Puccinia striiformis f.sp. tritici (Pst) is a major constraint to wheat production worldwide. The molecular events that underlie Pst pathogenicity are largely unknown. Like all rusts, Pst creates a specialized cellular structure within host cells called the haustorium to obtain nutrients from wheat, and to secrete pathogenicity factors called effector proteins. We purified Pst haustoria and used next-generation sequencing platforms to assemble the haustorial transcriptome as well as the transcriptome of germinated spores. 12,282 transcripts were assembled from 454-pyrosequencing data and used as reference for digital gene expression analysis to compare the germinated uredinospores and haustoria transcriptomes based on Illumina RNAseq data. More than 400 genes encoding secreted proteins which constitute candidate effectors were identified from the haustorial transcriptome, with two thirds of these up-regulated in this tissue compared to germinated spores. RT-PCR analysis confirmed the expression patterns of 94 effector candidates. The analysis also revealed that spores rely mainly on stored energy reserves for growth and development, while haustoria take up host nutrients for massive energy production for biosynthetic pathways and the ultimate production of spores. Together, these studies substantially increase our knowledge of potential Pst effectors and provide new insights into the pathogenic strategies of this important organism.Stripe rust caused by the fungus  Puccinia striiformis f.sp. tritici (Pst). In Australia, wheat stripe rust causes severe losses and is controlled mainly by fungicides, with about AUD$127 million expended per annum on chemical strategies Pst to cause disease is crucial for developing more effective control methods.Stripe rust is an important agricultural disease that constitutes a major challenge to wheat production worldwide. The causal agent is the fungus Pst, are obligate biotrophic pathogens that require living tissue to develop and reproduce. The biotrophic interaction may be sustained over many weeks, with important implications for the pathogenic lifestyle. For example, such prolonged interaction is usually associated with changes in photosynthate translocation patterns within the plant Rust fungi have the most complicated life cycles of all fungi Pst has adapted to source nutrients from living mesophyll wheat cells through a sophisticated cellular structure termed the haustorium per se but is separated from the host cytoplasm by an extrahaustorial membrane which is contiguous with the plant cell plasma membrane. The haustorium is thought to be the primary organ for nutrient transfer from the host cell to the fungal vegetative body As an obligate parasite, The effectors secreted by plant pathogens are believed to be essential to the parasitic lifestyle. Very little is known about the activities of plant fungal effectors, but broadly they are thought to manipulate the physiological and immune responses of host cells during infection The haustorium is a site of concerted host-pathogen interaction, and describing its functions is essential to understanding biotrophy. The presence of sugar transporters Puccinia graminis f.sp tritici (Pgt), Melampsora larici-populina (Mlp) Blumeria graminisPstPuccinia species Pst. The results derived from 454 and Illumina-based transcriptome sequencing allowed the prediction of haustorial secreted proteins (HSP), a set of genes enriched in effector candidates found to be specifically- or highly-expressed in haustoria. Additionally, the comparison of the haustorial and germinated spores transcriptomes revealed fundamental metabolic differences between the two pathogenic stages.The availability of genomic data for biotrophic organisms has increased dramatically in the last decade providing important insights into the infection strategies of these pathogens. The genome sequences for the plant pathogens Pst isolate (Pst-104E137A-)  and the germinated spore transcriptome generated in this study  resulting in 12,846 contigs representing the haustorial transcriptome. Assembled contigs ranged between 200 and 6,854 bp, with an average length of 704 bp (Transcriptomes corresponding to purified stripe rust haustoria and germinated spores were sequenced by single-read pyrosequencing on a 454 GS-FLX titanium platform (454). This produced 729,036 and 457,071 reads averaging 413 bp and 420 bp in length for haustoria and germinated spores, respectively. To remove contaminating wheat sequences from the haustoria data, the Pst-130 draft genome is study  were usef 704 bp  and S3.−25. One hundred and five genes were similar to hypothetical proteins in Pgt, and four were similar to previously identified Pst secreted proteins Pucciniales. Interestingly, Bhadauria et. al Colletotrichum truncatum (CtNUDIX) that is exclusively expressed during the late biotrophic phase and elicits the hypersensitive response in tobacco leaves, suggesting that this effector could be important for the transition from biotrophy to necrotrophy, or alternatively is recognised by the tobacco immune system. A secreted protein from Pst containing a NUDIX domain could potentially regulate redox homeostasis. Nevertheless, as observed previously for rust fungi The haustoria contigs were analyzed using CLC genomics to identify all possible ORFs, which were further analysed with SignalP 3.0 Blumeria graminis revealed that the most highly expressed candidate effector proteins contained a Y/F/WxC motif 1–30 aa after the predicted SP Pst HSPs, but the motif occurred within the first 30 aa for only 43 of these, which is similar to that number expected by chance (∼32). Thus similar to Pgt and MlpPst. Further attempts to detect novel motifs using the MEME analysis module within CLC failed to identify other conserved sequences.Genome and transcriptome sequencing of Extracellular proteins frequently contain elevated numbers of cysteine residues, which can participate in di-sulfide bonding to provide stability to the folded proteins in the protease-rich extracellular apoplastic space. Many known and predicted effector proteins from filamentous pathogens are small cysteine-rich proteins in silico predictions , 94 effector candidates were randomly chosen from the 437 HSP genes and tested for expression in vivo by non-quantitative reverse transcriptase-PCR (RT-PCR)  but were not amplified from genomic DNA because one or both primers span intronic regions. Cloning and Sanger sequencing of 25 candidate effectors confirmed that they corresponded to the sequence predicted from our 454 assemblies.To validate the  (RT-PCR). Primersde novo assembly of the combined germinated spores and haustorial 454 reads, giving a set of 12,282 transcripts . Flor-Parra et al.Ustilago maydis, complexing with the cyclin-dependent kinase Cdk5 for sustained polar growth Pcl12 homologs in our data  were found in our data, three of which were up-regulated in germinated spores. Septins constitute a cytoskeletal structure that is conserved in eukaryotes Pst  and three β-mannosidases  were up-regulated in haustoria. This suggests a requirement for GHs for entry into plant tissue, or alternatively, a role in haustorial wall maintenance. The higher expression of mannosidases in advanced stages of the infection has been reported in other rusts Pst transcripts encoded proteins similar to β-1,3-glucanases, four of which were up-regulated in germinated spores  and one in haustoria (PST79_5049). These could potentially play roles either in fungal cell wall remodelling or degradation of plant callose. The remaining GHs were similar to enzymes required for glucan synthesis and fungal cell wall remodeling , posttranscriptional modification of cell wall proteins, chitinases  and glycogen breakdown.Cell walls are complex polysaccharide structures composed mainly of cellulose, hemicellulose and pectin in plants Pgt and MlpLaccaria bicolorHemileia vastatrix suggested that two chitin deacetylases were most strongly expressed in the early stages of host invasion, coinciding with spore germination and tissue invasion Uromyces viciae-fabae exhibits massive chitin deacetylase activity when the fungus starts to penetrate through the stomata Related to cell wall modification, we also found genes with strong similarity to chitin synthases (mostly up-regulated in germinated spores) and carbohydrate deacetylases (including chitin deacetylases), which were massively up-regulated in germinated spores. Chitin deacetylases are of particular interest because they convert chitin into the less rigid chitosan, potentially avoiding recognition by plant chitin receptors Pst contigs set against the NCBI non-redundant protein and the Transport Classification databases (TCDB: http://www.tcdb.org). The initial cut off for evaluation was e-value <10−25, and the hits were further annotated manually to remove alignments of less than 100 amino acid residues. The major transporter classes that we found constituted those for amino acids and oligopeptides, sugars, small molecules and ions, and the vitamin co-factor nicotinic acid. Some of these were expressed differentially in a manner that gives insight to the pathogenic strategy of the fungus  that was expressed strongly in germinated spores but at low levels in haustoria. Similarly to U. fabaePst putative amino acid transporters; presumably the early expressed transporter should show very high affinity because of the scarcity of nutrients in the apoplast. In addition to this, eight predicted oligopeptide transporters (OPT) were found in our data, three up-regulated in haustoria , one in germinated spores (PST79_2988) and the other four did not show expression biases. The amplification of the OPT gene family in Pgt and Mlp was proposed to be a genomic adaptation of these pathogens to obtain amino acids, nitrogen and carbon from oligopeptides derived from their host Pathogenic fungi have a large requirement for nitrogenous compounds for macromolecule biosynthesis. The best characterized haustorial amino acid transporters are from Pst transcriptome, but only three of these showed differential expression , so no generalizations about biological function were possible. In other biotrophic pathogens such as Blumeria graminis f.sp. hordei, a marked reduction in the number of genome-encoded ABC transporters (only 20) has been correlated with the loss of secondary metabolic enzymes, reported as the lowest number known in fungi M. oryzae, especially in the appressorial stage where far more ABC transporters are expressed than in any of the studied stages of PstPgt and Mlp genomes respectively, however while the total number of such transporters in the Pst genome is presumably similar, only a few of them were expressed during the developmental stages and sampling conditions studied here. Nevertheless, most of the MFS transporters in Pst were preferentially expressed in germinated spores, which is the first infectious stage that encounters host defence. This could suggest that although rust pathogens are also equipped to export toxins or metabolites, the most important roles of these proteins is in growth and development.ABC transporters constitute a large superfamily of primary active transport systems that are present in all kingdoms of life, and play diverse physiological roles in trafficking of a wide range of substrates across internal and external membranes. These transporters represent one of the largest and most ancient transporter classes and derive their name from the shared highly conserved domain, the ATP binding cassette (ABC), which binds and hydrolyzes ATP Pst (PST79_113) shares 92% similarity at the amino acid level with the hexose transporter HXT1p of U.fabaeUf-HXT1, showed almost no expression in germinated spores. The expression pattern of Uf-HXT1p is very similar to the amino acid transporter Uf-AAT2p Pst homologs of these two transporters were also almost identical, suggesting that expression of these genes is coordinated across different rust species. We also identified three Pst homologs of the H+-ATPase transporter Uf-PMA1  which is believed to be important for establishment of a proton gradient for coupling substrate translocation into haustoria Uf-PMA1, all of the Pst homologs were expressed more strongly in germinated spores than