Systemic light chain amyloidosis (AL) is associated with a high cardiovascular morbidity and mortality. Cardiovascular involvement and determination of prognosis is underestimated by standard imaging parameters. Recently, cardiac deformation analysis of global circumferential and longitudinal strain has been shown to have great clinical impact on the assessment of prognosis and survival in this rare disease. For quantification of cardiac deformation analysis we applied a novel non-invasive post-processing feature tracking algorithm (FTI) on pre-acquired regular CMR SSFP images in healthy volunteers and in patients with AL and sought to investigate wall motion differences between both groups.65 patients  with biopsy proven systemic AL were scanned on a clinical 1.5 T CMR scanner (Philips Achieva). Short axis slices covering entirely both ventricles as well as 2-, 3- and 4-chamber were acquired using standard SSFP-sequences before initiation of specific pharmaceutical AL therapies. The control group consisted of 50 healthy subjects . Besides the standard CMR parameters for volumes, ejection fraction (EF) and myocardial mass and wall thickness we measured global circumferential and longitudinal strain on SSFP images by the application of the post-processing feature tracking algorithm.Global circumferential strain and global longitudinal strain correlated well with left ventricular ejection fraction . In patients with AL global longitudinal strain was significantly reduced compared to healthy subjects , whereas global circumferential strain was not . In the subgroup analysis of AL patients without cardiac involvement  global longitudinal strain showed significantly reduced values in comparison to healthy control subjects , whereas global circumferential strain did not show a significant difference. Patients with an ejection fraction ≥50% already had reduced global longitudinal strains , again global circumferential strain did not show a significant difference.FTI strain analysis derived from regular SSFP sequences offers the possibility for a retrospective or prospective fast quantitative wall motion assessment of myocardial deformation patterns without specific and time-consuming strain imaging techniques. FTI allows distinguishing between healthy subjects and subjects with AL. Further investigations are necessary to analyze the impact of this new method on clinical outcome in AL patients.none


The upper mini-sternotomy and right mini-thoracotomy can confer the advantages off a smaller surgical wound for aortic valve replacement.236 patients were included in our study, to whom aortic valve was replaced. The purpose of this study was to examine the potential benefit of double venous cannulation through superior vena cava (SVC) for venous drainage for aortic valve replacement (AVR) in upper mini-sternotomy and right mini-thoracotomy approaches. Five to 7 cm skin incision was made at the level of third intercostal space, through which the pericardium was opened longitudinally. Shortly after the patient was fully heparinized, venous drainage was achieved by vacuum assisted (40-60 mmHg) double venous cannulation through SVC (22 French) while arterial return was established by right femoral artery or ascending aorta (standard arterial cannula. We cross-clamped the aorta and infused antegrade cold blood cardioplegia into the ascending aorta and retrograde cold blood cardioplegia into the coronary sinus to achieve cardiac arrest. The aortic valve was approached through a transverse aortotomy. The native valve was removed completely and replaced with biological or mechanical valve, secured by Teflon pledges and sutures.Our study confirmed that even though technically challenging, upper mini-sternotomy and right mini-thoracotomy approaches for aortic valve replacement have potential advantages over conventional median sternotomy. They can offer easier exposure of ascending aorta and effective aortic valve replacement especially when using double drainage of SVC and when patients are reasonably selected for these procedures. These mini approaches were proved to be safe and efficacious.Upper mini-sternotomy and right mini-thoracotomy can be performed with standard equipment and instruments and are potentially compatible with new technologies such as suture-less aortic valve prosthesis which could be easily placed this way with a very short CPB time.


Ruling out Legionella sp. in patients presenting with community-acquired pneumonia (CAP) is important due to differences in treatment regimens. Yet antigen tests as well as blood cultures have low sensitivity and an important time delay, making empirical broad spectrum coverage necessary particularly in severe cases. Fiumefreddo and colleagues recently proposed a clinical score based on six clinical and laboratory variables  which allowed assessing the likelihood of Legionella . Yet theWe analyzed data from a large multinational database of patients with CAP (CAPO)  between Data for 8,278 CAP patients were analysed; the infectious organism was known in 2,321 cases (28%), including a total of 101 patients with urinary antigen-confirmed Legionnaires' disease and 983 patients with confirmed pneumococcal disease. All variables were predictors for Legionella with odds ratios ranging from 1.002 to 5.767. Combining the variables in a joint logistic regression model showed a high predictive accuracy with an AUC of 0.86.This analysis validates the Legionella score in an independent sample and shows high diagnostic accuracy. Interventional trials with adapted antibiotic regimes for non-inferiority in a real live population are warranted.


Our studies used human pancreatic carcinoma cells, cells stably transfected with human transporter cDNAs, or cells in which a specific transporter was knocked down by RNA interference. We have previously shown that 5-fluorouracil treatment affects the expression profile of relevant cellular transporters including multidrug resistance proteins (MRPs), and that MRP5 (ABCC5) influences chemoresistance of these tumor cells. Similarly, cell treatment with the nucleoside drug gemcitabine or a combination of chemotherapeutic drugs can variably influence the expression pattern and relative amount of uptake and export transporters in pancreatic carcinoma cells or select for pre-existing subpopulations. In addition, cytotoxicity studies with MRP5-overexpressing or MRP5-silenced cells demonstrate a contribution of MRP5 also to gemcitabine resistance. These data may lead to improved strategies of future chemotherapy regimens using gemcitabine and/or 5-fluorouracil.Pancreatic cancer ranks among the tumors most resistant to chemotherapy. Such chemoresistance of tumors can be mediated by various cellular mechanisms including dysregulated apoptosis or ineffective drug concentration at the intracellular target sites. In this review, we highlight recent advances in experimental chemotherapy underlining the role of cellular transporters in drug resistance. Such contribution to the chemoresistant phenotype of tumor cells or tissues can be conferred both by uptake and export transporters, as demonstrated by


Sir,Percutaneous tracheostomy has become an appropriate alternative to conventional surgical tracheostomy and has gained worldwide acceptance as a bedside procedure. Despite We describe a technique using a standard nasogastric tube (NGT) that allows precise and safe placement of tracheostomy tubes in patients under emergency situation of false passage. This method eliminates the need for specialized equipment and skills like fiberoptic which is usually not available at bedside where percutaneous tracheostomy actually takes place.A 22-year-old male patient operated for right fronto-temporo-parietal subdural hematoma required tracheostomy. Attempt to percutaneous tracheostomy using the Ciaglia’s Blue Rhino technique created a false passage. Repeated attempts to negotiate tracheostomy tube over guide wire failed, leading to airway emergency scenario. We devised a novel technique to overcome the false passage.in situ [An endotracheal tube (ETT) which had been withdrawn upto glottis was pushed caudally so that it occupied the distal end of the trachea, sealing off the rent created by dilation of the tracheostomy tract. A Ryle’s tube with its tip cut was inserted through the ETT already in situ . The ETTin situ . The NGTin situ . Initialin situ  and previn situ  Limitati


We provided a cross-tissue comparative analysis of between-subtype molecular commonality for ovarian cancer, breast cancer, hepatocellular carcinoma, glioma, lung squamous carcinoma and nasopharyngeal carcinoma. Our analysis showed that molecular subtypes with similar phenotype or similar clinical outcome could be correlated by their transcriptional profile and pathway profile. Pathway dysregulation across multiple cancer subtypes was also revealed by Gene Set Enrichment Analysis. Dysregulation of ‘complement and coagulation cascades’ was observed in a total of eleven subtypes across five tissues, implicating that the role of this process in personalized immune-based therapy may be worth further exploring. Based on high-through genomewide transcriptome data, molecular subtypes of human cancer have been identified and characterized by different biology. For example, distinct subtypes of breast cancer were associated with different patterns of therapeutic response , differe-low intrinsic subtype of breast cancer were characterized by expression of mesenchymal markers and immune response [A recent study depicted the molecular commonality between basal-like breast cancer and ovarian cancer by correlation analysis of transcriptional profile , but it response –8. TakinAll microarray datasets were downloaded from GEO. For Affymetrix data, we recalculated gene expression signal intensities by RMA  using DaThree published gene expression signature of Epithelial-Mesenchymal Transition (EMT) or Mesenchymal Transition (MT) were used in our analysis. The Taube’s ‘EMT core signature’ represent genes that shared by independent gene expression signatures (GESs) in human mammary epithelial cells (HMLE) induced to undergo an EMT by expressing Gsc, Snail, Twist, or TGF-β1 or by knocking down expression of E-cadherin . The GroData was median-centered by genes in each cohort separately at first . Then alThe median of spearman rank correlation of transcriptional profile between two subtypes was minus by 1 and then adopted as the dissimilarity distance. Average-linkage hierarchical clustering of the resulting dissimilarity matrix was performed in R.Hierarchical clustering of the pathway profile matrix (rows for pathway and columns for subtype) was performed in Cluster 3.0  using SpGSEA analysis  was perfWe first asked whether subtypes with similar phenotype or similar clinical outcome could be correlated by transcriptional profile and/or pathway profile. To answer this question, we measured between-subtype commonality by correlation analysis of transcriptional profile and pathway profile. A similar landscape was found for both two molecular profiles but the overall level of spearman rank correlation coefficients of pathway profiles was higher than that of transcriptional profiles . This maHere we demonstrated molecular commonality between cancer subtypes using breast cancer as an example  become available, we may expect more and more cancers being classified into molecular subtypes. Our method, and of course other improved and enhanced methods, could be applied to construct a more comprehensive map of cancer subtypes. With such a map, knowledge for a particular cancer subtype could provide clues for an extended understanding of its ‘friend’ subtypes in other cancers and bring potential novel therapeutic opportunities.Figure S1Scatter plot of correlation coefficient. A linear model was fit to the data using correlation coefficients by transcriptional profile as the independent variable. The resulting p-value and coefficient were showed.(TIF)Click here for additional data file.Figure S2Comparison of ovarian cancer and other cancer. Tumor samples were grouped by subtype. Yellow grids represented positive correlation while blue grids represented negative correlation. The number of samples per subtype was inside the brackets.(TIF)Click here for additional data file.Figure S3Comparison of glioma and other cancer. Tumor samples were grouped by subtype. Yellow grids represented positive correlation while blue grids represented negative correlation. The number of samples per subtype was inside the brackets.(TIF)Click here for additional data file.Figure S4Comparison of hepatocellular carcinoma and other cancer. Tumor samples were grouped by subtype. Yellow grids represented positive correlation while blue grids represented negative correlation. The number of samples per subtype was inside the brackets.(TIF)Click here for additional data file.Figure S5Comparison of lung squamous carcinoma and other cancer. Tumor samples were grouped by subtype. Yellow grids represented positive correlation while blue grids represented negative correlation. The number of samples per subtype was inside the brackets.(TIF)Click here for additional data file.Figure S6Comparison of nasopharyngeal carcinoma and other cancer. Tumor samples were grouped by subtype. Yellow grids represented positive correlation while blue grids represented negative correlation. The number of samples per subtype was inside the brackets.(TIF)Click here for additional data file.Table S1A brief description of all the microarray dataset used in this study. A "*" in the research type column indicated that the corresponding study defined the molecular subtypes.(XLSX)Click here for additional data file.Table S2GSEA results for KEGG pathways and published EMT signature. Only those with q-values less than 0.25 were included .(XLSX)Click here for additional data file.Table S3Results of DAVID functional analysis of published EMT signatures. (XLSX)Click here for additional data file.


