developed coMET, an R package and online tool for visualisation of EWAS results in a genomic region of interest. coMET generates a regional plot of epigenetic-phenotype association results and the estimated DNA methylation correlation between CpG sites (co-methylation), with further options to visualise genomic annotations based on ENCODE data, gene tracks, reference CpG-sites, and user-defined features. The tool can be used to display phenotype association signals and correlation patterns of microarray or sequencing-based DNA methylation data, such as Illumina Infinium 450k, WGBS, or MeDIP-seq, as well as other types of genomic data, such as gene expression profiles. The software is available as a user-friendly online tool from Our new software allows visualisation of EWAS results with functional genomic annotations and with estimation of co-methylation patterns. coMET is available to a wide audience as an online tool and R package, and can be a valuable resource to interpret results in the fast growing field of epigenetics. The software is designed for epigenetic data, but can also be applied to genomic and functional genomic datasets in any species. Epigenome-wide association studies (EWAS) systematically test for association between DNA methylation variation and human complex traits ,2. RecenEWAS findings can provide mechanistic insights into disease susceptibility or progression, and should be explored in functional genomic context. A comparison across multiple layers of epigenetic marks and chromatin domains can help define the functional context of a genomic region. Several R packages, such as Gviz , trackViWe have developed coMET, an R package and web-based tool to generate regional plots of EWAS data and results. coMET can be applied to visualise regional EWAS results from analyses of both single-CpG and region-based datapoints, for example using microarray-based technologies (such as Illumina 450k) or sequencing-based methods (such as whole genome bisulfite sequencing (WGBS) or DNA methylation capture by immuno-precipitation followed by sequencing (MeDIP-seq)), and to compute and visualise also the correlations between the CpG-sites or regions. The web-service  allows ucoMET is implemented in R to produce a multi-panel plot to visualise EWAS results, genomic annotations, and to estimate and plot DNA co-methylation patterns. The structure of the plots builds on snp.plotter , with exThe coMET R package is available for download from Bioconductor  or onlinThe coMET website  allows uData input for coMET includes a data file or matrix describing EWAS results at the DNA methylation CpG-sites or genomic regions to visualise ("mydata.file" and "mydata.type"), a data file or matrix describing the co-methylation dataset ("cormatrix.file" and "cormatrix.type"), and a configuration file containing the plot parameters ("config.file"). Currently, coMET can visualise the correlation of a maximum of 120 pre-defined features on the plot, due to limitations on the size of the plot. Optional input files can also be uploaded to include association P-values from a larger genomic region of interest in the upper plot or to include them as user-defined annotation tracks in the middle panel, but the lower panel is limited to visualizing only up to 120 CpG-sites or regions which can represent a subset of the larger genomic region in the top panel if required  includes six optional annotation tracks: genes or transcripts (Ensembl), CpG islands (UCSC), Broad ChromHMM domains (UCSC), DNaseI clusters (UCSC), Ensembl regulation tracks, and SNPs (UCSC). These tracks are obtained directly from the revelant online server or data repository (Ensembl BioMart or UCSC tracks) at the time of analysis. This allows for visualisation of up to date functional annotation data, but the analysis requires an active internet connection. In addition to the optional pre-defined tracks, user-defined annotation and data tracks can also be included in a format accepted by Gviz. Altogether, up to 6 annotation tracks can be viewed in the basic version of the package. The generic version of coMET (‘comet’) can visualise lists of customized annotation and data tracks using Gviz [The middle panel includes optional genomic annotation tracks, for example, functional annotations from Ensembl, ENCODE and UCSC for any species and versions available such as hg38, hg19 or GRCm38. By default, the pre-formatted version of coMET  for selected CpG-sites or regions, the selected correlation method , and the selected alpha level for the confidence interval . A user-provided correlation matrix can also be used. The colour scheme of the heatmap represents the correlation scale  to 1 (red) in Figure GATA4 gene, which is an a-DMR in whole blood, muscle, kidney, and brain samples [GATA4 gene on chromosome 8. The results are shown with respect to reference CpG site cg25216696, which is the most associated a-DMP in our data and in previously published whole blood datasets. These findings show for the first time that the GATA4 a-DMR is present in adipose tissue.To show the functionality of coMET, we explored previously published Illumina 450k DNA methylation profiles from adipose tissue in 648 individuals with available age at biopsy . We firs samples ,30. FiguRecently, Richmond et al.  also usecoMET is a user-friendly R-package and online tool that allows for quick and flexible visualisation of EWAS results, co-methylation patterns, and functional annotation. The software is designed for epigenetic data, but can also be applied to genomic and functional genomic datasets in any species.Project name: coMET Project home page: http://epigen.kcl.ac.uk/cometOperating system(s): Platform independent Programming language: R 3.1.1 or higher Other requirements: Bioconductor 3.1 or higher, Shiny License: GNU GPL 2 or higher Any restrictions to use by non-academics: none


Conservation of gene and protein sequence, and, therefore, conservation of the resulting molecular interactions that mediate biological processes, is foundational to our understanding of biology. This conservation allows discovery in one organism, such as worms or mice, to inform our understanding of the biology in another organism, such as humans. However, there is emerging recognition that many biological processes involve important non-conserved elements, and that de novo gene birth provides an important mechanism for functional evolution –3. UnderPristionchus pacificus identified strain-specific phenotypic differences in dauer formation, and argued that genetically distinct populations exhibit greater sensitivity to pheromone from other populations . The gePLOS Genetics [P. pacificus that exhibited marked differences in response to pheromone signaling: the RS2333/California strain, with low dauer formation in response to pheromone, and the RS5134/Ohio strain, with a high response. They then generated 911 recombinant inbred lines (RIL) between the strains, and used Quantitative-Trait-Loci (QTL) mapping to identify a novel gene with no apparent orthologs outside of Pristionchus, dauerless (dau-1), that had undergone a duplication event in the RS2333/California strain. This suggested that dau-1 functions as a repressor of dauer formation in a dose-dependent manner. To test this hypothesis, the authors created transgenic lines with multiple copies of dau-1, and created deletion mutants using CRISPR/Cas9 technology. Their data support the hypothesis as P. pacificus animals with more than two gene copies show no dauer formation in response to pheromone, whereas the deletion of one or both gene copies in RS2333 results in significantly higher dauer formation. The authors also show that this function is mediated by the CAN neurons. dau-1 is expressed in these cells, and ablation of these neurons results in increased dauer formation similar to that seen in dau-1 mutants. Finally, epistasis experiments show that dau-1 acts either downstream or in parallel to steroid hormone signaling, but is dependent on the nuclear hormone receptor gene daf-12  genes [Important questions remain. In particular, previous work showed that different  network . Whetherd) genes –17. Thes


We find that the topologies of real world networks, such as those formed within human societies, by the Internet, or among cellular proteins, are dominated by the mode of the interactions considered among the individuals. Specifically, a major dichotomy in previously studied networks arises from modeling networks in terms of pairwise versus group tasks. The former often intrinsically give rise to scale-free, disassortative, hierarchical networks, whereas the latter often give rise to single- or broad-scale, assortative, nonhierarchical networks. These dependencies explain contrasting observations among previous topological analyses of real world complex systems. We also observe this trend in systems with natural hierarchies, in which alternate representations of the same networks, but which capture different levels of the hierarchy, manifest these signature topological differences. For example, in both the Internet and cellular proteomes, networks of lower-level system components  are assortative and nonhierarchical, whereas networks of upper-level system components  are disassortative and hierarchical. Our results demonstrate that network topologies of complex systems must be interpreted in light of their hierarchical natures and interaction types. Networks occur in many contexts in the real world, and the topologies of these real-world networks have potentially large practical impact in areas of biological, social, and technical importance. Topological implications of human social networks, for example, influence public policies (such as for the development of effective vaccination schemes ) and busAs a consequence, network topologies have been extensively characterized with respect to their global topological properties, such as node degree distributions , node hiGlobal topological analyses of networks were performed as previously described for node degree distribution , assortaFig. 