However, under conditions that maximize CodY activity, braB expression was similar in wild-type and codY null mutant cells. Nonetheless, expression from the braB promoter was significantly elevated in cells containing partially active mutant versions of CodY or in wild-type cells under growth conditions leading to intermediate levels of CodY activity. This novel pattern of regulation was shown to be due to two opposing mechanisms, negative and positive, by which CodY affects braB expression. A strong CodY-binding site located downstream of the transcription start point conferred negative regulation by direct interaction with CodY. Additionally, sequences upstream and downstream of the promoter were required for repression by a second pleiotropic B. subtilis regulator, ScoC, whose own expression is repressed by CodY. ScoC-mediated repression of braB in codY null mutants cells was as efficient as direct, CodY-mediated repression in wild-type cells under conditions of high CodY activity. However, under conditions of reduced CodY activity, CodY-mediated repression was relieved to a greater extent than ScoC-mediated repression was increased, leading to elevated braB expression. We conclude that restricting increased expression of braB to conditions of moderate nutrient limitation is the raison d’être of the feed-forward regulatory loop formed by CodY and ScoC at the braB promoter. The increase in BraB expression only at intermediate activities of CodY may facilitate the uptake of BCAA when they are not in excess but prevent unneeded BraB synthesis when other BCAA transporters are active.The global transcriptional regulator, CodY, binds strongly to the regulatory region of the  Bacillus subtilis BraB, a branched-chain amino acid permease, is under both negative and positive control by a global transcriptional regulator CodY. The negative control is direct and the positive control is indirect and mediated by another B. subtilis pleiotropic transcriptional regulator, ScoC, which, in turn, is repressed by CodY. Thus, CodY and ScoC form a feed-forward regulatory loop at the braB promoter. In a very unusual manner, the interaction of CodY and ScoC results in high braB expression only at intermediate CodY activities; braB expression remains low both at high and low CodY activities. The novel regulation of braB shows that important, novel regulatory phenomena can be missed by analyzing null mutants in regulatory genes but revealed by using mutants with partial activity.Expression of  Bacillus subtilis   –11 and Ge (ILV)] , 11–14. braB gene proved to be directly repressed by two proteins, CodY and another pleiotropic regulator, ScoC (formerly known as hpr or catA)  and  and braBbraB242-gfp translational fusion was introduced into the wild-type strain, the codY null mutant, and a codY point mutant (R61K) strain with intermediate residual activity. In all cases, the level of braB expression was rather similar across the cell population (codY (R61K) mutant strain had elevated expression compared to the wild-type and codY null mutant strains.A pulation , eliminabraB regulation observed results from CodY binding independently to negative and positive regulatory sites within the braB regulatory region. If so, the positive and negative effects of fully active CodY might balance each other, but, at intermediate levels of CodY activity, positive regulation might dominate. The CodY-binding sites I and II are located upstream and downstream of the braB promoter in positions appropriate for positive and negative regulation, respectively. To determine their independent effects, we created additional lacZ fusions containing truncated versions of the braB regulatory region lacking the upstream CodY-binding site III (braB184-lacZ and braB162-lacZ) or sites III and I (braB144-lacZ) or the downstream site II (braB156-lacZ) We initially hypothesized that the very unusual pattern of 56-lacZ) .  (To confirm that site I is not involved in ctively) .braB242-lacZ fusion .The p3 mutation reduced the affinity of CodY for site II ≥10-fold . The p2 braB242-lacZ fusion 8-fold consistent with relief from CodY-mediated repression  than did CodY (KD≈5 nM) (braB-CodY complex as evidenced by formation of ScoC-specific complexes with higher mobility and the decrease in the amount of braB-CodY complexes with lower mobility (braB-ScoC complex cannot be recognized confidently because of the low mobility of CodY-specific complexes (complexes containing both proteins would have a similar low mobility). However, by comparing and braB-ScoC complexes than to free braB DNA, confirming competition between the two proteins for binding. A similar competition between CodY and ScoC was previously detected at the oppA promoter -ATP. oBB67 and oBB358 start 96 bp or 12 bp upstream of the XbaI site (and 112 bp or 28 bp upstream of the EcoRI site) used for cloning, respectively, and oBB102 starts 36 bp downstream of the HindIII site that serves as a junction between the promoters and the lacZ part of the braB fusion.The PCR products containing the regulatory region of the The procedures for gel shift and DNase I footprinting experiments were as described .braB PCR product, containing the entire braB regulatory region, was synthesized with oBB724 and oBB725 as primers. Plasmid pBB1845 (braB242-gfp) was created by cloning the EcoRI- and SalI-treated PCR product between the EcoRI and XhoI sites of an integrative plasmid pMMB759 (tet), containing a gene encoding a monomeric version (A206K) of GFPmut2 [braB insert within pBB1845 was identical to the insert in pBB1593 (braB242-lacZ). B. subtilis strain BB4082 carrying the braB242-gfp fusion at the lacA locus was isolated after transforming strain BB2263 (lacA::spc) with pBB1845, by selecting for resistance to tetracycline, conferred by the plasmid, and screening for loss of the spectinomycin-resistance marker, which indicated a double crossover, homologous recombination event.A 0.24-kb braB242-gfp fusion, were grown until mid- to late-exponential phase in TSS + 16 aa medium, centrifuged and resuspended in TSS medium at OD600≈3. The images were collected at a 1,500 ms exposure time using the 100x (1.3 N.A.) objective of the Zeiss Axio Observer.Z1 fluorescent microscope (Zeiss) with the Colibri.2 LED light source, and the ORCA-R2 digital charge-coupled device camera . Zen Pro 2012 software (Zeiss) was used to acquire, view, and analyze the images.Cells, containing the 5 and His6-ScoC were purified to near homogeneity as described previously [CodY-Hiseviously , 24.β-Galactosidase specific activity was determined as described previously .braB transcript abundance during exponential growth using oligonucleotides oSRB339 and oSRB340 as described [B. subtilis strain SMY chromosomal DNA to generate the standard curve. rpoC transcript was used to normalize mRNA abundance.RNA isolation, DNA depletion, and cDNA synthesis were performed as previously described . Quantitescribed , except


Tax is a powerful activator of the NF-kB pathway, a property shown to be required for HTLV-1-induced immortalization of primary T lymphocytes. A pivotal step in the stimulation of this pathway is the activation of the cytoplasmic IkB-kinase (IKK) complex, which consists of two catalytic subunits, IKK-alpha and beta and a regulatory subunit, IKK-gamma/NEMO. Previous studies showed that the ability of Tax to bind to and activate the IKK complex depends on its prior conjugation to ubiquitin. TRAF5, a member of the TNF Receptor-Associated Factor family, is an adaptor protein and E3 ubiquitin ligase which functions downstream various membrane receptors, notably for the activation of the NF-kB pathway. Interestingly, TRAF5 was also shown to interact with the Epstein Barr Virus (EBV)-encoded LMP1 oncoprotein and to contribute to LMP1-induced IKK activation. In this study, we investigated whether TRAF5 could also be a functional partner of Tax. We found that overexpressing TRAF5 significantly increases endogenous Tax ubiquitination while conversely endogenous Tax ubiquitination is reduced upon siRNA-mediated TRAF5 silencing. Surprisingly, preventing TRAF5-mediated Tax ubiquitination by siRNA depletion of TRAF5 does not affect Tax binding to endogenous NEMO. However, Tax-induced phosphorylation of IKK-alpha/beta is significantly decreased in the same setting, which coincided with a decreased ability of Tax to activate a NF-kB promoter. These findings reveal that TRAF5 mediates Tax ubiquitination for IKK activation and suggest that Tax binding to NEMO and Tax-induced IKK phosphorylation are regulated by distinct molecular determinants.


Protease inhibitors might be sufficient to maintain complete virological suppression when used as monotherapy for HIV-1-positive patients who have achieved sustained virological suppression on combination antiretroviral therapy (ART). The present study estimated the cost-effectiveness of a strategy of switching the ART to protease inhibitor monotherapy (PIM) with prompt return to combination therapy in the event of viral load rebound compared to continuing the ongoing triple-therapy (OTT) in the long-term management of HIV-1-positive patients.3. Outcomes were NHS costs (2012 UK pounds sterling) and quality-adjusted life-years (QALY) with comparative results presented as incremental cost-effectiveness ratios (ICERs).A within-trial cost-effectiveness analysis and modelling of lifetime cost-effectiveness was performed based on a randomized controlled trial of Protease Inhibitor monotherapy Versus Ongoing Triple-therapy (PIVOT). The setting was HIV outpatient care in the UK National Health Service, and the trial involved 587 patients, aged 18 years or more, who achieved sustained virological suppression and have a CD4+ cell count >100 cells/mmOverall, PIM was cost-effective compared to OTT. PIM was cost-saving due to large savings in the ART drug costs while being no less effective in terms of QALYs in the within-trial analysis and only marginally less effective with modelling. In the base-case within-trial analysis, the incremental total cost per patient was −£6,424.11  and the incremental QALY was 0.0051  making PIM dominant compared to OTT. Multiple sensitivity analyses were conducted to assess the importance of assumptions surrounding drug costs, missing data, trial protocol driven costs and mortality. In all sensitivity analyses, PIM was cost-saving and no marked difference in QALY was observed. Modelling of life time costs and QALYs showed significant cost-savings and marginally less effectiveness such that switching to PIM appeared cost-effective at accepted cost-effectiveness thresholds.The results suggest that PIM is a cost-effective treatment strategy compared to OTT for HIV-1-positive patients who have achieved sustained virological suppression.


Following infection and integration into the genome of host CD4+ T-lymphocytes, human T-lymphotropic virus type-1 (HTLV-1) persists by driving proliferation of infected T-cell clones which are counter-selected by HTLV-1-specific cytotoxic T-lymphocytes (CTLs). HTLV-1 Tax protein is the dominant target antigen recognized by these CTLs, and Tax is thought to cause the infected T-cell proliferation. In 40% of cases of adult T-cell leukaemia/lymphoma (ATLL) Tax protein is not expressed, suggesting that the provirus has down-regulated Tax to allow immune escape and viral persistence. Here, we tested the hypothesis that the genomic site of proviral integration determines the degree of clonal expansion and therefore determines the risk of ATLL. Proviral integration sites in 242 ATLL cases and 96 asymptomatic carriers were mapped and quantified using our recently described high-throughput technique . In the ATLL cases, we identified promoter deletions predicted to cause tax gene silencing, we sequenced the tax gene and quantified promoter methylation. The results showed that 65% cases of ATLL could silence Tax by promoter deletion, tax gene nonsense mutations or tax promoter hypermethylation. High-throughput sequencing data showed that ATLL cases were not invariably monoclonal and that the provirus is typically integrated into a transcriptionally active region of the host genome. The results also suggest that transcriptional activity of the flanking host genome contributes to tax gene silencing, allowing CTL escape and clonal expansion.