We previously identified miRNA promoter regions active in normal mammary cell types and here we analyzed which of these promoters are targets of aberrant DNA methylation in human breast cancer cell lines and breast tumor specimens. Using 5-methylcytosine immunoprecipitation coupled to miRNA tiling microarray hybridization, we performed comprehensive evaluation of DNA methylation of miRNA gene promoters in breast cancer. We found almost one third (55/167) of miRNA promoters were targets for aberrant methylation in breast cancer cell lines. Breast tumor specimens displayed DNA methylation of majority of these miRNA promoters, indicating that these changes in DNA methylation might be clinically relevant. Aberrantly methylated miRNA promoters were, similar to protein coding genes, enriched for promoters targeted by polycomb in normal cells. Detailed analysis of selected miRNA promoters revealed decreased expression of miRNA linked to increased promoter methylation for mir-31, mir-130a, let-7a-3/let-7b, mir-155, mir-137 and mir-34b/mir-34c genes. The proportion of miRNA promoters we found aberrantly methylated in breast cancer is several fold larger than that observed for protein coding genes, indicating an important role of DNA methylation in miRNA deregulation in cancer. MicroRNAs (miRNA) are short single-stranded RNA molecules that regulate gene expression at the posttranscriptional level by stimulating the degradation or inhibiting translation of target mRNAs Aberrant DNA methylation linked to silencing of individual miRNA genes has been found in many cancer types including breast cancer in vitro cell lines, LCM samples), they are more difficult to analyze in heterogeneous tumor specimens since, in addition to tumor cells, these specimens generally contain significant and variable proportions of normal epithelial and mesenchymal cells In this study we identified miRNA promoters that undergo aberrant DNA methylation in human breast cancer using 5-methylcytosine immunoprecipitation coupled to analysis by a custom designed miRNA tiling microarray. The active human mammary miRNA promoter locations were previously determined by H3K4me3 ChIP analysis and miRNA expression correlated with H3K4me3 status of the promoters Results from this study show that miRNA promoters are widely targeted for aberrant DNA methylation in breast cancer. Almost one third (55/167) of miRNA promoters active in mammary cells displayed hypermethylated DMRs in analyzed breast cancer cell lines. This percentage greatly exceeds the percentage of protein coding genes targeted for aberrant DNA methylation in cancer We have previously identified the promoter regions of miRNA genes expressed in normal breast cells represented by HMEC and HMF in vitro and in vivo samples form two clusters on the left side of the multidimensional scaling plot, while the tumor samples are more dispersed. The most separated are the pure cancer cell lines and the pure normal HMEC and HMF strains. Probably due to their heterogeneous nature, the distance between tumor and non-tumor tissues is smaller, nevertheless the direction of the change is the same, indicating similar DNA methylation differences in both cancer cell lines and tumor tissue. To discern possible effects of in vitro culture versus heterogeneity of in vivo tissues we also analyzed the in vitro and in vivo samples separately  of miRNA promoters active in normal mammary cells became hypermethylated in cancer cells , while oWe have previously shown that normal CTS miRNA expression is often regulated by epigenetic mechanisms including DNA methylation in vitro cultured cancer cell lines also occur in vivo in breast tumors and therefore may have clinical relevancy. To avoid possible confounding effects from different proportions of differentially methylated normal cell types (HMEC and HMF) in individual tissue samples, we also restricted this analysis to non-CTS miRNA promoters. Hypergeometric test revealed highly significant overlap between non-CTS miRNA promoters containing hypermethylated DMRs in cancer cell lines and those hypermethylated in a group of twelve tumor tissues  as well as those hypermethylated in individual tumor tissue samples only  were analyzed by Sequenom MassARRAY . In addiPolycomb targeted genes are frequent targets of aberrant DNA methylation in cancer Aberrant DNA methylation in cancer cells is often linked to gene silencing. We therefore analyzed the expression levels of the aberrantly methylated miRNA genes using quantitative real-time PCR. In general, negative or no correlation between miRNA expression and DNA methylation was observed , no posiin vitro and in vivo representations of normal and diseased mammary cells. We found a large portion of analyzed miRNA promoters to be hypermethylated in breast cancer cell lines, and the same promoters were targeted in breast tumor tissues, although less frequently. The hypermethylation of selected miRNA promoters was verified using MassARRAY technology. MassARRAY DNA methylation data combined with miRNA expression data revealed that in six out of twelve cases the hypermethylation of miRNA promoters was linked to silencing of miRNA expression; in the other six cases DNA methylation did not correlate with the miRNA level. Overall, our study has shown aberrant DNA methylation of a large portion of miRNA genes leading frequently to silencing of miRNA expression in breast cancer. Since miRNAs play an important role in the regulation and maintenance of cell identity, our observations suggest that the deregulation of miRNA expression due to aberrant DNA methylation is involved in human breast carcinogenesis.In this study, we sought to determine the DNA methylation status of miRNA promoters in breast cancer cells on the whole genome scale. To this end, we used 5-methylcytosine immunoprecipitation linked to a custom tiling microarray hybridization to analyze DNA methylation level in a variety of Almost one third of miRNA promoters active in normal mammary cells were found to contain hypermethylated DNA regions in breast cancer cells; this high proportion is several fold greater than usually observed for protein coding genes We have previously found a significant portion of miRNAs expressed in normal breast cells to be cell-type specific The incidence and magnitude of miRNA promoter hypermethylation found in breast tumor tissues was lower than in breast cancer cell lines . Two facOur study has shown that in six out of twelve miRNA genes analyzed in more detail the aberrant DNA methylation was linked to decreased levels of miRNAs in cancer. In addition to mir-137 and mir-34b/mir-34c genes, that were previously reported to be silenced by DNA methylation in various cancers in vivo breast tumor tissues and might be therefore clinically relevant. The miRNA targets of aberrant DNA methylation might be potentially used for diagnostic purposes. miRNA genes silenced by aberrant DNA methylation in cancer are potential targets for epigenetic drugs.Overall, our study reports the first systematic identification of aberrant DNA methylation of miRNA gene promoters in breast cancer. Aberrant DNA methylation found in breast cancer cell lines is also detectable in HMEC and HMF were previously described Genomic DNA was extracted using the DNeasy Blood and Tissue Kit  according to manufacturer protocol and quantified spectrophotometrically. Total RNA was extracted using the miRNeasy kit (Qiagen) according to manufacturer protocol except that Trizol  was used instead of Qiazol for the cell lysis. Each tissue sample was ground frozen in liquid nitrogen and resulting powder was then split for RNA and DNA extractions, respectively, to achieve homogenous representation of tissue components in both DNA and RNA samples.http://www.ncbi.nlm.nih.gov/geo/) under accession no GSE38254.The design of the microarray used in this study and MeDIP chip procedure was previously described H3K27me3 domain data from H1 ESC were downloaded from two sources: supplemental data from DNA methylation analysis by Sequenom MassARRAY  was performed as described Expression of microRNAs was performed in triplicate using EXIQON miRCURY LNA™ Universal RT kit and EXIQON miRCURY LNA™ Universal RT microRNA PCR, Pick-&-Mix, Ready-to-use Panels . Real-time PCR was conducted on an ABI Prism 7500 Sequence Detection System  using PerfeCta SYBR Green SuperMix, Low ROX  with a 95°C denaturation for 3 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 45 seconds. Differences in expression were determined using the comparative Ct method described in the ABI user manual. SNORD38B and SNORD49A expression was used for normalization of miRNA expression.The significance of promoter region overlaps was calculated in R using hypergeometric test as described previously Figure S1MicroRNA promoters hypermethylated in cancer are frequently occupied by polycomb specific H3K27me3 in normal cells. Left, Venn diagram showing the overlap between the non-CTS miRNA promoters occupied by H3K27me3 in ESC and those hypermethylated in tumor tissue samples (TT). Right, Venn diagram showing the overlap between the non-CTS miRNA promoters occupied by H3K27me3 in HMEC and those hypermethylated in TT. Both overlaps are highly significant (hypergeometric test).(PDF)Click here for additional data file.Table S1DNA methylation status for all analyzed miRNA promoters. The table shows the presence or absence of DMRs for individual miRNA promoters for groups of samples and individual samples. The first column shows the names of individual miRNA genes: non CTS genes have yellow background, HMEC-specific green background and HMF-specific red background. The second column shows the expression ratio of these miRNA between two normal mammary cell types: HMEC and HMF. The next three columns indicate whether the promoter region is occupied by polycomb specific H3K27me3 in three normal cell types: H1 ESC, HMEC and HMF. Then folows the DNA methylation data for all cancer cell lines compared to HMEC, all tumor tissue compared to nontumor tissue and for HMF compared to HMEC. The last section shows hypermethylated DMRs for individual cancer cell lines relative to HMEC and individual tumor tissue samples relative to nontumor tissue. The data are color coded, hypermethylated DMRs are indicated by brown color, hypomethylated DMRs by green color. The area of cell type specific miRNA promoters for tissue samples is shaded grey since this part of data is prone to missinterpretation.(XLS)Click here for additional data file.Table S2Positions of MassARRAY amplicons in hg18 and sequences of MassARRAY primers used for DNA methylation analysis.(PDF)Click here for additional data file.


