N-terminal) and a DNA binding Helix-Turn-Helix domain , but are not associated with a cognate N-acyl homoserine lactone (AHL) synthase coding gene in the same genome. Although a few LuxR solos have been characterized, their distributions as well as their role in bacterial signal perception and other processes are poorly understood. In this study we have carried out a systematic survey of distribution of all ABD containing LuxR transcriptional regulators (QS domain LuxRs) available in the InterPro database (IPR005143), and identified those lacking a cognate AHL synthase. These LuxR solos were then analyzed regarding their taxonomical distribution, predicted functions of neighboring genes and the presence of complete AHL-QS systems in the genomes that carry them. Our analyses reveal the presence of one or multiple predicted LuxR solos in many proteobacterial genomes carrying QS domain LuxRs, some of them harboring genes for one or more AHL-QS circuits. The presence of LuxR solos in bacteria occupying diverse environments suggests potential ecological functions for these proteins beyond AHL and interkingdom signaling. Based on gene context and the conservation levels of invariant amino acids of ABD, we have classified LuxR solos into functionally meaningful groups or putative orthologs. Surprisingly, putative LuxR solos were also found in a few non-proteobacterial genomes which are not known to carry AHL-QS systems. Multiple predicted LuxR solos in the same genome appeared to have different levels of conservation of invariant amino acid residues of ABD questioning their binding to AHLs. In summary, this study provides a detailed overview of distribution of LuxR solos and their probable roles in bacteria with genome sequence information.LuxR solo transcriptional regulators contain both an autoinducer binding domain (ABD;  N-terminal autoinducer binding domains (ABD) and C-terminal Helix-Turn-Helix (HTH) DNA binding domains are known to be involved in quorum sensing (QS) signaling in many proteobacteria (the presence of both domains is referred to as QS domain here on)  signaling molecules, the LuxI homolog. QS typically involves production of AHLs by a LuxI homolog and their sensing by the LuxR regulator in a cell-density dependent manner to regulate target genes , which contains sequences from member databases, PROSITE, Pfam, Prints, ProDom, SMART, and TIGRFAMs  if no gene coding for a LuxI homolog was found in the genome, (2) if no gene was found in the genomic locus near the gene coding for QS domain LuxR protein, or (3) if no unpaired or extra genes coding for LuxI homolog were present in the genome. The genomes carrying these QS domain LuxRs were assigned to three categories: LuxR solos, LuxR solo + QS (if a LuxI/R pair(s) was encoded in the genome in addition to a LuxR solo protein), or QS (if a LuxI/R pair(s) was encoded in the genome but no LuxR solo protein was found). In those cases where LuxR solo proteins were found, the number of genes coding for LuxR solo proteins in each genome was noted. These data were used to generate contingency tables, by using the dynamic table tool available in Microsoft Office 11 and graphs were plotted from the table data using GraphPad Prism.Agrobacterium tumefaciens)  was analyzed by CD-HIT  when two QS domain LuxR proteins occur in tandem near a gene coding for the LuxI homolog , Sphingobium baderi LL03 and S. lactosutens DS20, (2) when the gene coding for a LuxI homolog was located in a locus genetically unlinked from the locus coding for a QS domain LuxR homolog or two QS domain LuxR homologs adjacent to each other , and (3) when truncated LuxR proteins containing only the ABD without the DNA binding domain were present in a genome; genes coding for these proteins were often located near gene(s) coding for a QS domain LuxR protein (Supplementary Table The complete collection of QS domain LuxR proteins were sourced from Interpro database IPR005143) and analyzed to identify LuxR solos or orphans according to the criteria outlined in materials and methods. In total, 6030 QS domain LuxR protein sequences from 3540 sequenced genomes were analyzed and an inventory of 4860 LuxR solos and 1170 LuxR proteins that are part of complete QS systems generated , Chlamydiae/Verrucomicrobia (two LuxR solos), Nitrospirae (two LuxR solos), and Chrysiogenetes (one LuxR solo), respectively  LuxR solos form a robust clade that branches out from rest of the tree. Three other QS domain LuxRs, namely, RpaR, CinR, and VjbR form another group of this branch. (2) The LuxR solos of Methylacidiphilum fumariolicum, M. infernorum, Nitrospira defluvii, and Leptospirillum ferrooxidans form another separate group clustering together with LasR, LuxR, SinR, and OryR suggesting that they may be evolutionarily closer to these QS domain LuxRs when compared to Actinobacterial LuxR solos. (3) The third branch of the tree is made up of functionally characterized proteobacterial QS domain proteins CarR, EsaR, CviR, and BjaR, indicating high evolutionary relatedness of these proteins. (4) In the fourth major clade of the tree, a LuxR solo of Desulfurispirillum indicum was found to cluster with TraR and ExpR; the branching of this LuxR solo also revealed a shared common ancestor with other QS LuxR and LuxR solos like CepR2, PfsR, QscR, RhlR, SdiA, PluR, CepR, and PmlR. In summary, with respect to well-studied proteobacterial QS LuxRs and LuxR solos, the Actinobacterial LuxR solos are evolutionarily distant whereas other non-proteobacterial LuxR solos show a relatively higher degree of evolutionary relatedness.In order to determine the evolutionary relatedness of non-proteobacterial LuxR solos to Gram-negative canonical QS LuxRs and LuxR solos an unrooted phylogenetic tree was generated as described in methods Figure . Four maM. fumariolicum and M. infernorum carry W57L and W85R substitutions at the corresponding positions in their proteins. The LuxR solos of Mycobacterium sp., Streptomyces sp., and Rhodococcus sp. carry W57_ and W85D or R substitutions. D. indicum and N. defluvii solos also carry W85R or W85_ and W57F substitutions. The lack of conservation of these invariant amino acids raises the possibility that these LuxR solos may bind to ligands different from AHLs.It was of interest to analyze the sequence features of ligand binding domains of non-proteobacterial LuxR solos as there are no reported studies regarding these proteins. Our analysis revealed lack of conservation in at least two/nine amino acids in all these proteins; importantly the amino acid corresponding to W85 of TraR was changed Table . The LuxAgrobacterium sp., Sphingomonas sp., Roseivivax sp., Sagittula sp., Nitratireductor sp., Methylobacterium sp., Aurantimonas sp., Afipia sp., Paracoccus sp., Sinorhizobium sp., Rhodospirillum sp., Pelagibaca sp.), and Betaproteobacteria , showing high degree of similarity within each group but lesser similarity between them. A few LuxR solos of Gammaproteobacteria represented by Dickeya sp., Klebsiella sp., Alcanivorax sp., Pseudomonas sp., as well a LuxR solo from Streptomyces were also in cluster 1. Burkholderia sp. LuxR solos were distributed in all clusters. A step-wise increase in P-value cut off from 10−30 to 10−1 resulted in merging of these small clusters into a single cluster suggesting that these 65 LuxR solos are closely related. Decreasing P-value to 10−35 resulted in separation of clusters and at P-value of 10−200 all proteins separated and remained independent without any cluster formation. These results confirm the fact that LuxR solos from different taxonomic groups have low levels of sequence relatedness making it very difficult to compare across bacterial taxa.In order to group related LuxR solos, cluster analysis was carried out using CLANS as detailed in materials and methods. Results using the sub-set of 657 LuxR solo proteins revealed that majority of the LuxR solos remained ungrouped probably due to low sequence similarity. Only a small number (65 out of 657 LuxR solos) clustered together mainly into five groups; these groups are likely a reflection of their taxonomic relationships. Among these, only LuxR solos from closely related species showed conservation of flanking genes Figure . These cIn order to group LuxR solos in a biologically relevant manner, an alternate approach was used whereby the subset of representative LuxR solos were analyzed for the genomic context of genes encoding them and the conservation of invariant amino acids of ABD. By this approach LuxR solos could be divided into different groups consisting of known and unknown LuxR solos; members of each group are likely to be orthologs Tables , 4. Out P. aeruginosa) ExpR , CepR , AvhR , CarR , VjbR and BlxR (Brucella sp. and Ochrobactrum sp.), and PpoR (Pseudomonas spp) were more restricted in their taxonomical distribution in our analysis based on flanking gene conservation. As expected SdiA, PpoR, BlxR, ExpR group members showed no changes in the invariant amino acids of the ABD whereas XccR/OryR, VjbR, and some members of CepR2 group showed replacement of amino acids at positions W57, Y61, W85, and G113 with respect to TraR amino acid sequence. Since there are several studies describing the representative LuxR solos of groups mentioned above, and their functions, they will not be discussed further here.Some LuxR solos fell into groups, which had one or more members already well-studied Table . Known gP. aeruginosa were found to contain an ortholog of this LuxR solo, in addition to the well-studied QscR, which appears to be restricted to P. aeruginosa strains  were in separate transcriptional units. Group 4 LuxR solos occur near genes coding for flagellar motor protein, FliG, which is required for motility. Group 5 LuxR solo gene is flanked on either side by genes coding for thymidine kinase and a ribosomal methyl transferase, both enzymes involved in nucleic acid metabolism. Located upstream of the gene coding for LuxR solo of group 6 is iclR, encoding an IclR family of transcriptional regulator; these regulators are known to regulate degradation of QS signals, plant-bacterial interaction and secondary metabolite production  and H. phototrophica DHL-43 (yellow clade). Interestingly, one independent clade supported by high bootstrap values stems out from this tree and this clade includes ExpR and LuxR solos from Agrobacterium, H. phototrophica, E. adherens and R. freirei. In contrast most of the characterized LuxR proteins locate in a distinct branch suggesting lower levels of relatedness among these proteins and Alphaproteobacterial LuxR solos.According to our results, several proteobacteria species have more than three genes coding for LuxR solos. In order to determine the relatedness between LuxR solos carried by the same genome, phylogenetic analyses was carried out as detailed in materials and methods. Several species were included for the phylogenetic analyses of these proteins in Alphaproteobacteria Figure . SeveralBurkholderia species formed three major clades  Figure . ImportaThese observations suggest that multiple LuxR solos carried by a bacterial species may have different evolutionary origins and likely have different ligand binding properties.N-terminal ABD are well-studied with respect to their role in QS to regulate bacterial community behavior in proteobacteria. QS LuxRs are generally thought to bind to AHLs but recently several studies have identified novel AHL and non-AHL ligands for these proteins raising the possibility of unusual ligands and novel functions for these proteins  AG motif typical of known lux box sequences (Supplementary Table Genes coding for characterized LuxR solos are known to occur along with functionally linked genes and often regulate their expression. For example, QscR is known to regulate expression of the adjacent phenazine biosynthetic operon and XccR/OryR regulates adjacent proline imino peptidase (Pseudomonas sp., and Burkholderia sp. suggesting a role for these proteins in adaptation of these bacteria to diverse habitats. It is possible that LuxR solos of different ligand specificity were acquired by these bacteria from different sources by horizontal gene transfer as it is known to be highly prevalent in many of these species, especially those belonging to Pseudomonas sp. (Qiu et al., Pseudomonas fluorescens A506, which branch out with different characterized LuxR solos in the phylogenetic analyses (Figure Multiple LuxR solos present in the same genome showed different levels of relatedness to characterized QS domain LuxRs suggesting different ligand binding properties and different origins. In particular, multiple LuxR solos were found in several plant-associated Alphaproteobacteria, s Figure ; bootstrIn summary, the systematic analysis and functional grouping of LuxR solos carried out in this study provides new information regarding the taxonomic and niche specific distribution, evolutionary origins, variation in ligand binding domain and probable roles of these proteins in bacteria which could be studied in the future.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.


