Bombyx mori L.) is one of the most suitable lepidopteran insects for studying the molecular aspects of metamorphosis because of its large size, availability of mutants and genome sequence. Besides, this insect also has been amply studied from a physiological and biochemical perspective. Deep sequencing of small RNAs isolated from different stages of silkworm is a powerful tool not only for measuring the changes in miRNA profile but also for discovering novel miRNAs.In eukaryotes, microRNAs (miRNAs) have emerged as critical regulators of gene expression. The Silkworm  of four miRNAs are expressed at much higher levels than their corresponding miRNAs, and their expression profiles are distinct from their corresponding miRNA profiles during different developmental stages. Additionally, we detected two antisense miRNA loci  that are expressed in sense and antisense directions. Interestingly, miR-263 and miR-306 are preferentially and abundantly expressed in pupae and adults, respectively.We generated small RNA libraries from feeding larvae, spinning larvae, pupae and adults of We identified 101 homologs of conserved miRNAs, 14 species-specific and two antisense miRNAs in the silkworm. Our results provided deeper insights into changes in conserved and novel miRNA and miRNA* accumulation during development. Transcriptional regulation alone is insufficient to ensure tight control of gene expression in specific cells or tissues. Recently discovered microRNA (miRNA)-directed post-transcriptional regulation can provide an efficient fine-tuning of target gene expression in certain cell or tissue types and, thus, coordinate the spatial and temporal control ,2. FurthIn animals, most miRNA genes are transcribed by RNA polymerase II, yielding transcripts called primary miRNAs (pri-miRNAs), which are initially processed by a complex containing Drosha and then by Dicer-1 to excise miRNA:miRNA-star (miR:miR*) duplexes. One strand of the duplex (miR) is more stable and preferentially incorporated into an RNA-induced silencing complex (RISC). The miRNA then guides the RISC to regions of complementarity in the target site, where it downregulates the gene expression, often by blocking protein production or by degrading the target mRNA -16.Insect metamorphosis is a complex, highly conserved, and strictly regulated process of developmental events. During metamorphosis, diverse morphological, physiological, biochemical and molecular events result in distinct changes such as cell proliferation, programmed cell death, tissue remodeling and cell migration . The silDrosophilid species, and many miRNAs were discovered both by direct cloning [Drosophila, mosquito (A.gambiae) [A.mellifera) [Thus far, miRNAs cataloging in insects is primarily carried out in  cloning -23 and b cloning ,25. Besigambiae) ,27, honellifera) ,29, red llifera)  and locullifera)  are somellifera) -35. Suchllifera) , in whicllifera)  did not B. mori. Interestingly, the abundance of miR* species of several conserved miRNAs was greater than that of their miRNAs, and their levels varied greatly at various stages. We also discovered two antisense miRNAs in the silkworm. This study provides deeper insights into dynamic regulation of the conserved and novel miRNA and miRNA* levels during different developmental stages and suggests that both universal and silkworm-specific miRNAs play important roles in expression regulation.Deep sequencing of small RNAs can be used to reliably measure modest changes in miRNA abundance among different samples; such changes are unlikely to be identified by sequencing low numbers of clones  or hybridization-based methods such as small RNA blot and miRNA array analyses. The deep sequencing study is also well suited for the discovery of species-specific miRNAs expressed at low abundance. Indeed, in this study, by using deep sequencing we uncovered 15 novel miRNAs that appear to be silkworm-specific. Of the 101 conserved miRNAs identified in this study, most are dynamically regulated during different developmental stages of th and 5th instars) and the other from spinning larvae  during the larval-to-pupal molt. We constructed two additional libraries, from pupae and moths  ranging from 18 to 30 nt was obtained after trimming the adapter sequences. A significant number of reads  lacking the bar-code sequences were eliminated from the analysis because these could not be assigned to the source libraries. From the size distribution of total reads, we found a distinct bimodal distribution, with a peak around 20-22 nt representing miRNAs and another distinct peak around 26-29 nt mostly representing longer piRNA-like small RNAs in all four stages Figure  and 1c. Of 2.5 million total reads, 1,581,810 could be mapped to the available silkworm genome. We used the latest version of silkDB v2.0 to map and analyze these small RNAs. After eliminating redundant sequences from the dataset, only unique sequences represented by 739,119 reads were analyzed further  was identified in this study  were present at low levels in larvae relative to other stages  in ri Table . SpinninDrosophila adults [Anopheles stephensi, miR-989 is abundantly and specifically expressed in adults [B. mori, the expression of miR-989 was much higher in pupae than in other three stages  is transcriptionally active. Our sequencing efforts identified, for the first time, the prevalence of at least two new miRNA loci, miR-306 and miR-263, that generate miRNAs by convergent transcription from both strands in the silkworm genome. As well, we recovered eighteen reads of miR-iab-4AS, the first antisense miRNA reported in animals . AnalysiDrosophila, miR-iab-4-AS is functional in repressing the Hox family of genes [Comparing the mature sequences in each sense and antisense pair showed the seed region in miR-263 and miR263-AS to be almost identical, whereas miR-306 and miR-306-AS showed variations in several internal positions, including the seed region Figure . In Drosof genes ,51. miR-Mapping small RNA reads in this study revealed that certain small RNAs previously annotated as miRNAs may not qualify for such an annotation. In a recent study, two small RNAs were annotated as miR-1920 (one read) and miR-1921 (three reads) in silkworm . Our seqB. mori, that undergo well-defined developmental stages (metamorphosis). To gain insight into post-transcriptional gene regulation in silkworm development, determining a near-complete set of miRNAs and their expression patterns is essential.In order to build a normal animal, development of individual tissues and organs needs to be tightly coordinated with the developmental progression of the whole organism . This prRecent studies in Drosophilid species led to the suggestion that most novel miRNAs originated from non-miRNA sequences and only a small fraction (4%) is expected to be retained during evolution . Only 2.Bombyx diapause hormone receptor (BmDHR) is expressed in the prothoracic gland (PG), the organ, which synthesizes and releases the insect molting hormones, ecdysteroid [We have predicted over 1100 genes as targets for the newly identified silkworm-specific miRNAs and their stars using hitsensor software  (Additioysteroid . JH syntysteroid . Juvenilysteroid . JHE is A predicted target for novel miRNA (bmo-mir-2763) is nuclear receptor GRF (germ cell nuclear factor (GCNF)-related factor). Increasing evidence suggests that GCNF is important for both female and male reproduction . The devDuring the terminal stages of differentiation of the silkworm ovary, the follicular cells that surround the oocyte produce a large number of related polypeptides that are involved in the formation of the eggshell or chorion . MicroRNThe predicted target for bmo-miR-2998* is silk gland factor-1 (SGF-1). SGF-1 is a member of the fork head/HNF-3 family, which regulates transcription of tissue-specific genes . It playDrosophila is transcriptionally up-regulated by Twist and Mef2 during myogenesis [B. mori, miR-1 level is very high, miR-133 is low and miR-206 is extremely low, which suggests that miR-1 likely plays a major role in muscle development and physiology in insects.Evolutionarily, miR-1 is highly conserved and involved in muscle development and physiology ,66. The ogenesis ,67. In Breaper, grim and sickle) in Drosophila [C. elegans, plays a role in apoptotic and non-apoptotic cell death [B. mori, miR-34 is preferentially and abundantly expressed in pupae relative to the other three stages . By preventing the utilization of fat, needed for reproduction during the adult stages, these miRNAs would promote the utilization of glycogen stores.In tabolism . miR-278osophila  suggestsOf all four developmental stages we analyzed, the spinning-larval stage is important - silk is synthesized and a cocoon is built during this period. Strikingly, the overall miRNA abundance is almost doubled from feeding to spinning larval stages, which suggests a role for miRNAs in silk synthesis, besides their importance in developmental transition. The increase in miRNA levels is mostly quantitative and limited to conserved miRNAs. The levels of novel miRNAs in spinning larvae were very low. Similarly, miRNA levels are also sharply elevated in moths relative to the pupae. Again the increase could not be attributed to qualitative differences. Many conserved miRNAs along with a few species-specific miRNAs become more abundant and may regulate reproduction or aging.albeit their expression levels vary across different developmental periods. These abundantly expressed miRNAs accounted for most of the differences in miRNA abundance in different developmental stages of the silkworm. Variation in the level of a specific miRNA across developmental stages indicates stage-specific function, whereas a similar expression over a range of developmental stages points to a possible role in basal and/or constitutive processes. This analysis also revealed that many miRNAs are similarly regulated during development, suggestive of possible co-regulation of these miRNAs during silkworm development  but none from the larval or pupal libraries. Another miRNA, miR-277, was reported to be