Transcriptional regulation of protein-coding genes is increasingly well-understood on a global scale, yet no comparable information exists for long non-coding RNA (lncRNA) genes, which were recently recognized to be as numerous as protein-coding genes in mammalian genomes. We performed a genome-wide comparative analysis of the promoters of human lncRNA and protein-coding genes, finding global differences in specific genetic and epigenetic features relevant to transcriptional regulation. These two groups of genes are hence subject to separate transcriptional regulatory programs, including distinct transcription factor (TF) proteins that significantly favor lncRNA, rather than coding-gene, promoters. We report a specific signature of promoter-proximal transcriptional regulation of lncRNA genes, including several distinct transcription factor binding sites (TFBS). Experimental DNase I hypersensitive site profiles are consistent with active configurations of these lncRNA TFBS sets in diverse human cell types. TFBS ChIP-seq datasets confirm the binding events that we predicted using computational approaches for a subset of factors. For several TFs known to be directly regulated by lncRNAs, we find that their putative TFBSs are enriched at lncRNA promoters, suggesting that the TFs and the lncRNAs may participate in a bidirectional feedback loop regulatory network. Accordingly, cells may be able to modulate lncRNA expression levels independently of mRNA levels via distinct regulatory pathways. Our results also raise the possibility that, given the historical reliance on protein-coding gene catalogs to define the chromatin states of active promoters, a revision of these chromatin signature profiles to incorporate expressed lncRNA genes is warranted in the future. Evidence for important, including essential, cellular and organismal roles of lncRNA in mammalian systems began to emerge prior to the advent of high-throughput genome and transcriptome sequencing. These early examples included the demonstration that the lncRNA XIST Many lncRNA transcripts, similarly to mRNAs, are 5′-capped, polyadenylated, frequently spliced with conventional GT-AG intron excision, and readily evident in cytoplasmic polyA + RNA preparations; thousands of lncRNAs have been discovered from cDNA libraries in vivo human brain These diverse functional mechanisms summarily indicate that jointly with TFs, lncRNAs are key regulators of protein-coding genes – including those that encode TFs. A prerequisite toward understanding the biology of lncRNAs is their assignment into tractable gene regulatory networks. We previously showed In this study, our goal was to computationally test the hypothesis that the global transcriptional regulatory programs of lncRNA genes and protein-coding genes are different. We set this problem within the framework of machine learning classification of promoters of these two broad gene classes. Previous studies We compared DNA sequence promoter patterns of protein-coding and lncRNA genes. We found that A/T-rich mono-, di- and tri-nucleotide patterns are enriched at the promoters of lncRNA genes, relative to the promoters of protein-coding genes  . CpG-derPalindromes, widespread regulatory elements in the promoters of protein coding genes in silico predicted the incidence of known transcription factor binding sequences (TFBSs) at the promoters of both gene types, using the HOCOMOCO We cis-regulated by two lncRNAs: an lncRNA containing primate-specific repetitive elements provides transcriptional regulation cis-antisense transcript acts post-transcriptionally Several TFs regulated by specific lncRNAs emerge as potential global regulators of lncRNA transcriptome in our analysis. A representative example is PGR (progesterone receptor), a nuclear hormone receptor (NHR), whose predicted TFBSs are differentially enriched at lncRNA promoters. The human PGR gene itself is cis-antisense lncRNA at its own locus cis and in trans by multiple lncRNAs in silico predicted binding site results for homeobox TFs at the promoters of lncRNA genes are consistent with a recent evolutionary study We additionally found that 14 FOX-family TFs, 6 SOX-family TFs, 3 members of the HOXD homeobox family, 3 members of the CEBP family, 3 NKX-family TFs, and 2 PPAR TFs  demonstrab initio motif discovery to genome-wide promoters, in order to complement the HOCOMOCO results. Ab initio identified motif families (MFs) generated by the Dragon Motif Finder The human proteome harbors approximately 1500 TFs de novo mutations may also alter a sequence of a particular binding site complicating known-TFBS discovery ab initio MFs might reflect condition-driven, or protein complex-dependent, deviations from known TFBS models. We therefore compared the ab initio identified MFs to those already associated with known TFs. We confirmed five models and added one new TFBS model (NKX3-2) to our roster of lncRNA-promoter-enriched TFBSs  in eight human cell lines Protein-coding gene promoters more often overlapped CSs associated with active, weak or inactive/poised promoters, and were also more strongly enriched for Polycomb-repressed regions. Relative to protein-coding gene promoters, those of lncRNA genes more often overlapped CSs associated with insulators, regions of transcriptional transition (regions located between the initiation and elongation histone marks), elongation, weak transcription and heterochromatin . After tTo understand the biological context of the heterochromatin CS enrichment at lncRNA gene promoters, we analyzed histone modification marks (HMs) in the ENCODE Tier 1 cell line GM12878. LncRNA gene promoters were significantly depleted of almost all histone modification marks, except for H3K27me3 and H3K9me3 . H3K27meSeveral lines of evidence indicate that the transcriptional regulation of lncRNAs may differ substantially from that of protein-coding genes. To computationally test for any evidence of this phenomenon, we leveraged recent advances in machine learning to fit an integrative model based on the information from all analyzed data types to distinguish the promoters of protein-coding genes from those of lncRNAs. Our fitted ensemble model correctly classified the promoters (lncRNA or protein-coding) with more than 80% accuracy. Hence, across the majority of the genome sequence space, genetic and epigenetic information is sufficient to confidently separate these two classes of promoters . InterroSince we had originally considered the regions of  bp around the TSS  as a putLncRNAs show lower expression in almost all cell types as compared to mRNAs We aimed to assess the extent of experimental support for transcriptionally permissive chromatin configurations across all TFBSs enriched at lncRNA promoters. We reasoned that binding sites should have an open chromatin configuration in the cell or tissue types where binding occurs. We leveraged the empirical genome-wide catalog of DNase I hypersensitive sites, indicative of open chromatin, derived for 125 human cell types by the ENCODE Consortium In parallel, we overlapped lncRNA-promoter-enriched TFBSs with ENCODE ChIP-seq experimental evidence for the corresponding TFs across all ENCODE ChIP-seq datasets We present the first genome-wide demonstration of a significant difference in sequence characteristics between the promoters of human lncRNA and protein-coding genes, suggesting distinct regulation of the two gene groups. In view of the frequent bidirectionality of human promoters that simultaneously give rise to protein-coding and lncRNA genes in silico predicted TFBSs that we have linked to global catalogs of lncRNA promoters and to lncRNA regulatory programs modulated by specific TFs. By virtue of their TFBS localization, such variants may emerge as direct functional candidates.Our observation that lncRNAs might be selectively regulated by a distinct set of TFs has substantial implications for systems biology: cells are potentially capable of harnessing a defined subset of regulatory switches to toggle the expression levels of lncRNAs without altering mRNA levels. Most of the disease-associated sequence variants in the human genome are non-coding Our lncRNA gene collection is a composite of three previously published lncRNA sources – Gencode Until recently, only protein-coding gene sets were used in characterizing general promoter features. Therefore, some widely accepted promoter features and chromatin state signatures may be biased as a consequence of having been inferred from protein-coding genes. In this context, it is hardly surprising that certain sequence and epigenetic features, more specific for protein-coding genes, are less pronounced at lncRNA promoters, while the chromatin states associated with lncRNA promoters are predominantly labeled as inactive promoters. However, these labels were based on manual annotation by biologists, predominantly of protein-coding and intergenic regions http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/refGene.txt.gz, download date: 14 January, 2013) for the human genome (version hg19). Out of the 44,140 transcripts, we considered only the 34,475 that were clearly protein-coding (i.e. having an NM RefSeqID) and that were located on chromosomes 1–22, X, and Y. To construct a non-redundant (a single reference transcript per gene) set, we considered at least 1 bp overlap in the entire genomic span  among all transcripts located on the same strand in the same locus, and we randomly selected one transcript per locus. Through these filtering steps, we ultimately arrived at 18,789 protein-coding non-redundant representative transcripts conforming to our one-transcript-per-gene data structure (We used RefSeq transcripts from the UCSC Genome Database (tructure .www.gencodegenes.org), a manually curated list of coding and non-coding genes that are supported by full-length cDNA, EST, RNAseq, and targeted-RTPCR evidence from public sources as well as from Gencode's ongoing validation efforts. We enhanced this collection with non-redundant lncRNAs from three additional sources: 4) NCBI RefSeq (NR identifier) non-coding transcripts that do not host any known small RNAs according to the UCSC Genome Database sno/miRNA repository, 5) human ESTs from the dbEST division of Genbank (NCBI) We also assembled 18,498  experimewww.galaxyproject.org/).For each gene's representative transcript, we considered the  bp around the TSS as the putative promoter region, except in the specific analyses listed under Results where an alternate  bp TSS set was used. We chose relatively large promoter regions with the purpose of incorporating alternative TSS, which in turn allowed us focus on gene-based rather than transcript-based analysis, since alternative promoter usage is a widespread phenomenon in human transcriptome To identify the regulatory patterns which may facilitate the computational discrimination between the promoters of protein-coding genes and lncRNA genes, we extracted features from several broad categories. These include various frequency-based properties of the promoters such as k-mers, word commonality, skew, palindromes; regulatory elements such as CpG islands, repetitive elements, TFBS found within the promoter regions; epigenetic features such as chromatin states and separate