STK31 and MSRA with very high confidence. Several genes show a dual oncogene- and tumor suppressor-like behavior depending on the tumor type. Most notably, the well-known tumor suppressor RB1 shows strong oncogene-like signal in colon cancer. We applied OncoScape to cell lines representing ten cancer types, providing the most comprehensive comparison of aberrations in cell lines and tumor samples to date. This revealed that glioblastoma, breast and colon cancer show strong similarity between cell lines and tumors, while head and neck squamous cell carcinoma and bladder cancer, exhibit very little similarity between cell lines and tumors. To facilitate exploration of the cancer aberration landscape, we created a web portal enabling interactive analysis of OncoScape results (http://ccb.nki.nl/software/oncoscape).Although large-scale efforts for molecular profiling of cancer samples provide multiple data types for many samples, most approaches for finding candidate cancer genes rely on somatic mutations and DNA copy number only. We present a new method, OncoScape, which exploits five complementary data types across 11 cancer types to identify new candidate cancer genes. We find many rarely mutated genes that are strongly affected by other aberrations. We retrieve the majority of known cancer genes but also new candidates such as  Large-scale initiatives such as The Cancer Genome Atlas (TCGA)455et al. performed a network-based analysis of DNA mutations, gene expression, copy number and RNAi screening dataet al. combined gene expression with copy number data in the context of regulatory networksCancer is one of the biggest public health problems with an estimated number of 14.1 million new diagnoses and 8.2 million deaths worldwide in 2012 according to GLOBOCAN activation of specific pathways in cancer18In order to obtain a comprehensive characterization of the molecular aberration landscape of different cancer types, we developed OncoScape, an algorithm integrating gene expression, DNA copy number, DNA methylation and somatic mutation data, as well as shRNA knock-down screens. RB1, BRCA2 and PTEN.The main goal of our study was to provide a comprehensive map of the global aberration landscape across 11 human cancer types. To this end, we prioritized all genes in the human genome taking into account five different data types . Figure CA9 had the highest average oncogene score on Chromosome 9. CA9 is a known hypoxia marker and cancer drug targetArranging genes by chromosomal location revealed large aberration patterns corresponding to chromosome arms or whole chromosomes . Genes lOverall our results revealed a highly complex picture of the aberration landscape of human cancer. Integrating different data and cancer types allowed prioritizing a short list of highly relevant candidate cancer genes. We found evidence that frequent focal events highlight probable cancer gene candidates, thus suggesting that the genomic context of aberration patterns provides valuable information.To validate cancer gene prioritization by OncoScape, we analyzed the scores of genes in three types of datasets: 1) frequently mutated cancer genes453−16, −8, Second, we investigated the scores of the 513 genes in the Cancer Gene Census (CGC)RB1 and EGFR, the top-scoring tumor suppressor and oncogene, respectively  in our analysis and as oncogene in two other tissues (colon adenocarcinoma [COAD] and lung adenocarcinoma [LUAD]). Overall, we found 739 genes indicated as oncogene and tumor suppressor based on at least two different data types for different cancer types. A particularly interesting example was the well-known tumor suppressor gene RB1, which was scored as oncogene-like in COAD  , mostly because of DNA copy number and methylation changes (http://oncoscape.nki.nl) where users can explore the best candidates in each cancer type but also annotate a list of genes of interest with tumor suppressor and oncogene results.The scoring of known tumor suppressor genes demonstrated that our approach allows distinguishing between genes showing oncogene- and tumor suppressor-like behavior. However, we identified potentially activating aberrations for several known tumor suppressor genes. Some genes in TSGene, for instance, had higher oncogene scores than tumor suppressor scores , which c in COAD . This unes = 19) , but in  changes , but hasBRAF and KRAS leading to activation of the MAP kinase pathway40Cancer is a disease of pathways, which can be affected by aberrations in different genes18An interesting application of OncoScape is the identification of genes that are aberrated in specific subtypes of a disease. As an example, we performed an analysis of the recently published four consensus subtypes of colorectal cancerOncoScape was also used to identify drivers of two subtypes of invasive lobular breast cancer: the Hormone Related (HR) subtype and the Immune Related (IR) subtypeAs cancer cell lines are important models for studying the biology of cancer, we compared the molecular aberration landscape of cancer cell lines to that of tumor samples. To this end, we mapped cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE)The overall number of aberrated genes was similar between cell lines and tumor samples, as was the number of genes with aberrations in the different data types . Few genin vitro model system. Cell lines of several cancer types, such as GBM, BRCA and COAD, have a high overall correspondence with tumor samples implicating a good coverage of the tumor aberration space. Importantly, our analysis highlighted several cancer types  and pathways with only little similarity between tumors and cell lines which demands greater caution when choosing particular cell lines as model systems.Our results stress the importance of carefully selecting an http://oncoscape.nki.nl) for exploring OncoScape results interactively. It allows accessing detailed scoring results for all cancer types and visualizing them in different types of plots. A pre-defined gene list can also be uploaded and annotated with oncogene and tumor suppressor gene prediction scores in all cancer types. Additionally, the web portal provides functionality for directly comparing results from tumor samples and cell lines. All source code can be downloaded from GitHub (https://github.com/andreas-schlicker/OncoScape); result files can be accessed through Sage Bionetwork’s Synapse (doi:10.7303/syn2518467).We implemented a web portal .Gene expression, DNA copy-number and methylation data for primary tumor samples and matched normal samples were obtained from data freeze 4.7 of the TCGA Pan-Cancerhttps://github.com/andreas-schlicker/OncoScape. The package provides functionality to assess alterations of single genes for each data type and each cancer type individually  differentially expressed (lower or higher) in tumor samples, it received a +1 towards tumor suppressor or oncogene score, respectively.Segmented DNA copy-number data for tumor and normal samples were obtained using Affymetrix SNP6 arrays by TCGA. We compared log-ratio copy number values between tumor samples and matched normal samples using paired Wilcoxon tests and corrected p-values using Benjamini-Hochberg’s procedure. We required genes to have a mean copy number difference larger than 0.1 between tumors and normal to prevent small effects becoming significant because of a large number of samples. For genes with significant difference in copy-number (FDR < 0.05), we calculated the Spearman correlation between copy-number and gene expression . A gene We analyzed DNA methylation data in a probe-wise fashion. For each probe, we compared methylation values in tumor and tissue-matched normal samples using unpaired Wilcoxon tests and corrected p-values using the Benjamini-Hochberg procedure. For each probe with a mean difference of methylation beta values greater than 0.1 between tumors and normals and the difference being significant (FDR < 0.05), we computed the Spearman correlation between methylation level and expression of the genes annotated to that probe according to the Illumina annotation across all tumor samples. Correlations with FDR < 0.05 were regarded as significant . A gene Mutations were scored according to the 20/20 rule published by Vogelstein and colleaguesProject Achilles assessed cell viability after knocking down genes using different shRNA hairpins. In order to minimize off-target effects, Project Achilles integrated knock-down results of several hairpins targeting the same gene into so called “gene solutions”, providing a cell viability score for each gene and cell line combination. We used only genes for which only a single gene solution was provided. For each gene, we derived a distribution of viability values across all cell lines. A gene was scored as potential oncogene for one cancer type if at least 25% of the cell lines for this cancer type had a knock-down viability score that was lower than the 25th percentile across all cell lines. If at least 25% of the cell lines had a knock-down viability score greater than the 75th percentile for that gene, it was scored as potential tumor suppressor gene.In order to estimate which samples were affected by alterations in gene expression, DNA copy number or methylation of a specific gene, we first obtained the distribution of differences between tumor and normal samples. For paired analyses, we simply subtracted the value of the matched normal samples from the value of the tumor samples. For unpaired analyses, we subtracted the average value across all normal samples from the values of the tumor samples. A sample was called affected if its data value was more than one standard deviation away from the mean value of the distribution of differences.How to cite this article: Schlicker, A. et al. OncoScape: Exploring the cancer aberration landscape by genomic data fusion. Sci. Rep.6, 28103; doi: 10.1038/srep28103 (2016).