haustoria U. fabae ATPase showed that despite the higher transcript levels in germinated spores than in haustoria, its enzymatic activity is far greater in haustoria where it may act as a proton-substrate symport mechanism for Uf-HXT1p Pst and potentially outlines a conserved transport mechanism for glucose that is key to the biotrophic lifestyle of rusts.Three putative glucose transporters (MFS), seven unspecified sugar transporters (MFS), one mannose transporter and one oligosaccharide translocation protein were identified in our set of transcripts. One of the transcripts classified as a glucose transporter in M. oryzae expresses sugar transporters at an analogous stage; during appresoria formation but before tissue penetration, supporting our reasoning −101 with the Saccharomyces cereviseae gene), a glycerol-proton symporter that is inactivated by glucose The remaining two putative glucose transporters  were up-regulated in germinated spores, suggesting that the fungus prepares for tissue invasion and is poised to exploit host carbohydrates as a nutritional source, or alternatively that these could export substrates for cell wall synthesis. +/PO43− symporter Saccharomyces cerevisiae cells, response to phosphate starvation includes the up-regulation of the activity of a high-affinity phosphate uptake system Pst and its up-regulation in germinated spores could suggest that similar phosphate starvation sensing/signalling mechanisms exist in rusts, which could be crucial for cell cycle progression and growth as has been shown in yeast 2+, Cu2+, Fe2+, Mg2+, Mn2+, K+ and Zn2+ were identified in our Pst data. Transporters for Zn2+ and Mn2+ were up-regulated in haustoria, while transporters for K+ and Ca2+ were up-regulated in germinated spores.Inorganic phosphate is an essential nutrient required for the synthesis of nucleic acids, phospholipids, cellular metabolites, and protein modification, reactions that require high phosphate concentrations S. cereviseae nicotinic acid transporter TNA1  +), which can be reduced to NADH or the phosphorylated form NADPH. Generally, NADPH is a reductive intermediate for biosynthesis pathways, whereas NADH is an energy precursor and protects against oxidative damage. Thus uptake of host-derived niacin could be important in the haustorial stage which is greatly involved in energy production, biosynthesis of molecules for spore formation, and protection against oxidative stresses. Duplessis et al. in planta expression of four Mlp genes with similarity to S. cereviseae TNA1 permease at 96 hpi, supporting the idea that uptake of host nicotinic acid plays an important role during the advanced stages of the infection. We also searched for contigs that encode enzymes required for de novo synthesis of niacin from tryptophan. The known pathway in organisms that use tryptophan as a source for NAD+  involves six enzymes; we were able to identify five of these in our dataset, two of which were up-regulated in haustoria  encoding proteins with similarities to the U. fabaeBlumeria graminisPst genes with higher expression in germinated spores corresponded to the key enzymes of the glyoxalate cycle, which will be described later. In addition, a phosphate translocator of the inner mitochondria membrane (PST79_4938), which carries out the coupled transport of H2P04− and H+ for subsequent ATP synthesis via the electron transport chain, was massively up-regulated in haustoria, consistent with a high demand for ATP in this tissue. This could be a consequence of the different nutritional status of haustoria and germinated spores. While the stored energy resources are limited in spores, haustoria can derive sugars abundantly from the host to feed energy production pathways and drive biosynthetic pathways for subsequent development (Transcripts classified in this category were represented more in haustoria than in germinated spores  and 4. Telopment .STL1 (encoding a glycerol transporter) was also up-regulated in germinated spores, which suggests that glycerol derived from the host apoplast could be taken up and incorporated via the gluconeogenic pathway. Finally, genes encoding enzymes involved in glycogen breakdown were also up-regulated in germinated spores, which also results in glycerol production. In Pgt uredinospores, as well as spores of other plant fungal pathogens, glycogen is a stored energy source that is utilized during germ tube extension B. graminis and M. oryzaeHXT1 and enzymes of glycolysis, TCA cycle and oxidative phosphorylation in haustoria suggest that host-derived glucose is the primary carbon source.Acetyl-CoA is the basic currency of carbon metabolism within the cell. It is the immediate product of carbohydrate catabolism and β-oxidation of fatty acids, and when in excess, can be used for biosynthetic processes through the glyoxylate cycle, gluconeogenesis and glyceroneogenesis pathways. In our transcriptomic study, the gene for triacylglycerol lipase (PST79_8009) together with nearly all those for the enzymes of glyoxylate cycle, gluconeogenesis and glyceroneogenesis were significantly up-regulated in germinated spores compared with haustoria , or recycled for energy production via glycolysis (non-oxidative branch). An important product of the PPS is the reductant NADPH, which is used almost exclusively in anabolic pathways, for example biosynthesis of fatty acids, amino acids and nucleotides including ATP. We found evidence for expression of all enzymes of the PPS in both germinated spores and haustorial . Thus, tP. falciparumTHI1 (4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase) and TH2 (hydroxyethylthiazole phosphate synthase)(PST79_882) from U. fabae identified previously by Hahn and Mendgen A number of metabolic enzymes require the vitamin B1 derivative thiamine pyrophosphate as a cofactor. Examples from the pathways described here include alpha–ketoglutarate dehydrogenase (α–KGDH) in the TCA cycle; transketolase in the non-oxidative PPS; and pyruvate dehydrogenase (PDH) which connects glycolysis and the TCA cycle. We found that transcripts associated with the thiamine biosynthesis pathway were massively up-regulated in haustoria, with the exception of the genes for thiamine-phosphate kinase and thiamine pyrophosphokinase which could not be found in our data. These enzymes are responsible for phosphorylating thiamine phosphate and thiamine diphosphate respectively, and interestingly, the latter is also absent in the genome of the malarial parasite + to form glutamine catalyzed by glutamine synthetase. Subsequently, glutamate is used to synthesise the majority of amino acids, whereas glutamine serves as an amino-donor during other biosynthetic processes. Interestingly, the transcripts encoding genes for both glutamine (PST79_3032) and glutamate synthase (PST79_2575) identified here were up-regulated in haustoria, consistent with a greater role for biosynthesis in this organ. Genes encoding other enzymes that participate in subsequent steps including glutamate dehydrogenase (PST79_536), aspartate aminotransferase (PST79_7929), asparagine synthase (PST79_1116) and asparaginase (PST79_83), were all expressed in both tissues, suggesting nitrogen compounds obtained from the host can be converted freely into compounds required for pathogen growth and development.Previous genome studies of obligately biotrophic fungal plant pathogens have noted the absence of genes encoding enzymes for nitrate and sulfur assimilation Hyaloperonospora arabidopsidisB. graminisP. graminis f.sp. tritici2−) into cysteine, expressed at low levels in both tissues. Genes with high similarities with those involved in the interconversion of homocysteine and cysteine through the intermediary formation of cystathionine (transsulfuration pathways) were also identified. This pathway creates cysteine from methionine.Sulfur is an essential component of living cells as it is a fundamental component of the amino acids methionine and cysteine, Coenzyme A, and iron-sulfur enzymes. Most fungi take up sulfur as sulfate which is reduced to sulfide as a precursor of cysteine Glutathione provides redox buffering to cells, and plays a role in many cellular processes including iron metabolism M. larici-populina and P. graminis f. sp. triticiU. fabae as an in planta-induced gene Cytochrome P450s comprise a diverse superfamily of proteins containing a heme cofactor that catalyse the oxidation of organic substances. Among their most common substrates are steroidal molecules as well as drugs and toxins. The P450 system is important in fungal evolution for adaptation to ecological niches Ste12-like gene (PST79_9215), which was up-regulated in haustoria. Ste12 was first identified in a yeast sterile mutant, and multiple studies have shown that Ste12-like genes play major roles in regulating morphogenetic programs in response to environmental changes , a conserved transcriptional regulator from fungi and mammals, which controls dimorphism in certain fungi TUP1 plays a central role in controlling the expression of the genes implicated in the genetic control of mating, filamentation, and pathogenic development of Ustilago maydisTUP1 in rust fungi could provide a better understand of how it acts within the unique biological context in which these pathogens develop.A second interesting gene was the homolog of Cys3 (PST79_492) reported originally in Neurospora crassaCys3 controls the synthesis of a set of catabolic enzymes for utilization of secondary sulfur sources when sulfur-containing amino acids and inorganic sulfate are missing or limited Pst might suggest that secondary sulfur sources play a role in biotrophy, and upregulation of this gene in haustoria could suggest these sources are derived from the host.Lastly, five other genes associated with transcription factor activity were found in our data, encoding a putative CCAAT-box binding factor (PST79_5498), a putative activator of basal transcription (PST79_5377), an MCM-domain-containing protein (minichromosome maintenance ATPase) (PST79_3174), a putative bZIP transcription factor (PST79_8536) and a gene with similarity to the regulator Neurospora crassa. Os-1, also known as Nik-1, is required for adaptation to high osmolarity M. oryzae appresorium HIK1 (Os-1) in this pathogen caused no defect in growth on normal media or in pathogenicity to rice plants, the mutant strain acquired resistance to three groups of fungicides  Pst germinated spores suggests that the osmosensitive signal transduction pathway could play a role in the early stages of stripe rust disease.Cellular differentiation and filamentous growth are finely regulated in many fungi in response to environmental and nutritional signals. External stimuli must be transmitted within the fungus by signal transduction pathways to enable the appropriate response. Our transcriptomic analysis revealed high expression of signal transduction components in both germinated spores and haustoria. Transcripts encoding proteins related to two component response regulators, the Ras protein superfamily of guanine nucleotide exchange factors, and diverse protein kinases were most highly represented. In fungi, two-component signalling systems participate in processes such as environmental sensing, oxidative stress response, cell-cycle control, and switching between non-pathogenic and pathogenic states Ras proteins are a superfamily of guanine nucleotide exchange factors in which the phosphorylation status of the bound nucleotide operates a binary switch. They have numerous signaling roles in cellular functions including cytoskeletal integrity, proliferation, differentiation, cell adhesion, and cell migration. Ras proteins are active in the GTP-bound form, which is promoted by guanine nucleotide exchange factors (GEF) in an exchange reaction with GDP, and deactivated by GTPase activating proteins (GAP) that promote dephosphorylation of the bound GTP nucleotide. We identified evidence of expression of genes encoding proteins with similarity to Ras proteins called ADP ribosylation factors (ARF) in both sampled tissues, which is of particular interest to rust pathogenesis because they regulate vesicular traffic, phospholipid metabolism and actin remodelling. Other genes encoding other types of Ras protein as well as GEFs and GAPs were also found in