1 by introducing toy examples of two types of human social interactions similarly composed of 11 people (nodes) organized into three groups (indicated by node colors). Interpersonal relationships (edges) might be based on direct personal contact—the contact-centric model —such as for the cases of online dating [task-centric model —such as for sharing membership on company boards [Fig. 1C). In contrast, a task-centric network is characterized by group activities in which the pairwise interaction reflects the tendency for individuals to participate in the same groups. Note that individuals may participate in the same task but never actually directly contact each other (represented as dotted lines)—e.g., many coauthors for scientific papers do not have a personal relationship . The networks’ topologies can be characterized by various global topological properties [We illustrate this key distinction among the two network classes in e dating  or sexuae dating , or altey boards  or co-auy boards . In the operties . Here, wPc(k) = -γk, where Pc(k) represents the cumulative probability of having nodes with >k degree. The contact-centric network is expected to be more scale-free in nature, because there is no constraint limiting the number of links for individual nodes by this modeling perspective, permitting few nodes with many partners, while most nodes have only few partners . In contrast, the task-centric network is expected to be of single- or broad-scale [Fig. 1F). Single-scale networks are characterized by a fast-decaying tail of degree distribution, such as exponential decay. Broad-scale networks are often characterized by a power-law regime followed by exponential trimming of degree, represented by the function Pc(k) = -γexp(-k/k0k), where k0 is the cutoff degree for exponential decay. In general, networks following exponential decay or truncated power-law function exhibit an increased proportion of nodes with medium-connectivity, probably due to the constraint on the number of links by sizes of task-groups of the networks, resulting in reduced numbers of both hubs and nodes with low-connectivity. Because of the connections among entities performing the same tasks, the task-centric network shows a degree preference corresponding to the preferred sizes of the groupings. Thus, these two network classes intrinsically give rise to distinct node degree distributions.An examination of the topologies of these toy networks reveals striking differences. In general, the distribution of node degree of scale-free networks approximately follows a power-law , Pc(k) =ad-scale   of the degrees at either ends of an edge [r≤1 and −1≤r<0 indicate assortative and disassortative networks respectively, or by the statistical significance of the enrichment in connections between various ranges of node degrees as compared to permuted networks [r = −0.66, Fig. 1G), while the task-centric network example is assortative .We see an equally strong dichotomy for a second well-characterized topological property that of the correlation in degree between connected nodes. A given network is termed assortative if its hub-hub connections are enriched; if depleted, the network is termed disassortative . The ass an edge , where 0networks , and canC(k)[k increases in hierarchically organized networks. It is noticeable that this definition of hierarchy assumes that real world networks are in general scale-free and modular. Thus, disassortative networks are also expected to be hierarchical networks . There are other hierarchy models which are independent from network modularity [C(k) decreasing with k has been widely used for previous network topological studies we discussed in this paper. Many real world networks exhibit this signature of hierarchical organization [Fig. 1I. Nonhierarchical networks, however, exhibit roughly equal clustering coefficients, regardless of node degree. This trend suggests enriched connections between components with similar numbers of network neighbors, and the task-centric network example is accordingly nonhierarchical .The third major topological property that we consider is the hierarchical organization . In a hiC(k) of nodesdularity ,21. Howenization , as doesS1 Table).Thus, these two major classes of networks, characterized by the nature of the relationships among the interacting entities, show distinct topological properties: a contact-centric network is a scale-free, hierarchical, disassortative network; a task-centric one is a single- or broad-scale, nonhierarchical, assortative network. The analyses of these toy examples suggest that the contrasting topological properties of many real world networks might also stem from this intrinsic dichotomy of network type. We thus next examined real world networks to test this hypothesis (listed in ). The node degree distribution of the dating network showed more scale-free character, while that of the board of directors network showed single-scale . We observed contrasting assortativity between the two networks: the dating network is disassortative (r = −0.05), but the board of directors network is assortative (r = 0.27) . Similarly, we found a clearly hierarchical organization for the dating network but a nonhierarchical organization for the director board network . Thus, as for the toy network examples in Fig. 1, real world human social networks also show contrasting topologies according to the distinction between intrinsically pairwise vs. group activities. In the online dating network, most people online date only a few partners, while a few individuals make large numbers of online dating contacts. In contrast, membership on boards of directors carries significant obligations. Thus, directors typically participate in only a limited number of boards, and boards are limited in size; as a consequence, clustering coefficients are often similar between members belonging to large boards and those who belong to small ones.We first compared the three global topological properties for two human social networks, analyzing a contact-centric online dating network  and a tavia a network of routers, each implementing routing protocols. Internet scientists have considered two major networks, those composed of routers and those of domains (or autonomous systems) . A domain is an entity containing multiple routers under control of a network operator(s)  using a common routing policy to the Internet. Thus, each domain is considered to be an operational unit component of the Internet. For each domain, within-domain routing protocols and between-domain protocols such as the Border Gateway Protocol (BGP) can be implemented within one or more separate routers. Therefore, the Internet, when considered at the domain-level, maps only peer-to-peer connections between BGP routers, while the router-level maps all router connections including both within-domain and between-domain connections.The Internet represents another widely studied real world complex system comprising computers and other devices with IP addresses that communicate r = −0.17), and hierarchical, but the task-centric router network is broad-scale, relatively assortative (r = −0.01), and nonhierarchical . Disassortativity of a domain-level Internet has been also observed in a previous study [Fig. 2F, highest degree nodes). However, this pattern only affects a small number of routers at the highest level domain, and the overall two levels of organization share topological characteristics consistent with the trends expected for task-centric models. This trend arises because the Internet has top-level domains for mediating communication between lower-level domains. Between-domain routers of the top-level domains generally have connections to a large number of lower-level domains, resulting in much lower clustering coefficients for these few hub routers than for the rest of routers in the Internet. Thus, the Internet, considered at the level of organization for the entire router network, is nonhierarchical.Thus, the domain-level Internet model can be considered to be a contact-centric network, while the router-level Internet model resembles the task-centric model. Accordingly, analysis of published router network and domain network  revealedus study . Routers). We analyzed a high-confidence genome-wide Y2H map of yeast proteins, CCSB-YI1 [r = −0.12), and hierarchical [r = 0.45), and nonhierarchical . Thus, in protein networks, as in social and technology networks, the contact/task centric models explain the dominant topological properties.Similarly, biologists have measured interactions among cellular proteins, describing complex biological networks. These networks naturally fall into contact or task centric models. For example, measurements employing the yeast two-hybrid (Y2H) methodology map the direct physical pairwise contacts between proteins. In contrast, proteins participate in groups—in pathways and complexes—in order to fulfill their functional roles within cells, and physical interactions are but one indication of functional association. We can accordingly model PPI networks considering either direct physical contacts (contact-centric) or