Poribacteria is one of the most dominant and widespread members of the microbial communities residing within marine sponges. Cell compartmentalization had been postulated along with their discovery about a decade ago and their phylogenetic association to the Planctomycetes, Verrucomicrobia, Chlamydiae superphylum was proposed soon thereafter. In the present study we revised these features based on genomic data obtained from six poribacterial single cells. We propose that Poribacteria form a distinct monophyletic phylum contiguous to the PVC superphylum together with other candidate phyla. Our genomic analyses supported the possibility of cell compartmentalization in form of bacterial microcompartments. Further analyses of eukaryote-like protein domains stressed the importance of such proteins with features including tetratricopeptide repeats, leucin rich repeats as well as low density lipoproteins receptor repeats, the latter of which are reported here for the first time from a sponge symbiont. Finally, examining the most abundant protein domain family on poribacterial genomes revealed diverse phyH family proteins, some of which may be related to dissolved organic posphorus uptake.The candidate phylum  Poribacteria, symbionts of marine sponges, based on six single-amplified genome (SAG) sequences Poribacteria. Poribacteria were first discovered as highly abundant symbionts of marine sponges Poribacteria are one of the most predominant taxa inhabiting the extracellular matrix (mesohyl) of sponge species around the world Poribacteria showed a moderate phylogenetic relationship to Planctomycetes, Verrucomicrobia, and Chlamydiae (PVC superphylum) based on monophyletic clustering in 16S rRNA gene analysis Poribacteria were classified as members of the PVC superphylum although the exact position within the superphylum could not be completely resolved Poribacteria were also suspected to have a compartmentalized cell plan Poribacteria. We further reveal a large abundance and diversity of eukaryote-like domain containing proteins as well as phyH-like proteins in Poribacteria.Single-cell genomics is a powerful tool to describe genomes of as yet uncultivated organisms from diverse environments Candidatus Poribacteria WGA 3A, 3G, 4C, 4CII, 4E and 4G with Genbank accession numbers ADFK02000000, ASZN01000000, APGO01000000, ASZM01000000, AQTV01000000, AQPC01000000, respectively. These genomes were previously obtained by our group from uncultivated bacteria inhabiting the marine sponge Aplysina aerophoba by fluorescence activated cell sorting (FACS), multiple displacement amplification (MDA), and next generation sequencing Six poribacterial single-cell genome sequences were included in this study, these being Please also note that the initial version of genome WGA 3A (first published as WGA A3 with accession number ADFK00000000 version ADFK01000000) For clustering of pfam 05721-PhyH family proteins we used the fastclust algorithm in usearch Aerophobetes (CD12) and Hydrogenedentes (NKB19) were obtained by blast searches Sequences for 16S rRNA gene based phylogenetic analysis were selected from the SILVA 16S rRNA database version 108 Poribacteria were translated into all six reading frames and marker genes were detected and aligned with hmmsearch and hmmalign included in the HMMER3 package http://phylosift.wordpress.com/). Extracted marker protein sequences were used to build concatenated alignments of up 83 markers per genome. Alignments were included into the database constructed by Rinke and coworkers For the calculation of the bacterial phylogenetic tree we followed the procedure described by Rinke et al. Poribacteria SAGs clustered with the recently proposed phyla Aerophobetes (CD12) and/or Hydrogenedentes (NKB19) Elusimicrobia, formed in some tree calculations a sister clade to the PVC superphylum (Poribacteria with strong bootstrap support (Aerophobetes (CD12) and/or Hydrogenedentes (NKB19) (Poribacteria were placed (bootstrap support 91%) separately within a larger cluster of other phyla including the PVC superphylum as well as the candidate phylum WS3, recently renamed as LatescibacteriaAnalysis of phylogenetic interferences of up to 83 marker genes  showed that all poribacterial SAGs clustered, with 100% bootstrap support in all our tree calculations, in a monophyletic group distinct to the PVC superphylum , Fig. 1. support . However (NKB19) . InsteadPoribacteria in the tree of life will be further refined as more genome sequences of Poribacteria and of other candidate phyla become available. Importantly, the phylogenetic analyses performed in this study (whether 16S rRNA gene or marker genes based) did not support a clustering of Poribacteria with the PVC superphylum, which is in contrast to what was suggested earlier The inconsistency between phylogenomic and the 16S rRNA gene-based phylogeny might be due to the relatively low resolution provided by the single marker gene (16S rRNA) analysis compared to multiple genes analysis as has been suggested previously Poribacteria outside the PVC superphylum. However, these studies included only one poribacterial genome sequence available at that time, Candidatus Poribacteria sp. WGA A3 in its initial version (ADFK01000000). This version was later shown to be flawed by contaminating DNA and was replaced in Genbank (ADFK02000000) Poribacteria. Besides phylogenetic analysis, two marker proteins were described for members of the PVC superphylum Poribacteria.Previous studies based on concatenated alignments of protein data Poribacteria based on ring shaped fluorescence in situ hybridization (FISH) signals and the electron microscopic observations of compartmentalized prokaryotic cells in the mesohyl of the sponge Aplysina aerophobaPlanctomycetesGemmata obscuriglobus where the compartment was proposed to be a nucleus-like structure Poribacteria, we searched poribacterial SAGs for possible genomic evidence of such features. We were not able to find membrane coat like proteins or any genomic indication of large cell compartments. This is in accordance with a recent study which challenged the concept of the existence of these compartments even in other bacteria and confutes the existence of a nucleus-like structure in G. obscuriglobusCell compartmentalization is one characteristic that has been proposed for Poribacteria. Four of six poribacterial SAGs encoded for genes with hits to either one of two pfam domains namely, pfam00936 BMC or pfam03319 EutN CcmL , and some additional phyla  in utN CcmL . These dutN CcmL  that encutN CcmL . A detaiutN CcmL . This isutN CcmL . Poribacal phyla , appear The so far best described BMC functions are the carboxysome and BMCs containing enzymes for ethanolamine or propanediol utilization. Bioinformatic analysis of all available BMC shell protein encoding genomic regions at the time by Yeates et al. Poribacteria where the identified genomic regions with BMC shell protein genes (group A-C) appear scattered across the genome. For example, the different BMC shell protein genes (with pfam00936 and pfam03319) are generally in different genomic regions on poribacterial genomes and not encoded together within one region, as it is the case for many so far functionally characterized BMC types Poribacteria.It is suspected that novel BMC functions will be revealed in the future Eukaryote-like repeat domain containing proteins have received much recent attention in sponge microbiology and their involvement in mediating host-microbe interactions has been postulated. Especially ankyrin (ANK) and tetratricopeptide repeats (TPR) have been in focus of such investigations Vibrio, Colwellia, Bradyrhizobium and Shewanella. Pfam domain 07593- ASPIC and UNbV was also present on several poribacterial SAGs in multiple copies. ANK repeat domains were detected  in lower numbers on a total of 14 genes on SAGs 3G, 4C and 4E  but in lower abundances. We were also able to find Sel1 repeat like proteins domains encoded on poribacterial SAGs 3G and 4E  which have a similar structure to TPRs C and 4E . The freC and 4E .Poribacteria this domain has only been found in proteins of 14 bacterial genomes but not in archaeal genomes publically available at the IMG/MER database in July 2013. Most of these bacterial hits however do not show the tandem repeats that are characteristic for this domain in eukaryotes. Such tandem repeats were only detected in the poribacterial proteins and proteins of four other bacterial genomes. Amongst these were free-living marine cyanobacteria , the marine deep sea piezophile Mortiella sp. PE36, and the strictly anaerobic bacterium Paludibacter propionicigenes WB4, DSM 17365. The LDL receptor is best described in mammals where they transport ligands into the cell for degradation by lysosomes and plays a role in cholesterol homeostasis The occurrence of low-density lipoprotein (LDL) receptor repeat class B domains (pfam00058) on poribacterial genomes seemed noteworthy. We found these domains on one gene in each SAG 4C and 4E as well as on five genes in SAG 3G. Outside of Poribacteria since all of the discovered genes with these domains had predicted transmembrane helices (TMHs) (∼86%) with the majority of the protein located outside of the cell or signal peptides (SPs) (∼14%). TMHs and SPs were also frequently predicted on genes representing other eukaryote-like proteins of Poribacteria . These proteins are Fe(II) and 2-oxoglutarate dependent oxygenases that catalyze a wide range of oxidative reactions Among bacterial phyH genes are some potentially involved in quorum sensing Poribacteria.Among poribacterial genomes we found a remarkably high occurrence of genes encoding for proteins with pfam domain pfam05721-phyH . This pfPoribacteria-specific functions within the phyH family.A clustering analysis of poribacterial sequences showed that there was large diversity amongst poribacterial phyH family genes with 305 sequences clustering in 193 clusters with 60% aa id threshold . For thePoribacteria and elucidated one possible function of phyH superfamily genes in this candidate phylum.Although the majority of poribacterial phyH genes remained without further functional characterization, we were still able to make functional predictions in some cases. Poribacterial SAGs 3G and 4E encoded for phyH genes  with homologies (40% aa id each) to a 2-aminoethylphosphonate (2-AEPn) utilization gene (phnY) for which function was experimentally proven Poribacteria which are almost exclusively found in association with marine sponges. Here we show by use of phylogenetic and phylogenomic analyses that Poribacteria are not members of the PVC superphylum, but rather form a distinct monophyletic phylum in close proximity. We provide genomic evidence for bacterial microcompartments in Poribacteria that show no similarity to any previously described BMCs. Further novel functions might be hidden in the various eukaryote-like protein domains, which may be involved in mediating host-microbe interactions within the sponge holobiont. The high abundance of diverse phyH-domain containing proteins points to important and potentially specific functions in Poribacteria. Most of these functions remain to be revealed in future studies but some show the genomic potential for organic phosphorous utilization. Our analyses show how genome sequences can help to revisit past hypotheses and at the same time open the way for new investigations by revealing novel functional features. Challenges for the future will be to experimentally demonstrate function and to ultimately understand the implications for symbiosis.Our study demonstrates the power of single-cell genomics to reveal novel features of the candidate phylum Table S183 marker genes used for phylogenetic analysis.(PDF)Click here for additional data file.Table S2BMC group A genes with annotation.(PDF)Click here for additional data file.Table S3BMC group B genes with annotation.(PDF)Click here for additional data file.Table S4BMC group C genes with annotations.(PDF)Click here for additional data file.Table S5BMC group D genes with annotation.(PDF)Click here for additional data file.Table S6BMC shell protein pfam domain distribution on all genomes with either domain in IMG in July 2013.(PDF)Click here for additional data file.Table S7Overview of gene copy numbers (no) and percentage of genes per total genes on genome (%) of repeat proteins and eukaryote like protein domain genes on poribacterial SAGs and the maximum and average number of gene copies found on X finished genomes of marine free-living bacteria (n = 101).(PDF)Click here for additional data file.Table S8List of all repeat and eukaryote like domain protein encoding genes on poribacterial SAGs. Information is shown as available in IMG/MER system. THM: number of predicted transmembrane helicies. SP: signal peptide predicted yes (Y) or no (N).(PDF)Click here for additional data file.Table S9List of total repeat and eukaryote like protein domain encoding genes of poribacterial SAGs showing the number of genes (# genes), the number of genes with transmemebrane helicies (# TMH), percentage of genes with transmembrane helicies (% TMH), number of genes with signal peptide (# SP), and percentage of genes with signal peptide (% SP) for each domain.(PDF)Click here for additional data file.Table S10phyH domain distribution on publically available genomes.(PDF)Click here for additional data file.Table S11Poribacterial phyH gene clusters based on 60% amino acid identity.(PDF)Click here for additional data file.Text S1Genomic evidence for microcompartments in Poribacteria. (extended description of genomic architecture)(PDF)Click here for additional data file.