both tissues, suggesting complete representation of these complex molecular switches that regulate cell fate.Transcriptome studies such as the current work considerably enhance analysis of genome information. The 454 platform used here gives significantly longer reads than Illumina, which both helps with transcript assembly and demonstrates gene expression. In contrast, small-read based assemblies are subject to assembly errors and the uncertainties of gene prediction, although these also benefit from annotation with transcriptomic data. One important aspect of defining transcriptomes, as described here, is that it cuts through the complexity of the genome. Thus, while the predicted coding capacity of rust genomes is ∼22,000 genes, we identified ∼12,000 expressed contigs in the sampled tissues. Presumably, many of the remaining genes are expressed in different contexts , or were simply undetected or unassembled in our study. Although isolation of haustoria is a very powerful way to track the pathogenic stage of the fungus, we did not analyse infected tissue which includes the infectious hyphal stage that presumably also secretes effector proteins. Another important aspect of our study is quantification of expression using Illumina data, which considerably enhances the accuracy and reliability of our expression measurements. Thus, use of 454 and Illumina together provides a robust expression data set. This allowed us to predict a suite of 437 potential effector genes, defined chiefly as genes upregulated in haustoria that encode secreted proteins. We also note that not all secreted proteins will be virulence effectors, and some within our dataset show clear hallmarks of roles in fungal cell wall modification.M. oryzae, but with adaptation to the particular pathogenic lifecyle of wheat rusts. Haustoria and spores are clearly working in different ways. The germinating spores prioritises polarised growth from stored energy until such time as it is able to mine its own resources, whereas haustoria are a sessile stage devoted to nutrient extraction and defence suppression. The uredinospore contains vast stores of energy in the form of lipid bodies, sugar alcohols and proteins The current data provide a comprehensive view of the stripe rust transcriptome in germinated spores and haustorial stages. Overall, the fungus seems to use similar biochemical pathways to those described in better characterised species such as Pst-susceptible wheat cultivar Morocco were grown in the greenhouse under 70% relative humidity at 21°C and 16∶8 light:dark cycle. Seven-day old seedlings were inoculated with fresh uredinospores of P. striiformis f.sp. tritici strain 104E137A- Seedlings of the g for 5 min, frozen in liquid nitrogen and store at −80°C prior to RNA isolation.For 454-based transcriptome analysis, haustoria were isolated from stripe rust-infected Morocco leaves at 9 dai by Concanavalin A affinity chromatography  [122]. Fg for 15 min at 4°C and the resulting pellet was resuspended in 20 ml of 1× isolation buffer  containing 30% Percoll. The suspension was divided into four tubes and centrifuged at 25,000 g for 30 min at 4°C. The first 10 ml of each tube was recovered, diluted 10 times with 1× isolation buffer and centrifuged at 1080 g for 15 min at 4°C. The pellets were resuspended in 20 ml of 1× isolation buffer containing 25% Percoll and centrifuged at 25,000 g for 30 min at 4°C. The first 10 ml of each tube was recovered, diluted 10 times with 1× isolation buffer and centrifuged at 1080 g for 15 min at 4°C. The final pellet was frozen in liquid nitrogen and stored at −80°C prior to RNA isolation.For Illumina RNAseq expression profiling, haustoria were purified by a Percoll gradient method. Twenty g of infected wheat leaves were treated as above, but after passing through the 20 µm mesh, the filtrate was centrifuged at 1080 −1 FITC-conjugated wheatgerm agglutinin (Sigma) or Concanavalin A, Alexa Fluor 594 conjugate 10 µM (Invitrogen), by incubating them for 30 min at room temperature in phosphate-buffered saline. Viability tests were done on ultrapure haustoria isolated on Percoll gradients combined with flow cytometry  by using CellTracker™ Orange CMRA (Invitrogen) following the manufacturer’s instructions.Bright field and fluorescence images of haustoria isolated by affinity chromatography were collected on a Leica DMR epifluorescence microscope using a 40× objective. Cells were labelled with 100 µg ml18  according to the manufacturer’s instructions. cDNA was produced and fragmented by nebulization, and 454-GS-FLX titanium pyrosequencing (single-end strategy) was carried out on a Roche 454 Titanium sequencer at the Biomolecular Resource Facility . Half a plate was used following standard procedures recommended by Roche. Initial quality filtering of the raw reads was performed using Roche proprietary analysis software Newbler (software release 2.0.00.22) to remove poor quality reads and adapter sequences.Frozen haustoria and germinated spores samples were ground in liquid nitrogen, and total RNA was isolated using the QIAGEN  Plant RNeasy kit following the manufacturer’s instructions. RNA was eluted in RNase-free water and checked for integrity on an Agilent 2100 Bioanalyzer. For 454 sequencing, approximately 5 µg of total RNA from isolated haustoria and 20 µg of total RNA from germinated spores were used to isolate mRNA using Dynabeads Oligo(dT)For Illumina sequencing, approximately 10 µg of total RNA per biological replicate of isolated haustoria and germinated spores were processed with the mRNA-Seq Sample Preparation kit from Illumina to produce the sequencing libraries. Quality and quantity controls were done on Agilent 2100 Bioanalyzer using a DNA 1000 chip kit and each library was diluted and used for sequencing using an Illumina Genome Analyser GX II platform. Libraries from haustoria samples and germinated spores samples were sequenced with 100-base paired-end reads.http://www.clcbio.com/) was used for de novo assembly of 454 reads, prediction of ORFs, Illumina read mapping against reference contigs, computation of normalized counts , allowing up to two base mismatches. Reads that mapped to multiple sites were assigned randomly and proportionally to one of the mapped sites. The random distribution was done proportionally to the number of unique matches that the genes to which it matches have, normalized by the transcript length.CLC Genomic Workbench 4.0 software  correction to obtain an adjusted P-value. Additionally, BLASTn and BLASTx screening against NCBI non-redundant nucleotide and protein databases were used for manual analysis. Only the transcripts that belonged to one of the functional categories were analysed manually to verify if more than one transcript represented the same gene, in which case it was annotated in For the gene ontology (GO) classification the reference transcripts set created by pooling raw reads from both transcriptomes was analyzed using BLAST2GO 2.5.1 18 and Milli-Q water up to 11 µl and heated at 70°C for 10 min, followed by cooling on ice for one minute. DTT was added to a final concentration of 1 mM together with 1 µl of dNTPs (10 mM), 4 µl of 5× superscript II buffer (supplied with superscript) and 0.5 µl of SuperScript III reverse transcriptase . After incubation for 1 h at 42°C, the reaction was stopped by heating 15 min at 70°C and 1 µl of RNase H (1 U/µl) was added to each tube and incubated for 20 min at 37°C. Target cDNAs were amplified using a dilution of 1∶15 of the synthesized cDNA as template, specific primers forward and reverse and 2× PCR Master Mix (Promega). PCR was performed using a MiniCycler (MJ Research) and consisted of 30 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec and extension at 72°C for 1 min.Total RNA was isolated from infected (9 dai) and uninfected wheat leaves ground in liquid nitrogen and extracted using QIAGEN  Plant RNeasy kit according to the manufacturer’s instructions. For cDNA preparation, 2 µg of total RNA were mixed with 1 µl of oligo(dT)Figure S1Number of 454 reads per assembled contig for germinated spores and haustorial samples.(TIF)Click here for additional data file.Figure S2Length of contigs for germinated spores and haustorial samples assembled from 454 data.(TIF)Click here for additional data file.Figure S3Coverage of 454 contigs assembled from 454 data from germinated spores and haustoria samples.(TIF)Click here for additional data file.Figure S4Isolated haustoria by affinity chromatography. A. Stripe rust haustoria isolated by affinity chromatography. Hautoria isolated by ConA affinity chromatography using a 20 µm mesh to filter homogenized tissue and remove plant cell debris. Bright field image with differential-interference contrast, haustoria (black arrows) and contaminating chloroplasts can be seen. B. Fluorescence microscopy of isolated haustoria by affinity chromatography. Haustoria were isolated from infected tissue by ConA affinity chromatography as described in (TIF)Click here for additional data file.Table S1Transcripts reference set. 12,282 EST contigs assembled from germinated spores and haustorial datasets were used as references to map Illumina RNA-seq data from the same tissues. Three biological replicates for haustoria  and spores  were used for sequencing. Differences between each tissue were established by RPKM values for each replicate which were subjected to Baggerley’s test to provide statistical support for differences in gene expression.(XLSX)Click here for additional data file.Table S2Haustorial secreted proteins (HSPs). A compilation of analyses of the 437 predicted haustorial secreted proteins. The table shows values for expression levels based on Illumina data , differential expression, cysteine content, BLASTx analysis against the NCBI-nr protein database, BLAST2GO analysis, PFAM motif analysis and Y/F/WxC motif content. In the Y/F/WxC column, 1b30 indicates a single of these motifs before the 30th amino acid, whereas 1a30 represents a single motif after the 30th amino acid.(XLSX)Click here for additional data file.Table S3General metabolism. Transcripts encoding proteins with similarities to enzymes involved in metabolic pathways, or with particular roles as described in the text. Expression levels based on Illumina data are presented  as well as BLASTx analysis against the NCBI-nr protein database.(XLSX)Click here for additional data file.Table S4Transporters. Transcripts encoding proteins with similarities to different transporter categories were identified by BLASTx against the NCBI-nr protein database and the Transport Classification Database (TCDB). Expression levels based on Illumina data are presented .(XLSX)Click here for additional data file.Table S5Energy metabolism. Transcripts encoding proteins with similarities to the enzymes of glycolysis, citric acid cycle, and oxidative phosphorylation, and their in silico expression levels based on Illumina data are presented .(XLSX)Click here for additional data file.Table S6Gene expression values for the ontological analysis presented in . Transcripts analysed with BLAST2GO and classed into different metabolic categories summarised in in silico expression levels based on Illumina data are presented . Transcripts highlighted in brown were excluded from the analysis due to low RPKM values (<10).(XLSX)Click here for additional data file.Table S7Illumina sequencing data before and after mapping against the transcripts reference set. Three biological replicates were sequenced with Illumina for isolated haustoria (H) and germinated spores (S). The table shows the millions of reads obtained per replicate and the percentage of reads mapping against the transcripts reference set assembled from 454 haustoria and germinated spore data.(DOCX)Click here for additional data file.Table S8List of primers to validate candidate effector gene expression by non-quantitative RT-PCR.(XLSX)Click here for additional data file.Data S1Germinated spores transcriptome.(ZIP)Click here for additional data file.Data S2Haustoria transcriptome.(ZIP)Click here for additional data file.Data S3437 HSP ORFs.(ZIP)Click here for additional data file.Data S4PST79 combined transcriptome.(ZIP)Click here for additional data file.Text S1Supplementary text.(DOCX)Click here for additional data file.