functional associations (task-centric) between proteins . We also examined the distribution of shortest path lengths of the real world networks, and observed significantly higher ranges for the shortest path lengths and network diameters of task-centric real-world networks . Taken together, we conclude that the task-centric modeling perspective generated more community structures, which increased frequencies of 5-node graphlets and shortest path lengths of the networks.We also observed a dichotomy among the real world networks for additional topological properties. One useful characteristic of network topology is the frequency distribution of the complete set of graph topologies with three, four, and five nodes, dubbed 3,4,5-node graphlets. The graphlet frequency distributions (GFD)  of the rThese models can account for contrasting observations from previous topology studies for a variety of real world networks. Typically, many PPI networks have been implicitly modeled from the contact-centric view, while the task-centric view has been implicitly adopted for modeling many human social networks. Thus, PPI networks are generally thought to be scale-free disassortative hierarchical networks, and the human social networks as broad-scale assortative nonhierarchical networks. However, there have been reports of noncanonical topological properties for protein functional networks ,26 and oFig. 1 immediately suggests that different representations of the associations among the same entities could give rise to networks with either topology. We see that this is indeed the case in real-world networks and can be easily illustrated for both the internet and protein networks. In both cases, the topological properties of the resulting alternate network models match those expected for contact and task centric networks.A study of the toy examples in Fig. 2D-F; reprinted in Fig. 3A for clarity), the domain network is scale-free, disassortative, and hierarchical, but the router network is broad-scale, assortative, and nonhierarchical. This observation of contrasting Internet topology between router- and domain-level also has been reported by [r = −0.15)  and hierarchical . Regardless of node degree, most proteins and routers showed clustering coefficients near the average clustering coefficient for the entire protein or router network. Notably, our findings were consistent when biological process networks were created using alternate methods . Thus, these observations thus suggest common rules-of-organization between the Internet and the yeast proteome at equivalent system levels—assortative and nonhierarchical organization among lower-level components, and disassortative and hierarchical organization among upper-level components .For example, for the case of the internet, the router and domain networks differ in not only edge representation but also node representation—the task-centric router-level Internet has nodes of individual routers, whereas the contact-centric domain-level Internet has nodes of domains composed of multiple routers. As seen above  . This te nation . Similare nation . Howeverde novo by arbitrarily connecting randomly chosen node pairs; this approach essentially implements a contact-centric generative model, and we would expect the resulting random network’s topological properties to reflect this mechanism of generation.Finally, we note that although our focus here is on real world networks, researchers often generate random networks for study or to serve as background models for comparing to real world networks. Such randomized graphs can of course be generated by many different algorithms. We would expect the method employed to dictate the resulting topological properties. For example, one common approach is to construct graphs In summary, we describe two broad classes of network models whose intrinsic topological properties explain observations on many real world networks, as we have shown with examples from human societies, the Internet, and cellular proteins. Contact-centric networks are characterized by intrinsically pairwise interactions, generating scale-free, disassortative, hierarchical networks. Task-centric networks are characterized by interactions within and between groups, and give rise to single- or broad-scale, assortative, nonhierarchical networks. Alternative representations of the same entities, such as by grouping individuals together hierarchically, can also give rise to such networks, with lower-level representations generating assortative, nonhierarchical networks, and upper-level, clustered representations generating disassortative, hierarchical networks. The intrinsic topologies of these two classes of networks may account for previous paradoxical observations among real world networks, and imply similar organizational principles among human societies, the Internet, and cellular proteomes.S1 FigThree contact-centric (indicated by the letter “C”) networks and three task-centric (indicated by the letter “T”) networks were analyzed, highlighting the degree distribution tails.(TIF)Click here for additional data file.S2 FigFour contact-centric (indicated by the letter “C”) networks and three task-centric (indicated by the letter “T”) networks and their randomized networks were analyzed.(TIF)Click here for additional data file.S3 FigFour contact-centric (indicated by the letter “C”) networks and three task-centric (indicated by the letter “T”) networks and their randomized networks were analyzed.(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Figth level GO-BP annotations based on sharing of annotated proteins; labeled “Process level – GO” in the plot) and the network of biological processes inferred by MCL clustering of a functional protein network (“Process level – MCL”) show hierarchical organization, while the functional protein network itself (“YeastNet core”) shows a nonhierarchical organization, in which clustering coefficients for a given degree connectivity (C(k)) trend near the average clustering coefficient for the entire network (<C>).Both a network of previously defined Gene Ontology biological processes (in which edges are defined between 7(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


The rate of sway within local-control was significantly higher in the DPN group by 49% , which suggests a compromised local-control balance behavior in DPN patients. Unlike local-control, the rate of sway within central-control was 60% smaller in the DPN group , which suggests an adaptation mechanism to reduce the overall body sway in DPN patients. Interestingly, significant negative correlations were observed between central-control rate of sway with neuropathy severity  and the history of diabetes . Results suggest that in the lack of sensory feedback cueing, DPN participants were highly unstable compared to controls. However, as soon as they perceived the magnitude of sway using sensory feedback, they chose a high rigid postural control strategy, probably due to high concerns for fall, which may increase the energy cost during extended period of standing; the adaptation mechanism using sensory feedback depends on the level of neuropathy and the history of diabetes.Poor balance control and increased fall risk have been reported in people with diabetic peripheral neuropathy (DPN). Traditional body sway measures are unable to describe underlying postural control mechanism. In the current study, we used stabilogram diffusion analysis to examine the mechanism under which balance is altered in DPN patients under local-control  and central-control . DPN patients and healthy age-matched adults over 55 years performed two 15-second Romberg balance trials. Center of gravity sway was measured using a motion tracker system based on wearable inertial sensors, and used to derive body sway and local/central control balance parameters. Eighteen DPN patients (age = 65.4±7.6 years; BMI = 29.3±5.3 kg/m It is estimated that some 382 million people had diabetes in 2013, and this number will increase 55% to 592 million people by 2035 ,5. TherePrevious research has demonstrated more body sway during upright quiet standing in those with DPN, compared to those without ,7. To bePrevious studies have reported marked reductions in strength, reflexive responses, and sensing function of lower extremity muscles in diabetic patients ,14. It iParticipants with type 2 diabetes and DPN were recruited from the Southern Arizona Limb Salvage Alliance (SALSA), at the University of Arizona. Healthy age-matched controls were recruited from a community-dwelling aging adults in a southwestern tertiary academic medical center, Tucson, Arizona. All participants were aged 55 years or above, with no major mobility disorder (able to perform 15-second upright standing). Participants were excluded if they had any clinically significant medical or psychiatric condition (including Dementia or Alzheimer’s), dizziness and nausea symptoms, or a sensation of a spinning motion in the head while standing upright, or a laboratory abnormality that could, in the judgment of the investigators, interfere with the ability to participate in the study. DPN participants were excluded if they had disorders other than diabetes leading to severe balance deficits (including stroke or Parkinson’s disease), an active foot ulcer, major foot deformity (e.g. Charcot neuroarthropathy), or major foot amputation. All participants signed a written informed consent form, specifically approved for this study by the University of Arizona institutional review board, and presented by trained clinical research coordinators.Peripheral neuropathy was confirmed using the American Diabetes Association criteria based on insensitivity to 10-g Semmes-Weinstein monofilament ,18. AddiEach participant performed two 15-second trials of balance assessment, within which participants stood upright with their feet together as close as possible without touching each other, and with their arms crossed across their chest. Participants were instructed to keep their eyes open (no target was specified) in the first trial, and to keep their eyes closed in the subsequent trial. Two sensors , each in(AP) sway, COG (ML) sway, and COG sway. The ranges of sway in the AP and ML directions were defined as COG (AP) sway and COG (ML) sway, respectively, after excluding outliers as described in previous work [(AP) sway × COG (ML) sway).Traditional body sway parameters from COG plots include: COG ous work ,23. The slope, central-controlslope-Log, and local-controltime-interval.  