MYC, -Runx2) develop rapid onset tumours that can be accelerated and rendered polyclonal by neonatal Moloney murine leukaemia virus (MoMLV) infection. RIM/DS analysis of 28 polyclonal lymphomas identified 771 common insertion sites (CISs) defining a ‘progression network’ that encompassed a remarkably large fraction of known MoMLV target genes, with further strong indications of oncogenic selection above the background of MoMLV integration preference. Progression driven by RIM was characterised as a Darwinian process of clonal competition engaging proliferation control networks downstream of cytokine and T-cell receptor signalling. Enhancer mode activation accounted for the most efficiently selected CIS target genes, including Ccr7 as the most prominent of a set of chemokine receptors driving paracrine growth stimulation and lymphoma dissemination. Another large target gene subset including candidate tumour suppressors was disrupted by intragenic insertions. A second RIM/DS screen comparing lymphomas of wild-type and parental transgenics showed that CD2-MYC tumours are virtually dependent on activation of Runx family genes in strong preference to other potent Myc collaborating genes . Ikzf1 was identified as a novel collaborating gene for Runx2 and illustrated the interface between integration preference and oncogenic selection. Lymphoma target genes for MoMLV can be classified into (a) a small set of master regulators that confer self-renewal; overcoming p53 and other failsafe pathways and (b) a large group of progression genes that control autonomous proliferation in transformed cells. These findings provide insights into retroviral biology, human cancer genetics and the safety of vector-mediated gene therapy.Retroviral insertional mutagenesis (RIM) is a powerful tool for cancer genomics that was combined in this study with deep sequencing (RIM/DS) to facilitate a comprehensive analysis of lymphoma progression. Transgenic mice expressing two potent collaborating oncogenes in the germ line  events but the advent of cancer genome sequencing highlights the growing challenge of separating important (driver) from irrelevant (passenger) mutations. Retroviruses that induce cancer by inserting into host DNA and thereby altering key genes are valuable tools because they act as ‘tags’ to identify the critical targets. In this study we combined retroviral tagging with next generation sequencing to achieve a comprehensive description of lymphoma development and progression in transgenic mouse model systems. Our study suggests that three events may be sufficient for lymphoma development and identifies a genetic bottleneck at a small gene set that regulates tumour cell self-renewal, including the  The oncogenic potential of murine γ-retroviruses (MLVs) stems from proviral integration into host DNA, a mutagenic process which can result in activation or disruption of critical host cell genes Myc and Runx2, could overcome the need for genetic inactivation of the p53 pathway, despite the fact that both oncogenes evoke p53 growth suppression and collaborate strongly with p53 deficiency Pim1, a gene that accelerates tumour onset when combined with MYC/Runx2 in the germ-line Moloney murine leukaemia virus (MoMLV) is an oncogenic γ-retrovirus that has been widely used in RIM studies MYC, which is highly dependent on Runx gene activation. Comparison with other transgenic models of Myc over-expression shows that these each display potent selection from a small pool of master collaborating genes. These genes share the capacity to suppress the p53 pathway but are differentially recruited according to lymphoid lineage and developmental stage. The identification of a small gene set that confers the lymphoma initiating cell phenotype and is conserved in human disease has significant implications for targeted interventions.In this study we have conducted a further screen on the same progressing lymphomas, using a deep sequencing method (splinkerette/454) which is orders of magnitude more sensitive than previous shotgun cloning methods. Sequencing at this depth raises another potential concern, as γ-retroviruses including MoMLV display preferential integration at transcriptional start sites and other chromatin feature that may also entail a bias towards proto-oncogenes MYC and CD2-Runx2 transgenic mice are displayed in Relevant features of the CD2-Here, a panel of 28 lymphomas was analysed by RIM/DS (splinkerette/454). Processing of reads as described in Pim-1 insertions were also detectable as rearrangements by Southern blot analysis  map to metanodes that include many of the less frequent targets . The typical pattern of insertions distributed across the target genes is suggestive of a tumour suppressor role, although a role for oncogenic truncated isoforms is also plausible While orientation bias is useful to identify oncogenic selection on a background of preferential integration, we noted that there was a second frequent CIS group defined by intragenic insertions that displayed no statistical bias in orientation. Evidence that these are also under oncogenic selection is provided by the fact that 17 of the 20 most frequent targets have been observed in end-stage lymphomas  and by tMyc/Pvt1, Mycn and Runx family genes in the progression set. However, there was also a marked loss of selection for some major targets recorded by Kool et al. including Gfi1 and Notch1. It appears that the combination of MYC and Runx2 in this context also renders these insertions redundant, which is intriguing as insertions at Gfi1 have been shown to be positively selected in some CD2-Runx2 lymphomas Although the majority of top ranking MLV target genes were shared between our progression dataset and the Kool meta-analysis of end-stage lymphomas, there were also notable differences. This was evident from comparison of CIS peak heights and relative rank order of CISs between the datasets where the most discordant examples are listed . OncogenOtx2, a homeobox transcription factor which plays a major oncogenic role in medulloblastoma Myo16, an atypical nuclear myosin with links to survival, cell cycle progression and PI3K signalling Endou (Pp11), a placental poly-U endonuclease over-expressed in ovarian adenocarcinomas Xrra1, which has been shown to modulate the response to X-ray irradiation Ttc28 (Tprbk), encoding a large tetratricopeptide domain protein that is regulated by p53, complexes with BRCA1 and suppresses the growth of Ras-transformed cells Also of interest was the large number of novel CISs in the progression set . The mosRunx2/MYC and control thymus at 10 days of age, several weeks before clonal tumours emerge. Ccnd1 showed significant up-regulation compared to control thymus. Moreover, the frequent MoMLV targets that were not enriched in the progression network showed a similarly wide distribution with regard to expression levels. The exquisite selection by RIM of specific members of multigene families  also appeared to be poorly correlated with expression level, strengthening evidence for post-integration selection as the predominant force shaping the progression network.A previously published analysis of preferential integration targets in early passage CD34+ cells showed a good correlation between basal transcriptional levels and integration frequency Ccr7, and to a lesser extent Ccr9, is interesting in view of their central roles in mediating T-cell progenitor homing to thymus Ccl21a, Ccl19, Ccl25), are highly expressed in normal thymus, but intriguingly were significantly down-regulated genes in premalignant organs . A number of high abundance RIS mapped far upstream of Runx2, adding this gene to the list of those subject to long-range activation. Only two tumours displayed no detectable Runx insertion.The most striking features of the single transgenic tumours were evident when the most abundant RISs were sorted according to gene family . High coMYC, CD2-Runx2 and wild-type mice appeared to be selected from a broad cross-section of the progression network, with the ‘winners’ of the progression race largely recapitulating the expansion rate measured by earlier analysis of the progression network  and Myc family targets  conformed to this pattern, being selected in CD2-MYC and CD2-Runx2 respectively and effectively disappearing from the double transgenic tumours  positive selection in lymphomas of single transgenic mice compared to wild-type and (b) loss of selection or reduction to background levels in double transgenics. As expected, the  tumours . Surprist clones . Intrigupression .Gfi1 and Notch1 insertions on the CD2-MYC background which directly mirrors findings on the progression set compared to common MoMLV targets  mice Ikzf1 illustrates the principle whereby a gene may be selectively targeted by γ-retroviral integration but leads to clonal expansion in the presence of a complementary oncogenic programme provided in this case by Runx gene activation. It has been demonstrated recently that γ-retroviral integration at transcriptional start sites is a consequence of interaction with BET chromatin regulators that direct the process towards genomic regions rich in acetylated histones In this study we examined an established system of oncogene cooperation and retroviral acceleration using a deep sequencing (DS) platform. RIM/DS increases sensitivity of RIS detection by almost two orders of magnitude over earlier methodologies Ikzf1).The implications of our analyses are also interesting for retroviral vector-based gene therapy. As the most potently selected insertions mediate enhancer-mode gene activation, the removal of enhancer elements in self-inactivating vectors Otx2, Myo16) is not merely due to their up-regulation in the background of the Runx2/MYC model. These findings suggest that it will be of value to employ RIM/DS to probe the growth checkpoint networks in tissues and cell lineages that have been less well explored to date.While intrinsic preference for integration at transcriptional start sites and other chromatin features Ccr7 and Ccr9, which in their normal developmental roles promote T-cell homing to thymus and ligand-dependent survival and proliferation Ccr7 is stimulated by Notch signalling