Crohn's disease (CD) is an inflammatory bowel disease of complex etiology, although dysbiosis of the gut microbiota has been implicated in chronic immune-mediated inflammation associated with CD. Here we combined shotgun metagenomic and metaproteomic approaches to identify potential functional signatures of CD in stool samples from six twin pairs that were either healthy, or that had CD in the ileum (ICD) or colon (CCD). Integration of these omics approaches revealed several genes, proteins, and pathways that primarily differentiated ICD from healthy subjects, including depletion of many proteins in ICD. In addition, the ICD phenotype was associated with alterations in bacterial carbohydrate metabolism, bacterial-host interactions, as well as human host-secreted enzymes. This eco-systems biology approach underscores the link between the gut microbiota and functional alterations in the pathophysiology of Crohn's disease and aids in identification of novel diagnostic targets and disease specific biomarkers. Humans live in close association with communities of microorganisms (the human microbiota) that inhabit every exposed surface and cavity in the body The next step is to determine how the human microbiome varies with disease. As part of a demonstration project funded through the NIH Human Microbiome Project (HMP) we have focused on the impact of the inflammatory bowel disease (IBD), Crohn's disease on the gut microbiota. Although most human host-microbe associations are beneficial, several studies using both culture-dependent and molecular approaches have suggested that there is a dysbiosis in the gut microbiota of patients with Crohn's disease (CD) compared to healthy subjects identities of microbial community members (obtained from 16S rRNA gene-based measurements), metabolic potential (obtained from metagenome sequence data) and expression (obtained from metaproteome data) should enable exploration of the gut microbiota at multiple molecular levels simultaneously.Although recent advances in DNA sequencing and proteomics technologies have opened the door to investigation of the structure and function of the gut microbiota without the necessity for cultivation, there have been very few efforts to date that have used a multi-“omics” approach to study the complex ecosystem in the human gut Faecalibacterium prausnitzii.This study was focused on a subset of stool samples collected from a large Swedish twin cohort with inflammatory bowel disease (IBD) that was previously characterized with respect to their bacterial community composition by deep 16S rRNA pyrotag sequencing Here our aim was to further explore a subset of the same Swedish twin cohort for functions that were correlated to CD by applying non-targeted, shotgun metagenomics The specific questions that we set out to address in this study were: (1) What genes are actually expressed as proteins in the gut and could play a functional role in the gut environment? (2) Are there specific genes and proteins that could help to explain the previously observed differentiation of the samples according to Crohn's disease etiology?Shotgun metaproteomics is a relatively new technology in its' application to complex and highly diverse microbial communities, such as the human gut, and only recently have there been reports about protein compositions in the gut and from only a few healthy subjects We generated shotgun metagenomic (Table S2 in expressed gene products (proteins) under the conditions studied. Therefore, to directly address gene function and protein abundance, we performed database searches with tandem mass spectra (MS/MS) of peptides from the same samples collected via multi-dimensional liquid chromatography tandem mass spectrometry (2d-LC-MS/MS). These extensive MS/MS datasets were searched either against their corresponding matched metagenome (MM) , 850 (ICD), and 788 (CCD) orthologous protein clusters were identified with MM searches and 2,904 , 1,928 (ICD), and 2,241 (CCD) proteins using HMRG searches. Together, these data represent the largest metaproteome analysis of the human gut to date (Tables S3 and S4). To gauge the overlap in protein sequence coverage between the MM  and HMRG databases, we compared the assigned, non-redundant spectra with high mass accuracy (±10ppm) with PSMs from both searches. Of the total spectra that have peptide assignments to microbial and human proteins, 64% and 33% of the PSMs were unique to the MM and HMRG databases, respectively . Greater than 60% of the metagenomic sequence reads in the samples from healthy subjects could not be assigned at the phylum, family or genus level, compared to ∼40% of the reads in ICD or CCD subjects, potentially reflecting the reduced bacterial diversity in the gut of CD patients. Of the metagenomic reads for which a taxonomic assignment could be made, 396 genera were represented in all of the samples, and nine of those were present at >5% of reads, representing the core taxa. Some members of the Firmicutes phylum, such as  p<0.05; , a resulFaecalibacterium, Roseburia, Dialister and Coprococcus were significantly less abundant in ICD relative to healthy subjects  . This fiEach dataset contained a subset of genes and proteins of unknown function. For example, ∼17% of predicted ORFs in the metagenomic data were either conserved with no known function or were not homologous to any known proteins. Approximately 31% of the proteins identified with the HMRG database  and 29% There were significant differences in several COG categories when comparing the metaproteomes of ICD to healthy, primarily due to a decrease in abundance of proteins in ICD . GeneralAt a finer scale of resolution, there were 116 statistically significant differentiating specific COGs between disease categories in the metaproteome data  of ≤0.05; The metaproteome data indicated significant differences in carbohydrate degradation pathways between ICD and healthy . SimilarFaecalibacterium was even lower than would be expected by the abundance of this organism  production pathways in ICD in both the metagenome  and metaorganism , suggestBacteroides proteins  had a higher representation in the ICD microbiota compared to healthy . Based oproteins  and 6A. proteins  and 6A. both bacterial and human proteins in the same samples using metaproteomics, a total of 1,646 human proteins were experimentally identified in addition to the microbial proteins discussed above. Gene ontology (GO) analysis revealed that human proteins found in all 3 subject groups (core) are enriched in functions associated with the structural integrity of the mucosal epithelium such as regulation and activity of actin cytoskeletal components. Proteolysis, digestion, and carbohydrate catabolism were also among the most abundant ‘core’ functional terms, as would be expected in the human GI-tract  how the composition and function of the gut microbiota changes over time with respect to disease inflammation and ii) how the microbiota is impacted by other factors including drug therapy and surgery.The Swedish twin cohort was previously described in several studies Stool samples were shipped to the Orebro University Hospital, Orebro, Sweden, at most one day after sample collection and immediately frozen at −70°C upon arrival. The samples were stored continuously frozen until use and small portions were excised and thawed immediately prior to DNA extraction to avoid freeze-thaw damage. DNA was extracted from 250 mg of each stool sample in duplicate using the MoBio Power Soil DNA Kit , as previously described DNA isolation from stool samples yielded 3–5 ug of purified metagenomic DNA from each of twelve samples. Each sample was subjected to picogreen and gel-based QC assays prior to library construction. Unpaired, shotgun fragment sequencing libraries were constructed using our customized, automated library construction procedure. Our method modifies the manufacturer-provided protocol by adjusting enzymatic reaction volumes and replacing gel-based fragment size-selection steps with AMPure SPRI magnetic beads to enable automation of the process using liquid-handling robotics. Following library construction, each sample was subjected to emPCR amplification and 454 sequencing according to manufacturer specifications. Raw sequence data was processed using the Roche/454 run processing software to filter short, mixed, and low-quality reads. Whole metagenomic shotgun sequencing generated a total of 15,307,850 reads and more than 5,428,202 kilobases (or 5 Gbp) of high-quality, passed-filter sequence data  for taxonomic assignment. In cases where reads did not match reference genomes taxonomic classification was made using sequence comparison against known proteins in NCBI NR using BLASTX (90% id). In cases where reads had high identity matches to multiple sequences, the taxonomic nearest neighbor was chosen. Taxonomic classification for each MS spectrum was determined by the protein sequence predicted from metagenomic contig sequences, where the taxonomy of a contig is based on the nearest neighbor classification of the read sequences composing the contig. In cases where no classification was obtained, the ‘human gut microbiome classification’ was given. Family assignments are based on the NCBI taxonomic tree. Potential 16S sequences were identified using RNA-HMM and classified using RDP 2.0. Clustering of samples by taxonomy was done using Ginko, with a log10(X+1) normalization, euclidean distances and Ward's method for hierarchical clustering.Sequences were assembled with the Newbler Assembler (v2.0.01.14) and genes were predicted on contigs greater than 500 bp using METAGENE An all-vs-all BLASTP −6 and bits per position cutoff of 1. COG and NOG functional assignments were assigned based on this comparison. In addition sequences were searched against a library of HMMs consisting of TIGRFAMS ORFs were searched against the eggNOG 18 solid phase extraction, concentrated, solvent exchanged into 100% water/0.1% formic acid, filtered (0.45 um filter), and aliquoted.Approximately 10 g portions of the same stool samples used for DNA extractions were processed by differential centrifugation to enrich the bacterial cell fraction as previously described 2O, 5% acetonitrile, 0.1% formic acid) to 50% solvent B . The last salt pulse was followed with a gradient from 100% solvent A to 100% solvent B. During a single chromatographic separation (22 hr run), mass spectral data acquisition was performed with Xcalibur software . Precursor full MS spectra (from 400–1700 m/z) were acquired in the Orbitrap with resolution r = 30,000 followed by five data-dependent MS/MS scans at 35% normalized collision energy in the LTQ with dynamic exclusion enabled (repeat count 1).All samples were analyzed in technical duplicates via two-dimensional (2D) nano-LC MS/MS with a split-phase column (RP-SCX-RP) The first database, referred to as the matched metagenome (MM), was created per sample by directly predicting ORFs from raw sequencing reads to prevent loss of sequence diversity when collapsing unrelated sequencing reads for metgenome assembly , was utilized in a complementary database search and also contained human reference sequences and common contaminants. While this reference genome database is not exactly representative of each sample, it can provide definitive species/protein identifications, which were used to support and complement the MM searches. This database was created by concatenating 51 human-derived reference isolate genomes from the JGI IMG human microbiome project (IMG-HMP) into a single FASTA-formatted protein sequence database. The criteria used to select 51 human-derived microbial isolates were based on genera that have been previously found in the 16S data from the same samples All MS/MS from individual runs were searched with the SEQUEST (v.27) algorithm www.proteomecommons.org and assigned the hash identifier: rji3fAXT1XG0PxdrWWrM1M4XXznm6i7XKW2ZMVbfyYvo2G44eBimTcv4osnXHyhDvoCOA1av4EywiTFqX8PfJI9SP4EAAAAAAAChfg.The acquired mass spectrometry data (mzXML format) from this publication have been submitted to the Proteome Commons Tranche repository at A target-decoy database The spectral count for a microbial protein cluster (“CLST…”) was calculated as the number of unique peptide identifications that can be attributed to proteins from that cluster and not from any other cluster. Because proteins with high sequence similarity were grouped in clusters, the majority of peptide identifications from the metagenomic read databases (RMPS) can be uniquely attributed to only one cluster. The spectral counts for human proteins were calculated from both unique and non-unique peptide identifications using DTASelect with default settings as described above.i, was calculated for every run as αi  =  N/ni, where N is the average number of total MS/MS spectra per run and ni is the MS/MS spectral number of run i. The spectral counts for all proteins in a single MS run were then normalized by multiplying them with the run's scaling factor. The reference isolate genome database results were also normalized using the same scaling factor and approach.Spectral counts for both human proteins and microbial protein clusters from an MS/MS run were normalized by the total numbers of tandem mass spectra (MS/MS) of this run. A scaling factor, αThe 24 MS runs were grouped into the following three sample sets for both databases (MMs and HMRGs): healthy subjects: 6a, 6b, 16b, and 18b; CCD subjects: 9a and 9b; and ICD subjects: 10a, 10b, 15a, 15b, 16a, and 18a.The metagenomic microbial protein clusters (MM databases) with differential expression between two sample sets were identified using label-free quantification. We only considered microbial protein clusters that have more than five spectral counts in four or more of the runs in the two