Sepsis is a syndromic illness that has traditionally been defined by a set of broad, highly sensitive clinical parameters. As a result, numerous distinct pathophysiologic states may meet diagnostic criteria for sepsis, leading to syndrome heterogeneity. The existence of biologically distinct sepsis subtypes may in part explain the lack of actionable evidence from clinical trials of sepsis therapies. We used microarray-based gene expression data from adult patients with sepsis in order to identify molecularly distinct sepsis subtypes.We used partitioning around medoids (PAM) and hierarchical clustering of gene expression profiles from neutrophils taken from a cohort of septic patients in order to identify distinct subtypes. Using the medoids learned from this cohort, we then clustered a second independent cohort of septic patients, and used the resulting class labels to evaluate differences in clinical parameters, as well as the expression of relevant pharmacogenes.We identified two sepsis subtypes based on gene expression patterns. Subtype 1 was characterized by increased expression of genes involved in inflammatory and Toll receptor mediated signaling pathways, as well as a higher prevalence of severe sepsis. There were differences between subtypes in the expression of pharmacogenes related to hydrocortisone, vasopressin, norepinephrine, and drotrecogin alpha.Sepsis subtypes can be identified based on different gene expression patterns. These patterns may generate hypotheses about the underlying pathophysiology of sepsis and suggest new ways of classifying septic patients both in clinical practice, and in the design of clinical trials. The protean illnesses of the ICU are syndromic in nature, defined by a number of clinical, laboratory and radiologic criteria, rather than specific pathologic findings. Examples of this include acute respiratory distress syndrome (ARDS), acute kidney injury (AKI) and sepsis. In such cases, the lack of specificity of the diagnostic criteria may lead to the inadvertent grouping together of physiologically disparate disease states under the same rubric ,2. By faGene expression microarray data may be useful in identifying sepsis subtypes based on differential expression of key genes . In pediMicroarray data were obtained from two previously published, prospectively designed studies of gene expression in sepsis. Patient enrollment, data collection, RNA extraction and gene-expression profiling were carried out in the same manner for both studies, and are described in detail elsewhere ,9. BriefData from two separate studies conducted using the same tissue and the same microarray platform were used. In both studies, sepsis was defined as the presence of systemic inflammatory response syndrome (SIRS) and infection, where the diagnosis of infection required the presence of clinical, as well as laboratory or pathological, evidence of infection. The first study included 72 critically ill patients, 55 of whom met diagnostic criteria for sepsis (derivation cohort). The second study included 94 critically ill patients, 71 of whom met diagnostic criteria for sepsis . The latter study included a larger number of missing values from gene expression profiling, and one patient from the sepsis group with > 80% missing data was removed.We used partitioning around medoids (PAM) clustering based on Euclidean distance, in order to identify sepsis subtypes within the gene expression profiles. From the derivation cohort, we identified the set of genes with the greatest differences in expression levels between subtypes and evaluated these as the gene signature.k).In order to reduce the dimensionality of the dataset and improve the likelihood of discovering stable clusters, we used a multi-stage approach to feature selection. First, we searched Genbank for relevant genes using the terms "sepsis", "severe sepsis" and "septic shock". We then reduced the candidate genes to the intersection of the complete set of genes and the sepsis-specific set. Next, we carried out an enrichment step to identify the most discriminatory genes from within this subset, as well as the optimal number of clusters . We chose the value of To identify a specific gene signature, we again used a process of randomly selecting one-third of the sepsis-specific genes, and used these as the basis for PAM clustering. One hundred cluster solutions based on random thirds were carried out. Each time the gene set producing the highest average silhouette width was added to a list of high-value genes. This process was repeated 100 times, after which we created a tally of the number of times each gene had been included in the high-value list. The 100 most frequently identified genes were taken as the enriched subset.We used PAM clustering with the enriched subset of sepsis-specific genes to determine class labels for each of the patients in the derivation cohort. Using these labels, we then returned to the complete set of genes, and used significance analysis of microarrays (SAM) to identify genes that showed differential expression between groups . This prk identified in the preceding steps. We examined the distribution of individual gene expression values between cluster pairs, to determine whether these were bimodal, as would be expected if the clusters were distinct. This was done using the bimodality index [As an additional verification step, we performed hierarchical clustering using Minkowski distance as a measure of similarity and Ward's method for agglomeration, and compared the cluster results to those obtained by PAM. We also carried out a number of statistical analyses to assess the internal validity of the clustering solutions derived from the above procedures. At each stage of enrichment, we evaluated the silhouette width of each cluster. We also carried out a bootstrapping cluster analysis to see how stable the solutions were, given the value of ty index , which yFor both the derivation and validation cohort, we used hierarchical clustering to identify the subset of genes that were differentially co-expressed between groups. We conducted a pathways analysis of this gene signature using the PANTHER classification system , with thWe used the clustering solution derived from the method described above to identify sepsis subtypes within a second, independent dataset  that was based on the same microarray platform as the first. We limited the genes in this dataset to those that were included in the gene signature. To classify the patients in the validation cohort, we determined which of the derivation medoids they were closest to, and assigned them to the corresponding subtype.t-test. To investigate the potential role of genetic differences in accounting for some of the negative or conflicting clinical evidence for sepsis therapies, we looked at genes implicated in the action of a select group of drugs that have been studied in large-scale randomized controlled trials (RCTs) in sepsis. Using the Pharmacogenomics Knowledge Base (PharmGKB) [Using these labels, we evaluated a limited number of clinical attributes using Fisher's exact test and Student's harmGKB) , we idenharmGKB)  to expanAll analyses were performed using the R software environment for statistical computing and graphics, with functions from the samr, clValid, cluster, e1071 and ClassDiscovery libraries .k = 2, regardless of whether PAM or hierarchical clustering was used. Results for the validation cohort were similar.The Genbank search returned a total of 450 unique genes, 365 of which were included in the microarray platform. Using this subset of genes, the best silhouette values were achieved with a value of Graphical representations of the derivation clusters through successive stages of enrichment are shown in Figure k = 2 was stable . Internal measures showed cluster stability similar to that achieved with the derivation cohort, including an average silhouette width of 0.26 Figure .Hierarchical clustering revealed 178 co-expressed genes in the derivation cohort, and 171 co-expressed genes in the validation cohort. All but one of the validation genes was also found in the derivation set, and the 170-gene intersection was taken as the gene signature  to pure yellow (the samples are in the same branch 100% of the time). (A) Result using the?initial gene set derived from Genbank. (B) Results following the gene enrichment stages. Analysis carried out in R using the ClassDiscovery package.Click here for fileDistributions of gene expression values. Density plots for the 50 genes with highest bimodal index.Click here for fileSepsis subtype gene signature . Gene signature derived from the overlap of co-expressed genes in the derivation and validation cohorts.Click here for file


The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of  Saccharomyces cerevisiae. The sequence specificities of TFs are generally represented as position specific weight matrices (WMs) and using these WMs in combination with sophisticated comparative genomic algorithms for transcription factor binding site (TFBS) prediction, it is now possible to obtain fairly comprehensive annotations of the TFBSs occurring across yeast promoters Large-scale ChIP-chip and protein-microarray experiments, e.g. Using data from several high-throughput methods, comprehensive annotations of transcription start sites (TSSs) in yeast have also become available recently Saccharomyces cerevisiae, are well-known core promoter elements which are known to have very specific positional preferences relative to TSS. In particular, in mammalian promoters the distribution of TATA sites is sharply peaked at about TATA sites, also called TATA boxes, which are recognized by TATA-binding protein in mammals and by SPT15 in Here we show that, besides the TATA motif, there are Saccharomyces cerevisiae and the sensu stricto Saccharomyces species that have been sequenced, we predicted TFBSs for the As we have described previously Finally, using experimentally determined TSSs Interestingly, families : The welfamilies , and theGATA motif, i.e. GATGAG and GATAAG. The FKH and ROX1 motifs share a common AACAA core, and the CACAA motif of NDT80 differs in only 1 letter from this core. In general, all motifs contain runs of purines interspersed by either a single thymine or a single cytosine. We will see below that the PPMs share these features with the initiator motif found at TSSs.Besides showing the positional distribution of the To investigate whether the positional preferences that we observe could simply be a result of the sequence composition of promoters relative to TSS we performed binding site predictions on a set of randomized alignments. These randomized alignments are constructed by permuting the original alignment columns in such a way as to conserve the di-nucleotide frequencies as a function of position relative to TSS, the exact gap patterns, and the cross-species conservation patterns of the original alignments see . For exaAs we mentioned in the Comparisons of the expression profiles of TATA-containing and TATA-less promoters have indicated that TATA-less promoters are enriched for house-keeping genes that are expressed in a fairly constitutive manner, whereas TATA-containing promoters show more variability in expression, and are often induced in response to various stresses We next investigated the distribution of TFBSs for individual TFs in TATA and TATA-less promoters. Consistent with the pattern shown in The bottom two panels of For each TF we also investigated nucleosome coverage in the subset of promoters that contain a binding site for the TF, again separately for TATA and TATA-less promoters. To this end we averaged nucleosome coverage patterns over all promoters, weighting each promoter with the probability that at least oter see . Strikinoter see . That isThe results so far have shown that there is a clear difference in the promoter architecture of TATA-containing and TATA-less promoters. In particular, TFBSs are positioned further upstream in TATA promoters, and the region of lowest nucleosome coverage closely matches the region of highest TFBS occurrence. To investigate the potential role of the PPMs in TATA-containing and TATA-less promoters we measured their binding site positioning in both classes of