Wolbachia pipientis causes complex patterns of crossing sterility between populations of the Culex pipiens group of mosquitoes. The molecular basis of the phenotype is yet to be defined. In order to investigate what host changes may underlie CI at the molecular level, we examined the transcription of a homolog of the Drosophila melanogaster gene grauzone that encodes a zinc finger protein and acts as a regulator of female meiosis, in which mutations can cause sterility. Upregulation was observed in Wolbachia-infected C. pipiens group individuals relative to Wolbachia-cured lines and the level of upregulation differed between lines that were reproductively incompatible. Knockdown analysis of this gene using RNAi showed an effect on hatch rates in a Wolbachia infected Culex molestus line. Furthermore, in later stages of development an effect on developmental progression in CI embryos occurs in bidirectionally incompatible crosses. The genome of a wPip Wolbachia strain variant from Culex molestus was sequenced and compared with the genome of a wPip variant with which it was incompatible. Three genes in inserted or deleted regions were newly identified in the C. molestus wPip genome, one of which is a transcriptional regulator labelled wtrM. When this gene was transfected into adult Culex mosquitoes, upregulation of the grauzone homolog was observed. These data suggest that Wolbachia-mediated regulation of host gene expression is a component of the mechanism of cytoplasmic incompatibility.Cytoplasmic incompatibility (CI) induced by the endosymbiont  Wolbachia are maternally inherited bacteria that manipulate invertebrate reproduction. Cytoplasmic incompatibility is embryo death that occurs when males carrying Wolbachia mate with females that do not, or that carry a different Wolbachia variant; its mechanism is poorly understood. In Culex mosquitoes, in the presence of Wolbachia a gene related to a Drosophila melanogaster gene, grauzone, which has been shown to act as a regulator of the meiotic cell cycle, showed an elevated level of expression. When lower levels of expression were achieved through RNA interference, embryo hatch rates were affected and the stage of development at which embryo death occurs was altered. To find Wolbachia genes that influence cytoplasmic incompatibility, we compared the genomes of two variants of Wolbachia from Culex that produce cytoplasmic incompatibility with one another. Although most segments of these genomes were very similar, one newly identified gene is predicted to be a regulator of gene transcription. We cloned this gene into a plasmid, expressed it in adult mosquitoes and found higher levels of expression of the Culex grauzone homolog. This suggests that the Wolbachia transcriptional regulator may play an important role in manipulating the host in order to induce cytoplasmic incompatibility. The intracellular maternally inherited bacterium Wolbachia pipientis, a widespread endosymbiont of invertebrates Wolbachia (wPip) is present in Culex pipiens, complex crossing types including both unidirectional and bidirectional CI C. pipiens lines. Understanding the basis of this complexity has been a long standing problem.The Culex pipiens group of sibling species of mosquito, in which CI was first discovered, provides a model system with useful features for examining the genetic differences that underlie CI. Even though only one designated strain of Several studies have shown that Wolbachia and its products can influence host gene transcription, notably a major upregulation of immune genes in transinfected naïve mosquitoes grau is one of only two genes known to be involved in regulation of metaphase II. Given the parallels between abnormal chromosome segregations in both CI embryonic arrest and in grau mutant embryos, and the similarities between metaphase-anaphase transition in meiosis II and mitosis, we considered this gene a candidate for involvement in CI generation.Cytological studies in Drosophila and Nasonia have revealed that aberrant events in the male pronucleus in an incompatible fertilization include abnormal histone H3.3 and H4 deposition, prolonged or incomplete DNA replication, delayed chromosome condensation/segregation and nuclear envelope breakdown Wolbachia genes are responsible for the manipulation of host early embryogenesis. The lack of a transformation system for this intracellular symbiont, which cannot be cloned or cultured outside of host cells, has meant that systematic testing of the phenotypic effects of candidate genes has not been possible. The Pel line of Culex quinquefasciatus from which a wPip genome sequence was generated Culex molestus line Wolbachia strains could therefore be used to determine differences between them that may be involved in the generation of different crossing types. Comparisons with the JHB line wPip genome Culex and test its potential involvement in the mechanism of CI, and in parallel to purify, sequence and analyse the wPipMol genome in order to attempt to identify Wolbachia genes that may be involved in manipulation of the host, particularly in transcriptional modification.Not much is known about which We identified homologs of the D. melanogaster gene grau in Culex quinquefasciatus (a member of the C. pipiens complex) by interrogation of the genome database Relative transcription of CPIJ005623 was measured in members of the C. pipiens complex using quantitative reverse transcription PCR (qRT-PCR). We first looked at the transcription of the gene in whole adults over time to understand its expression dynamics in individual mosquitoes that carry Wolbachia and their antibiotic-treated Wolbachia-free genetic counterparts. In Wolbachia-infected C. quinquefasciatus Pel adult females, CPIJ005623 transcription showed a peak of over two-fold upregulation compared to the Wolbachia-cured line PelU at 4 days post pupal eclosion (dpe), which then decreased to similar levels as the PelU line by 8 dpe . DiffereWe then focused our analysis at single time points where the expression was found to be highest, separately in ovaries (4 dpe) and testes (2 dpe) (the tissues where CI is expressed). A similar pattern of upregulation (Wilcoxon rank-sum: p<0.05) of CPIJ005623 in Wolbachia-infected ovaries  and testGiven the upregulation of CPIJ005623 detected in the Italy Wolbachia-infected line compared to uninfected females, we knocked down the gene in infected females using dsRNA to investigate whether this change may be involved in the rescue function