as suggested by previous work [time-interval represents the maximum time interval in which the central-control mechanism is added to the local-control.To assess the performance of local- and central-control balance strategies, we used the stabilogram diffusion analysis as described by Collins et al. ,11. Thisous work . Local-cr). Pearson correlations were also used to assess associations between stabilogram diffusion parameters with age, height, weight, and BMI. Summary results are presented as means (standard deviation—SD). All analyses were done using JMP , and statistical significance was concluded when p ≤ 0.05.Separate analyses of covariates (ANCOVAs) were used to compare the effect of DPN on balance parameters, considering age, body mass index (BMI), and gender as covariates; Cohen’s effect size was calculated. Correlations between balance parameters with neuropathy and history of disease were assessed using linear regression-ANOVA models, considering age, BMI, and gender as adjusting variables; correlation results were reported as Pearson correlations  and 34.6 ± 7.0 mV (from 26 to 52 mV), respectively, for DPN participants. As expected a higher fear of falling (as measured by FES-I) and a larger number of fallers  were determined among DPN participants compared to healthy controls (P < 0.05). Demographic details are reported in Thirty-six participants  were recruited and completed all tests. Mean age and BMI of DPN participants were 65 ± 8 years and 29.3 ± 5.4 kg/mtime-interval was not significantly different among the two groups  in the DPN group was significantly higher than controls, respectively by 74% and 87% in eyes-open and eyes-closed conditions Tables . Local-cs Tables , which ms Tables . Interesy Tables . This suslope-Log (AP) , central-controlslope-Log (ML) , and central-controlslope-Log  within the eyes-closed condition. Similarly, significant correlations were determined between disease history and central-controlslope-Log (AP) in eyes-open  and eyes-closed  conditions. These correlations represent an overall reduction in the rate of body sway within the central-control stage with the VPT score and disease history. On the other hand the rate of sway within local-control stage increased with neuropathy severity and disease history, approaching significance in our sample P = 0.07; r = 0.57). No significant correlation was found between stabilogram diffusion parameters with age (r ≤ 0.45), height (r ≤ 0.54), weight (r ≤ 0.47), or BMI (r ≤ 0.51) in either DPN or healthy groups (p > 0.06).Significant negative correlations were observed between the VPT score and central-controlThe results of this study suggest that DPN affects the amount of body sway in short time-intervals or local-control stage when comparing with healthy controls. This could be related to inability of lower extremity and postural muscles to provide sufficient activity level and joint stiffness in patients with DPN. It has been previously claimed that the strength of lower extremity muscles drops with neuropathy. Anderson et al. reported 17% and 14% less strength in ankle flexor and extensor muscles of DPN patients, respectively, when compared to age-matched healthy controls . The redCompromised reflexive responses of lower extremity muscles have also been associated with DPN –30. It hIn addition to deterioration of reflexive responses and reduction in strength, previous work suggested that spindle sensation of muscles  also compromises with DPN. van Deursen et al. used a tracking performance protocol with and without vibration to assess the muscle spindle function of the ankle . Their rslope-Log in the older group compared to younger participants, which was interpreted as an adaptation to compensate for a worsen local-control strategy with aging [Unlike local-control, our results demonstrated small sway rate within the central-control stage in DPN group, even smaller than healthy controls during both eyes-open and eyes-closed conditions. Similar findings were reported by Collins et al. when they compared healthy young and older adults. They discovered smaller central-controlth aging . Within th aging , or may To assess changes in postural control strategies with disease severity, additional analyses were performed to explore the association between local- and central-control performance with the level of neuropathy and history of DPN. Interestingly, we observed that by increasing the VPT score and history of DPN, the rate of body sway within the central-control stage decreases. This reduction in the rate of body sway within central-control balance was mainly observed in the eyes-closed condition. Accordingly, it can be concluded that sensory units other than visual feedback may involve in the central-control adaptation among the DPN group; however, this hypothesis requires further investigation of the effect of sensory feedback mechanisms in order to separately assess vestibular, extroceptive, and visual balance regulation performance.A convenient sample of DPN and healthy controls were recruited for the current study, and, therefore, the current results, while encouraging, should be confirmed in a larger sample size. However, we managed to recruit DPN participants with different history of diabetes and covered a wide range of neuropathy levels to better associate balance parameters with the disease severity. Moreover, in the implemented approach, two distinct stages of local-control and central-control are considered, which means that at one specific time interval sensory feedback is added to control the balance. However, Newell et al.  criticizIn summary, our findings from the current research provide insights regarding balancing strategies in DPN patients based on dependency on postural muscles and sensory feedback. For the first time, using stabilogram diffusion analysis, we were able to explain the large amount of body sway in DPN patients during quiet standing according to local- and central-control balancing behaviors. First, we concluded that in the lack of sensory feedback cueing, rate of sway was on average ~49% higher in DPN participants compared to healthy controls in both eyes-open and eyes-closed conditions. This compromised local-control balance control may be explained by the poor local muscle control response . Second, we observed that after receiving central cueing, rate of body sway dropped substantially even beyond the range of healthy controls balance behaviors, suggesting an adaptation mechanism in DPN patients that depends on history of disease and the level of neuropathy. Overall, these new findings introduced a new approach of postural balance assessment, which may be helpful in better understanding of balance alterations with DPN, and ultimately implement more efficient strategies to improve balance performance using exercise routines or medical treatments.ri)2 between successive COG data points separated in time by a specified time-interval (Δt) were calculated. The squared displacements (Δri)2 were then averaged over the specified time-interval (Δt), ranging from 0 to 10 seconds (0 ≤ Δt ≤ 10 sec) to provide a plot of mean square COG displacement (Δr)2 versus Δt according to the following formula:t, m is the number of data intervals. Using this approach a stabilogram diffusion plot is developed (r)2 as a function of time-interval (Δt). Two distinguishable regions exist in this plot, named as short time-interval  and long time-interval  regions, which are separated by identifying the critical time-interval . Local-controltime-interval is estimated by identifying two best linear fits, which represent, respectively, the short time-interval linear fit  and long time-interval linear fit  of the stabilogram diffusion plot as illustrated in slope) and central-control  conditions. Similar to local-controlslope and central-controlslope, the scaling exponents, local-controlslope-Log and central-controlslope-Log were calculated from short time-interval and long time-interval slopes of the log-log stabilogram diffusion plot. Based on fractional Brownian theory, the following equation exist between squared of displacement and time-interval:slope-Log in the current study represent scaling exponent H within the long time-interval region. The scaling exponent determines the correlation between the past and future increments. If H > 0.5, the COG moving in a particular direction, will tend to continue in the same direction. On the other hand, for H < 0.5 the correlation between past and future movement is negative. Based on previous work [To identify local-control and central-control strategies, the square of displacement Click here for additional data file.