We are able to detect subclinical post-irradiation changes to the heart with left atrial late gadolinium enhanced magnetic resonance imaging (LGE-MRI).There are over 11 million cancer survivors, yet many long-term cancer survivors experience lasting changes including cardiac functional and anatomical abnormalities following external beam radiation and chemotherapy. One of the well-established pathological and histological sequelae of radiation exposure to the heart is intense post-irradiation injury with fibrotic changes. Here, we have explored the ability to apply LGE to identify the extent of left atrial uptake after radiation therapy.A total of 20 patients were enrolled in this study; 10 patients (ages 29 to 64 years) who had survived 9.2 ± 25 years after thoracic external beam radiation for lymphoma and 10 control patients (ages 55 to 66 years) recruited from the University colonoscopy center. All patients underwent a MRI study including cine imaging, left ventricular LGE, and high-resolution LGE imaging of the left atrium. The extent of late gadolinium enhancement was calculated as a relative percent of total left atrial wall using a threshold-based algorithm based on pixel intensity distribution.As seen in Figures Patients with thoracic radiation for lymphoma have greater LGE reflecting radiation-induced injury than in an older group of controls without thoracic radiation therapy. LGE-MRI shows promise for finding and screening for prevalent myocardial tissue changes in this group of patients. Further studies with larger patient populations post thoracic radiation therapy with and without chemotherapy  and with longitudinal follow up would be useful for correlation with external beam radiation dosimetry and the development of future cardiovascular events in long-term cancer survivors.University of Utah Seed Grant.


One third of all spinal injuries involve cervical vertebrae, and the impact of injury to the cervical spinal cord is profound and requires systemic treatment. The role and timing of surgical decompression after an acute spinal cord injury (SCI) remains one of the most controversial topics pertaining to spinal surgery. Lack of controlled, prospective, multicenter clinical studies has contributed to confusion in optimal treatment methods for patients with injuries of the cervical spinal cord. Clinically, the question of whether surgical decompression improves motor recovery following SCI remains surrounded by controversy.Two cases with a 32-year-old man who developed right -sided Brown-Séquard Syndrome Following a motor to car accident with the large right paramedian C3–C4 disc herniation, and ipsilateral spinal cord compression and a 30-year-old man with the syndrome of acute central cervical spinal cord injury with motor impairment involving only upper extremities due to central C2–C3 disc herniation following a pedestrian accident are reported. Discectomy and anterior cervical fusion with the polyetheretherketone (PEEK) cage were performed. A complete motor deficit recovery and a marked sensitive deficit improvement were obtained.The need for and timing of surgical decompression in post traumatic spinal cord injury is controversial. Surgery may expedite neurological recovery in some patients and may provide additional neurological recovery when clinical improvement has plateaued or worsened. In our patient a complete motor deficit recovery was observed. Cervical, Disc herniation, Spinal cord injury


Arterial stiffness had been proposed as the cause of sub-clinical left ventricular dysfunction in patients with COPD. Vascular studies in COPD patients were mainly assessed by carotid-femoral (aortic) pulse wave velocity using micromanometer applanation tonometry. The evaluation of pulmonary artery (PA) elasticity in COPD has not been assessed. This study seeks to evaluate indices of PA stiffness in patients with COPD. We hypothesized that patients with COPD would have increased pulmonary artery stiffness compared to healthy individuals. To test this we determine the pulmonary artery area change (distensibility or pulsatility in %) by cardiac magnetic resonance imaging (MRI) and related PA distensibility to the severity of COPD.100%x(maximum area-minimum area)/minimum area.MRI studies were performed using a 1.5T scanner  on 71 participants recruited to date into an ongoing nested case-control study of current and former smokers, age 58-79 years, free of clinical cardiovascular disease. COPD presence and severity was defined on post-bronchodilator spirometry by GOLD criteria . To measure left ventricular function, the heart was imaged with MRI in short-axis orientation using a retrospectively gated steady-state free precession sequence. Phase-contrast images perpendicular to the pulmonary trunk were obtained using a segmented fast gradient echo sequence  without breath hold. Pulsatility of the pulmonary vessels (in %) was measured from the largest systolic and the smallest diastolic areas derived from the magnitude images as: Participants with COPD had increased PA stiffness compared to normals as shown in Table Patients with COPD may have increased pulmonary artery stiffness measured by MRI PC imaging. Ongoing recruitment into this study is may confirm these preliminary findings.


A discrete transition of wall stiffness at the interface of a compliant aorta and stiffer carotid arteries creates a reflection site for the aortic pulse wave and consequently limits excessive pulsatile energy transmission towards the brain. Arterial wall stiffness can be expressed by the pulse wave velocity (PWV) and dual-slice one-directional through-plane velocity-encoded (VE) MRI is well-validated for accurate PWV-assessment. The hypothesis is that with aging, leveling of PWV across the aortic arch and carotid artery will occur with negative effect on the flow towards the brain. The purpose of this study was to evaluate PWV and volume flow in aorta and carotid artery in younger and older volunteers using 3T VE MRI.Sixteen younger volunteers with age<30 years (mean 25±3years) and sixteen older volunteers with age>45 years (mean 56±6years) underwent 3T MRI (Philips) to assess PWV in the aortic arch and the left carotid artery based on the transit-time method. PWV in the aortic arch was acquired from a single VE MRI acquisition Figure  and fromAll measurements are presented in Table PWV leveling between the aortic arch and the left carotid artery at older age represents increased stiffening of the aortic arch and is associated with a reduction in volume flow towards the brain. High-field velocity-encoded MRI is well-suited for PWV evaluation across vascular trajectories.This study is supported by an unconditional grant provided by Dr. Rainer Bluemm .