Ccr7 insertions is also in accord with the relatively low read/RIS ratio in primary thymic lymphomas. Identification of Ccr7 as a major target highlights the complementary value of RIM screening, as this gene does not appear to be subject to mutation or amplification in human cancer, yet is required for CNS metastasis of human leukaemia cells While most of the functionally annotated progression network genes are predicted to confer autonomous proliferation, an exception to this rule was provided by the frequent activation of Myc and Runx family members were massively under-represented in the progression set. Moreover, while most major targets overlapped strongly, a few prominent targets including Gfi1 and Notch1 were also greatly diminished in the progression network. Our second RIM/DS of parental transgenic mice shed further light on this observation, as the CD2-MYC parental transgenic system in particular did not select for these targets but instead showed virtual dependence on activation of a Runx family gene with the order Runx2>Runx3>Runx1 in targeting frequency in accord with previous observations MYC, Eμ-Myc, Mmtv(d)-Myc) shows that these have massively divergent preferences for collaborating genes detected by RIM, presumably reflecting the lineage and stage-specificity of Myc expression control Comparison of the progression network with a large scale meta-analysis of MoMLV targets in T-cell lymphomas from various genetic backgrounds The foregoing observations invite the model presented in Bmi1 is largely a feature of B-cell lymphomas in the mouse Runx allele Notch1 or Gfi1 pathways are not available for RIM targeting at this stage and that Runx2, the ‘bone-specific’ family member, which is also transcriptionally active in early haematopoietic development Runx family members are downstream of Notch signalling in expression control and effector functions Runx and Myc supplants the need for activation of Notch. The model we propose has implications for therapeutic targeting of Notch signalling with γ-secretase inhibitors Runx and Myc may represent another pathway to resistance.Why do the major collaborating gene targets vary so markedly between Myc transgenic models? The most obvious rationale is presented by the lineage and stage-specificity of Myc expression. RIM targeting of Runx2 selects strongly for activation of Myc family genes by RIM Ikzf1 is also favoured as a collaborating target on this background. Notably, Ikzf1 is a haplo-insufficient tumour suppressor that has been reported to act as a transcriptional suppressor of Myc Ikzf1 targeting that leads to its co-selection with Runx2. It would interesting in this regard to test whether lymphomas of Runx2 transgenic mice with reduced Ikzf1 function Myc family genes and Ikzf1.Although CD2-Myc, Runx, Ikzf1, Gfi1, Notch1, and Bmi1) that act in pairwise combinations to confer lymphoma self-renewal and overcome failsafe responses via the p53 pathway. It seems likely that this network operates under normal physiological conditions to licence cell growth and is co-ordinately subverted in cells carrying mutations in the pathways. The recent description of Gfi1 as an ‘oncorequisite’ factor that is rarely directly mutated but nevertheless required for growth of ALL cells This analysis has wider implications for the genetics of human lymphomas and other cancers. It appears that the final step in lymphoid transformation by MLV can be accomplished by a wide range of genes with the common functional end-point of growth factor-independent proliferation. As the progression network also includes numerous genes that are mutated, amplified or deleted in human cancer , it is tAnimals were routinely monitored and sacrificed when showing signs of ill health in line with the UK Animals (Scientific Procedures) Act, 1986.MYC, CD2-Runx2, and CD2-MYC/CD2-Runx2 transgenic animals and maintenance were described previously 5 infectious units of MoMLV as previously described CD2-DNA was extracted from approximately 20 mg of frozen enlarged lymphoid/tumour tissue using Gentra Puregene Genomic DNA Purification Kit  according to the manufacturer's instructions.Isolation of the retroviral insertion sites from the tissues was performed using splinkerette PCR to produce barcoded PCR products that were pooled and sequenced on 454 GS-FLX sequencers (Roche Diagnostics platform) as described previously Sleeping Beauty transposons on which the bioinformatics processing method was developed). Reads from the same sample whose start genomic locations aligned within three nucleotides of each other were merged together. Reads from the same sample that were more than three nucleotides apart were considered independent integration events. CISs were identified using the multi-scale GKC approach Processing of 454 reads, identification of insertion sites, and Gaussian kernel convolution (GKC) statistical methods used to identify common insertion sites (CISs) have been described previously In order to determine whether the MLV screen had reached some level of saturation, the Gaussian Kernel Convolution (GKC) CIS calls from all 28 samples were analysed using the ACT software package ACT considers genomic locations generated by multiple samples for specific biological phenomenon under study (e.g. ChIP-seq peaks) to determine the saturation of a screen. The program considers the various combinations in which samples can be added so that the increase in base pair coverage is a range of values based on all the samples. The results can be depicted as a series of boxplots showing the increase in base pair coverage, where the boxplot at each position n on the x-axis shows the coverage values of all combinations of n samples. Boxplots that approach a horizontal asymptote indicate that the coverage has reached saturation.For the GKC CISs generated by all 28 samples, the insertion sites that contributed to CISs were extracted, resulting in a set of 7,485 sites. The insertion sites were then selected per sample and pseudo-kernels of 7.5k nucleotides either side of each insertion were applied to mimic GKC kernels of 15k nucleotides. Overlapping kernels within each sample were merged into continuous genomic regions. These 28 modified insertions files were then analysed using ACT. For each combination of samples the median values, and 25th and 75th percentiles were plotted using ggplot2 As a control, the 28 samples were re-analysed where the same number of insertion sites per sample were selected at random across the mouse genome. The pseudo-15k nucleotide kernels were applied.While the analysis does not produce a clear-cut asymptote this is to be expected due to the type of data under consideration. ACT was designed to analyse such data as ChIP-seq arrays for predicting transcription factor binding sites. In these scenarios ChIP-seq replicates should ideally report the same key binding sites/genomic locations. Hence across multiple samples the same locations should be reported.For MLV screens however, while insertions in the same gene will be found from different samples, the locations of the insertion sites will not overlap perfectly, even with the addition of the 15k nucleotide pseudo kernels. Hence each sample will introduce novel regions, such that the overall coverage will continue to increase even if the screen has truly reached a ‘saturation’ point. Also not all samples will contribute to all CISs. Different combinations of samples will thereby result in varying coverages, causing the coverage profile not to asymptote perfectly.The genomic coordinates of the ‘UCSC Genes’ set was downloaded via the UCSC genome browser for mouse assembly mm9. Each of the 12,485 MoMLV integration sites was then mapped relative to the transcription start site (TSS) of its closest UCSC-defined ‘known’ gene.http://mutapedia.nki.nl/) The Kool set of 19,923 mouse retroviral insertions sites was downloaded from the Mutapedia website  with mechanical lysis using a pellet pestle in a microfuge tube (Sigma). RNA purity was assessed using a Nanodrop 2000 Spectrophotometer (Thermo Scientific), and integrity verified using the Agilent 2100 Bioanalyser with RNA 6000 Nano Reagents kit (Agilent Biotechnologies) as per the manufacturer's protocol. Whole genome expression profiling was performed using Affymetrix mouse GeneChip microarrays (MoGene-1) in triplicate as per the manufacturer's protocol . Data analysis was carried out using the Partek Genomic Suite . Briefly, after Robust Multichip Average normalisation RNA was isolated and purified from the thymuses of 10 day old wild type and CD2-Figure S1a) Basic features of the lymphoma model. Expression of either Runx2 or MYC under the control of the CD2 locus control region leads to a low lifetime incidence of T-cell lymphoma. This appears to be due to the variegated activation of the transgenes and counter-selection for expressing cells which either die by apoptosis (MYC) or grow slowly with impaired differentiation (Runx2). The combination of both transgenes cancels these failsafe responses and leads to early onset lymphoma in a 100% of mice b) The clonal nature of CD2-Runx2/MYC lymphomas is demonstrated by rearrangements of the T-cell receptor β-chain. Southern blot analysis of 20 mg samples of DNA digested with HindIII and analysed with a Cβ probe. The virtual disappearance of the unrearranged Cβ1 is due to the replacement of non-lymphoid cells by lymphoid cells carrying deletions or rearrangements of Cβ1. As TCR rearrangement can result in productive rearrangement or deletion of Cβ1, dominant clones may be represented by one or two bands. As can be seen, spontaneous tumours in these mice typically display a single major clone, although some evidence of minor clones is present in some cases (-MoMLV). In MoMLV accelerated tumours, there is typically a more complex pattern indicative of greater clonal complexity. Due to the limited sensitivity of Southern blot analyses, clones representing less than 5% of the tumour mass are not detectable. c Phenotypic analysis of CD4 and CD8 expression in primary thymic lymphoma CD2-Runx2/MYC mice. Note that normal thymocytes were almost completely replaced by the characteristic bi-modal tumour cell population (>96–99%). No phenotypic difference was observed in MoMLV-accelerated lymphomas.