Concern has been raised regarding the potential contributions of veterinary antimicrobial use to increasing levels of resistance in bacteria critically important to human health. Canine pyoderma is a frequent, often recurrent diagnosis in pet dogs, usually attributable to secondary bacterial infection of the skin. Lesions can range in severity based on the location, total area and depth of tissue affected and antimicrobial therapy is recommended for resolution. This study aimed to describe patient signalment, disease characteristics and treatment prescribed in a large number of UK, primary-care canine pyoderma cases and to estimate pyoderma prevalence in the UK vet-visiting canine population.Of 54,600 dogs presented to 73 participating practices in 2010, 683 (1.3%) had a pyoderma diagnosis recorded in available electronic patient record (EPR) data. Antimicrobials were dispensed in 97% of cases and most dogs were prescribed systemic therapy (92%). Agents most frequently prescribed were amoxicillin-clavulanate, cefalexin, clindamycin and cefovecin. Systemic antimicrobials were prescribed for fewer than 14 days in around 40% of study cases reviewed in detail. Prescribed daily doses were below minimum recommended daily dose (MRDD) in 26% of 43 dogs with sufficient information for calculation of minimum dose.Antimicrobial prescribing behaviour for treatment of canine pyoderma was variable but frequently appeared inconsistent with current recommendations. Use of clinical data from primary practice EPRs can provide valuable insight into common clinical conditions and associated prescribing.The online version of this article (doi:10.1186/s12917-014-0240-5) contains supplementary material, which is available to authorized users. S. pseudintermedius. Other staphylococcal  and non-staphylococcal bacteria  can also be isolated from affected skin in some cases. While these species can act as primary pyoderma pathogens, particularly in immune-compromised patients, they are usually secondary infectious agents or simply lesion contaminants ) was used to select a subset sample of at least 10% of the dogs identified as pyoderma cases by the initial search criteria. These selected cases were reviewed in depth, including evaluation of disease and treatment details, follow-up of clinical progression and validation of the initial case-finding approach. Queries were designed to extract all clinical entries associated with these dogs from the original database on dates up to and including the 31st December 2011. Chronological review of all available free-text clinical history was undertaken for each dog to assess the validity of selection as a pyoderma case and to describe pyoderma lesion depth, underlying cause and treatment-related information in greater detail.Pyoderma depth was categorised as either ‘surface’ , ‘superficial’ , ‘deep’ , or ‘mixed’ based on lesion description statements recorded by the examining clinician . Cases wAdditional treatment-related data extracted for this subset included the daily dosing instructions and total number of units (tablets/capsules/volume of injectable formulation) of any initially prescribed systemic antimicrobial agent dispensed across all consecutive prescribing events during the studied pyoderma episode. Based on calculations using patient weight (as recorded on the date of diagnosis) and drug manufacturers’ recommended daily dose (MRDD) rates for treatment of canine pyoderma , dogs wed. Concurrent prescription of shampoos or liquid washes (including non-medicated products) was noted to reflect the intention for regular physical bathing of the skin and coat.Non-antimicrobial treatments prescribed or administered to study dogs concurrently with the systemic antimicrobial agent of interest were also recorded. Where appropriate, prescribed products were classified  into one or more of the following categories, highlighted due to their potential relevance in cases of skin infection: Systemic steroids, antifungal agents and anti-ectoparasitic agentsData were cleaned in Microsoft Excel . Statistical analysis was carried out using Stata . Variables were categorised where required. Descriptive statistics were calculated for all study dogs with respect to signalment factors and antimicrobial product/s prescribed within 24 hours of recorded diagnosis of the pyoderma episode of interest.Additional descriptive statistics were calculated for the randomly selected subset of pyoderma cases described in greater detail, including lesion depth, duration of systemic antimicrobial treatment prescribed and adequacy of prescribed daily dose compared with MRDD. The extent of missing or insufficiently specific data was summarized. Mean and standard deviation or median and range were used for the description of quantitative data as appropriate while qualitative data were described using frequencies with percentage of stated totals.aRVC Ethics Committee, March 2009.bThe recording format of the PMS used by participating practices included opportunities for standardised terminology based on the VeNom codes.cStudy protocol included a list of terminology accepted as compatible with pyoderma for study purposes, based on clinical codes defined by the VeNom Coding group. Some notable exceptions were also specified, e.g. pyotraumatic dermatitis.dFull category listings are provided in Additional file


We examined the usefulness of dual-energy computed tomography (DECT) in the evaluation of symptomatic systemic sclerosis (SSc)-related calcinosis of the hands. We performed DECT scan of the hands in 16 patients with symptomatic SSc-calcinosis to better characterize the calcinosis, their locations within the soft tissues, and exclude monosodium urate (MSU) crystal deposition. We also compared their computed tomography (CT) images to plain radiographs of one hand. Pertinent clinical information from this cohort was collected. Sixteen SSc patients underwent DECT and plain X-ray imaging of the hands. Five of the 16 SSc patients in our cohort had overlap disease, either rheumatoid arthritis (RA) and/or myopathy. Calcinosis symptoms included painful deposits (88 %), soft tissue swelling (66 %), and recurrent infections (44 %) from these deposits. On DECT, calcinosis deposits had the same color and density as the bone and no MSU was found. However, their CT images showed better details of the calcinosis locations in the soft tissues as well as the bone destruction, especially if there was overlying bulky deposits or flexion contractures. These deposits were most commonly found in the subcutaneous fat pads of the fingertips, along tendon and muscle groups, or within the carpal tunnel. DECT did not confirm MSU in our cohort with calcinosis. However, CT imaging was superior to plain radiographs in locating these deposits within the soft tissues and may be a useful tool to study SSc-calcinosis affecting the hands, particularly in the setting of progressive hand deformities. Calcinosis, the deposition of calcified material in the skin and subcutaneous soft tissues, is frequently seen in patients with symptomatic systemic sclerosis (SSc). It has been reported that approximately 40 % of patients with limited SSc have calcinosis , 2. CalcIn this IRB-approved study, we performed DECT imaging with a 64-slice dual-source CT (Siemens Definition), using dual-energy application in patients with symptomatic SSc-calcinosis, to better characterize the location and radiographic details of SSc-calcinosis and exclude MSU. We also compared the DECT to plain X-ray radiographs in all 16 patients. All had known calcinosis confirmed by prior imaging or physical exam and met the American College of Rheumatology (ACR) criteria for definite SSc –11, eithThe clinical, demographic, and serologic characteristics of our SSc patient cohort are summarized in Table DECT confirmed calcinosis had the same density and color as the bone, and no MSU was found within or near any of these tumorous deposits. Common locations  patients reported pain  at the site of their deposits, even if the area was not draining, which often affected their activities of daily living. Ten patients reported soft tissue swelling near the calcinosis site(s) just prior to spontaneous draining. We found that plain X-ray imaging of the hands was sufficient to view the size and distribution of calcinosis in most of our cohort, although in five subjects CT imaging showed better details due to extensive flexion deformities or osteopenia with nearby large calcinosis deposits. CT imaging found more destructive bone and joint disease in our four SSc patients with rheumatoid arthritis and calcinosis. Their calcinosis was otherwise similar in distribution, size, and extent as the rest of our cohort. Interesting, although their arthritis appeared well controlled clinically, all 4 SSc patients reported progressive and disabling hand deformities over the years. We found CT showed better 3-D details of calcinosis locations within the soft tissue structures and demonstrated more clearly how these could contribute to joint contractures or muscle atrophy. Thus, standard CT imaging should be considered in the evaluation of any SSc patient with progressive hand deformities, especially in the presence of bulky calcinosis, while DECT could be applied if MSU was suspected. Newer software is currently being developed to better differentiate between calcinosis and bone on standard CT, which would be very useful to quantify bulky calcinosis using volumetric measurements.To our knowledge, we are the first group to use DECT to analyze calcinosis in a cohort of scleroderma patients. Our limitations include the small population of patients, five of whom also had overlap disease. However, their calcinosis did not appear any different or any more extensive than in those without overlap disease, and further studies are needed to determine the differences in pathophysiology between those with and without overlap disease.Although no MSU crystals were confirmed by DECT in our cohort, CT imaging is a more useful tool to study patients with bulky SSc-calcinosis affecting the hands.