promoters.We first determined binding site positioning for the TATA motif itself in TATA-containing and TATA-less promoters. We next considered TFBS positioning of the other PPMs. Strikingly, in contrast to other TFs , the PPMAs a further support of the preference of PPMs for TATA-less promoters we also investigated directly to what extent the PPMs avoid TATA-containing promoters. For each of the PPMs other than SPT15, and for each TSS, we assigned the PPM to the TSS when a binding site with posterior probability of at least romoters . For RPNAs mentioned in the Besides the TATA motif, another core promoter motif that has attracted considerable attention is the initiator motif, see e.g. The fact that there is no difference between the initiator in TATA-containing and TATA-less promoters suggests that there is no systematic difference in the mechanism of initiation site selection in TATA and TATA-less promoters. Furthermore, we reasoned that just as TF motifs represent the binding specificities of TFs, so the initiator motif may represent the binding specificity of the PIC. Therefore, by systematically comparing the matches to the initiator motif of the sequences upstream of TSS, both in TATA and TATA-less promoters, we may identify which locations upstream of TSS have higher and lower affinity for the PIC, and maybe gain insight into where the PIC is initially recruited.For every known TSS, from both data sets, we extracted the DNA sequence from Importantly, the locations of these second maxima clearly distinguish TATA-containing promoters from TATA-less promoters, with the maximum occurring significantly further upstream in TATA promoters. The affinity profiles are suggestive of a process in which the PIC is initially recruited at the upstream peak, after which it would quickly scan through the low affinity region, to finally ‘lock in’ at the strong initiator motif at the TSS. Most importantly, however, from the point of view of PPM analysis, is the fact that the location of the upstream peak in PIC affinity in the TATA promoters corresponds to the position where the highest density of TATA sites is found see , and theFinally, we note that, although not very specific, there is a clear similarity between the initiator motif and the PPMs: all of these motifs contain runs of purines interrupted by a pyrimidine see . This isAs a final comparison of the PPMs we classified promoters according to the PPMs that they contain. For each TSS we calculated a score for each PPM based on the quality and the position of sites for that PPM see . We thenWe then determined the overall frequency, positional distribution of TFBSs, and nucleosome coverage, for each set of TSSs assigned to one of the PPMs, and for the set of TSSs that are not assigned to any of the PPMs. Strikingly, the profiles that we obtain clearly identify three classes of promoters . PromoteSaccharomyces cerevisiae promoters. We uncovered that TATA-box containing and TATA-less promoters have significantly different architectures. Compared to TATA-less promoters TATA promoters show an overall lower number of TFBSs per promoter, these TFBSs occur further upstream of TSS on average, and show a wider distribution of distances with respect to TSS. We find that the TFBS profiles closely mirror nucleosome coverage profiles, i.e. TATA promoters have higher nucleosome coverage, the region of lowest nucleosome coverage occurs further upstream, and the region of lowest nucleosome coverage is more sharply defined in TATA-less promoters.Starting from a genome-wide annotation of TFBSs for There recently has been a large amount of investigation into the mechanisms that determine nucleosome positioning, and the extent to which nucleosome positioning is determined by intrinsic sequence preferences of the nucleosomes is currently actively disputed, see Whereas the position of overall highest TFBS density occurs more than A key result of this study is that, besides the TATA motif, there are an additional Our results establish that many of the TATA-less promoters are characterized by the occurrence of alternative PPMs, and suggest that these play a crucial role in regulating transcription at these TATA-less promoters. The main question that now arises is what the precise functional role of these alternative PPMs is, and how their function relates to that of the TATA-box. With respect to the latter, although only about The simplest hypothesis for the functioning of the alternative PPMs, which is consistent with all our results, is to assume that they ‘replace’ the TATA site in TATA-less promoters, i.e. that these PPM sites are directly involved in recruiting the PIC. However, a review of the literature on the PPM motifs is at odds with this simple interpretation.First, GATA sites are generally found upstream of genes that are subject to nitrogen catabolite repression The NDT80 motif is another example of a motif where a repressor and an activator TF compete for binding to target sites. NDT80 is a meiosis-specific TF that is required for exit from pachytene and that activates middle sporulation genes. During mitosis and in the vegetative state the same binding sites, which are also called middle sporulation elements (MSEs), are bound by the repressor SUM1, i.e. SUM1 acts as a brake on meiosis A similar function applies to the third proximal promoter motif, ROX1. ROX1 is a heme-dependent repressor of hypoxic genes, i.e. under aerobic conditions ROX1 binds to its target sites while under anaerobic conditions its targets are derepressed Fourth, the forkhead transcription factors FKH1 and FKH2 are key regulators of the cell-cycle in yeast, targeting the CLB2 cluster of genes which includes the downstream TFs SWI5 and ACE2 Fifth, the PAC (Polymerase A and C) motif is found in the promoters of ribosome biogenesis and rRNA genes and it has recently been shown to be bound by the TFs PBF1 and PBF2 Finally, RPN4 is an activator of 26S proteasome genes which is itself rapidly degraded by the proteasome, generating a negative feed-back loop that controls proteasome homeostasis In summary, it appears that all PPMs are involved in responding to environmental stresses, often involving nutrient availability, either releasing  their target genes in response to the stress or  repressing their targets when stresses are present. Another feature shared by the PPMs is that, through competition of both activating and repressing TFs binding to the site, the PPM sites are essentially always bound. These features are consistent with their preferred targeting of TATA-less promoters.Previous studies have shown that TATA promoters in yeast are characterized by closed chromatin, regulation through chromatin, and that many of the associated genes are upregulated upon various stresses. In contrast, ‘house-keeping’ genes tend to have TATA-less promoters Saccharomyces cerevisiae orthologous intergenic regions from four other sensu stricto Saccharomyces species were obtained using the ORF annotations of The binding site annotations were performed as described previously The randomized alignments were constructed as follows. Since we want to maintain the exact conservation patterns of the original alignments we first checked which subset of species have orthologs for each of the alignments and find that for We used a combination of the TSS annotations from To determine the most preferred position of a motif in a way that is robust to the noise in the positional profiles we proceeded as follows: We first determined the position unique TSS in all data-sets used To start constructing initiator motifs we wanted to initially focus on genes that have the same romoters  which leequences . We iteress TSSs .Finally, after we determined that there were no systematic differences between the initiator motifs in TATA and TATA-less promoters . This gave For the assignment of PPMs to TSSs we consider only TFBSs, for each PPM, that occur near the preferred position for the PPM. For each PPM, we considered TFBSs within a range of In addition to the standard MotEvo settings which yielded the ‘specific set’ of TFBS predictions, we also produced a set of more ‘sensitive’ TFBS predictions. In making its predictions, MotEvo considers that each region within the multiple alignment can either contain a binding site for one of the WMs (which has been under selection in one or more of the species), neutrally evolving background DNA, or a regulatory element of unknown function, i.e. a TFBS for a TF for which we currently have no WM Figure S1Average GC-content of the true (red) and randomized (green) promoter sequences as a function of position relative to TSS. The figure shows that, in line with the way the randomized promoters were constructed Click here for additional data file.Figure S2Reverse-cumulative distribution of the number of predicted binding sites across motifs for predictions on the original alignments (red symbols) and on the randomized alignments (green symbols). The ‘number’ of predicted binding sites is defined as the sum of the posterior probabilities of all binding sites that lie within the least predicted sites on the true alignments.(TIFF)Click here for additional data file.Figure S3Cumulative distribution of the position of highest TFBS density across motifs for binding site predictions done on the true (red) and randomized (green) alignments. Each symbol corresponds to one motif. The blue symbols indicate the PPMs on the randomized alignments. As the figure shows, on the randomized alignments the positions of highest TFBS density for the PPMs vary greatly indicating that their preference for proximal locations in the true alignments is not a consequence of di-nucleotide composition of the promoters.(TIFF)Click here for additional data file.Figure S4Positional distributions of TFBSs for the proximal promoter motifs on the true (red lines) and randomized (green lines) alignments. Each panel corresponds to one of the proximal promoter motifs. In each panel, the position relative to TSS is indicated along the horizontal axis and the density of TFBSs is shown along the vertical axis. The figures show that PPMs show little evidence of preferred positioning on the randomized alignments.(TIFF)Click here for additional data file.Figure S5Positional distributions of all TFBSs for promoters with the region of minimal nucleosome coverage (MNO) up to 100 base pairs upstream of TSS (red), between 100 and 200 base pairs upstream (green), between 200 and 300 base pairs upstream (blue), and more than 300 base pairs upstream (purple). The inset shows the total number of binding sites in each of the promoter classes. The results demonstrate the locations of highest TFBS density match the locations of the region of minimal nucleosome occupancy.(TIFF)Click here for additional data file.Figure S6Positional distributions of the TFBSs for each proximal promoter motif, separately in promoters with the region of minimal nucleosome occupancy (MNO) up to 100 base pairs upstream of TSS (red), between 100 and 200 base pairs upstream (green), between 200 and 300 base pairs upstream (blue), and more than 300 base pairs upstream (purple). Each panel corresponds to one proximal promoter motif . In each panel the horizontal axis shows position relative to TSS and the vertical axis shows TFBS density. The insets show the total numbers of predicted TFBSs in each promoter class for the corresponding motif. With the exception of the TATA motif, which shows highest density of TFBSs in promoters with MNOs more than 200 base pairs upstream, all other PPMs show highest densities of TFBSs in promoters with MNOs more proximal to TSS.(TIFF)Click here for additional data file.Figure S7Affinity profiles for the initiator WM exclusively for TATA promoters. To construct the red profile, the TSS was taken as reference point in each promoter, whereas for the green profile the TATA box was taken as a reference point. To align the green and red profiles, we set the TATA-box reference point at the position where the highest density of TATA sites is observed ((TIFF)Click here for additional data file.Figure S8Upper panels: Seed WMs for initiator motifs in TATA and non-TATA promoters. Lower panels: Iterated WMs obtained from (TIFF)Click here for additional data file.