specifically expressed in the moth stage [Recently, miRNA microarray was used to profile their expression during silkworm metamorphosis ,36. The th stage . We deteth stage ,36 needsB. mori. Our study represents the first exhaustive expression profiling of miRNAs in the major developmental stages. We discovered 14 novel miRNAs in the silkworm that are potentially species-specific, identified 101 conserved miRNAs, monitored the dynamic changes in miRNA levels during metamorphosis, and identified miR* species that accumulate at higher levels than their corresponding miRNAs. The accumulation of miR*appears to be regulated in a stage-specific manner. In addition, we found novel antisense miRNA loci in silkworm. Future identification of the miRNA targets should enable us to integrate the data into post-transcriptional regulatory networks important for silkworm metamorphosis, as well as other economically important traits such as silk synthesis.Deep sequencing of small RNAs from four distinct developmental stages of the silkworm , each with distinct anatomy, morphology and physiology, allowed us to comprehend changes in the miRNA expression profiles during development and to discover novel miRNAs in B. mori eggs and diet were purchased from Carolina Biological Supply  and larvae were reared at ambient temperature. Whole insects in the feeding larval (4th and 5th instars), spinning larval, pupal, and adult stages were separately frozen in liquid nitrogen and grinded for total RNA extraction using Trizol reagent . Total RNA was dissolved in de-ionized formamide for effective RNA denaturation. Small RNAs in the desired size range (15-30 nt) were purified from denaturing 15% polyacrylamide gel and sequentially ligated with the 3' and 5' adapters as described previously [eviously . ReverseB. mori.Our computational methods for analyzing the small RNA library was reported previously . In brieB. mori downloaded from Silkworm Genome Database [B. mori.The remaining unique small RNAs were aligned to genome sequences of Database  and for Database . CandidaR.S. designed the research, analyzed the data, coordinated the project and wrote the paper. G.J. constructed the small RNA library. Y.Z. and W.Z. performed the computational analysis. N.S and H.J. reared the insects and isolated total RNA used for library construction. H.J. also edited the manuscript. J.L.S. and E.A helped with the analysis and interpretation of the computational dataset. All authors read and approved the final manuscript.Predicted targets for the newly identified miRNAs and miRNA*s in silkworm. For each predicted target, accession number, annotation and hit scores are provided.Click here for fileTop 20 most abundantly expressed miRNAs in silkworm during different developmental stages. Proportion of reads recovered for miRNAs in each of the four developmental stages, i.e., feeding larvae, spinning larvae, pupae and moth are provided.Click here for file


Cyanobacteria are model organisms for studying photosynthesis, carbon and nitrogen assimilation, evolution of plant plastids, and adaptability to environmental stresses. Despite many studies on cyanobacteria, there is no web-based database of their regulatory and signaling protein-protein interaction networks to date.Synechocystis sp. PCC 6803. It predicts the protein-protein interactions using public interaction databases that contain mutually complementary and redundant data. Furthermore, SynechoNET provides information on transmembrane topology, signal peptide, and domain structure in order to support the analysis of regulatory membrane proteins. Such biological information can be queried and visualized in user-friendly web interfaces that include the interactive network viewer and search pages by keyword and functional category.We report a database and website SynechoNET that provides predicted protein-protein interactions. SynechoNET shows cyanobacterial domain-domain interactions as well as their protein-level interactions using the model cyanobacterium, Synechocystis proteins. SynechoNET is freely available at  or directly at .SynechoNET is an integrated protein-protein interaction database designed to analyze regulatory membrane proteins in cyanobacteria. It provides a platform for biologists to extend the genomic data of cyanobacteria by predicting interaction partners, membrane association, and membrane topology of  Synechocystis sp. PCC 6803 (henceforth referred to as Synechocystis) was determined at Kazusa DNA Research Institute [Synechocystis has been highlighted to integrate "omics" data in systems biology field [Cyanobacteria are prokaryotic microorganisms that perform plant-like photosynthesis as well as carbon and nitrogen assimilation to obtain intracellular energy. Since cyanobacteria are believed to be a prototype organism that changed the ancient anoxygenic environment to oxygenic by photosynthesis, many scientists have used cyanobacteria as an ideal model organism to study adaptation to various abiotic environmental stress . Furthernstitute , which anstitute -8, Synecgy field . HoweverSynechocystis  and were aligned with SCOP (version 1.69) domains using the PSI-BLAST algorithm with a common expectation value  cutoff of 0.001. By applying SCOP domain interaction pairs obtained from the PSIMAP-based interaction information database, PSIbase (build 3) [Synechocystis.3,672 build 3) , 12,748 Synechocystis proteins were collected from SwissPfam [Synechocystis were mapped to Pfam domain-interacting partners from iPfam (version 19), resulting in the construction of 13,448 predicted protein-protein interactions involving 1,541 proteins. They account for 42% of all proteins in Synechocystis. Likewise, Synechocystis proteins were mapped to Pfam domain-interacting partners from InterDom, resulting in 80,319 predicted protein-protein interactions involving 1,760 proteins. They account for 48% of all Synechocystis proteins. Furthermore, 2,658 proteins comprising 72% of all Synechocystis proteins were involved in 26,805 protein-protein interactions directly obtained from STRING (version 6.3). Taken together, SynechoNET revealed that 2,930 proteins participate in 109,532 protein-protein interactions. It is noteworthy that they comprise 79% of all proteins in Synechocystis.Pfam domains of all the wissPfam . All proin silico prediction of protein-protein interactions, we used the number of databases that provide supporting evidence for each interaction as well as the reported reliability scores from InterDom and STRING. As a filter, we selected 509 Synechocystis proteins participating in 1,591 high-confidence protein-protein interactions that were commonly found in all the databases encompassing PSIMAP, iPfam, InterDom, and STRING. Those were further rescaled into the confidence range from 0.0 to 1.0 using the arithmetic means of InterDom and STRING scores. The resultant high-confidence protein-protein interaction network was dynamically visualized in Java applet viewer, a modified version of the public Integrator program [To denote the confidence of the  program .In addition to the interaction information of SynechoNET, it was reinforced to contain the information on membrane proteins that includes transmembrane topology, signal peptide, and domain structure information provided by Phobius and the prokaryotic version of Localizome program. The Localizome program gives an advantage for users to see the transmembrane topology and domain structure of cyanobacterial proteins at a glance.SynechoNET provides user-friendly web interfaces by (i) keyword search Figure  includinSynechocystis positive phototaxis using yeast two-hybrid analysis. The result showed that the hybrid sensory kinase Sll0043 strongly interacts with cognate response regulators, Sll0038 and Sll0039 (data not shown). These experimental protein-protein interactions were consistent with the high-confidence prediction result of SynechoNET. On the other hand, in the analysis of membrane protein complexes of Synechocystis, we found evidence that photosystem II D2 protein (Sll0849) and cytochrome b6 protein (Slr0342) interact directly with photosystem D1 protein (Sll1867) and cytochrome b6f complex subunit 4 (Slr0343), respectively. Furthermore, the experimentally-verified nine transmembrane helices of MntB protein encoded by sll1600 [in silico protein-protein interaction and transmembrane topology information provided by SynechoNET is useful and reliable for the functional genomics study of Synechocystis.To validate SynechoNET, we examined the interactibility between histidine kinase and response regulators involved in  sll1600  was alsoSynechocystis. SynechoNET can be used for the analysis of regulatory membrane proteins by predicting transmembrane topology and domain structure. In particular, approximately one third of the Synechocystis proteome are left to be fully annotated. Thus, SynechoNET can help biologists to annotate them by analyzing their predicted interaction partners, membrane association, and membrane topology.SynechoNET is a database and website that provides predicted protein-protein interactions. It integrates public protein-protein interaction databases that contain mutually complementary as well as redundant data. It is designed for biologists who are interested in the unicellular cyanobacterium  and directly at . All the generated protein-protein interaction lists in tab-delimited and Cytoscape [. The dynamic interaction viewer based on Java applet technology requires Java-enabled web browsers.SynechoNET is freely available at ytoscape  formats The authors declare that they have no competing interests.WK constructed the database, developed the website, and helped to draft the manuscript. SK designed the system, coordinated the project, and wrote the manuscript. BK helped to extract the interaction list. JO manually validated the interaction list. SC modified and improved the Java applet viewer. JB conceived and directed the study and helped to draft the manuscript. JC directed the project, participated in the system design, and helped to draft the manuscript. All authors read and approved the final manuscript.