histone modification marks (see http://www.cbrc.kaust.edu.sa/hocomoco/Download.php) in the promoters of both protein-coding and lncRNA genes. Since the extent to which the original nucleotide composition of promoters is a cause or a consequence of the possible TFBS repertoires present in these promoters is unclear, we used the same strategy for both protein-coding and lncRNA promoters. For each PWM the threshold was set in the following way: for a random word generated by a background model (independent nucleotide distribution with nucleotide frequency of hg19) there was a fixed probability of 0.0005 to obtain the PWM score no less than the threshold. We generated 426 features using the binary value 0 or 1 (zero or non-zero hits above the threshold in a given promoter sequence in both strands). We selected significantly overrepresented TFBSs in promoters of protein-coding vs. promoters of lncRNA (and vice versa) gene sets  We predicted TFBSs using 426 position weight matrices (PWMs) for 401 human TFs from the HOCOMOCO http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeCshlLongRnaSeq/) and we quantified the expression levels as RPKM (read per kilobase of exon per million mapped reads) We used RNA-seq data from Gm12878, H1-hESC, K562 and HUVEC cell lines to check the model performance, when expression levels of lncRNAs and protein-coding genes are similar. We used the mappings, provided by ENCODE (www.uniprot.org) and the GeneCards resource (www.genecards.org) to compile a comprehensive list of human transcription factors that accounts for all name multiplicity, synonymity, and redundancy between the abbreviated transcription factor names used by HOCOMOCO (since Uniprot naming is in one-to-one relationship with the HOCOMOCO naming system) and ENCODE. We manually curated this list. We identified 106 . Green line corresponds to promoters of protein-coding genes; black line corresponds to lncRNA gene promoters. Sub-figure. a-d show distribution of the feature in a sliding window of 100 bp with a step of 50 bp, resulted in 39 windows on the plot. Sub-figure. e–f show the percentage of promoters where features were found. Transparent regions correspond to 5–95% bootstrap confidence interval of the statistics. WC: word commonality, PALIN: palindromes, CGI: CpG Islands, RE: repetitive elements. The enrichment score was calculated using right-sided exact Fisher's test Click here for additional data file.Figure S2Logos for over-represented ab initio identified motif families (MFs) from promoters of a) protein-coding genes in CPS, b) lncRNA genes in CPS, c) protein-coding genes in REFPS and d) lncRNA genes in REFPS. Logos for ab initio motif families (MFs), corresponding reverse complement (RC) MF and known TFBS match by TOMTOM system from promoters of e) lncRNA genes in CPS, f) lncRNA genes in REFPS.(PDF)Click here for additional data file.Figure S3Distribution of chromatin states in cell lines with normal karyotypes across promoters of protein-coding and lncRNA genes. Blue bar corresponds to promoters of coding genes from repeat-filtered promoter set (REFPS), green bar corresponds to promoters of coding genes from complete promoter set (CPS), red bar corresponds to promoters of lncRNAs from REFPS, and black bar corresponds to promoters of lncRNAs from CPS. This figure demonstrates fraction of all promoters overlapping with chromatin states. At the end of each bar 5–95% bootstrap confidence interval of the statistic is shown. AP: Active Promoter, WP: Weak Promoter, IP: Inactive Promoter, SE: Strong Enhancer, WE: Weak Enhancer, I: Insulator, TT: Transcriptional Transition, TE: Transcriptional Elongation, WT: Weakly Transcribed, PR: Polycomb Repressed, HC: Heterochromatin low signal, RP: Repetitive/Copy number variation.(PDF)Click here for additional data file.Figure S4Distribution of histone modification marks, modified histone H2AZ, CTCF, and the Polycomb-group protein (PRC2 complex component) EZH2 in cell lines across lncRNA and protein-coding gene promoters.(PDF)Click here for additional data file.Figure S5Distribution of chromatin states in cell lines with normal karyotypes across promoters of protein-coding and lncRNA genes with similar expression. Green bar corresponds to promoters of coding genes from complete promoter set (CPS), black bar corresponds to promoters of lncRNAs from CPS. This figure demonstrates percentage of all promoters overlapping with chromatin states. At the end of each bar 5–95% bootstrap confidence interval of the statistic is shown. AP: Active Promoter, WP: Weak Promoter, IP: Inactive Promoter, SE: Strong Enhancer, WE: Weak Enhancer, I: Insulator, TT: Transcriptional Transition, TE: Transcriptional Elongation, WT: Weakly Transcribed, PR: Polycomb Repressed, HC: Heterochromatin low signal, RP: Repetitive/Copy number variation.(PDF)Click here for additional data file.Figure S6Boxplot and Quartile-Quartile plot for expression value of protein-coding genes and lncRNA genes from complete promoter set (CPS) in different cell lines.(PDF)Click here for additional data file.Table S1Mono-, di-, and tri-nucleotides frequency and observed/expected ratio for both complete promoter set (CPS) and repeat-filtered promoter set (REFPS).(PDF)Click here for additional data file.Table S2Transcription factor binding sites overrepresented in promoters of protein-coding and lncRNA genes for complete promoter set (CPS) and repeat-filtered promoter set (REFPS) and support provided by DNAseI and ChIP-seq peaks.(PDF)Click here for additional data file.Table S3P-values of overrepresentation for chromatin states, CpG islands, repetitive elements and palindromes for complete promoter set (CPS) and repeat-filtered promoter set (REFPS).(PDF)Click here for additional data file.Table S4a. Summary of the results for separation of promoters of protein-coding and lncRNA genes using different combinations of features for the complete promoter set (CPS) and repeat-filtered promoter set (REFPS). For REFPS, we used all types of repeats except “simple repeats”, “low complexity regions” and “satellite repeats”. k-mer: mono-, di-,tri-nucleotide frequencies, CS: chromatin states, WC: word commonality, TFBS: transcription factor binding sites, CGI: CpG Islands, SKEW: A/T and C/G skews, PALIN: palindromes, RE: repetitive elements, COMBINE: combination of all types of features. b. Summary of the cross validation (CV) results for separation of promoters of protein-coding from lncRNA genes using all features (COMBINE) for completer promoter set (CPS) and repeat-filtered promoter set (REFPS).(PDF)Click here for additional data file.Table S5Results from execution of the computational model from promoters considering only upstream  of TSS, as well as from promoters considering only upstream  of TSS having no overlap with CGI.(PDF)Click here for additional data file.Table S6P-values of overrepresentation for chromatin states for similarly expressed genes promoter in complete promoter set (CPS).(PDF)Click here for additional data file.Text S1Supporting information for the methods applied and results obtained. The details of methods are described under Methods section. The details of results are described under Results section.(DOCX)Click here for additional data file.Dataset S1Set of a) RefSeq and b) lncRNA transcripts with hg19 human genome assembly coordinates in BED format for complete promoter set (CPS). Set of c) RefSeq and d) lncRNA transcripts with co-ordinates from hg19 in bed format for repeat-filtered promoter set (REFPS). Set of e) RefSeq and f) lncRNA promoters ([−250…+250]) with co-ordinates from hg19 in bed format for complete promoter set (CPS). Set of g) RefSeq and h) lncRNA promoters ([−250…+250]) with co-ordinates from hg19 in bed format for repeat-filtered promoter set (REFPS). i) “ENCODE_HOCOMOCO_mapping” - excel sheet contains mapping of ENCODE transcription factor name and HOCOMOCO V.8 motif name. Excel sheet “track_MEF2A_chip”, “track_GATA3_chip”, “track_ARI3A_chip” contains the track information for ENCODE ChIP-seq supported TFBS in CPS for MEF2A,GATA3 and ARI3A respectively.(ZIP)Click here for additional data file.Dataset S2Promoters, considering only upstream , of a) RefSeq and b) lncRNA transcripts with hg19 coordinates in BED format for complete promoter set (CPS). Promoters, considering only upstream , of c) RefSeq and d) lncRNA transcripts, having no overlap with CpG islands, with hg19 coordinates in BED format for CPS.(ZIP)Click here for additional data file.Dataset S3RNA-seq expression value in RPKM (read per kilobase of exon per million mapped reads) for lncRNA genes and protein-coding genes in complete promoter set (CPS).(ZIP)Click here for additional data file.


The viral G protein-coupled receptor (vGPCR) is proposed to act as one of the predominant mediators of Kaposi’s sarcoma (KS), a human herpes virus 8 (HHV8)-elicited disease. The actions of vGPCR manifest pathogenesis, in part, through increased permeability of endothelial cells. Endothelial cell-cell junctions have indeed emerged as an instrumental target involved in the vasculature defects observed within the tumor microenvironment. The pathway leading to adherens junction destabilization has been shown to involve the activation of the small GTPase Rac, in the context of either latent infection or the sole expression of vGPCR. However, the precise molecular mechanisms governed by vGPCR in vascular leakage require further elucidation.in vitro.Guanine exchange factors (GEFs) function as critical molecular switches that control the activation of small GTPases. We therefore screened the effects of 80 siRNAs targeting GEFs on vGPCR-driven endothelial permeability and identified switch-associated protein 70 (SWAP70) as necessary for its elevating effects. Pull-down experiments further showed that Rac activation by vGPCR was dependent on SWAP70. Examination of tissues and cells from HHV8-positive patients revealed that SWAP70 was ubiquitously expressed. Furthermore, SWAP70 was found to be crucial for vGPCR-driven endothelial tube formation and endothelial sprouting SWAP70 appears to act as a molecular intermediate between vGPCR and endothelial activation. Because of the important role of vGPCR-mediated endothelial plasticity in KS pathogenesis, inhibition of SWAP70 function could be of interest for blocking vGPCR-driven activities in HHV8-defined diseases.The online version of this article (doi:10.1186/s12964-015-0090-1) contains supplementary material, which is available to authorized users. Of the multiple genes encoded by the human herpes virus 8 (HHV8), the viral G protein-coupled receptor (vGPCR) is one of the key instigators of Kaposi’s sarcoma (KS) pathogenesis . vGPCR i3 (PIP3), which binds its pleckstrin homology (PH) domain and recruits the protein to the membrane [We aimed to identify molecular switches involved in Rac-dependent vGPCR-driven endothelial permeability. Because vGPCR hijacks IL-8-induced intracellular signaling pathways ,4,10, wemembrane . InteresWe first confirmed that silencing SWAP70 impaired vGPCR-driven endothelial permeability using an independent siRNA sequence Figure A. As expTo examine whether SWAP70 expression may be associated with HHV8 infection, its gene expression levels were measured in KS patient-derived tissues, HHV8-positive