of CI. Restoring transcription to uninfected levels in these females might result in developmental arrest in embryos from crosses with Wolbachia-infected males . Knockdo2 = 28.957, Df = 1, p<0.0001), with a higher proportion of embryos arresting at stage I in iCPIJ005623 females  or late (stage II+III) stage in embryo development  females .2 = 23.841, Df = 1, p<0.0001). Although in Pel females unhatched rates were not affected after grau homolog KD, the shift from late to early embryo mortality is conserved in the two Culex lines.A lower level of upregulation was observed for CPIJ005623 in Pel Wolbachia-infected versus uninfected females . To undeSince CPIJ005623 was also observed to be upregulated in infected versus uninfected males, we tested if this upregulation had a role in inducing CI in crosses between infected males and uninfected females. We failed to obtain an antibiotic-treated Wolbachia uninfected C. molestus line due to the low egg production of this blood meal independent autogenous line. As the upregulation detected was fairly similar and not statistically different between the Wolbachia infected lines tested  we focus2 = 65.975, Df = 1, p<0.0001, T1-2 = 33.079, Df = 1, p<0.0001, T2-LacZ injected controls  in all crosses where CPIJ005623 transcription was reduced irrespective of the gender of the individual (A double knockdown (double-KD) experiment in both Italy males and Pel females was also performed, since upregulation of CPIJ005623 was observed in Pel-infected vs Pel-uninfected ovaries . A KD ofdividual , Table 2wPipMol strain present in C. molestuswPipPel wPipJHB C. quinquefasciatus is bidirectionally incompatible with Mol, while JHB and Pel are compatible with each other in both crossing directions  were identified in total, in twenty-three genes  repeats, and ankM2 encodes ANK and Tetratricopeptide (TPR) repeats, both of which function in mediating protein-protein interactions ankM1 gene is a homolog of WD0596 from Wolbachia strain wMel of Drosophila melanogasterwtrM, contains DNA binding domains associated with transcriptional regulators. Five inserted or deleted regions each comprising one to five genes were identified in wPipPel and wPipJHB but absent in wPipMol  C. molestus mosquitoes, expression of wtrM was strongly localised to the ovaries in females   .Several lines of evidence support the hypothesis that the differential regulation of a Culex homolog of the D. melanogaster gene grau plays a direct role in CI generation. The presence of Wolbachia induced changes in CPIJ005623 transcription levels and the degree of upregulation of CPIJ005623 varied between incompatible C. pipiens group females. A decrease in embryo hatch rates was detected in a C. molestus line when CPIJ005623 expression was knocked down in females of an otherwise compatible cross. This increase in levels of CI suggests a role for CPIJ005623 in CI rescue in C. molestus females. The knockdown of CPIJ005623 also extended the mean developmental stage reached by embryos in incompatible crosses. When reversal of CI might have been expected , more embryos arrested at a later stage and when increased levels of CI might have been expected , more embryos arrested at an early stage.It has been hypothesized based on its mutant-sterile phenotype that D. melanogaster grau could be involved in egg activation, act as a negative regulator of the cyclin B complex stabilizing factor involved in meiotic cell cycle regulation, and/or regulate microtubules; these are not mutually exclusive possibilities Given that there was no complete reversal of embryo hatch rates in the rescue cross following CPIJ005623 knockdown, it is likely that differential regulation of CPIJ005623 is a component of CI but not the only molecular change that underlies the phenotype. It also seems likely that different Wolbachia strains produce different combinations of molecular manipulations, given the mutual incompatibilities that can exist among Wolbachia strains. The differences in grau homolog upregulation and KD analysis between the two C. pipiens group lines presented in this study reflect that. Further work is needed to determine what other factors along with grau are involved in this complex phenotype and whether other completely independent pathways are targeted in different Wolbachia strain-host associations where CI is induced. In light of the female-sterile phenotype produced by grau mutations, it would also be interesting to search for and examine the expression of grau homologs in Asobara tabida, where Wolbachia has become essential for the completion of this wasp's oogenesis Wolbachia gene that modifies transcription levels of a non-immune host gene is a significant step forward in understanding the molecular methods by which this endosymbiont manipulates its hosts. This Wolbachia gene is one of only a small number that vary between two incompatible populations. Although it was not possible to directly assay the effects of wtrM on crossing patterns using transfection, since the plasmid did not enter the ovaries, the upregulation of CPIJ005623 in wtrM-transfected females does provide clear support for the hypothesis that wtrM has a role in the generation and/or rescue of CI between these Culex lines. The fact that the natural expression levels of wtrM in C. molestus were highest in ovaries is of interest given that CPIJ005623 knockdown in C. molestus females changed embryo development and hatch rates whereas CPIJ005623 knockdowns in males produced little effect. It should be noted that although wtrM appears to play a role in this particular Wolbachia - host interaction, there may well be a number of other Wolbachia genes beyond this family of homologous transcriptional regulators that interact directly with host genomes.The identification of a wPip strains makes this a promising system for better elucidating the molecular mechanism of CI. All the genes identified here that vary between mutually incompatible wPip substrains provide new avenues for investigation; their concentration in the wPip prophages also underlines the importance of these hypervariable regions in wPip recent genome evolution Wolbachia transformation may be required in order to produce a direct phenotypic confirmation of implicated Wolbachia genes in generating/rescuing CI. Despite considerable effort this remains a highly challenging goal, since it is an obligately intracellular microbe. Transformation of the host insect to express Wolbachia genes could be carried out, as was