In recent years, the maturation of microarray technology has allowed the genome-wide analysis of gene expression patterns to identify tissue-specific and ubiquitously expressed ('housekeeping') genes. We have performed a functional and topological analysis of housekeeping and tissue-specific networks to identify universally necessary biological processes, and those unique to or characteristic of particular tissues.We measured whole genome expression in 31 human tissues, identifying 2374 housekeeping genes expressed in all tissues, and genes uniquely expressed in each tissue. Comprehensive functional analysis showed that the housekeeping set is substantially larger than previously thought, and is enriched with vital processes such as oxidative phosphorylation, ubiquitin-dependent proteolysis, translation and energy metabolism. Network topology of the housekeeping network was characterized by higher connectivity and shorter paths between the proteins than the global network. Ontology enrichment scoring and network topology of tissue-specific genes were consistent with each tissue's function and expression patterns clustered together in accordance with tissue origin. Tissue-specific genes were twice as likely as housekeeping genes to be drug targets, allowing the identification of tissue 'signature networks' that will facilitate the discovery of new therapeutic targets and biomarkers of tissue-targeted diseases.A comprehensive functional analysis of housekeeping and tissue-specific genes showed that the biological function of housekeeping and tissue-specific genes was consistent with tissue origin. Network analysis revealed that tissue-specific networks have distinct network properties related to each tissue's function. Tissue 'signature networks' promise to be a rich source of targets and biomarkers for disease treatment and diagnosis. The issue of tissue and cell-type specificity of gene expression is central to human biology and biomedicine. It impacts such fundamental problems as tissue ontogenesis, evolution and carcinogenesis. It is generally believed that the most relevant disease biomarkers and drug targets predominantly are found among proteins specific for the tissue the disease affects. In 1965 Watson et al. described genes universally expressed to maintain cellular functions as 'housekeeping' (HK) genes -8, identHere we report the identification of 2374 HK genes, based on a novel study of gene expression in 31 human tissues on a whole-genome ABI array. We set up a definitive 'HK baseline' for gene expression across tissues, and compared previously published HK data sets with ours. We also identified gene sets uniquely expressed in individual tissues and groups of tissues, and generated tissue-specific merged metabolic/signaling 'signature networks'. Both HK and tissue-specific genes were subjected to a comprehensive, three-phase functional analysis, including gene set enrichment across four ontologies, network topology analysis and tissue-specific network analysis. We clustered tissues into groups according to expression patterns and network parameters, and revealed associations between HK and tissue-specific genes with human diseases and drug targets.Whole-genome gene expression was analyzed in 31 human tissues using an ABI human genome array with probe sets for 27,868 individual human genes. A relatively conservative ten-fold 'signal-to-noise' ratio (SN10) across all three replicate arrays for each tissue was applied to identify the transcripts present in all tissues . The SN10 HK set comprised 2374 genes  were defined as tissue specific. Tissue-specific sets varied in size from four genes for thymus to 484 genes for testis, with an average size of 43.8 genes, or 30.9 genes for somatic tissues only (excluding testis) Table . The gen cut-off t-test, was also applied to identify tissue-specific genes. Functional analysis showed striking similarities in the biological processes encompassed in the genes specific to each tissue between the t-test and the threshold-based methods . This vital, ATP-yielding pathway is the terminal part of cellular respiration, in which electrons are transferred through the electron transport chain (ETC). Almost all subunits of the ETC protein complexes were expressed in the HK set . Ubiquitin-proteasomal proteolysis was among the highest scored GG process networks (p < 10–26), with virtually all essential proteins included in the HK gene set  and breast neoplasm (p < 10–14) were the top-scored diseases  processes, GeneGo (GG) process networks, and diseases, using the hypergeometric distribution  and Genet Figure . The sect Figure . GG proct Figure , with mas p < 10–6, with ves Table .Using the GSEA procedure  across tp-value distributions of top-scored pathways, processes and diseases were strikingly consistent with the tissue of origin. For instance, 190 retina-specific genes were enriched for eye-specific categories in all four ontologies  and slightly lower in the IN and OUT component than a random gene set of the same size ; intestine and adult kidney in OUT component (p < 0.03 and p < 0.07), and skin (p < 0.1) in IN component . Intestine, liver and adrenal gland featured the highest fraction of enzymes. Fetal tissues were highly enriched in transcription factors, and retina and brain in membrane receptors  network algorithm in MetaCore. AN is a version of the 'shortest path' algorithm, optimized for larger data sets. AN generates overlapping sub-networks of up to 50 nodes and calculates enrichment of the networks with input data and canonical pathways ,18. UnliTissue gene expression patterns were clustered by Euclidean distance across the average normalized probe intensities of the replicate hybridizations for each tissue  Figure . Most tiWe compared the distribution of drug targets between HK and tissue-specific protein sets. Two sets of drug targets were compiled: 'therapeutic targets' – a set of 104 direct protein targets of commercially available drugs, and 'xenobiotic targets' – 518 proteins known to interact with xenobiotics. Therapeutic drugs are defined as key proteins or genes directly affected by a marketed or withdrawn drug from the market. Xenobiotic targets are proteins and genes known for physical interactions with a large set of bioactive compounds including drugs, drug candidates and lead compounds.On average, therapeutic targets were twice as prevalent among tissue-specific proteins than HK proteins   and statistical measures such as ANOVA and nly used ,20. Renenly used ,22 follonly used . It was t-test) methods to determine tissue specificity of gene expression. Although the resulting datasets did not overlap well in gene content (6% on average), they were highly similar by functional analysis. Importantly, the intersections between distributions of entities within functional ontologies for SN10 and t-test sets were substantially larger than between genes  and statistical , and many of these genes belong to our HK set, confirming the functional integrity and completeness of the HK set.Recently it was shown in line with our results that the number of HK genes is larger than previously reported ,25. Our p-values or GSEA analyses were employed. This important observation adds confidence to the conclusions drawn from the analysis of tissue-specific gene expression, and can be applied similarly to other studies of differentially expressed genes and proteins. It also will facilitate the development of a comprehensive set of 'meta-ontologies' derived from an understanding of the interplay and crossover between the different types of ontological categories available, leading to improved understanding of biological systems, and the identification of biologically meaningful biomarkers and new therapeutic targets.Ontology enrichment in tissue-specific genes was strikingly different from HK genes, and in most cases strikingly consistent with the tissue's function. Retina genes were enriched with such processes as visual perception, detection of visible light, detection of light stimulus and eye disease-related genes, and testis genes scored highest for reproductive processes, cell cycle, cell division and male reproductive diseases, for example. Importantly, almost all tissues featured a high level of consistency between the highest-scored categories in different