sets under comparison. P-values were calculated using the Wilcoxon rank sum test. The p-values were then used to compute q-values The proteomics results were also analyzed using hierarchical clustering. We only considered proteins with median absolute deviations greater than 1. Normalized spectral counts were log2 transformed by adding a pseudo-count of one. Hierarchical clustering on both proteins and samples were performed using the hclust function in the R stat library and the heatmap was plotted using the heatmap.2 function in the R gplot library.Non-metric multidimensional scaling (nMDS) was performed using normalized spectral abundances of proteins derived from 24 MS runs searched against 51 human-associated bacterial isolates. nMDS was performed in PCORD v5 using the Bray-Curtis distance measure KEGG modules analysis was performed to highlight differences in metabolism between healthy and CD. The bulk of metaproteomic KOs were mapped to the KEGG modules reference database in addition to the butyrate production module. Only modules that had more than 30% coverage were considered for downstream analysis. Then differential expression between modules was tested using Wilcoxon's rank-sum test in R and p-values were corrected for multiple testing using Benjamini-Hochberg's false discovery rate (FDR). A module was considered significantly different if the median difference between the two groups was more than 5 with FDR set to 10% under a two-sided alternative hypothesis. Modules and KOs that were significantly down regulated in ICD were visualized within iPATH LBNL has an approved Federal-wide assurance on file with HHS that covers this activity: OHRP Federal-wide Assurance Number FWA 00006253. Certification of Human Subjects Committee review: This activity has been reviewed and approved by the HSC in accordance with requirements sent forth in the DHHS regulations at 45 CFR 46.103(f), which requires that each application or proposal for HHS-supported human subject research be reviewed and approved by the Institutional Review Board. Date of Approval: April 30, 2010; Approval Number: 272H01-30APR11.The consent procedure was approved by the ethical research committee at Örebro University Hospital, where the samples were collected. The study was approved by Örebro Lans Landsting on December 17, 2003 (D-nr 167/03).I have been informed in writing about this actual study and have had time in peace and quiet to read through the information and to ask questions by telephone. I have also been provided with a copy of the written information and my written consent.Through my signature I provide my consent to:participate in the study.that my personal information can be used as in the written information.that my samples are treated as in the written information.that Jonas Halfvarson, gastroenterologist at USÖ, can request copies of my medical journal.I am aware that participation is voluntary, and that at the same time I may at any time and without excuse cancel my participation without influencing my future care.(Direct translation from Swedish).Supporting Information S1Additional figures, tables, a note regarding technical and twin reproducibility in the metaproteomes and peptide-level false discovery rates.(PDF)Click here for additional data file.Table S5Normalized total spectra counts across all subjects and 24 MS runs for the matched metagenome (MM) database searches.(XLS)Click here for additional data file.Table S6Normalized total spectra counts across all subjects and 24 MS runs for the human microbial isolate reference genome database (HMRG) searches.(XLS)Click here for additional data file.Table S7Human microbial isolate reference genome database (HMRG) database components. 51 bacterial isolates were downloaded from the JGI IMG human microbiome project (IMG-HMP) into a single FASTA-formatted protein sequence database.(XLSX)Click here for additional data file.Table S8Distribution of all normalized ‘unique’ spectra counts (worksheet 1) for a metaproteome genus-level comparison of all 24 MS runs against the HMRG database. Three comparisons (worksheet 2–4) between different phenotypes  were performed with Wilson rank sum: Q value  less than 0.05, difference between medians of the two conditions greater than 5, and more than 4 runs with greater than 5 spectral counts. Only the ‘healthy’ versus ‘ICD’ comparison have several genera that are significantly changed.(XLSX)Click here for additional data file.Table S9Core and unique microbial protein clusters identified in the metaproteomes. Common core microbial protein clusters (worksheet 1) identified in the metaproteomes of all subjects included in the study . Microbial protein clusters that were identified as unique to one phenotype, healthy (worksheet 2), ICD (worksheet 3), and CCD (worksheet 4).(XLSX)Click here for additional data file.Table S10Metaproteomic statistical comparison of spectra assigned to COGs identified across all samples. Three comparisons (worksheet 2–4) between different phenotypes  were performed with Wilson rank sum. A COG labeled with significant UP or DOWN has to satisfy these criteria: Q value  less than 0.05, difference between medians of the two conditions greater than 5, more than 4 runs with greater than 5 spectral counts.(XLSX)Click here for additional data file.Table S11False discovery rates estimated at the peptide level (≥2 peptide level) for the HMRG database searches for three MS experiments: 6b, run 1 (worksheet 1), 9a, run 2 (worksheet 2), and 18a, run 2 (worksheet 3).(XLS)Click here for additional data file.Table S12False discovery rates of read-based peptides from MM metaproteomic database searches for three MS experiments: 6b, run 1 (worksheet 1), 9a, run 2 (worksheet 2), and 18a, run 2 (worksheet 3) at a 1-peptide level with and without high mass accuracy. FDRs estimated post-database mapping of all read-based peptides to assembled contigs at a 2-peptide level are provide in the final worksheet.(XLS)Click here for additional data file.


Studies that analyzed single nucleotide polymorphisms (SNP) in various genes have shown that genetic factors are strongly associated with age-related macular degeneration (AMD) susceptibility. Copy number variation (CNV) may be an additional type of genetic variation that contributes to AMD pathogenesis. This study investigated CNV in 4 AMD-relevant genes in Korean AMD patients and control subjects.VEGFA, ARMS2/HTRA1, CFH and VLDLR), were selected based on the outcomes of our previous study which elucidated common CNVs in the Asian populations. Real-time PCR based TaqMan Copy Number Assays were performed on CNV candidates in 273 AMD patients and 257 control subjects.Four CNV candidate regions located in AMD-relevant genes . Comparisons of the raw copy values (RCV) revealed that 3 of 4 candidate genes showed significant differences .The predicted copy number  of each region was called using the CopyCaller program. All candidate genes except CNVs located in AMD-relevant genes may be associated with AMD susceptibility. Further investigations encompassing larger patient cohorts are needed to elucidate the role of CNV in AMD pathogenesis. Age-related macular degeneration (AMD) is a major cause of visual loss in the elderly in industrialized countries, estimated to affect more than 50 million people worldwide CFH) and 10q26 (ARMS2/HTRA1) CFH and ARMS2/HTRA1, genes associated with inflammation, oxidative stress, angiogenesis and lipid metabolism have been reported to be responsible for AMD susceptibility Genome-wide linkage and association studies have successfully identified several major chromosomal regions, including 1q31 (We have previously discovered common Asian copy number variations (CNVs) through genotyping 30 individuals from 3 Asian populations - Korean, Chinese and Japanese. Using microarrays with 24 million probes, we have discovered 5,177 CNVs, of which 3,547 are putative Asian-specific CNVs To verify if the CNVs of AMD-relevant genes are associated with AMD, we selected candidate CNVs discovered in the Asian populations and analyzed their status in Korean AMD patients and control subjects.Consecutive, unrelated AMD patients and control subjects were recruited from the outpatient clinic at a research hospital from August 2008 to February 2010. The institutional review board of the Seoul National University Hospital approved this study, and written informed consents were obtained from all participants prior to study entry.Color photographs of fundi were evaluated using the Age-Related Eye Disease Study (AREDS) grading system by graders blinded to patient diagnoses Ten milliliter (10 mL) of peripheral blood sample was collected from each study subject, and centrifuged for 5 min at 2000 g to separate the serum. DNA was extracted using FlexiGene DNA kit  according to the manufacturer's protocol and stored at −70°C. DNA concentrations were measured by Nanodrop ND-1000 spectrophotometer . Samples from AMD patients and control subjects were collected during the same period and processed using the same method.VEGFA, ARMS2/HTRA1, CFH and VLDLR  was chosen as a reference gene, of which every human is known to possess 2 copies. Every reaction was duplicated in 384 well plates, and each plate was comprised of almost an equal number of disease and control samples. Real-time PCR was performed using the Applied Biosystems 7900HT Fast System and Sequence Detection Systems Software v2.3. The thermal cycling condition was as follows: holding at 95°C for 10 min, 40 cycles for 15 s at 95°C for denaturation and 60 s at 60°C for amplification.We performed real-time PCR to determine CNV status on each sample. TaqMan probes, which were designed by the manufacturer , were used to target the specific regions. Applied Biosystems CopyCaller™ Software v1.0 was used to determine the copy number status of each target region, and calculation was performed according to a maximum-likelihood algorithm of the software. Raw copy value (RCV) represents a non-integer number of copy calculated, whereas predicted copy number (PCN) is defined as an integer number of copy determined by the algorithm . Copy number (CN) gain is defined as PCN higher than 2, and PCN lower than 2 would be regarded as CN loss.p-value was lower than 0.05 (p<0.05).We performed a two-tailed t-test to evaluate the difference in mean values of RCV between the disease and control groups. A chi-square test was used to compare the frequency of CN gain and loss respectively between the two groups. For both analyses, statistical significance was deemed to have been reached when the The clinical characteristics of AMD patients and control subjects are summarized in ARMS2/HTRA1 showed either CN gain or loss. CN gains in VEGFA and VLDLR genes had been reported in the previous study on 30 Asian samples. However, CN losses in VEGFA and VLDLR genes, which were detected in 3 and 29 subjects respectively, were novel findings in the Asian population.We attempted to predict the integer value of CN (PCN) at each region and the results were summarized in VEGFA gene was not detected and one CN gain was found. The patient with CN gain of VEGFA gene had neovascular AMD in the left eye and intermediate AMD in the right eye , while CN loss was not significantly different between the patient and control groups.In AMD patients, CN loss of ight eye . Three soth eyes . CN gainVEGFA and CFH , and lower RCV at ARMS2/HTRA1  is a major molecular mediator of neovascularization. Intraocular VEGF expression was found to be increased in neovascular AMD patients, which led to the development of VEGF inhibition therapies with anti-VEGF antibodies for neovascular AMD ARMS2/HTRA1 gene encodes a member of a family of serine proteases expressed in both mouse and human retinas, and its expression in human fibroblasts increases with aging ARMS2/HTRA1 alters the integrity of the Bruch's membrane, favoring the invasion of choroid capillaries across the extracellular matrix, as occurs in wet AMD ARMS2/HTRA1 was lower in AMD patients compared to control subjects. Since there was no CN gain or loss predicted, studies to elucidate the genuine CNV status of ARMS2/HTRA1 in AMD patients, and its possible effect on ARMS2/HTRA1 expression would be needed.The CFH) on chromosome 1q32 CFH gene in one AMD patient but it could not find any association of CFH variation with AMD. Other complement factor genes and complement factor related regions may find significant genetic variations in complement factor related genes in AMD patients.Genes encoding complement factors have been identified as AMD susceptibility loci with convincing statistical evidence. They include complement factor H gene (VLDLR gene knockout (VLDLR−/−) mice were shown to develop sub-retinal neovascularization VLDLR showed associations with AMD prevalence VLDLR was very common in control subjects and in AMD patients. It may be due to its regional complexity in the human genome, and therefore this region should be interpreted with caution.Very low density lipoprotein receptor (VLDLR) is involved in lipid transportation and chronic inflammation through the Wnt pathway C2/BF, TIMP, CFHR1 and CFHR3, were not included in our target regions.In this study, we selected target CNVs out of Asian-specific CNVs discovered in our previous study. Consequently, several genes which have been thought to play important roles in AMD pathogenesis and widely studied in other ethnic groups, such as in vivo remains unelucidated. Further CNV-oriented research is required to improve our understanding of the mechanism that underlies AMD.In conclusion, the outcome of this study suggests that some CNVs might be associated with AMD development, although the actual action of these CNVs Table S1General information of TaqMan Copy Number Assays used for this study.(DOC)Click here for additional data file.