Preventing falls and bone fractures in hospital care is an important issue in geriatric medicine. Use of hypnotics is a potential risk factor for falls and bone fractures in older patients. However, data are lacking on the association between use of hypnotics and the occurrence of bone fracture.We used a national inpatient database including 1,057 hospitals in Japan and included dementia patients aged 50 years or older who were hospitalized during a period of 12 months between April 2012 and March 2013. The primary outcome was the occurrence of bone fracture during hospitalization. Use of hypnotics was compared between patients with and without bone fracture in this matched case-control study.P<0.001), ultrashort-acting non-benzodiazepine hypnotics , hydroxyzine , risperidone and perospirone . Other drug groups were not significantly associated with the occurrence of in-hospital fracture.Of 140,494 patients, 830 patients suffered from in-hospital fracture. A 1:4 matching with age, sex and hospital created 817 cases with fracture and 3,158 matched patients without fracture. With adjustment for the Charlson comorbidity index, emergent admission, activities of daily living, and scores for level walking, a higher occurrence of fractures were seen with short-acting benzodiazepine hypnotics (odds ratio, 1.43; 95% confidence interval, 1.19–1.73; Short-acting benzodiazepine hypnotics and ultrashort-acting non-benzodiazepine hypnotics may increase risk of bone fracture in hospitalized dementia patients. Bone fracture following falls in hospitalized patients is an unresolved problem in geriatric medical management. Older patients, especially dementia patients, have higher risks of delirium, insomnia and day—night reversal with environmental changes related to hospital admission. Hypnotics and psychoactives are sometimes used to relieve these symptoms.Studies have examined the risk of hypnotic drug use on fall and fracture. In a meta-analysis of 22 studies, fall was significantly associated with use of sedatives and hypnotics, neuroleptics and antipsychotics, antidepressants, and benzodiazepines, but was not associated with narcotics . A retroFor this study, we used the Diagnosis Procedure Combination database. The details of the database have been described elsewhere –20. BrieAmong approximately 7 million inpatients over 12 months between April 1, 2012 and March 31 2013, we identified patients aged 50 years or older. Of these, we selected patients who were diagnosed with dementia or dementia-related diseases, including dementia in Alzheimer disease , vascular dementia (F01), dementia in Pick disease , dementia in Creutzfeldt—Jakob disease , dementia in Huntington disease , dementia in Parkinson disease , dementia in human immunodeficiency virus disease , dementia in other specified diseases classified elsewhere (F02.8), unspecified dementia (F03), alcoholic dementia (F107), dementia in cerebral lipidosis , Lewy bodies dementia , and mild cognitive disorder (F06.7).kampo herbal medicine used as a hypnotic and in the treatment of behavioral and psychological symptoms of dementia; and other neurological drugs used as hypnotics.We extracted data on the following 17 types of hypnotics (or sedatives used as hypnotics) for each patient: benzodiazepine anxiolytics; diazepam; ultrashort-acting benzodiazepine hypnotics; short-acting benzodiazepine hypnotics; middle- to long-acting benzodiazepine hypnotics; ultrashort-acting non-benzodiazepine hypnotics; melatonin-receptor agonists; hydroxyzine; phenothiazine antipsychotics; haloperidol; sulpiride; risperidone and perospirone; multi-acting-receptor-targeted antipsychotics used as a hypnotic; antidepressants used as a hypnotic; Japanese Comorbidities were assessed by ICD-10 codes and converted into scores to calculate the Charlson comorbidity index (CCI) based on Quan’s algorithm . ADL scoThe outcome in this study was in-hospital fracture. In the database, comorbidities already present at admission are clearly differentiated from complications that occurred after admission. In-hospital fracture was defined as fracture that occurred after admission and was determined according to the following ICD-10 codes: fracture of skull and facial bones (S02); fracture of neck (S12); fracture of rib(s), sternum and thoracic spine (S22); fracture of lumbar spine and pelvis (S32); fracture of shoulder and upper arm (S42); fracture of forearm (S52); fracture at wrist and hand level (S62); fracture of femur (S72); fracture of lower leg, including ankle (S82); fracture of foot, except ankle (S92); fractures involving multiple body regions (T02); fracture of spine, level unspecified (T08); fracture of upper limb, level unspecified (T10); and fracture of lower limb, level unspecified (T12).We performed a matched case—control study. First, we identified cases with in-hospital fracture. For each case, we selected four controls of similar age (±5 years) and the same sex from the same hospital. When there were more than four matched-control candidates to each case, we randomly selected four control patients. Specifically, control cases were sorted by randomly generated values from a Microsoft SQL server and the top four were selected. There are two ways to conduct matching: matching with replacement; and matching without replacement . MatchinDescriptive statistics were presented for the matched patients. Categorical variables were compared using the chi square test. We performed multivariable logistic regression for the occurrence of in-hospital fractures fitted with a generalized estimating equation to account for the clustered nature of the cases and controls. There are two ways to cluster in matched case-control studies: generalized estimating equations (GEEs) and conditional logistic analysis. Both methods can make consistent estimates. As GEE is more robust in terms of the specification of matching effect, we chose GEEs . The depAmong 140,494 eligible patients, 830 patients suffered from in-hospital fracture.P = 0.582) or sex (P = 0.728) between the case and control groups. To exclude the possibility that controls may have been matched to cases with a larger age difference than to cases with a smaller age difference, we also compared the distribution of age in the case and control groups. Mean, median, standard deviation, range and interquartile range of age (years) in the case and control groups were 81.5 vs. 81.8, 82.0 vs. 82.0, 7.9 vs. 7.5, 50–103 vs. 50–103, 11.0 vs. 10.0, respectively. The age distributions of the case and control groups were also similar. No significant difference in CCI or emergent admission was present between the cases and controls. The ADL score for walking on a flat floor on admission was significantly different between the groups.Using 1:4 matching, we obtained a case group of 817 patients and a control group of 3158 patients. P<0.001) (P<0.001). The case group was significantly more likely to use benzodiazepine anxiolytics; ultrashort-acting benzodiazepine hypnotics; short-acting benzodiazepine hypnotics; middle- to long-acting benzodiazepine hypnotics; ultrashort-acting non-benzodiazepine hypnotics; melatonin-receptor agonists; hydroxyzine; phenothiazine antipsychotic; haloperidol; sulpiride; risperidone and perospirone; multi-acting-receptor-targeted antipsychotics used as a hypnotic; and an antidepressant used as a hypnotic. The proportion of patients who used Japanese kampo herbal medicine was not significantly different between the cases and controls.P<0.001) . The prokampo herbal medicine was associated with the occurrence of in-hospital fracture.The present study showed an increased risk for in-hospital fracture with several hypnotics and psychoactives used as hypnotics in dementia patients who were admitted to acute care hospitals. In-hospital fracture risk was associated with a short-acting benzodiazepine hypnotic, an ultrashort-acting non-benzodiazepine hypnotic, and risperidone and perospirone.A previous US study of nursing home residents demonstrated a fracture risk with ultrashort-acting non-benzodiazepine hypnotics . The preTo our knowledge, the present study is the first to show that hydroxyzine may increase the risk of fracture in dementia patients. Hydroxyzine has been shown to be