cell lines, and KS patient-isolated peripheral B cells Figure A. SWAP70in vitro tubulogenesis was tested under serum-free conditions [To further evaluate the role of SWAP70 in endothelial homeostasis in the context of vGPCR expression, previously established vGPCR-positive SV40-transformed mouse endothelial cells (SVEC-vGPCR)  were furnditions . While tnditions  was alsoIn this study, SWAP70 was identified as a protein required for vGPCR-driven endothelial permeability through a genetic screen using siRNAs against 80 referenced human GEFs. In keeping with its conserved role as a GEF ,20, it wSWAP70 was also necessary for vGPCR-mediated endothelial tubulogenesis and sprouting. These two assays measure the migratory and invasive capacity of endothelial cells as an indicator of angiogenic potential and plasticity. Thus, SWAP70 appears to be essential for the angiogenic phenotype imposed on endothelial cells by vGPCR ,2,7. In It should be mentioned that five additional GEFs were identified in the screen that also impaired vGPCR/IL-8-induced permeability. Owing to their lack of reported Rac GEF activity, they were not further explored. However, it is interesting to speculate that they potentially play additive roles in vGPCR-promoted permeability via Rac-independent pathways. Thus their role in vGPCR-promoted permeability should be deciphered in future studies.In summary, this study identifies the Rac GEF SWAP70 as a mediator of permeability downstream of both vGPCR and IL-8. Blockade of SWAP70 function could be of therapeutic value to prevent permeability and activation of endothelial cells and thus hinder tumor angiogenesis.The data sets and methods description supporting the results of this article are included within the article and its Additional file


Intraplatelet vasodilator-stimulated phosphoprotein (VASP) analysis is a commonly used laboratory approach for monitoring of the anti-platelet therapy with adenosine diphosphate (ADP) receptor blocking agents; however, it’s testing in clinical laboratory needs a high level of experience and proficiency. The ability to recognize how the pre-analytical variations can change the results would be helpful for the interpretation of data from intraplatelet VASP analysis. The aim of this study was to describe the possible differences of intraplatelet phospho-VASP expression between washed and platelet rich plasma (PRP) samples, both at baseline levels and following experimentally induction of VASP phosphorylation. 157-VASP or anti P-Serine239-VASP, the samples were subjected to flow cytometric analysis to monitor the levels of intraplatelet phospho-VASP expression.PRP and washed platelet samples were treated with different inducers of VASP phosphorylation, including forskolin (10 µM), prostaglandin E1 (PGE1) (50 nM) and sodium nitro-prusside (SNP) (100 µM). Untreated PRP and washed platelet samples were also included in study as baseline controls. After labeling of platelets with either anti P-Serine157-VASP at baseline state and also more expression of P-Serine157-VASP and P-Serine239-VASP in response to forskolin and SNP, compared with PRP samples. Though, reduced levels of PGE1-induced VASP phosphorylation at both residues were detected for washed platelets.  Washed platelet samples tend to show increased expression of intraplatelet P-SerineIn this study we have provided some background information required for performing of intraplatelet VASP analysis on differently handled platelet samples and interpretation of the obtained results. Vasodilator-stimulated phosphoprotein (VASP) is a regulator of actin reorganization in platelets. VASP-actin interaction prevents capping proteins from binding to actin, therefore allows growing of the actin polymers . Phospho in-vitro approach for monitoring of anti-platelet therapy with adenosine diphosphate (ADP) receptor antagonists  containing freshly added 50 u/mL heparin. After centrifugation at 800 x g for 10 min at 25 °C, supernatant was removed and the pellet was rinsed without resuspension by gently adding of citrate wash buffer and removing slowly. This type of rinsing step was applied to improve recovery of platelet number at the end of the procedure. Then modified Tyrode’s-HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer containing 0.02 u/mL apyrase was added on platelet pellet. The sample was then incubated for 15 min at 37 °C before resuspension. The concentration of platelets in washed samples was adjusted to the equal level of PRP samples and allowed to rest at least for 1 hour at 25 °C prior to be used in experiments.According to the ethical committee guidelines of our institute, blood samples were obtained with consent from healthy donors who denied taking anti-platelet drugs for at least two weeks in plastic tubes containing sodium citrate 3.8 %. PRP was prepared from whole blood samples according to our in house protocol . Each PRExperimental treatments on washed and PRP samplesPRP and washed platelet samples were incubated in the presence of 50 nM PGE1 (Sigma), 10 µM forskolin , or 100 µM SNP (Merck). Untreated PRP and washed platelet samples were also included in each run of the experiments as baseline controls. After 10 min incubation at room temperature on shaker, the treatments were terminated by adding Para-formaldehyde .Flow cytometry analysis6 platelets were then incubated with a specific primary antibody  for 14 hour at 4 °C with gentle shaking. Primary antibodies utilized were polyclonal rabbit anti-P-Ser157-VASP and polyclonal rabbit anti-P-Ser239-VASP . After a washing step, FITC-conjugated goat anti-rabbit polyclonal IgG  was used as secondary antibody. Flow cytometric analysis of 10,000 events in platelet area / sample was performed using a flow cytometer . Platelets were gated by forward (FSC)/ side (SSC) scatter characteristics, the median fluorescence intensity (MFI) was quantified for gated events using Flomax instrument software. In each run of experiments, a platelet sample labeled only with secondary antibody was included as negative control to exclude the fluorescence due to non-specific binding of secondary antibodies.The platelet samples which were fixed in Para-formaldehyde, permeabilized in 0.5% Triton X-100 in PBS for 30 min at 4 °C. Samples containing 20x10After each run of experiments on either PRP or washed platelet samples, MFI value obtained for untreated sample was considered as baseline and the percent of MFI shift from baseline, representing the percent of induced change in P-VASP expression, for a forskolin-, PGE1-, or SNP-treated sample was calculated, using formulaStatistical analysisStatistical significance was analyzed using non-parametric analysis of variance.157-VASP and anti-P-ser239-VASP, were subjected to flow cytometric analysis. The gating strategy which was applied has been described in PRP and washed platelet samples, labeled by anti-P-ser157-VASP and P-ser239-VASP were quantified by flow cytometry software, as median fluorescence intensity value (MFI). From the graphs in 157-VASP at baseline level for washed platelets, however, no significant differences were found between two types of the samples in expression of P-Ser239-VASP at their baseline levels, graph (B). The graphs also indicate more levels of forskolin- and SNP-induced VASP phosphorylation at Serine 157, 239 for washed platelets, whereas, reduced levels of PGE1-induced VASP phosphorylation at both residues were detected for washed platelets.Expressions of intraplatelet P-serResearch and clinical laboratory experiments on platelets may be conducted on whole blood, PRP, or washed platelet samples. Variati157-VASP expression at baseline for washed platelets, compared with the platelets existed in PRP samples. The elevation of Ser157-VASP expression in thrombin and collagen activated platelets has been also reported, previously . Our results indicated more levels of P-Sereviously , 22. Plape again . In this239-VASP expression at baseline state between washed and PRP samples. This finding is consistent with the data from previous studies, indicating unchanged levels of P-Ser239-VASP in agonist activated platelets  cascade . In spitVariability of intraplatelet P-VASP expression observed between PRP and washed platelet samples might be related to the mechanical stress, which could be exerted on platelets by centrifugation and other procedures of washing step, however, possible effects of matrix components within different group of samples cannot be also ignored. One other caveat that must be noted here is that the results from this study may have been influenced by test-specific properties of applied method (flow cytometry). Further investigations using ELISA-based P-VASP analysis are required to be undertaken for estimating the effect of those factors.Our results suggested that the dynamics of phospho-VASP expression in platelets is sensitive to be changed due to variation in pre-analysis samples preparations. The Patterns of observed changes were different, regarding the type of P-VASP inducer and also the amino acid residue of phosphorylation on VASP molecule. The data provided from this study might be considered as an integral part of background information that is required for performing of intraplatelet P-VASP analysis and interpretation of the results. An extensive attention to avoid platelet stimulation during blood sampling and processing might be important to prevent artifactual variations of P-VASP data in platelet pharmacology.


One form of meditation training, the integrative body-mind training (IBMT) has been shown to improve attention, reduce stress and change self-reports of mood . Here, wp < .01] and a session main effect  for TTCT. These results indicated that short-term (30 min per day for 7 days) IBMT improved creativity performance in the divergent thinking task than RT. The ANOVAs also revealed a group (IBMT vs. RT) × session (pre-training vs. post-training) interaction effect and a session main effect for positive affect (PA) and negative affect (NA) , indicating better emotional regulation than RT. In addition, the cross-lagged models [Forty healthy Chinese undergraduates were randomly assigned to short-term IBMT group or a relaxation training (RT) control group. Mood and creativity performance were assessed by the Positive and Negative Affect Schedule  and Torrd models  were useAs predicted, PA had a positive cross-lagged impact on TTCT, which indicated a causal influence from positive mood changes to the creativity changes in IBMT group but not in RT group. In addition, NA had a negative cross-lagged impact on TTCT, which indicated a causal influence from negative mood changes to the creativity changes in the IBMT group but not in RT group.Consistent with our previous research, the IBMT group significantly outperformed the RT group in TTCT scores and emotion after training. The cross-lagged analyses indicated that both positive and negative mood changes may contribute to the creativity changes following IBMT. Our results suggested that emotion-related creativity-promoting mechanism may be attributed to short-term meditation. Modeling emotion-creativity interaction using cross-lagged analysis may open up an important avenue for studying meditation-emotion-creativity relationships.