Silene latifolia with the aim of studying gene expression differences between the two sexes in both reproductive and vegetative tissues. The catalogue contains more than 15 reference genes with both stable expression intensities and a range of expression intensities in flower buds and leaf tissues. These reference genes were used to normalize expression differences between reproductive and vegetative tissues in eight candidate genes with sex-biased expression. Our results suggest a trend towards a reduced sex-bias in sex-linked gene expression in vegetative tissues. In this study, we report on the systematic identification and validation of internal reference genes for adequate normalization of qRT-PCR-based analyses of gene expression differences between the two sexes in S. latifolia. We also show how RNA-seq data can be used efficiently to identify suitable reference genes in a wide diversity of species.Quantitative real time (qRT)-PCR is a precise and efficient method for studying gene expression changes between two states of interest, and is frequently used for validating interesting gene expression patterns in candidate genes initially identified in genome-wide expression analyses, such as RNA-seq experiments. For an adequate normalisation of qRT-PCR data, it is essential to have reference genes available whose expression intensities are constant among the different states of interest. In this study we present and validate a catalogue of traditional and newly identified reference genes that were selected from RNA-seq data from multiple individuals from the dioecious plant  RNA-seq, a transcriptome-based next-generation sequencing technique, has revolutionised genome-wide gene expression analysis and has massively increased the number of studied genes and species. In comparison to microarrays for example, RNA-seq has two major strengths For accurate expression estimation of target genes, expression estimates are normalised relative to internally expressed reference genes. These must be equally expressed among the states of interest for normalization to reduce non-specific variation. Such variation may be the result of different amounts of template RNA or different efficiencies in cDNA synthesis, for example. In addition, suitable reference genes should be expressed at intensities similar to those of the target genes. Typically, two to four references genes are recommended for adequate qRT-PCR data normalization de novo reference transcriptomes Reference genes in species with uncharacterized genomes have been identified using microarray data www.leonxie.com/referencegene.php?type=reference) takes into consideration results from all algorithms to arrive at a robust stability estimate and thus further facilitates the identification of suitable reference genes.Several different algorithms are available for assessing the stability of reference genes using qRT-PCR, such as geNorm Silene latifolia Poiret), a dioecious plant with evolutionarily young sex chromosomes, has become a model system for studies of plant sex chromosomes and the evolution of separate sexes S. latifolia plants.Novel reference genes have recently been characterized in several plant species including model de novo reference transcriptome) in male and female flower buds of S. latifolia. The qRT-PCR expression stability of these newly developed reference genes was then compared to traditional reference genes in flower buds and leaf tissues. Finally, we used the best reference genes to estimate the percentages of sex-biased genes in reproductive and vegetative tissues of S. latifolia by comparing a set of eight randomly chosen sex-linked genes with sex-biased expression patterns In the present study, we used RNA-seq data from multiple male and female individuals to identify the most stably expressed genes  Arabidopsis thaliana entries of the Uniprot database actin 11 (SL_ACTIN), putative ubiquitin-conjugating enzyme E2 21 (SL_UBCE), tubulin beta-5 chain (SL_TUB), eukaryotic translation initiation factor (SL_EF1), ubiquitin 11 (SL_UBQ), and glyceraldehyde-3-phosphate dehydrogenase (SL_GAPDH).Candidate reference gene sequences were annotated using the t) values exceeded 35. In cases of signals in NTC, the difference between the Ct of NTC and that of the template had to be ≥8 Ct values. Primer assays that produced ≥10% missing data were excluded. The remaining gene assays were ranked according to their expression stability between males and females in flower buds and leaves . We used the web tool RefFinder to assign robust ranks based on the results from the four widely used stability estimators geNorm, Δct, NormFinder, and BestKeeper. We used all data, including the technical replica, to calculate stabilities. Results from one leaf sample were consistently identified as outliers and were therefore removed. Consequently, we randomly removed one leaf sample of the other sex to maintain a balanced design. qRT-PCR efficiencies were calculated across all samples and tissues using LinReg Four flower buds (5–6 mm) without the calyx and one disc (1 cm diameter) of a young, fully developed rosette leaf were collected from three males and three females of an 11-generation inbred line (U11); similarly, four males and four females were collected from the C1 cross. All samples were snap-frozen in liquid nitrogen directly after sampling. Total RNA was extracted from both tissue types separately using the RNeasy Plant extraction kit  with a BeadRuptor . RNA was treated in tube (see manufactory's instruction) with a RNase-Free DNase Set  and purified over a second column because we observed that on-column digestions were not sufficient to eliminate genomic DNA contaminations. RNA was checked for genomic DNA contamination by amplifying an intron-spanning PCR product whose fragment size was checked on agarose gels. The integrity of the RNA was assessed on an Agilent 2100 Bioanalyzer Plant RNA chip (RIN ≥7.5 for leaves and ≥9 for flower buds) and RNA concentrations were determined using a Qubit Fluorometer . 0.5 μg total RNA was then reverse transcribed to cDNA according to the standard protocol using the QuantiTect Reverse Transcription Kit  including the genomic DNA Wipeout buffer. qRT-PCR reactions were performed in triplicates on a 7500 Fast Real Time PCR system  using SYBR FAST Universal 2X qPCR mix with low ROX concentration  in a total volume of 11 μl. To assess the quality of our primer assays, we tested for dimer formation and the absence of amplification products in NTCs (no template controls), and Sanger sequenced  all amplified products to confirm amplification of target transcripts. Transcripts were considered undetermined if their cycle threshold (Ct) and were therefore not used further in the statistical analysis. Outliers were removed (<25th or >75th percentile) and fold changes and mean standard errors over the 5-7 biological replicates per sex were calculated using R. We considered contigs to be significantly differentially expressed between males and females if fold changes were ≥1.5 and Wilcoxon tests remained significant after performing Benjamini-Hochberg corrections for multiple testing (p≤5%) We used the same cDNAs as for the reference genes and eight randomly selected sex-linked genes that were previously found to have sex-biased gene expression patterns , based on RNA-seq data S. latifolia flower buds., We could not design primer assays for two of these contigs, and one of the 22 validated candidate reference genes was excluded because it did not pass our quality control  observed for most contigs . We fountrol see   reference genes . The sucSeven of the eight contigs that were originally inferred to have sex-biased expression based on RNA-seq data RNA-seq data allow the analysis of thousands of genes in one single experiment and make it possible to screen the transcriptome for candidate genes showing expression patterns of interest. qRT-PCR is an effective, robust and inexpensive method for the subsequent validation of such RNA-seq experiments and for detailed studies of identified candidate genes. Medium and high throughput qRT-PCR experiments typically allow inclusion of more biological and technical replica compared to RNA-seq based transcriptome analyses. For an appropriate normalization of the qRT-PCR data it is, however, essential to have two to four suitable reference genes available in order to reduce non-specific expression variation that can otherwise lead to incorrect biological conclusions de novo assembly of reference transcriptomes that can be searched for the most stably expressed contigs. In this study, we observed that only a small proportion of contigs could be used as candidate reference genes because expression of the great majority of genes varied substantially between biological replica and the two sexes. It is therefore important to evaluate a large number of expressed genes for their suitability as reference genes. The approach we used here to identify reference genes was straightforward. We used two different RNA-seq datasets from multiple individuals to reduce technical variation, and population effects to identify candidate reference genes that span a broad expression range. These genes  individuals from an inbred line (U10) and (B) individuals from an intraspecific cross. Low CV values indicate high expression stabilities between the sexes. The 1% most stably expressed contigs are highlighted in green (bottom left of A and B).Genome-wide expression stability estimates of genes in flower buds of male and female (TIF)Click here for additional data file.Figure S2t) and stabilities of candidate reference genes ordered by decreasing stability across both sexes and tissue types. (A) Boxplots of tC values across all tested samples and tissues for candidate reference genes arranged by decreasing (left to right) stability rank as inferred from RefFinder. (B) Average expression stability (M) estimates for all candidate reference genes across all samples and both tissue types. Traditional reference genes are indicated in bold.Expression values (C(TIF)Click here for additional data file.Figure S3Gene expression stabilities of candidate reference genes depend on RNA quality. Average expression stabilities of candidate reference genes in cDNA from flower buds after long-term storage (two years) at −80°C (A) and short-term storage (one year) at −80°C (B).(TIF)Click here for additional data file.Table S1Traditional reference genes found among the 10%, 5% and 1% most stably expressed contigs between male and female flower buds based on RNA-seq data.(XLSX)Click here for additional data file.