((TIF)Click here for additional data file.Figure S2a) Evidence that expanding clones in virus-accelerated Runx2/MYC lymphomas contain a single provirus. The top 40 RISs (in rank order by number of reads) shows few insertions at isolated RIS far from known target genes(5/40), although these predominate (85%) in the total population of 12,485 RISs. If clonal expansion required two or more hits of proviral insertion, we would expect many more instances of co-amplification of passenger RIS (grey bars). (b) There is a correlation between splinkerette 454 sequence reads and Southern blot detection of rearrangement, with insertions at Pim1 in expanded tumours clones in tumours 20i and 13i being detected by both methods at similar relative efficiency (compare to (a)). G: germ line; R: retrovirus insertion.((TIF)Click here for additional data file.Figure S3Additional MLV insertion patterns at other biased and non-biased CISs. Each vertical bar represents an individual RIS, red indicates reverse orientation compared to the+strand, green the same orientation. The positions of exons and introns were extracted from the UCSC genome browser (NCBI37/mm9).(TIF)Click here for additional data file.Figure S4KEGG Cytoscape plot. Genes with RIS counts of 3 or more are visualized in the context of their KEGG pathway interactions using Cytoscape. The KEGG network is based on metanodes. A metanode is a collection of genes that share similar function. Some metanodes only contain a single gene. Links in the KEGG network denote a functional interaction between any of the genes in the two metanodes connected by the link. For visualization purposes the metanodes themselves are not displayed. Consequently, in the resulting graph a link between two genes is present if there is a link between the metanodes in which these genes reside. Blue links are KEGG pathway links, red links connect genes that are in the same metanode in KEGG. Genes that are in the same metanode share functionality (according to KEGG). Note that metanodes are not necessarily consistent across different pathways, which is why some genes that are in the same metanode have a different set of interaction partners. Node colour and size represents the number of RIS attributed to that gene: blue and small circle: 3 RIS, red and large circle: up to a maximum of 127 RIS.(TIF)Click here for additional data file.Figure S510 p-values for all pathways in the KEGG database. The legend lists the pathways with a significant p-value (at the 1×10E-5 level) for at least one of the discovery set definitions, with the minimum p-value between parentheses. The leftmost box depicts the log10 p-values of the pathway enrichment when the discovery set is defined as all genes associated with five or more RISs. For the second box from the left the discovery set is defined as all genes with at least one associated RIS. This is also the case for box 3 through 6, but in those discovery sets the top 20, 50, 100 and 500 most frequently targeted genes are removed from the discovery set, respectively. The horizontal red line indicates the 10−5 significance level. Surprisingly, enrichment is more significant when the entire ‘integrome’ is analysed than when restricted to genes that are frequently targeted (by 5+ RIS). Moreover, removal of 20 to 100 ‘top hit’ genes which includes genes common to many of the annotated pathways  has relatively modest effects on significance scores, while enrichment for pathways in cancer and others survives even the removal of the top 500 genes. These results strongly indicate that either a) the majority of RISs, including those that are not common across multiple tumours, have been subjected to oncogenic selection or b) viral targeting of these pathways is an underlying phenomenon based on integration preference.KEGG pathway enrichment analysis. The effects on pathway analysis of limiting gene sets by number of hits or removal of most prominent CISs. This analysis was conducted to test the extent to which oncogenic selection is present throughout the detected RISs. Box plots represent log(TIF)Click here for additional data file.Figure S6a) Quantitative real-time PCR validation of key gene changes observed in the microarray. Quantification is relative to house-keeping gene TBP for genes in the CC chemokine family, with fold changes and significance as determined by two-tailed unpaired student's T-test shown in (b) Error bars represent S.E.M. Genes with fold differences reaching a q<0.05 significance threshold in the microarray are noted with an asterisk (*). N/A = gene not present on the microarray.((TIF)Click here for additional data file.Figure S7a) Ccr7 and ligands (b) Ccr9 and ligand. (c) Significance of down-regulation of CC ligands compared to receptors in T-cell lines, determined by two-tailed unpaired student's T test. Errors represent standard error (SEM).Lack of expression of CC chemokine genes in lymphoma cells from Runx2/MYC and other genetic backgrounds. Quantitative real-time PCR analysis of CC chemokine receptors and ligands for a number of T-cell lymphoma lines from Runx2/MYC (GIM) or p53null/MYC (p/m) backgrounds, expressed relative to adult normal thymus, with HPRT as control. ((TIF)Click here for additional data file.Table S1Master list of all CISs showing CIS chromosomal location and peak height, plus associated genes. Also shown is the number of insertions in each CIS.(XLSX)Click here for additional data file.Table S2.bed file of all GIM1 alignments, showing chromosomal locations, tumour identity, read counts and strand polarity.(XLSX)Click here for additional data file.Table S3The top 25 progression CISs, ordered by the number of insertions. Gene names and annotations are shown, as are the total number of insertions, the number of tumours with insertions and the average number of reads per RIS for each gene. Genes in bold were previously identified as targets by shotgun cloning.(TIF)Click here for additional data file.Table S4et al. screen, showing CIS chromosomal locations and gene identities.Overlapping CISs found in both this screen and the Kool (XLSX)Click here for additional data file.Table S5et al. screen.CISs with significant orientation bias, showing gene identities, the total number of insertions, the percentage bias and p values with Benjamini multiple testing correction. Also shown is the rank order of the CIS in the Kool (TIF)Click here for additional data file.Table S6et al., Nucleic Acids Res., 2004, 32: D523–527), and the rank-order position of the gene in terms of peak height from a total of 823 CIS in the Kool et al. data set. N/A denotes the absence of the gene in the Kool et al. CIS list.Most frequent intragenic insertions without orientation bias. Unbiased/intragenic CIS locations and target genes identified from the RIM screen, ordered by number of insertions are shown. Also shown are the number of hits in the Retrovirus and Transposon Tagged Cancer Gene Database Click here for additional data file.Table S7et al. meta-analysis of 956 lymphomas are notably under-represented in the Progression CISs. This is illustrated in the table where the most discordant examples are compared by CIS peak height (a measure of number hits and degree of clustering) and rank order. A nil entry (-) means that no CIS was recorded in the progression dataset. For comparison, several prominent targets that are shared are listed below. Grey shading denotes reduced selection compared to the meta-analysis.CISs displaying loss of selection in Runx2/MYC lymphoma progression compared to end-stage lymphomas. Some of the most strongly selected target genes in the Kool (TIF)Click here for additional data file.Table S8et al. dataset showing gene name and function, peak height and evidence of that gene's role in human cancer.Top targets unique to Huser (PPTX)Click here for additional data file.Table S9List of CISs from GIM set 2, showing chromosomal location and associated genes.(XLSX)Click here for additional data file.Table S10.bed file of all GIM2 alignments, showing chromosomal locations, tumour identity, read counts and strand polarity.(XLSX)Click here for additional data file.Table S11Number of insertions in end-stage lymphomas of three genotypes  for the top 25 progression CISs . The numbers of expanded clones (RIS with >100 reads) are listed in brackets.(TIF)Click here for additional data file.Table S12The number of insertions in end-stage tumours of three genotypes  is shown for the most frequent targets for intragenic insertions in the progression network. Numbers in brackets denote expanded RIS (>100 reads).(TIF)Click here for additional data file.Table S13Ikzf1 between WT and MYC (P = 0.034) and between Runx2 and MYC (P = 0.04) (Mann-Whitney U test).CIS biased by genotype, sorted by genotype in end-stage lymphomas. Number of RIS are shown alongside number of reads/RIS separated by |. Shading denotes apparent positive selection (reads/RIS>10). In some cases there are clear qualitative differences that are not amenable to statistical comparison due to small numbers of RISs (e.g. Mycn). Significant differences for insertions * at Runx2 between Runx2 and MYC genotypes (P = 0.021) and ** at (TIF)Click here for additional data file.Table S14et al. genomic regions that are amplified or deleted in TKO mouse and human T-ALL with Huser et al. CIS regions. Shown are the chromosome locations of the syntenic regions  from Maser et al., and the start, end and peak locations as well as peak height of CIS coinciding with these regions. Also shown are gene annotations for CIS and whether the CIS is intragenic or not. The CIS at Gpr132 is both intragenic and upstream of the gene and is labelled +/−. 43/771 CISs map to the syntenic regions which encompass 79.7 Mb of genomic sequences. To assess the probability of this overlap, the mouse genome was considered as a series of 30 kb ‘bins’, the average size of the defined CISs. On this basis, the syntenic regions occupy 2613/89796 bins. The probability of the observed overlap based on the contingency table below is P = <0.0001 (http://vassarstats.net/tab2x2.html).Comparison of Maser (TIF)Click here for additional data file.