Native T1 mapping is promising non-invasive method for quantitative assessment of diffuse myocardial processes. The aim of the current study was to interrogate normal values for T1 measurement within native myocardium for age and gender using most commonly used sequences and field strengths.Ninety-nine subjects with no history of cardiovascular or systemic disease or regular medication underwent native T1 imaging with modified  and shortened modified look-locker inversion recovery sequence  at either 1.5T or 3T. The images were analyzed using an Osirix plug-in with manual motion correction of the ROI placed conservatively within the septal myocardium. For comparison, lateral myocardium and blood values were also derived.There was similar representation for age and gender across both field strengths Table . Mean vaWe report age-gender normal values for native myocardial T1 mapping by the two most commonly used sequences and field strenghts. We show that there are neither gender differences in a cohort as a whole nor within separate age groups.NIHR


Atlantic Bluefin Tuna (ABFT) shows complex demography and ecological variation in the Mediterranean Sea. Genetic surveys have detected significant, although weak, signals of population structuring; catch series analyses and tagging programs identified complex ABFT spatial dynamics and migration patterns. Here, we tested the hypothesis that the genetic structure of the ABFT in the Mediterranean is correlated with mean surface temperature and salinity.We used six samples collected from Western and Central Mediterranean integrated with a new sample collected from the recently identified easternmost reproductive area of Levantine Sea. To assess population structure in the Mediterranean we used a multidisciplinary framework combining classical population genetics, spatial and Bayesian clustering methods and a multivariate approach based on factor analysis.ST analysis and Bayesian clustering methods detected several subpopulations in the Mediterranean, a result also supported by multivariate analyses. In addition, we identified significant correlations of genetic diversity with mean salinity and surface temperature values revealing that ABFT is genetically structured along two environmental gradients. These results suggest that a preference for some spawning habitat conditions could contribute to shape ABFT genetic structuring in the Mediterranean. However, further studies should be performed to assess to what extent ABFT spawning behaviour in the Mediterranean Sea can be affected by environmental variation.F Assessing the correlation between landscape features with the genetic variation of populations might lead to identifying environmental factors that are involved in the adaptive divergence of populations Thunnus thynnus) shows a spatial population structure, which is stable over short and long time periods The Mediterranean is a temperate sea with sharply different oceanographic conditions in the western and eastern parts Multidisciplinary seascape genetics sensu  addresseHere, we test the hypothesis that dispersal and reproduction of the ABFT populations in the Mediterranean correlate with the physical environmental heterogeneity. We do that by comparing measures of ABFT genetic structure at microsatellite loci with surface temperature and salinity variation. For this purpose, we numerically and geographically expanded the ABFT sampling We also used the spatial Bayesian clustering models implemented in the software GENELAND Thunnus thynnus used in this study were collected from Mediterranean individuals caught during scientific research programs under the permission of the Italian Ministry of Agricultural and Forestry Policies  and by commercial long-liners and purse-seiners within the Total Allowed Catch quotas assigned by the International Commission for the Conservation of Atlantic Tunas (ICCAT) to National Governments . No specific approval of this vertebrate work is required since the Bluefin tuna individuals sampled in this study were obtained from scientific and commercial fishing activities.Tissue samples of the Atlantic Bluefin tuna In this study we have reanalysed the genetic variation of six ABFT Mediterranean population samples (N = 256) previously genotyped at potentially neutral microsatellite loci by STST under the hypothesis of panmixia. A global HWE test was performed by GENEPOP 4.0 Allelic richness was estimated using FSTAT version 2.9.3.2 The software STRUCTURE The spatially explicit Bayesian clustering program GENELAND 3.2.4 http://gher-diva.phys.ulg.ac.be/web-vis/), a Pan-European infrastructure for managing, indexing and providing access to ocean and marine data sets and data products, acquired via research cruises and other observational activities, in situ and remote sensing. Temperature data were averaged over the period 1985–2007, while S data were averaged over the period 1900–2009.Seawater salinity  and surface temperature  data from the sampled sites were obtained from SeaDataNet Climatologies Pan-European Infrastructure for Ocean and Marine Data Management  Multilocus genotype data were finally analysed under a model of isolation by distance (IBD). A matrix of geographical distances was obtained considering the shortest sea-paths between each pair of sampling sites using Google Earth version 6.0.2 OOB; genetic distances between populations were expressed by the ratio FST values were significantly greater than 0 except four , suggesting that different Mediterranean areas roughly correspond to distinct ABFT subpopulations. The LIG-SAR FST became insignificant after the sequential Bonferroni correction  implemented in GENELAND did not reach convergence and this poor MCMC mixing could be due to departures from model assumptions . On the contrary the spatially explicit model with correlated allele frequencies reached the convergence of MCMC and the most likely value of K identified was for K = 3. Individual assignments performed in GENELAND with K fixed to 3 identified distinct subpopulations splitting the southernmost samples  and STY from the central (SAR) and northernmost samples  . These fThe Correspondence Analysis (CA) displayed a spatial pattern of differentiation largely consistent with those detected by the Bayesian clustering analysis. The CA separated the Central Northern Mediterranean ADR and LIG samples form the other samples, identified the group SAR-ALB and revealed the genetic similarity between STY and CYP .A significant correlation between genetic distances and both geographical and mean-S distances, was revealed by the Mantel test . On the The CCA analysis was carried out using mean-S and mean-t data as constraining factors and correlations at both axes were significant at the ANOVA 95%-significance test , from the central-southern samples  and differentiating the SAR sample from all the others .STs. Nevertheless the level of genetic divergence detected by STs’ range 0.011–0.021) was higher than in previous surveys ST = 0.02) showing that such structuring of the Mediterranean ABFT is probably stable through time. These results, along with the evidence for a recent colonization of the Mediterranean Click here for additional data file.Table S2Thunnus thynnus samples.Summary statistics at the seven microsatellite loci of (DOC)Click here for additional data file.Table S3Thunnus thynnus samples.Gene diversity and Hardy-Weinberg equilibrium deviation test of (DOC)Click here for additional data file.Table S4Percentages of variations explained by CA and CCA axis.(DOC)Click here for additional data file.Table S5Ordination scores of mean temperature (Mean-t) and mean salinity (Mean-S) values.(DOC)Click here for additional data file.Text S1DNA extraction, Microsatellite marker analysis protocols, Correspondence Analysis (CA) and Canonical/Constrained Correspondence Analysis (CCA) results and references.(DOC)Click here for additional data file.Dataset S1Microsatellite dataset in gtx format.(GTX)Click here for additional data file.


MLH1 mutation, developed early-onset GI cancer. In contrast to LS with clear GI screening and surveillance recommendations, there are no recommendations for surveillance of individuals with biallelic mutations and no literature describing the long term outcome.Lynch syndrome (LS) is caused by heterozygous germline mutations in the DNA mismatch repair (MMR) genes and is a highly penetrant autosomal dominant condition. A novel childhood cancer syndrome caused by biallelic germline MMR gene mutations and characterized by brain tumors, leukemias, gastrointestinal (GI) polyposis, GI cancer and café-au-lait spots (CALS) has been described. We reported the first biallelic kindred in which 2 of 3 siblings proven to have a homozygous germline To prospectively describe long-term outcome of our two young patients with biallelic MMR mutations, and to develop a generic cancer screening protocol for other patients with biallelic MMR mutations.On the basis of the molecular results, the 2 surviving sisters and parents of a deceased child with metastatic duodenal cancer began a surveillance protocol based on our crude estimates of cancer risks and available cancer screening modalities.During endoscopic screening the youngest sister developed colonic polyps with high grade dysplasia and underwent a subtotal colectomy with ileorectal anastomosis. At 11 years she developed polyps in the duodenum with low grade dysplasia which were excised endoscopically. At 13 years of age, surveillance MRI revealed a left parieto-occipital anaplastic astrocytoma enabling total resection. Endoscopy in the older sister who had a subtotal colectomy with ileorectal anastomosis at 9 years of age for metastatic colorectal cancer has low grade polyps in the duodenum, ileum and rectum which were excised endoscopically. Gynecological follow-up of both sisters with annual pelvic ultrasounds have shown functional ovarian cysts. All neoplastic lesions identified during surveillance were asymptomatic at diagnosis. The sisters are currently fifteen and seventeen years old with no evidence of disease 10 years after their brother’s diagnosis. The parents (43 and 44 years) have annual colonoscopy with no evidence of polyps or cancer. Gynecological screening for the mother has not revealed any abnormalities. Based on our experience to date and review of available literature, we have developed the screening guidelines shown in Table Biallelic carriers who participated in oncologic surveillance had presymptomatic neoplasms identified and treated. These siblings are alive with no evidence of disease at 10-year follow-up. Aggressive surveillance in biallelic MMR carriers is feasible, allows early detection and improves long-term survival.


Our laboratory is examining the profiles of microRNA expression in ATLL cells and infected T-cell lines using microarrays and small RNA libraries.Microarray analysis of ATLL samples revealed 6 upregulated and 21 downregulated microRNAs in ATLL cells compared to CD4+ T-cell controls. Potential targets for deregulated microRNAs were identified by integrating microRNA and mRNA expression profiles. Current experiments are aimed at verifying these predicted microRNA-target interactions.Mass sequencing of small RNA libraries prepared from normal CD4+ cells and two chronically infected T-cell lines yielded panels of known and candidate new microRNAs for each library. Comparison of frequencies of known microRNAs led to the identification of a small number of microRNAs differentially expressed in both infected cell lines compared to controls. Most of the candidate new microRNAs were intragenic with poor species conservation, suggesting that they might have particular roles in human T-cell function. Two sequences mapped to the HTLV-1 genome, suggesting that the virus may produce its own microRNAs. Further analyses of the new cellular and viral microRNA candidates are in progress.