Gene set analysis is moving towards considering pathway topology as a crucial feature. Pathway elements are complex entities such as protein complexes, gene family members and chemical compounds. The conversion of pathway topology to a gene/protein networks (where nodes are a simple element like a gene/protein) is a critical and challenging task that enables topology-based gene set analyses.Unfortunately, currently available R/Bioconductor packages provide pathway networks only from single databases. They do not propagate signals through chemical compounds and do not differentiate between complexes and gene families.graphite, a Bioconductor package addressing these issues. Pathway information from four different databases is interpreted following specific biologically-driven rules that allow the reconstruction of gene-gene networks taking into account protein complexes, gene families and sensibly removing chemical compounds from the final graphs. The resulting networks represent a uniform resource for pathway analyses. Indeed, graphite provides easy access to three recently proposed topological methods. The graphite package is available as part of the Bioconductor software suite.Here we present graphite is an innovative package able to gather and make easily available the contents of the four major pathway databases. In the field of topological analysis graphite acts as a provider of biological information by reducing the pathway complexity considering the biological meaning of the pathway elements. A great deal of effort has been recently directed towards the study of gene sets (hereafter GSA) in the context of microarray data analysis. The aim is to identify groups of functionally related genes with possibly moderate, but coordinated, expression changes. Several GSA tests, both univariate and multivariate, have been recently developed. See  for a coSPIA) attempting to capture several aspects of the data: the fold change of differentially expressed genes (DEGs), the pathway enrichment and the topology of signaling pathways. In particular, SPIA enhances the impact of a pathway if the DEGs tend to lie near its entry points. Massa et al. [1] "acetylation and deacetylation of rela in nucleus"[2] "actions of nitric oxide in the heart"[3] "activation of camp-dependent protein kinase pka"> biocarta[["ras signaling pathway"]]"ras signaling pathway" pathway from BioCartaNumber of nodes = 18Number of edges = 22Type of identifiers = nativeRetrieved on = 2011-05-12or its position in the list of pathways:> p <- biocarta [[175]]> p@title[1] "ras signaling pathway"In the network, nodes represent genes and edges functional relationships among them. Nodes can have heterogeneous IDs  and edges can be characterized by multiple functional relationships.pathwayGraph builds a graphNEL object from a pathway p:The function > g <- pathwayGraph(p)> gA graphNEL graph with directed edgesNumber of Nodes = 18Number of Edges = 23> edgeData(g) [1]$ 'EntrezGene:10928 | EntrezGene:5879'$ 'EntrezGene: 10928 | EntrezGene:5879 '$weight[1] 1$ 'EntrezGene:10928 | EntrezGene:5879 '$edgeType[1] "catalysisOut (ACTIVATION)"converterIdentifiers allows the user to map such variety of IDs to a single type (Entrez-Gene or Gene Symbol). For the ID conversion graphite uses the data provided by the Bioconductor package org.Hs.eg.db. This mapping process, however, may lead to the loss of some nodes  and has an impact on the topology of the network (one ID may correspond to multiple IDs in another annotation or vice versa).The function > pEntrez <- convertIdentifiers > pEntrez"ras signaling pathway" pathway from BioCartaNumber of nodes = 20Number of edges = 20Type of identifiers = Entrez GeneRetrieved on = 2011-05-12> nodes(pEntrez)[1:10][1] "10928" "1147" "3265" "387" "4303" "5295" "572" "5879" "5894" "5898"graphite uses the Rcytoscape package to export the network to Cytoscape [Several pathways have a huge number of nodes and edges, thus there is the need of an efficient system of visualization. To this end ytoscape . Cytosca> cytoscapePlot)The network will be automatically loaded into Cytoscape. See Figure In order to verify our signal propagation strategy we perform a simulation study. Using the insulin signaling pathway of the KEGG database we select as differentially expressed 22 genes lying on the signal paths highlighted in Figure FC μ~ U ; 2) we randomly generate log fold change values  of the differentially expressed genes as i δ~ N  ; 3) we run the SPIA[Our simulation is based on the following steps: 1) we randomly generate  the SPIA algorithAs shown in Figure The dataset, recently published by , characthttp://www.bioconductor.org/help/publications/2003/Chiaretti/chiaretti2/) Expression values, appropriately normalized according to rma and quantile normalization, derived from Affymetrix single channel technology, consist of 37 observations from one experimental condition  and 41 observations from another experimental condition . Probes platform have been annotate using EntrezGene custom CDF version 14 [Data is available at the Bioconductor site  on the entire list of genes belonging to a pathway. Here, differentially expressed genes required for SPIA package have been identified using RankProd test [F D R <0.01), while the test on the mean has been chosen for topologyGSA package.We report the results obtained by SPIA and topopologyGSA on the grod test  , the results testify the feasibility of performing analyses using Our results highlight that the hierarchical pathway structure and the reduced dimension of the pathways characterizing respectively the Reactome and Biocarta databases jointly with the specialized cancer pathways of the NCI databases allow the user to have deeper insight into the data.graphite networks identified as significantly altered in the previous analysis.To highlight the usefulness of topological analysis in the context of transcriptomic data interpretation, we report two topologyGSA. Figure graphite network from KEGG database with differentially expressed genes mapped with different colors according to fold change sign. It is interesting to note the presence of several OR groups , single members of which resulted to be differentially expressed. Two clear deregulated paths starting from BCR and ABL1 genes towards apoptosis and NFKB pathways highlight the power of topological analysis to deeper investigate signal cascades within large pathways.Chronic myeloid leukemia pathway includes both genes, BCR and ABL1, and was identified as differentially expressed between BCR/ABL positive and negative patients by KEGGgraph[NCIgraph that imports BioPax models from Cytoscape [Currently there are two Bioconductor packages that try to convert pathway to the SIF model. KEGGgraph that parytoscape  without graphite, on the other hand, is an innovative package able to gather and make easily available the contents of the four major pathway databases. In the field of topological analysis graphite acts as a provider of biological information by reducing the pathway complexity considering the biological meaning of the pathway elements.It is evident that gene set analysis is moving towards considering pathway topology as a crucial feature. A correct conversion of the pathway topology to a gene network becomes therefore important. Available packages are not able to correctly reconstruct the signal transduction in most cases. graphite• Project name: http://www.bioconductor.org/packages/devel/bioc/html/graphite.html • Operating system(s): Platform independent• Project home page: • Programming language: R• Other requirements: Bioconductor• License: GNU AGPL• Any restrictions to use by non-academics: noneGS, EC and CR jointly define the concept proposed. GS, EC developed the methods proposed and performed the analysis on gene expression data. CR supervised the work and wrote the paper. DC Participated in the critical definition of the concept proposed and participated in drafting and commenting critically the manuscript. All Authors read and approved the final manuscript.Example of KGML and owl. Additional file 1: bmc-supp.pdf, 116 K. http://www.biomedcentral.com/imedia/1501537976613594/supp1.pdfClick here for file


The random nature of transgene integration harbours various pitfalls for development of production cell lines including clonal variation in expression level and growth characteristics. The CEMAX system is an expression system for targeted integration of expression cassettes via DNA double-strand break induced homologous recombination. Stable high producers are available within 4 weeks without the need of extensive clone screening and process development.Stable and high expression rates are ensured with the CEMAX system due to targeted integration of a single copy of the gene of interest at a transcriptionally highly active site in the host cell genome. Producer cell lines for various therapeutic product candidates were established. These cell lines produced antibodies and highly glycosylated antibody fusion proteins and showed high clonal similarity. This made a platform fed-batch process profitable.The CEMAX expression system is based on a genetically modified CHO cell line bearing a tag at a highly active genomic transcription site. The gene of interest (GOI) is integrated via site-directed DNA double-strand break-induced homologous recombination. The target site comprises a screening and selection cassette, which was used for initial development of the high producer host cell. This exchangeable cassette is flanked by elements that facilitate site-directed integration by homologous recombination. These elements include regions of homology for recombination with the CEMAX vector, rudimentary selection markers that are activated during recombination, and cleavage sites for the homing endonuclease I-SceI (Meganuclease).Transfection of the CEMAX vector comprising the GOI along with transient Meganuclease activity triggers a chain of events after cotransfection: DNA gets cleaved by I-SceI and cellular DNA repair machinery is induced by free DNA double-strand breaks. The CEMAX vector comprising the GOI functions as a repair matrix using recombination between homologous elements flanking the DNA lesion. The GOI gets integrated and the rudimentary selection markers become activated upon homologous recombination. CEMAX producer cells were selected at multi-well plate scale followed by analysis for outgrowth of stable producer cells. The platform process for fed-batch cultivation of various CEMAX producer cells was applied after expansion to the scale needed for production of protein material.The idea of a platform process that is suitable for all CEMAX producer cells was based on theoretical consideration about similarity of cells due to site-directed integration and the observation that cell growth and metabolism of CEMAX cell lines were comparable. This was verified by the results of this study. Applying the fed-batch process cell growth of CEMAX host cells and CEMAX producer cells was comparable Figure . The devThe fed-batch process comprises a chemically defined and commercially available basal medium and the chemically defined feed solution CeloFeed (Celonic) supplemented according to the improved feeding regime. The glucose level was maintained between 4.5 g/L and 7.5 g/L. Glutamine was kept at a concentration above 0.8 mM. By applying the platform fed-batch process product concentrations up to 690 mg/L were achieved with CEMAX cell lines producing a glycosylated Fc fusion protein after site-directed integration of the GOI Figure . AchievaThe platform fed-batch process was developed by optimization based on commercial available and proprietary basal and feed media formulations. It was suitable for all producer cell lines derived of a particular CEMAX host cell due to highly similar growth behavior after site-directed integration of the gene of interest. The platform process made product concentrations of up to 0.7 g/L achievable. Animal derived component free chemically defined medium and feed solution ensure a robust and regulatory compliant process. By combining the platform process with site-directed integration timelines in cell line development and process development were shortened for faster entry in first-in-man studies.


Blunt chest trauma (BCT) is a common clinical presentation seen in emergency departments. Few cases of cardiac conduction abnormalities due to BCT have been reported in the medical literature. This dysrhythmias may present as permanent conduction defects requiring permanent pacemaker or may have temporary conduction abnormalities requiring temporary pacemaker or supportive care. We present the case of a young woman who suffered from BCT after being kicked by a horse with the development of a significant substernal hematoma. She developed temporary atrioventricular block, which was completely resolved with the decrease in the size of the substernal hematoma suffered. Blunt cardiac injury (BCI) inducing cardiac conduction abnormalities is not a common medical occurrence and probably underreported in the medical literature. An initial review by Dolara and Pozzi in 1966 presented 23 cases of atrioventricular block (AVB) from BCI [μg/L (0.00–0.08 μg/L). These were felt to be secondary to a blunt cardiac contusion. Her elevated cardiac enzymes reverted back to normal within 48 hours.A 25-year-old woman with no past medical or surgical history was brought to the Emergency Department (ED) after being kicked by a horse in her chest. The patient was found to have a midsternal fracture with underlying hematoma on computed tomography (CT) chest see . Her bloThe patient's electrocardiogram (EKG) on admission revealed normal sinus rhythm with rate of 65 bpm (beats per minute) with first-degree atrioventricular block (AV Block) and left anterior hemiblock as depicted in According to the American Association for the Surgery of Trauma, the effects of BCI can range from being asymptomatic with minor EKG abnormalities to rupture of the cardiac anatomy, heart failure, and even traumatic avulsion of the heart . ConductMost conduction abnormalities are postulated to arise from myocardial infarction or ischemia, stunning of the conduction system, or excessive cholinergic activation . Basic aDysrhythmias (including AVB) following blunt chest wall injury can be self-limited or may require permanent pacemaker. Despite the resolution of our patient's AVB, most sources suggest close observation and followup in case delayed complications arise.