PSD95 Dlg ZO-1 (PDZ) domain spanning residues 106–212 (CPAF106-212). This PDZ domain is unique in that it bears minimal sequence similarity to canonical PDZ-forming sequences and displays little sequence and structural similarity to known chlamydial PDZ domains. We show that the CPAF106-212 sequence is homologous to PDZ domains of human tight junction proteins.The need for more effective anti-chlamydial therapeutics has sparked research efforts geared toward further understanding chlamydial pathogenesis mechanisms. Recent studies have implicated the secreted chlamydial serine protease, chlamydial protease-like activity factor (CPAF) as potentially important for chlamydial pathogenesis. By mechanisms that remain to be elucidated, CPAF is directed to a discrete group of substrates, which are subsequently cleaved or degraded. While inspecting the previously solved CPAF crystal structure, we discovered that CPAF contains a cryptic N-terminal  Chlamydia trachomatis is one of the leading bacterial pathogens, infecting over 100 million people worldwide annually [C. trachomatis is an obligate intracellular pathogen, with fifteen different serovars affecting various mucous membranes in the body. C. trachomatis invades epithelial tissues including those in the eyes and reproductive tract, where it affects the single outer layer of columnar cells. Currently, azithromycin and doxycycline are the antibiotics of choice to combat chlamydial infections. However, because infections are asymptomatic in nearly 70 percent of cases, patients frequently fail to seek treatment [C. trachomatis stands to pose a significant public health threat in the near future, similar to that currently posed the recent drug-resistant Neisseria gonorrhoeae.annually . C. tracreatment , 4. As areatment . Based oC. trachomatis has a biphasic developmental cycle, in which infectious elementary bodies (EB) invade host cells. From there, the resulting endosomal compartment evades lysosomal degradation pathways and forms an intracellular parasitophorous vacuole termed an inclusion. From within this inclusion, C. trachomatis is effectively cloaked and protected from host immunological defenses. Chlamydial EB differentiate into metabolically active reticulate bodies (RB) that undergo replication and initiate protein synthesis [C. trachomatis secretes effectors into the host cytoplasm that perform numerous functions to maintain the infected cell in a state where the immune defenses are dampened, cell viability is enhanced, and pro-apoptotic signaling is inhibited [ynthesis . During nhibited –10. The Chlamydia that cleaves host proteins including the cytoskeleton intermediate filament vimentin, the nuclear envelope lamin-associated protein (LAP1), and also disrupts cell division [Chlamydial effector proteins are translocated into the host cell cytoplasm and dynamically remodel the inclusion, interfere with host cell apoptosis, dampen immune responses, and inflammation, as well as abrogate or co-opt signal transduction pathways. Of the known chlamydial effectors, the serine protease chlamydial protease-like activity factor (CPAF) has been identified as a factor involved in the intracellular biology of division –15. HowePSD95 Dlg ZO-1 (PDZ), Src Homology 2 (SH2), Src Homology 3 (SH3), and WW domains [Assembly of many functional signal transduction complexes are facilitated by protein-protein interaction motifs such as  domains . In part domains –20. PDZ  domains , 22. PDZ domains –25. Addi domains .106-212 sequence is most similar to PDZ domains of human proteins involved in cell polarity and epithelial tight junction formation.While inspecting the CPAF crystal structure, we discovered that CPAF contains a cryptic N-terminal PDZ domain. This PDZ domain is unique in that it bears almost no sequence similarity to canonical PDZ-forming sequences and displays minimal sequence and structural similarity to known chlamydial PDZ domains. We found that the CPAF106-212). The coordinates corresponding to residues CPAF106-212 were extracted from the mature CPAF structure and analyzed by the methods outlined below.All protein structures were retrieved from the RCSB Protein Data Bank repository . Upon in106-212 structure was compared to those of canonical PDZ domains known to bind to class I, II, and III ligands, as well as to those of unclassified PDZ domains (PDB ID): (Class I) PSD95-PDZ3306-402  [9-91  [112-193 and syntenin-PDZ2195-273  [1318-1412  [(Class II) CASK487-572  [69-153  [380-480  [362-454  [(Class III) nNOS14-100  [653-741  [1001-1096  [(Unclassified) PS2-D1P158-249  [287-379  [136-219  [6-92  [354-453  [The CPAF306–402) , NHERF2-195–273) , and erb77–1371) ; (Class  69–153) , HtrA138380–480) , and Htr01–1096) ;   and HtrA106-212 using the STructural Alignment of Multiple Proteins (STAMP) feature of the Visual Molecular Dynamics (VMD) software suite [106-212 to the representative canonical PDZ domains.Each set of PDZ domain coordinates was extracted from the full PDB coordinates and aligned to CPAFre suite , as wellre suite . Root me106-212 were submitted to the Dali Protein Structure Database server [The PDB coordinates for CPAFe server  in ordere server  to retriHomo sapiens, 13 (20%) from mouse and rat, 3 (5%) from Escherichia coli, and 2 (3%) from Drosophila melanogaster.The UniProtKB accession numbers for the resulting proteins were cross-referenced with the NCBI Conserved Domain Database (CDD)  to map a106-212 using the NCBI Protein BLAST (blastp) server [The 65 proteins mapped to 170 PDZ domain sequences, which were retrieved from the UniProtKB database and limited to the residue ranges provided by the CDD search. The PDZ domain sequences were aligned against CPAF) server , 48 to i) server . The res) server  for figuChlamydia. The chlamydial PDZ domains were cross-referenced against the UniProtKB database and the proposed function(s) for each protein was obtained from the database.We searched across all chlamydial strains and species for proteins containing annotated PDZ domains using the Pfam database . The res106-212). Canonical PDZ domains share a common fold consisting of five antiparallel beta strands and two alpha helices, in which the ligand-binding pocket is formed by the β2 strand and α2 helix, as depicted by the class I PDZ domain from PSD95 (PSD95-PDZ3)  . Variati2 domain . Additio2 domain .The residues lining the ligand-binding pocket of PDZ domains contribute significantly to peptide ligand binding and specificity. Thus, sequence similarity along the PDZ binding site hotspots is frequently a strong predictor of ligand specificity , 53. Can106-212) to representative structures of all three canonical PDZ domain classes, as well as to unclassified domains   to ident106-212, we aimed to address to what extent these domains retain sequence homology with the CPAF PDZ domain. The 170 PDZ sequences that were previously identified were retrieved from the UniProtKB database using residue ranges supplied by the NCBI CDD. These sequences were submitted for alignment against CPAF106-212 using the NCBI Protein BLAST (blastp) server [106-212. These sequences were submitted for multiple sequences alignment using the Clustal Omega webserver [Having identified a set of proteins containing PDZ domains with high structural homology to CPAF) server , 48. We ebserver , and theebserver  Fig 3)..106-212,106-212 within the PDZ domain recognition pocket. The conserved residues lie toward the core of the CPAF PDZ domain and binding pocket and may suggest that the residues are involved in establishing the PDZ fold to mimic host domains or in recognizing PDZ ligands  originate from C. trachomatis, making it the chlamydial species with the highest PDZ domain occurrence. Interestingly, all 40 of the PDZ-containing proteins identified in C. trachomatis are classified as hydrolases, in particular tail-specific and HtrA class peptidases.Having identified a cryptic PDZ domain in CPAF, we used the Pfam database to address whether PDZ domains are commonly annotated in chlamydial species and to which proteins they are coupled. We searched the Pfam database for known chlamydial PDZ domain types  and identified 86 unique PDZ-containing proteins from all species of 106-212 was shown to exhibit significant structural homology with unclassified PDZ domains, particularly that of human MPP7. The CPAF PDZ domain also shares structural and sequence homology with human PDZ domain-containing proteins involved in epithelial tight junction maintenance, including those from the MAGUK  and LAP (SCRIB) families.We have discovered through bioinformatics analysis that CPAF contains what represents a cryptic PDZ domain within the N-terminal subunit of the enzyme, spanning residues 106–212. When compared to canonical exemplary domains from each major PDZ class, CPAFCPAF is a secreted S41 protease in the chlamydial arsenal that recognizes a broad range of substrates within the active site. S41 proteases that exhibit broad active site substrate specificity will frequently utilize secondary specificity elements to restrict enzyme activation or catalysis to a defined subset of targets. This phenomenon is demonstrated by proteins in the tail-specific, DegP and HtrA protease families. In many of the S41 proteases, including DegP, HtrA2, HtrA3, and tail-specific proteases, secondary regulation of enzyme activity is achieved through communication between the catalytic serine protease domain and a nearby PDZ domain , 54, 55.106-212 is composed of a Tyr109-Leu110-Pro111-Tyr112 (YTPY). This YTPY sequence maintains the typical hydrophobic nature of the GLGF-loop despite the fact that the sequence is divergent from canonical PDZ domains  motif in the binding site loop for ligand recognition . The CPA domains .106-212 to the PDZ domains in human Dlg1, MPP7, and SCRIB revealed that Arg209 occupies an analogous region for the conserved basic residues from known PDZ domains  that occludes the protease domain active site [491, and CPAF106-212  substrate binding at the CPAF PDZ site induces conformational changes, similar to other S41 PDZ-containing proteases, after which the inhibitory sequence is presented to the active site in a catalytically competent conformation or (2) the singly cleaved inhibitory sequence obtains increased conformational flexibility, but remains bound to the CPAF PDZ domain to facilitate catalysis of the second and third cleavage events. Once the inhibitory segment is removed, the N- and C-terminal CPAF subunits associate to form the activated catalytic triad [This orientation of the inhibitory sequence argues for a possible role in simultaneously preventing ligand binding to the PDZ domain in addition to blocking substrates from reaching the protease active site. Removal of the inhibitory sequence is required for enzyme activity and may be facilitated by the PDZ domain. We propose that, if the CPAF PDZ domain is involved in zymogen activation, one of several outcomes may occur upon cleavage at the first activation site between Metic triad .172 and Ser176 may leave CPAF susceptible to hyperactivation as a result of activating PDZ ligands having easier access to the PDZ binding site.Interactions between the inhibitory helix and both the protease and PDZ domains highlight the potential for communication between the CPAF active site and PDZ domain. Such communication may form the basis for regulation of protease activation and/or substrate recognition. Distal regulation of protease activity is an established mechanism for the DegP and HtrA family hydrolases, as both classes exhibit PDZ-mediated oligomerization and activation , 40, 60.Following activation of the CPAF zymogen and formation of the active enzyme, substrates are either cleaved in discrete positions or completely degraded, further demonstrating the broad activity of the enzyme. This catalytic duality suggests that CPAF may operate as both an endo- and exo-peptidase. These different modes of cleavage may be the result of active site pocket selectivity or may be linked to contributions from secondary substrate specificity binding sites. The dichotomy of CPAF proteolysis mechanisms and the lack of a rigid active site consensus motif suggest that CPAF targeting may be dictated by distal regulatory elements, one of which may be the putative, N-terminal PDZ domain.E. coli tail-specific protease was shown to utilize a PDZ domain to assist in substrate binding. Mutations to the PDZ binding site resulted in a higher KM and lower kcat/KM for a peptide substrate [The concept of PDZ domains as secondary substrate recognition sites in proteases is well established. The HtrA and DegP proteases utilize the PDZ domains to form higher order oligomers and as secondary binding sites for substrates . In partubstrate . Chlamydubstrate .106-212 closely resembles human PDZ domains, we speculate that it may have evolved to hijack host protein-protein interactions, exposing them to potential CPAF-mediated proteolysis or directing CPAF to specific locales within the host cell. Alternatively, CPAF106-212 may intercept endogenous host PDZ ligands or PDZ-containing proteins to aid in molecular targeting of substrates. Others have shown that CPAF is released from the inclusion in late stages of infection [Because CPAFnfection , where iChlamydia infection disrupts tight junctions and adherens junctions in the single columnar layer of epithelial cells in genital tract and ocular infections, similarly to the sexually transmitted pathogen, N. gonorrhoeae [Many viral and bacterial pathogens have established a precedent of utilizing host-like PDZ domains or PDZ ligands as decoy pathogenesis effectors , 22, 26.orrhoeae .106-212 has been shown to be similar to human Dlg, MPPx, MAG1, and SCRIB, PDZ-containing proteins, which are known to be components of cell polarity and tight junction assembly in epithelial cells. MPP7, for example, co-localizes in epithelial cells with tight junction proteins Dlg1, E-cadherin, occludin and CASK, and builds assemblies at the interface of the outer membrane that act as scaffolding for formation of tight junctions [N. gonorrhoeae [106-212 to known PDZ-containing junction proteins indicates that CPAF may be involved in disruption of epithelial cell junctions to promote chlamydial infection.CPAFunctions . Chlamydunctions  and keraunctions , as wellunctions . Such a orrhoeae . Comprom106-212 may facilitate localization of the enzyme to interact with vimentin, vimentin binding proteins, or associated cytoskeletal filaments. Vimentin has also been implicated as a component of epithelial tight junctions and may be a cellular target for CPAF proteolysis late in chlamydial infections [While CPAF may directly intercept host PDZ-mediated signaling, it may also indirectly influence additional PDZ-associated proteins. CPAF has been postulated to degrade nectin-1, a protein required for assembly of adherens junctions in epithelial cells . We, andfections . FurtherMus musculus and Rattus norvegitus were detected through our bioinformatic searches. The CPAF protein sequences between C. trachomatis and C. muridarum are highly conserved, suggesting that the C. muridarum may contain a homologous PDZ domain Click here for additional data file.S2 Fig106-212 against 170 candidate PDZ domains are shown with the E-value and bit score cutoffs shown in blue and red, respectively. Any results with E-value greater than 1 were deemed insignificant and removed from analysis. In the inset plot, the concentration of data points in the left, upper quadrant indicates a population of homologous sequences that exceed the cutoffs for both alignment parameters.The E-values and bit scores for the results of the NCBI-BLAST alignment of CPAF(TIF)Click here for additional data file.