The extend of lymph node involvement in patients NSCLC is the cornerstone of staging and influences both multimodality treatment and final outcome. We studied safety, accuracy and characteristics of intraoperative ultrasound guided systematic mediastinal nodal dissection in patients with resected NSCLC.Prospective randomized trial of intraoperative surgical staging after radical surgery for NSCLC was carried out. Intraoperative hand held ultrasound probe was used in systematic mediastinal nodal dissection in 124 patients after radical surgery for NSCLC and compared with 120 patients who underwent radical surgery followed by standard systematic mediastinal nodal dissection. Mapping of the lymph nodes by their number and sation followed by histopathologic evaluation was performed. Patients data were statistically analyzed.The surgical procedure used was comparable in both groups of patients. Operating time was prolonged for 10 ( 6-20 ) minutes in patients with US guided mediastinal nodal dissection, but number and stations of evaluated lymph nodes were significantly higher ( p<0.001 ) at the same group of patients. Skip nodal metasteses were found in 24 % of patients without N1 nodal involvement. We upstaged 12 ( 10 % ) patients using US guided mediastinal lyphadenectomy. Median follow-up was 38 ( range 10-52 ) months. Standard staging system seemed to be improved in US guided mediastinal lyphadenecetomy patients. Complication rate showed no difference between analyzed groups of patients.Higher number and location of analyzed mediastinal nodal stations in patients with resected NSCLC using hand held ultrasound probe siggested to be of great oncological significance. Procedure showed absolute safety and high accuracy. Our results indicate that intraoperative US may have important staging implication. Further clinical studies should be performed in order to improve intraoperative staging in NSCLC patients.


Cardiac imaging has been identified as a potential use of hybrid PET-MRI. This new technology allows simultaneous Positron Emission Tomography (PET) and MR scanning to occur.This case study features a 34 year old male with known pulmonary sarcoidosis which was diagnosed in 2006 and initially treated with immunosuppressants. He presented with NYHA functional class 2 symptoms. ECHO showed asymmetrical LV anterior and lateral wall hypertrophy with a maximum wall thickness of 18mm in the basal and mid anterior and lateral wall.The patient was referred for a cardiac18F Fluorodeoxyglucose (FDG) PET-MR study to determine the cause of the hypertrophy. The possible differential diagnoses included hypertrophic cardiac myopathy with pulmonary sarcoidosis or cardiac sarcoidosis.Prior to the scan, the patient followed a high protein, low carbohydrate diet for one day. This was then followed by a 12 hour fast to suppress physiologic glucose metabolism by the heart.The patient was administered with 355mBq of 18F FDG 180 minutes prior to the PET-MR study.A simultaneous PET-MR study was acquired. A standard CMR protocol was undertaken in conjunction with a simultaneous 10 minute single PET bed using a Siemens Biograph mMR hybrid PET-MR scanner. This is a 3T magnet containing a 260mm field of view PET detector located at the isocentre.The scan demonstrated mediastinal 18F FDG avid lymphadenopathy with anterio-lateral myocardial thickening with mid-myocardial LGE and focal 18F FDG uptake compatible with active myocardial inflammation within an area of "scar" in keeping with active cardiac sarcoidosis.The oedema sensitive STIR sequence did not differentiate between the hyper-metabolic active disease and the chronic "burnt out" disease.This patient is one of the first examples of imaging active cardiac sarcoidosis using a hybrid PET-MR technique. It demonstrates the potential for differentiating between active and chronic cardiac sarcoidosis during one scan. This new imaging technique could have a substantial impact on the diagnosis and management of cardiac sarcoidosisUCL\UCLH receives a proportion of funding from the Department of Health's NIHR Biomedical Research Centres funding scheme.


More than 240 million people worldwide are infected with hepatitis B virus (HBV) and are at risk of developing liver cirrhosis and hepatocellular carcinoma (HCC). Reducing this pool of infected people is a necessity since despite an effective prophylactic vaccine, about 2% of the vaccinated individuals in high endemic areas still develop chronic HBV infection (CHB). A battery of antiviral drugs based on nucleoside or nucleotide analogues that target the HBV reverse transcriptase are available. They efficiently suppress HBV replication and reduce liver inflammation linked with cirrhosis but rarely achieve virus eradication. HBV is present in hepatocytes in a mini-chromosomal form  that is untouched by reverse transcriptase inhibitors. As a consequence, response to the treatment is hardly durable and a majority of patients experience HBV reactivation when antiviral therapy is withdrawn 6 copies/ml) Efficient control of HBV infection is associated with the induction and persistence of helper and cytotoxic T cells targeting different HBV proteins and production of anti-HBV envelope antibodies. This antiviral immune response is composed of T cells able to secrete Th1 cytokines, proliferate and lyse HBV infected hepatocytes After years of exposure to HBV antigens, HBV-specific T cells are deleted or functionally exhausted in chronic patients. Virus-specific T cells express inhibitory molecules like PD-1 Blocking inhibitory pathways associated with T cell exhaustion has shown therapeutic efficacy in cancer patients Vaccine therapy aims to induce functionally efficient HBV-specific T cells on the background of virus-specific T cell exhaustion. Several strategies have been tested in clinical trials with disappointing results. Often, vaccine therapy did not induce an HBV-specific T cell response, or when such response was boosted, it did not have a therapeutic effect However, more radical approaches could be needed to circumvent HBV-specific T cell deletion in patients with high viral loads. Engineering HBV-specific T cells through transfer of HBV-specific T cell receptors or HBV-specific chimeric antigen receptors (CAR) showed encouraging results in vitro and in animal models Several other factors have also hampered the restoration of efficient adaptive immunity in CHB. Epigenetic programs were suggested to preserve the T cell exhaustion state in chronic viral infection The duration of exposure and antigen burden in chronic HVB infection are believed to be the primary factors affecting virus-specific T cell function. Thus, modulating the quantity of viral antigens secreted by HBV might have a beneficial effect as has been observed in HBV transgenic mice The important role played by innate immunity in the early stages of infection has stimulated therapeutic strategies to specifically target this branch. IFN-α therapeutic efficacy is associated with NK cell activation + T cells Other approaches in clinical development seek to induce intrahepatic IFN-α production through oral administration of TLR agonists. TLR7 agonists stimulate robust IFN-α production in plasmacytoid dendritic cells. However, prolonged efficacy in TLR-7 agonist–treated, HBV-infected chimpanzees was not only related to the production of IFN-α, as its production was transient and not liver specific. Antiviral efficacy was more consistent with a boost of intrahepatic NK, NKT, and T cell responses associated with production of IFN-γ The immunotherapeutic strategies we reviewed aim to control HBV infection by increasing antiviral immunity and as such terminate the chronic inflammatory process. However, activation of intrahepatic adaptive or innate cellular immunity requires tight control because it could also exacerbate liver inflammation. Even though mechanisms like IL-10 production, release of arginase from hepatocytes, and even the dampening of T cell responses by activated NK cells A radically different perspective in chronic HBV treatment is to consider CHB a necro-inflammatory rather than viral disease. Recent data in HBV transgenic mice clearly indicate that suppressing intrahepatic CTL activity in the liver using anti-platelet therapy prevents hepatocellular carcinoma. Platelets promote the accumulation of CD8 T cells in the liver and anti-platelet therapy blocks this process, reducing hepatocellular injury and fibrosis In conclusion, the current immunotherapeutic strategies designed to suppress and control HBV replication have strong scientific support. However, they are restricted by our limited knowledge, and further understanding of the relationship with the virus in the unique environment of the human liver will almost certainly provide opportunities to enhance them or perhaps develop totally novel approaches.


Somatic embryogenesis (SE) in forest trees is considered as one powerful approach in cloning elite genotypes. However, SE is difficult to achieve in tissue or cell beyond the mature embryo phase in trees and its physiological process involved are also poorly understood. Recently metabolic profiling studies have been contributed to understand the mechanism involved in SE process of plant -3. In ad


Caenorhabditis elegans are free-living in nature and serve as model organisms for studying parasitic infections. Excretory/secretory proteins play an important role in parasitic helminth infections which make these proteins attractive targets for therapeutic use. In the case of helminths, large volume of expressed sequence tags (ESTs) has been generated to understand parasitism at molecular level and for predicting excretory/secretory proteins for developing novel strategies to tackle parasitic infections. However, mostly predicted ES proteins are not available for further analysis and there is no repository available for such predicted ES proteins. Furthermore, predictions have, in the main, focussed on classical secretory pathways while it is well established that helminth parasites also utilise non-classical secretory pathways.Helminths are important socio-economic organisms, responsible for causing major parasitic infections in humans, other animals and plants. These infections impose a significant public health and economic burden globally. Exceptionally, some helminth organisms like We developed a free Helminth Secretome Database (HSD), which serves as a repository for ES proteins predicted using classical and non-classical secretory pathways, from EST data for 78 helminth species  ranging from parasitic to free-living organisms. Approximately 0.9 million ESTs compiled from the largest EST database, dbEST were cleaned, assembled and analysed by different computational tools in our bioinformatics pipeline and predicted ES proteins were submitted to HSD.http://estexplorer.biolinfo.org/hsd, for checking the sequence similarity of new protein sequences against predicted helminth ES proteins.We report the large-scale prediction and analysis of classically and non-classically secreted ES proteins from diverse helminth organisms. All the Unigenes (contigs and singletons) and excretory/secretory protein datasets generated from this analysis are freely available. A BLAST server is available at  Caenorhabditis elegans (the most studied helminth till date) serve as models to understand parasitism   unverified}. Each annotated ES protein is also tagged as verified or unverified based on the presence or absence of sequence similarity to experimentally determined parasitic helminth ES proteins  technologies, there are many transcriptomic studies completed especially for individual helminth species with good coverage but we have focussed on the coverage of a large number of helminth organisms for secretome analysis. The earlier analysis from our group using the EST2Secretome pipeline has now been extended to cover non-classical secretory proteins, with validation against experimentally known excretory/secretory proteins. We plan to carry out further prediction of ES proteins using more comprehensive helminth transcriptomic datasets from NGS platforms and provide the results through HSD.Several billion people worldwide are afflicted by infections caused by parasitic helminths. Infections from parasitic helminths, especially from nematodes also results in heavy economic losses worth billions of dollars due to agricultural crop and livestock infection each year. In this study, we have predicted and analysed ES proteins from the largest freely available EST data of several helminth organisms from dbEST.Fasciola hepatica . The. The13].Our bioinformatics approach made possible the large scale prediction and analysis of ES proteins. As a result of our analysis we develop a unique resource HSD (Helminth Secretome Database) of ES proteins for the parasitology/infectious diseases/pharmacy communities. Our approach can be used on new large-scale transcriptomic data sets from NGS platforms, for rapid prediction and annotation of ES proteins. The approach can be applied to any organism but its main application is for neglected organisms with limited knowledge.BRITE: Biomolecular Relations in Information Transmission and Expression; KEGG: Kyoto Encyclopedia of Genes and Genomes; KAAS: KEGG automatic annotation server.The authors declare that they have no competing interests.SR directed the study. GG developed the database and carried out the analysis. SR and GG contributed to writing the manuscript.Summary of large scale helminth EST analysis. Statistics of excretory/secretory proteins and Unigenes across different helminth species (Table S1)Click here for fileGene Ontology distribution of helminth ES proteins. Statistics of Gene Ontology distribution across different helminth species (Table S2)Click here for fileHelminth ES protein domain mapping. Represented Interpro domains found in helminth ES proteins. (Table S3)Click here for fileKEGG BRITE objects mapping of helminth ES proteins. Represented KEGG BRITE objects found in ES proteins predicted by KAAS (Table S4)Click here for fileC. elegans (Wormpep) and S. mansoni proteinsComparison of putative helminth ES proteins with . Statistics of sequence similarity results of helminth ES proteins with C. elegans (Wormpep) and S. mansoni proteins using BLASTP across different helminth species (Table S5)Click here for fileComparison of putative helminth ES proteins with NR database proteins. Statistics of sequence similarity results of helminth ES proteins with NR database proteins using BLASTP across different helminth species (Table S6)Click here for fileS. japonicum, human proteinsComparison of putative helminth ES proteins with . Statistics of sequence similarity results of helminth ES proteins with S. japonicum, human proteins using BLASTP across different helminth species (Table S7)Click here for fileComparison of putative helminth ES proteins with interaction databases proteins. Statistics of sequence similarity results of helminth ES proteins with interaction databases proteins using BLASTP across different helminth species (Table S8)Click here for file