CTNS gene, encoding a cystine efflux channel in the lysosomal membrane. In Ctns knockout mice, the pathologic intralysosomal accumulation of cystine that drives progressive organ damage can be reversed by infusion of wildtype bone marrow-derived stem cells, but the mechanism involved is unclear since the exogeneous stem cells are rarely integrated into renal tubules. Here we show that human mesenchymal stem cells, from amniotic fluid or bone marrow, reduce pathologic cystine accumulation in co-cultured CTNS mutant fibroblasts or proximal tubular cells from cystinosis patients. This paracrine effect is associated with release into the culture medium of stem cell microvesicles (100–400 nm diameter) containing wildtype cystinosin protein and CTNS mRNA. Isolated stem cell microvesicles reduce target cell cystine accumulation in a dose-dependent, Annexin V-sensitive manner. Microvesicles from stem cells expressing CTNSRed transfer tagged CTNS protein to the lysosome/endosome compartment of cystinotic fibroblasts. Our observations suggest that exogenous stem cells may reprogram the biology of mutant tissues by direct microvesicle transfer of membrane-associated wildtype molecules.Cystinosis is a rare disease caused by homozygous mutations of the  CTNS gene on the short arm of chromosome 17q, encoding a ubiquitous cystine-selective transport channel in the lysosomal membrane Cystinosis is a rare disorder caused by homozygous mutations of the 2/day typically reduces leukocyte cystine levels to about 15% of baseline. However, the odour and gastric side-effects of oral cysteamine make adherence extremely difficult. Furthermore, even well-treated patients eventually require kidney transplantation and develop profound distal myopathy, cerebral atrophy and other complications as young adults In the late 1980's, it was discovered that the pathologic accumulation of cystine could be substantially reduced by cysteamine therapy. This drug attacks the internal disulfide bond of cystine to form mixed disulfides that are able to efflux from the lysosome via alternative transport channels Ctns −/− knockout mice et al infused allogeneic wildtype bone marrow stem cells into Ctns −/− knockout mice after partial ablation of the bone marrow. The exogenous stem cells were progressively retained by cystinotic organs, resulting in 90% decrease in tissue cystine levels and, in the case of the kidney, leading to normalization of organ dysfunction Ctns transcript level rose to only 10% of normal It was with great interest, therefore, that the medical community learned in 2009 of successful bone marrow stem cell therapy of et al reported that cancer stem cells shed microvesicles from the cell surface and that endocytotic uptake of the microvesicles transfers epidermal growth factor receptors to nearby endothelia; this induces proliferation of the normal endothelial cells, by rendering them responsive to local transforming growth factor-alpha in vitro co-culture model to demonstrate that human stem cells reverse cystine accumulation in cystinotic cells in paracrine fashion. We demonstrate that mesenchymal stem cells shed microvesicles containing wildtype cystinosin protein and CTNS mRNA into the culture medium and that uptake of isolated microvesicles transfers CTNS protein to the lysosomal compartment of cystinotic fibroblasts and reverses pathologic cystine accumulation.Stem cells are known to exert a number of useful paracrine effects, including the release of soluble factors that suppress inflammation or stimulate proliferation of endogenous cells following acute organ injury The cells used in this study were obtained from the Cell Repository from the Montreal Children's Hospital with ethical approval from the Montreal Children's Hospital Research Ethics Board. For cell lines previously published, ethical approval was obtained and stated in the corresponding reference.Red was previously described CTNSCTNS(f) 5′-gcagtcacgctggtcaagta-3′, and CTNS(r1) 5′-caggaaagtggccttcagag-3′ or CTNS(r2) 5′-cgaagacgatggagaagacc-3′; GAPDH(f) 5′-gagtcaacggatttggtcgt-3′, GAPDH(r) 5′- gatctcgctcctggaagatg -3′. Genotyping for CTNS mutation was performed with primers LDM1 flanking the common 57 Kb deletion or CTNS Exon 4 Total RNA was isolated with RNeasy kit (Qiagen). Purity and concentration of RNA were determined with a NanoDrop ND-1000 . Transcripts were amplified (35 cycles) using: Normal human amniotic mesenchymal stem cells  and adult bone marrow cells  −8 mol/l; Sigma-Aldrich) for 4 weeks and then stained with 1% Alizarin Red S (Sigma-Aldrich). In order to test for adipogenic differentiation of MSCs, cells were exposed to complete media supplemented with insulin (5 µg/ml), dexamethasone , 3-isobutyl-methylxanthine , and indomethacin  for 14 days, and then stained with Oil Red O (Sigma-Aldrich).One million cells were incubated for 30 minutes at +4°C with the following monoclonal antibodies: phycoerythrin-labeled HLA-DR (clone LN3) from eBioscience, allophycocyanin-labeled CD34 (clone 581), phycoerythrin-labeled CD44 (clone G44-26), phycoerythrin-labeled CD31 (clone WM59), phycoerythrin-labeled CD105 (clone 266), phycoerythrin-labeled CD45 (clone HI30), fluorescein isothiocyanate-labeled CD90 (clone 5E10), phycoerythrin-labeled CD73 (clone AD2) from BD Biosciences . Cells were analyzed with CellQuest Pro software on a FACSCalibur flow cytometer (BD Biosciences), using antibody isotype controls. In order to test for osteogenic differentiation of MSCs the cells were exposed to complete media supplemented with β-glycerol phosphate , ascorbic acid 2-phosphate , and dexamethasone  equipped with a 1.4 NA 63× oil lens, Leica DMI6000 B microscope, Yokowaya CSU-10 spinning disc confocal head and a Hamamatsu ImagEM (EM-CCD) camera. Images were acquired using 0.2 micron optical sectioning and analysed using the Improvision Volocity software. Images were registration corrected and deconvolved. Briefly, cells were grown on Lab-tek II chambered coverglass slides . In some experiments, MV bearing CTNSRed were added to mutant cell monolayers 4 hours before confocal microscopy. LysoTracker probe  was added 10 min before image capture.CTNSStatistical analysis were performed using paired, two tailed Student's t-test. Data bars and error bars indicate mean ± standard deviation., right panels).To study the paracrine effects of wildtype stem cells on nearby adjacent mutant cell targets, we first isolated mesenchymal stem cells from bone marrow of healthy adults (bmMSC)  containing high intracellular levels of cystine . After 4 days in co-culture, we measured the cystine content of the mixed cell population and calculated the cystine attributable to the subset of mutant target cells. As seen in We then co-cultured amniotic mesenchymal stem cells (amMSC) with various proportions of mutant fibroblasts from a cystinosis patient with homozygous deletion of the GFP) after co-culture with a variety of donor cells. The target ciPTEC(−/−)GFP cells were from a cystinosis patient bearing the common homozygous 57 kb deletion of the CTNS gene GFP targets compared to baseline. In another set of experiments, ciPTEC(−/−)GFP cell cystine was unaffected by co-culture with untagged mutant proximal tubular ciPTEC(−/−) donor cells; on the other hand, co-culture of ciPTEC(−/−)GFP with genetically corrected (transfected with wildtype CTNS) ciPTEC(−/−) cells again showed significant reduction of cystine content et al suggested that an important characteristic of stem cells is that they shed plasma membrane-derived microparticles into the local environment and these are taken up by neighboring cells Al-Nedawi CTNS mutant target cells, we isolated microvesicles from bmMSC or amMSC and transferred them to separate monolayer cultures of CTNS mutant fibroblasts (−/−) for 24 hours (1–2 mg microvesicles/mg fibroblast protein). The cells were then harvested and assayed for cystine content as above. As seen in To ascertain whether MSC microvesicles can reduce cystine content of Red GFP fibroblast targets; there was no evidence of fusion between donor and target cells. As seen in Red particles are visible in a single confocal plane within a cluster of amMSC GFP fibroblasts, indicating transfer of the tagged protein from amMSC to the target cell.To test the possibility that microvesicles mediate direct CTNS protein transfer between donor and recipient cells, we stably transfected amMSC with CTNSRed vesicles ( inset) applied directly to cultured CTNS(−/−) fibroblasts stained with a green-fluorescent endosomal/lysosomal dye. As seen by confocal microscopy GFP fibroblasts with either amMSC or unlabelled CTNS(−/−) fibroblast donors for 48 hours and then separated the CTNS(−/−)GFP fibroblasts by FACS. As seen in CTNS transcripts were detectable by RT/PCR in the mutant recipient target cell co-cultured with amMSC, but not with the mutant fibroblast donors.Other investigators have shown that microvesicles contain mRNA and miRNA as well as protein Long regarded as inactive cellular debris, microvesicles shed from the surface of viable cells are increasingly recognized as important vehicles for the transfer of complex biologic information between neighboring cells. Microvesicles arise either through vesiculation of the plasma membrane to generate 100–500 nm diameter particles exposing phosphatidyl serine residues shifted to the outer leaflet (ectosomes) or through redirection of endosomal structures to multivesicular bodies that disgorge their 40—80 nm diameter particles (exosomes) into the extracellular environment Ctns knockout mice While many cells shed microvesicles, flux from the surface of stem cells and cancer cells is thought to be especially high and may exert important influence on the stem cell niche or on tumour invasiveness. Here, we show that mesenchymal stem cells shed microvesicles containing wildtype cystinosin protein and mRNA. Uptake of these microvesicles by mutant CTNS(−/−) fibroblasts or proximal tubule cells delivers CTNS to the endosomal/lysosomal compartment and reduces the pathologic accumulation of cystine. This provides a plausible mechanism to explain the clinical effects of bone marrow stem cell infusion on et al, optimal clinical effect was obtained by infusion of 20 million wildtype hematopoietic stem cells (HSC) into Ctns knockout mice Ctns(−/−) mouse tissues noted by Syres et al could be explained by microvesicle shedding.In the study by Syres Our studies show that the majority of microparticles shed by amMSC or bmMSC monolayers into culture medium containing 0.5% BSA are between 100–400 nm in diameter, characteristic of microvesicles (ectosomes). Unlike exosomes (<100 nm diameter) which arise during endosomal recycling within the cell, microvesicles arise through regulated outward blebbing of lipid raft-associated areas of the plasma membrane et al have shown that MSC infused into mice with glycerol-induced renal injury ameliorate tissue damage by suppressing apoptosis and enhancing proliferation of endogenous cells Bruno et al have reported a second cystinosin isoform (CTNSLKG), constituting about 15% of total cellular cystinosin, which lacks the lysosomal targeting sequence and readily appears at the plasma membrane CTNS gene and the capacity to shed microvesicles should be able to reduce cystine content of adjacent mutant cells. Indeed, we found that mutant cell cystine accumulation was reduced by co-culture with either amMSC or genetically corrected proximal tubular cells and by microvesicles from either amMSC or bmMSC.In our studies, transfection of amMSC with an expression vector containing Red-tagged CTNS generated microvesicles richly supplied with the tagged fusion protein. Wildtype cystinosin contains a 3′sequence which targets most of the protein to the lysosomal membrane CTNS mRNA, it is uncertain whether de novo CTNS synthesis in the mutant cell contributes to the rescue effect. It is likely that some cells shed more microvesicles than others and this could influence therapeutic efficacy. Furthermore, delivery of wildtype protein to the plasma membrane could limit the rate of protein transfer to the mutant cells. Steinherz showed that the efflux half-time of cystine dimethylester from heterozygous cystinotic lysosomes is 86.3+/−5 vs normal 43.1+/−3 minutes Microvesicles are known to contain proteins as well as mRNA and miRNA A wide range of lysosomale storage diseases have been treated with allogeneic hematopoietic stem cells derived from umbilical cord For years, the therapy of genetic disease has focused on: a) vector-mediated gene transfer to mutant tissues; b) transplantation of long-lived wildtype cells that can metabolize systemic load of toxic metabolites; c) transplantation of stem cells that can transdifferentiate and become integrated into mutant organs. Our observations suggest an additional type of cell-based therapy for genetic diseases in which microvesicle-mediated transfer of membrane-bound proteins may correct the defect in nearby mutant cells. This shifts the primary therapeutic consideration toward the ability of stem cells to shed microvesicles that transfer molecules to the mutant tissue rather than their transdifferentiation potential.