In contrast, phase-amplitude coupling between slow and alpha oscillations correlated with drug concentrations in blood. Our findings highlight novel markers that prognosticate individual differences in susceptibility to propofol and track drug exposure. These advances could inform accurate drug titration and brain state monitoring during anaesthesia.Accurately measuring the neural correlates of consciousness is a grand challenge for neuroscience. Despite theoretical advances, developing reliable brain measures to track the loss of reportable consciousness during sedation is hampered by significant individual variability in susceptibility to anaesthetics. We addressed this challenge using high-density electroencephalography to characterise changes in brain networks during propofol sedation. Assessments of spectral connectivity networks before, during and after sedation were combined with measurements of behavioural responsiveness and drug concentrations in blood. Strikingly, we found that participants who had weaker alpha band networks  Though scientific understanding of how brain networks generate consciousness has seen rapid advances in recent years, application of this knowledge to accurately track transitions to unconsciousness during general anaesthesia has proven difficult due to considerable variability in this gradual process across individuals. Using high-density electroencephalography, we studied changes in these networks as healthy adults were sedated using propofol. By measuring their behavioural responsiveness and amount of sedative in their blood, we found a striking pattern: the strength of their brain networks before sedation predicted why some participants lost consciousness while others did not, despite registering similar blood levels of drug. By uncovering underlying signatures of this variability, our findings could enable accurate brain monitoring during anaesthesia and minimise intra-operative awareness. Understanding how the human brain reversibly generates and loses consciousness, through complex interactions of neural activity at multiple spatial and temporal scales, is a grand challenge for modern neuroscience. Recent theoretical advances have argued that consciousness changes when the balance between integrated and differentiated neural activity is affected –4. HowevThe process of reversibly inducing unconsciousness using anaesthetic drugs like propofol is commonplace in clinical medicine . HoweverTo better understand the factors underlying this variability, we combined the measurement of high-density resting state EEG from healthy volunteers sedated with propofol with measurement of drug concentrations in blood, in addition to objective assessment of behavioural responsiveness. With this aim in mind, we administered propofol at dosages expressly aimed at engendering varying degrees of mild to moderate sedation across our participant group, rather than complete unconsciousness in all of them. Employing modern functional EEG tools to assess spectral power and connectivity, we identified key changes in brain networks using graph-theoretic tools, and linked these changes to individual variability in drug concentrations and loss of behavioural acuity during sedation. Drawing upon previous research –21, we hdrowsy (green triangles) and responsive (blue triangles) in the following descriptions. As expected, we found a highly significant interaction between group and sedation level in hit rates , meso-scale (modularity and participation coefficient) and macro-scale properties (characteristic path length) of each participant’s network at each level of sedation  and moderate sedation (p = 0.01). Further, the clustering coefficient , 26, whih length ), was sih length . Crucialh length  in the dThese changes in alpha networks can be understood more visually with Spectral connectivity in the alpha band identified a prospectively valuable determinant of the variability in susceptibility to propofol seen in the behavioural data. During the baseline period before sedation, though there were no differences in the topography or relative strength of alpha power between the responsive and drowsy groups , there wTo explicate this result further, We found that, at baseline, participants in both responsive and drowsy groups had similar temporal coupling between the phase of slow oscillations and alpha power, with negative values of phase-amplitude coupling PAC;  over occJuxtaposed with previous research, our findings are convergent with existing evidence for characteristic changes in PAC alongside propofol induction. Trough-max slow-alpha PAC has been shown to accompany transitions to unconsciousness in frontal EEG channels, which then switches to a peak-max pattern in the same channels following loss of consciousness during deep sedation , 35. WhiOur experimental design used propofol sedation to engender transitional states of responsiveness that varied across participants. The levels of drug administered produced a variable pattern that spread the participant group along a spectrum of varying behavioural impairment, rather than resulting in complete unconsciousness in all of them. Using EEG to track brain activity and measuring actual levels of drug in blood alongside this spectrum of impairment has enabled us to identify neural markers that dissociate conscious report from drug exposure , and makWe have built upon previous research that has shown that while occipital alpha power progressively drops as participants become behaviourally compromised as measured by reaction times, the qualitatively dissimilar onset of frontal alpha power is a characteristic marker of the loss of consciousness , 32, 36.Measurement of drug concentrations at each level of sedation dissociated a principal clinical pharmacodynamics target per se  from incidental pharmacodynamic consequences of drug exposure during propofol sedation. The considerable individual variability in the susceptibility to anaesthesia has been documented , and is before sedation predicts susceptibility to propofol. Specifically, given two behaviourally indistinguishable individuals undergoing administration of sedative, the one with the more robust, small-worldy alpha network with well-connected hubs is likely to require a greater amount of drug to render them unresponsive to the same degree. It is important to note that this latent variability in the state of alpha connectivity at baseline could be detected despite the lack of any significant differences in behavioural performance or alpha power at that time. Orthogonally, slow-alpha PAC complements this predictive capability by tracking the concentration of propofol in blood plasma. This set of results, if replicated and verified in the clinical context, could contribute to reliable applications of brain monitoring for tracking and accurately modulating consciousness with anaesthetics during routine surgery.Finally, by tracking individual brain networks across levels of sedation, we have shown that the quantifiable robustness of alpha connectivity networks in the awake state Declaration of Helsinki.All healthy controls gave written informed consent. Ethical approval for testing healthy controls was provided by the Cambridgeshire 2 Regional Ethics Committee. All clinical investigations were conducted in accordance with the A convenience sample of 22 neurologically healthy adults participated in the study. Data from two participants could not be used due to technical issues, leaving 20 participants   whose data were analysed.Each experimental run began with an awake baseline period lasting 25–30 minutes  followinTIVATrainer pharmacokinetic simulation software revealed that plasma concentration of propofol would approach zero in 15 minutes leading to behavioural recovery; hence behavioural assessment was recommenced 20 minutes after cessation of sedation. Blood samples of 1cc each were taken at the beginning and end of the mild and moderate sedation states, and once at recovery, as indicated in Three blood plasma levels were targeted– 0.6μg/ml (mild sedation), 1.2μg/ml (moderate sedation), and recovery from sedation. The state of mild sedation was aimed to engender a relaxed but still responsive behavioural state. At each target level, a period of 10 minutes was allowed for equilibration of plasma propofol concentrations to attain a steady state, following which behavioural tests and EEG measurements were commenced. After cessation of infusion, plasma propofol concentration exponentially declined toward zero. Computer simulations with the hit rates, i.e., percentage of correct responses. In addition, we measured reaction times based on the delay between auditory tone onset and correct button press.At each of the 4 steady-state levels above, participants were requested to perform a simple behavioural task involving a fast discrimination between two possible auditory stimuli . Specifip, the probability of a correct response, and its 95% confidence intervals were estimated. For a given participant, if the confidence interval at moderate sedation was lower than and non-overlapping with that at baseline, they were considered to have become significantly impaired, and we designated them as drowsy. If the confidence intervals overlapped, we designated them as responsive.We employed binomial modelling to distinguish participants who became behaviourally impaired during moderate sedation, from those who remained responsive, albeit with slower reaction times. Specifically, we fitted a binomial distribution to each participant’s hit rates at baseline and during moderate sedation. With each fitted model, the distribution parameter From each participant, approximately 7 minutes of 128-channel high-density EEG data were collected at each level of sedation. EEG was measured in microvolts (uV), sampled at 250Hz and referenced to the vertex, using the Net Amps 300 amplifier . Participants had their eyes closed in a resting state during data collection.Data from 91 channels over the scalp surface  were retpwelch method. At each channel, power values within canonical frequency bands, namely delta (0–4Hz), theta (4–8Hz), alpha (8–15Hz), beta (12-25Hz) and gamma (25–40Hz), were converted to relative percentage contributions to the total power over all five bands. Alongside, cross-spectrum between the time-frequency decompositions (at frequency bins of 0.49Hz and time bins of 0.04s) of every pair of channels was used to calculate debiased weighted Phase Lag Index . For a PLI, see , and hasPLI, see  and pharPLI, see , dwPLI iDirect PAC estimator formally defined by Ozkurt and Schnitzler [trough-max to peak-max PAC during propofol sedation, as determined by whether the slow oscillation is at its trough (at a phase angle of pi) or its peak (phase angle of 0) when alpha power is maximal, respectively. Such variations were measured by assigning a negative or positive sign to the amplitude of the complex-valued Direct PAC estimator depending on whether its phase angle was closer to pi or 0 radians, to indicate trough-max and peak-max coupling respectively.Phase-amplitude coupling (PAC), also referred to as cross-frequency coupling , was usehnitzler  and implhnitzler . Purdon,hnitzler  and Mukahnitzler  previousN), where N is the number of nodes in the network . This lower boundary guaranteed that the resulting networks could be estimated [connection densities have been shown to be the most sensitive to the estimation of ‘true’ topological structure therein [The 91x91 subject-wise, band-wise dwPLI connectivity matrices were thresholded to retain between 50–10% of the largest dwPLI values. They were then represented as graphs with the channels as nodes and non-zero values as links between nodes. The lowest threshold of 10% ensured that the average degree was not smaller than 2 * log measure and one repeated (level of sedation) measure. The obtained p-values were corrected for violations of sphericity using a Greenhouse-Geisser correction. Pairwise tests between groups were corrected for multiple comparisons using Tukey’s HSD test. The ability of graph metrics of individual participants to predict their behaviour was tested using robust linear regression  to calcuS1 Fig(A) Delta power topography in the drowsy group showed prominent increases over occipital channels during moderate sedation, but there were no group differences in connectivity (B). Theta power decreased in both groups with sedation (C), with no differences in connectivity (D). In contrast, beta and gamma band power over bilateral frontocentral channels (E and G) and small-worldness (F and H) increased in the responsive but not the drowsy group. However, unlike in the alpha band, neither baseline power nor connectivity in any of these bands predicted later loss of responsiveness during moderate sedation.(TIFF)Click here for additional data file.S2 FigCoupling between ongoing slow phase and alpha power over frontal channels delineated in (TIFF)Click here for additional data file.