Non-replicating virus-like particles (VLPs) have been suggested as a promising platform for viral vaccines. Recently several studies have introduced VLPs produced in insect cells as a vaccine candidate against influenza infection. Neuraminidase (NA), one of the two membrane glycoproteins of influenza A viruses has been employed to construct such influenza VLPs to induce protective immunity.In this study, the human influenza virus ) was propagated in MDCK cell culture. Viral RNA was extracted using RNX-plus solution. Complementary DNA synthesis was carried out using uni-12 primer. NA open reading frame (1413 bp) was amplified by RT-PCR using high fidelity Taq DNA polymerase. The amplicon was purified from the gel and cloned into pFastBac1 plasmid through SalI/XhoI sites. After verification of cloned NA by restriction analysis, it was subjected to automated sequencing bidirectionally. The recombinant pFastBacNA vector was transformed to E.coli DH10Bac cells which harbor bacmid DNA and helper plasmid to create NA recombinant bacmid. Restriction map analysis and sequencing results confirmed the fidelity of NA sequence. Homologous recombination between pFastBacNA and bacmid and creation of NA recombinant bacmid was verified by PCR using NA specific and M13 universal primers.In an ongoing study, the cultured Sf9 (Spodoptera frugiperda) insect cell line will be transfected with NA recombinant bacmid to produce recombinant baculovirus expressing NA gene. This production is in its native highly glycosylated form appropriate to use in vaccine research projects. Recombinant baculovirus expressing NA gene can be also used with other individual recombinant baculoviruses expressing HA and M1 genes in production of influenza VLPs.


Pneumothorax is a rare complication of percutaneous venous access and blind subclavian puncture. The incidence of pneumothorax related to blind subclavian puncture is reported to be 1% [A 78-year-old male patient underwent implantation of cardiac resynchronization devise with defibrillator (CRT-D) for dilated cardiomyopathy and congestive cardiac failure. Patient received a Guidant CRT-ICD. RV lead during implantation, subclavian access was obtained by venogram assisted extra thoracic puncture of the subclavian vein. Right atrial (RA) lead was initially attempted in the right atrial appendage (RAA), but the sensing and pacing thresholds were found unacceptable and right atrial free wall was chosen after few initial trials in RAA. The atrial lead was an active fixation lead, Guidant Flextend SN 4087. Right ventricular (RV) lead (Guidant Endotak Reliance SN 184420) was positioned at RV apex and left ventricular lead (Guidant easy track 2) was positioned in posterolateral tributary of coronary sinus. Patient tolerated the procedure well and there were no acute complications. Immediate post operative X ray of the chest showed satisfactory lead positions without any evidence of any pleural pathology 2 hours after the procedure patient experienced pleuritic chest pain and shortness of breath. His chest X Ray revealed moderate right-sided pneumothorax .  A draiA CT scan of the chest was done to identify the location of the lead tip. Right atrial lead was identified along the lateral aspect of the RA without any gross displacement. . There wSince the pneumothorax got resolved and there were no pericardial effusion or indicators of major lead displacement right atrial lead was not repositioned.Pneumothorax is usually a complication of subclavian venous access .  Lead pThe lead parameters especially pacing thresholds and sensing thresholds will show significant changes in case of lead perforation . In micrMany operators prefer Atrial screw in leads as they reduce the chances of lead dislodgement. However screw in leads increase the chance of perforation of thin walled atrium. Acute lead related complications were 2.4% in one series .In conclusion, contralateral pneumothorax in atrial based pacing systems is a rare complication and almost always is caused by atrial lead perforation. Pericardial effusion can also be a part of the problem in case of a macro perforation. This etiology has to be investigated by a CT scan in a suspected case. Extra caution should be taken when selecting RA lateral wall for deployment of screw in atrial lead especially in patients with associated lung pathology like bullous emphysema. Minimum number of turns to deploy the helix and extra post procedure vigilance should be considered in these patients.


We report a young male patient who developed plasma cell myeloma/plasmacytoma 11 years after having received an allogeneic hematopoietic cell transplantation for AML. The patient received a second transplantation from the same donor without immunosuppression and developed graft-versus-host disease (GVHD). Our observation has two aspects that warrant attention: first, insufficiency of long-term tolerance to prevent GVHD in the absence of immunosuppression and second, a stromal or genetic susceptibility to develop hematologic malignancies despite of a complete donor-type chimerism. Allogeneic hematopoietic cell transplantation (HCT) is the only therapy successful in achieving cure for hematological malignancies. However, long-term survivors of allogeneic HCT face the risk of developing second malignancies, mainly the posttransplant lymphoproliferative diseases (PTLDs), secondary MDS/AML, and secondary solid malignancies . Posttra++ and CD117+ atypical plasma cell infiltration with kappa monoclonality. He had IgG kappa monoclonal gammopathy on immune electrophoresis and an elevated erythrocyte sedimentation rate along with a mild anemia and normal renal function tests. Bone marrow (BM) examination revealed 9% plasma cells with CD38+CD138+CD19−CD56−CD44+CD28−CD20− immunophenotype and kappa predominance. The cytogenetic study did not detect any abnormality. Bone marrow chimerism analysis was consistent with 100% donor type in both T-and non-T-cell lineages. Chimerism study was also done with the DNA extracted from the plasmacytoma and revealed 18% donor and 82% recipient cells  and received an allogeneic peripheral blood stem cell transplantation from his 37-year-old HLA identical brother in 1997. The conditioning regimen consisted of busulfan and cyclophosphamide, and the GVHD prophylaxis was done with cyclosporin and short-term methotrexate. He did not develop any acute or chronic GVHD and remained in CR with complete donor-type chimerism until 2008 when he was admitted to the neurosurgery clinic with back pain. The vertebral MRI revealed a tumor invading the 6th thoracic vertebrae causing pathological fracture. The tumor was completely excised, and the pathological evaluation was consistent with CD38nt cells . The pre Posttransplant lymphoproliferative diseases are a heterogenous group of disorders which develop after solid organ or hematopoietic stem cell transplantation as a result of immunosuppression. In contrast to the high incidence of posttransplant lymphomas, PTLDs manifesting as plasma cell myeloma/plasmacytoma are extremely rare. Most of the reported cases have developed after solid organ transplantation, mainly renal transplantation. The incidence of PTLD after allogeneic HCT is 1-2%, mostly diagnosed within the first year and strongly associated with EBV. On the other hand, EBV-negative PTLDs usually occur later . The ris Although the patient was in complete donor-type chimera when he was retransplanted from the same donor 11 years later with fresh peripheral blood stem cells, he still developed GVHD and required immunosuppression. We were not able to find any reports to guide us in our decision making regarding the immunosuppression strategy at the time of retransplantation. Spontaneous occurrance of autologous GVHD in myeloma patients undergoing autologous HCT has been recently reported . On the


Early detection of subclinical manifestations of obesity is important to initiate early effective treatment aiming at reversing the process. Recent studies using echocardiography tissue doppler or strain rate imaging have suggested the potential of motion tracking methods to characterize subtle systolic changes that may be associated with obesity in children .The aim of the present study was to investigate LV global and intra-myocardial regional systolic mechanics using CMR tagging in obese and non-obese adolescents in order to better characterize the subclinical early changes associated with obesity in children.InTag post-processing toolbox  implemented in OsiriX software  to perform quantitative myocardial strain analysis. Motion estimation is based on the Sine Wave Modeling approach(3). Regional and global peak circumferential(Ecc), longitudinal(Ell) strains as well as peak rotation and torsion were calculated.Standard CMR global and regional LV function study and cine-tagging with complementary myocardial tagging(CSPAMM) were performed in 25 obese children and 25 lean controls. Inclusion criteria were: no previous diagnosis of hypertension and treatment with antihypertensive/anti-diabetic drugs or drugs known to affect glucose and lipid metabolism; no history of familial hypertension/dyslipidemia; absence of diabetes or other chronic disease. The study protocol has been approved by the Mother&Child Ethics Committee. Magnitude CSPAMM images were processed using cc displayed only a trend to lower values in basal level  similarly to what was observed for Ell . Torsion was also not significantly increased in obese children .Gender distribution was not different between the two groups but obese children were older. BMI(30.4±5.4vs18.9±1.9),Z-score, systolic  and diastolic blood pressure  were greater whereas heart rate was lower in the obese group. Measurements of LV mass  and EDV  were significantly greater in the obese children. Although global systolic function was not different among the two groups, obese children exhibited a significantly increased stroke volume . In obese children, EIf our data confirm that obese children have early morphological changes in LV mass, EDV and SV, regional circumferential and longitudinal strains are not yet altered from normal. The trend to increased torsion and increased wall thickness may be an adaptation to increased LV ED volume and may lead to early impairment of LV diastolic function.