Staphylococcus aureus cell walls and controls autolysis largely via the major autolysin AtlA whose activity is known to decline at acidic pH values. Compounds that increase or decrease the activity of the respiratory chain, a main source of protons in the cell wall, modulated autolysis rates in WTA-producing cells but did not affect the augmented autolytic activity observed in a WTA-deficient mutant. We propose that WTA represents a cation-exchanger like mesh in the Gram-positive cell envelopes that is required for creating a locally acidified milieu to govern the pH-dependent activity of autolysins.Wall teichoic acid (WTA) or related polyanionic cell wall glycopolymers are produced by most Gram-positive bacterial species and have been implicated in various cellular functions. WTA and the proton gradient across bacterial membranes are known to control the activity of autolysins but the molecular details of these interactions are poorly understood. We demonstrate that WTA contributes substantially to the proton-binding capacity of  The bacterial cell envelope governs vital processes including maintenance of cell shape, cell division, and protection against environmental challenges. In addition, it has a crucial role in the respiratory energy metabolism because the cytoplasmic membrane harbors the electron transport chain components, which generate a proton gradient across the membrane that is used to generate ATP or energize transport processes Staphylococcus aureus and Bacillus subtilisB. subtilis cells has been shown to be protonated during respiratory metabolism The majority of Gram-positive bacteria contain polyanionic cell wall glycopolymers (CWGs) in their cell envelopes, which are covalently linked to either peptidoglycan  or membrane glycolipids (e.g. lipoteichoic acid [LTA] or succinylated lipoglycans) S. aureus, or of the corresponding AtlE of Staphylococcus epidermidis is not lethal but the mutants form huge cell clusters as a result of incomplete cell separation S. aureus we observed that a WTA-deficient ΔtagO mutant (ΔtagO) B. subtilisS. aureusPeptidoglycan is built from long glycan strands that are cross-linked by peptide side chains. Peptidoglycan must be continuously synthesized to maintain cell integrity and viability and to allow for cell division. Peptidoglycan hydrolytic autolysins are critical for separating daughter cells after cell division S. aureus autolysis largely via the major autolysin AtlA. Compounds that modulate the activity of the respiratory chain, a main source of protons in the cell wall, affected autolysis in the presence of WTA but not in its absence, which supports the notion that WTA contributes to the control of autolysin activity by governing the abundance of protons in the cell wall.Here, we show that WTA contributes substantially to the concentration of proton-binding sites in the cell wall and impacts on S. aureus wild type and isogenic WTA-deficient tagO mutant (ΔtagO) were determined by Fourier Transform Infrared (FTIR) spectroscopy and a whole-cell titration approach.We hypothesized that the polyanionic properties of WTA may contribute to the binding of protons thereby affecting the local pH in the cell wall. In addition to WTA the cell envelopes of Gram-positive bacteria contain other negatively charged residues in LTA, peptidoglycan, phospholipid head groups, and surface-associated proteins. To assess if WTA contributes significantly to the overall density of negative charges in the cell envelope, the proton-binding capacities of tagO. Accordingly, the proton-binding sites in the mutant were 23% reduced compared to the wild type . The three strains had similar membrane potentials around −120 mV  to augment respiration.How the respiratory activity affects autolysis in S. aureus wild type  containing appropriate antibiotics when necessary unless otherwise noted.-1 and 256 interferograms with an optical range of 4000 to 400 cm−1. To prepare S. aureus wild type and ΔtagO samples for FTIR measurement, 2 mg of freeze-dried bacterial sample were homogeneously mixed with 250 mg of dry potassium bromide  to prepare a KBr pellet. The samples were freeze-dried in order to remove water for FTIR measurements. Slight changes in structure duo to this procedure can, however, not be ruled out.FTIR spectra were recorded using a Bruker Vertex 80v FTIR spectrometer. The spectral resolution of the spectrometer was set to 2 cm3 background electrolyte at a concentration of 2 mg/ml for titration experiments. A 40 ml aliquot of the bacterial suspension was then transferred to a 150 ml Metrohm glass titration vessel. Bacterial suspensions were acidified to pH 3 and then covered with an air-tight lid fitted with an N2 gas line interface to prevent carbonate formation during the course of the titration and a pH electrode connected to an autotitrator (Metrohm Titrando 806) to monitor changes in H+ activity as a function of added titrant. The titrator settings were programmed as described previously Bacterial cells were grown until exponential phase and washed twice using ultrapure water. Cells were lyophilized and resuspended in 45 ml of 0.01 M NaNO−+H+, where L− corresponds to a proton-binding functional group on the cell surface and H+∼H3O+ represents the hydronium ion species in solution. The apparent equilibrium constant for the proton dissociation mechanism described previously, Ka, can be defined as function of measured, [H+], and adjustable, [L−], parameters as follows: Ka = [H+].[L−]/[HL] where [H+] refers to the proton concentration calculated by the relationship [H+] = {H+} γH where, {H+} is the proton activity measured using the pH electrode, and γH is the proton activity coefficient for I = 0.01 M (KNO3). A multi-site Langmuir isotherm coupled to a linear programming optimization method (LPM) was used to calculate the concentration of functional groups on the cell surface and generate pKa (−log Ka) spectra where pKa is a measure of functional group acidity as described previously To account for the deprotonation of bacterial cell surface functional groups as a function of increasing solution pH the following dissociation mechanism is proposed: HL = L+ whose equilibrium across the cytoplasmic membrane is indicative of membrane potential as described previously 600 of 1, harvested, and then resuspended 1∶3 in fresh medium. [3H]TPP+  was added to a final concentration of 1 µCi/ml to cell suspensions and incubated for 10 min. Subsequently, aliquots were filtered onto 0.2-µm-pore-size cellulose acetate membranes  and washed twice with 50 mM phosphate buffer (pH 7.0). The filters were dried and radioactivity was measured using a Packard 1900CA liquid scintillation counter in the presence of Filtersafe  scintillant.were determined with the small lipophilic cation [3H]TPPAutolysis assays were performed essentially as described recently


Yersinia pestis and Bacillus anthracis which feature on the Center for Disease Control and Prevention’s list of potential biothreats. Here, a colorimetric sensor array was used to continuously monitor the volatile metabolites released by bacteria in solid media culture in an Advisory Committee on Dangerous Pathogen Containment Level 3 laboratory. At inoculum concentrations as low as 8 colony-forming units per plate, 4 different bacterial species were identified with 100% accuracy using logistic regression to classify the kinetic profile of sensor responses to culture headspace gas. The sensor array was able to further discriminate between different strains of the same species, including 5 strains of Yersinia pestis and Bacillus anthracis. These preliminary results suggest that disposable colorimetric sensor arrays can be an effective, low-cost tool to identify pathogenic bacteria.A colorimetric sensor array is a high-dimensional chemical sensor that is cheap, compact, disposable, robust, and easy to operate, making it a good candidate technology to detect pathogenic bacteria, especially potential bioterrorism agents like  Yersinia pestis requires only 10 organisms to infect a human with pneumonic plague Bacillus anthracis spores resist standard sterilization techniques, such as UV radiation, heat, and various chemicals, as well as environmental damage Certain pathogenic bacteria that present high transmission and/or mortality rates are considered high-priority bioterrorism agents because of their potential for a major public health impact. For example, Current bacterial identification methods can be slow and require highly trained personnel to operate sophisticated instruments that are generally too expensive, delicate, or bulky to deploy outside of a dedicated laboratory facility. Culturing remains a standard technique for identifying microbiological species, but typically requires 24 to 72 hours to support an initial diagnosis Yersinia and Bacillus, and found that the two organisms could be clearly differentiated even by visual inspection of their chromatograms 6 CFU/plate Recent advances in colorimetric sensor array (CSA) technology open a promising new path to rapid and low-cost bacterial identification via analysis of the volatile metabolites that are outgassed by living microorganisms Unlike methods such as GC-MS that involve component-by-component analysis, electronic-nose technologies like the CSA can classify complex chemical mixtures without ever explicitly identifying their individual components Bacillus anthracis and Yersinia pestis.In this study, we examined the ability of a current-generation CSA to discriminate bacterial species and strains at substantially lower inoculum concentrations than previously reported, even under the strict biosafety protocols required to handle dangerous organisms like Bacillus anthracis strains Ames and Vollum and Yersinia pestis were handled at ACDP containment level 3. The ACDP containment level 2 bacteria, Bacillus anthracis UM23CL2 and Yersinia pseudotuberculosis, were also handled at containment level 3 to avoid any bias due to the operating environment. Escherichia coli and Bacillus anthracis were pre-cultured overnight in L-broth at 37°C with shaking at 180 rpm. Yersinia species were pre-cultured for 48 h at 28°C in blood agar base (BAB) broth. Cultures were diluted with PBS to achieve the desired optical density (OD600nm). Serial dilutions in PBS were performed to enable the desired number of CFU per plate to be delivered in 100 µL. Culture was spread over the surface of TSA +5% sheep blood agar plates  at a concentration of 10–106 CFU, and the plates were then incubated at 37°C. All bacterial strains were tested in triplicate at each concentration, except Yersinia pestis CO92 and Bacillus anthracis Vollum which had triplicate runs repeated on different days for a total of six repetitions at each concentration.Bacterial strains were purchased from the National Type Culture Collection  or obtained from culture collections maintained at the Defense Science and Technology Laboratory (Dstl). CSAs were fabricated by Specific Technologies as previously reported Immediately after bacteria plating, the CSA was unsealed and affixed to the Petri dish cover. As illustrated in All manipulations and incubations were carried out in an ACDP containment level 3 facility.#/C++, to manually position and orient a grid over the spot array using a mouse, automatically segment each grid cell into spot and background pixels using a global contrast threshold, and estimate each spot’s center using a flood-fill centroid of its segmented pixels. Color vectors could also be generated using ImageJ, a public-domain image-processing program available from the U.S. National Institutes of Health that was used to verify a representative subset of SpotFinder output Using an ordinary flatbed scanner (Epson Perfection V600), the CSA was imaged before initial exposure to volatiles and at 20 minute intervals after exposure, for up to 48 hours after exposure. For each image, we extracted a 240-dimensional vector of 12-bit RGB colors by taking the median of all pixels within a 20-pixel-radius circle centered in each of the 80 indicator spots. Color vectors were extracted using SpotFinder , a semi-automated spot-finding program written in CTo construct a kinetic profile for each culture, we first subtracted the color vector of a reference image of that culture from the vectors of all subsequent images, creating a time series of color differences which we smoothed with a Gaussian kernel (σ = 20 min). We ignored all images captured prior to the reference image, which was taken 4 hours after initial exposure; this choice gave most indicators enough time to finish changing color in response to the headspace out-gas of the growth medium itself. Finally, we subsampled the slopes of the smoothed color differences for each of the 240 colors at 11 evenly spaced times starting 4 h before and extending 6 h after the peak slope of a trigger indicator that was preselected for its strong and nonselective response to generic bacterial growth, creating an initial kinetic profile with 2640 features. Time to detect bacterial growth was taken to be the time of the peak trigger indicator slope. Color vectors were processed in Python using the NumPy and SciPy packages −5 for species, p<0.01 for strain) for every pair of separable classes, and then trained a support vector machine (SVM) to classify the reduced feature vectors by species or strain. The trained classifier was evaluated on the kinetic profile of the corresponding test trial after projecting it onto the features selected by the training set. Validated classifier accuracy was the average misclassification rate across classes for each test trial, with 95% confidence intervals computed using Wilson’s score test. Statistical analysis was performed in R, using the stats (t-test), e1071 (support vector machine), and binom  packages We validated the classifier performance using leave-one-out cross-validation (LOOCV) to construct a training set for each trial. For each training set, we reduced the feature set by applying a two-tailed t-test to each feature Click here for additional data file.