ontologies. Further, consistency was observed whether hypergeometric Network analysis  showed tp-values  should be considered valid, although such ad hoc cut-offs may eliminate many condition-relevant genes from analysis. Functional analysis provides a different level of validation for such datasets by consideration of functional biological units. For instance, the retina-specific gene set is enriched in vision-related pathways, GO and GG ontologies, and diseases. The same set is enriched with OUT network component, and the protein class 'receptors'. Self-assembling retina-specific networks reconstruct rhodopsin-stimulated signal transduction and key steps in the metabolism of rhodopsin and its co-factors. Although the retina gene set is too small to achieve p-values < 0.01 in most of these analyses, the combination of independent functional evidence builds a strong case for its relevance and importance as a highly likely source of specific biomarkers for eye conditions and ophthalmic drug response. The comprehensive biological interaction data in MetaCore allows multiple 'high-content' data types to be mapped onto networks. Gene expression data, proteomic, single nucleotide polymorphism and metabolomic data all can be addressed, visualized and used in network construction and analysis, independently or in concert ['Multi-dimensional' functional analysis of gene expression data adds to the ongoing debate on proper statistical procedures for gene set selection. A 'rule of thumb' opinion is that only gene sets and distributions with low  concert , making Tissue-specific gene sets  were twice as enriched in drug targets as the HK set. 'Therapeutic' targets comprised up to 25% of mammary gland and thymus-specific proteins. Most of the identified tissue-specific drug target genes are targets to drugs whose mechanism are consistent with the tissues. For example, three brain-specific proteins identified to have at least one drug target are GABA-A receptor beta-2 subunit, Na (V) I alpha and SCN10A. GABA-A receptor beta-2 subunit is a target for clomethiazole (sedative and anticonvulsant), Na (V) I alpha is a target for drugs such as levetiracetam (epilepsy), tetrodotoxin (anesthetics), toprimate (anticonsulvant) and SCN10A is target for bupivacaine racemic (anesthetics) and lidocaine (anesthetics).Tissue-specific proteins and their pathways and networks are therefore a potentially rich source of targets and biomarkers for disease treatment and diagnostics. The addition of data on which network components are detectable in blood and other readily accessible body liquids makes tissue-specific networks exceptionally attractive for the identification of putative biomarkers of tissue-specific disease, drug-response or toxicity.RNA from 31 normal human tissue RNAs (Table The Applied Biosystems Human Genome Survey Microarray (P/N 4337467) contains 31,700 60-mer oligonucleotide probes representing 27,868 individual human genes. Digoxigenin-UTP labeled cRNA was generated and amplified from 1 μg of total RNA from each sample using Applied Biosystems Chemiluminescent RT-IVT Labeling Kit v 1. 0 (P/N 4340472) according to the manufacturer's protocol P/N 4339629). Fifteen micrograms of DIG-labeled cRNA was hybridized for 16 hrs at 55°C and chemiluminescence detection, image acquisition and analysis were performed using Applied Biosystems Chemiluminescence Detection Kit (P/N 4342142) and Applied Biosystems 1700 Chemiluminescent Microarray Analyzer (P/N 4338036) following the manufacturer's protocol (P/N 4339629). Images were auto-gridded and the chemiluminescent signals were quantified, background subtracted, and finally, spot- and spatially-normalized using the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer software v 1. 1 (P/N 4336391). Probe signals were normalized using the Limma method [. FifteenA 'stringent' set of HK genes expressed in each of the 31 tissues was identified by applying cut-off to the ABI-calculated signal-to-noise (S/N) for each probe. The S/N is a metric that captures the confidence of the measurement 'detectability' above all known sources of noise. S/N is commonly used to bin genes or probes as 'Present' or 'Absent' at a desired level of confidence. Since the S/N expresses the number of standard deviations, the associated confidence can be looked up from a probability table for a normal distribution. For example, signals with S/N ≥ 3 have > 99.9% confidence in the measurement. For our purposes in determining whether a gene was expressed in a given tissue, we additionally took into account the inflection points where the slope of the S/N curve significantly changes. We applied the threshold across all three replicate hybridizations on a probe-by-probe basis to identify the genes expressed in each tissue with a high level of confidence. The overlap between all 31 sets, that is, genes consistently expressed in all tissues, defines the HK set /2 is the maximum number of such links, the clustering coefficient is a number between 0 and 1. The average clustering coefficient is obtained by averaging over the clustering coefficient of individual nodes. A network with high clustering coefficient is characterized by highly connected sub-graphs.where The enrichment levels of the HK genes and tissue-specific genes in the different parts of the network  and in different protein classes were calculated using the hyper-geometric distribution:p-value corresponding to the enrichment-level according toWe calculated the k or more marked elements in a sample of size n by random selection.which gives the probability of having The authors declare that they have no competing interests.TN, YN and ZD conceived the study and designed research. ZD drafted and YN wrote the final manuscript. RJB contributed to writing and editing of the manuscript. ZD, WS and ES performed the statistical analysis of the data. TS, DD, AB, AG, ES, ZD and YN performed research. ER contributed with making the figures in the manuscript. KL, JB, RRS designed and carried out the experimentList of all housekeeping genesClick here for fileList of all tissue-specific genesClick here for fileTable for intersection between genes, ontologiesClick here for fileComplete enrichment analysis for all housekeeping setsClick here for fileCanonical pathway maps and GeneGo networks essential for growth and viabilityClick here for fileEnrichment analysis of the unique parts of the housekeeping gene setsClick here for fileComplete enrichment analysis of all tissues in all four ontologiesClick here for fileGene Set Enrichment Analysis for selected tissues, ontologiesClick here for fileNetwork topological properties of all housekeeping setsClick here for fileTissue-specific connectivityClick here for fileTable for interactions mechanismsClick here for fileTable for component analysis, protein class analysis for all tissuesClick here for fileMC legend with protein classesClick here for fileList of genes specific for tissue pairs and tripletsClick here for fileDrug targetsClick here for fileTissue-specificity distribution of all genesClick here for fileDefinition of tissue specific and housekeeping genesClick here for file


Anti-inflammatory actions of two anti-allergic drugs, alone or with dexamethasone (Dex) were examined in two models, because inflammation is claimed to be important for allergic events, especially for asthma. Cromoglycate and nedocromil were tested in ischaemic- and histamineinduced paw oedema models of mice. These antiallergic drugs  failed to suppress these oedemata, but enhanced the suppressions by a low dose of dexamethasone  at 3–8 h after Dex injection. The mode of effects by anti-allergic drugs resembled that of a natural antioxidant , and was different from that of an immunosuppressant like FK506. The enhancing potencies of the two anti-allergic drugs were similar at 6 h after Dex in both oedemata, and were diminished by superoxide dismutase (SOD) or catalase (i.p.). Cycloheximide completely abolished suppressions. Nedocromil, but not cromoglycate, inhibits inflammatory events. Therefore, there are common unknown actions by which the two anti-allergics enhance suppression by Dex. A possible mechanism of this action was supposed to enhance the superoxide and/or hydrogen peroxide-dependent glucocorticoid receptor (GR) signalling in the target cells.