This article addresses the boundary layer flow and heat transfer in third grade fluid over an unsteady permeable stretching sheet. The transverse magnetic and electric fields in the momentum equations are considered. Thermal boundary layer equation includes both viscous and Ohmic dissipations. The related nonlinear partial differential system is reduced first into ordinary differential system and then solved for the series solutions. The dependence of velocity and temperature profiles on the various parameters are shown and discussed by sketching graphs. Expressions of skin friction coefficient and local Nusselt number are calculated and analyzed. Numerical values of skin friction coefficient and Nusselt number are tabulated and examined. It is observed that both velocity and temperature increases in presence of electric field. Further the temperature is increased due to the radiation parameter. Thermal boundary layer thickness increases by increasing Eckert number. There is a substantial interest of the recent researchers in the flows of non-Newtonian fluids. Such motivation in these fluids is mainly because of their use in the industrial and technological applications. Many materials like mud, pasta, personal care products, ice cream, paints, oils, cheese, asphalt etc. are non-Newtonian fluids. Most biological fluids with higher molecular weight components are also non-Newtonian in nature. The usual properties of polymer melts and solutions together with the desirable attributes of many polymeric solids, have given rise to the world-wide industry of polymer processing. The non-Newtonian fluids in particular have key importance in geophysics, chemical and nuclear industries, material processing, oil reservoir engineering, bioengineering and many others. Rheological properties of all the non-Newtonian fluids cannot be predicted using single constitutive equation (unlike the case of viscous fluids). Therefore many models of non-Newtonian fluids are based either on “natural” modifications of established macroscopic theories or molecular considerations. The additional rheological parameter in the constitutive equations of non-Newtonian fluids are the main culprit for the lack of analytical solutions. The resulting equations are more complex and higher order than the Navier-Stokes equations. Hence these equations have been attracted from modelling as well as solutions point of view. The advancement in the study of non-Newtonian fluids has been made even by the recent investigators  third grade fluid over a porous stretching surface. Here the fluid is electrically conducting in the presence of applied magnetic If The local skin friction coefficient is defined asThe velocity and temperature can be expressed in the set of base functionsThe initial guesses The zeroth order problems areSetting th order of approximation in We note that the series solutions (33) and (34) contain the non-zero auxiliary parameters 1E and Eckert number This section illustrates the impact of physical parameters. The results are displayed graphically in the The flow of third grade fluid and heat transfer in the presence of thermal radiation and Ohmic dissipation are examined. The graphs are prepared to study the influence of the pertinent flow parameters including the second grade parameter (The effect of third grade parameter The maximum velocity is attained for higher values of electric parameter Effect of suction parameter, unsteadiness parameter and Reynolds number on boundary layer thickness is similar in a qualitative sense.Effects of The velocity field Magnitude of skin friction coefficient Electric parameter


Histopathologic assessment of liver tissue is an essential step in management and follow-up of non-alcoholic fatty liver disease (NAFLD) while inter- and intra-observer variations limit the accuracy of these assessments.The aim of this study was to assess the inter- and intra-observer reproducibility of histopathologic assessment of liver biopsies based on NAFLD activity score (NAS) scoring system.The anonymous liver biopsy samples of 100 consecutive NAFLD suspected adults were randomly assigned to four pathologists. Then, the samples were randomly reassigned to the pathologists for the second time in a way that each sample would be evaluated by two different pathologists. Biopsies were revisited by their first evaluator after two months. The results were reported based on NAS scoring system.Inter-observer agreement of the pathology scores based on NAS scoring system was acceptable for steatosis, lobular inflammation, and fibrosis, but not for hepatocyte ballooning. The intra-observer agreement was acceptable in all scales, with lowest intra-class correlation observed for lobular inflammation.NAS scoring system has good overall inter- and intra-observer agreement, but more attention should be given to defining the hepatocyte ballooning and lobular inflammation, and training the pathologists to improve the accuracy of pathology reports. Considering the increasing prevalence of non-alcoholic fatty liver disease (NAFLD) world-wide, it is essential to have methods and procedures for accurate diagnosis of the cases, as well as to identify patients with non-alcoholic steatohepatitis (NASH) . The diaHistopathologic assessment of liver tissue is an essential step in the management and follow-up of chronic liver conditions , 4. It iAlthough liver biopsy is the gold standard of staging and evaluating the progress of the disease, it is a risky procedure and there are a few limitations that affect the clinical acceptance of the process . InvasivSampling error is another issue which affects the acceptance of liver biopsy and it mainly results from sampling variability and observer variation. Sampling variability reflects the uneven distribution of histologic lesions in the liver tissue and may result in misdiagnosis and staging inaccuracy , 7. InteNAFLD comprises a wide morphological spectrum which makes the pathologic evaluation and distinction difficult. NAFLD is histologically further categorized into NAFL and NASH. The diagnosis is made based on the degree of steatosis, hepatocellular ballooning, and lobular inflammation . HistopaInterventions such as image review by the pathologists and use of scoring sheet with written diagnostic criteria for different NAFLD phenotypes have been applied to improve the agreement . Also, vSome researchers believe that NAS is a valuable tool in clinical trials, while its generalizability and diagnostic accuracy should be studied . AlthougThe aim of this study was to help the clinicians better judge the results of the liver biopsies both on diagnosis and follow-up of NAFLD patients.In this cross-sectional study, 100 consecutive liver samples of adult cases suspected to NAFLD whom were biopsied in 6 different hospitals in Tehran, Iran, between 2010 and 2012 were included. All of the cases were diagnosed for NAFLD based on clinical evaluation and evidence of steatosis in ultrasonography after ruling out the other etiologies of fatty liver like excessive alcohol consumption and other chronic liver disease. The cases were Iranian and older than 18 years old.Ultrasound-assisted percutaneous liver biopsy was performed using Tru-Cut biopsy needles.The biopsy samples were sectioned in 3 different levels and stained by hematoxylin and eosin, trichrome, and reticulin methods. For the purpose of the study, these anonymous biopsy section samples were randomly assigned to four pathologists  who had agreed to evaluate the samples. The pathologists were not aware of patient identity or open label pathology report, and reported the samples based on NAS scoring system . The samWhen all samples were evaluated, they were randomly reassigned to the pathologists for the second time in a way that each sample would be evaluated by two different pathologists. The pathologists were blinded to the result of the prior evaluation and the identity of the first evaluator. The agreement between the 2 raters was evaluated by intra-class correlation (ICC). From 100 biopsies, 91 cases were revisited by their first evaluator after 2 months and reported based on NAS scoring system. The agreement between the 2 evaluations was evaluated by ICC to assess the intra-observer agreement.The study was approved by the ethical committee of Middle East Center of Liver Disease (MELD). The results were analyzed using  Mean and standard deviation were used to describe the data. ICC was applied to assess the correlation between the pathologists' evaluations in different scales. One hundred liver biopsies of adult cases were evaluated. Four cases were excluded due to technical problems in staining. The mean age of participants was 41.5 (SD: 9.74) years and the range was 18 to 58 years. Sixty four cases were male.The results of the NAS total score based on the first pathologists' assessment is presented in The ICC of the total NAS scores and steatosis, lobular infiltration, and ballooning scores are presented in The intra-observer agreement of the pathologists in different scales is demonstrated in In our study, highest agreement was observed in steatosis scale both in inter-observer and intra-observer assessments. The agreement for hepatocyte ballooning and lobular inflammation was lower compared to other scales. Research has shown that ballooning and lobular inflammation are important pathologic features that enable discrimination of NASH . The stuWe observed moderate inter-rater agreement and good intra-rater agreement in fibrosis score, while Kleiner et al.  showed hPrevious studies have shown lower agreement in pediatric cases compared to adults , 15. OurVariability in the distribution of hepatic lesions within the liver reduces the accuracy of liver biopsy as the gold standard . ConsideExperts have suggested a few methods for reducing the risk of misclassification. Interpretation of biopsies by experienced liver pathologists is one of these methods . Others Despite the importance of histopathologic findings in the management of liver diseases and their increasing prevalence, most of the pathologists have not received enough formal training and have little experience with liver biopsy . Some researchers have shown that in evaluation of liver samples from chronic viral hepatitis, the level of experience of the pathologist in terms of specialization, duration, and location of practice has a stronger effect on the agreement compared to the characteristics of the specimen . Many non-invasive methods have been proposed for evaluation of NAFLD–suspected cases. Fibroscan® is one of these methods which has gained popularity in the clinic. Accuracy of Fibroscan® is variable especially in health conditions such as steatosis, metabolic syndrome, high body mass index, hepatic hemangioma, and heart failure . AnotherAlthough many experts believe that histological assessment of liver in NAFLD cases “is far from being both accurate and precise” , it is sFinally, NAS scoring system has good overall inter-observer and intra-observer agreement, but more attention should be paid both in defining the hepatocyte ballooning and lobular inflammation and training of the pathologists to improve the accuracy of the pathology reports.