Tarebia granifera, on nitrogen (N) cycling in aquatic systems on the island of Trinidad. We quantified snail biomass, growth, and N excretion in locations where riparian vegetation was present or removed to determine how snail demographics and excretion were related to the condition of the riparian zone. In three Neotropical streams, we measured snail biomass and N excretion in open and closed canopy habitats to generate estimates of mass- and area-specific N excretion rates. Snail biomass was 2 to 8 times greater and areal N excretion rates ranged from 3 to 9 times greater in open canopy habitats. Snails foraging in open canopy habitat also had access to more abundant food resources and exhibited greater growth and mass-specific N excretion rates. Estimates of ecosystem N demand indicated that snail N excretion in fully closed, partially closed, and open canopy habitats supplied 2%, 11%, and 16% of integrated ecosystem N demand, respectively. We conclude that human-mediated riparian canopy loss can generate hotspots of snail biomass, growth, and N excretion along tropical stream networks, altering the impacts of an invasive snail on the biogeochemical cycling of N.Non-native species and habitat degradation are two major catalysts of environmental change and often occur simultaneously. In freshwater systems, degradation of adjacent terrestrial vegetation may facilitate introduced species by altering resource availability. Here we examine how the presence of intact riparian cover influences the impact of an invasive herbivorous snail,  Species introductions and habitat degradation are two major components of environmental change Pomacea canaliculata) Previous studies have shown that individual species can play important roles in nutrient cycling, even in tropical aquatic systems with diverse communities Contemporary species invasions occur in the context of rapidly changing landscapes. Habitat degradation caused by land conversion is a prevalent agent of environmental change that can occur simultaneously with invasive animal dispersal and establishment, often with facilitating or unknown effects on invaders Tarebia granifera), on nitrogen (N) cycling in tropical streams. Nitrogen is an important and potentially limiting or co-limiting nutrient for primary production in tropical streams T. granifera biomass, growth rates, and N excretion in locations where riparian vegetation was present or removed to determine how these variables were related to the condition of the riparian zone. Phosphorus (P) excretion by snails can also be significant in aquatic systems T. granifera in our study system was not detectable from background levels of dissolved P in streamwater . We also compared total snail N excretion to ecosystem N demand to estimate the degree to which T. granifera influences N cycling in tropical streams, and how that influence may be mediated by riparian canopy cover. Lack of riparian vegetation could be hypothesized to have opposing impacts on food resource availability and, subsequently, on snail biomass, growth, and excretion rates - it may augment sunlight availability and lead to increases in algal productivity and biomass, but also inhibit subsidies of allochthonous food resources from terrestrial ecosystems. We determined the overall quantity and quality of food resources available to invasive snails in habitats where riparian vegetation was present and removed, and discuss the potential implications for snail demographics, metabolism, and impact on ecosystem-scale N cycling.Here we studied how riparian canopy cover alters the impacts of an introduced herbivore/detritivore, the quilted melania snail (Tarebia granifera (Gastropoda: Thiaridae) to the West Indies occurred via both accidental and intentional pathways , Aripo River (ARI), and Yarra River (YAR)–from separate drainage basins in the Northern Range Mountains on the island of Trinidad, West Indies . Nutrient diffusing substrates a and ash-free dry mass) and carbon:N composition of epilithon, respectively. Epilithon was collected by scrubbing the entire surface area of rocks on the stream bottom with a plastic bristle brush, generating a slurry of organic and inorganic matter . A subsample of known volume was removed from the slurry using a pipette and filtered  for analysis of chlorophyll a  and ash-free dry mass . Upon filtration, chlorophyll a samples were extracted in 90% buffered ethanol for 24 hours and analyzed using standard fluorometric techniques Percent riparian canopy cover over streams was determined using a densiometer . “Open canopy” habitat was designated as 25% canopy cover or less, “closed canopy” habitat was 75% canopy cover or greater, and remaining values (26–74% canopy cover) were categorized as “partially closed canopy”. Quantity and quality of food resources for invasive snails were characterized in open and closed canopy habitats by measuring biomass of organic matter (chlorophyll Tarebia granifera densities were estimated for each habitat type from randomly assigned transects (RAM) or quadrats (ARI and YAR) using a hand net . Cross-stream transects of a constant width (0.15 m) from the edge to the midpoint of stream wetted width were used in RAM (n = 16). Quadrats (0.33 m2) in ARI (n = 16) and YAR (n = 12) were divided evenly between random locations across the width of streams to account for edge effects. Total length of individual snails was measured to the nearest 0.1 mm using digital calipers. Snails smaller than 2 mm were not included in the study. Snail biomass was measured as mg of ash-free dry mass (AFDM) and calculated using a length-mass regression  or an aperture-mass regression if shell tips were eroded or broken .T. granifera N excretion rates using methods modified from 4 concentration using fluorometric methods described in 4 because it is the dominant N compound excreted by aquatic snails and is readily available for uptake by primary producers and microorganisms. Sampled snails represented the size distribution of T. granifera found in each stream . We controlled for background NH4 concentration in stream water and any vial effects by subtracting the background NH4 concentration in vials incubated without snails . Some fraction of calculated NH4-N excretion rates may have been due to leaching from egested fecal matter. Egestion was not quantified but fecal production was low over the course of incubations. We estimated areal N excretion by multiplying mass-specific excretion rates by average snail density per unit streambed area for each snail size class, then aggregating across all size classes.We measured T. granifera growth rates differed between canopy types, we measured growth rates of snails using a reciprocal transplant design in RAM. We chose an open canopy and closed canopy site separated by 30 m. This was considered sufficient distance between independent open and closed canopy sites as previous work determined that average longitudinal movement rates for T. granifera individuals in RAM were 3.7±0.15 m/week  tM – ln 0M)/t where tM was the mass of an individual snail after incubation and 0M was the mass of the same snail before incubation tX/cX), where tX is the mean growth rate for transplanted snails and cX is the mean growth rate for control snails To determine if T. granifera NH4-N excretion into the context of ecosystem-level NH4-N demand, we measured area-specific NH4-N uptake rate in RAM in February 2008 and 2010 4-N demand was measured using a short-term solute addition 4 (as NH4Cl) and a conservative tracer (NaCl) were simultaneously released at a constant rate via a peristaltic pump. The decline in concentration of NH4 was measured after correction for background concentration and dilution (using the decline in conservative tracer as an estimate). Solutes were released until conductivity reached plateau at the downstream end of the study reach (∼1 h). Sampling stations were set up every 10 m along a 100 m study reach; NH4 samples and conductivity measurements were taken at all stations before solutes were added (to establish background concentrations) and once conductivity had reached plateau (n = 3 per station). Conductivity was measured using a YSI 85 meter . Water samples were analyzed fluorometrically within 4 hours of sampling. Mass of NH4 added to the stream was calibrated to be detectable using fluorometric techniques but not in sufficient quantities to alter pathways of uptake over the time period of the addition (∼2 h). We used data from the solute addition to calculate NH4-N uptake length (the length an NH4+ ion travels downstream before biotic uptake), uptake velocity (the velocity at which biotic uptake removes N from the water column), and areal uptake rate . Data on T. granifera biomass density and N excretion rates were log10-transformed to satisfy ANOVA assumptions of normality and equal variance.Tests for significance of riparian canopy effect across streams were