Identifying the determinants of health-related quality of life (HRQOL) in patients with systolic heart failure (CHF) is rare in primary care; studies often lack a defined sample, a comprehensive set of variables and clear HRQOL outcomes. Our aim was to explore the impact of such a set of variables on generic and disease-specific HRQOL.In a cross-sectional study, we evaluated data from 318 eligible patients. HRQOL measures used were the SF-36  and four domains of the KCCQ . Potential determinants (instruments) included socio-demographical variables , clinical ), depression (PHQ-9), behavioural (EHFScBs and prescribing) and provider (e.g. list size of and number. of GPs in practice) variables. We performed linear (mixed) regression modelling accounting for clustering.Patients were predominantly male (71.4%), had a mean age of 69.0 (SD: 10.4) years, 12.9% had major depression, according to PHQ-9. Across the final regression models, eleven determinants explained 27% to 55% of variance : Depression ; age ; multimorbidity ; list size ; SES ; and each of the following once: no. of GPs per practice, NYHA class, COPD, history of CABG surgery, aldosterone antagonist medication and Self-care (0.1/-0.2/-0.2/0.1/-0.1/-0.2).HRQOL was determined by a variety of established individual variables. Additionally the presence of multimorbidity burden, behavioural (self-care) and provider determinants may influence clinicians in tailoring care to individual patients and highlight future research priorities. Chronic systolic heart failure (CHF) is a common clinical syndrome, with increasing incidence at older age, and is associated with high mortality rates, and compromised health-related quality of life (HRQOL) . MoreoveThe objectives of CHF treatment are to maximise life expectancy, improve HRQOL and prevent disease progression and admissions . OptimalHRQOL is a multidimensional concept comprised of several domains, including physical/biological factors, symptom status, functional status, health perceptions, and overall well-being . In rese2, multimorbidity, BNP [Many previous studies have deepened the understanding about what factors can determine HRQOL in CHF, not least to identify intervention targets for improved outcome. They have been performed in various sectors and settings, mostly in secondary care or post-discharge setting -22, someity, BNP -17,20-25ity, BNP ) determiity, BNP ,25 vs. sity, BNP ,23,24) lGiven there is little evidence for patients with CHF recruited in primary care, our aim was to identify and explore the impact of determinants of generic and disease-specific HRQOL with respect to a wide set of individual and provider variables. We focused on an exploratory comparison between generic and disease-specific HRQOL.subproject 10 "quality of life" within the German "Competence Network Heart Failure", sponsored by the Federal Ministry of Education and Research [This study was conducted as a cross-sectional study of pooled baseline data of Research . Within Research -30. BothResearch  and wereInterested GPs were eligible for participation if they were certified as a primary care physician or equivalent and practiced as a statutory health insurance affiliated physician. Fifty general practitioners (GPs) from 48 practices participated in the two studies in one region of Northern Baden, Germany.Eligible patients were adults ≥ 40 years with confirmed systolic heart failure (CHF) with stable symptoms at the time of inclusion, and diagnosis of a chronic, irreversible CHF at least 2 weeks prior to inclusion. CHF diagnosis was based on dyspnea (NYHA) and objective measurement (e.g. by echocardiography) of impaired left ventricular systolic dysfunction (left ventricular ejection fraction ≤ 45%). Criteria slightly differed between the TTT-trial and HICMan-trial regarding the cut-off and actuality of the determination of left ventricular ejection fraction  and dyspnea . Exclusion criteria were: Primary valvular heart diseases and relevant hemodynamic effects, hypertrophic obstructive/restrictive cardiomyopathy (HOCM/RCM), and people with a concomitant terminal illness, addictive disorders , dementia or severe psychological illness ,30.All GPs and patients gave written informed consent. 367 eligible patients were recruited, 168 within TTT (enrolment of patients and data collection: March to September 2005) and 199 within HICMan (enrolment of patients and data collection: June 2006 to January 2007), details have been described elsewhere ,30. WithGPs received an initiation visit by a study nurse including an introduction to the trial's investigator file. GPs collected and documented organisational , physicians' individual (e.g. years in practice) and patients' individual clinical data  on pre-specified case report forms (CRFs) according to the Basic Clinical Dataset (BCD) of the Competence Network Heart Failure . The docParallel to the clinical baseline assessment, patient-reported questionnaires were handed out by practice personnel.A sociodemographic dataset was obtained from all patients . For genDepression was assessed using the German version of the Patient Health Questionnaire depression module (PHQ-9) . It consThe European Heart Failure Self-Care Behaviour Scale  is a 12-item, self-administered questionnaire regarded as a valid, reliable and practical scale to measure the self-reported self-care behaviour of heart failure patients, for example, daily weighing, fluid restriction, exercise or contacting a health care provider . Scores To allow comparisons across the two HRQOL measures we decided a priori to focus on summary measures, i.e. the PCS and MCS (SF-36), and Functional status,  Quality of life, Self efficacy and Social function (KCCQ), all distinct domains that differentiate most adequately between 'physical' and 'psychosocial' aspects of HRQOL. Therefore, we omitted KCCQ-os as it represents aggregated scales of both aspects, rendering a comparison with generic HRQOL (PCS and MCS) difficult.Our choice of variables to be analysed with respect to their predictive value was based on the literature and clinical reasoning. We selected variables of the provider and individuals respectively as shown in tables Dummy variables were built for all ordinal variables . We aggregated NYHA functional class I with II and II with IV accounting for the low number of observations in the classes I and IV. As NT-proBNP had a skewed distribution, logarithmic transformation was performed as described and performed previously ,56. AlcoPearson correlation coefficients were calculated for analyses of relationships between numerical explanatory variables and the dependent variable. In the following, Creatinin-clearance was omitted due to collinearity with age, which correlated consistently higher with dependent variables.2) we present the results of the unified regression models. For analyses we used Stata/MP version 10.1, SPSS version 16.0.2 (SPSS Inc.) and SAS 9.2 (PROC MIXED).Variables with a p-value less than 0.05 were entered into multiple regression analyses using the forward selection algorithm. As this procedure uses only those individuals who have complete information on all explanatory variables results were validated in unified regression models. All explanatory variables remained within the models, except for NYHA functional class for PCS and Hypertension for  Quality of life (KCCQ). To account for the clustering of data (intraclass correlation within each practice attributable to clustering) we performed linear mixed effects regression models with the physician as a random effects model nested within groups. Regression coefficients were the same; the few exceptions regarding explanatory variables are reported. Given the possibility to inform about the amount of explained variance , KCCQ domains and summary scores are shown in Table Table S1  variables, i.e. a greater (patient) list size impacted negatively on the MCS and Self efficacy, whereas a higher number of physicians per practice determined MCS positively.Among sociodemographic factors, age remained in four of the overall six models , and a higher socio-economic status in PCS and Self efficacy (β: 0.14 to 0.23).NYHA functional class contributed only to the explained variance of the disease-specific summary scale Functional Status (β: -0.16), whereas COPD for the generic summary PCS (β: -0.15).A history of CABG surgery determined the generic HRQOL summary MCS positively (β: 0.09) and the prescription of an aldosterone antagonist the KCCQ domain Social limitation negatively (β: -0.13). Aggregated physician-rated multimorbidity (CIRS-G) contributed to the explained variance in generic and specific scales  Quality of life and Social limitation; β: -0.09 to -0.14).Better self-care behaviour  contributed significantly to the KCCQ domain Self efficacy (β: -0.21).Depression severity (PHQ-9 summary score) was a significant determinant of generic and disease-specific HRQOL in all models, and had the strongest impact (β: -0.3 to -0.72) on each summary scale of HRQOL in comparison to other determinants.In a clearly defined convenience sample of outpatients with stable systolic chronic heart failure (CHF), we could explore determinants of generic and disease-specific health-related quality of life (HRQOL) using