Heterogeneity of neuronal responses is abundant in sensory systems. For example, spiral ganglion neurons (SGN), which are postsynaptic to mechanosensory inner hair cells (IHCs), show high variability in spontaneous and sound-driven discharge . Recent 2+ channels with voltage-dependent gating rates. The vesicle fusion rate is proportional to the calcium concentration [Ca2+] at the release site. We approximated [Ca2+] changes as instantaneous upon the Ca2+ channel opening and closing, and set the spatial profile as a linear approximation of buffered diffusion. The recovery rate of empty docking sites was assumed to be constant. As a quantitative response characteristic of the synapse we considered adapted rate-level (RL) functions, one of the experimentally most studied response features, of SGN. We analyzed the temporal pattern of release events at the synapse as a proxy of the SGN spike pattern, which is justified by experimental evidence  levels at the release sites. Changes in RL function slopes resulted either directly from changes in slopes of Ca2+ channel voltage activation curves or varying levels of heterogeneity among different release sites of the same active zone. In spite of the relative freedom of the parameter choice in reproducing the main effects, some of the choices resulted in more efficient stimulus encoding, than the others. We discuss information-transfer-optimal organizations with particular emphasis on the biologically most plausible active zones that consist of single channel driven release sites.We found that the model is capable of reproducing the SGN RL function heterogeneity semi-quantitatively including the experimentally known correlations between the activation thresholds, spontaneous and maximum rates, and dynamic ranges. These experimental requirements partially constrain the active zone organizations. Nevertheless, various parameter sets can result in similar RL functions. For example, the shift of the stimulus dependence of RL functions was achieved either by shifting the voltage dependence of Ca


Slow oscillations are a hallmark of slow wave sleep. They provide a temporal framework for a variety of phasic events to occur and interact during sleep, including the expression of high-frequency oscillations and the discharge of neurons across the entire brain. Evidence shows that the emergence of distinct high-frequency oscillations during slow oscillations facilitates the communication among brain regions whose activity was correlated during the preceding waking period. While the frequencies of oscillations involved in such interactions have been identified, their dynamics and the correlations between them require further investigation. Here we analyzed the structure and dynamics of these signals in anesthetized rats. We show that spindles and gamma oscillations coexist but have distinct temporal dynamics across the slow oscillation cycle. Furthermore, we observed that spindles and gamma are functionally coupled to the slow oscillations and between each other. Following the activation of ascending pathways from the brainstem by means of a carbachol injection in the pedunculopontine nucleus, we were able to modify the gain in the gamma oscillations that are independent of the spindles while the spindle amplitude was reduced. Furthermore, carbachol produced a decoupling of the gamma oscillations that are dependent on the spindles but with no effect on their amplitude. None of the changes in the high-frequency oscillations affected the onset or shape of the slow oscillations, suggesting that slow oscillations occur independently of the phasic events that coexist with them. Our results provide novel insights into the regulation, dynamics and homeostasis of cortical slow oscillations. Slow oscillations  Act, 1986 (UK) and the Society for Neurosciences policy on the use of animals in neuroscience research, under the Project License 30-2639 approved by the Home Office and the local ethical committee of the University of Oxford.Experiments were carried out on 7 adult male Sprague-Dawley rats . Anesthesia was induced with 4% v/v isoflurane in The electrocorticogram (ECoG) was recorded via a 1 mm diameter steel screw juxtaposed to the dura mater above the frontal cortex  and immediately after the infusion of carbachol (post-carbachol). After reviewing the recordings, for every animal and condition (pre- and post-carbachol) at least 200 s segments  of continuous recordings were selected for further analysis.Welch periodograms  and Hilbert transform, and used this as the threshold for defining the existence of an active oscillation (an oscillation above the noise level) Cross-frequency interactions between different frequency bands were assessed by means of the modulation index (MI), as outlined by Tort Here we analyzed the cross-frequency coupling between slow oscillations and fast frequencies (spindles and gamma) and between spindles and gamma oscillations. For the coupling between the slow oscillations and fast frequencies, we first calculated the phase signal by filtering raw data from 0.2 to 3.5 Hz  in the vicinity of the transition from the DOWN to the UP state. Then, we computed the unwrapped phase for each detected spindle from its onset 8 cycles forward and 4 backward. As a result, the phase of each spindle is obtained in the interval To evaluate whether the spindle-gamma coupling is different as a function of the phase of the slow oscillation, we extended the MI concept from two to three signals and defined a constrained cross-frequency coupling index, Data are expressed as means and standard error of the means (SEM) or standard deviation (SD). The effects of carbachol on the spectral content of the SWA were assessed by means of a cluster-based, non-parametric permutation paired test. Additionally, differences in the pre-post carbachol energy values for each of the three selected frequency bands  were assessed by means of paired t-test. Paired t-tests were also used to assess differences in the average duration and amplitude of the slow oscillation before and after carbachol administration.Prior to any statistical analysis, values were normalized using the transformation described by van Albada and Robinson Cluster-based nonparametric permutation paired-tests Carbachol effects on the interaction between frequencies were studied in different ways. First, we used cluster-based nonparametric permutation paired tests again to compare the distributions of the mean amplitude of the instantaneous envelope across the slow oscillation cycle. Then, we used paired t-tests to compare the pre/post MI values  for the slow oscillation/spindles, slow oscillation/gamma and spindles/gamma oscillations pairs. Finally, effects of carbachol on the preferred phase and degree of modulation were assessed by means of bivariate-paired Hotelling's T-square tests. Differences in the pre/post carbachol constrained cross-frequency coupling MI values were assessed by means of paired t-tests, while differences in the cross-frequency coupling pattern We recorded the ECoG activity from the frontal cortex of urethane- and ketamine/xylazine-anesthetized rats during stable cortical SWA. SWA was characterized by a prevalent slow rhythm in the 0.3 to 3 Hz frequency range. Periods of light cortical activation (lower amplitude and higher frequency oscillations) spontaneously alternated with SWA, as described previously for urethane recordings Previously we reported that the injection of a low carbachol dose , and observed that the slow oscillation cycle typically has an average amplitude of approximately 0.8 mV  and average duration of approximately 1s  . Followiobserved . Howeverserved ; . To furtWe then evaluated the dynamics of the faster frequency oscillations  across the slow oscillation cycles to detect possible differences in their organization or distribution. Spindles showed their highest energy at the beginning of the UP state and then tended to decrease progressively. In contrast, gamma oscillations were higher at the peak of the slow oscillation (zero phase), coinciding with the UP state . After tUP state . These dWe next investigated whether spindles and gamma activity coexist during slow oscillation cycles. We evaluated the probability of occurrence of each of the following combinations and analyzed in four categories: 1) slow oscillation intervals  with spindles alone (‘spindles-only’), 2) slow oscillation intervals with gamma alone (‘gamma-only’), 3) slow oscillation intervals with both spindles and gamma (‘spindle-associated gamma’), and 4) slow oscillation intervals with neither spindles nor gamma . ‘SpindlFollowing the carbachol injection, no qualitative changes in the pattern of distribution of spindles and gamma within the slow oscillation cycle were observed : spindlemodified . In contbability . The probability , nor wasbability . Thus, tOnce we determined that spindles and gamma activity co-exist within slow oscillations, we tested whether there is any interaction between these oscillations. We analyzed the cross-frequency coupling between the phase of the slow oscillations and the amplitudes of the spindle and gamma activities to determine whether there is any dependency of fast frequencies on the slow frequencies. We observed that the phase of the slow oscillation significantly determined the amplitude of both spindles , and observed that the amplitude and emergence probability of gamma oscillations was increased, whereas the same measures were reduced for spindles. In addition, we detected significant changes in the functional coupling between spindles and gamma. Our results provide fundamental insights into the composition and dynamics of the slow oscillations, and provide a detailed framework to understand the contribution of subcortical structures to the regulation of cortical oscillations during slow wave activity.The origin of the cortical slow oscillations is determined by the synchronization of multiple cortical neurons whose membrane potentials fluctuate and alternate between hyperpolarized (DOWN states) and depolarized (UP states) configurations The analysis of the structure, organization and distribution of high frequency oscillations reveal that spindles and gamma coexist and interact during cortical UP states, supporting findings from sleeping humans Increasing evidence supports the notion of organized subcortical activity during SWA. Distinct structures in the midbrain and brainstem with upstream projections to thalamic and cortical targets remain active during cortical slow oscillations and their activity is highly structured. Such is the case of the pedunculopontine nucleus In contrast to the above, the co-occurrence of slow oscillations and ‘spindle-associated gamma’ activity was unchanged. This suggests that the activation of subcortical structures, driven by the carbachol injection, has an impact on ‘gamma-only’ activity, but not on gamma activity that is associated with the spindles. We also revealed that ‘spindle-associated gamma’ depends on the phase of the spindles, indicating a functional dependence. We did not observe any effect of carbachol on the probability of emergence of ‘spindle-associated gamma’ or its energy, as observed in the difference between pre- and post-carbachol for the gamma energy see . The lacSpindles and gamma activities are hallmarks of the cortical slow oscillations, and together are hallmarks of slow wave sleep. Recent evidence shows that both spindles and gamma play an important part in the functional role of the slow oscillations and have been associated with plasticity and learning processes