Retrospective molecular profiling of untreated Diffuse Large B-Cell Lymphoma (DLBCL) samples has recognised distinct sub-classifications of this disease, each with unique biological features and clinical outcomes.(i) To demonstrate superior clinical efficacy, of bortezomib in combination with rituximab and CHOP (RB-CHOP) versus R-CHOP for the treatment of previously untreated patients with DLBCL.(ii) To assess whether the molecular phenotype determines benefit from the addition of bortezomib.The trial uses an adaptive design and aims to recruit 940 patients across 100 sites in the UK and Switzerland (currently recruited 398 patients). After being profiled within real time (during cycle 1), ABC, GCB and unclassifiable molecular phenotype patients are randomised to receive RB-CHOP or R-CHOP with equal allocation. Two interim analyses will be carried out to stop the good prognosis GCB group early if required using a Case Morgan [(i) The first will be a safety analysis performed after the first 55 GCB RB-CHOP patients have been followed for 6 months. If 6 month PFS<80%, the GCB group will close.(ii) The second will be for futility in the GCB group and performed after the first 73 GCB RB-CHOP patients have been followed for 1 year. If 1 year PFS<85%, the GCB group will close.If the GCB group closes the trial will then be modified to a randomised Phase II trial in ABC patients and sample size calculations will be revised.


The tenuous blood supply of traditional flaps for wound cover combined with collateral damage by sacrifice of functional muscle, truncal vessels, or nerves has been the bane of reconstructive procedures. The concept of perforator plus flaps employs dual vascular supply to flaps. By safeguarding perforators along with supply from its base, robust flaps can be raised in diverse situations. This is achieved while limiting collateral damage and preserving nerves, vessels, and functioning muscle with better function and aesthesis.The perforator plus concept was applied in seven different clinical situations. Functional muscle and fasciocutaneous flaps were employed in five and adipofascial flaps in two cases, primarily involving lower extremity defects and back. Adipofascial perforator plus flaps were employed to provide cover for tibial fracture in one patients and chronic venous ulcer in another.All flaps survived without any loss and provided long-term stable cover, both over soft tissue and bone. Functional preservation was achieved in all cases where muscle flaps were employed with no clinical evidence of loss of power. There was no sensory loss or significant oedema in or distal to the flap in both cases where neurovascular continuity was preserved during flap elevation. Fracture union and consolidation were satisfactory. One patient had minimal graft loss over fascia which required application of stored grafts with subsequent take. No patient required re-operation.Perforator plus concept is holistic and applicable to most flap types in varied situations. It permits the exercise of many locoregional flap options while limiting collateral functional damage. Aesthetic considerations are also addressed while raising adipofascial flaps because of no appreciable donor defects. With quick operating times and low failure risk, these flaps can be a better substitute to traditional flaps and at times even free tissue transfers. The cornerstone of reconstructive work is the provision of wound flap cover which requires a delicate balance between vascularity and reach, while limiting collateral damage. It is desirable to provide wound cover without sacrifice of functional muscles, neurovascular supply, or aesthetics.2Traditional flaps, at times, suffer the disadvantage of flap ischemia and loss over critical areas. Free flaps offset this problem but are technically demanding, time consuming, and equipment intensive. Perforator anatomy and mapping allow diverse locoregional fasciocutaneous flaps albeit with limitations imposed by perforator location and size. The concept of perforator “plus” flaps aims to offset the disadvantages of traditional and perforator flaps while increasing applicability. Traditional fasciocutaneous flaps have vascular supply only from the base whereas true perforator flaps are island flaps based on a single perforator. By marrying the two concepts, perforator “plus” flaps are those which are designed based on a perforator input “plus” additional vascular supply from a retained base. These arThe perforator plus concept was applied to various flap types including adipofascial, muscle, and myocutaneous. In all cases, primary defect closure was obtained without flap failure in any patient. Morbidity and collateral damage were minimized without compromising on end result, wound stability, or fracture union.The defects were assessed clinically and possible traditional flaps in the vicinity were considered based on the availability of regional muscle, adipofascial, or skin. Flaps were chosen based on the principle of minimal morbidity and collateral damage. It was attempted to avoid functioning muscle sacrifice, preserve cutaneous nerves/vessels, and follow esthetic needs. Known perforators within the flap circumference were located by a handheld Doppler. Limb surgeries were performed under tourniquet control without exsanguination. Perforator dissection was performed by the naked eye/loupe using microsurgical instruments from the defect margins till the selected perforators were identified, mobilized, and flap raised. The flap base was always retained and narrowed if required to permit tensionless inset. Detached muscles were reinset after mobilization and achieving wound cover to minimize/avoid functional deficit. Creation of secondary defects was avoided, and immediate grafting of residual defects was done. Subcutaneous vessels and nerves underlying the flap were preserved in continuity while retaining inputs to the flap itself. Suction drains were employed on need basis. No special flap monitoring techniques or drugs were employed in any case.Seven patients underwent perforator plus flaps based on posterior trunk, inguinal region, and lower extremity trauma. The age ranged from 28 to 67 years with a mean of 44 years. Follow-up was on an average of 11 months . Three flaps were myocutaneous perforator plus flaps where the muscle origin/insertion were retained as the additional blood supply along with perforator inputs. One sliding muscle flap employed tibialis anterior (TA) and flexor digitorum longus (FDL), retaining segmental perforators while shifting muscle origins. Two lower extremity perforator fasciocutaneous flaps were raised without sacrifice of saphenous neurovascular bundles passing through their substance. There was no sensory loss distal to the flap and no clinically apparent distal limb oedema. Retained neurovascular inputs to the flap limited their oedema and resulted in sensate flaps. The remaining two cases underwent perforator plus adipofascial flaps with subdermal dissection and no consequent secondary defects. The adipofascial flap peninsular base was retained while the preserved perforator provided the main vascular input. None of the flaps exhibited loss and stable wound cover was achieved in all on follow-up. One adipofascial flap exhibited minor graft loss due to β-hemolytic streptococcus wound infection. Stored graft was applied with subsequent successful graft take. Operative time averaged 130 min . Exposed fracture sites exhibited sound bony union with no evidence of osteomyelitis and none required re-operation. One patient expired 1 month postoperatively due to uncontrolled iliofemoral bleed from metastatic penile carcinoma.2 defect overlying thoracolumbar spine of 6 months duration resulting postexcision and radiotherapy for soft tissue sarcoma. Two attempts elsewhere for wound closure and grafting failed due to the ischemic ulcer. A muscle “fill” and overlying stable sensate skin cover was deemed essential. Lateral paraspinal perforators were marked by Doppler, the medial row of lumbar perforators being sacrificed during tumor extirpation. The ulcer was excised and a V-Y skin flap was raised over the superior segment of Latissmus dorsi. The paravertebral muscle origin was detached and intercostal perforators and nerves dissected intramuscularly. Segmental dissection preserved thoracodorsal vessels and nerve and permitted movement of the entire superior segment of latissmus with the overlying skin paddle into the defect. The muscle origin was resutured across the midline and the skin closed in a V-Y advancement while leaving the humeral insertion intact. The blood supply to the Latissmus was retained both from the thoracodorsal trunk as well as lateral paraspinal perforators. Preservation of intercostals nerves resulted in sensate cover. Healing was uneventful, and the patient noted no discernable deficit of shoulder power or movement. On follow-up at 12 months, the cover remained stable [A 48-year veteran presented with a 15 × 7 cmd stable A 28-year soldier sustained a compound comminuted fracture lower third left leg in a road accident and was referred 7 weeks later with exposed bones at the fracture site. Two posterior tibial perforators were identified and a distally based perforator plus flap planned due to a deep proximal laceration . The ski2 extending from penile base till right ilium. The femoral vessels were exposed and covered with slough with risk of impending rupture. Histopathological examination confirmed metastatic penile carcinoma. Doppler revealed two anterolateral thigh perforators in close proximity. After careful debridement of the ulcer, an anterolateral thigh flap was raised including the tensor fascia lata origin. The proximal perforator was dissected along its pedicle after ligation of the distal one. Adequate perforator length permitted flap rotation by 110° based on vascular supply both from the perforator and muscle origin. The secondary defect was split skin grafted [A 36-year soldier was referred with a progressively enlarging malignant ulcer in the right groin of 25 × 9 cm grafted . The patA 47-year old diabetic suffered multiple fractures right leg due to collapse of a wall under construction. The compound comminuted lower third tibial fracture was stabilized with an external fixator and wound debrided. He was referred 4 weeks after the injury with an exposed fracture site. A fasciocutaneous medial leg V-Y perforator advancement flap was planned based on posterior tibial artery perforator. The flap was elevated on the perforator while preserving the underlying saphenous neurovascular bundle . DissectA 48-year security personnel sustained a fracture of the right patella following a fall. Tension band fixation was done following which he had wound breakdown. He was referred 5 weeks later with a 2″ × 2″ open wound over the knee with exposed patella and hardware. A medial knee perforator was marked and proximally based fasciocutaneous flap elevated. The underlying perforator was safeguarded while preserving and maintaining the continuity of the great saphenous vein, saphenous artery, and nerve . Neurova2 indolent ulcer with pale granulation. The lower medial tibial perforator was localized just above the ulcer. A turnover fascial flap was dissected subdermally from the medial leg skin and based distally. The perforator and distal flap base of the adipofascis were retained. This dual supply flap was inset into the defect and grafted. The elevated skin flaps were primarily closed leaving no secondary defect [A 67-year diabetic presented with a nonhealing venous ulcer over left medial ankle of 8 months duration. He had refused surgery for varicose veins and was on conservative therapy for the same with no deep vein thrombosis. Conservative therapy for the ulcer and split skin grafting elsewhere had been unsuccessful. He had a 5 × 3 cmy defect . Graft t2 raw area in the lower leg with exposure of 5 cm tibia devoid of periosteum. Wound culture was positive for β-hemolytic streptococcus. Two perforators were identified by Doppler along the medial leg. The patient was operated 5 weeks after the injury after a negative culture report. An adipofascial perforator flap was planned and subfascial dissection done along the medial wound edge till the perforators were identified and mobilized. Dissection was then completed till the earmarked flap margins. Subcutaneous dissection separated the medial skin from the underlying fascia. Scissors inserted through stab incisions at flap margins permitted adipofascial flap mobilization without devascularization of the skin. A superiorly based adipofascial transposition flap was raised and inset after ligating the lower perforator [A 37-year soldier fell from a truck and sustained a comminuted fracture of the lower end right tibia. After debridement and skeletal fixation, he was referred 3 weeks after the injury with a 6 × 8 cmrforator . The fasAttention to regional flap applied anatomy has been refocused with the advent of perforator flaps. These fl6Latissmus dorsi is a versatile reliable flap with varied uses. Large spinal defects closure has been variously described by Desprez et al.,[et al.,[et al.[The z et al., Moore et,[et al., and McCr.,[et al. VariatioEssential to lower extremity salvage trauma protocols is adequate and early soft tissue coverage requiring use of vascularized locoregional or free flaps. Exposureen masse is achievable as in case 3. Extensor digitorum longus and peroneus longus with overlying skin were employed by Bloch et al., but they not only divided both the tendons but advocated ligation of several lower perforators making the flaps tenuous.[Muscle flaps have been traditionally favored for providing cover at exposed lower limb fracture sites with a view to providing additional blood supply and tolerance to infection. The gast14 tenuous. We effacThe anterolateral thigh flap has been described as an ideal soft tissue flap by Chen and Tang. Our patiFasciocutaneous flaps are back in favor and studies prove an equal efficacy and long-term results of fracture union compared to muscle flaps. Numerous recent articles have questioned the role of musculocutaneous flaps and advocate fasciocutaneous flaps with good results.1220 Ther1224et al. in their series of fascial feeder and perforator based V-Y advancement flaps advocate preservation of cutaneous veins to prevent congestion since the fascial perforating artery is not accompanied by draining veins in all cases.[Adipofascial flaps have been of limited use in lower limb trauma due to to the inherently imposed limit on their length to base ratio in order to remain viable. This restricted their utility despite favorable outcomes. The knowledge of perforator anatomy has given us the freedom to manipulate adipofascial flap designs. With perforator plus approach, adipofascial flaps are planned over Doppler identified perforator sites. Fascial dissection is done safeguarding the supplying perforators. The peninsular base of the fascia serves as an additional source of arterial input and helps in venous drainage. Niranjanll cases.The present approach of “perforator plus flap” allows fascial flaps to be used in 1:4 or larger ratios with good results. These flaps have numerous advantages over the muscle and fasciocutaneous flaps. There is no sacrifice of any muscle mass with full preservation of function in an already traumatized limb. No skin requires to be moved thus avoiding any additional defect. The flap being pliable can be turned over while perforator dissection permits a wide reach allowing coverage with esthesis.The “perforator plus” approach lends itself to diverse uses employing muscles, skin or adipofascia either alone or as composite tissues. Central to the approach is the preservation of dual vascular inputs via perforators and flap base. Minimal limitations on flap axiality, position of base, length to breadth ratios, or use in the presence of scarring/infection are the result of double blood supply. Functioning muscle and neurovascular preservation while minimizing secondary defects are advantageous outcomes, which are feasible without compromising flap requirements. This versatility allows functional preservation of muscles and nerve while permitting esthesis. Stable wound cover is achievable with minimal collateral damage.