performed in Drosophila with several Wolbachia Ankyrin repeat-encoding genes Wolbachia, for example in post-translational processing, folding and in epistatic interactions with other Wolbachia proteins within the bacteria, failure to generate a sterility phenotype does not rule out the involvement of candidate genes in CI.The small number of differences between the genomes of incompatibility-generating Aedes mosquito vectors of dengue virus, the introduction of several non-native Wolbachia strains produced a strong inhibitory effect on dengue virus transmission. Given its population invasion capacity, Wolbachia-based strategies seem very promising as a new dengue control tool Recently, Wolbachia has attracted considerable scientific and public interest as a control tool for mosquito-borne diseases. In the C. pipiens complex laboratory lines Pel  20) throughout larval development for 5 consecutive generations. Adults were not treated with antibiotic. The line was subsequently reared in the absence of antibiotic for at least 5 generations more before experiments were performed. Removal of Wolbachia was confirmed by PCR and the line has been kept Wolbachia free since 2011. Cpq-T generation is described in detail in Wolbachia free since 2010.Wolbachia uninfected PelU was generated by treating PelA with rifampicin  and incubated for 10 min at 95–99°C. Samples were centrifuged for 5 minutes at maximum speed at 4°C. Supernatant, containing gDNA, was kept and used for Polymerase Chain Reaction (PCR); all oligonucleotides are listed in table S5.Adult female and male RNA was extracted from 4–5 adult mosquitoes using TRIzol Reagent (Invitrogen-Life Technologies) following manufacturer's instructions. TRIzol-extracted RNA was DNase I treated for genomic DNA elimination and purified via standard phenol chloroform extraction. RNA from both mosquito ovaries and testes , was extracted and purified via the RNeasy Mini protocol for animal tissue (Qiagen). All purified RNA samples were quality checked via Nanodrop analysis and only highly pure (A260:230 and A260:280 ratios of >2.00) were kept. Exception was made for testes RNA for which the maximum A260:230 achieved were 1∶50-1.80 even when using a highly optimised protocol.t) values were determined and background fluorescence was subtracted. Transcription levels of target genes were calculated by the standard curve method, as described in technical bulletin #2 of the ABI Prism 7700 Manual (Applied Biosystems-Life Technologies), relative to the endogenous reference genes and RpS7.cDNA synthesis was performed in 10 µl (adults) or 20 µl (ovaries and testes) reaction volumes with 100, 300 or 900 ng of total RNA for the testes, ovaries and adult  samples respectively, using the iScript cDNA synthesis kit (BioRad). qRT-PCRs were performed on 1∶10, 1∶20 and 1∶30 dilutions of the cDNAs from the testes, ovaries and adult samples respectively, using iQ SYBR Green PCR mastermix (BioRad), a DNA Engine thermocycler (MJ Research) with a Chromo4 real-time PCR detection system (Bio-Rad) and the following cycling conditions: 95°C for 3 minutes, then 41 cycles of 95°C for 10 s, 60°C for 30 s, with fluorescence acquisition at the end of each cycle and a melting curve analysis after the final cycle. The cycle threshold (CCPIJ005623 transcript. Amplifications were performed using primers RpL32 (F-R) and CPIJ005623 (F1-R1) for Culex Pel, RpLS7 and CPIJ005623 (F3-R3) for Culex Italy as described above.T7-tailed primers were used to amplify a PCR template for CPIJ005623 and the control LacZ gene from Culex cDNA and E.coli DNA respectively. DsRNAs were synthesized using the T7 Megascript Kit (Ambion-Life Technologies) following manufacturer's advice. Purified dsRNAs were diluted to a concentration of 3 µg/µl and 69 nl were injected into the thorax of cold anesthetized mosquitoes using a Nanoject microinjector (Drummond Scientific). After dsRNA injections and at the indicated time points, testes and ovaries from 30 male and female mosquitoes were dissected. RNA was extracted by using the RNeasy Mini protocol for animal tissue (Qiagen) in order to verify the successful knockdown of the 1 generation progeny from crosses were characterised by i) total number of rafts; ii) total number of eggs, iii) mean proportion of hatched eggs and iv) for unhatched embryos mean proportion of developed embryos.Crossing experiments following knockdowns were carried out using 50 virgin individuals of each sex obtained by sexing and isolating pupae for mosquitoes. On the day of pupal eclosion, 50 male or 50 female mosquitoes were injected with dsRNA. Injected individuals were allowed to recover and on the dates represented in the experimental design diagrams mating partners were added to the injected individuals for the designated times indicated  and 5A. Transcription levels were analysed using a non-parametric Wilcoxon rank-sum test in the case of 2 sample variables and by a two-way ANOVA (with replication) analysis of variance for 3 sample variables. Embryo developmental results were analysed by using generalized linear models (GLM) with binomial error structures (where reaching SII or beyond is classed as a success). Differences between hatched and unhatched embryos were determined via a Welch two sample t-test. All calculations were performed using the R software .Wolbachia wPip DNA from Mol early embryos, library construction, general PCR conditions and crossing experiments were as previously described The purification of Phylogenetic trees were constructed based on Maximum Likelihood using PhyML wPip genome as a reference using mummer. SNPs were called where the mapped sequences differed from the reference sequence. The contig and scaffold SNPs were mostly at contig boundaries and phage regions, where mapping was difficult. Only SNPs which were agreed upon by both the contig and scaffold mapping were considered strongly supported.Contigs and scaffolds were mapped against the C. pipiens group lines used for these PCR experiments were as previously described For PCR analysis, genomic DNA was extracted from whole mosquitoes as above. Primers were designed to flank SNP regions and amplified using standard PCR conditions . PCR products were purified using a Qiagen PCR purification kit and sequenced using GATC Biotech sequencing. The wtrM was PCR amplified from the strain of Wolbachia found in Culex molestus. DNA was extracted as above. PCR was conducted with Phusion Taq polymerase (Finnzymes) according to manufacturer's