a wide set of explanatory variables. Generic and disease-specific HRQOL varies but is considerably impaired in these patients. They demonstrated good Heart failure self-care behaviour but a considerable burden of (multi-)morbidity. We explored the impact of sociodemographic variables, objective measures of heart failure severity, somatic and depressive comorbidity, behavioural and provider variables on the variance of generic and disease-specific HRQOL in patients with CHF using multiple linear regression analyses. Eleven determinants were independently associated with generic and/or disease-specific HRQOL: These were depression severity, physician-rated morbidity burden, increasing age, bigger list size, higher NYHA functional class, COPD, prescription of aldosterone antagonist determined worse, whereas more GPs (per practice), a higher socio-economic status, better Self-care and history of CABG surgery determined better HRQOL.A considerable number of studies have led to conceptual models of HRQOL in relation to CHF that describe the interactive relationships between pathophysiology, symptoms , functional and psychological aspects . AccordiOur results regarding the impact of objective measures such as EF and BNP on HRQOL are similar to previous literature: For example, while BNP correlated bivariately with HRQOL scales, it did not remain in the multivariate regression analyses with included (known) correlates such as NYHA functional class. Therefore, decreased EF or elevated BNP seems not to be sensed by the individual, but the associated symptoms or functional status ,17.Depression severity as assessed by the summary score of the PHQ-9 had by far the greatest impact on HRQOL variance in all six investigated summary scores or domains, a finding that is in line with previous findings: Gott et al. found depression, measured by a geriatric depression scale, determining generic and specific HRQOL (summary scores of SF-36 and KCCQ) in a cross-sectional study with 542 elderly (mean age 77 years) patients in primary care, a sample where the diagnosis of CHF was validated by the GP . In our In this study, NYHA functional class determined KCCQ Functional Status, but not the Physical Component summary of the SF-36. There, COPD, a disease characterised also by 'dyspnea' was independently associated. These findings are supportive for the higher specificity of the KCCQ than the SF-36 with regard to the cardinal symptoms of CHF.Moreover, our results regarding the role of disease severity (NYHA class) and Depression (PHQ-9 summary score) in disease-specific HRQOL (KCCQ) are in line with Faller et al. . They in2 0.18), but not within the MCS [Notably, physician-rated overall morbidity burden (multimorbidity) was considerable (CIRS summary score) and determined HRQOL in 4 of 6 investigated models, i.e. in the PCS of the SF-36, but not MCS, and in all KCCQ models, except for Self efficacy. Studies that investigate co-/multi-morbidity in relation to HRQOL of patients with CHF usually account for single diseases and/or count the no. of conditions and rarely used the CIRS instrument: We included the CIRS additionally to certain single conditions as a physician-rated disease severity aggregate accounting for all body systems would better reflect the patients' disease burden. Regarding generic HRQOL, our results are consistent to a study in primary care with a sample of 238 patients with chronic diseases: The CIRS played a role within the PCS  impacted on KCCQ Self efficacy and SF-36 PCS. Little is known regarding this aspect in relation to CHF. In the study of Gott et al. lower SES impacted negatively on MCS and KCCQ overall scores , and eduA larger practice list size was associated with worse HRQOL (MCS and Self efficacy), whereas a higher number of GPs per practice counterbalanced this observed association (in MCS), which is a new finding, as there is little evidence for the impact of organizational aspects, i.e. practice factors, on HRQOL in CHF in routine care. Moreover, our variables on organization  were not comprehensive.However, some literature on practice performance and patient satisfaction shows associations within the organization of primary care that cannot be fully explained -70: In asureness on what to do if heart failure worsens and about the patient's understanding of the ability to prevent worsening of CHF . According to the European Clinical Practice Guideline for CHF, HF self care can be defined as action aimed at clinical stability, avoidance of behaviour that can worsen the condition, and early detection of symptoms of deterioration. Self care management is regarded as a key issue of successful treatment and can significantly impact on health outcomes [The EHFScBS scores that aggregate the actual patient-reported self-care behaviour 12 items) were associated with KCCQ Self efficacy (2 items), as expected, although the two instruments ask not completely the same concepts: The instruments differ in that the EHFScBS asks for patient's agreement on defined 12 behaviours, and the KCCQ Self efficacy asks about patient's  items weoutcomes . The autA number of limitations should be stated: The cross-sectional design of our study implies that no cause-effect relationship between variables can be established. The explorative approach rather implies the identification of independent associations, whereas predictive value needs to be validated in consecutive studies. A known dilemma exists between internal validity and generalisability: Participants of the parental trials might not be representative for the population, e.g. because elderly patients may decline to participate because of effort. On the other hand, it can be regarded as strength - contrary to most other studies in primary care - the sample consisted of patients with defined CHF, so more valid statements can be made regarding this patient group.Most complete models were obtained for KCCQ Functional Status and  Quality of life, whereas most missings pertained to SF-36 summary scores, socio-economic status and self-care. We abstained from imputation methods as they have their own limitations. We did not perform a non-responder analysis, but regard our approach in the context of exploration as appropriate.Considerably impaired and varying health-related Quality of life (HRQOL) of patients with systolic heart failure in primary care could be explained by known determinants in different patterns and to various extents across generic and disease-specific concepts of HRQOL, such as age, social status, depression symptoms and disease severity. Patients' perceptions of disease severity  were the strongest determinants, whereas, physician-rated multimorbidity (morbidity burden) also impacts independently on HRQOL - more than findings in previous studies suggest. New potentially relevant variables, i.e. organisational aspects of primary care, need to be confirmed in future studies. Our results also corroborate the ongoing challenge of holistic care for elderly (multimorbid) patients (with CHF) in primary care. Information about patient and provider-related determinants of HRQOL in patients with CHF may help in providing individually tailored care.A2RA: Angiotensin-2 receptor antagonist; ACE: Angiotensin converting enzyme; CCCT: Coordination Centre Clinical Trials; CABG: Coronary artery bypass graft surgery; CHD: Coronary heart disease; CHF: Chronic (systolic) heart failure; CI: Confidence interval; CIRS-G: Cumulative illness rating scale, geriatric; COPD: Chronic obstructive pulmonary disease; EHFScBS: European Heart Failure Self-care Behaviour Scale; GFR: Glomerular filtration rate; GP: General practitioner; HICMan: Heidelberg Integrated Case management; HRQOL: Health-related quality of life; ICD: Implantable cardioverter defibrillator; KCCQ: Kansas City Cardiomyopathy Questionnaire; LVEF: Left ventricular ejection fraction; LVSD: Left ventricular systolic dysfunction; NT-proBNP: N-terminal Brain Natriuretic Peptide; NYHA: New York Heart Association; MCS: Mental Component Summary (of SF-36); PAD: Peripheral arterial disease; PCS: Physical Component Summary (of SF-36); PHQ-9: Depression module of the Patient Health Questionnaire; PTCA: Percutaneous Transluminal Coronary Angioplasty; RCT: Randomised controlled trial; SF-36: MOS 36-item short-form health survey; TTT: Train the trainer The authors declare that they have no competing interests.FPK, CUK and TMT designed the study. CUK, GL and FPK analysed the results. All authors interpreted the results. FPK wrote the manuscript, and all authors contributed to writing revisions and approved the final manuscript. FPK is the guarantor.Table S1: Determinants of generic (SF-36) and disease-specific (KCCQ) health-related quality of life (HRQOL)Click here for file