conducted using a mixed effects ANOVA model using canopy cover as a fixed effect and stream as a random effect. Tests of canopy effects within individual streams were conducted using Student’s a) and organic matter (AFDM), was significantly greater in open canopy habitats  and YAR  but not ARI. Epilithon molar C:N ratios did not differ among canopy types in RAM and ARI, but were greater where canopy was removed in YAR  in RAM and ARI showed no difference among canopy types  to 1424 individuals/m2 . Canopy cover negatively influenced T. granifera biomass across the three study streams , and the range of snail size distributions varied among streams . In 2010, no open canopy sites remained in the RAM study site due to riparian vegetation growth; however, snail densities were higher in habitats that were only partially closed compared to those with a fully closed canopy .Average snail density ranged from 23 individuals/m streams . Within-F1,141 = 86.9, p<0.001). Within-stream comparisons indicated that snails in open canopy habitat exhibited greater excretion rates than closed canopy snails in RAM and ARI , but not YAR.Nitrogen excretion rates of individual snails ranged from 0.33 to 2.58 ug N/h. Mass-specific excretion of individual snails decreased with increasing snail size and ranged from 0.02 to 0.76 ug N/mg AFDM/h. Snail N excretion rates showed allometric size scaling and differed significantly among canopy types . Response ratios were similar for both transplants  and initial snail size (before incubation) explained 23% of the variation in growth rates for the entire data set.Mass-specific growth rates of T. granifera in 2008  ranged from 0 to 900 ug N/m2/h and were 3 to 9 times greater in open relative to closed canopy habitats. Differences in areal N excretion across canopy types for all three streams were significant ; within-stream analyses indicated areal excretion rates were greater in open canopy habitat across all streams .Areal excretion rates of 4-N uptake rate in RAM in 2008 and 2010 from NH4 release data showed a significant decline in background-corrected NH4-N with distance downstream from the nutrient addition site , uptake velocity , and area-specific uptake . Mean areal NH4-N excretion rates in fully closed (2008 and 2010), partially closed (2010 only), and open canopy (2008 only) sites supplied 2%, 11%, and 16% of integrated areal NH4-N demand, respectively. In 2010, partially closed canopy sites were used instead of open canopy sites for estimates of areal excretion rates and ecosystem N demand, as riparian vegetation regrowth precluded the presence of open canopy sites.Estimates of NHT.granifera in open canopy sites was linked to increased snail density and, to a lesser extent, increased mass-specific excretion rates relative to shaded sites. Studies have shown that invasive mollusks can alter nutrient cycles in aquatic systems in temperate Our results suggest that invasive snails were heterogeneously distributed along streams due to differences in light availability. Human-mediated removal of riparian vegetation can therefore create hotspots of invasive snail excretion and subsequent impacts on aquatic N cycling. The greater influence of Differences in invasive snail impact among sites with different canopy cover were likely driven by variation in food resources. In two out of the three study streams, estimates of algal and organic matter standing crop were significantly greater in open canopy sites where direct sunlight reaches the streambed and primary producers may be released from growth constraints posed by light limitation . In the We found that increased light availability was more often associated with quantity as opposed to quality of snail food resources. Yarra River was the only one of three study streams in which food quality was significantly greater in closed canopy habitat, and interestingly, the only stream for which the effect of canopy type on mass-specific excretion rates was not significant. In this stream, increased algal quality in closed canopy sites may have diminished the benefits of open canopy habitat where food was more plentiful but of poorer quality. These results partially support the light-nutrient hypothesis, which states that increased light intensities should be associated with decreased nutrient content in algal communities T. granifera likely has altered a suite of ecosystem properties within our study area. Lack of preinvasion datasets or comparable reference sites uninvaded by snails prohibited direct analysis of ecosystem-level effects of invasion. However, results from this study have shown that primary production in Trinidadian lotic systems can be co-limited by availability of N and phosphorus. Changes in absolute and relative availability of these nutrients mediated by invasive snail excretion may influence the identity of the nutrient that limits primary productivity, as well as the growth and community composition of primary producers T.granifera in Trinidadian streams despite dense and conspicuous aggregations , these primary consumers may act as a trophic dead end for aquatic communities, diverting energy fixed by primary producers away from higher trophic levels Invasion by Snail size distributions influenced aggregate N excretion among study streams. Small-bodied organisms generally exhibit greater mass-specific excretion rates due to higher metabolism relative to large-bodied organisms T. granifera. If snail biomass is associated with food quantity as our results suggest, areal snail excretion rates will be more pronounced during the dry season. In contrast, hydrologic variability due to frequent rain events in the wet season can diminish the impact of snail excretion through dilution and dislodgment of snails, reducing snail biomass and eroding spatial structure by washing individuals downstream. An analysis of shrimp N and P excretion Temporal dynamics likely moderate the impact of invasive snails on N cycling. This study was conducted during the dry season in the Caribbean, when stream discharge is relatively low and flooding events are typically infrequent. These conditions facilitate growth of primary producers that provide food resources for T. granifera can have a variable influence on nutrient cycling in tropical systems, dependant on light availability. Mitigation efforts focused on curtailing habitat degradation in adjacent riparian corridors may therefore ameliorate the magnitude of impacts. In this case, a management strategy involving restoration and/or protection of riparian buffer zones along stream corridors may decrease the biogeochemical impacts of an established non-native mollusk. Further investigation into interactive effects of human-mediated habitat degradation and introduced animals on nutrient cycles may uncover pragmatic management opportunities.Understanding direct links between impacts of species introduction and habitat degradation is important for understanding the spatial dynamics and spread of invasive species as well as for prioritizing use of limited funding for control efforts. Eradication–if possible–is often arduous and costly, and careful examination is necessary to identify the most urgent cases that have potential for success. Habitat degradation can unintentionally create patches of favorable habitat that act as spatial resource subsides to invasive species at landscape scales. Our study suggests that populations of Figure S1Nutrient limitation in Ramdeen Stream. Mean chlorophyll a on nutrient diffusing substrates after a two-week incubation in RAM. Asterisk indicates significantly greater chlorophyll a relative to controls using a randomized block ANOVA (p<0.001). Results revealed that algal accrual was co-limited by N and P availability, as substrates containing both N and P were the only treatment with significantly greater algal biomass than controls.(TIF)Click here for additional data file.Figure S24 addition in Ramdeen stream in 2008 and 2010.Short-term NH Tracer NH4-N and conductivity are concentrations at plateau corrected for background concentrations. Distance from injection site indicates location downstream from site where solutes were added using a peristaltic pump.(TIF)Click here for additional data file.Table S1Stoichiometry of T. granifera body tissue. Mean (±1SE) body tissue C:N and C:P of T. granifera (shell removed) in open and closed canopy habitat. RAM  =  Ramdeen Stream, ARI  =  Aripo River.(DOCX)Click here for additional data file.Supporting Information S1Nutrient limitation in Ramdeen Stream as measured using nutrient diffusing substrates.(DOCX)Click here for additional data file.Supporting Information S24-N uptake length, uptake velocity, and areal uptake rate in Ramdeen Stream.Calculation of NH(DOCX)Click here for additional data file.Supporting Information S3Measurement of C, N, and P stoichiometry of T. granifera body tissue.(DOCX)Click here for additional data file.