As cell-free circulating DNA exists predominantly as mono- and oligonucleosomes, the focus of the current study was to examine the interplay of circulating nucleosomes, DNA, proteases and caspases in blood of patients with benign and malignant breast diseases.®3/7 Assay, respectively.The concentrations of cell-free DNA and nucleosomes as well as the protease and caspase activities were measured in serum of patients with benign breast disease (n = 20), primary breast cancer , metastatic breast cancer , and healthy individuals (n = 28) by PicoGreen, Cell Death Detection ELISA, Protease Fluorescent Detection Kit and Caspase-GloPatients with benign and malignant tumors had significantly higher levels of circulating nucleic acids in their blood than healthy individuals , whereas these levels could not discriminate between benign and malignant lesions. Our analyses of all serum samples revealed significant correlations of circulating nucleosome with DNA concentrations (p = 0.001), nucleosome concentrations with caspase activities (p = 0.008), and caspase with protease activities (p = 0.0001). High serum levels of protease and caspase activities associated with advanced tumor stages (p = 0.009). Patients with lymph node-positive breast cancer had significantly higher nucleosome levels in their blood than node-negative patients (p = 0.004). The presence of distant metastases associated with a significant increase in serum nucleosome (p = 0.01) and DNA levels (p = 0.04), and protease activities (p = 0.008).Our findings demonstrate that high circulating nucleic acid concentrations in blood are no indicators of a malignant breast tumor. However, the observed changes in apoptosis-related deregulation of proteolytic activities along with the elevated serum levels of nucleosomes and DNA in blood are linked to breast cancer progression. Every year, more than one million women are worldwide diagnosed with breast cancer, the second most common tumor entity after lung cancer. At the time of initial diagnosis approximately 5% of patients are found to have advanced or metastatic disease . AlthougThe quantification of cell-free DNA, which circulates in high concentrations in blood of patients with various malign and benign lesions , could sThe elevated levels of circulating DNA in blood of cancer patients have been shown to reflect the characteristics of tumor DNA, and to harbor genetic and epigenetic alterations . PreviouAn essential aspect for cancer development and progression is the deregulation of caspases involved in the regulated apoptotic cascade which consequently, leads to the release of DNA or nucleosomes into the blood circulation . CaspaseThe aim of the present study was to investigate the influence of protease and caspase activities on the levels of circulating nucleosomes and DNA in blood of breast cancer patients, and to correlate the obtained data with the clinicopathological parameters of these patients. Despite the small sample sizes of the cohorts, the broad range of preliminary results shows that the quantification of these determinants could be used as a rapid, non-invasive and blood-based screening tool for the early detection of breast tumor progression.The present study was conducted at the Clinic of Gynecology of the University Medical Center Hamburg-Eppendorf, at the Ludwig Maximilians University of Munich and at the Department of Gynecology and Obstetrics of the University Hospital of Essen. Between April 2006 and September 2008 blood serum was obtained from 20 patients with benign breast disease . During January 2008 to August 2009, blood serum was taken from 31 patients with primary breast cancer (M0) after surgery before initiation of adjuvant therapy (Ludwig Maximilians University of Munich). During January to April 2009, blood serum from 32 patients with metastatic breast cancer (M1) was collected 1 to 13 years after surgery of the primary tumor . Additionally, 28 female controls aged between 21 and 67 with no history of cancer were recruited. The median ages of patients with benign and malignant breast disease was 49 (range 18-79) and 56 (range 34-82), respectively. The data of the tumor patients and healthy controls are summarized in Table Informed written consent was obtained from all diseased and healthy individuals, and the study was approved by the Local Research Ethics Committee.For quantification of the relative amounts of cell-free DNA and nucleosomes in serum, the Quant-iT PicoGreen dsDNA Kit  and the Cell Death Detection ELISA plus kit  were performed, respectively, and carried out according to the manufacturer's protocol.For quantification of the relative protease activity, the Protease Fluorescent Detection Kit  was used, and carried out according to the manufacturer's protocol. This kit is based on the proteolytical hydrolysis of a Fluorescein Isothiocyanat (FITC)-labeled casein-substrate detecting all four proteases . A three-point standard curve of 1:10, 1:20 and 1:40 dilutions of Trypsin Control was prepared.®3/7 Assay  was carried out according to the manufacturer's instructions. This kit is based on the cleavage of the DEVD sequence of a luminogenic substrate by the caspases 3 and 7 resulting in a luminescent signal.For measurements of the activities of caspases 3 and 7, the Caspase-GloThe statistical analyses were performed using the SPSS software package, version 18.0 . The chi Square or two-tailed Fischer's exact test was used to identify possible associations of the measured parameters. For non parametric comparisons, univariate analyses of the Mann Whitney-U test and the T test of two independent variables, and bivariate analyses of the Spearman's rank correlation were performed. Diagnostic power of the single markers was analysed by receiver operating characteristic (ROC) curves. Areas under the curves (AUC) were calculated. A p-value ≤ 0.05 was considered as statistically significant. All p-values are two-sided.We compared the levels of circulating nucleosomes Figure  and DNA As shown in the box plots of Figure As shown in the box plots of Figure As shown in a scatter plot of Figure To determine the impact of protease and caspase activities on the levels of circulating nucleosomes and DNA, we measured these activities in blood serum of the patient subgroups and healthy individuals Figures  and 5. TIn contrast to benign breast tumor and M1 patients, a broad range of protease activities was observed in healthy individuals and M0 patients Figure . In M1 pAs shown in Figure The Spearman rank test of all analyzed blood samples revealed significant correlations between the circulating nucleosome levels and caspase activities  was too small and the distribution of patients within the populations with high and low values was too heterogeneous Table . Of the In the current study we examined the relationship of the serum levels of circulating nucleic acids with the activities of proteases and caspases. In addition, we explored the interplay and the changes in release of these determinants in the blood circulation of breast cancer patients, patients with benign breast disease and healthy individuals.Although patients with benign and malignant breast tumors had significantly higher levels of circulating nucleosomes and DNA in their blood than healthy individuals, these elevated yields could not discriminate between benign and malignant lesions as documented by our present study. Thus, cell-free nucleic acids may be a molecular biomarker for detecting both, benign and malignant breast diseases in contrast to the circulating protease and caspase activities which were specifically increased in blood of breast cancer patients.Cell-free DNA may circulate in human blood in diverse forms, as naked DNA, associated with histones as nucleosomes, bound to other plasma proteins or packed in apoptotic bodies . Our detBesides the elevation of caspase activities, we also observed a significant upregulation of protease activities, including the various serine, cysteine, metallo and aspartic proteases, in blood of patients with advanced tumor stages and metastatic disease. Our findings substantiate the active role of proteases in tumor progression and metastases. The degradation of extracellular matrix represents a key event in the multistage process of tumor invasion and metastasis. Such a degradation requires the concerted action of proteases which are involved in the proteolysis of many different components of the extracellular matrix . The cleOur data suggest that the measurement of circulating nucleosome concentrations may be used for the risk assessment of breast cancer patients who develop metastases, because the elevated nucleosome levels significantly associated with regional and distant metastatic breast disease spreading. However, to sustain the prognostic relevance and the clinical utility of serum nucleosomes, as well as DNA, long-term follow-up analyses have to be performed.Although we found significantly higher nucleosome levels in blood of lymph node-positive and metastatic breast cancer patients than in patients without overt metastases or with benign breast disease, serum DNA yields were lower in cancer patients than in patients with benign breast disease. However, we detected a significant association between the DNA and nucleosome values of all patients and healthy individuals. This ostensible discrepancy can be explained by the dramatic increase in the nucleic acids in blood of patients with benign disease. Nevertheless, the rise in nucleosome and DNA levels leading from healthy individuals over M0 patients to M1 patients was similar, albeit the inclination of the DNA levels of these three cohorts was somewhat more gently than that of the nucleosome levels. Why the DNA and nucleosome concentrations are so high in our cohort of patients with benign disease is unknown. It is well-known that an increased amount of circulating DNA is not only related to cancer, but is also found in blood of patients with severe inflammatory processes . Since aIn breast cancer the serum tumor markers cancer antigen (CA) 15-3 and carcinoembryonic antigen (CEA) play a role in tumor growth and spreading. Particularly in advanced stages of cancer patients these markers have been shown to be elevated . While iIn conclusion, based on the significant association between DNA and nucleosomes, DNA may predominantly circulate in form of nucleosomes in human blood. Although serum levels of nucleosomes and DNA in breast cancer patients are significantly elevated when compared with healthy controls, they are not useful for cancer diagnosis, owing to the increase in blood of patients with benign breast tumors. Nevertheless, the quantification of circulating nucleic acids as a rapid, non-invasive and blood-based screening tool could be used for the early detection of breast tumor progression, but larger studies with follow-up blood samples are required to corroborate this conclusion. The increased activities of caspases 3 and 7 observed in blood of the patients with advanced tumor stages and metastatic disease may contribute to the rise in nucleic acid concentrations in blood of cancer patients, but they do not seem to be relevant for the high serum levels of nucleic acids in patients with benign breast disease. Here, other caspases seem to be responsible for this release. The striking upregulation of the protease activities in patients with advanced tumor stages and metastatic disease reflects their role in tumor progression and metastases.Considering the small sample sizes of our cohorts and the broad range of our data, additional studies to these preliminary results are warranted to obtain further information on the tumor activity and to determine the clinical relevance of the circulating nucleic acids in blood of breast cancer patients.M0: patients with primary breast cancer; M1: patients with metastatic breast cancer; MMPs: matrix metalloproteinases; TIMPs: tissue inhibitors of metalloproteinasesThe authors declare that they have no competing interests.CR and HS performed all experiments. HS and CR performed the statistical analysis. HS drafted the manuscript and KP revised the manuscript. BR, VM, SKB and WJ prepared the clinical material. CR summarized the clinical parameters. HS and CR were involved in conception and design of the study and participated in the discussion and interpretation of the results. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/4/prepub