Establishment of sinus rhythm (SR) following radiofrequency ablation (RFA) for longstanding persistent atrial fibrillation (AF) is not necessarily an equivalent to physiological atrial mechanical activity. We aimed to determine the influence of atrial ultrastructural remodeling on the recovery of its mechanical transport following restitution of SR.We enrolled 32 patients operated for severe mitral regurgitation. Half of the patients had no history of AF constituting the SR group. The remaining half had concomitant RFA for longstanding persistent AF, the RFA group. Intraoperative transesophageal echocardiography was used for tissue Doppler indices (TDI) data acquisition at the left atrial (LA) lateral wall. During post-procedural data acquisition all patients were in SR. LA biopsies were obtained during surgery and quantified for fibrosis after Mallory’s trichrome staining.Atrial mechanical contraction was noted in both groups following surgery, although TDI values of late diastolic strain rate (SRI A’) and tissue velocity (TVI A’) were superior in the SR group . Post-procedural atrial filling was compromised in the RFA  and the SR group . Negative correlation between fibrosis and the pre-procedural SRI A’ was noted in the SR group , although not significant throughout both groups .We have shown that the restoration of SR following RFA and mitral valve surgery is not equal to normal atrial mechanical transport. Interstitial fibrosis, a symbol of ultrastructural remodeling, is a negative precursor of LA mechanical activity. Our data did not corroborate clear difference in the extent of fibrosis between groups, although a tendency towards more severe ultrastructural remodeling was noticed in patients with history of AF.


Diastolic dysfunction is often used as a marker for allograft rejection and may even precede systolic abnormalities. However, diastolic indices provided by echocardiography can be of inconsistent quality with low sensitivities. Cardiac magnetic resonance (CMR) with high temporal resolution cine imaging offers an alternate noninvasive method for evaluating ventricular function. The purpose of this study is to describe CMR LV diastology indices in a cohort of heart transplant patients, comparing CMR results to echocardiography.A retrospective search was performed for cardiac transplant patients with echocardiograms and CMR examinations with high temporal resolution steady state free precession (SSFP) cine images in long axis orientation (average TR: 11.86 ms) obtained within 1 week of each other. The study cohort comprised 18 patients . SSFP cine images were analyzed using prototype software evaluating deformation fields to automatically identify and track the mitral base plane at lateral and septal insertions, extracting lateral e' values . Lateral e' values were obtained for each patient from available echo data as well. Additionally, in 13 patients, 3-chamber high temporal resolution SSFP cine images were adequate in orientation for calculation of time elapsed between aortic valve closure and mitral valve opening (IVRT). Bland-Altman analysis was performed. Lateral e' values were reviewed between methods and across estimates of LV filling pressure with E/lateral e' ratios obtained from echo to determine a threshold CMR e' value suggestive of elevated LV filling pressures.Representative lateral mitral annular velocities are shown in Figure High temporal resolution SSFP cine imaging enabled semiautomated quantification of LV diastolic function parameters in heart transplant patients. While CMR consistently underestimated lateral e' values in our cohort, high temporal resolution SSFP cine imaging provided acceptable estimates of IVRT. Additionally, there may be a threshold lateral e' value at CMR  that is suggestive of elevated LV filling pressures. Our conclusions are limited due to the small number of patients in this retrospective study; work is ongoing to confirm our findings in a larger cohort.Dr. Collins has funding from RSNA Research & Education Foundation.


Cardiac MRI imaging showed a very large ventricular septal cystic mass most consistent with a primary cardiac teratoma causing mild LVOT obstruction.CMR is an established method for evaluation of cardiac masses. It is superior to other modalities as it can provide tissue characteristics, thus allowing highly accurate differentiation of benign from malignant tumors, as well as local mass effects such as outflow compression.Using Cardiac MRI for assessment of etiology of a massive, septal tumor as well as its effect on LVOT flow and LV filling.A 5 day old full term female with a prenatal diagnosis of a large intracardiac tumor underwent post-natal echocardiography. Echo confirmed the suspected diagnosis as a large cystic mass compressing both ventricles without significant outflow tract obstruction. Cardiac MRI was performed specifically for further morphologic characterization. Imaging was performed using non-breatheld technique with multiple signal averages while under general endotracheal anesthesia on a 1.5 Tesla GE Excite magnet utilizing an 8-channel phased-array cardiac coil. The CMR sequences included SSFP 2, 3, and 4 chamber views and short axis multislice cine stacks; tagged SPAMM cines in 3 short and 2 long axis planes; gadolinium-enhanced myocardial first pass perfusion and delayed enhancement imaging in both short and long axis planes. Pre- and post-contrast T1 FSE single phase short axis stacks. Both right and left ventricular volumes and tumor volume were measured at end systole and end-diastole using MEDIS QMass software.All the sequences show a large heterogeneous intraventricular cystic mass surrounded by thinned myocardium (Fig.CMR provided exquisite anatomic localization and tissue characterization of this pediatric cardiac tumor. It also delineated the extent of viable function within the heart chambers allowing for proper patient management. Based on the MR imaging, no interaction was undertaken. At the time of the last evaluation at 4 months of age, the patient was doing well and was without symptoms. The tumor size was unchanged and the cardiac function remains normal.There are no financial disclosures.


Cor triatriatum sinister is a rare congenital cardiac anomaly with an incidence of between 0.1 and 0.4%  whereby A 64 year old man with a 4 year history of long-lasting persistent drug refractory AF and previous failed attempts at cardioversion was referred for catheter ablation. There was no history of hypertension, coronary artery disease or obstructive sleep apnea. Previous transthoracic echocardiogram had been reported as demonstrating mildly dilated left atrium at 4.2cm with a membrane within the left atrium. There was mild mitral regurgitation with no evidence of impaired trans-mitral flow. The left ventricle was undilated (left ventricular end diastolic diameter 4.9cm) with normal systolic function (ejection fraction 70%). These findings were confirmed by a pre-operative multi-slice CT scan which clearly demonstrated a membrane dividing the left atrium into 2 chambers. The supero-posterior chamber received the 4 pulmonary veins whilst the infero-anterior chamber contained the left atrial appendage and was in direct continuity with the mitral valve  and 1b.The patient was in AF at the commencement of the procedure. Under general anaesthesia, bilateral femoral venous access was obtained. A 6Fr quadrapolar catheter was advanced from the left femoral vein to the His position and a 7Fr steerable decapolar catheter was advanced from the right femoral vein to the coronary sinus. Intra-operative transesophageal echocardiogram (TEE) was performed to further delineate left atrial anatomy and aid trans-septal access to the left atrium.Standard and 3-dimensional TEE confirmed the diagnosis of cor triatriatum with a fenestrated membrane arising from the anterior roof of the left atrium extending partly down the chamber in the antero-inferior direction  . The seAccess to the infero-anterior chamber was gained via the PFO using an SL1 sheath advanced from the right femoral vein. A decapolar circular catheter  was advanced into this chamber via the sheath and a 4mm irrigated catheter  was passed alongside using a single wire, dual access approach. Intravenous heparin was used to maintain an ACT of 300-350 seconds. Both catheters were inserted into the antero-inferior chamber. The supero-posterior chamber was accessed relatively easily by rotating the catheters posteriorly under the membraneA 3 dimensional multichamber geometry of the left atrium and pulmonary veins was created using the Ensite NavX system  without CT integration. Given that the left atrium was functionally and anatomically divided by the membrane, the supero-posterior and infero-anterior aspects were created as separate chambers  and 1d. The strategy employed was pulmonary venous antral ablation with isolation as the end point. Further ablation within the supero-posterior chamber was performed with linear ablation across the roof connecting the right and left superior pulmonary veins. Complex fractionated electrograms were then targeted within the two left atrial chambers, the right atrium and within the coronary sinus  and 3b. During 7 months of follow up the patient has remained symptomatically free of atrial fibrillation without any antiarrhythmic drugs.  24 hour holter monitoring 3 and 6 months post procedure did not show any evidence of atrial fibrillation.Cor triatriatum sinister is commonly thought to result from failure of the common pulmonary vein to become fully incorporated into the left atrium during foetal development. This results in a fibro muscular septum like structure which divides the left atrium into a postero-superior chamber receiving the pulmonary veins and an antero-inferior chamber in direct continuity with the mitral valve. It usually presents in childhood, often in association with other cardiac abnormalities. However with improvements in diagnostic imaging it is being diagnosed with increasing frequency in adults  either iAlthough there are two previous case reports of AF ablation in the literature, these involved only single chamber ablation with isolation of the pulmonary veins and no attempt at additional substrate modification. It is generally recognized that pulmonary vein ablation with isolation alone is insufficient in the treatment of persistent atrial fibrillation and that additional substrate modification is required -12. To tThe use of a 3 dimensional mapping system and good pre- and intra-operative imaging were essential in the planning and execution of the ablation procedure. Our experience would suggest that these patients can be effectively treated with a good outcome.