MicroRNAs (miRNAs) are short regulatory RNAs derived from longer precursor RNAs. miRNA biogenesis has been studied in animals and plants, recently elucidating more complex aspects, such as non-conserved, species-specific, and heterogeneous miRNA precursor populations. Small RNA sequencing data can help in computationally identifying genomic loci of miRNA precursors. The challenge is to predict a valid miRNA precursor from inhomogeneous read coverage from a complex RNA library: while the mature miRNA typically produces many sequence reads, the remaining part of the precursor is covered very sparsely. As recent results suggest, alternative miRNA biogenesis pathways may lead to a more diverse miRNA precursor population than previously assumed. In plants, the latter manifests itself in e.g. complex secondary structures and expression from multiple loci within precursors. Current miRNA identification algorithms often depend on already existing gene annotation, and/or make use of specific miRNA precursor features such as precursor lengths, secondary structures etc. Consequently and in view of the emerging new understanding of a more complex miRNA biogenesis in plants, current tools may fail to characterise organism-specific and heterogeneous miRNA populations.Arabidopsis thaliana and the Volvocine algae Chlamydomonas reinhardtii; the latter organism has been shown to exhibit a heterogeneous and complex precursor population with little cross-species miRNA sequence conservation, and therefore constitutes an ideal model organism. Furthermore we identify novel miRNAs in the Chlamydomonas-related organism Volvox carteri.miRA is a new tool to identify miRNA precursors in plants, allowing for heterogeneous and complex precursor populations. miRA requires small RNA sequencing data and a corresponding reference genome, and evaluates precursor secondary structures and precursor processing accuracy; key parameters can be adapted based on the specific organism under investigation. We show that miRA outperforms the currently best plant miRNA prediction tools both in sensitivity and specificity, for data involving https://github.com/mhuttner/miRA.We propose miRA, a new plant miRNA identification tool that is well adapted to complex precursor populations. miRA is particularly suited for organisms with no existing miRNA annotation, or without a known related organism with well characterized miRNAs. Moreover, miRA has proven its ability to identify species-specific miRNAs. miRA is flexible in its parameter settings, and produces user-friendly output files in various formats . It is freely available at The online version of this article (doi:10.1186/s12859-015-0798-3) contains supplementary material, which is available to authorized users. Caenorhabditis elegans ..8]. CorrWe identify Chlamydomonas miRNA precursors using miRA, and determine recall rates based on the list of known miRNAs from . We use %. The larger number of novel miRNAs derived from the data in [Results based on miRA, miRDP and miR-PREFeR for both data sets are summarised in Table  data in  compared data in  is relatWe are able to identify many novel Chlamydomonas miRNA precursors, many of which show expression profiles consistent with the generation of two and more mature miRNAs from the same precursor. Corresponding precursor structures range from up to 500 nt long single hairpin structures, and up to 700 nt long multiple hairpin structures. Examples of complex miRNA precursors are given in Figure S2 of the Additional file Lds,max), length of the primary (i.e. most strongly expressed) mature miRNA (L(mature)), and length of the miRNA precursor (L(precursor)) for the identified (known and novel) Chlamydomonas miRNA precursors based on the data from [A complete list of identified Chlamydomonas miRNA precursors is provided in Table S3 of the Additional file ata from  are summArabidopsis thaliana (Arabidopsis) library containing two samples that was used in the miR-PREFeR publication [We use an lication . Detailslication . Pooled lication  using toLds,max, L(mature) and L(precursor) in the top three panels of Fig. We compare the performance of miRA, miRDP and miR-PREFeR in Table It is interesting to note that in comparison to results for Chlamydomonas, the Arabidopsis precursor population is more homogeneous, showing a narrower length distribution and fewer complex secondary structures. The length distribution of mature miRNAs in Arabidopsis shows two characteristic peaks at 21 nt and 24 nt. Only a small fraction (∼15 %) of the loci corresponding to the 24 nt long sequences are repeat element-associated, which may support the association of these sequences with siRNAs/ta-siRNAs. This suggests that the majority of identified mature miRNAs of different lengths should be attributed to alternative miRNA biogensis pathways such as miRNA precursor processing by different members of the Dicer-like enzymes . ThVolvox carteri (Volvox). We use small RNA sequencing data derived from Volvox somatic cells during their vegetative cycle (GEO accession number GSE58703). Reads were mapped to the Volvox reference genome version 9.0  sequencing data of plants and plant-like organisms . miRA is particularly suited to investigate heterogeneous miRNA precursor populations. Identification of miRNA precursors occurs through an evaluation of corresponding secondary structures and subsequent precursor processing accuracy. Our method has three key features: First, it allows for the identification of miRNAs in species with little or no miRNA conservation. Second, it enables a consistent investigation of both species-specific and homologous miRNAs in different organisms. Third, it allows for the identification of miRNA precursors with complex and heterogeneous secondary structures, such as precursors including e.g. additional sub-hairpins or multiple mature/star miRNA duplexes.Project name: miRAProject home page:https://github.com/mhuttner/miRAOperating system(s): Any Unix-based system Programming language: COther requirements: Optional requirements: Java 1.6+, LaTeX, gnuplotLicense: GNU GPL1 RNAfold is invoked from the main program, with parameters being passed directly to RNAfold as part of miRA’s main routine.2 Values given in the following sections correspond to default values, and may be changed by the user.