as effective as bromazepam, one of the benzodiazepines, in the treatment of generalized anxiety disorder . BecauseA receptor-independent pathway in contrast to most of hypnotics that act on the GABAA receptor. The present study suggests that a melatonin agonist may be safer than other hypnotics in terms of fall and fracture risk.Our results showed that a melatonin agonist was not significantly associated with the occurrence of in-hospital fracture. Melatonin-receptor agonists including ramelteon are new types of hypnotics. They act on the GABAkampo herbal medicines, which are often used as sedatives or hypnotics in Japan, have beneficial effects on the behavioral and psychological symptoms of dementia [Japanese dementia –30. Our This study has several limitations. First, recorded diagnoses in an administrative claims database are less well validated than those in planned prospective studies. Second, the time interval between drug administration and related in-hospital fracture cannot be identified from the database and its causal relationship remains to be clarified. We have information on the timing and use of these agents, but not on the timing of fracture. Consequently, we are not certain as to whether the agent was prescribed before or after the fracture, or if it was not used for a short period of time for weeks to months prior to fracture. Third, it is difficult to distinguish the deleterious effect of hypnotic use itself from underlying conditions, including night delirium and insomnia requiring prescription of hypnotics. Fourth, there was no information about previous falls, and so we were unable to examine this relationship owing to the lack of data.kampo herbal medicine may be preferable to these drugs.In light of these findings, it is preferable to avoid prescribing short-acting benzodiazepines and ultrashort-acting non-benzodiazepine hypnotics, risperidone or perospirone, hydroxyzine, or multi-acting-receptor-targeted antipsychotics to in-hospital dementia patients. Melatonin-receptor agonists or Japanese kampo herbal medicine.Short-acting benzodiazepines and ultrashort-acting non-benzodiazepine hypnotics were associated with an increase in in-hospital fractures in dementia patients, while no significant association with an increase in in-hospital fractures was seen with middle- to long-acting benzodiazepine hypnotics, melatonin-receptor agonists, or Japanese


Objective: The emergence of extended-spectrum β-lactamase (ESBL)-producing bacteria is now a critical concern. The ESBL-producing Klebsiella pneumoniae constitutes one of the most common multidrug-resistant (MDR) groups of gram-negative bacteria involved in nosocomial infections worldwide. In this study we report on the molecular characterization through whole genome sequencing of an ESBL-producing K. pneumoniae strain, LAU-KP1, isolated from a stool sample from a patient admitted for a gastrointestinal procedure/surgery at the Lebanese Amrican University Medical Center-Rizk Hospital (LAUMCRH) in Lebanon.Methods: Illumina paired-end libraries were prepared and sequenced, which resulted in 4,220,969 high-quality reads. All sequence processing and assembly were performed using the A5 assembly pipeline.Results: The initial assembly produced 86 contigs, for which no scaffolding was obtained. The final collection of contigs was submitted to GenBank. The final draft genome sequence consists of a combined 5,632,663 bases with 57% G+C content. Automated annotation was performed using the RAST annotation server. Sequencing analysis revealed that the isolate harbored different β-lactamase genes, including blaoxa−1, blaCTX−M−15, blaSHV−11, and blaTEM−1b. The isolate was also characterized by the concomitant presence of other resistance determinants most notably acc(6′)-lb-cr and qnrb1. The entire plasmid content was also investigated and revealed homology with four major plasmids pKPN-IT, pBS512_2, pRSF1010_SL1344, and pKPN3.Conclusions: The potential role of K. pneumonia as a reservoir for ESBL genes and other resistance determinants is along with the presence of key factors that favor the spread of antimicrobial resistance a clear cause of concern and the problem that Carbapenem-non-susceptible ESBL isolates are posing in hospitals should be reconsidered through systematic exploration and molecular characterization. Enterobacteriaceae  are enzymes produced by many bacterial species as a means for defense against β-lactam drugs with the genes encoding for those enzymes being mainly located on mobile genetic elements  groups of gram-negative bacteria worldwide  are also common  or the class D OXA-type enzymes .The aim of this study is to characterize an ESBL-producing The University's Institutional Review Board approved the study (UMCRH.AF.12/Dec/2012).Enterobacteriaceae by means of three consecutive rectal swab or stool cultures, as per regular screening for ESBL detection. The first fecal sample was collected at the same time as the other preoperative tests. The second and third samples were collected later, latest day 2-post surgery. K. pneumoniae LAU-KP1 sequenced in this study was isolated from a 62-year-old woman working as practitioner nurse or nurse's assistant. She was admitted for radical resection of an abdominal tumor and repair of abdominal hernia. The patient had other than her cancer, recurrent urinary tract infections. She had been using amoxicillin-clavulanate, metronidazol, and cefriaxon in the 3 months prior to presentation. She had been admitted to hospital in the year prior to surgery, but was not known to be a carrier of multidrug resistant organism and no one in her household was known to be a carrier of multidrug resistant organisms.Samples were collected from the University Medical Center-Rizk Hospital (UMC-RH). All patients included in the study were screened for the presence of ESBL-producing E-test method using double sided strips containing on one side ceftazidime or cefotaxime and on the other the same antibiotic combined with clavulanate was used to assess for screening and confirming ESBL production in all strains according to the recommendations of the CLSI. E. coli ATCC25922 and K. pneumoniae ATCC700603 were used as the quality control strains.The standard disk diffusion method with combined discs (ceftazidime/clavulanate and cefotaxime/clavulanate) was used for screening. The standard Antimicrobial susceptibility test by the disk diffusion method was performed to determine the resistance patterns of the isolates to 17 antibiotics: cefotaxime, ceftriaxone, ceftazidime, aztreonam, ceftazidime/clavulanate, cefotaxime/clavulanate, cephalothin, cefuroxime sodium, cefepime, gentamicin, amikacin, ciprofloxacin, tetracycline, ampicillin, amoxicillin/clavulanate, piperacillin, piperacillin/tazobactam, imipenem, meropenem, ertapenem, and sulfamethoxazole/trimethoprim . All antimicrobial testing was performed on Mueller-Hinton agar by the flooding technique and data interpreted according to the CLSI guidelines.Bacterial DNA was extracted using the Nucleospin kit (Macherey-Nagel) and following the manufacturer's instructions.Genomic DNA (gDNA) was used as input for library preparation using the Illumina TruSeq DNA library preparation kit (Illumina). Bioruptor® NGS was used to sonicate 1 μg of sample DNA  for 8 min of 30 s on/off cycles at 4°C. Sheared DNA was used as input for library preparation using the Illumina TruSeq DNA library preparation kit (Illumina). The gDNA was subjected to end-repair, A-tailing, ligation of adaptors including sample-specific barcodes as recommended in the manufacturer's protocol. After ligation of the adaptors, and to reduce the number of fragments smaller than 500 bp, fragments between 500 and 1000 bp were selected using a Pippin Prep™ DNA size selection system (Sage Science). The resulting library was quantified by quantitative PCR in triplicate at 1:1000 and using the Kapa library quantification kit  and as recommended by the manufacturer's protocol. The resultant library size was assessed using an Agilent Bioanalyzer with the High Sensitivity DNA Kit. The library was multiplexed, clustered, and sequenced on an Illumina MiSeq with paired-end 500 cycles protocol to read a length of 250 bp.de novo using A5 with default parameters . The service identified protein-encoding, rRNA and tRNA genes, assigned functions to the genes, and predicted which subsystems are represented in the genome  was used to identify acquired antimicrobial resistance genes in the WGS data, using a threshold of 98.00% identity (ID)  using progressiveMauve  . Screening patients for rectal carriage by Baraniak et al. lb-cr, qnr, SHV-11, and CTX-M-15. CTX-M-15 detection was consistent with the recent predominance of CTX-M and CTX-M-15 in particular . Harbouring the qnrB1 (quinolone resistance) was another important finding that additionally distinguished LAU-KP1. Previously Breurec et al. . These enzymes hydrolyze all β-lactams including monobactams  ST258 and its variants  dicitrate transport system and a class I integron carrying trimethoprim resistance, both of which were also detected in LAU-KP1.Resistance to carbapenems involves multiple mechanisms including alterations in outer membrane permeability mediated by the loss of porins, upregulation of efflux systems along with hyper production of AmpC β-lactamases or more commonly the production of carbapenemases  . Screening patients for rectal carriage by Baraniak et al.  in Baltimore, MD (Hazen et al., K. pneumoniae ATCC BAA-2146, which is the first U.S. isolate found encoding the gene for the broad-spectrum and carbapenem-active metallo-β-lactamase NDM-1 (Hudson et al., K. pneumoniae isolate with a complete genome, Ecl8, in order to visualize the genomic organization (Figure Furthermore, the phylogenetic analysis of LAU-KP1 showed that it falls within a clade of other known multidrug resistant K. pneumoniae emphasizes the potential for transmission of highly resistant gram-negative pathogens that are difficult to treat. This merits more detailed future studies directed toward characterization of molecular mechanisms, environmental factors, and selection pressures promoting the spread of such strains. Detection of IncFIIK a virulence plasmid (Dolejska et al., K. pneumoniae as a reservoir for ESBL genes and other resistance determinants. The spread of antimicrobial resistance in this region could be attributed to different factors including: uncontrolled consumption of antimicrobial agents through self-medication, inappropriate antibiotic prescription, the substandard quality of some drugs, and a lack of effective measures to prevent nosocomial infections. The virtual lack of physical barriers between the community and hospital settings in the countries in this region and the increasing pressure from war immigrants along with poor living conditions may be also important contributory factors. High-resolution genotyping studies of isolates collected over larger spatial and temporal scales will be necessary to decipher the dynamics of the emergence of MDR K. pneumoniae clonal groups.The detection of such a multidrug resistant strain of K. pneumoniae. This will provide a better insight into the emergence and spread of these multidrug resistant strains and hopefully lead to information essential in preventing infections and limiting the spread of such organisms.Therefore, we urgently need well-designed epidemiological and molecular studies to understand the dynamics of transmission, risk factors, and reservoirs for The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The recent FDA approval of ramucirumab  has added a third agent to our existing armamentarium of angiogenesis inhibitors (bevacizumab and ziv-aflibercept) for the second-line treatment of metastatic colorectal cancer, which may have some impacts in the current clinic practice. The recent FDA approval of ramucirumab , in combination with FOLFIRI  [Angiogenesis plays a crucial role in colorectal tumorigenesis, and the VEGF pathway is the only target that has been validated to date . The VEGP = 0.0219) and progression-free survival  in patients with mCRC whose disease had progressed during or after first-line treatment with bevacizumab, oxaliplatin, and a fluoropyrimidine [ML18147  was the first study to demonstrate the benefit of continuing anti-angiogenic agent bevacizumab (in combination with chemotherapy) as a second-line therapy, even after previous exposure to the agent . The VELA careful review of results indicates noteworthy similarities between these three studies. All of these trials demonstrated a benefit of combining an anti-VEGF agent  with chemotherapy beyond initial progression in patients with mCRC. Although cross-trial comparison suffers from inherent limitations and should be interpreted with caution, it is interesting to note that the three anti-VEGF agents evaluated in these trials exhibited a similar improvement in OS  and PFS . The stratified HR for OS were also quite similar in the TML (0.83), VELOUR (0.82), and RAISE (0.84) trials. Moreover, the toxicity profiles of these agents overlapped, with a higher incidence of anti-VEGF-associated adverse events  in the anti-angiogenesis agent arms, as was expected.There were, however, a few important dissimilarities noted as well, which were primarily related to the treatment regimens used in these studies. In the VELOUR and RAISE trials, all patients received oxaliplatin- and fluoropyrimidine-based regimens as first-line treatment. In the ML18147 study, approximately 60 % of patients received irinotecan-based, and the remaining 40 % received oxaliplatin-based regimen as the first-line therapy. All patients in the ML18147 and RAISE trials had received previous treatment with bevacizumab, as compared with only 30 % of patients in the VELOUR trial. The anti-VEGF agents used in these trials also differ with respect to their mechanism of action and pharmacokinetic properties. For example, bevacizumab targets VEGF-A to cause ligand sequestering; ziv-aflibercept blocks VEGF-A, VEGF-B, and PlGF using the IgG1 Fc-VEGF receptor construct; and ramucirumab targets VEGFR-2 to prevent receptor activation by VEGF-A. Despite these differences, data from these three trials provide confirmatory evidence that inhibition of tumor angiogenesis beyond initial disease progression is an effective treatment strategy in mCRC. However, questions remain: potential predictive markers for these VEGF-A/VEGFR-2 pathway inhibitors, whether colorectal cancer may develop crossover tolerability/resistance to these treat different agents, potential opportunities to the possible mechanisms of moderate benefits  of these anti-angiogenic agents in mCRC, and possibilities of these anti-angiogenic agents in combination with other biologic target oriented agents, in particular immune checkpoint inhibitors.The efficacy of bevacizumab and aflibercept may be affected by the local concentration of targeted ligand(s). Expression of VEGF-A and other ligands may increase after the loop blockage as a physiological response to rebalance the pathway, e.g., aflibercept benefits less in those patients previously exposed to bevacizumab (Velour data). Biologic efficacy of ramucirumab may be less influenced by the local concentration of VEGF-A because of binding in the VEGFR-2 extracellular domain. A limited benefit of this class of anti-angiogenic agents may be partly due to the fact that no selection criteria have been used to enrich for those patients who may be likely to derive a durable benefit from treatment. Based on the heterogeneous nature of colorectal cancer, to identify predictive biomarkers for VEGF signaling inhibitors may potentially broaden the clinical impact of these agents and might even distinguish the clinic application of each agent. Many biomarker studies have been developed and others are ongoing; however, there has been no prospective validation reported. For example, low VEGF(165)b:VEGF ratio may be a predictive marker for bevacizumab in metastatic colorectal cancer , and indSo far, no selection criteria have been used to enrich for patients that might be more likely to derive durable responses from a particular anti-VEGF therapy. Consequently, optimal utilization of the available anti-angiogenesis options remains an unfulfilled goal at this time. In current clinical practice, decisions regarding the choice of agent are usually guided by physicians’ familiarity with the drug and the cost of therapy. Identification of robust clinical and molecular biomarkers to predict sensitivity, resistance, or toxicity from VEGF signaling inhibitors would be considered a significant breakthrough, as it might help distinguish the clinical application of each agent . Transla