instructions. PCR products were digested and ligated into the NotI-SacI site of insect cell expression vector, pIEX8 (Novagen). Plasmids were purified using endotoxin free maxi preps (Qiagen).The transcriptional regulator C. quinquefasciatus Pel line mosquitoes less than 24 hours post eclosion, infected with Wolbachia wPipPel were intrathoracically injected with 200 ng plasmid DNA and Cellfectin II (Invitrogen) transfection reagent according to a previously published protocol wtrM or a control plasmid with no insert and left to recover. Adults were isolated for total RNA extraction in groups of 2 to 4 individuals: 10 replicates of 3–4 individuals (n = 32) in pIEX and 10 replicates of 2–3 individuals (n = 27) for pIEX wtrM. cDNA synthesis was performed in 10 µl reaction volumes with 1000 ng of total RNA using the iScript cDNA synthesis kit (BioRad). qRT-PCRs were performed on 1∶20 dilutions of the cDNA using DyNAmo Colorflash (Fisher), a DNA Engine thermocycler (MJ Research) with a Chromo4 real-time PCR detection system (Bio-Rad) and the cycling conditions as above. Transcription levels were calculated by the standard curve method relative to the endogenous Culex reference gene RpL32.Adult Figure S1Comparative analysis between CPIJ005623 and CPIJ015950. A. Schematic representation of the gene architecture of CPIJ005623 and CPIJ015950. The two predicted genes are virtually the same with the exception of 1 nucleotide (nt) change leading to a P-S substitution at the N-terminus of the predicted proteins, 1 nt deletion near the exon1-exon2 border of CPIJ015950 leading to two amino acid substitutions and the 56 nt deletion leading to the loss of a Zinc-finger C2H2 type domain signature (PS00028). B. Analysis of 56 nt indel region of CPIJ015950. Specific primers flanking the 56 nt deletion region of CPIJ015950 that amplify an 84 basepair (bp) and a 141 bp fragment of CPIJ015950 in predicted cDNA and gDNA, respectively and a 141 bp fragment of CPIJ005623 in predicted gDNA or cDNA were used in electrophoresis analysis following PCR. Only a fragment around 140 bp, consistent with the presence of only CPIJ005623, was amplified in these two C. pipiens group lines. RpS7 PCR with specific primers spanning an intron were run in parallel to check the quality of gDNA, cDNA and PCR reaction; No T = no template control.(TIF)Click here for additional data file.Table S1C. pipiens group lines (at least 800 embryos counted per cross); other crossing data between these lines are listed in Crossing relationships between a selection of (DOC)Click here for additional data file.Table S2Wolbachia wPip Mol and Pel genomes are compared. The nucleotide sequence of each SNP is shown for Pel and Mol (column 5) and the amino acid that corresponds to that sequence is also shown (column 6). The nucleotide sequence of the SNPs in JHB is shown where the sequence is the same as either Pel or Mol.Genes containing at least one non- synonymous SNP when the (DOC)Click here for additional data file.Table S3SNPs in Italy and Thai lines compared to Pel and Mol. Sequence data obtained for SNPs that showed the same nucleotide in JHB as for Pel were assessed in the Italy and Thai lines which have the same crossing types where tested as Mol and Pel/JHB respectively (table S1). The nucleotide at each SNP position is shown and whether it corresponds to the SNP sequence for Pel or Mol.(DOC)Click here for additional data file.Table S4wPipPel and wPipMol genomes in a sample of C. pipiens group lines.PCR tests for presence (+) or absence (−) of inserted or deleted region genes that vary between the (DOC)Click here for additional data file.Table S5List of oligonucleotides used.(DOC)Click here for additional data file.


Interestingly, the phenotype of the ICAD−/− mice was not associated with a significant increase in the precancerous aberrant crypt foci suggesting a potential link to tumor progression rather than initiation. More importantly, ICAD deficiency was associated with severe genomic instability as assessed by array comparative genomic hybridization. Such genomic instability consisted most prominently of amplifications but with sizable deletions as compared to the wild-type counterparts affecting several cancer-related genes including RAF-1, GSN, LMO3, and Fzd6 independently of p53. Altogether, our results present a viable case for the involvement of ICAD deficiency in colon carcinogenesis and show that apoptosis and genomic instability may comprise the means by which such deficiency may contribute to the process of increasing susceptibility to carcinogen-induced tumorigenesis.We previously showed that DNA fragmentation factor, which comprises a caspase-3-activated DNase (CAD) and its inhibitor (ICAD), may influence the rate of cell death by generating PARP-1-activating DNA breaks. Here we tested the hypothesis that ICAD-deficient colon epithelial cells exhibiting resistance to death stimuli may accumulate additional genetic modifications, leading to a tumorigenic phenotype. We show that ICAD deficiency may be associated with colon malignancy in humans. Indeed, an examination of ICAD expression using immunohistochemistry in an array of both colon cancer and normal tissues revealed that ICAD expression levels were severely compromised in the cancerous tissues. Upon DNA damage caused by a low dose of irradiation, ICAD cells acquire a tumorigenic phenotype. Colon epithelial cells derived from ICAD mice showed a significant resistance to death induced by the colon carcinogen dimethylhydrazine in vitro and in mice. Such resistance was associated with a decrease in PARP-1 activation. In an animal model of dimethylhydrazine-induced colon tumorigenesis, ICAD We also demonstrated the importance of DFF in the fragmentation of DNA into 50-kb pieces during apoptosis −/− fibroblasts to generate 50-kb DNA fragments in response to TNF-α protected the cells from excessive activation of PARP-1 and thereby prevented depletion of intracellular NAD Fragmentation of DNA is thought to be an important step in disposal of the genome of apoptotic cells. Concomitant with cleavage of nuclear lamin, DNA is first degraded into large fragments of 50 to 300 kb, which are then processed into internucleosomal repeats ICAD gene were recently associated with a number of human malignancies as well as with an animal model of skin cancer ICAD expression and mutations in the The current study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Maintenance (IACUC protocol: BC0015) and experimental protocol (IACUC protocol: 2227) were approved by the LSUHSC Animal Care and Use Committee. The colon cancer mid-density tissue array  and the 23 human specimens of colon cancer and normal tissues (LSUHSC pathology core) fall under IRB exemption type 4.