Octodon degus), which are a species of small caviomorph rodents, could be trained to use a T-shaped rake as a hand tool to expand accessible spaces. To elucidate the neurobiological underpinnings of this higher brain function, we compared this tool use learning task with a simple spatial  memory task and investigated the changes that were induced in the hippocampal neural circuits known to subserve spatial perception and learning. With the exposure to an enriched environment in home cage, adult neurogenesis in the dentate gyrus of the hippocampus was augmented by tool use learning, but not radial maze learning, when compared to control conditions. Furthermore, the proportion of new synapses formed in the CA3 region of the hippocampus, the target area for projections of mossy fiber axons emanating from newborn neurons, was specifically increased by tool use learning. Thus, active tool use behavior by rodents, learned through multiple training sessions, requires the hippocampus to generate more novel neurons and synapses than spatial information processing in radial maze learning.We previously demonstrated that degus ( The degus lived with their littermates (or their family) in a wooden sleeping den and tunnel with a metal running wheel. These so-called “enriched environments” were mandatory for proper maintenance of the degu colony, as previous reports showed that chronic isolation and poor environmental living conditions induces a significant decrease in the density of NADPH-disphorase-reactive neurons Adult male degus (weight 180–260 g) from five litters were used in this study. Degus, which have a similar taxonomic classification as Guinea pigs, are diurnal rodents that are native to the South American Andes Mountains in Chile. Their ecology and social systems are unique and quite different to laboratory-raised nocturnal rats and mice. Degus live in tight-knit extended family units and display complicated social behaviors, including combined maternal paternal rearing and rich repertoires of vocal communications Eight degus were used for tool use experiments  and 7 were used for radial maze experiments . There were no differences between control and two experimental groups in genetic characteristics, hair colors and body weight , tool use: 216.2±16.0 g (n = 4), Radial maze: 228.7±6.2 g (n = 3), one-way ANOVA, p = 0.31) when behavioral analyses initiated at 8–9 months old. We selected these animals for the present experiments, as 1) training procedures for tool-use has been established Tool use training was carried out on a training platform consisted of a training stage (20×30×7 cm) and a transparent fence (45×55×0.5 cm) that separated the animal from the areas where the food rewards (peeled sunflower seeds) and tools were placed. The rake-like tools were T-shaped, with a 4 cm long wire shaft, a rectangular plane (3 cm wide×1 cm high) made of acrylic resin, and a spherical grip 1 cm in diameter. The tool weighed 2.1 g Figure 1CD). Training sessions were monitored with two video cameras (Sony DCR-PC110) placed at the top and the upper-left side of the platform, and sessions were recorded with a camcorder (Panasonic VDR-M30). After approximately two weeks of habituation, the training sessions were performed using the successive approximation method detailed in our previous report D. TrainiThe radial maze consisted of eight equally spaced arms that radiated from an octagonal central platform. Each arm was 64.5 cm long×40 cm high×11 cm wide. A food cup of 3 cm diameter was placed at the end of each arm. Spatial cues  were placed on the wall of the experimental room . Degus aImmediately after the acquisition of training was completed in either the tool use or radial maze task, bromodeoxyuridine  was intraperitoneally injected for 4 consecutive days. Then, 10 days after the final injection of BrdU, animals (12–15 month old) were deeply anesthetized by pentobarbital and transcardially perfused with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). The same procedures were applied to control animals . After postfixation by 4% PFA in PBS and subsequent cryoprotection in 20% sucrose in PBS, coronal sections (35 µm thickness) were cut with a sliding microtome. Free-floating sections were permeabilized for 30 min in 0.1% Triton X-100 in PBS and were then subjected to immunohistochemistry. Brain sections were stained with antibodies against PSA-NCAM (a marker of immature neurons) and BrdU (a marker of newly generated cells) by an indirect immunofluorescent method. Adjacent sections were then immunostained with antibodies against PSA-NCAM and Synapsin-I (a marker of presynaptic vesicles).stratum lucidum of the CA3 region. Synapsin-I is a synaptic vesicle-associated protein We used antibodies against PSA-NCAM and BrdU to detect adult neurogenesis in the hippocampal dentate gyrus. BrdU is incorporated into the DNA of dividing cells and can then be detected by immunohistochemistry in all of the progeny of these dividing cells. PSA-NCAM is expressed during an early phase of adult neurogenesis in the dentate gyrus and is a marker of immature neurons. We also used antibodies against PSA-NCAM and Synapsin-I to detect both newly generated and exsisting synapses in the To detect neurogenesis in the dentate gyrus, the primary antibodies used for immunohistochemistry were: mouse anti-polysialic acid-NCAM (PSA-NCAM) monoclonal antibody  and rat anti-BrdU mAb . The secondary antibodies used for immunohistochemistry were: goat anti-mouse IgM antibody Alexa Fluor 546 Conjugate (Molecular Probes) (1∶500) and goat anti-rat IgG antibody Alexa Fluor 488 Conjugate (Molecular Probes) (1∶500). For CA3 presynaptic puncta detection, the primary antibodies used for immune- histochemistry were: mouse anti-polysialic acid-NCAM (PSA-NCAM) mAb (Millipore Chemicon) (1∶400) and rabbit anti-Synapsin I pAb (Sigma) (1∶300). The secondary antibodies used for immunohistochemistry were: goat anti-rabbit IgG antibody Alexa Fluor 488 Conjugate (Molecular Probes) (1∶500) and goat anti-mouse IgM antibody Aleza Fluor 546 Conjugate (Molecular Probes) (1∶500).For analyses of neurogenesis in the dentate gyrus, two to three confocal images of double-positive signals in the dentate gyrus  were acquired randomly from each slice in a blind fashion using a FV500 laser scanning microscope system (Olympus). Two to four sections were used for each animal. Ten confocal images were acquired along the z-axis (1.2 µm×12) for each visual field with an oil-immersion objective lens . Double-positive signals were counted in a blind fashion by a researcher who was not involved in acquisition of the confocal images. Proper alignment and correct image registration of both the laser lines  and the detection channels were verified with double-labeled fluorescent beads . To avoid cross detection of green and red signals, images were sequentially acquired at 488 and 543 nm, respectively. Because the corner of the dentate gyrus, where the upper and lower blades intersect, was the location where adult newborn neurons were most highly clustered, we acquired two to three images per field of the dentate gyrus to avoid wide scattering.stratum lucidum  were acquired randomly from each slice, in a blind fashion, using a FV500 system. Two to four slices were used for each animal. Proper alignment and correct image registration of both the laser lines and the detection channels were verified with double-labeled fluorescent beads . Eighteen confocal images were acquired at a z-step of 0.6 µm with an Olympus FV1000 microscope equipped with an oil-immersion objective lens  and a digital zoom of ×2.5. To avoid cross detection of green and red signals, images were sequentially acquired at 488 and 543 nm, respectively. Colocalization was quantified using Image Pro Plus 4.5.1 software (MediaCybergenetics). Synapsin I-positive puncta (Green) and PSA-NCAM-positive fiber (red) images were merged, and colocalized signals (yellow) were automatically detected in a double-blind fashion.For the analyses of presynaptic puncta in CA3, two to three confocal images of double-positive signals in the In previous reports stratum lucidum in CA3 subfield), and no significant differences were found among slices from the same groups. When assessing the visual fields, the large Synapsin-I-positive puncta were situated directly adjacent to the apical dendrites of the pyramidal neurons, which was consistent with the expected distribution of large MF boutons in vivo. Because the large puncta were invariably located in the hilus and the stratum lucidum of the CA3 subfield, but never in the CA1 subfield stratum lucidum were used to validate data.Visual fields were randomly selected to count adult neurogenesis (subgranular layer of dentate gyrus) and synaptogenesis (t-test) confirmed that there were no significant differences between these two learning curves (P = 0.16).A total of 15 degus were used in this study. This number included degus exposed to tool use (n = 4) and radial maze (n = 3) task trainings, as well as control groups . The procedure that was used for tool use training was reported in our previous study BrdU was injected into the animals subjected to training once per day for 4 days after task acquisition . These animals were then fixed 10 days after the final BrdU injections . In the right panels of Figures 2AB, BrdU injections occurred at day “0″, and both the tool use and radial maze trainings continued throughout the 14 days shown (trainings were performed 4 days per week). The success rates of the tool use and radial maze tasks were greater than 80% both before and after the BrdU injections. The control animals were exposed to the same number of training days, but with control conditions, and were given the same amount of food in the same experimental apparatuses as the trained animals. Similar to the trained animals, control animals were fixed 14 days after the initial BrdU injections. Instead of using idle naïve controls, as is done in many other rodent experiments employed to detect enhanced neurogenesis in various learning paradigms Five brain sections were randomly picked from 50 total sections that covered the entire dentate gyrus, and were subjected to double-blind confocal microscopic analyses to detect adult neurogenesis by double-labeling of BrdU and Polysialic acid-neural cell adhesion molecule (PSA-NCAM), BrdU is a marker of newly-generated cells and PSA-NCAM