Simulation-based-mastery-learning (SBML) is an effective method to train nephrology fellows to competently insert temporary, non-tunneled hemodialysis catheters (NTHCs). Previous studies of SBML for NTHC-insertion have been conducted at a local level.Determine if SBML for NTHC-insertion can be effective when provided at a national continuing medical education (CME) meeting. Describe the correlation of demographic factors, prior experience with NTHC-insertion and procedural self-confidence with simulated performance of the procedure.Pre-test – post-test study.2014 Canadian Society of Nephrology annual meeting.Nephrology fellows, internal medicine residents and medical students.Participants were surveyed regarding demographics, prior NTHC-insertion experience, procedural self-confidence and attitudes regarding the training they received. NTHC-insertion skills were assessed using a 28-item checklist.Participants underwent a pre-test of their NTHC-insertion skills at the internal jugular site using a realistic patient simulator and ultrasound machine. Participants then had a training session that included a didactic presentation and 2 hours of deliberate practice using the simulator. On the following day, trainees completed a post-test of their NTHC-insertion skills. All participants were required to meet or exceed a minimum passing score (MPS) previously set at 79%. Trainees who did not reach the MPS were required to perform more deliberate practice until the MPS was achieved.Twenty-two individuals participated in SBML training. None met or exceeded the MPS at baseline with a median checklist score of 20 . Seventeen of 22 participants (77%) completed post-testing and improved their scores to a median of 27 . All met or exceeded the MPS on their first attempt. There were no significant correlations between demographics, prior experience or procedural self-confidence with pre-test performance.Small sample-size and self-selection of participants. Costs could limit the long-term feasibility of providing this type of training at a CME conference.Despite most participants reporting having previously inserted NTHCs in clinical practice, none met the MPS at baseline; this suggests their prior training may have been inadequate. Simulation-based-mastery-learning (SBML) is an effective method to train nephrology fellows to insert temporary, non-tunneled hemodialysis catheters (NTHCs). Prior studies of SBML for NTHC-insertion have been conducted at a local level.SBML for NTHC-insertion can be effective when provided in the setting of a continuing medical education (CME) conference.The ability to competently insert a temporary non-tunneled hemodialysis catheter (NTHC) is a requirement of nephrology training in Canada . NonetheSimulation-based-mastery-learning (SBML) is a rigorous form of competence-based education in which learners complete pre-testing, deliberate skills practice with feedback and post-testing until they meet or exceed a predetermined minimum passing score (MPS) ,7. LearnThe Canadian Society of Nephrology (CSN) annual meeting provides a unique opportunity to gather current and future nephrology fellows for specialized training during its pre-conference educational program. As such, we sought to determine if SBML for internal jugular (IJ) NTHC-insertion would be effective in this CME setting. The specific aims of this study were to: 1) evaluate the feasibility and effectiveness of NTHC-insertion SBML sessions at the CSN annual meeting; 2) describe the correlations of demographic factors, prior experience with NTHC-insertion and procedural self-confidence with simulated performance of the procedure; and 3) assess learners’ perceptions of SBML as part of a national CME conference.The study was a pre-test – post-test (before-after) design , assessiStudy participants provided demographic information including age, gender, year of post-graduate medical training; prior NTHC-insertion clinical experience (number of prior IJ NTHC-insertions) and; a rating of their self-confidence to perform NTHC-insertion competently using a scale of 0-100 . Subsequently, all participants underwent an NTHC-insertion clinical skills baseline examination (pre-test). Pre-test clinical skills examinations were conducted using a previously described IJ NTHC-insertion checklist . Over apWe conducted the NTHC SBML course using the CentraLineMan™  simulator that provides a realistic representation of the anatomy of the right upper torso and head. This simulator has IJ and subclavian (SC) veins and carotid and SC arteries. In addition, the simulator features an arterial pulse, different coloured venous and arterial ‘blood’, appropriate venous and arterial blood pressures and realistic tissues with self-sealing veins and skin into which needles, dilators and guidewires may be inserted. It is compatible with ultrasound so as to produce highly realistic ultrasound images. A portable ultrasound machine  was made available at each practice and testing station. In addition, NTHC kits, sterile gowns, sterile gloves, sterile drapes and sterile CVC-insertion trays  were available in unopened packaging at each practice and testing station.To ensure standardization of training and grading of checklist items, faculty instructors  underwent a four-hour ‘train-the-trainers’ session led by an instructor with extensive NTHC SBML experience (JHB) the day before the trainees’ precourse. During this ‘train-the-trainers’ session, instructors reviewed the SBML curriculum and approaches to providing feedback, and practiced scoring simulated NTHC-insertions using the 28-item checklist.A 28-item skills checklist was used to complete clinical skills evaluations Table . This waTo assess inter-rater agreement, video-recorded clinical skills evaluations were rescored by one of two authors  who were blinded to pre- or post-testing status and the original learners checklist score.We evaluated differences between pre- and post-test scores using the Wilcoxon signed-rank test to assess the impact of the intervention. Spearman’s rank correlation coefficients were calculated to assess relationships between demographic factors, prior procedural experience and self-reported procedural-confidence with pre-test scores. The difference between pre-test scores of nephrology fellows versus those of the other trainees was assessed using the Wilcoxon rank-sum test. Inter-rater agreement was assessed by evaluating the percentage of items upon which the two raters agreed. A Kappa coefficient was not calculated because the checklist was previously validated and there was complete agreement on several items. We evaluated differences between the original scores of the video-recorded clinical evaluations with the scores obtained through video review using the Wilcoxon rank-sum test. We performed all statistical analyses using IBM SPSS version 22 .p = 0.001) to 27  with all participants achieving the MPS on their first attempt. Figure Twenty-two participants  enrolled in the precourse and completed pre-testing and training. Table n = 11) compared with trainees who had less medical training (n = 11): the fellows’ median score was 14  compared to 19  for the residents and student (p = 0.44).The 5 participants who did not return for post-testing were nephrology fellows who each reported performing at least 20 prior IJ NTHC-insertions in clinical practice. Their median pre-test score of 19  was similar to overall group performance. Overall, no significant difference in pre-test scores were observed for nephrology fellows , gender , years of post-graduate medical training , clinical experience  or procedural self-confidence .Spearman’s rank correlation coefficients demonstrated no significant correlations between pre-test scores and age (n = 13), raters agreed on 331/364 (91%) of the rescored checklist items. The difference between the median of scores obtained by initial grading ) and the median of scores obtained from video review ) was not significant (p = 0.88).For the evaluations video-recorded in order to assess inter-rater agreement  and 3 ‘agreed’  that SBML-training improved their ability to competently insert NTHCs. As well, 15 ‘strongly agreed’ and 2 ‘agreed’ that this type of training should be a required component of nephrology fellowship training.This study shows that SBML can be used to train current and future nephrology fellows in NTHC insertion at a national CME meeting. Consistent with the findings of earlier research , this prWe believe that one of the strengths of this training intervention was the maintenance of a high level of realism during pre-testing, post-testing and deliberate-practice sessions. Anecdotally, many participants commented that they had received ‘partial’ simulation training for NTHC-insertion in the past, typically involving the use of a patient-simulator and ultrasound machine to repeatedly insert the finder needle into the IJ vein. Forcing participants to complete the entire procedure, using all necessary supplies in their original packaging, allowed them to encounter and overcome difficulties they will face in clinical practice but not during less realistic simulations: for example, the challenge of maintaining sterile technique while preparing the cover for the ultrasound probe only becomes evident during highly realistic simulation.In addition to failing to achieve the MPS at pre-test, most trainees committed potentially serious errors. Notably, over-advancing the guidewire beyond 20 cm) can provoke serious ventricular arrhythmias, particularly in patients with acute kidney injury . This is0 cm can SBML has many potential clinical benefits for Canadian nephrology training programs. SBML for CVC insertion reduces the number of needle passes per insertion ,13, arteThis study has several other important limitations. The small sample size and self-selection of participants limit the generalizability of our findings. It is unknown if those who participated are more or less likely to be proficient at NTHC-insertion than their colleagues who did not elect to participate. We did not assess skill retention although this has been reported elsewhere . AdditioNTHC-insertion SBML during a national CME conference is an effective method to train current and future nephrology fellows. The long-term feasibility of providing this type of training at a national conference on a recurring basis would depend upon a formal evaluation of its cost effectiveness. Most participants reported having significant experience previously inserting NTHCs. However, pretest performance on a clinical skills examination was poor. SBML should be considered for incoming nephrology fellows prior to performing actual NTHC-insertions on patients.