−/− mice, both maintained on a C57BL/6×129/sv background, were bred in a specific-pathogen free facility at LSUHSC, New Orleans, LA, and allowed unlimited access to sterilized chow and water ad libitum and were maintained on a 12-hour light-12-hour dark schedule. At 5 weeks of age, mice  received an i.p. injection of 20 mg/kg DMH  in saline containing 1 mM EDTA once a week for eight weeks. The animals were then sacrificed by CO2 asphyxiation 12 or 24 weeks later. For acute exposures, mice received a single i.p. injection of 20 mg/kg of the drug and sacrificed either 24 or 48 h later. Colons were removed and fixed with formalin. Some mice were utilized to isolate primary CECs as described below.WT and ICAD2 for 24 h, after which floating crypts were transferred to a fresh plate in complete medium. The efficiency of cell isolation was evaluated by the degree of purity of the crypts and the low number of dead cells. The epithelial nature of the cells was assessed by immunofluorescence with antibodies to pan-cytokeratin  as described previously −/− mouse fibroblasts  with constant gentle agitation. The digest was washed with HBSS by centrifugation and then resuspended in complete medium without enzymes. Isolated colon crypts were then plated and incubated at 37°C in 5% CO−/− cells failed, in general, to display obvious positive signals upon labeling. The colon cancer mid-density tissue array (103 cases/208 cores) was purchased from US Biomax Inc. . Twenty three specimens of colon cancer and adjacent normal tissues were acquired from the LSUHSC pathology core. Tissue sections were subjected to IHC with primary antibodies to ICAD , poly(ADP-ribose) , PCNA , MCP-1 , COX-2  as described previously Serial sections from paraffin-embedded colonic tissues were prepared using standard methods. Every fifth section was subjected to H&E staining for routine histological evaluation. Pycnotic/apoptotic cells were determined by nuclear condensation, cellular fragmentation, shrinkage, and/or detachment from the mucosa, aided with TUNEL assay . The TUNEL assay was not used as a primary means to determine pycnosis/apoptosis given that ICADK. Fallon). ACF with 2 or more glands were included in the assessment. Diameter of tumors was assessed using a stereomicroscope.ACF were identified in H&E-stained serial sections on the basis of the presence of atypical nuclei, size, increased pericryptal area, and thickened layer of epithelial cells aided by the assistance of a pathologist . The reference controls were DNA isolated from colon tissue of age-matched naïve WT or ICAD−/− mice. The reference and test DNAs were digested with Alu I and Rsa I , and purified with the QIAprep Spin Miniprep kit . Test DNA (1 µg) and reference DNA (1 µg) were labeled by random priming with Cy5-dUTP and Cy3-dUTP, respectively, using the Agilent Genomic DNA Labeling Kit Plus. The individually labeled test and reference samples were concentrated using Microcon YM-30 filters  and then combined. Following probe denaturation and pre-annealing with mouse Cot-1 DNA, hybridization was performed at 65°C with rotation for 40 h at 20 rpm. Four steps were done with Agilent Oligo CGH washes: Wash buffer 1 at room temperature for 5 min, wash buffer 2 at 37°C for 1 min, an acetonitrile rinse at room temperature for 1 min and a 30 sec wash at room temperature in Agilent's Stabilization and Drying Solution. All slides were scanned on an Agilent DNA microarray scanner. Data including Copy Number Variations were obtained by Agilent Feature Extraction software 9 and analyzed with Agilent Genomic Workbench software 5.0, using the statistical algorithms z score and ADM-2 according to sensitivity threshold respectively at 2.5 and 6.0 and a moving average window of 0.2 Mb.The profile of chromosome aberrations in colonic tissue (isolated from paraffin-embedded tissues) from DMH-treated WT or ICADAll data are expressed as means ± SD of values from at least six mice per group or from 4 to 6 replicates of the same treatment of the cultured cells. PRISM software  was used to analyze the differences between experimental groups by one-way ANOVA followed by the Dunnett's multiple comparison test. The analysis of the aCGH data was described above.We initiated our studies by conducting a direct examination of ICAD expression using immunohistochemistry in an array of both colon cancer and normal tissues. The expression of ICAD appeared to be more prominent in epithelial cells of normal colonic mucosa with a primary nuclear subcellular localization . ICAD ex−/− fibroblasts promoted their transformation after induction of DNA damage, we examined the ability of these cells to form colonies in soft agar. Wild-type (WT) and ICAD−/− cells were first exposed to a low-dose (2 Gy) IR, after which they were suspended in agar with complete medium and colonies were counted after incubation for 3 weeks. We previously showed that ICAD deficiency reduces sensitivity to (TNF-α)-induced cell death in vitro and in vivo. To begin testing our hypothesis, we examined the effect of ICAD gene knockout on the response of primary colon epithelial cells (CECs) to inducers of cell death, for instance, TNF-α, cycloheximide, and butyric acid (TNF+CHX+BA) or to the colon carcinogen DMH. We have previously shown that the TNF+CHX+BA combination is effective in promoting cell killing in CECs in vivo upon an acute exposure. Mice were treated with a single dose (20 mg/kg) of DMH for 24 or 48 h. −/− CECs to DMH-induced death was associated with a decrease in PARP activation. −/− mice. A quantification of these results is depicted in Given the potential tumorigenic effect of ICAD deficiency upon DNA damage, we examined this possibility in the colon setting −/− mice were injected intraperitoneally once a week for 8 weeks with 20 mg/kg DMH or vehicle . Mice were then sacrificed 12 or 24 weeks after the last injection. −/− mice developed tumors, confirming the tumorigenic potential observed in vitro. Whereas the average tumor per mouse in WT mice was ∼0.5, the average of tumors in ICAD−/− mice was ∼3.5 about 7-fold higher, suggesting that ICAD deficiency not only increased the susceptibility of mice to DMH but also increased tumor multiplicity. The tumors detected in DMH-treated ICAD−/− mice varied in