The benefits and use of low-dose corticosteroids (LDCs) in severe sepsis and septic shock remain controversial. Surviving sepsis campaign guidelines suggest LDC use for septic shock patients poorly responsive to fluid resuscitation and vasopressor therapy. Their use is suspected to be wide-spread, but paucity of data regarding global practice exists. The purpose of this study was to compare baseline characteristics and clinical outcomes of patients treated or not treated with LDC from the international PROGRESS  cohort study of severe sepsis.Patients enrolled in the PROGRESS registry were evaluated for use of vasopressor and LDC  for treatment of severe sepsis at any time in intensive care units (ICUs). Baseline characteristics and hospital mortality were analyzed, and logistic regression techniques used to develop propensity score and outcome models adjusted for baseline imbalances between groups.P <0.001). Overall hospital mortality rates were greater in the LDC than in the non-LDC group . After adjusting for baseline imbalances, in all mortality models (with vasopressor use), a consistent association remained between LDC and hospital mortality (odds ratios varying from 1.30 to 1.47).A total of 8,968 patients with severe sepsis and sufficient data for analysis were studied. A total of 79.8%  of patients received vasopressors, and 34.0%  of patients received LDC. Regional use of LDC was highest in Europe (51.1%) and lowest in Asia (21.6%). Country use was highest in Brazil (62.9%) and lowest in Malaysia (9.0%). A total of 14.2% of patients on LDC were not receiving any vasopressor therapy. LDC patients were older, had more co-morbidities and higher disease severity scores. Patients receiving LDC spent longer in ICU than patients who did not (median of 12 versus 8 days; Widespread use of LDC for the treatment of severe sepsis with significant regional and country variation exists. In this study, 14.2% of patients received LDC despite the absence of evidence of shock. Hospital mortality was higher in the LDC group and remained higher after adjustment for key determinates of mortality. Much iciency) . The oveiciency) . The dif studies ,14 resul studies . The rec studies  suggeste studies ,18 evalu studies . In cont results , a recen results  found noThe global PROGRESS  registry was developed and designed to provide a description of the management and outcomes of severe sepsis in intensive care units, reflecting everyday clinical practice . AlthougPROGRESS was an international, non-interventional, multi-center, prospective, observational study of all age patients with severe sepsis treated in ICUs. Criteria for study entry included a diagnosis of severe sepsis defined as a suspected or proven infection and presence of one or more acute sepsis-induced organ dysfunctions, and treatment for severe sepsis in a participating ICU. Treatment was the standard of care at each participating ICU. Evaluations, procedures, or treatment beyond those used at each institution's standard of care were not performed. As a result, ethical review board approval and informed consent were not a uniform requirement, however, most countries obtained ethics review or approval to confirm that informed consent was not required. Data for routine clinical practice parameters were collected for qualifying patients. Clinical data collected, via a secure website, included patient demographics, co-morbid conditions, clinical features of severe sepsis patients, characteristics of infection, therapy and support care, and ICU outcomes. There were no study-specific interventions and no attempt was made to alter the treatment that patients received. The study was conducted at 276 study centers in 37 countries and data were collected from December 2002 until December 2005 with 12,570 adult patients with severe sepsis entered into the database. An independent international medical advisory board was involved in study development, decisions surrounding data use, and publications. The PROGRESS website was developed and maintained by Eli Lilly and Company. The Progress Advisory Board was responsible for the oversight of the publication of results from this study, and provided approval to access and retrieve data from the study database.Patients could be enrolled in the study only if they had a diagnosis of severe sepsis and were treated in the ICU. The definition of severe sepsis used in PROGRESS, previously described , includeData for each patient in the study were entered electronically by the participating physician or other investigative site personnel with an electronic data form via a dedicated, secure website. Patient identities were kept anonymous. Patients with records that remained incomplete due to data or technical limitations (n = 388) were not included in the reporting database. Safety information was not captured.The purpose of this sub-study is to describe the use of low-dose corticosteroids in adult patients with severe sepsis across ICUs globally, comparing baseline characteristics, as well as the hospital mortality rates in these patients. Patients who were identified as chronic steroids recipients, or who received high doses of steroids, were excluded from the sub-study.Summary statistics for demographic and clinical characteristics, co-morbid conditions, and supportive care were compared for low-dose corticosteroids use versus non-low-dose corticosteroids use overall, and for patients with and without vasopressors. Continuous variables were compared across treatment groups using non-parametric analysis of variance (ANOVAs) and qualitative variables were compared using the chi-square test.P-values less than 0.10 were selected for the logistic propensity model. A patient's propensity score is the conditional probability of receiving low-dose corticosteroids given their observed values of the 12 selected predictors in the propensity score model. The propensity score is a single number which synthesizes the effect of the 12 covariants on the probability of receiving low-dose corticosteroids. Patients were subdivided into quintiles based on their propensity scores and the propensity score quintile was used in logistic regression models of mortality. Additional details and discussion concerning propensity score development can also be viewed in Additional file Because of the non-randomized nature of this observational study, there could be baseline imbalances between the low-dose corticosteroid and non-low-dose corticosteroid treatment groups. This could lead to bias estimates of the effect of low-dose corticosteroids on mortality unless methods are instituted to control for potential confounders. To implement these adjustments, a two-step bias-removing procedure was performed. The first step of this procedure was to estimate a propensity score for each subject using logistic regression of treatment received on covariates ,24, withP-values are presented.In the second step of the statistical adjustment process, a set of logistic models were developed to assess the effect of treatment  on hospital mortality. In addition to treatment, models included propensity score quintiles, and factors that were significantly associated with mortality as additional covariates. In these multivariate logistic models, adjusted odds ratios of the effect of low-dose corticosteroid treatment on hospital mortality with corresponding 95% confidence intervals, and P-values for the propensity quintiles were tabulated alongside the P-values from the unadjusted baseline comparisons.To assess the degree to which the propensity score method was successful in the correction of the imbalance between the two treatment groups, the Kolmogorov-Smirnov test and the chi-square test were performed within each propensity score quintile, facilitating comparisons of the distributions of the continuous and qualitative variables within said quintiles. A total of 12,570 adult patients with severe sepsis were entered into the PROGRESS study database. Of these patients, 12,510 had complete data for both low-dose corticosteroid and vasopressor use. Of these patients, 8,968 did not receive chronic or high-dose steroids, and made up the patient population described in this sub-study  are presented in Table Table P <0.001), and spent more days on vasopressors , as shown in Table A description of the intensive care therapies that patients received is given in Table P <0.001), respectively, in patients receiving vasopressors and 27.4% (66/241) and 23.9% , respectively, in patients not receiving vasopressors. All patient mortality rates were greater in the low-dose corticosteroids group than in the non-low-dose corticosteroids group at 58.0%  versus 43.0% .Table Because of the noted imbalances in baseline characteristics  between those with and without low-dose corticosteroids therapy, mortality results for the two cohorts are not directly comparable. Therefore, multiple logistic regression models were developed utilizing propensity scores and independent mortality risk factors in an attempt to ameliorate the impact of observed differences between the two cohorts in this non-randomized comparison. Eleven models are presented in Table P = 0.248). It is interesting to note that the odds ratios were very similar in models with fewer factors  and in models with more factors included . All models also showed non-significant P-values (P > 0.05) for the Hosmer and Lemeshow Goodness of Fit test, indicating that there is insufficient evidence to reject the logistic regression models for lack-of-fit even in a very large dataset, thus implying that the models provide adequate fits to the data. Within each propensity score quintile, mortality was always higher in the low-dose corticosteroid group than in the