is a marker of immature neurons stratum lucidumstratum lucidum. Synapsin-I is a protein associated with small synaptic vesicles stratum lucidum. A representative image is shown in Hippocampal adult neurogenesis alters network functions through the continuous addition of new neuronal units to the network. These new neurons extend long axonal projects along the mossy fiber pathways that innervate the target CA3 pyramidal cell layer and make new synapses with dendrites of pyramidal cells that are located in the The hippocampal dentate gyrus is known to be a location where new neurons are generated throughout adulthood. These new neurons are sparsely integrated throughout the granular cell layer, and their numbers are enhanced by exposure to an enriched environment A previous report showed that reduction of adult neurogenesis by toxin affect fear conditioning paradigm but did not contextual fear conditioning or spatial navigation learning in Morris water maze What are the differences between radial maze and tool use learning? There are three major components of tool use learning that may be more demanding than radial maze learning and may require additional hippocampal circuit resources. One difference would be that tool use learning requires quantitatively greater numbers of items and spatial structures to be learned. For example, the locations of the beams and food need to be learned in the radial maze task, while the tools, baits, hand movements, and variations in different situations need to be learned in the tool use task. A second and more critical qualitative difference exists between the two tasks. In the tool use task, spatial structures change dynamically in time. When a tool is used to move food, then the locations of the items in space are changed and the neuronal circuitry needs to encode the new spatial locations in order to predict the future spatial relationships of the items The present study demonstrated that tool use learning induces adult neurogenesis in the hippocampal dentate gyrus of dexterous rodents (degus). These newborn neurons create novel hippocampal neural circuits by projecting their axons via the mossy fiber tract to the CA3 subfield and by forming new synapses in this target region. These enhancements in adult neurogenesis and synaptogenesis in the hippocampus may be necessary to accommodate the greater information processing demands placed on the circuitry by tool use learning relative to simple spatial learning, and also to accommodate the unique relationships between self and spatial structures that change over time. The results indicate that the central nervous system can adapt to new requirements that emerge from physical conditions (degus have peculiar hands that enable tool use), and that these adaptations may be dependent on the enhancements of existing neural resources. The data presented here may ultimately be relevant for understanding much higher human intelligence and cognitive functions.


HIV-1 infection generates numerous abnormalities in the B cell compartment which can be partly reversed by antiretroviral therapy. Our aim was to evaluate the effects that re-exposure to HIV antigens might have on the frequency and functionality of antibody secreting cells (ASC) in patients undergoing structured treatment interruptions (STI). As re-exposure to viral antigens may also boost the production of  antibodies, we also assessed the neutralizing activities during STI cycles.Retrospective study of 10 patients undergoing 3 cycles of STI with 2 weeks on and 4 weeks off HAART. ASC frequencies were determined by flow cytometry in samples obtained at the beginning and the end of STI. Neutralization capacity, total IgG concentration and anti-gp120-IgG titres were evaluated.nd STI.As expected, median viral loads were higher at the end of STI compared to on-HAART time points. The level of CD27 and CD38 expressing ACS followed the same pattern; with ASC being elevated up to 16 fold in some patients (median increase of 3.5% ± 4.13). Eight out of 10 patients maintained stable total IgG levels during the study. After purifying IgG fractions from plasma, HIV-neutralizing activity was observed in the two subjects with highest anti-gp120 titers. In one of these patients the neutralizing activity remained constant while the other showed elevated neutralizing Ab after first STI and once treatment was reinitiated after the 2Our data suggest that STI and its associated transient increases in viral load drive the frequencies of ASC in an antigen-specific manner. In some subjects, this re-exposure to autologous virus boosts the presence of neutralizing antibodies, similar to what is seen after influenza vaccination. STI may not boost clinically beneficial nAb levels but offers opportunities to isolate nAb producing cells at considerably higher levels than in subjects with completely suppressed viral replication. Clinical parameters to assess HIV disease progression focus largely on CD4 T cell counts and CD4/CD8 T cell ratio, yet the B cell compartment of HIV infected subjects can be severely impacted by chronic HIV infection as well. This includes hypergammaglobulinaemia ,2 and maThere is currently a great interest in the field to elicit broadly neutralizing antibodies (NAb) by different HIV-1 vaccine regimens as well as to identify, isolate and characterize novel monoclonal nAb. There are also encouraging results from the RV114 phase III vaccine trial, which has associated IgG responses against the V1-V2 region of gp120 and specific viral mutations in these regions with relative control from infection -17. Howe+ plasmablastic B cell  that peaked at day 7 after vaccination and which could reach up to 6% of all peripheral B cells. This significant expansion allowed them to isolate and characterize more than 50 novel nAb that were able to bind with high affinity to several Flu vaccine strains. We here followed the same rational and hypothesized that HIV infected individuals undergoing structured HAART treatment interruption would be similarly exposed to viral antigen and may boost at least transiently their HIV-specific ASC response. These expectations were supported by data showing unusually high titers of neutralizing antibodies in patients who were intermittently adherent to therapy and showed brief viremic periods as well as by earlier studies by Montefiori et al., where neutralizing antibodies emerged after treatment interruption  and 615  for the first STI, <20 [<20-654] and 3655 [<20-45900] (p = 0.015) for the second STI and <50 [<20-482] and 290 [<20-17400] (p = 0.031), respectively for the third STI  seven out of ten patients had normal (<15 mg/ml) total IgG concentrations in plasma  and one patient presented median neutralization activity  (data not shown). The remaining four patients had high neutralization titers  in at least one of the samples tested  was determined in all 10 subjects for all three STI cycles. Neutralization assays were carried out using two laboratory viral strains NL4-3 and BAL and two clinical viral isolates (AC10 and SVBP16) in the TZM-bl cell line based neutralization assay . Five ouThe presence of HIV-specific neutralizing antibodies has been associated in several studies with at least partial viral control after treatment interruption ,27. HoweSalmonella typhi oral vaccine, ASC were found to appear in the peripheral blood a few days after vaccination, reaching the highest levels on day 7 and were again undetectable 2 weeks after vaccination [-CD19+CD20-/lowCD27hiCD38hi plasmablast phenotype were indeed increased in HIV infection and accounted for the vast majority of HIV-specific ASC (up to 92% of gp-120-specific ASC) [In previous studies, with immunizations using a cination . The samcination . The trific ASC) . This, tWhen assessing gp120 specific Ab production upon STI, we did not observe a correlation between ASC frequencies and anti-gp120 IgG levels in the plasma. As all patients showed detectable anti-gp120 IgG levels in the baseline sample, only mild increases in their concentration upon STI may have been masked by anti-gp120 IgG produced by long-term memory plasma cells that continuously produce antibodies without the need of antigen exposure . AlternaOverall, 20% (2/10) of the patients showed neutralizing activity in purified IgG fractions. These subjects were among the 5 individuals who showed robust reactivation of their virus during all STI cycles and who had a reduced CD4 T cell count at the start of first STI (median CD4 count 801/ul) compared to the five subjects that did not show consistent viral reactivation (median CD4 count 1122/ul). Of note, the fluctuations in neutralization activities are not uniformly paralleled in these two subjects, possibly reflecting variable parameters that govern their kinetics, including growth kinetics of autologous virus, existing T-helper cell immunity  and the presence of memory B cells in lymph nodes -47. In aIn summary, our results suggest that antigen re-exposure after STI can lead to substantial increases in the frequencies of ASC and, at least in some patients, this increase is associated with the enhancement of HIV-specific IgG-mediated neutralizing activities. While additional factors that limit such a recovery in neutralizing activity to few individuals remain to be identified, analytical STI in therapeutic vaccine trials or the screening of extensive sample banks from past STI trials may offer opportunities to isolate ASC producing nAb at significantly elevated levels, facilitating the identification and characterization of new nAb against HIV, and thereby providing crucial information for future HIV vaccine design.The authors declare that they have no competing interests.CB conceived the experiments, participated in its design and collaborated to draft and to write the manuscript. ALL designed the experiments, carried out the flow cytometry, and the IgG purifications assays, performed the statistical analysis, analyzed the data and wrote the manuscript. JC carried out the ELISA assays and participated in the data interpretation. JB participated in experiments design, data discussion and manuscript revision. EY, VS, BM and BC helped in data discussion and manuscript revision. LR helped in selecting the samples and in drafting the manuscript. SM and EG carried out the neutralization assays and helped in their analysis. All authors read and approved the final manuscript.