A 42 year male presented with episodic headache and vomiting. Two years earlier he had similar symptoms when a diagnosis of tuberculous meningitis was made after CSF examination. He was treated with antitubercular drugs and a ventriculoperitoneal shunt was placed with good relief. One year later he had recurrence of headache when second scan showed persistent of hydrocephalus . ConsideIn India, both NCC and tuberculosis of central nervous system (CNS) are common endemic problems. Obstructive hydrocephalus by CNS tuberculosis is common, but hydrIt is important to consider that hydrocephalus in the absence of obvious obstruction might not be due to CNS tuberculosis. New imaging such as three-dimensional constructive interference in steady state 3D-CISS) or heavily T2W sequences could improve the diagnosis. In casesD-CISS or


Mesocricetus auratus as an important model for dyslipidaemia because it mirrors many aspects of human disease and pharmacological responses. We show that complementary use of two independent next generation sequencing technologies combined with mapping to multiple genome databases allows unambiguous transcript annotation and quantitative transcript imaging. We refer to this approach as “triple match sequencing” (TMS).Whole transcriptome analyses are an essential tool for understanding disease mechanisms. Approaches based on next-generation sequencing provide fast and affordable data but rely on the availability of annotated genomes. However, there are many areas in biomedical research that require non-standard animal models for which genome information is not available. This includes the Syrian hamster Contigs assembled from a normalized Roche 454 hamster liver library comprising 1.2 million long reads were used to identify 10’800 unique transcripts based on homology to RefSeq database entries from human, mouse, and rat. For mRNA quantification we mapped 82 million SAGE tags (SOLiD) from the same RNA source to the annotated hamster liver transcriptome contigs. We compared the liver transcriptome of hamster with equivalent data from human, rat, minipig, and cynomolgus monkeys to highlight differential gene expression with focus on lipid metabolism. We identify a cluster of five genes functionally related to HDL metabolism that is expressed in human, cynomolgus, minipig, and hamster but lacking in rat as a non-responder species for lipid lowering drugs.The TMS approach is suited for fast and inexpensive transcript profiling in cells or tissues of species where a fully annotated genome is not available. The continuously growing number of well annotated reference genomes will further empower reliable transcript identification and thereby raise the utility of the method for any species of interest. Mesocricetus auratus has become an important animal model for pre-clinical dyslipidaemia research because HDL-c levels are raised in response to drugs designed for human target proteins. M. fascicularis liver expression data came from a previous study[http://www.reactome.org/), because their functional interactions are well documented. Hierarchical clustering generated six distinct clusters of genes with similar gene expression patterns across all species  are missing in the draft genomes of the pig[M.auratus AMN receptor gene (cluster 5) was not present in our long read library.The primary motivation for our study was to compare liver gene gene expression in hamster with humans and model organisms used in metabolic disease research. For this study we constructed SAGE-based liver RNA libraries from us study. The gen the pig or the mWe show that triple match sequencing (TMS) allows robust deep sequencing based transcript imaging in absence of an annotated genome. Using a SAGE based comparison of liver gene expression across five species we show that TMS derived data are comparable to mRNA quantification data using conventional genome based mapping. Similar to other vertebrates about 10’800 genes are expressed in hamster liver at comparable levels  an animal model for experimental hepatitis B infection and hepatocellular carcinoma[http://www.ensembl.info/blog/2011/05/24/human-bodymap-2-0-data-from-illumina). TMS does not attempt to annotate species-specific transcripts because the approach relies on mapping to known mRNAs in other species. We note that any unmapped long reads are candidates for species specific transcripts of hamster. Normally, unique 454- reads or contigs matched the same RefSeq transcript from more than one species  had only limited value for our project[C. griseus focused mainly on pathways relevant for protein production and modification. Furthermore, the evolutionary distance between C. griseus and M.auratus is comparable to that between mouse and rat[As it turned out, the genome sequence or the partial transcriptome analysis of the  project,15, beca and rat. FinallyTriple match sequencing is a robust and rapid method for annotation and quantification of transcripts in the absence of a reference genome. The method is especially useful in projects where reliable mRNA quantification rather than primary sequence information is important. TMS does not require implementation of specialized hard- or software which makes it fast, affordable and user friendly. It might even contribute to the identification and selection of novel animal models prior to a genome sequencing effort.Male Syrian hamsters, weighing 125 ± 5 g were obtained from Charles River Germany (Sulzfeld). The animals were kept at standard housing conditions . They were supplied with standard laboratory chow and water ad libitum, and left to acclimatize for 1 week before the experiments. The harvested liver was cut in small pieces (approximately 3 × 3 mm) and snap frozen immediately for further processing. All experimental procedures were carried out in accordance with international guidelines for care and use of laboratory animals.http://www.ellegaard.com). Normal human liver RNA was provided by the Human Tissue and Cell Research foundation (project-nr: 2012–13 approved 06/25/2012).RNA was isolated from Syrian hamster liver samples using RNeasy Mini Kit from Qiagen (No.74104). RNA integrity was analyzed using an Agilent Bioanalyzer (RNA 6000 Nano Kit) and all samples reached a quality score (RIN) above 9.7. Total Wistar rat liver RNA was a gift from Dr T. Heckel and snap-frozen minipig liver tissue was a gift from Dr Niels-Christian Ganderup (The Applied Biosystems SOLID 3 System SAGE kit (No.4443756AB) was used for library construction. Presence of the expected 100-base pair SAGE templates was confirmed using an Agilent Bioanalyzer (High Sensitivity DNA Assay). The emulsion PCR (ePCR) library was constructed using DNA at a concentration 0.75 pM. The ePCR was carried out on a SOLID EZ Bead amplifier and the enriched beads were quantitated using a NanoDrop 2000 spectrophotometer. Sequencing was performed on a SOLiD4 instrument with DOLF-TOP sequencing chemistry Frag-Lib F3 Tag MM35 (No. 445352).′-kit primer is replaced by polTdeg (5′-AAG CAG TGG TAT CAA CGC AGA GTA CTT TTG TTT TTT TTT CTT TTT TTT TTV N -3′) The kit PCR primer M1 is substituted by polTM1 (5′-AAG CAG TGG TAT CAA CGC AGA GTA CGG-3′). The product of the full-size preparation of ds cDNA is used for normalization with the TRIMMER kit using 700-1300 ng of purified cDNA. The normalization is performed according to the supplied protocol without any changes. emPCR was carried out with 5cpb input on SOLID EZ Bead Amplifier. Typically a GS FLX+ Sequencing Run generated about 800’000 reads with an average length of 410 bases.To normalize the levels of cDNA prior to library construction we used a competitive hybridization protocol provided with commercial kits . For 454 sequencing the use of special cDNA primers is required. The 3de novo using the software Trinity with the option “inchworm” as assembly method (release 2012-01-25)[bona fide transcripts. Re-aligning the raw reads to the set of transcripts with Roche-454’s Newbler software (version 2.7) yielded a mapping efficiency of 97%.Following depletion of ribosomal sequences, 454-sequencing reads were assembled 2-01-25). Unassem′-UTRs  with the tag length set to 22 bases and a maximum of two mismatches allowed. The read frequencies per transcript for all species included in this study are available in Additional file.10 with SAGE: Serial analysis of gene expression; CDS: Protein coding sequence; NGS: Next generation sequencing; emPCR: Emulsion polymerase chain reaction; qPCR: Quantitative polymerase chain reactionThe authors declare that they have no competing interests.RS and MB carried out deep sequencing data processing and transcript analysis and quantification. EK performed library construction and sample normalization. SG provided liver samples from matched hamsters. SL and WT selected a well annotated human liver sample and provided high-quality total RNA. ME designed the workflow and experimental strategy. UC lead the project and drafted the manuscript. All authors have read and approved the final version of this manuscript.Read frequencies of transcripts detected in human, cynomolgus, minipig hamster and rat liver samples. Given are the absolute read frequencies without normalization for the species indicated using standard gene symbols.Click here for file


The authors wish to add the following funding source to the financial disclosure information: "E.C. is recipient of the "Teresa Ariaudo Fellowship" from Istituto Pasteur-Fondazione Cenci Bolognetti".