size but reached sizes as large as 1 cm as shown in To further test our abovementioned hypothesis, we examined the effect of ICAD gene deletion in a model of colon tumorigenesis. WT and ICAD−/− mice trended higher than those detected in the WT counterpart, the difference did not reach statistical significance  analyses of tumor-containing colon tissues of ICAD−/− mice and compared them with the WT counterparts. −/− mice were extensive and consisted chiefly of amplifications. CAD deficiency was recently associated with genomic instability in skin carcinogenesis and in irradiated cell lines p53 remained intact, a number of genes whose products are known to alter its function or associated signaling were modified. For instance, LMO3, whose gene was amplified more than 5-fold, was shown to interact with p53 and inhibit its transcriptional activity GSN), whose gene was amplified more than 3-fold in modifications in colonic tissues of DMH-treated ICAD−/− mice, was very recently shown to bind p53, leading to inhibition of p53-induced apoptosis by anchoring of p53 in the cytoplasm RAS gene was not altered in DMH-treated ICAD−/− mice, RAF-1, a critical kinase that influences RAS function with an important function in colon carcinogenesis Fzd6 have been recently suggested to constitute a risk for papillary thyroid cancer with a potential involvement in maintenance of genomic integrity −/− mice to DMH-induced colon tumorigenesis.Analysis of the sequences that were modified in both experimental groups shows a substantial amplification of genes necessary for cell growth or that have been associated with colon carcinogenesis. fibroblast growth factor gene (fgf)7, whose product is a member of the FGF family of proteins that signal through FGF receptors fgf7 gene remained unaltered in colonic tissues of DMH-treated ICAD−/− mice. These distinct and numerous differences between the susceptibility of WT and ICAD−/− mice to genomic instability may explain the different susceptibility of the two strains to colon tumorigenesis.Although the genes of critical relevance to colon carcinogenesis were not affected by DMH treatment in WT mice, the The development of resistance to apoptosis remains an important mechanism for the initiation of tumorigenesis. This resistance allows some cells to acquire new genetic modifications, which permit them to become more proliferative than normal cells. These cells will then have a growth advantage leading them to contribute to tumor progression. In this study, we have established an association between ICAD deficiency and colon malignancy in specimens from patients with colon cancer. We demonstrated, in the present work as well as in others −/− mice. Konishi et al. ICAD gene was mapped to chromosome region 1p36.2, which is a region alleged to involve one or several tumor suppressor genes in neuroblastoma tumors. This group showed that three different coding alterations were found in the region encoding the ICAD N-terminal regulatory domain ICAD expression and mutations were recently linked to some human cancers and to an animal model of skin cancer in vitro to some apoptotic stimuli −/− CECs exhibited a higher resistance to DMH than to the TNF+CHX+BA combination. Additionally, ICAD−/− fibroblasts show a substantial resistance to a combination of TNF and cycloheximide as shown in our previous reports −/− cortical neurons exhibited a significant resistance to staurosporine, whereas these cells showed little or no resistance to etoposide −/− mice upon repeated treatment with DMH compared with the WT counterparts; however, it was statistically insignificant. Although these results may suggest that resistance of ICAD−/− CECs to DMH-induced death may not contribute to ACF formation and initiation of tumorigenesis, it is highly likely that this resistance plays an important role in progression of ACF to tumors. With the added role of DFF in genomic stability, ICAD deficiency appears to affect the number and size of tumors in DMH-treated mice. By decreasing susceptibility to apoptosis, CECs may accumulate additional mutations leading to clonal selection Although the resistance to cell death conferred by ICAD deficiency remains unresolved because few reports have indicated that such deficiency caused no significant changes in sensitivity of cells in vivo compensatory mechanisms that take place in response to gene deletion may modify tissue responses to carcinogens such as DMH. An ideal approach would be to examine the relationship between ICAD deficiency and colon cancer in mice with a colon-specific ICAD gene deletion.Although it is increasingly evident that DFF plays a role in genomic stability, the mechanism by which the apoptotic endonuclease complex participates in such a vital and continuous process is largely unclear. A more puzzling issue is the fact that ICAD deficiency was associated with a large number of amplifications upon DMH treatment. It is tempting to speculate that the endonuclease activity intrinsic in CAD may participate in preventing amplification of genomic DNA. However, it is widely established that the function of CAD requires caspase activity and inactivation of ICAD RAS, p53, APC, or β-catenin genes. However, several genes that can affect the aforementioned factors have been altered, namely RAF-1, GSN, LMO3, Fzd6, and several others. The alteration in the latter genes may constitute the driving forces for increased susceptibility of the ICAD−/− mice to tumorigenesis in response to DMH treatment. Although the confidence in the selected modifications is high given the very low p-values associated with these changes, their validation by immunohistochemistry may be necessary particularly with respect to amplifications because the levels of some gene products can be regulated at the translation level or post-translationally. Additionally, an intact p53 gene does not necessarily indicate an intact protein with a normal function given the possibility that ICAD deficiency could alter, directly or indirectly, the fate, phosphorylation, or overall function of the tumor suppressor. This is also true to the other genes including RAS, APC, and β-catenin. The mild genomic instability induced by DMH in wild type mice is not surprising as similar results were reported by Rosenberg's group in which the A/J mice, a strain with a normal level of wild type ICAD (data not shown), exhibited a low level of genomic instability upon exposure to azoxymethane, the byproduct of DMH Our aCGH analysis revealed that a number of genes that have high relevance to overall carcinogenesis and colon cancer have been modified. It is interesting that all these modifications occurred in the absence of any detectable effects on