We show that electric field imprinting technique allows for patterning of metal nanoparticles in the glass matrix at the subwavelength scale. The formation of glass-metal nanocomposite strips with a width down to 150 nm is demonstrated. The results of near-field microscopy of imprinted patterns are in good agreement with the performed numerical modeling. Atomic force microscopy reveals that imprinting also results in the formation of nanoscale surface profile with the height going down with the decrease of the strip width. The experiments prove the applicability of this technique for the fabrication of nanoscale plasmonic components. Nowadays, plasmonic materials and structures are the subject of wide-scale studies. In addition to metals, new materials like wide bandgap semiconductors ,2 and glThe required dimensions of the majority of plasmonic structures -10 are iIn , nanoimpSilver-based GMN sample was prepared in a plate of commercial 1-mm thick soda-lime glass using silver-to-sodium ion exchange followed by hydrogen-assisted reduction of silver ions and metal clustering as it was reported elsewhere . Accordiμm. The widths and depths of the grooves were checked with scanning electron microscopy (SEM), Zeiss Leo 1550 Field Emission Scanning Electron Microscope . The stamp was used as the anode in the EFI of both the GMN sample and the plate of virgin glass. The imprinting was carried out at 250°C under 600 V DC.To find the linewidth achievable in the EFI, a profiled glassy carbon  stamp wiThe imprinted structure was studied using AFM and SNOM techniques using AIST-NT SmartSPM scanning probe microscope and AIST-NT CombiScope Scanning Probe Microscope with optical fiber probe . Numerical modelling was carried out using COMSOL Multiphysics®; package .The measured optical spectrum of the GMN exhibits strong surface plasmon resonance (SPR) absorption centered at 415 nm, and the SPR peak drops after the electric field imprinting  [Generally, the observed modulation could be due to local light absorption in the strips, to the interference of incident light wave with the wave scattered by the surface humps, and to the light wave phase shift difference in poled (out of strips) and unpoled regions of the glass sample. The latter effect may come from the refractive index change in poled glass, which amounts to 03−0.09) . Basing f=0.01 was used for the modeling of GMN optical parameters. In the calculations, we used a 300-nm GMN layer buried at 150-nm depth. One can see good correspondence of the experimental data and our modeling. It is worth to highlight that the nanocomposite fill factor was assumed to be the same for all imprinted strips. Thus, the comparison of the model and the experiment bear evidence that even in the 150 nm imprinted strip, the concentration of the nanoparticles is roughly the same as in the initial GMN sample; the lower magnitude of the light modulation as compared to the thicker strips is due to geometrical factor only.To interpret the obtained experimental results numerical modelling has been used. The results of near-field intensity calculations at 100-nm distance above the glass plate with GMN strips corresponding to the stamp used in EFI are shown in Figure Finally, in this study, we used a scanning near-field optical microscopy to characterize the spatial resolution of the EFI technique applied to the glass-metal nanocomposites. For this purpose, we replicated a set of nanostrips differing in width to the silver-based glass-metal nanocomposite sample using a profiled glassy carbon stamp as the anodic electrode. Our near-field measurements showed significant dependence of optical transmission of the imprinted strips on the excitation wavelength. In contrast to relatively low modulation of optical signal at 633- and 532-nm wavelengths, the transverse scan of the intensity profile at 405 nm contained sharp dips corresponding to the silver nanoparticle surface plasmon resonance absorption in the imprinted strips. Numerical simulations of near-field signal under the assumption that the nanoparticle concentration is equal in all of the strips showed good agreement with our experiment. Finally, this study proved that glass-metal nanocomposite elements with linewidth down to at least 150 nm can be fabricated with electric field imprinting technique.The authors declare that they have no competing interest.ISS conducted SNOM, AFM, and spectroscopy measurements. AKS supervised the experiments and participated in data processing. MIP developed the models used. VVR prepared the samples from ion exchange until their annealing in hydrogen and performed the numerical calculations. AAL supervised the whole work starting from sample preparation to analysis of data. All authors read and approved the final manuscript.ISS is a Masters degree student of St. Petersburg Academic University and an assistant at the National Research University of Information Technologies, Mechanics and Optics. MIP is a former PhD student of the University of Eastern Finland; he defended the thesis in April 2013. AKS is a PhD degree holder and is a junior research fellow at the National Research University of Information Technologies, Mechanics and Optics; he defended his thesis at Ioffe Institute in December 2011. VVR has graduated from St. Petersburg Academic University in 2012. AAL holds a DrSci degree and Professor positions in St. Petersburg Academic University and St. Petersburg State Polytechnical University.


Pigmented villonodular synovitis (PVNS) is a proliferative benign lesion originating from the synovium and commonly affects large joints of the extremities. PVNS can arise from any synovium in the whole body and rarely affects the zygapophyseal joints of the spine. Spinal PVNS is diagnosed mostly after resection of the mass. In our case we present a 22-year-old male patient showing progressive spastic paraparesis with insidious onset of back pain and difficulty of walking in a relatively short period of 1 month. After gross excision of the mass, diagnosis was established through histopathology. Two years of follow-up period reveals complete resolution of the patient's complaints and no recurrence on radiologic images. Pigmented villonodular synovitis (PVNS) is a benign proliferative and locally aggressive tumor of the synovium , 2. PVNSA 22-year-old male was admitted to our hospital with a short history of back pain with progressive difficulty in walking. The patient had spasticity in both lower extremities and after hospitalization neurology progressed to paraplegia. Powers of the lower limbs got worse to grade 2-3. Routine hematologic investigations were within normal ranges, and there was no history of trauma, family, and neurological diseases. Immediate MRI and CT scans revealed a tumorous mass that originated from the posterior elements of thoracic 7th vertebra protruding through the spinal cord canal  showed there was no recurrence of the tumor Figures  and 7.PVNS arises from the zygapophyseal joint synovium, and its etiology is unknown; tumor growth rate is low, but PVNS usually is locally aggressive. When affecting the spine the first symptom is usually back pain, and progressive neurologic deficit occurs after the spinal cord is compromised . NeuroloIn our patient's radiological workup, a lytic, expansile mass having visible borders with 25 × 18 × 33 mm dimensions arising from the T6-7 left facet joint invading transverse process and lamina was visualized. The central canal diminished in size, and spinal cord was compromised. IV contrast material stained the lesion homogeneously. Radiological findings of PVNS of the spine are obscure. Aneurismal bone cysts (ABC), osteoid osteoma, and osteoblastoma of the spine should be differentiated, because they all involve the posterior vertebral elements . ABC of the spine mimics PVNS as it arises from the neural arch, and does not show matrix mineralization. PVNS differs from ABC by absence of cystic components and does not present liquid-liquid levels, septa are not seen, and also a peripheral rim contrast cannot be visualized .Osteoid osteoma of spine presents itself with painful scoliosis mostly, and affected vertebrae are usually the cervical and lumbosacral vertebrae. Pain at night sometimes diminishes with salicylate consumption .Osteoblastoma of spine is mostly seen in the second and third decades while aneurismal bone cysts presents mostly in the first twenty years of life . PVNS inOn MRI, the intensity of PVNS is not definite, but the demonstration of continuity between the mass and the facet joint helps support the diagnosis of PVNS .PVNS of the spine is seen mostly in cervical and lumbosacral region while in our case a thoracal vertebra involvement was seen. Of the 54 patients reported only 12 of them were reported to involve the thoracic vertebrae .The management options for spinal PVNS include surgery, radiation therapy, and radioisotope infusion. The role of radiation therapy has not been clearly defined. Surgical resection is the primary treatment for this lesion. However, radiotherapy in diffuse lesions may be justified if surgery fails to control the process .Pigmented villonodular synovitis of the spine is rarely seen, and a histopathological workup is necessary for definitive diagnosis. Acute neurological findings with a relatively short time of symptoms should alert the physician for locally aggressive tumors of the spine, and a radiological workup using MRI and CT may differentiate the lesion before surgery. Age group, clinical findings, absence of matrix mineralization, and homogenous contrast enhancement of the lesion in CT and MRI findings with absence of liquid-liquid levels and relationship of the lesion with facet joint may help surgeon preoperatively. Local recurrences may occur, so gross resection of the tumor and close followup of the patient are mandatory.