non-low-dose corticosteroid group, with an increasing mortality trend across the propensity score quintiles  showed a consistent and significant association between low-dose corticosteroids and hospital mortality with odds ratios varying from 1.301  in Model 8 to 1.470 , in Model 6. The exceptions are Models 5 and 10, based only on the sub-populations of patients who did not receive vasopressors, with an odds ratio of 1.115  and 1.194 , respectively. This result is consistent with the unadjusted mortality results from Table Other Region group containing a small sample size (n = 162) and the least low-dose corticosteroid use (17.3%).Given the large regional and country variation in low-dose corticosteroids use and relative mortality rates, regional mortality comparisons are shown with low-dose corticosteroid and non-low-dose corticosteroid use by region. Results are indicated in Figure Figure PROGRESS is one of the largest global severe sepsis registries ever completed with 12,570 adult patients in 37 countries identified as having severe sepsis. Given the recent controversy over the use of low-dose corticosteroids for this deadly disease, our study provides important novel information on the use of low-dose corticosteroids in everyday clinical practice over several years, in addition to providing information on treatment variation across regions and countries. These results indicate widespread adoption of low-dose corticosteroids for the treatment of severe sepsis with significant regional and country variation, and increased hospital mortality in patients treated with low-dose corticosteroids, even after adjustment for baseline imbalances in disease severity.The emerging picture from the PROGRESS registry regarding low-dose corticosteroids use is an important one particularly when the controversy on their use remains present after contradictory results from trials, guidelines and meta-analyses -28. Hithet al. 2002 steroids results [It is likely that the use of low-dose corticosteroids increased in clinical practice following the publication of the Annane  results  and thei results , althoug results  did find results  may have results  and new  results .The present sub-study also demonstrates significant differences in most baseline characteristics between patients receiving low-dose corticosteroids and those that did not. Low-dose corticosteroid-treated patients were older, more likely to have undergone surgery (if receiving vasopressor therapy), have more organ dysfunction, and receive higher levels of therapeutic organ support and severe sepsis therapies, including drotrecogin alfa (activated). Patients receiving low-dose corticosteroids also received vasopressors for longer and spent longer in ICU than patients who did not receive low-dose corticosteroids. These differences point to a greater severity of illness in patients treated with low-dose corticosteroids. This was also seen from the results of steroid use in the PROWESS trial  which inet al. 2009 study [P = 0.02) and hospital mortality (P = 0.05) with low-dose corticosteroids given for ≥5 days [In-hospital mortality was higher in the low-dose corticosteroid group. Other epidemiological studies ,14 have 09 study  agree wi09 study ,17 demon ≥5 days , and in et al. 2009 meta-analysis [An important finding of this study was the relatively high incidence of low-dose corticosteroid use 14.2%) in patients with severe sepsis which did not require vasopressor agents. It is likely that the low cost of corticosteroids and physician comfort prescribing this therapy are significant factors in this inappropriate usage of low dose corticosteroids. Recommendations have stated that these patients should not receive steroids ,15. Beca4.2% in panalysis  showed nreal world clinical practice. It also included rigorous statistical model development, including propensity scores, to try and compensate for observed differences in disease severity. As with any observational study, there are inherent weaknesses with the PROGRESS study. The lack of a randomized control group clearly sets limitations to any inferences that might be drawn about the effect of low-dose corticosteroid use on clinical outcomes and the study was not prospectively designed to examine mortality by low-dose corticosteroid use. Other limitations include the fact that the PROGRESS registry was designed to describe global patterns of care in severe sepsis, not an assessment of low-dose corticosteroid use, and the exact timing, dose and duration of low-dose corticosteroid treatment and adrenal status was not assessed. Similarly, the exact timing, dose and duration of vasopressor treatment was also not recorded. It was perhaps surprising that fluid resuscitation was not reported in 32% of the vasopressor-no group receiving low-dose corticosteroids, raising the possibility that although sites were requested to record 'low dose steroids as treatment for severe sepsis' that in fact low-dose corticosteroid treatment was not being used as a specific treatment of severe sepsis in some patients. However, in the models including only vasopressor-no patients (Models 5 and 10), results were consistent with other models suggesting that the overall results were not driven by the vaspressor-no group. Although PROGRESS involved large patient numbers, significant levels of missing values resulted in small numbers for certain parameters. Country variability in the standard of care, the severity of cardiovascular organ dysfunction (except vasopressor requirements), and some characteristics, such as timing of organ dysfunction and of various treatments received, were not evaluated. Also relevant is that the various centers and countries were involved in the three-year study over different periods of time, which could have affected the standard of care with evolving practice . RegardThe PROGRESS registry has helped document information on the use of low-dose corticosteroids in severe sepsis in everyday clinical practice and on treatment variation across regions and countries. Approximately 14% of severe sepsis patients received low-dose corticosteroids despite never receiving vasopressors during their ICU stay. Low-dose corticosteroids patients were older, had more co-morbidities and higher disease severity scores. When adjustments were made for imbalances, mortality remained significantly higher in the group of patients receiving low-dose corticosteroids.• There is widespread adoption of low-dose corticosteroids for the treatment of severe sepsis with significant regional and country variation.• Approximately 14% of severe sepsis patients received low-dose corticosteroids despite never receiving vasopressors during their ICU stay.• PROGRESS registry patients treated with low-dose corticosteroids were older, had more co-morbidities and higher disease severity scores.• Mortality was higher, and remained higher after adjusting for key determinants of mortality, in the low-dose corticosteroids group.• Low-dose corticosteroids should not be used without careful patient selection.ANOVA: analysis of variance; APACHE II: Acute Physiology and Chronic Health Evaluation II; DAA: Drotrecogin alfa (activated); ICU: intensive care unit; LDC: low-dose corticosteroids; MAP: mean arterial pressure; OD: organ dysfunction; PROGRESS: Promoting Global Research Excellence in Severe Sepsis; SBP: systolic blood pressure; SOFA: Sequential Organ Failure Assessment; SSC: Surviving Sepsis Campaign; VTE: venous thromboembolism.Drs. Beale, Levy, Martin and Profs. Dobb, Ramsay, Vallet, Vincent and Reinhart have all served as consultants to and participated in Eli Lilly and Company sponsored trials. Prof. Brunkhorst received research grants from Eli Lilly Deutschland GmbH. Dr. Martin's institution received funding for Dr. Martin to conduct clinical trials with Eli Lilly and Company. Dr. Silva has served as a consultant for Eli Lilly, Brazil, and Prof. Sprung has received lecture fees and served as a consultant for Eli Lilly and Company. Drs. Costigan, Leishman, Williams and Janes are employees and stockholders of Eli Lilly and Company.RB, FB, GD, ML, GM, GR, ES, BV, J-LV, MW and KR participated in the conception and design of the PROGRESS registry. RB, JJ, CS, TC and KR contributed to the development and conduct of the principle analyses in this sub-study. All authors contributed to drafting and critically revising the manuscript and read and approved the final version of the manuscript.Supplementary tables S1 to S3. A Word file containing further clinical information, and the following tables: Table S1: Organ Dysfunction Definitions; Table S2: Chronic Steroids, High-Dose Corticosteroid and Drotrecogin Alfa (Activated) Patient Hospital Mortality Outcomes for Low-Dose Corticosteroid use (yes/no) and Vasopressor use (yes/no); Table S3: In Hospital Mortality Rates Across the Propensity Quintiles.Click here for fileSupplementary tables S4 to S5. A Word file containing further statistical information on Propensity Score and Model Development and the following tables: Table S4: Summary of Baseline Characteristics Used in Mortality Models and Associated Hospital Mortality, a Univariate Analysis; Table S5: Improvement in Baseline Imbalance Using Propensity Score Analysis.Click here for file