Integrated Discrete Multiple Organ Co-culture (IDMOC) is emerging as an in-vitro alternative to in-vivo animal models for pharmacology studies. IDMOC allows dose-response relationships to be investigated at the tissue and organoid levels, yet, these relationships often exhibit responses that are far more complex than the binary responses often measured in whole animals. To accommodate departure from binary endpoints, IDMOC requires an expansion of analytic techniques beyond simple linear probit and logistic models familiar in toxicology. IDMOC dose-responses may be measured at continuous scales, exhibit significant non-linearity such as local maxima or minima, and may include non-independent measures. Generalized additive mixed-modeling (GAMM) provides an alternative description of dose-response that relaxes assumptions of independence and linearity. We compared GAMMs to traditional linear models for describing dose-response in IDMOC pharmacology studies. However, failure of animal models to accurately predict a response in humans for many compounds, cost, and ethical concerns associated with the use of animal resources are limiting factors to the utility of animal models  was the expected florescence, ujk was a random intercept for well j in plate k, s1 was a smoothing function , β1:3 were coefficients associated with each linear term, Conc was the concentration of each agent, and Co was the co-cultured vs. mono-culture category.For each endpoint response of the fibroblasts for each toxicant, the following models were compared:Non-independence in preparation heterogeneity was controlled using the random intercept terms . A GaussAIC was used to select models for inference, and weights were tabulated for model comparisons across all candidates . First, predict [AIC preferred models were rendered graphically for interpretation, along with selected graphic comparisons between competing models. Graphs were produced by predicting response values and confidence intervals across a range of fixed concentration values, using the R function predict .For illustrative purposes, GAMMs compared to ANCOVA and ANOVA, and response to cyclophosphamide were also weighted and graphed. Pearson residuals for the 3 models were compared using a LOESS smoother with span of 0.5 .in vivo [in vitro [The categories of toxicants chosen in the experiments described herein are as follows: hepatotoxic (ticlopidine), generally cytotoxic (paraoxon), activated by hepatocytes (cyclophosphamide), and detoxified by hepatocytes (4-aminophenol). Ticlopedine is an anti-platelet drug that has been shown to induce hepatotoxicity both in vivo  and in vin vitro , while iin vitro , 23, andin vitro , 25. In in vitro . The toxin vitro , 27, andwi) of only 0.52 for the model with interactions. Preferred models for 4-aminophenol and paroxon included a smoothed term for concentration. Model preference for smoothed concentration only in paroxon, with no term for co-culture category, was somewhat weak, with a weight 0.64 over a weight of 0.36 for a model including the linear category term. The preferred model for 4-aminophenol included a linear term for co-culture category, with no interaction between co-culture category and concentration, smoothed or linear.For cyclophosphamide and ticlopedine, preferred models included full interactions between smoothed concentration and culture category (co-culture vs. monoculture). However, for ticlopedine, model preference was weak, with a model weight (Cyclophosphamide and ticlopedine exhibited separate trajectories for co-cultured and monocultured fibroblasts . This inwi = 0.0). While ANCOVA captured the transition from compensatory to toxic response with increasing concentration (wi = 0.004), due to non-linearity in the response. A comparison of residuals  in those cases where almost all of the AIC weight is attributed to the preferred model Click here for additional data file.


With the goal of reducing exposure to secondhand smoke, the state of Minnesota (MN), enacted a smoke-free law  in all workplaces, restaurants, and bars in 2007. This retrospective cohort study analyzes emergency department (ED) visits in Olmsted County, MN, for chronic obstructive pulmonary disease (COPD) and asthma over a five-year period to assess changes after enactment of the smoke-free law.We calculated the rates of ED visits in Olmsted County, MN, with a primary diagnosis of COPD and asthma in the five-year period from January 1, 2005 to December 31, 2009. Analyses were performed using segmented Poisson regression to assess whether ED visit rates declined following enactment of the smoke free law after adjusting for potential underlying temporal trends in ED visit rates during this time period.Using segmented Poisson regression analyses, a significant reduction was detected in asthma-related ED visits  but not for COPD-related ED visits following the enactment of the smoke-free law. The reduction in asthma related ED visits was observed in both adults  and children .In Olmsted County, MN, asthma-related ED visits declined significantly after enactment of a smoke-free law. These results add to the body of literature supporting community health benefits of smoke-free policies in public environments and their potential to reduce health care costs. Tobacco smoke is one of the most common asthma triggers. Asthma may be caused by smoking, and childhood exposure to secondhand smoke (SHS) increases the risk for asthma exacerbations . ChronicProtecting the public from exposure to tobacco smoke is a key component of the World Health Organization (WHO) Framework Convention on Tobacco Control . After fThe 2013 population estimate of Olmsted County, located in southeastern MN, was 147,066  . Within The MN smoke-free law that was passed on May 16, 2007, and enacted on October 1, 2007, included all workplaces (including bars and restaurants) . The datInternational Classification of Diseases, Ninth Revision codes 491–492 and 494–496) or asthma  were captured. When multiple ED visits occurred for a single person, all visits were counted since our focus was to assess changes in ED utilization over time for a defined geographic population.All ED visits with the primary diagnosis of COPD , a total of 5,293 ED visits occurred with a primary diagnosis of COPD and 5,906 ED visits with a primary diagnosis of asthma  for the younger age group compared to overall asthma-related ED visits (RR = 0.85) . This isThe findings that asthma-related ED visits were significantly reduced after the smoke-free law implementation, but the COPD-related visits were not significantly reduced, is not surprising. The lack of findings with COPD-related visits confirms findings from a recent meta-analysis . TypicalOur finding of decreased asthma-related ED visits following implementation of a statewide smoke-free law is biologically plausible and consistent with the known causal relationship between cigarette smoke exposure and exacerbation of pulmonary disease symptoms  as well Our study has several limitations. First, we did not measure actual SHS exposure in different environments around the community. Second, our study data was limited only to ED visits which were for the most part, ambulatory and did not include hospital admissions for the local hospitals. Third, other tobacco control efforts were occurring during the study period, which could have contributed to the decrease in smoking prevalence and to reductions in SHS exposure. For example, from 1999 through 2010, the per capita cigarette sales in MN declined by 40% and smoke-free homes increased from 64.5% (1999) to 87.2% (2010) . Other aFollowing the enactment of a smoke-free workplace law we observed a reduction in asthma-related ED visits for both adults and children. This finding underscores the importance of comprehensive smoke-free laws and policies for improving public health and reducing health care costs. Communities need to continue to advocate for smoke-free laws and policies in order to reduce the burden of disease caused by SHS.