By detecting perfusion-like abnormalities, early-phase 11C-PiB-PET might add diagnostic value. Early-frame (1–6min) 11C-PiB-PET was obtained in 11 non-demented patients with probable CAA-related symptomatic lobar intracerebral haemorrhage (70±7yrs), 9 age-matched healthy controls (HCs) and 10 HCs <55yrs. There was a significant decrease in early-phase atrophy-corrected whole-cortex SUV relative to cerebellar vermis (SUVR) in the CAA vs age-matched HC group. None of the age-matched controls fell below the lower 95% confidence limit derived from the young HCs, while 6/11 CAA patients did . Combining both early- and late-phase 11C-PiB data did not change the sensitivity and specificity of late-phase PiB, but combined early- and late-phase positivity entails a very high suspicion of underlying Aβ-related clinical disorder, i.e., CAA or Alzheimer disease (AD). In order to clarify this ambiguity, we then show that the occipital/posterior cingulate ratio is markedly lower in CAA than in AD (N = 7). These pilot data suggest that early-phase 11C-PiB-PET may not only add to late-phase PiB-PET with respect to the unclear situation of late-phase positivity, but also help differentiate CAA from AD.Although late-phase (>35min post-administration)  The halease (AD). Clinicaease (AD) relies o11C-Pittsburgh compound B (PiB) has been developed as a PET ligand for imaging cerebral fibrillar β-amyloid (Aβ), using either kinetic modelling of the tissue pharmacokinetics over 60–90mins following tracer administration, or late-phase uptake images [11C-PiB-PET correlates with post-mortem fibrillar Aβ deposition extent in the brain associated with AD, but also with cerebrovascular Aβ deposits[11C-PiB binding in the cognitively healthy elderly[y >40min),4. Brain deposits,5,6. Amyy elderly, a well-y elderly,9.Improving the specificity of late-phase PiB-PET for the diagnosis of probable CAA by means of a second imaging variable would therefore be of clinical utility. One widely used imaging approach in neurodegenerative and age-related neurological conditions is resting-state perfusion or glucose metabolism imaging using SPECT and FDG-PET, respectively. which are highly inter-correlated,11 and rPerforming both PiB and FDG-PET in suspected CAA patients is one option, but this would be time-consuming, costly and demanding on patients, and would increase radiation exposure. It would therefore be advantageous to obtain, from a single amyloid tracer administration, indexes of both perfusion and amyloid load. It is w11C-PiB as an index of brain perfusion in sLICH patients diagnosed with probable CAA based on current Boston criteria, as compared to age-matched healthy controls. Our general aim was to determine if early-phase PiB could point towards potential diagnostic utility, particularly if combined with late-phase 11C-PIB.Here we assessed early-phase Specifically we tested the following three hypotheses. Our first hypothesis is that whole-cortex early-PiB uptake will be reduced relative to aged HCs as a result of reduced perfusion from the widely distributed cortical  and white matter ischemic changes (undercutting the cortex) present in CAA. Our secHowever, as discussed extensively elsewhere, even ifThe HCs and CAA patients used in this study have been described previously. Eleven post hoc due to the presence of three lobar microbleeds on MRI as this might indicate incipient CAA. The final age-matched HC group therefore comprised 9 subjects .Twenty medication-free healthy controls (HCs) with no memory or cognitive complaints and normal Mini-mental State Examination results (MMSE ≥29) were also recruited through advertisements posted at the University of The Third Age and the hospital advert board. This sample comprised of a ‘young’ subset of 10 HCs ≤55yrs which was used to derive normative values (see below), and a subset of 10 HCs aged 60 or older and matching in age the CAA group for comparison. One aged HC was excluded p = 0.03 relative to CAA, p<0.001 relative to HCs); they were diagnosed with probable AD according to the NINCDS-ADRDA criteria [Seven patients with mild-to-moderate AD were also recruited , occipital and PCC ROIs, and normalized by the CSF-corrected SUV of the cerebellar vermis[Based on our objectives and main hypotheses, a whole cortex ROI was computed as the sum of all AAL cortical ROIs. In addition, as per our third hypothesis, the occipital and posterior cingulate GM ROIs were also obtained. For each patient, any ROI directly affected by the lobar ICH was excluded ar vermis .The lower 95% confidence limit for Whole Cortex SUVR derived from the subgroup of young healthy controls  was 0.94. No aged control fell below this cut-off, while 6/11 CAA patients did .p<0.05) differences among the three groups for both ROIs. The post-hoc tests comparing among the three patient groups revealed results entirely consistent with our hypotheses. Thus, the CAA group had significantly lower occipital SUVR than both the age-matched healthy controls and the AD groups, while the AD group did not differ from the healthy controls. Likewise, regarding the PCC, only the AD group had significantly lower early PiB uptake as compared to the aged controls. Accordingly, the Occipital/PCC ratio was strongly significantly lower in the CAA relative to AD group  (The mean (and 1SD) SUVR values for the occipital and PCC ROIs are shown in Whitney) , Fig 1. Consistent with our hypothesis, whole-cortex early-phase PiB uptake was significantly decreased in the CAA group relative to aged-matched controls, suggesting that as predicted overall cortical perfusion is reduced in CAA. No aged control fell below the 95% confidence limit for whole-cortex early-phase PiB derived from young controls, resulting in a 100% specificity. However, only 6/11 CAA patients were early-PiB positive, resulting in a sensitivity of 55%. Combining the early- and late-phase whole-cortex PiB data did not nominally improve sensitivity and specificity, but since, contrary to late-phase PiB, no aged HC was early-phase PiB positive while both early- and late-phase PiB positivity was present only in CAA patients, this double-positivity points to a very high suspicion of underlying Aβ-related clinical disorder, i.e., CAA or AD . In ordepost hoc the analysis after removing all AAL ROIs from the hemisphere affected by ICH across the whole sample. Doing this removed the statistical significance, suggesting this mechanism plays a major role in the observed reduction in whole-cortex early-phase PiB uptake. Note however that this post-hoc analysis did not remove the strong difference in occipital/posterior cingulate ratio between the CAA and AD groups, which remained highly significant (p<0.001).Our finding of significantly reduced whole-cortex early-phase PiB uptake in probable CAA is novel. To the best of our knowledge, there is no published study of FDG uptake in CAA to compare our results to. Regarding perfusion, a brain SPECT study reported focal hypoperfusion in various locations in CAA but this was in non-ICH, atypical presentation patients, many with mixed diagnoses. As prevEarly-phase PiB SUVR was found to have a sensitivity of 55% and a specificity of 100% for CAA vs aged HCs. Although preliminary as based on a small sample and not generalizable to other presentations of probable CAA, these results are nevertheless of interest because using late PiB uptake in the same sample, the opposite results were found, namely a very high (91%) sensitivity but a low specificity (55%) due to the occurrence of late-phase PiB positivity in four of the nine healthy elderly subjects Table 1Table 1. In order to clarify the CAA vs AD ambiguity in case of late-phase positivity, we assessed whether early-phase PiB can help to differentiate between these two possibilities. As predicted, the early-phase PiB occipital uptake and the occipital/PCC ratio were both markedly lower in CAA relative to AD . Thus, iThe only previous study reporting baseline occipital perfusion in probable CAA-related ICH used MR-based arterial spin labelling and found no difference in absolute blood flow, but relOne potential confounder in the above paradigm is Lewy Body disease (LBD), a neurodegenerative disorder partly overlapping with AD neuropathologically, where amyloid PET is frequently positive and that is characterized by reduced occipital perfusion and FDG uptake, as well as positive DaT-scan,36. Howepost-hoc the analysis without CSF correction, which did not substantially change the overall results. Notably the occipital/PCC ratio, which is largely independent of CSF correction, continued to highly significantly discriminate between CAA and AD (data not shown).In the present study we corrected the regional PET data for cortical atrophy, using the MR-derived fraction of CSF in each voxel to increase the voxel PET count, according to the widely used Meltzer method,37,38. T11C-PiB may be useful in the diagnostic work-up of ICH patients suspected of CAA, particularly in case of late-phase PiB positivity. Further studies in larger cohorts of probable CAA patients, if possible including pathological diagnosis, are required.Our findings are based on small samples and lack a direct validation against FDG or perfusion PET, and are therefore preliminary. Nonetheless, they do support the idea that combining both early- and late-phase


Due to drug-drug interactions of HIV- and HCV-specific antivirals when initiating an HCV-therapy, the antiretroviral therapy (ART) often has to be changed. The spectrum of applicable antiretrovirals is small, therefore many patients were switched to raltegravir/truvada (RAL/TVD) in our cohort. Due to the relatively low genetic barrier of RAL, this regimen may be endangered to fail, if the NRTI backbone is not fully active because of pre-existing NRTI resistance. We investigated the long-term follow-up and safety of RAL/TVD in co-infected patients after hepatitis C virus (HCV) therapy was stopped and the protective antiretroviral effect of interferon ended.Twenty patients initiated a direct-acting antiviral (DAA) containing HCV therapy  between 11/2011 and 01/2013. Seventeen were switched to RAL/TVD, three patients were not treated before, but started with the regimen. Diagnosis of HIV infection was dated between 1985 and 2010. The HI-viral suppression was monitored retrospectively to date.Thirteen of the twenty patients (65%) remained on RAL/TVD after finishing HCV treatment, for seven patients, no data about their ART continuation was available, after HCV therapy had stopped. All remaining thirteen patients showed an HI-viral load below detection limit up to date . Only for four patients, historic resistance data were available but none showed NRTI mutations.Switch to RAL/TVD as HIV ART due to initiating HCV therapy was safe for the observed small cohort even in long-term follow-up without switchback or a second ART switch. However, resistance data for the cohort was little, showing no NRTI mutations, indicating a relatively safe setting. Since no further data is available, physicians should keep in mind ART history, historical therapy failure and HIV-resistance while switching ART to treat HCV in co-infected patients. Further investigation in larger cohorts is needed, especially thinking of upcoming interferon-free HCV regimen in heavily pre-treated co-infected patients.