Cardiovascular diseases estimate to be the leading cause of death and loss of disability-adjusted life years globally. Conventional risk factors for cardiovascular diseases only partly account for the social gradient. The purpose of this study was to compare the occurrence of the most frequent cardiovascular diseases and cardiovascular mortality in two close cities, the Twin cities.We focused on the total population in two neighbour and equally sized cities with a population of around 135 000 inhabitants each. These twin cities represent two different social environments in the same Swedish county. According to their social history they could be labelled a "blue-collar" and a "white-collar" city. Morbidity data for the two cities was derived from an administrative health care register based on medical records assigned by the physicians at both hospitals and primary care. The morbidity data presented are cumulative incidence rates and the data on mortality for ischemic heart diseases is based on official Swedish statistics.The cumulative incidence of different cardiovascular diagnoses for younger and also elderly men and women revealed significantly differences for studied cardiovascular diagnoses. The occurrence rates were in all aspects highest in the population of the "blue-collar" twin city for both sexes.This study revealed that there are significant differences in risk for cardiovascular morbidity and mortality between the populations in the studied different social environments. These differences seem to be profound and stable over time and thereby give implication for public health policy to initiate a community intervention program in the "blue-collar" twin city. Both Conventional risk factors for cardiovascular diseases (CVD) such as high blood pressure and smoking only partly account for the social gradient for CVD that has been widely demonstrated ,8. StudiLike most western societies, Sweden, is becoming a more multicultural society with a mix of natives and first or second-generation immigrants, which also has implications for the occurrence of cardiovascular diseases in the population. A Swedish study has found that immigrants have an increased incidence of acute myocardial infarction compared to natives. Immigrants also have a higher incidence of myocardial infarction including non-fatal as well as fatal out-of-hospital cases, than Swedish born people after adjustment for age and socio-economic group .To better understand disease occurrence, treatment, health outcomes, and prevention it has for centuries been evident in medicine to also include different social determinants . The rolNeighbourhood of residence has been suggested to affect cardiovascular risk above and beyond personal socio-economic status ,25. In aIn order to address an upstream perspective of cardiovascular disease occurrence in the population we conducted a study focusing on the population in two comparable and equally sized Swedish cities, the Twin cities.The aim of this study was to compare the occurrence of the most frequent cardiovascular diseases and cardiovascular mortality in two close but different social environments, the Twin cities.A description of some indicators underpinning the differences in social environments of the two twin cities is given in table Today the cities are quite similar but their social history differs. We therefore generalize the urban identity of the cities based on their history. The term "white-collar" is generally used to refer to work that does not require strenuous physical labor. The term "blue-collar", conversely, refers to workers whose work requires manual labor. We use the terms to describe the two different social environments and geographical areas. Manual laborers mostly inhabit a "blue-collar" community and a "white-collar" community contains mostly administrators.During the 17th century, the "blue-collar" twin city received its rights to foreign trade and an international port. This was the beginning of a development that made the "blue-collar" twin city a centre for textile industry, "The Manchester of Sweden". Over time, the textile industry became more mechanised and the number of unskilled workers increased in the "blue-collar" city. Only in recent decades, have non-manual occupations become dominant in "the blue-collar" city and it has also become a university town in the last decade. Meanwhile the "white-collar" twin city was a regional and agricultural centre dominated by a quite life of pupils from the cathedral school. While the industrial revolution went on in the "blue-collar" city the "white-collar" city remained a rural market town, ruled by both the church and the state. For a long time there has also been a significant military presence, with several regiments. After World War II the "white-collar" city grew in terms of population and became industrialized, mainly due to the aviation industry expansion. Today, high technology companies and a university dominate the "white-collar" city.Although these twin cities today have an extrinsic resemblance in terms of culture, environment and climate, there are tendencies of differences in public health between them which are today manifested in public health indicators such as; life expectancy, prevalence of cardiovascular diseases, sick leave, perceived health and lifestyle factors .There is a long tradition especially in the Scandinavian countries of documenting diseases in registers. In Sweden each inhabitant is assigned a unique personal code based on birth date and gender . By law,The administrative Health Care Register (HCR) used in this study is based on computerised data files linked by a unique personal code to birth date and gender of all inhabitants in the region. The same personal code is used for all visits and diagnoses in HCR. The International Classification of Diseases (ICD 10-code) for the CHD disorders presented in this study was: E78 (high cholesterol), I10 (hypertension), I20.9 (angina pectoris), I21 , I25 (ischemic heart disease), I50 (cardiac insufficiency), I61.9 , I63 (stroke). Physicians in hospitals as well as in primary care assigned these diagnostic codes. The morbidity data presented are cumulative incidence rates. The numerator for the cumulative incidence rates was the number of the first diagnosed cases identified during the six-year study period (2002-2007). The denominator was the number of inhabitants calculated as the mean number in each age group of the population during the six-year study period. The cumulative incidence rates are presented as numbers per 1000 inhabitants.Swedish national data on mortality for ischemic heart diseases (mainly heart infarction) in the largest Swedish cities  was also included. It represents early mortality in the population from the age of 15 and above in each city between 2002 and 2006 and are age standardised against the national Swedish population . The hisData was stored in a shared database and statistically analysed using SPSS 17.0 . Risk Ratios (RR) and 95% confidence intervals (CI) were calculated based on crude data with the "white-collar" city as the reference group. A p-value of p < 0.05 was considered statistically significant.Recent Swedish national data 2002-2006) on mortality in connection with ischemic heart diseases in males and females living in the largest Swedish cities are presented in Figure 002-2006 The cumulative incidence of different cardiovascular diagnoses in younger men and women (aged 45-64 years) is presented in table The cumulative incidence of different cardiovascular diagnoses in elderly men and women aged 65-79 years) is presented in table -79 yearsThis study revealed that the risk for cardiovascular morbidity and mortality is very different between the population in a "blue-collar" and a "white-collar" city. These differences seem to be profound and stable over time. The two studied cities are quite comparable and referred to as twin cities, although historically they represent different social environments, a "blue-collar" and a "white-collar" city. By this study design we want to uncover the relative importance of the social environment for cardiovascular risks in a defined population.The strength of this study is that it is based on computerised data files (HCR) of all inhabitants linked by birth date and gender. The same personal code is used for all visits and diagnoses in HCR. An individual can thus be followed retrospectively or prospectively through the health care system. The health care institution where the patient was diagnosed represents all health care levels, primary care, outpatient hospital care, and/or inpatients hospital care. General limitations in register data is that misclassifications do occur, including cases that are not recorded because they are overlooked or given incorrect clinical codes. There could also potentially be some referral bias. However, according to The National Board of Health and Welfare, analysis of the quality and content of the Swedish Hospital Discharge Register indicates up to 98% coverage of main diagnostic codes in in-patient care in this region . ValidatA close connection between socio-economic factors and cardiovascular morbidity has long been known . Low socIndividual behaviour and lifestyle and thereby socio-economic factors are of importance when analysing cardiovascular risk determinants ,39,40. TAssociations between neighbourhood socioeconomic characteristics in the population and hypertension risk have in other studies found to be mediated by differences in body mass index . This inIn general, the health situation with regard to morbidity, mortality and self-reported health in the "blue-collar" twin city is in general notably worse than in the "white-collar" twin city. These differences have not diminished over time from the late 1970s and onwards.There are noticeable socio-economic and socio-cultural differences between the two studied cities that might contribute to the explanation of the cardiovascular differences found in this study. Although the traditional industrial worker hardly exists today, not even in the "blue-collar" city, the older generation in particular still identifies with that role. In many respects, the "blue-collar" twin city has gradually lost its leading role in the region during recent decades in terms of population, political weight, strategic investments etc. Widespread factory close-downs in the 1960s and 1970s resulted in high unemployment in the "blue-collar" twin city. In addition, relatively poor working conditions as well as the powerlessness of people in this situation are detrimental for health. Until the last 10-15 years the "blue-collar" twin city was "a town of sorrow", with an old structure and a history of closed factories. All these factors might have an impact on the risk for cardiovascular disease in the population. However, trends that could have positive effects on health have also emerged in the last decade since the university has also established a university campus in the "blue-collar" twin city. Today, many social structures in the "blue-collar" twin city are approaching the structures of the neighbouring "white-collar" twin city, but public health differences seem to prevail.The "white-collar" twin city has experienced a strong economical and population growth throughout the 20th century especially in the post-war period. This is especially due to the establishment of an aerospace industry with guaranteed governmental orders of airplane production, which created a demand for engineers and thereby manifested the "white-collar" city as a city of high technology. This city managed to turn a precarious situation in the 1960's, mainly through the establishment of the Technical University College and eventually a fully-fledged university. A successful collaboration between the University and the County Council also led to the establishment of a medical faculty in the 1980s. The economic and political success of a city is probably a health-promoting factor in itself. A differentiated industry leading to a diverse labour market in the "white-collar" municipality is certainly even more important. The transformation from an industrial society to technological information society was faster than in the neighbouring "blue-collar" twin city. However, the other side of the coin reveals a rising income gap and increasing ethnical segregation in the "white-collar" twin city in the last decade.It is possible to change the risk of cardiovascular disease not only on the individual level but also in a population and in a whole community. There are some good international examples of programmes how community-based intervention actually reduces the risk of cardiovascular disease in a community. Since the early 1970s, community-based programmes have shown to be effective in the field of cardiovascular disease prevention. A major intervention program, The North Karelian project, was launched in eastern Finland, where morbidity and mortality incidence was among the highest in Europe ,44. The However, these projects did not take into account the voices of ordinary people living in the area of prevention programmes. Ways that people who suffers from heart diseases understand and make sense of their disease has been reported in medical anthropological research . AnotherThere are profound differences in cardiovascular morbidity and mortality between the studied twin cities representing two close but different social environments. These differences seem not to diminish but to be stable over time, although both cities have undergone social and economic changes from industrial to post-industrial cities. Health behaviours and lifestyles seem to prevail in both of these social environments casting a shadow of higher risk of cardiovascular disease in the population of the "blue-collar" city. This data give strong public health implications with an upstream approach to initiate a long-term community intervention programme in the "blue-collar" twin city.The authors declare that they have no competing interests.CW, A-KJ, TF participated in the study design and coordination and completed the data collection. CW, A-KJ, TF drafted the manuscript. All authors contributed to analysis and interpretation of data, and read and approved the final manuscript.