Inferoseptal crypts have been previously reported as a marker of hypertrophic cardiomyopathy (HCM) as well as genotype positive/phenotype negative HCM. We hypothesized that the presence of such crypts would have incremental predictive value for the presence of a disease-causing mutation compared to prediction based solely on left ventricular morphologic pattern of hypertrophy.Cardiovascular magnetic resonance (CMR) data sets of 300 consecutive unrelated HCM patients who underwent genetic testing were reviewed independently by two experienced clinical CMR readers who were blinded to results of genetic testing. Readers assessed for presence or absence of deep basal inferoseptal crypts (DBISC) on cine steady state free precession and late gadolinium enhancement sequences and documented the imaging plane in which DBISC were convincingly visualized. Imaging and genetic testing results were correlated.Deep basal inferoseptal crypts occurred more commonly in patients with disease-causing mutations than in patients without them  and the presence of DBISC was a stronger predictor of the presence of disease-causing mutations than reverse septal curvature morphologic phenotype (p=0.025). 91.2% of patients with both reverse septal curvature and DBISC had disease-causing mutations . Mutations discovered in patients with DBISC had a higher likelihood of being disease-causing than non-disease-causing (p<0.001). DBISC were more commonly identified in patients with reverse septal curvature than in all other morphologies combined (p<0.001). In approximately half of cases, DBISC were missed on short-axis cines.Presence of deep basal inferoseptal crypts in HCM patients demonstrate incremental predictive value for genotype-positive status over that of left ventricular morphologic phenotype based prediction. Assessment for DBISC is best performed with the use of multiplanar CMR data sets. Additional imaging planes double the sensitivity for detection of deep basal inferoseptal crypts. Where genetic testing is not available, or a limited resource for economic reasons, the presence of CMR-detectable crypts may represent one factor among several that may guide decision-making for genetic testing. They may also have a role in investigating patients with modest degrees of hypertrophy (13-15mm).None.


A major challenge in HIV vaccinology is the development of appropriate immune monitoring models to determine vaccine efficacy. Studies of HIV-specific CD8 T cells (CTL) suggest that these responses play an important role in infected individuals capable of spontaneous viral control (HIV elite controllers) and that they will likely play a role in immune interventions. However, no single CTL assay is uniquely associated with the controller phenotype.We compared functionality of HIV-1-specific CTLs in individuals with spontaneous viral control and subjects with treated or untreated progressive infection. A model integrating multiple features of epitope-specific CTL responses that delineate HIV controllers from subjects with treated or untreated progressive infection was built.Area Under the Receiver Operating Characteristic (ROC) Curve showed that proliferative capacity, absolute early cytokine production and kinetics of cytokine secretion were all associated with HIV control. However, only integrated modeling of these different dimensions of data allowed reaching the remarkable 90% accuracy, which was validated in separate cohorts.Our results suggest that while the search for a common determinant of protective immunity remains elusive, combining parameters generated by various well-established assays in models that can be iteratively refined may have important applications for predicting disease outcome and for immune monitoring of HIV-1 vaccine trials.


Vibrio cholerae, the enteropathogenic gram negative bacteria is one of the main causative agents of waterborne diseases like cholera. About 1/3rd of the organism's genome is uncharacterised with many protein coding genes lacking structure and functional information. These proteins form significant fraction of the genome and are crucial in understanding the organism's complete functional makeup. In this study we report the general structure and function of a family of hypothetical proteins, Domain of Unknown Function 3233 (DUF3233), which are conserved across gram negative gammaproteobacteria . Profile and HMM based sequence search methods were used to screen homologues of DUF3233. The I-TASSER fold recognition method was used to build a three dimensional structural model of the domain. The structure resembles the transmembrane beta-barrel with an axial N-terminal helix and twelve antiparallel beta-strands. Using a combination of amphipathy and discrimination analysis we analysed the potential transmembrane beta-barrel forming properties of DUF3233. Sequence, structure and phylogenetic analysis of DUF3233 indicates that this gram negative bacterial hypothetical protein resembles the beta-barrel translocation unit of autotransporter Va secretory mechanism with a gene organisation that differs from the conventional Va system. Vibrio cholerae, Vibrio parahaemolyticus, Vibrio splendidus and Vibrio vulnificus are pathogenic to human and aquatic life. Vibrio cholerae causes seasonal outbreaks of cholera of epidemic proportions in developing countries with high mortality rates Domain of Unknown Function (DUF) 3233 (PFAM: PF11557) is a family of uncharacterised hypothetical proteins conserved among gram negative gammaproteobacteria. Representative members of this domain include marine bacteria from genus Vibrio, Shewanella, Colwellia and Alcanivorax of which th release lists about 3000 DUF families. Many of these DUF families are kingdom specific , limited/shared between kingdoms  or restricted/specific to certain organisms . The specific and ubiquitous nature of these domains suggests their functional importance in organism specific niches or a common biological role.Significant fraction of genomes of Vibrio species lack structure function annotation and most of these gene products are classified as hypothetical proteins or domains of unknown function In silico structure prediction methods together with sequence similarity detection methods assist the annotation of fold-function space. Fold recognition methods like I-TASSER Identifying homologous protein families through sequence based search marks the first step in the annotation of DUFs, providing an initial broad picture of the protein's probable family and function. Sequence homology search becomes increasingly powerful when we advance from normal sequence-sequence based searches to methods that uses profile or HMM information like HHsenser In this study we analyse the sequence and structural characteristics of DUF3233 using computational approaches and try to infer various properties of this domain. Sequence search by HHsenser identifies similarity with the beta-barrel translocation unit of autotransporter Va secretory proteins. Sequence homology combined with secondary structure prediction indicates a beta-barrel domain of 12 beta-strands. The predicted 3D model from I-TASSER shows the structure with an overall beta-barrel topology with an N terminal helix running along the central barrel axis perpendicular to the 12 antiparallel strands that form the barrel. Amphipathicity and membrane barrel discrimination analysis suggest the domain is a potential outer membrane gram negative beta-barrel protein.Autotransporter translocation units belong to the transmembrane beta-barrel fold in SCOP database Vibrio cholerae DUF3233 (RefSeq: NP_232949) against the NCBI nr database with a threshold 0.005, reached convergence at the 4th iteration. The resulting sequences identified were hypothetical proteins conserved among gram negative proteobacteria. For improved search and better coverage of homologous sequence space, information from aligned regions of DUF3233 sequences in the form of a multiple sequence alignment profile was queried with HHsenser. From the resulting sequences in the permissive alignment list we were able to infer homology between DUF3233 and the outer membrane beta-barrel translocation domain of autotransporter proteins. DUF3233 shares sequence similarity with outer membrane beta-barrel domain of Ochrobactrum intermedium autotransporter , Rhizobium leguminosarum adhesin autotransporter  and Yersinia aldovae AidA adhesin autotransporter . Interestingly, a number of gram negative hypothetical proteins were picked up as potential homologues, which showed fair amount of similarity to the autotransporter beta-domain . This cluster comprises profile HMMs of autotransporter sequences whose 12 stranded beta-barrel transmembrane domains conform to the translocation unit of autotransporter NalP Position specific scoring matrix (PSSM) profile based discrimination analysis using TMBETADISC-RBF Proteins targeted for transport across membranes posses leader sequence or signal peptide at their N-terminus, which directs translocation. We analysed DUF3233 sequences using a combination of artificial neural networks and HMMs implemented in SignalP We browsed DUF3233 genomic region of all representative organisms with STRING N) followed by 12 consecutive β-strands (β1–β12) interspersed by two short turns of α-helices α1 and α2 predicted to occur between β1–β2 and between β5–β6 respectively. With no suitable template with significant sequence homology available, we used the fold recognition algorithm implemented in I-TASSER to predict a 3D model of DUF3233. The translocation unit of NalP from N. meningitidis  was identified by I-TASSER in the top four threading templates to model V. cholerae DUF3233. The predicted structure of V. cholerae DUF3233  fetched results with a maximum alignment length covering 51 residues. Secondary structure assignment by PSIPRED  DUF3233  resemble DUF3233 . Using aTo infer evolutionary relation with type V secretory proteins, we analysed DUF3233 representatives with members of both Va and Vb family . The thiA vital part in the survival and adaptation mechanism of bacteria lies in the constant interaction with their extra-cellular environment. Bacteria secrete a wide range of molecules into the extra-cellular milieu that includes enzymes, which break down carbohydrates, proteins and lipids, and virulence factors such as adhesins and toxins by those involved in pathogenesis. Transport of these molecules is mediated by protein complexes through conserved secretory pathways. Of the 6 types of secretory mechanisms known in gram-negative bacteria (type I to type VI), type V represents the simplest transport system. Proteins of the type V secretory system fall under the autotransporter (Va), two partner secretion (Vb) and the AT-2 (Vc) families, and share a similar domain organisation: an N-terminal signal peptide for inner membrane translocation followed by a passenger protein which is normally a virulence determinant and a C-terminal translocation unit for transporting the upstream passenger protein Proteins of the autotransporter (Va) family were the first to be described The present work describes sequence and structure based characterization of proteobacteria DUF3233 as a beta-barrel transmembrane domain of autotransporter proteins. DUF3233 packs an average 312 amino acid residues  and is currently classified as a domain of unknown function.DUF3233 is encoded as a single domain protein, homologous to the translocation unit of autotransporters. One aspect of DUF3233 that distinguishes it from other main class autotransporters is that it lacks a covalently linked N-terminal passenger domain, to which C-terminal translocation units of all autotransporters are committed to transport. Few autotransporter representatives of two-polypeptide architecture V. cholerae are organised in clusters or operons Few representative members from the Vibrio genus express DUF3233 and upstream putative GGT or response regulatory proteins in a single operon . Over exet al., H. influenzae Hap translocation unit. Deletion of terminal 12 residues proved detrimental to the outer membrane localisation; while the stability and/or outer membrane localisation of the translocation unit was affected with the deletion of all three terminal residues, point mutations of these residues showed no effect on the outer membrane localisation or secretion of the mature protein The translocation units of autotransporters exhibit conserved amino acid consensus motif at their carboxy terminus, the barrel closing beta-strand displays alternate arrangement of polar and hydrophobic residues terminating with a conserved aromatic amino acid at the barrel terminus which is usually a phenylalanine or a tryptophan DUF3233 exhibits certain features that are in common with the translocation units of type Va secretory proteins and yet possesses characteristics that are not typical to the proteins of the above system. DUF3233 represents a translocation unit that is devoid of a secretable passenger unit. Considering its location in the representative genomes alongside other virulence genes, we hypothesize that this domain is involved in pathogenesis. However, the mechanism apparently looks new and different than a typical type Va secretion system.Our study on the proteobacterial protein DUF3233 with a combination of methods like sequence similarity searches, outer membrane beta-barrel discrimination, phylogenetic analysis, and fold recognition has led us to a consensus at annotating fold and function to this domain.Sequence similarity search suggests that DUF3233 has remote homology with the translocation unit of autotransporter proteins of the type V secretory system. The domain's outer membrane beta-barrel nature was further emphasised by signal peptide, outer membrane beta-barrel discrimination and amphipathicity analysis. Secondary structure prediction and alignment with translocation units, and inputs from the predicted model suggests that DUF3233 and the translocation unit of autotransporter proteins share a similar domain organisation.in silico prediction methods it appears that DUF3233 is a cluster of remote homologues of autotransporters. This is the first report of an autotransporter like protein family in Vibrio species, and though within the realm of bioinformatics we were able to infer its probable family and fold, the pathogenic mechanism still remains to be explored and seeks further experimental studies.Drawing a consensus from various 20 DUF3233 genes from NCBI comprising one sequence each from Aliivibrio, Colwellia and Ferrimonas, five sequences from Shewanella and twelve from Vibrio species were fetched using their corresponding RefSeq ids. YP_001366070 and YP_001555810 were excluded because of their short domain size and a total of 18 DUF3233 sequences were included in this study. The signal peptides of DUF3233 sequences have not been included in various analyses of this study unless mentioned. PSI-BLAST The presence of N-terminal signal peptide and the putative cleavage sites were predicted with SignalP 3.0 V. cholerae DUF3233 (RefSeq: NP_232949) was predicted using I-TASSER fold recognition method Secondary structure assignments were made using PSIPRED V. cholerae (RefSeq: NP_232949), C. psychrerythraea (RefSeq: YP_269983) and S. loihica (RefSeq: YP_001095898) along with beta-barrel domain sequences of the Va, Vb secretory systems were analysed with MEGA 4 Amino acid sequences of translocation unit of autotransporters, and pore forming beta-domain of two-partner secretion (TPS) family members, reported in Figure S1Sequence alignment of DUF3233 representative sequences. DUF3233 has an average 23 amino acid N-terminal signal sequence (gray) which guides inner membrane translocation. The signal peptidase I cleavage site is marked by a red arrow. Cps_Colwellia psychrerythraea (YP_269983.1), Vfi_Vibrio fischeri (YP_206466.1), Vpa_Vibrio parahaemolyticus (NP_800436.1), Vvu_Vibrio vulnificus (NP_762291.1), Vch_Vibrio cholerae (NP_232949.1), Vsp_Vibrio splendidus (YP_002395311.1), Sba_Shewanella baltica (YP_001052604.1), Slo_Shewanella loihica (YP_001095898.1), Spi_Shewanella piezotolerans (YP_002312093.1), Fba_Ferrimonas balearica (YP_003912385.1).(TIF)Click here for additional data file.Table S1List of DUF3233 homologous sequences. Sequence search by HHsenser (permissive) picked gram-negative and cyanobacterial (Cyn) hypothetical proteins that share significant homology with DUF3233. These hypothetical proteins posses sequence characteristics that apparently resemble the autotransporter beta-domain. # Insignificant Pfam A hits, $ Significant Pfam A hits.(DOC)Click here for additional data file.Table S2List of representative sequences used for phylogeny analysis. C-terminal translocation unit sequences of Autotransporter (Va) (DOC)Click here for additional data file.