Increased expression of the pro-fibrotic protein connective tissue growth factor (CTGF) has been detected in injured kidneys and elevated urinary levels of CTGF are discussed as prognostic marker of chronic kidney disease. There is evidence that epithelial cells lining the renal tubular system contribute to uptake and secretion of CTGF. However, the role of different types of tubular epithelial cells in these processes so far has not been addressed in primary cultures of human cells.Tubular epithelial cells of proximal and distal origin were isolated from human kidneys and cultured as polarized cells in insert wells. The pro-fibrotic stimuli lysophosphatidic acid (LPA) and transforming growth factor β (TGF-β) were used to induce CTGF secretion.LPA activated CTGF secretion in proximal tubular cells when applied from either the apical or the basolateral side as shown by immunocytochemistry. CTGF was secreted exclusively to the apical side. Signaling pathways activated by LPA included MAP kinase and Rho kinase signaling. TGF-β applied from either side also stimulated CTGF secretion primarily to the apical side with little basolateral release.Interestingly, TGF-β activation induced different signaling pathways depending on the side of TGF-β application. Smad signaling was almost exclusively activated from the basolateral side most prominently in cells of distal origin. Only part of these cells also synthesized CTGF indicating that Smad activation alone was not sufficient for CTGF induction. MAP kinases were involved in apical TGF-β-mediated activation of CTGF synthesis in proximal cells and a subset of epithelial cells of distal origin. This subpopulation of distal tubular cells was also able to internalize recombinant apical CTGF, in addition to proximal cells which were the main cells to take up exogenous CTGF.Analysis of polarized human primary renal epithelial cells in a transwell system shows that vectorial secretion of the pro-fibrotic protein CTGF depends on the cell type, the stimulus and the signaling pathway activated. In all conditions, CTGF was secreted mainly to the apical side upon TGF-β and LPA treatment and therefore, likely contributes to increased urinary CTGF levels in vivo. Moreover, CTGF secreted basolaterally may be active as paracrine pro-fibrotic mediator. Connective tissue growth factor CTGF, CCN2) is a secreted matricellular protein which has been associated with fibrotic diseases, often mediating the pro-fibrotic effects of transforming growth factor β (TGF-β) ,2. Eleva is a secUrinary CTGF is a marker of chronic kidney disease such as progressive hypertensive nephrosclerosis , diabetiIn vivo, epithelial cells are polarized with structurally and functionally distinct basolateral and apical domains. Polarization of epithelial cells in vitro can be achieved by culturing the cells on transwell membranes where they have access to nutrients and growth factors from two sides. MDCK cells derived from distal tubular cells readily establish dense polarized monolayers characterized by high transepithelial electrical resistence (TEER), whereas cell lines obtained from proximal parts of the nephron show lower TEER in line with the higher capacity for paracellular transport of proximal nephron segments ,16. CorrWhile TGF-β is the most important pro-fibrotic stimulus, other mediators add to inflammatory and fibrotic reactions in the kidney as, for example, lysophosphatidic acid (LPA). LPA is released from platelets upon stimulation but is also produced from phosphatidylcholine by extracellular phospholipases upon cell damage . By bindIn this study, we addressed the question whether epithelial cells of proximal or distal tubular origin respond to the pro-fibrotic stimuli LPA and TGF-β by vectorial stimulation of CTGF synthesis and secretion. For this purpose, we used our recently established cell culture model of primary human tubular cells . We show2 were obtained after 8 days and increased when the cells became denser  from the apical or the basolateral side. Apical secretion of CTGF was rapidly induced within 2 to 4 h as shown by Western blotting  further endorsing the specificity of CTGF secretion.Based on the data obtained with LPA, it was clear that proximal tubular epithelial cells secreted CTGF to the apical side. However, as long as the molecular mechanisms of CTGF secretion have not been deciphered, we cannot rule out that other stimuli may activate also basolateral secretion of CTGF from proximal tubular cells.LPA stimulated CTGF expression applied from both sides. In line with these data CTGF receptors have been characterized in both, apical and basolateral membranes of polarized gastrointestinal Caco-2 cells . By contAn additional level of complexity arises from the diversity of TGF-β receptor signaling, which implies Smad dependent and independent pathways (summarized in ). FurtheBasolateral stimulation of tubular epithelial cells by TGF-β rapidly activated Smad signaling in all distal tubular cells and also part of the proximal cells. By contrast, only distinct cell populations stained positive for intracellular CTGF at any given time point after TGF-β stimulation. This may in part be due to the fact that intracellular CTGF can only be detected when it accumulates in the secretory pathway. However, more likely it reflects the fact that activation of the Smad pathway is not sufficient for CTGF synthesis and secretion. For instance, induction of CTGF by TGF-β in epithelial cells cultured in dishes was strongly dependent on cell density, whereas Smad translocation was density-independent . The sigCellular uptake of CTGF has been observed in different cell types, mesangial cells , fibroblIt was interesting to note that upon stimulation with TGF-β, CTGF was not exclusively secreted to the apical side but also detectable at the basolateral side. Extrapolating to the in vivo situation, this may imply that the secreted CTGF is not lost in the urine but may be potentially active as a paracrine mediator. This aspect is supported by in vivo studies showing a role for tubular CTGF in the model system of remnant kidney disease in TGF-β1 transgenic mice . ActivatIn this study we provide evidence that non transformed polarized human epithelial cells secrete the pro-fibrotic protein CTGF in a cell-type and stimulus-specific manner. Furthermore, CTGF secretion was distinguishable from other secreted proteins indicative of specific molecular mechanisms regulating vectorial secretion. Most interestingly, CTGF was not only secreted but also taken up by proximal tubular cells. Approximating the in vivo situation, our model system is thus suitable for further investigation of secretion and uptake of proteins that are relevant markers for renal injury.DMEM/Ham’s F12 medium was purchased from Biochrom AG , DMEM medium and Hank’s BSS from PAA Laboratories , insulin-transferrin-selenium supplement from Gibco , fetal calf serum (FCS) from PAN Biotech , triiodothyronine from Fluka , hydrocortisone from Sigma , epidermal growth factor from PeproTech , TGF-β1 from tebu-bio , lysophosphatidic acid (LPA) ; Rho kinase inhibitor Y27632, TGF-β RI Kinase Inhibitor VI, SB431542, MEK kinase inhibitor U0126, p38 kinase inhibitor and brefeldin A were obtained from Calbiochem, Munich, Germany.3 pieces and digested with collagenase type II  and DNase I grade II  for 60 min. Next, cell suspension was sieved through 100 μm and 70 μm meshes. After a washing step with HBSS, cells were seeded in epithelial cell selective medium  in the presence of 0.5% FCS. After 1–2 days, medium was replaced by FCS-free medium. Cells were subcultured by application of trypsin. For experiments, hPTEC were seeded in medium containing 2.5% FCS to facilitate cell attachment, and medium was replaced after 24 h to FCS-free epithelial cell selective medium. Bright field pictures were recorded by Olympus CK40 microscope  using Leica DC Viewer software .Human primary tubular epithelial cells (hPTEC) were isolated from renal cortical tissues collected from healthy parts of tumor-nephrectomies essentially as described previously . IsolatiAbout five days after isolation at passage one cells were routinely analyzed for the content of proximal and distal cell by staining for N- and E-cadherin, respectively. The ratio of distal and proximal cells varied with most preparation containing a higher percentage of distal cells (about 70%).Polarized tubular epithelial cells were obtained by culturing primary epithelial cells for 8 days on permeable transwell inserts .Transepithelial electrical resistance was measured using a commercially available epithelial vpltohmeter and a set of so-called chopstick electrodes . All measurements were corrected for resistance of culture medium and empty inserts.Cell culture supernatants were precipitated with ethanol. Western blot analyses were performed essentially as described before  using thRNA of cells cultured in inserts was isolated and analyzed as described previously .Cells were fixed with paraformaldehyde (3.5% in PBS) for 10 min and afterwards permeabilized by 0.5% Triton X-100 in PBS for 10 min. After washing three times with PBS, cells were blocked in 1% BSA in PBS. Antibodies used for Immunocytochemistry: E-cadherin (Abcam), N-cadherin (Santa Cruz), CTGF (Santa Cruz), acetylated tubulin (Sigma), Smad2/3 . Alexa Fluor 488- or 555-conjugated secondary antibodies were from Invitrogen.After mounting, slides were viewed using a Nikon Eclipse 80i fluorescent microscope and digital images recorded by Visitron Systems 7.4 Slider camera  using Spot Advanced software (Diagnostic Instruments). Three dimensional images were evaluated by epifluorescence microscopy including Apotome technique .A full length human CTGF cDNA, kindly provided by M. Bauer, Med. Clinic 1, Erlangen, was expressed in HEK293 cells. His-tagged CTGF was purified from cell culture supernatants using Ni-columns . Purity of the protein was shown by silver staining of SDS polyacrylamide gels and estimated to be over 90%.To compare multiple conditions, statistical significance was calculated by one-way ANOVA with Dunnett’s post hoc test using GraphPad software. A value of P < 0.05 was considered to indicate significant differences.hPTEC: Human Primary Tubular cells; LPA: Lysophosphatidic Acid; TEER: Transepithelial Electrical Resistance; TGF-β: Transforming Growth Factor Beta; UUO: Unilateral Ureter Obstruction.The authors declare that they have no competing interests.JZ and AE performed the experiments, BK analyzed data and wrote the manuscript; MGS designed the study, analyzed data, drafted and wrote the manuscript. All authors read and approved the final version of the manuscript.


Peripheral haemodynamic response during adenosine stress CMR perfusion does not vary among those with or without perfusion defects identified during routine clinical practice.Haemodynamic response to adenosine infusion during CMR stress perfusion continues to be used as surrogate to determine adequacy of hyperaemia in clinical setting. Our aim was to determine the proportion of patients who achieve pre-specified adequate response to adenosine (increase in heart rate (HR) by ≥ 10 beats or a drop in systolic blood pressure (SBP) by 10mm Hg from baseline reading) and if this varies in patients identified with and without perfusion defect in a clinical setting.We prospectively identified 94 consecutive patients undergoing A-CMRP, 7 of who were excluded because of missing data. 87 patients underwent first pass CMRP on a Philips Achieva CV 1.5 T MR scanner , with standardised SCMR acquisition protocol infusing adenosine at 140 μg/kg/min for 3 minutes with HR and SBP recorded at baseline, one, two and three minutes. All patients confirmed abstinence from caffeine and chocolate for >24 hours and no patients were on theophylline or dipyridamole. Descriptive statistics and logistic regression were performed.67 (77%) patients achieved the pre-specified adequate haemodynamic response and this was mainly driven by increase in heart rate.The OR (adjusted for age) for predicting perfusion defect by adequate haemodynamic response in patients with and without CAD was 2.5  and 3.26  respectively.Our study shows that in patients undergoing adenosine (140 μg/kg/min) stress perfusion, there is no significant difference in peripheral haemodynamics in patients identified with or without perfusion defect and challenges their use as a marker of hyperaemic response. It remains to be seen whether high dose adenosine will influence these findings and change clinical practice.No funding was obtained for this study.