Computertomographic and magnetic resonance imaging revealed the envelopment of the left 7th and 8th spinal nerve along with the anterior displacement of the left vertebral artery. The interdisciplinary surgical strategy consisted of a partially incomplete cyst resection, subtotal spondylectomy with posterior screw-and-rod fixation from C6-Th1, iliac crest bone grafting and anterior plating from C6-Th1. With regard to the high rate of recurrence after incomplete resection published in the recent literature, the patient was postoperatively treated by megavoltage radiotherapy with a total dose of 30Gy (daily dose of 1.8 Gy for 3 weeks). The clinical and radiographic follow-up showed complete recovery of all neurologic impairments and no signs of tumour recurrence at 3, 6 and 12 months after surgery. This case highlights diverse treatment regimens and shall outline the challenge and the problems of the interdisciplinary decision-making in adolescents presenting with ABC in high-demanding anatomical regions.The aneurysmatic bone cyst (ABC) is a benign primary bone tumour. If located in the cervical spine, its expansive growth and destructive behaviour may lead to instability and serious neurological impairment. We report a case of a 16-year-old boy with an aggressive ABC in the 7 Aneurysmatic bone cysts (ABC) are described to be benign, but locally destructive bony lesions. Representing approximately 1% of all primary bone tumours, 10-30% of ABC cases involve the spine, with approximately 22% of these located in the cervical spine region [To date, the etiology of ABCs remains uncertain. Possible explanations include a posttraumatic intralesional hematoma, whereupon ABCs may be classified into primary (idiopathic) and secondary ABC, either evolving from a bleeding into a preexisting traumatic, infectious or malignant lesion . RespectAccording to Enneking's classification, ABC may be subdivided into three developmental stages:1. State of latency without progressive growth and the tendency to spontaneous remission.2. Active state with progressive growth, but without extravagating anatomic borders.3. Aggressive state with expansive and osteolytic growth, thereby extravagating diverse tissue layers.Radical surgical debridement and complete excision of the cystic wall should be the primary therapeutic option. When complete excision of the cystic wall is achieved, the overall prognosis is good and tumour recurrence is highly unlikely. The risk of local recurrence, however, correlates well both with the preoperative state and the radicalness of resection . Therefoth cervical vertebra (stage 3 according to Enneking).In the following, we report our therapeutic regimen and surgical strategy in the case of a 16 year old boy who presented with an expansive growing bone cyst of the 7A 16 year old patient presented with a 4-month history of cervical pain, which was accompanied by a progressive radicular pain of his left arm. Within this time period he remarked a slight swelling of soft tissue in the neck and a weakness of his left arm when performing weight training.His previous medical history was without comorbidities or surgical interventions. His family history was clear of malignancies.On clinical examination there was a soft tissue swelling of the left anterior and lateral region of the neck and atrophy of the left biceps muscle. Neurologic examination revealed a slight weakness (4 out of 5 according to the Janda classification) and numbness of the left forearm and hand.th cervical vertebra in both lateral and ap views  demonstrate an expansive growth and destructive behaviour with a high rate of recurrence. Neurologic impairment and pain may be a clinical prodrome for the manifestation within the spinal column. In conventional radiographs ABC appear as expansive osteolytic bone lesions with a round-oval and polycystic structure, delineated from the surrounding tissue by a thin radiopaque layer. The three-dimensional expansion of the cyst is best evaluated by CT scan. The typical CT images outline a fluid-filled cavern, often confirmed and more precisely detected on MRI, hence with improved sensitivity for the diverse fluid layering .The differential diagnosis of the ABC includes the giant cell tumour, hemangioma, fibrotic dysplasia, the osteosarcoma and spinal metastasis of any other distant tumour.The primary goal in the therapeutic regimen of spinal ABC is to completely resect, decompress neurologic and vascular structures and to re-establish and secure biomechanical stability. However, these goals may provoke ambivalent thinking when the sufficient degree of surgery for an otherwise benign tumour and the protection of sensitive neurologic structures and their anatomical surrounding are considered.Cervical spine ABC's often involve the posterior column, which then require posterior decompression and/or resection in terms of a laminectomy, respectively. In order to prevent postlaminectomy kyphosis, additive fusion is highly recommended .The low incidence of cervical spine ABC, the small and inhomogeneous number of reports and the absence of applying any form of classification make a comparison among different surgical techniques and treatment concepts difficult. Moreover, most studies involve long follow-up periods and therefore bear the risk of changing therapeutic modalities within the study itself.Therefore, with regard to the case reported here, we aim to discuss the different modalities prophylaxis of tumour recurrence, even in cases where a complete resection is not feasible due to the expansion of the bone lesion.Recent studies have reported a high incidence of tumour recurrence in cases of incomplete primary resection, most of which appeared within the initial 6 to 12 months after surgery ,8,9. PerABC at the spine requires an angiography in the diagnostic set-up to analyse the vascularity and to support the decision making process regarding the proper therapeutic approach. As mentioned above, embolization as alternative treatment option has to be taken into account. This procedure has shown to be successful in case of ABC located at the extremities. There are cases described in the literature, where embolization alone was able to heal SAE localized at the spine without further complications . If ABC Other reports have suggested postoperative radiotherapy with a high-energy low-dose application in cases with incomplete resection -12. To dPast studies have demonstrated the effective use of megavoltage radiotherapy in both a monotherapeutic or combined surgical and radiotherapeutic approach. Yavuz and colleagues  demonstrth and 8th spinal nerve, as well as a long section of the vertebral artery was necessary allowing only for intralesional/incomplete resection.Thus, we believe that megavoltage radiotherapy for the prophylaxis of tumour recurrence in ABC is justified in cases, where the anatomical region is highly demanding, especially in case of possible ABC recurrence. As presented in our case, careful preparation of both the 7The decision should then be based on an interdisciplinary approach amongst others evaluating body height of both parents and the skeletal maturation of the patient to estimate possible growth disturbances. In our case, this evaluation revealed a state assuming skeletal growth completion and the postoperative megavoltage radiotherapy discussed and agreed upon as a decision by consensus of the interdisciplinary tumour conference.Postoperative megavoltage radiotherapy applied within the range of today's standardized protocols can be an effective adjuvant in the treatment of ABC of the cervical spine. Exact preoperative staging and multiple biopsies to confirm the diagnosis, along with the careful evaluation of all risk factors, should be mandatory. This also implies the need for a comprehensive and continuous follow-up of patients receiving such complex treatments. More particular, this case demonstrates the challenges of the decision-making process in the treatment strategy of stage 3 ABC to be a rather complex and interdisciplinary issue as the case arises. Therefore, each case is to be evaluated individually.The authors declare that they have no competing interests.JR and SKT wrote the manuscript. JG and MW collected the data pre- and postoperatively. UE and CEH operated the patient and revised the manuscript critically. CEH and GVS-S gave input regarding the design and gave the final approve of the version to be published. All authors read and approved the manuscript.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.