Crystalloids have different electrolyte composition and therefore different strong ion differences (SIDinf). The aim of the study was to investigate the response of the kidney to plasmatic acid-base changes induced by dilution with three crystalloid solutions at different SID.2 constant. A urinary catheter was placed and connected to a urinary analyzer  [Six pigs (22 ± 4 kg) were anesthetized and mechanically ventilated. The respiratory rate was adjusted to maintain pCO, Italy) . Pigs wein vitro studies [Plasmatic changes are consistent with previous  studies . dSID waThe quality of infused fluids affects greatly the acid-base and electrolyte equilibrium of plasma. This in turn alters the quality of urine (pHu).


Unipolar brush cells (UBCs) are excitatory glutamatergic interneurons of the cerebellar granular layer receiving both primary and secondary vestibular inputs through mossy fibers (excitatory input) and Golgi axon (inhibitory input). The brush like structure of the dendrite allows to form a giant synapses in the glomerulus and to produce an all or none post synaptic response with short delay and protracted kinetics. The excitable response of UBCs can be either a tonic discharge or a high-frequency burst of action potentials. When injected with progressively increasing depolarizing currents from a negative membrane potential, the UBC generates a burst sustained by a calcium spike Figure , +20pA aHere we present a biologically realistic multi-compartmental mathematical model of the UBC realized with the NEURON simulator. According to literature -4, ionic


Pulmonary pulse wave velocity has proven to be an excellent indicator of pulmonary vascular wall stiffness. In this study a novel MRI method was evaluated, showing rapid assessment of pulmonary pulse wave velocity with excellent repeatability and observer reproducibility.Pulmonary pulse wave velocity has proven to be an excellent indicator of pulmonary vascular wall stiffness. The main objective of this study was to evaluate a novel magnetic resonance imaging (MRI) method to rapidly assess pulmonary pulse wave velocity (PWV) and to test repeatability and observer reproducibility of the proposed technique.In 15 healthy volunteers  pulmonary PWV was prospectively assessed between the proximal main pulmonary artery (MPA) and just before the first branching of the distal left pulmonary artery (LPA). One-directional through-plane velocity-encoded (VE) MRI using breath holds was used to assess the pulmonary flow at the levels of MPA and distal LPA with a temporal resolution of 15 ms. Pulmonary PWV was calculated by dividing the pulmonary path length between the measurement sites by the transit time between the arrival of the systolic wave front at these sites. The volunteers were studied twice to test reproducibility in PWV-assessment.Results are expressed as mean±SD, Pearson correlation coefficient (PCC) and intraclass correlation (ICC). Acquisition time of the MRI examination was 2±1 minutes and image analysis lasted for 2±1 minutes. Both observers showed good agreement for pulmonary distance  and pulmonary PWV . Reproducibility of pulmonary PWV was high for both observers .Rapid non-invasive VE MRI assessment of pulmonary pulse wave velocity shows low interobserver and intraobserver variability and can be determined with high reproducibility.No disclosures.


Many natural and engineered biofilm systems periodically face disturbances. Here we present how the recovery time of a biofilm between disturbances (expressed as disturbance frequency) shapes the development of morphology and community structure in a multi-species biofilm at the landscape scale. It was hypothesized that a high disturbance frequency favors the development of a stable adapted biofilm system while a low disturbance frequency promotes a dynamic biofilm response. Biofilms were grown in laboratory-scale reactors over a period of 55-70 days and exposed to the biocide monochloramine at two frequencies: daily or weekly pulse injections. One untreated reactor served as control. Biofilm morphology and community structure were followed on comparably large biofilm areas at the landscape scale using automated image analysis  and community fingerprinting (single-strand conformation polymorphisms). We demonstrated that a weekly disturbed biofilm developed a resilient morphology and community structure. Immediately after the disturbance, the biofilm simplified but recovered its initial complex morphology and community structure between two biocide pulses. In the daily treated reactor, one organism largely dominated a morphologically simple and stable biofilm. Disturbances primarily affected the abundance distribution of already present bacterial taxa but did not promote growth of previously undetected organisms. Our work indicates that disturbances can be used as lever to engineer biofilms by maintaining a biofilm between two developmental states. Pseudomonas biofilm  using low-magnification microscopy and community fingerprinting revealed the dynamic development of mixed-culture biofilm systems over several weeks.•Weekly disturbed biofilms showed a resilient morphology and community at the time-scale of the disturbance frequency. A week-long recovery was sufficient to recover the initial biofilm morphology and community structure before recurring disturbances. Even after several weeks, biofilm responses to monochloramine were comparable and no adaptation became apparent. •Aquabacterium dominated the microbial community. Preferential growth of this bacterium after disturbance events may serve as explanation for the observed community dynamics.At high disturbance frequencies, biofilm morphology and community composition continued to simplify. A seemingly resistant bacterium of the genus •Within days, biofilm systems at both disturbance frequencies redeveloped a complex structure when disturbances were stopped.•Monochloramine pulse injections could be used as lever to engineer morphology and community structure in biofilm ecosystems.Figure S1A time-series of top view images of biofilms. Images are acquired with opaque illumination before and after the second (days 27 - 31) and third (days 34 - 38) monochloramine pulse injection in the weekly treated reactor of the first experiment  and four days after the treatment (images to the right); least complex morphologies are observed on images immediately following the treatment. The contrast in the images was globally enhanced in order to improve the display on screen and in print. periment . Darker (EPS)Click here for additional data file.Figure S2Six descriptors with experimental time for the three treatment scenarios in the first experiment . The data were derived from images that were analyzed using the Spatial Gray Level Dependence Matrix (SGLDM) approach. Treatment scenarios are color-coded in each panel. Color-coded arrows indicate monochloramine pulse injections in the treated reactors. Panels A, D and E show results for images taken with transmitted illumination; panels B, D and F show results for images taken with oblique illumination. Mean gray levels from the images can be extracted by transforming the descriptor f6 (see r f6 see : (f6/2)-(EPS)Click here for additional data file.Figure S3A time-series of CE-SSCP profiles during the period of weekly treatment of the first experiment. Arrows indicate the weekly pulse injections of monochloramine. Experimental time in days is given for each profile. A simplification of the profiles is noticeable after each pulse injection. Over the days following a disturbance, profiles diversify once more. (EPS)Click here for additional data file.Figure S4Correlation of PCoA 1 scores with the respective –log Simpson diversity indices for both experiments.(EPS)Click here for additional data file.Figure S5Proof on concept for image analysis method. Correlation between average gray levels from transmitted illumination images and gravimetrical measurements of biofilm biomass as total solids at the same sampling dates. Data from the first experiment described in this publication and an additional control experiment under identical conditions were presented in the figure. Error bars in x-direction indicated the standard deviations of 14 image repetitions. Error bars in y-direction indicated standard deviations of repetitions for total solids measurements that were done in three cases. The linear regression is flanked by 95% confidence intervals. (EPS)Click here for additional data file.Figure S6Morphology and community development with experimental time for the three reactors in the second experiment. Treatment periods were shaded where appropriate (panels C-F). Each monochloramine pulse injection was indicated by an arrow. Vertical gray lines mark Mondays. By performing Principal Component and Principal Coordinate Analyses, most of the variability in the data sets could be summarized in the first Principal Component (PC1) for morphology data and the first Principal Co-ordinate Axis (PCoA1) for the community data. More negative values on PC1 (note inverted axis labels) were related to more complex biofilm morphology. Similarly, the higher the value on PCoA1, the more complex was the community structure. Note the inverted axis in panels (EPS)Click here for additional data file.Figure S7Linear regressions between morphology data PC1 in  and comm Only filled points in panels B and C were considered for the regressions as these data were sampled during the treatment periods. (EPS)Click here for additional data file.Figure S8Development of bacterial diversity over time. We used the – log Simpson diversity index for individual molecular fingerprints and the Raup-Crick index to compare the composition (presence-absence of peaks) in a moving window of two subsequent samples from the same reactors. The solid lines with symbols represent – log Simpson diversity. The height of bars indicates the value of the Raup-Crick index for the comparison of the two samples at the left and right edge of the bars. Bar width stands for time between two samples and may vary. Raup-Crick values smaller than -0.95 indicate significantly more similar community compositions than expected from a random draw. Values around 0 indicate that observed differences could be explained by random effects while values greater than 0.95 indicate significant dissimilarities in composition. Shaded areas and arrows show treatment periods and monochloramine pulse injections. Dashed lines are the -0.95 and 0 levels for the Raup-Crick index. (EPS)Click here for additional data file.Figure S9CE-SSCP profiles of majority clones (profiles A-F) and low abundance clones (A'-C'). At the top of the figure, reference profiles for biofilm samples from the first experiment are shown. DNA from these samples was taken for cloning.(EPS)Click here for additional data file.File S1Integrates the supplemental figures in a text document.(PDF)Click here for additional data file.