Impairment of intestinal epithelial barriers contributes to the progression of HIV/SIV infection and leads to generalized HIV-induced immune-cell activation during chronic infection. Rhesus macaques are the major animal model for studying HIV pathogenesis. However, detailed characterization of isolated rhesus epithelial cells (ECs) from intestinal tissues is not well defined. It is also not well documented whether isolated ECs had any other cell contaminants from intestinal tissues during the time of processing that might hamper interpretation of EC preparations or cultures. In this study, we identify and characterize ECs based on flow cytometry and immunohistochemistry methods using various enzymatic and mechanical isolation techniques to enrich ECs from intestinal tissues. This study shows that normal healthy ECs differentially express HLA-DR, CD23, CD27, CD90, CD95 and IL-10R markers. Early apoptosis and upregulation of ICAM-1 and HLA-DR in intestinal ECs are thought to be the key features in SIV mediated enteropathy. The data suggest that intestinal ECs might be playing an important role in mucosal immune responses by regulating the expression of different important regulatory and adhesion molecules and their function. This study shows that ECs are positive for HLA-DR, CD23, CD27, CD90, CD95 and IL-10R phenotypes. Early apoptosis and upregulation of ICAM-1 and HLA-DR in intestinal ECs are thought to be the key features in SIV mediated enteropathy. The data suggest that intestinal ECs might be playing an important role in mucosal immune responses by regulating the expression of different important regulatory and adhesion molecules and their function.Epithelial cells from jejunum and colon were isolated using several enzymatic and isolation techniques that have been diagrammatically represented in There was no colocalization either between epithelial (cytokeratin) and Ham56 (macrophage marker), epithelial and CD11c (dendritic cell marker), and epithelial and CD54 (cell adhesion molecule) . Few douTo confirm the observations detected by immunohistochemistry, single-cell suspensions of isolated cells were generated from intestinal mucosa followed by DTT wash as described in the CD23, the low-affinity IgE receptor is widely distributed on B cells in the follicular mantle, on resting B cells. CD23 is also detected in monocytes and eosinophils and has influence on cell differentiation and growth of both B and T cells. CD23 expression in jejunum ECs  was significantly higher than CD23 expressed by CD45 leukocytes , however their function is not well understood .Surface receptor CD27, a type 1 glycoprotein and a member of the tumor necrosis factor receptor family was shown on a subset of B and T cells and it was thought that their expression may be required for the generation of memory and long term maintenance of B and T cell immunity Intracellular adhesion molecule-1  has an important role to play in the intestinal immune response CD90, a glycophosphatidylinositol-linked glycoprotein was initially described as a differentiation marker expressed predominantly in the mouse brain and thymus CD95 is a member of the tumor necrosis factor family and induces apoptosis when cross-linked by its natural ligand, CD95L. High percentages of ECs express CD95 (mean 74.9%±12.9%). However CD95 expression on ECs is significantly lower compared to intestinal CD45+ leukocytes  and chronically (288 days post infection) SIV infected RMs were stained with anti-active caspase-3 (AC-3) antibodies. Confirmation of ECs apoptosis was performed using anti-cytokeratin antibodies in all acute and chronically SIV infected RMs . As predIntestinal epithelium primarily takes part in the digestive system. However, they play an important part in the immune system, both as a barrier and as a first-line pathogen recognition system in vitro will allow us to further characterize these progenitor cells both phenotypically and functionally and study their distribution in different tissues including the airway, lung, parenchyma, skin, bone marrow and buffy coat.Combinations of mechanical preparation and enzymatic digestion methods to enrich RM intestinal ECs from tissues obtained from necropsied animals were compared. Using these techniques, large numbers of intestinal ECs, with a viability of 90–96%, were obtained. Bacterial contamination is rarely a problem in these isolation procedures, as the majority or bacterial flora are removed by several washing steps and percoll separation. Cells isolated from directly after DTT wash may contain more bacterial contamination that needs further investigation. The use of DTT or EDTA was found to be best for isolating intestinal ECs as the isolation procedure is faster, yields increased percentages of ECs, and takes fewer steps to identify ECs. Further collagenase enzymatic treatment reduces the yield of ECs and increases the percentages of leukocytes. Overall in all isolation protocols, none of the protocols yielded pure populations of ECs in our experiment and there was contamination with fibroblast/myofibroblast cells (CK−CD90+CD45−) (data not shown) Epithelial cells provide a shield of the intestinal mucosa from other mononuclear cells below the epithelium, and express HLA-DR molecules in the normal, noninflammed intestinal mucosa. Our data confirms the earlier reports where expression of HLA-DR in ECs has been shown in both small and large intestines A wide variety of isolation methods for human and animal ECs have been used including purely mechanical procedures The most important costimulatory receptor expressed by a majority of T cells is CD28, which interacts with CD80 and CD86 ligands on antigen presenting cells and plays a major role in T cell activation. Our studies have shown the lack of CD80 and CD86 expression on intestinal ECs as previously reported for human colonic ECs IL-10 bears close resemblance to IFNγ and their receptor complexes also belong to the same cytokine receptor family Despite the lack of CD3 and CD4 expression (data not shown) on ECs, there was increased apoptosis of intestinal ECs in both acute (21days pi) and chronically SIV-infected RMs . Our datIn summary, either DTT or EDTA chemical treatment yields increased percentages of intestinal ECs compared to other protocols tested. Furthermore, normal healthy rhesus ECs did not express CD80 or CD86 costimulatory molecules. However, they did express CD23 and HLA-DR suggesting they may play a role in antigen presentation, which needs further verification. Combined these results are consistent with the interpretation that ECs are highly activated cells and capable of modulating immune responses. Interestingly, a small percentage of CK+CD45+ ECs in intestinal ECs were always present in normal intestinal mucosa, which invites further exploration to discover the significance of these potential “stem cells” and to define their role in restoration of EC damage and SIV pathogenesis. Early apoptosis and increased expression of ICAM-1 and HLA-DR in ECs in SIV infected RMs are thought to be the key features of intestinal enteropathy that is also associated with local inflammation, and increased gut permeability. The immunoregulatory cells and their cytokines that might play an important role in regulating ECs apoptosis and their activation are the subject of our future studies. The functional implication of CD23, IL-10R and CD90 receptors in the pathophysiological mechanisms of HIV/SIV gut enteropathies, clearly deserves further investigation.Approval for all veterinary procedures in this study was obtained from the Tulane University Animal Care and Use Committee (Protocols #3567 & 3568), Animal Welfare Assurance A-4499-01. All the animals in this project were housed at the Tulane National Primate Research Center (TNPRC) and under the full care of TNPRC veterinarians in accordance with the standards incorporated in the Guide to the Care and Use of Laboratory Animals (NIH) 78-23 . All veterinary procedures were performed only with sedated animals. Animal welfare and steps were taken to ameliorate suffering in accordance with the recommendations of the Weatherall report, “The use of non-human primates in research”.Macaca mulatta) either male or female between 1.9–9.9 years of age, which were negative for HIV-2, SIV, type D retrovirus and STLV-1 infection were used in this study. Six RMs were infected either through intravenous or intravaginal route with 300–500 TCID50 SIVMAC251. One SIV infected animal was euthanized at 21 days (acute) post infection and five SIV infected animals were euthanized within 167–401 days (chronic) post infection. The remaining 10 macaques were SIV uninfected normal controls. Intestines (jejunum and/or colon) and blood were collected from all animals at the time of necropsy for phenotyping experiments.Sixteen Indian RMs  in PBS, and frozen in cryomolds containing OCT compound . Tissue sections of 8 µm thick were processed for immunofluorescence staining as described previously Immunoperoxidase staining of jejunum and colon ECs were performed in 5 µm thick section. The slides were first deparaffinized and heated at 95°C for 30 min in citrate buffer for epitope retrieval. Slides were allowed to cool and finally rinsed in PBS (pH 7.4). Slides were further incubated with endogenous peroxidase block solution (Dako) followed by biotin blocking system (Dako) according to manufacturer's protocol. Slides were then incubated with cytokeratin-large spectrum monoclonal antibody (KL-1). Biotinylated mouse IgG and Vectastain ABC reagent  were used for detection. Liquid DAB (Dako) were used as chromogen for developing slides.2).Intestinal samples (6–10 cm long pieces of jejunum and colon) were collected in 50 ml tubes with ice-cold calcium and magnesium free HBSS (Fisher Scientific) and immediately transported to the laboratory. Fat, blood vessels, and mesenteric lymph nodes were trimmed. Mucus and gross debris were quickly removed by covering the specimen with dry paper towels. Specimens were further washed twice in cold PBS (pH 7.4). Intestinal samples were cut into small pieces    [21] folemicals)  as repor7 cells/ml and 100 µl aliquots or 100 µl of whole blood samples were incubated with appropriately diluted concentrations of antibodies for 30 min at 4°C. Whole blood samples from normal RMs were used for fluorochrome compensations. Whole blood cells were then lysed using a whole blood lysis protocol as previously described For flow cytometry staining, cells were adjusted to 10Graphical presentation and statistical analysis of the data were performed using GraphPad Prism . Results between experimental groups were compared using a one-way ANOVA and nonparametric Mann-Whitney t test. Those p values<0.05 were considered statistically significant.