Rear (warm) edge populations are often considered more susceptible to warming than central (cool) populations because of the warmer ambient temperatures they experience, but this overlooks the potential for local variation in thermal tolerances. Here we provide conceptual models illustrating how sensitivity to warming is affected throughout a species' geographical range for locally adapted and non-adapted populations. We test these models for a range-contracting seaweed using observations from a marine heatwave and a 12-month experiment, translocating seaweeds among central, present and historic range edge locations. Growth, reproductive development and survivorship display different temperature thresholds among central and rear-edge populations, but share a 2.5 °C anomaly threshold. Range contraction, therefore, reflects variation in local anomalies rather than differences in absolute temperatures. This demonstrates that warming sensitivity can be similar throughout a species geographical range and highlights the importance of incorporating local adaptation and acclimatization into climate change vulnerability assessments. Low latitude populations are often thought to be more vulnerable to climate change due to warmer ambient conditions. Here, Bennett et al. show that populations of seaweed from different areas of their range have distinct thermal-tolerance thresholds, but share remarkably similar thermal safety margins to warming. Climate change is altering species distributions across the globe134346Along thermal gradients, however, selection pressure from the environment can lead to local adaptation and acclimatization of thermal-tolerance limits among populations89912910Here we apply ‘locally adapted' and ‘non-adapted' models of warming sensitivity to test whether a warm range edge (RE) population undergoing range contraction is indeed more vulnerable to warming and extreme events than central populations living under cooler climatic conditions. Specifically, we observed whether thermal-safety margins varied among populations, by assessing the absolute temperature and the maximum thermal-stress anomaly at which biological processes  ceased to function. Thermal-stress anomalies  were defined as temperatures that exceed the climatologically (long-term average) warmest day of the yearScytothalia dorycarpa, a dominant habitat-forming seaweed, underwent a ∼100 km range contraction from its former warm RE (FRE) in Jurien Bay (30°S) to Lancelin (31°S), where only a few scattered individuals remainedScytothalia still forms canopies, such as the new functional RE  across 1,000 km of the west coast of Australia's Great Southern Reef1819Scytothalia from Ce to RE, from Ce to FRE and from RE to FRE , because of the low thermal tolerance of pre-settlement stagesLike growth, reproductive development declined with increasing stress anomalies , Table 2The decline in growth and reproductive development under high-stress anomalies coincided with an increase in putative disease symptoms and epiphytic fouling  and 4. TScytothalia exhibit different absolute temperature tolerances, but have similar thermal-safety margins among populations  and between genetic and non-genetically based variation in thermal-tolerance limits12The narrow thermal-safety margins observed in this study may reflect the remarkably stable climatic history in South-Western Australia. Since the formation of the Leeuwin Current during the Eocene2815An important challenge for climate change research will be to find generalities about the nature of thermal-safety margins among and within populations, taxa and systems, to enable a more precise understanding of climate change impacts and identify the areas of greatest vulnerability. We provide a conceptual framework to help address these challenges and demonstrate that central and rear-edge populations can be equally vulnerable to temperature perturbations, in contrast to the assumption underpinning many current climate change impact predictions. Our findings highlight that we may currently be underestimating the vulnerability of central populations to warming, particularly in light of increasing extremes, and/or overestimating the sensitivity of rear-edge populations.Translocations took place among Hamelin Bay  to Marmion  and Jurien Bay  in the western region of Australia's Great Southern ReefEcklonia radiata, Sargassum spp. (subgen. Arthrophycus) and Scytothalia, except in Jurien Bay, where Scytothalia has been absent since the 2011 marine heatwaveScytothalia cover-up until 2011 (ref. 2 steel mesh quadrants (five mesh per translocation treatment) were fixed to the reef. All mesh were separated by at least 4 m and situated within the canopy, to maximize the survival and growth of the young Scytothalia33Within each location three wave exposed sites between 9–12 m depth were selected, each separated by at least 1.8 km. All sites were topographically flat limestone reefs, with mixed canopies dominated by 011 ref. . Within Scytothalia, respectively. Within each source site, 30 young Scytothalia  were chiselled from the reef ensuring a fragment of limestone was retained beneath the holdfast. For each translocation, individuals were collected from three source sites, kept in buckets of aerated seawater during the day and then transported to their destination in cool, damp calico bags and stored in the dark. Deployments within each destination were staggered so that seaweed from each site spent no longer than 24 h in transit before being returned to the reef. Each individual was measured  and the maximum circumference (C)) and labelled. Within each translocation site, six individuals were transferred to each of the five replicate mesh per treatment using latex-covered cable tiesTranslocations were carried out during the period of coolest seawater temperatures (August and September 2012), to minimize the translocation shock on the seaweeds. Translocations were performed from Ce to RE, Ce to FRE, RE to FRE and procedural control sites were set up among the three RE and within the three Ce sites using self-transplants and naturally attached Scytothalia were measured in Hamelin Bay instead of self-transplants. Handling effects were highly unlikely to have been the cause of the observations for three main reasons. First, self-transplants, that is, transplants with the same source and recipient location that otherwise underwent the same handling procedures (including 24 h out of the water) were conducted in Marmion. Both Marmion and Hamelin Bay controls experienced low-temperature stress throughout the translocation experiment and both locations demonstrated the same results in terms of growth, reproductive development and tissue health. The consistency in results between low-anomaly sites that used natural populations and self-translocated population indicates that handling effects did not influence the observed temperature-induced results. Second, the translocation experiment was conducted over 12 months. In the first month of the experiment, some of the translocated seaweeds lost biomass, most likely as a result of handling effects (E. radiata) dominated canopies of densities between 8 and 12 sporophytes per m2, described above.Because of logistical constraints natural populations of  effects . After t effects . MoreoveL) and circumference (C) were converted to fresh weight (WF) by WF=LC2  represented repeated bi-monthly measures of mean seaweed growth within each mesh (n=75), site and location combinationThe relationships between physiological performance metrics  and temperature metrics  were tested using linear mixed-effects models. Separate nested intercept models were used to test the relationship between each performance and temperature metric combination. The relationship between growth rates and each temperature metric  was first analysed across the entire year to assess growth responses across a broad range of temperatures and stress anomalies. Analyses were structured so that ‘time'  measured in March–May 2013 following the warmest temperatures  growth rates at each treatment/site combination were analysed in response to stress anomalies and maximum temperatures to the test growth responses of the year . The releratures  and corrHow to cite this article: Bennett, S. et al. Central and rear edge populations can be equally vulnerable to warming. Nat. Commun. 6:10280 doi: 10.1038/ncomms10280 (2015).Supplementary Figures 1-3 and Supplementary References.


The battle between plants and microbes is evolutionarily ancient, highly complex, and often co-dependent. A primary challenge for microbes is to breach the physical barrier of host cell walls whilst avoiding detection by the plant’s immune receptors. While some receptors sense conserved microbial features, others monitor physical changes caused by an infection attempt. Detection of microbes leads to activation of appropriate defense responses that then challenge the attack. Plant cell walls are formidable and dynamic barriers. They are constructed primarily of complex carbohydrates joined by numerous distinct connection types, and are subject to extensive post-synthetic modification to suit prevailing local requirements. Multiple changes can be triggered in cell walls in response to microbial attack. Some of these are well described, but many remain obscure. The study of the myriad of subtle processes underlying cell wall modification poses special challenges for plant glycobiology. In this review we describe the major molecular and cellular mechanisms that underlie the roles of cell walls in plant defense against pathogen attack. In so doing, we also highlight some of the challenges inherent in studying these interactions, and briefly describe the analytical potential of molecular probes used in conjunction with carbohydrate microarray technology. The dynamic interplay between pathogen and plant host is the product of millions of years of co-evolution. This struggle is often described in terms of an “arms race,” a fitting term considering the investment required and the significance for both sides. The goal for the plant is to keep healthy and fertile, but for pathogens the strategy varies. Biotrophic and hemibiotrophic microorganisms rely on keeping their host alive and unaware of their presence, at least until later stages of infection. In contrast, necrotrophs live and feed on dead and dying cells and can therefore use a more forceful attack strategy . The froinfectious-self and non-self, whereas for ETI it is the ability to sense microbe-mediated modifications inferred on points of vulnerability in the host. By guarding these weak points or even setting up decoys to confuse invaders, ETI is an efficient safety net for more progressed infections , is a warning of impending attack . Consequfections .Brassicaceae family, while in rice recognition of EF-Tu, occurs via the EFa50 region  EFR in Solanaceae species that cannot perceive EF-Tu is sufficient to confer resistance to a broad range of phytopathogenic bacteria, suggesting high conservation of the responses downstream of PAMP recognition (Pathogen-associated molecular pattern perception is mediated by ligand-binding surface-exposed transmembrane pattern-recognition receptors (PRRs) of either the receptor-like kinase (RLK) or receptor-like proteins (RLPs) families. Both types of modular proteins are single-pass transmembrane proteins with extracellular domains, but where RLKs have an intracellular kinase domain; RLPs lack this cytosolic signaling domain . The arc0 region . Interesognition . PTI indognition .Figure 1). Chitin, a homopolymer of β-(1→4)-linked N-acetylglucosamine (GlcNAc) units, is the major structural component of fungal cell walls, and is also a main constituent of insect exoskeletons, crustacean shells, and the eggs and gut linings of nematodes  and activates the hypersensitive response (Oryza sativa) by a dual recognition system consisting of the lysine motif (LysM)–RLK CHITIN ELICITOR RECEPTOR KINASE-1 OsCERK1 and the LysM-RLP CHITIN OLIGOSACCHARIDE ELICITOR-BINDING PROTEIN OsCEBiP  during infection. Ecp6 is a scavenger of chitin fragments released by chitinases and out-competes chitin-PRR binding to avoid fungal detection . The low basal cytosolic concentration allows rapid spatial and temporal changes in the calcium flux. Such calcium signatures are sensed by stimulus-specific Ca2+-binding sensors that can mediate the downstream signal transduction. Deciphering the calcium signatures enables the affected cells to respond appropriately to diverse stimuli . The four functionally redundant Arabidopsis CDPKs, CPK4, CPK5, CPK6, and CPK11 define a sub-clade required for flg22 induced PTI responses . In Arabidopsis, the ROS burst is facilitated by the RESPIRATORY BURST OXIDASE HOMOLOGUE D (RbohD), an NADPH oxidase (Solanum tuberosum) StRbohB is phosphorylated by the plasma membrane-localized potato StCDPK5, and expression of constitutively active StCDPK5 in Nicotiana benthamiana leads to induction of ROS (An oxidative burst of reactive oxygen species (ROS) such as ·O oxidase . The act oxidase , and int oxidase . In potan of ROS .Figure 2; Arabidopsis MAP kinases MPK3, MPK4, MPK6 as well as the recently documented MPK11 are all activated by PAMP treatment  phosphorylate MAP kinase-kinases (MAPKKs), which in turn phosphorylate MAP-kinases , the predominant synthase responsible for defense-induced callose deposition  that is recognized as a PAMP (Lycopersicum esculentum), EIX is sensed by the cell wall-derived RLPs LeEix1 and LeEix2 -linked backbone of mannose, glucose, or xylose. The central role of hemicelluloses is to fortify the cell wall by interaction with cellulose and sometimes lignin . Xylans nd GlcpA . The arand GlcpA . These fnd GlcpA . Some phnd GlcpA . Hemicelnd GlcpA . Fungi os a PAMP . In tomaArabidopsis, and rwa2 knock-out mutants have  has been shown to be responsible for the acetylation of numerous pectic and non-pectic polymers in nts have  increase cinerea . Further cinerea .D-GalpA residues  or repeating dimers of α-(1→4)-D-GalpA-α-(1→2)-L-Rhap residues . Within the pectin complex especially HGA appears to have special significance in the context of defense responses. HGA can be methyl esterified at C6 and acetylated at C2–C3 and regulation of these substitutions enable plants to fine-tune the functionality of HGA to suit prevailing local requirements. For example non-esterified HGA carrying negatively charged free carboxyl groups may be subject to cross-linking via calcium leading to the formation of gels that are required for cell adhesion and other structural roles , thereby degrading cell wall integrity and aiding pathogen access . Moreover, another class of microbial enzymes – the pectin lyases act preferentially on highly methyl esterified HGA.Some of the first enzymes that phytopathogenic fungi secrete during infections are endo-polygalacturonases (PGs) which cleave HGA  implies a less generic and more subtle effect. For example, it is possible that alterations in pmr6 cell wall composition make these mutants poor hosts specifically for Erysiphe spp. It certainly seems highly unlikely that a plant gene would have evolved to serve the needs of a pathogen, and the pleotropic effects observed in pmr6 are consistent with a role as a susceptibility factor. It is plausible that pmr6 resistance is a specialized form of disease resistance, possibly based on the loss of a host susceptibility gene required by the pathogen for growth and development  and carbohydrate binding modules (CBMs) have been invaluable for advancing our understanding of plant cell walls mponents , but thimponents .One technique that appears to offer considerable promise for plant/pathogen interaction research is based on carbohydrate microarrays . This teOur current knowledge of the interactions between plants and pathogens at the plant cell wall level, and how this affects downstream signaling is still limited. To obtain more insight we need to expand our understanding of the full repertoire of cell wall modifications that occurs during microbial interactions. Carbohydrate microarrays could be used to monitor the complex interplay between microbes and plants, and may reveal additional, perhaps more subtle, cell wall modifications to those documented so far. This technique enables rapid and multiplexed analysis of multiple changes in wall composition and could be used as a tool to provide new insight into the dynamic nature of host/pathogen interactions.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.