Purpose. The aim of this study was to assess the effect of water and saliva contamination on the shear bond strength and failure site of orthodontic brackets and lingual buttons. Materials and Methods. 120 bovine permanent mandibular incisors were randomly divided into 6 groups of 20 specimens each. Both orthodontic brackets and disinclusion buttons were tested under three different enamel surface conditions: (a) dry, (b) water contamination, and (c) saliva contamination. Brackets and buttons were bonded to the teeth and subsequently tested using a Instron universal testing machine. Shear bond strength values and adhesive failure rate were recorded. Statistical analysis was performed using ANOVA and Tukey tests  and Chi squared test (ARI Scores). Results. Noncontaminated enamel surfaces showed the highest bond strengths for both brackets and buttons. Under water and saliva contamination orthodontic brackets groups showed significantly lower shear strengths than disinclusion buttons groups. Significant differences in debond locations were found among the groups under the various enamel surface conditions. Conclusions. Water and saliva contamination of enamel during the bonding procedure lowers bond strength values, more with orthodontic brackets than with disinclusion buttons. The procedure of bonding orthodontic brackets to enamel requires completely dry and isolated fields to obtain clinically acceptable bond strengths, because of hydrophobic properties of bonding materials . Many clPrevious studies that evaluated the effect of water and saliva contamination on the bond strengths of brackets bonded with light-cured composites showed a significant reduction in bond strength values , 6–9. InAccordingly, the aim of the present investigation was to measure and compare shear bond strength and adhesive remnant index (ARI) score of a conventional orthodontic bracket and a disinclusion button bonded onto dry, water- and saliva-contaminated enamel. The null hypothesis of the study was that there is no significant difference in shear bond strength values and debond locations among the various groups.One hundred and twenty freshly permanent extracted bovine mandibular incisors were collected from a local slaughter house and stored in a solution of 0.1% (wt/vol) thymol. The criteria for tooth selection included intact buccal enamel with no cracks caused by extraction and no caries. The teeth were cleansed of soft tissue and embedded in cold-curing, fast-setting acrylic . Metal rings (15-mm diameter) were filled with the acrylic resin and allowed to cure, thus encasing each specimen while allowing the buccal surface of enamel to be exposed. Each tooth was oriented so that its labial surface was parallel to the shearing force. Teeth were randomly divided in six groups of 20 specimens.Orthodontic stainless steel maxillary central incisor brackets  and stainless steel orthodontic buttons  were used. Both brackets and buttons were tested under 3 different enamel surface conditions: (1) dry, (2) water contamination, (3) saliva contamination.The labial surface of each incisor was cleaned for 10 seconds with a mixture of water and fluoride-free pumice in a rubber polishing cup with a low-speed handpiece. The enamel surface was rinsed with water to remove pumice or debris and then dried with an oil-free air stream. Before bonding, 4 scanning electron microscope photographs were taken using a scanning electron microscope  to observe differences between orthodontic bracket and disinclusion button bases. For each appliance, microphotographs were taken in the most prominent and in the deepest parts of the bracket base (magnification 2500x) .Bonding procedures are described in Brackets were then light-cured with a visible light-curing unit  for 10 seconds on the mesial side of the bracket and for 10 seconds on the distal side . After bonding, all samples were stored in distilled water at room temperature for 24 hours and then tested in a shear mode on a universal testing machine . Specimens were secured in the lower jaw of the machine so that the bonded bracket base was parallel to the shear force direction.Specimens were stressed in an occlusogingival direction at a crosshead speed of 1 mm per minute, as in previous studies –13. The This scale ranges from 0 to 3. A score of 0 indicates no adhesive remaining on the tooth in the bonding area; 1 indicates less than half of the adhesive remaining on the tooth; 2 indicates more than half of the adhesive remaining on the tooth; 3 indicates all adhesive remaining on the tooth. The ARI scores were used as a more complex method of defining bond failure site among the enamel, the adhesive, and the bracket base.Statistical analysis was performed with Stata 9.0 software . Descriptive statistics, including the mean, standard deviation, median, minimum and maximum values were calculated for all groups. post hoc. The chi-square test was performed to determine significant differences in the ARI scores among the different groups. Significance for all statistical tests was predetermined at P < 0.05.An analysis of variance (ANOVA) test was applied to determine whether significant differences in debond values existed among the groups. The Tukey test was used as P < 0.05). Post hoc test underlined that groups 1 and 4 (brackets and buttons onto dry enamel) had the highest shear bond strength (P < 0.001) and exhibited no significant difference between them (P > 0.05). Groups 5 and 6 (disinclusion button onto contaminated enamel) had lower bond strengths (P < 0.001) and showed no significant difference between them (P > 0.05). Groups 2 and 3 (orthodontic bracket onto contaminated enamel) presented the lowest bond strengths and no significant difference among them (P > 0.05).Descriptive statistics for the shear bond strength (MPa) of the different brackets are illustrated in P < 0.05) that showed no significant difference between them (P > 0.05). Groups 2 and 3 presented higher frequency of ARI score of “0” and no significant difference was found between them (P > 0.05). Groups 2 and 3 exhibited higher frequency of ARI score of “1” and no significant difference was found between them (P > 0.05).The results of ARI Scores are illustrated in The null hypothesis of the study has been rejected. In the present