The purpose of this study was to develop an aggressive locally advanced orthotopic prostate cancer model for assessing high-dose image-guided radiation therapy combined with biological agents. For this study, we used a modified human prostate cancer (PCa) cell line, PC3, in which we knocked down a tumor suppressor protein, DAB2IP (PC3-KD). These prostate cancer cells were implanted into the prostate of nude or Copenhagen rats using either open surgical implantation or a minimally invasive procedure under ultrasound guidance. We report that: i) these DAB2IP-deficient PCa cells form a single focus of locally advanced aggressive tumors in both nude and Copenhagen rats; ii) the resulting tumors are highly aggressive and are poorly controlled after treatment with radiation alone; iii) ultrasound-guided tumor cell implantation can be used successfully for tumor development in the rat prostate; iv) precise measurement of the tumor volume and the treatment planning for radiation therapy can be obtained from ultrasound and MRI, respectively; and v) the use of a fiducial marker for enhanced radiotherapy localization in the rat orthotopic tumor. This model recapitulates radiation-resistant prostate cancers which can be used to demonstrate and quantify therapeutic response to combined modality treatments. Prostate cancer is one of the most common male cancers, representing 28% of all male cancers in 2010  in the treatment of prostate cancer. Because the prostate is a mobile organ, inter and intra fraction movement, as well as random deformation of the organ, can occur throughout the course of daily radiation treatment . Crevoisoutcomes . ProstatWhile the use of image-guidance has improved the accuracy of radiotherapy, allowing safe and effective dose escalation in the treatment of primary prostate cancer, there is still no effective therapy for aggressive or recurrent disease. To develop effective therapies for aggressive prostate cancer, whether novel or a combination of several agents and treatment modalities, it is imperative to first develop a model of radiation resistant prostate cancer.To facilitate the investigation of resistant aggressive disease after radiation in a preclinical environment it would be best to have a locally advanced orthotopic rodent model. In this study we report the development of such a model for resistant tumors after IGRT in rat prostates using a novel tumor suppressor knockdown prostate cancer cell line.et al and co-transfected with luciferase reporter gene as described previously by Kong et al. Cells w5 PC3-KD cells were diluted to a final volume of 30 μl. Male rats, either nude or Copenhagen, were anesthetized using 1–2% isoflurane mixed with 100% O2. Cells were injected into the right lobe of prostate and a gold fiducial marker was placed into the prostate for subsequent image guided therapy. All the experiments were conducted under UT Southwestern Institutional Animal Care and Use Committee-approved guidelines for animal welfare.A total of 1×10® 770 Imaging System, Amsterdam, The Netherlands). Once the prostate was visualized, a gold seed was implanted into the prostate using an 18G needle mounted on a trocar. The needle was aligned parallel to the transducer and inserted parallel to the urethra. After confirming proper placement of the fiducial, the needle was left in place and cells were implanted using a 1cc syringe. Imaging was performed on the VisualSonics Vevo 770 Imaging System using the real-time Micro Visualization scan head RMV716 (11–24 MHz) specific for rats. Rats were anesthetized and the transducer head was placed transverse to the pelvis of the rats in supine position. Images were obtained by resolving various depths of tissue into the center of optimum resolution plane to ensure clear images throughout the tumor volume.Ultrasound imaging was employed for i) minimally invasive implantation of tumor cells; ii) measuring tumor volume in the rat prostate. For implantation, rats were anesthetized and the pelvis was shaved and sterilized. Animals were secured to the handling table to ensure no movement. The prostate and bladder was identified in the field of view by using an RMV716 transducer head . Rats were anesthetized by using isoflurane inhalation mixed in pure oxygen followed by an i.p. injection of D-luciferin (80 mg/kg). BL images were acquired 10 min after luciferin injection using various exposure times.et al(Surviving fraction (SF) analysis was performed using PC3 Con (DAB2IP proficient) and PC3-KD (DAB2IP deficient) as described by Kong et al,6. In br3 isotropic voxel).MRI studies were conducted using a 3 Tesla whole-body human scanner  with a small animal solenoid radio-frequency (RF) coil . Under anesthesia, animals were placed supine where the thigh of the rat centered with respect to the center of the RF coil. A volume containing the entire tumor was subsequently obtained using T2 weighted multi-slice fast spin echo sequences  and three dimensional gradient sequences  were prepared for standard H&E staining. Briefly, sections were deparaffinized, rehydrated, stained with Harris’s hematoxylin for 20 sec followed by treatment with Scott’s solution. After washings, sections were stained with eosin for 2 min, rinsed, dehydrated in ethanol and xylenes and mounted using permount.Prostate tumors were removed and fixed in 4% formalin. Tumors were mounted in paraffin and sections . Hypoxyprobe-1 (pimonidazole HCl) was administered i.p. (120 mg/kg) in tumor bearing rats. Two hours later, the animal was sacrificed; tumor tissue was collected and fixed in 4% formalin solution for 48 h. For detection, tumor sections were incubated with FITC-conjugated mouse monoclonal antibody against pimonidazole (1:50) for overnight at 4°C. After incubation with primary antibodies, tumor sections were washed thoroughly and visualized using a Zeiss Axio Imager 2 microscope  using the FITC filter.To appropriately facilitate the study of IGRT for aggressive PCa, it is necessary to have a model that closely mimics human disease, ideally with a tumor that is initially limited to one lobe of the prostate. In this model, we implanted a human prostate tumor cell line which is deficient in a tumor suppressor protein DAB2IP (PC3-KD). This protein is a member of the Ras-GTPase activating family and the loss of DAB2IP has been associated with PI3K-Akt hyperactivation , increasPC3-KD cells are highly aggressive and demonstrate significant radio-resistance . We alsoPC3-KD cells were implanted into the prostate of Copenhagen (immune competent) and athymic nude rats (immune deficient). Tumor growth was followed by BLI as described above. Once the tumor size reached a certain size (approximately 5–7 mm in diameter) based on ultrasound imaging, animals were split into either a treatment  or control arm (n=2 for both arms). 5 as shown in Control group tumors demonstrated aggressive, but predictable growth (5 to 1×106)  . These rTo correlate the imaging with biological events we performed IHC analysis. H&E stained sections confirm the placement of the tumor into the rat prostate; PC3-KD cells are highly anaplastic and aggressive . OrthotoWhile several other OT prostate tumors have been reported in the literature –13, therThere are several requirements necessary for the development of a locally advanced prostate tumor specially for IGRT or combined modality therapy. Current OT prostate models for preclinical studies are primarily developed in mouse, however, to delineate tumors and track tumor response animals with larger prostates need to be used, hence we used rats. Secondly, the major difficulty in creating an OT model that accurately demonstrates human disease, specifically a tumor that is radiation resistant, is reliability. In order to facilitate resistance we used a DAB2IP knockdown prostate cell line. By using the DAB2IP knockdown cell line resistant tumors are reliably formed. Orthotopically implanted cells were able to grow large tumors in immune-competent male Copenhagen rats as well as nude rats.in vitro but could not find correlating aggressive radiation resistant tumors in vivo 2 Gy of 60 Gy, a dose that is clinically relevant in the treatment of human PCa. Furthermore, rapidly growing tumors often display heterogeneous areas of necrosis as a result of insufficient vascular supply . InsuffiIn radiation resistant models, the ability to track tumor growth and response to therapy is essential. BLI was the primary imaging modality in this study and has been correlated with both CT as well as MRI . We furt


To the Editor: Verani et al. (Acanthamoeba keratitis (AK) in persons wearing soft contact lenses who used Complete MoisturePlus (CMP) multipurpose contact lens solution . They noted similarities between the AK outbreak and the Fusarium keratitis (FK) outbreak of 2004–2006, including the concomitant time frame and association with a particular solution, ReNu with MoistureLoc . Both solutions were new products introduced within 1 year before the respective outbreaks.In neither outbreak was the solution contaminated; in both outbreaks, implicated bottles were from multiple lots, suggesting that each outbreak resulted from insufficient antimicrobial activity. However, in the FK outbreak, all reported cases involved bottles produced at 1  of 4 multinational Bausch & Lomb manufacturing plants  associated with the Fusarium keratitis (FK) outbreak during 2004–2006 (Ancanthamoeba keratitis (AK) outbreak that we reported  multipurpose contact lens solution .We obtained lot numbers for 22 bottles of CMP that AK case-patients used before symptom onset. Because no lot number was repeated, intrinsic contamination was unlikely as the source of the AK outbreak; the geographic and temporal distribution of cases further argued against a point-source outbreak. All 17 lot numbers for which AMO plant of origin was determined were manufactured in Spain . According to a press release from AMO in November 2006, the “vast majority of AMO's contact lens solution products distributed in the U.S.” were manufactured in the company’s production facility in Spain, 1 of its 2 international manufacturing plants (CMP was produced and used internationally at the time of the US multistate outbreak (