Implants trigger an inflammatory response, which is important for osseointegration. Here we studied neutrophil extracellular trap (NET) release of human neutrophils in response to sandblasted large-grit acid etched (SLA) implants using fluorescent, confocal laser scanning and scanning electron microscopy. Our studies demonstrate that human neutrophils rapidly adhered to SLA surfaces, which triggered histone citrullination and NET release. Further studies showed that albumin or acetylsalicylic acid had no significant effects on the inflammatory response to SLA surfaces. In contrast to bioinert materials, which do not osseointegrate, the bioactivity of SLA surfaces is coupled with the ability to release NETs. Further investigations are necessary for clarifying the role of NETosis for osseointegration. Endosteal implants are sterile foreign bodies surgically inserted into bone with an associated inflammatory host’s response –3. In caIn the initial stage of osseointegration, spaces around titanium implants are filled with blood coagulum infiltrated with leukocytes ,3 PolymoFor this purpose, we comprehensively studied (i) whether PMNs and thrombocytes attach to SLA and poly-D-lysine-coated surfaces, (ii) whether NETosis occurs on such surfaces, and if so, (iii) whether platelets, albumins, acetylsalicylic acid (ASA) and IgG contribute to implant-induced NETosis. Therefore, we quantified the responses of PMNs and platelets after contact with SLA surfaces of standardised titanium samples in a short-term incubation system. Our studies demonstrate that PMNs form NETs in response to SLA implants. Understanding this NET release by bioactive SLA implants might contribute to new possibilities for modulating osseointegration.2 for 4 hours. The same procedure was applied to the set of uncoated (control) plates. Subsequently, the bulk of coagulated blood was removed from the SLA surfaces with the help of fine tipped forceps, thus leaving only the directly surface-attached layer in place. The plates were then rinsed in phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde in PBS. For immunocytochemistry-based quantification of PMNs and NETs, two samples on SLA plates were prepared per subject, session and pre-treatment regime  were selected for donation of capillary blood. All blood donors gave written consent to take part in the study. These studies were approved by the ethical committee of the University of Tuebingen. Blood sampling from each volunteer was performed in two sessions with an interval of at least one week. Capillary blood was collected under sterile conditions from finger pricks using safety lancets and processed ,27. Sampt regime , resultiFor unambiguous identification of PMNs and for the detection of platelets, immunofluorescence staining was performed as follows: Samples were washed in phosphate buffered saline , blocked with 10% normal goat serum in PBS containing 10mM glycine and 0.2% Tween 20, and then incubated with primary antisera against appropriate molecular markers. Antisera against citrullinated histone H3  and neutrophil elastase  were employed for detection of PMNs, rat anti-human CD41 IgG1  for detection of platelets. Goat anti-rabbit DyLight 488 , goat anti-mouse TRITC  and goat anti-rat Alexa Fluor 488  were applied as secondary antibodies. DNA was stained with DAPI (Sigma Aldrich) or propidium iodide . Stained plates were mounted on glass slides and coverslipped using GelMount (BioMeda) mounting medium, and viewed and photographed in a Reichert Polyvar microscope equipped for fluorescence microscopy.ImageJ. Cell numbers per stack were assessed using the cell counter plugin. PMNs were identified by positive staining for NE, nucleated but NE-negative cells were classified as ‘other cells’. For the quantification of NETs, the outlines of areas covered by extracellular DNA were traced and the total area per stack was measured in μm2. The software SPSS 20 was used for data processing and statistical analysis. The three types of surface treatment  were compared by univariate analysis of variance with subject and sampling session as covariates, and by Tukey HSD post hoc test.For photo sampling, groups of 30 randomised non-overlapping photos per plate were taken along two parallel transects across the entire surface, thus enabling adequate representation of the central and peripheral areas. Image stacks of each group were created and analysed using the software CLSM was employed to support qualitative screening for the presence of platelets. Samples on poly-D-lysine-coated cover slips, either incubated for 4 h, or only for 5 min, and immuno-stained for CD41 as described above, were analysed and photographed in a Zeiss LSM 510 meta UV CLSM .SEM served to detect NETs and platelets. PFA-fixed samples on SLA plates and glass cover slips were, dehydrated in a graded series of ethanols, critical point dried and sputter-coated with gold, and finally viewed in a ESEM XL30  scanning electron microscope operating at 25 kV.Examination of blood cell attachment to the surfaces of the SLA titanium plates under the fluorescence microscope revealed that after 4 hours of incubation, several types of nucleated cells had attached to the titanium surfaces of the plates of all three pre-treatment schedules  , Table 1A subset of PMNs attached to the SLA surface displayed diverse stages of NETosis as defined by previous studies ,28. AccoIn order to test a possible interaction of platelet and PMN adhesion, the SLA surfaces of subsets of plates from three pre-treatment regimes were screened for the presence of these characteristic disc-shaped cell fragments. This was done by fluorescence microscopy after immunostaining for CD41, and on separately prepared specimens under the SEM see . After fThe inflammatory host response to bioactive implants is poorly understood, particularly the role of PMNs. Here we demonstrate that human neutrophils rapidly adhered to SLA surfaces and release extracellular DNA structures with characteristics of NETs. This finding also suggests that some NET components might be beneficial for osseointegration, supported by the inability of bioinert materials to osseointegrate at all  and by tActivated platelets can induce NETosis ,17,25 anPlatelet lysis through ROS production has been previously reported ,36 and tOur findings concerning the neutrophil adhesion are in accordance with earlier reports concerning the neutrophil response to both smooth titanium –9 and ot2-coated titanium surfaces, which accelerate the osseointegration in comparison with the smooth titanium surfaces [2 particles have been demonstrated to activate human PMNs [in vitro and to cause a pronounced inflammatory reaction in animal models marked by neutrophilia [2 particles. Indeed, both SLA surfaces and nano-particles [SLA surfaces are micro/nano-textured TiOsurfaces –43. Indesurfaces ,45. Inteman PMNs  in vitrorophilia –49. By varticles  are chararticles  between articles –40 and Sin vivo role of NETosis for osseointegration remains to be defined in future studies.Collectively, our results demonstrate that the initial inflammatory response to bioactive implants is marked by NETosis, which is not the case for bioinert poly-D-lysine-coated surfaces. NETosis is the first reported mark of initial response to a bioactive implant, so far as the bioactivity of endosteal implants has been defined only as the ability to osseointegrate. The deterioration of osseointegration through administering anti-inflammatory drugs during the initial stages of osseointegration –32 and tS1 Dataset(XLS)Click here for additional data file.S2 Dataset(XLS)Click here for additional data file.S3 Dataset(XLS)Click here for additional data file.S4 Dataset(XLS)Click here for additional data file.S1 FigOverview of the SLA micro/nano-textured surface. Scale bars: 5μm.(TIF)Click here for additional data file.