O-linked glycosylation within lubricin’s mucin-rich domain is critical for its boundary lubricating function; however, it is unknown exactly how glycosylation facilitates cartilage lubrication. Here, we find that the lubricin glycome is enriched with terminal β-galactosides, known binding partners for a family of multivalent lectins called galectins. Of the galectin family members present in synovial fluid, we find that galectin-3 is a specific, high-affinity binding partner for lubricin. Considering the known ability of galectin-3 to crosslink glycoproteins, we hypothesized that galectins could augment lubrication via biomechanical stabilization of the lubricin boundary layer. We find that competitive inhibition of galectin binding results in lubricin loss from the cartilage surface, and addition of multimeric galectin-3 enhances cartilage lubrication. We also find that galectin-3 has low affinity for the surface layer of osteoarthritic cartilage and has reduced affinity for sialylated O-glycans, a glycophenotype associated with inflammatory conditions. Together, our results suggest that galectin-3 reinforces the lubricin boundary layer; which, in turn, enhances cartilage lubrication and may delay the onset and progression of arthritis.Lubricin is a mucinous, synovial fluid glycoprotein that enables near frictionless joint motion via adsorption to the surface of articular cartilage and its lubricating properties in solution. Extensive  O-linked oligosaccharide chains911O-linked β(1–3)Gal-GalNAc oligosaccharidesLubricin is a large, mucinous glycoprotein critical for boundary lubrication of articular cartilage. Lubricin protects against cartilage damage, with intra-articular lubricin supplementation mitigating the severity and progression of post-traumatic arthritis in animal models1234O-linked). Site-specific glycan analysis of lubricin identified 168 independent O-glycosylation sites14Glycans are in large part responsible for the boundary lubricating properties of lubricin. Mucinous glycoproteins, like lubricin, are defined by their densely clustered pendant glycans that initiate with N-acetylgalactosamine saccharides covalently linked to the polypeptide through the oxygen molecules of serine and threonine side chains (O-linked β(1–3)Gal-GalNAc oligosaccharides10Changes in glycosylation are a hallmark of disease states characterized by chronic inflammationO-linked glycosylation plays in mediating boundary lubrication, little effort has been devoted to characterizing the glycosylation of administered recombinant lubricin or the glycoprotein product of lubricin gene therapy approaches. Thus, it is unknown what glycan composition of lubricin could be used clinically to derive the optimal therapeutic benefit.A considerable body of evidence is accumulating to suggest that lubricin may be an effective therapy for the treatment of osteoarthritis (OA). Lubricin synovial fluid concentrations are decreased in experimental rodent models of post-traumatic OA, including the anterior cruciate ligament (ACL)-deficient guinea pig stifle34In biology, a major function of glycans is to mediate biomolecular interactions; however, little attention has been focused on elucidating mechanisms by which glycans may facilitate lubrication distinct from repulsive or hydration mechanisms. Glycans mediate protein-protein interactions through lectins such as siglecs, selectins and galectins. Lectin glycan interactions play a critical role in several biological contexts, ranging from lattice formation to receptor signalling to glycoprotein clustering and assembly2324Therefore, our objective was to examine the lubricin glycome in order to identify potential synovial fluid binding partners for lubricin. Based upon the enrichment of terminal β-galactoside residues and the high density of β(1–3)Gal-GalNAc T-antigens in normal equine synovial fluid lubricin, combined with the abundant expression of galectins-1 and -3 in cartilage and synovial fluid26O-glycans, intensely stained the cartilage boundary layer. Maackia amurensis lectin II (MALII), which preferentially binds to α2–3 sialylated core-1 O-glycans, and jacalin, which binds to both sialylated and nonsialylated core-1 O-glycans, also labelled the surface layer of articular cartilage. Although there was faint staining of the superficial zone interterritorial matrix with succinylated wheat germ agglutinin (S-WGA) and phytohemagluttinin-leucoagglutinin (PHA-L), no appreciable boundary staining was present for either of these lectins, indicating that core-2 O-glycans and complex, branched N-glycans do not contribute substantially to the oligosaccharide population of the cartilage boundary layer. Weak staining of the cartilage surface by Sambucus nigra (SNA) was detected, indicating the presence of low quantities of α2–6 linked sialic acids. Taken together, these results suggest that the articular cartilage boundary layer is composed of a significant proportion of nonsialylated core-1 O-glycans.In order to determine what glycans were present on healthy cartilage, adult equine articular cartilage explants were incubated with fluorophore-conjugated lectins followed by nuclear staining with Hoechst 33342 and combined confocal and multiphoton microscopy . Peanut O-linked glycans using mass spectrometry (O-glycans predominated (57.2%), followed by approximately similar concentrations of nonsialylated (22.8%) and disialylated (19%) structures. Core-2 O-glycans were detected as <2% of the population of O-glycans.Lubricin is the predominant carrier of O-linked glycans within the cartilage boundary layer. The glycosylation profiles of lubricin from human patients with OA and RA have been described1018trometry . MonosiaO-linked glycans present within the cartilage lamina splendens, we performed immunohistochemical staining for both lubricin and galectins. Immunostaining of osteochondral sections obtained from healthy equine carpal joints demonstrated intense staining of both lubricin and galectin-3 on the surface of articular cartilage  of 51 nM  for full-thickness bovine cartilage explants using a cartilage-on-glass custom tribometerGalectin-3 has been shown to rapidly precipitate as pentamers in the presence of multivalent ligandsTo assess whether multivalent galectin-3 could stabilize surface-adsorbed lubricin by increasing the residence time of adsorbed lubricin, we evaluated articular cartilage lubricin staining after a 12-hour incubation with 100 mM β-lactose. In the presence of β-lactose to compete for galectin binding, lubricin surface staining was diminished by 32.6%  , suggestThe glycosylation of lubricin is essential to its lubricating function. Besides contributing to the overall net negative charge and hydration of the articular cartilage surface layer91026. Our studies suggested that equine synovial fluid lubricin is uniquely glycosylated, rich in terminal monosialylated and non-sialylated β-galactoside sugars. Therefore, we hypothesized that the predominant synovial fluid galectins-1 and -3 could be crosslinking lubricin to facilitate lubrication in synovial joints.Galectins comprise a family of at least 15 multivalent lectins with specificity for β-galactoside sugars. Prototypical galectins, such as galectin-1, possess a single carbohydrate recognition domain and can form homodimers through N-terminal interactions. Galectin-3 is unique in that it forms pentamers, resulting in a functionally pentavalent carbohydrate-binding molecule. Through glycan binding, galectins can crosslink glycoproteins to form complex lattices on the cell surface or within the extracellular matrix404227O-linked glycophenotype of lubricin consists of primarily core 1 O-linked oligosaccharides that are either mono-, di- or nonsialylated, with monosialylated structures predominating. Glycomic analysis of synovial fluid from horses with normal articular cartilage, structural OA lesions, and osteochondral fragments have also revealed a predominance of monosialylated core 1 O-glycansO-glycans, and both galectins27274243Because lubricin and galectin-3 interact in a carbohydrate-dependent manner, glycosylation changes in arthritis may have significant effects on galectin-3 stabilization of the lubricin boundary layer. We are just beginning to understand how lubricin is glycosylated and how that glycosylation is altered in the inflammatory context of arthritis. Early studies revealed that 2/3 of the O-GalNAc-Gal oligosaccharide chains on lubricin were capped with terminal sialic acid residuesThe impact of galectins on cartilage lubrication and joint mechanics has never been investigated. Herein, we describe an extracellular role for galectins in joint lubrication and cartilage protection in synovial joints. These findings are important to understand the role of galectins in joint biology. Prior reports have primarily focused on galectins within the synovial environment as mediators of inflammation in RA, juvenile idiopathic arthritis and antigen-induced arthritis in animal models27424849Galectins function distinctly through intracellular and extracellular pathways, with intracellular signalling resulting from N-terminal protein-protein interactions and extracellular functions arising primarily from carbohydrate-dependent interactions on the cell surface or within extracellular matrices2748N-glycans on the cartilage surface as demonstrated by lectin staining suggests that O-linked glycans are the primary targets for galectin binding. Binding of galectin-3 to the core 1 O-glycan structure has been characterized previously, with the CRD demonstrating a unique Glu-water-Arg-water motif-based mode5′AHGDAO-glycan bindingOur studies revealed that galectin-3 localized to the boundary layer of healthy articular cartilage and within superficial and middle zone chondrocytes. Although galectin-3 staining has been previously demonstrated in synovial tissues5122Herein, we provide a mechanism to explain how synovial fluid galectin-3 may enhance the biomechanics of cartilage lubrication and protect against the development of cartilage damage through stabilization of the lubricin boundary layer via lattice formation. Future investigation into how lubricin glycosylation is altered in arthritis and how these alterations impact galectin-3 binding and cartilage lubrication will be critical in fully understanding the role of galectins in cartilage biomechanics.All experimental protocols were approved by the Cornell University Institutional Animal Care and Use Committee (Protocol Number: 2011-027), and all methods were carried out in accordance with approved guidelines.Synovial fluid was obtained from healthy joints of two young horses and purified using diethylaminoethyl (DEAE) affinity chromatography using previously described methodologyRNA was purified from equine kidney tissue, and gene-specific primers were designed against the NCBI predicted sequences of equine galectin-1 and galectin-3. Recombinant human galectin-1 and galectin-3 constructs were obtained from C. Bertozzi and, along with the galectin-3C mutant and equine galectins, were recombinantly expressed and purified using β-lactosyl sepharose affinity chromatography similar to previously described methodsLubricin purified from normal equine synovial fluid or bovine asialofetuin was coated to high-binding, 96-well ELISA plates. Recombinant, biotinylated equine galectin-1 or galectin-3 was added to each well at serial concentrations, and affinities were measured using streptavidin-HRP, TMB and a monochromator.® PNGase F, ii) Sialidase ATM, or iii) O-glycanase®  + prO-LINK ExtenderTM (β(1–4) Galactosidase + β-N-Acetylglucosaminidase)  listed in FPLC-purified equine synovial fluid lubricin was digested with the following combinations of deglycosylation enzymes: i) N-glycanaseArticular cartilage explants were aseptically harvested from the femoropatellar groove of young bovine stifles and incubated in PBS or recombinant human galectin-1, galectin-3, or galectin-3C. Tribological testing was performed using a previously described custom friction apparatusHow to cite this article: Reesink, H. L. et al. Galectin-3 Binds to Lubricin and Reinforces the Lubricating Boundary Layer of Articular Cartilage. Sci. Rep.6, 25463; doi: 10.1038/srep25463 (2016).


Drosophila; the SPD-5 and SPD-2 proteins appear to play a similar function in C. elegansDrosophila larval brain cells. Spd-2 fluxes outwards from the centrioles in both brains and embryos in a microtubule-independent manner. In contrast, although Cnn is initially incorporated into the region of the PCM occupied by Spd-2 in both brains and embryos, Cnn fluxes outwards along microtubules in embryos, but not in brain cells, where it remains concentrated around the centrosomal Spd-2. Thus, the microtubule-independent centrosomal-flux of Spd-2 occurs in multiple fly cell types, while the microtubule-dependent outward flux of Cnn appears to be restricted to the syncytial embryo.Centrosomes are major microtubule organising centres comprising a pair of centrioles surrounded by pericentriolar material (PCM). The PCM expands dramatically as cells enter mitosis, and we previously showed that two key PCM components, Centrosomin (Cnn) and Spd-2, cooperate to form a scaffold structure around the centrioles that recruits the mitotic PCM in  Drosophila larval brain cells using fluorescence recovery after photobleaching (FRAP). Both Spd-2–GFP and GFP–Cnn fluorescence recovered at centrosomes after photobleaching the centrosomal GFP signal, although Spd-2–GFP recovered faster than GFP–Cnn and both recovered more slowly than at embryonic centrosomes . Clearly it will be interesting to determine whether Spd-2 homologues exhibit centrosomal-flux in other species.Thus, Spd-2 in flies exhibits a genuine centrosomal flux that is microtubule-independent, while Cnn can build a supporting scaffold around the centrosomal Spd-2 without fluxing outwards; the microtubule-dependent outward flux of Cnn is only generated in specific cell types. Interestingly, SPD-5, which appears to perform a similar phospho-dependent scaffolding role to Cnn in worm embryos