Nanog locus. These results suggest that chondrogenic cells induced by this approach are free from a risk of teratoma formation which associates with cells prepared through generation of iPS cells followed by redifferentiation into the target cell type. The dox-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. This approach could lead to the preparation of hyaline cartilage directly from skin, without going through pluripotent stem cells, in future regenerative medicine.Repair of cartilage injury with hyaline cartilage has been a challenging clinical problem. Articular cartilage damage sometimes heals with fibrocartilage, which is different from hyaline cartilage. Fibrocartilage is a type of scar tissue that expresses types I and II collagen. In contrast, hyaline cartilage does not express type I collagen. When aiming to induce hyaline chondrogenic cells directly from dermal fibroblasts, in addition to activation of cartilage-specific matrix genes, elimination of expression of type I collagen is needed for generation of hyaline cartilage. Otherwise, the presence of type I collagen impairs cartilage extracellular matrix architecture, which leads to formation of fibrocartilage. The generation of induced pluripotent stem cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. We found that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts; the promoters of type I collagen genes were extensively methylated. Transduction of c-Myc, Klf4, and SOX9 produced two types of cells: chondrogenically reprogrammed cells and partially reprogrammed intermediate cells. Chondrogenically reprogrammed cells generated stable homogenous hyaline cartilage-like tissue without tumor formation when subcutaneously injected into nude mice. Hyaline cartilage-like tissue expressed type II collagen but not type I collagen. On the other hand, partially reprogrammed intermediate cells expressed type I collagen and produced tumor when injected into nude mice. Induced chondrogenic cells did not undergo pluripotent state during induction from dermal fibroblast culture, as time-lapse observation did not detect GFP reporter expression during induction from dermal fibroblasts prepared from transgenic mice in which GFP is inserted into the


We report a case of a persistent left superior vena cava (PLSVC) with absent right superior vena cava (RSVC). It is a very rare congenital anomaly also known as isolated PLSVC.This venous malformation was identified in a 75-year-old woman during cardiac magnetic resonance imaging (MRI), which was performed with the suspicion of a paracardiac mass.We performed an MRI and a multislice computed tomography (MSCT) evaluation.Cardiac MRI revealed a persistent left superior vena cava which descended on the left side of the mediastinum and drained into the right atrium (RA) via a markedly dilated coronary sinus (CS) which mimicked a paracardiac mass. The RSVC was absent.These findings were confirmed by MR and MSCT venography.The patient had no additional cardiac abnormality.Although PLSVC is usually asymptomatic, it is important to be aware of its existence, since it may cause problems performing central venous catheterization, pacemaker implantation and cardiothoracic surgery.This anomaly is also associated with high incidence of congenital heart disease, arrhytmias and conduction disturbances.Modern imaging techniques including computed tomography and magnetic resonance imaging provide precise diagnosis of this anomaly.


This study sought to demonstrate the potential for myocardial arterial spin labeling (ASL) to identify the ischemic myocardial segments due to stenosis in coronary arteries as detected by X-ray angiography.stress/MBFrest), which is a common indicator for the severity of coronary artery disease. In healthy myocardium, perfusion reserve is known to be approximately four [Myocardial ASL is a technique for the assessment of myocardial blood flow (MBF) without contrast agents. It can be safely applied to patients with end-stage renal disease who are not candidates for first-pass imaging with contrast agents. Myocardial ASL perfusion imaging performed at rest and during adenosine stress provides perfusion reserve (MBFely four .Twenty nine patients were recruited from those scheduled for routine cardiac MR (CMR) and X-ray angiography. Myocardial ASL measurements were obtained from a single mid short-axis slice at rest and during adenosine infusion (dosage: 0.14 mg/kg/min) on a GE Signa 3T scanner. The ASL sequence was composed of flow-sensitive alternating inversion recovery (FAIR) tagging and balanced steady-state free precession (SSFP) imaging . PerfusiTen of the twenty-nine patients were found to have significant stenosis on X-ray angiography. Table There was visual agreement  and no statistically significant difference between ischemic myocardial segments identified by ASL perfusion reserve maps and by X-ray angiograms. This suggests that myocardial ASL with vasodilation may have a potential to identify ischemic myocardial segments in patients with stenosis.