Fouramong 10 patients undergoing coronary artery bypass graft surgery (CABG) are diabetic inthe United States.Furthermore, insulin-dependent diabetics undergoing CABG reaches 20% in São PauloRegistry of Cardiovascular Surgery (REPLICCAR) .Coronary artery disease (CAD) is a leading cause of mortality and morbidity in diabeticpatients. Over time, one of the reasons that made the CABG overlappercutaneous techniques was the anastomosis of the internal thoracic artery (ITA) on theleft anterior descending artery. Long-term benefits would be related to higher patencyof the ITA graft compared to the saphenous vein graft. Later studies in large databases were able toshow that the use of bilateral internal thoracic artery (BITA) increased further patientsurvival. Inthis respect, only one prospective and randomized study showed no difference in one yearand awaited long term outcomes.Currently, there is a progressive increase in the prevalence of diabetes in patientsreferred for CABG. This is mainly in response to FREDOOM trial that showed that diabeticpatients have a higher survival rate when they undergo surgical myocardialrevascularization showed that even in insulin-dependent diabetic patientadvantagens with BITA is over. This means that the longer survival achieved with BITA isnot affected by a higher incidence of deep sternal wound infection (DSWI) in the shortterm. In orderto justify the risk of this complication, patients should have a life expectancy of>10 years. Therefore, they are contraindicated for the most part of the time indiabetic individuals.Therefore, which could make us desist from using BITA? Demand for technical dissection ofBITA and risk of deep sternal infection especially in diabetic patients would be themost frequent reasons. Gatti et al.. These changes may contribute to the less favorablepostoperative outcomes after cardiac surgery. Complications and DSWI were significantlyhigher in uncontrolled (HbA1c >7%) than with controlled (<7%) diabeticpatients.Hyperglycemia in perioperative CABG increases up to 10 times the risk of morbidity andmortality. Thus, using protocols with continuous insulin drip to control hyperglycemiaduring the perioperative period (<180 mg/dl) reduced mortality, morbidity, incidenceof DSWI, length of stay and increased long-term survival.CABG to insulin-dependent diabetic patient comprises various quality parameters. Thisbegins with the classification in controlled and uncontrolled diabetics by glycosylatedhemoglobin (HbA1c). It has been demonstrated that poorly controlled diabetes patientsbut not well-controlled diabetes significantly impairs endothelium-dependent andendothelium-independent relaxation of human peripheral microvasculature as compared withnon-diabetes. Because of diffuseatherosclerotic disease and complex coronary anatomy, complete revascularization in CABGis recommended for diabetic patients. Even more than that, the use of BITA plus incompleterevascularization is better than single AIT plus complete revascularization in atwenty-year-survival. Thereby, BITA is the most important to CABG in diabeticpatients.Skeletonization technique instead of pedicle preparation confers a protective benefitagainst sternal wound infection in patients receiving BITA and this should be thetechnique of choice for diabetics in whom BITA harvest is desired. In addition, the graftpatency after 1 year was significantly lower in the off-pump CABG (83.1%) when comparedto on-pump CABG (88.4%). Raza et al. shows that the hospital outcomes and long-term survivalof patients undergoing off pump or on-pump surgery are similar.With respect to CABG be performed with or without the use of cardiopulmonary bypass(CPB), a subanalysis of the ROOBY trial show higher rates of incompleterevascularization in diabetic patients operated with off-pump surgery recommends that the use of BITA should be considered inpatients <70 years (Class IIa). Also, it provides that BITA in diabetic patientsshould be considered. Thus, all diabetic patients under 70 years, without morbid obesityand HbA1c <7% should receive BITA. On the other hand, skeletonized dissection of ITAsis recommended in these patients. After this support, the use of BITA as a qualitystandard in insulin-dependent diabetic patients is advised after CABG.The Guideline ESC / EACTS 2014 in CABGand around 10% in the European Database, contradict the recommendations given by the respectivehost societies. REPLICCAR showed better results, and it is the first multicenterprospective cohort of patients undergoing cardiovascular surgery in São Paulothat has 10% of BITA.However. it might be best to avoid BITA grafting in obese diabetic women with diffuseatherosclerotic, the use of BITA becomes the best cost-effectiveness revascularizationstrategy to long term survival even in insulin-dependent diabetic patients. This isreinforced by a growing incidence in coronary artery disease in young individuals andincrease in life expectancy in developing countries. Therefore, underutilized BITA, 4.4%in the STS databaseIn conclusion, BITA grafting is recommended for patients with diabetes undergoing CABGand efforts should be made to complete revascularization. DSWI, because of its rareoccurrence, had little effect on morbidity and mortality increase. Accordingly, thestate-of-the-art coronary artery bypass surgery in insulin-dependent diabetic patientsconsidering the use of BITA is a quality parameter of health care.


Bronchial arteries originate from the descending aorta at the level of the T5-T6 vertebrae following an intrapulmonary course along the major bronchi. When bronchial arteries take off from a vessel other than the descending aorta, the anatomy is defined as an anomalous origin of the bronchial artery.A 3-day-old boy from Kosovo with dextro-transposition of the great arteries who developed progressive heart failure required an emergency arterial switch operation. Because of persistent pulmonary edema after completion of the arterial switch operation at our institution, the patient could not be weaned off mechanical ventilation. Transthoracic echocardiography revealed an anomalous accelerated flow, indicating an anomalous systemic pulmonary shunt. Arterial catheterization revealed an abnormal bronchial artery originating from the left subclavian artery and bifurcating to both lungs. The anomalous ectatic bronchial artery was successfully occluded by coil embolization. The improvement of the patient’s hemodynamic status resulted in an uneventful post-operative course.A coil embolization procedure was successfully performed to treat an anomalous bronchial artery originating from the left subclavian artery after a switch operation in a patient with transposition of the great arteries. When clinically indicated, catheter-based therapy with coil embolization can be performed to successfully treat anomalous bronchial arteries by reducing as such the pulmonary overflow. Bronchial arteries (BAs) normally originate from the descending thoracic aorta at the level of the T5-T6 vertebrae with an intrapulmonary course along the major bronchi . Anomalo1, dopamine, oxygen and antibiotics was initiated. The following day, owing to hemodynamic instability, urgent corrective cardiac surgery was necessitated. The intra-operative findings included the following. The sinus 2, according to the Leiden convention [A 3-day-old boy from Kosovo, weighing 2.4kg, was admitted to our hospital with severe dyspnea and cyanosis. Transthoracic echocardiography revealed d-TGA with intact ventricular septum, left aortic arch and a 4mm patent ductus arteriosus. Medical therapy with prostaglandin EThe most common complications after an ASO are the supravalvular pulmonary stenosis, neoaortic valve dysfunction and myocardial ischemia related to coronary artery translocation ,6, such Hypoxemic respiratory failure happens as a result of atelectasis, infection, pulmonary immaturity or surfactant deficiency, pulmonary hypertension and ventilation/perfusion mismatch. The presence of a left-to-right shunt due to enlarged BAs might be another cause of respiratory failure . In patiAbout half of patients with TGA undergoing ASO present with anomalous bronchial collateral arteries detected during post-operative cardiac catheterization studies . Flow thAbnormally enlarged BAs might not be diagnosed in patients with simple TGA, as pre-operative angiography is rarely performed. Unexplained pulmonary hypoxemia with pulmonary hypercirculation after ASO could be an indicator of anomalous enlarged BAs. Post-operative cardiac catheterization permits the clinician to visualize the pulmonary veins and left atrium after injection of contrast medium into the aorta. The presence of enlarged BAs should be differentiated from the major aortopulmonary collateral arteries (MAPCAs), which supply blood to the lungs when the native pulmonary circulation is underdeveloped, which carries a pathophysiology different from simple TGA. The MAPCAs’ sites of origin are from the descending aorta (70% of cases), aortic arch (15% to 20% of cases) and ascending aorta (10% to 15% of cases) [Previous studies have documented enlarged BAs in patients with TGA ,9; howevet al. [The most interesting finding in our patient was the anomalous origin of a single-trunk BA originating from the left subclavian artery that furnished both lungs simultaneously. Such an abnormality is scarcely reported in the literature ,12 and het al. .Late surgical ligation or coil embolization of these collateral arteries has been reported following ASO . AlthougIn this report, we describe a case of a pediatric patient with a combination of an anomalous BA originating from the left subclavian artery and TGA, whom we treated successfully with coil embolization. Catheter-based therapy with coil embolization can be performed with good results when clinically indicated, reducing pulmonary overperfusion and avoiding the risks of a surgical procedure.Written informed consent was obtained from the patient's legal guardian for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.