investigation conventional brackets and disinclusion buttons bonded onto dry enamel had significantly higher shear bond strength values than other groups and exhibited no significant differences between them. The similar strength values of the two appliances in dry state is probably due to the similar mesh pad design of the devices tested . PreviouUnder water- and saliva-contaminated conditions both orthodontic brackets and disinclusion buttons showed significantly lower shear bond strength values. In fact the properties of an adhesive resin can be diminished by various intraoral factors that include high humidity in the oral cavity , 17, agiIn the present investigation water and saliva contamination of enamel has been investigated. In fact, enamel can be contaminated with water during bonding procedure after conditioning followed by insufficient drying. On the other hand saliva is always present in the patient's oral environment, and it contains antioxidants, immunoglobulins, antibacterial enzymes, growth factors, and mucous secretion to protect epithelial cells from mechanical and chemical challenges .When the etched enamel becomes wet, most of the porosities become plugged, and the penetration of the resin is impaired, which results in resin tags of insufficient number and length . ContamiAs blood , 13, 20,A minimum bond strength of 6 to 8 MPa is reported to be adequate for most clinical orthodontic needs . These vIn the present investigation permanent bovine lower incisors were used. Bovine deciduous and permanent enamel has been reported to be a reliable substitute for human enamel , 27 in bMoreover in the present investigation ARI scores have been recorded. Uncontaminated enamel groups reported a higher frequency of ARI score of “2.” Under contamination, conventional brackets exhibited higher frequency of ARI score of “0,” whereas disinclusion buttons showed higher frequency of ARI score of “1.” This is in agreement with previous investigations that evaluated ARI scores of orthodontic brackets bonded onto water- and saliva-contaminated enamel that all showed lower scores for moistened groups , 9, 24. As a high number of orthodontic products is present on the market, future further studies are needed to test also other orthodontic devices bonded onto dry and contaminated enamel.Noncontaminated enamel surfaces showed the highest bond strengths for both brackets and buttons. Under water and saliva contamination all groups showed significantly lower shear bond strength values.Under contamination orthodontic brackets groups exhibited significantly lower shear strengths than disinclusion buttons groups. Noncontaminated groups showed higher frequency of ARI score of “2,” both for orthodontic brackets and disinclusion buttons. For both water- and saliva- contaminated groups, orthodontic brackets reported higher frequency of ARI score of “0,” whereas disinclusion buttons displayed higher frequency of ARI score of “1.”This study demonstrated the following.


Sediment grain-sizes affected shorebird community spatial patterns indirectly by influencing benthic macroinvertebrate community compositions. Furthermore, combining sediment and macroinvertebrate information produced a 27% increase in correlation (ρs = 0.71) with shorebird assemblage patterns over the correlation of the bird community with the macroinvertebrate community alone. Beyond its indirect effects acting through prey distributions, granulometry of the flats influenced shorebird foraging directly by modifying prey availability. Our study highlights the importance of habitat heterogeneity, showing that no single patch type was ideal for the entire shorebird community. Generally, shorebird density and diversity were greatest at lower elevations on flats when they became exposed; these areas are at risk from human intervention by inlet sand mining, construction of groins and jetties that divert sediments from flats, and installation of seawalls on inlet shorelines that induce erosion of flats.Worldwide declines in shorebird populations, driven largely by habitat loss and degradation, motivate environmental managers to preserve and restore the critical coastal habitats on which these birds depend. Effective habitat management requires an understanding of the factors that determine habitat use and value to shorebirds, extending from individuals to the entire community. While investigating the factors that influenced shorebird foraging distributions among neighboring intertidal sand flats, we built upon species-level understandings of individual-based, small-scale foraging decisions to develop more comprehensive guild- and community-level insights. We found that densities and community composition of foraging shorebirds varied substantially among elevations within some tidal flats and among five flats despite their proximity . Non-dimensional multivariate analyses revealed that the changing composition of the shorebird community among flats and tidal elevations correlated significantly (ρ Worldwide declines in shorebird populations, driven by coastal development and increasingly by direct and indirect effects of sea-level rise The Ideal Free Distribution (IFD) optimal foraging model Here, we ask whether the distribution of a community of foraging shorebirds conforms with expectations from the IFD; that is, whether the density distribution of foraging guilds matches the distributions of prey and presumed influential environmental variables. We build upon individual- and species-level understandings of foraging decisions in order to reveal more comprehensive guild- and community-level insights. Because the value of a habitat to foraging shorebirds can depend upon both local characteristics of the habitat patch and also the landscape-scale context in which that patch is located Marine Corps Base Camp Lejeune is located on the North Carolina coast midway between Cape Lookout and Cape Fear. Onslow Beach, Camp Lejeune’s 12 km-long, southeast-facing barrier island, borders the Atlantic Ocean and is bounded on the southwest by the New River Inlet . Our stuFlats ranged in area from about 0.5 to 2 ha at spring low tide, and were within 5–250 m of each other . “Semi-EGPS location and elevation surveys were conducted using a Trimble Real Time Kinematic unit on 12 November 2008 and supplemented by additional measurements on 9 February 2009. Survey points were recorded at 0.5 m intervals along transects spaced approximately 10 m apart, perpendicular to the low-tide water line of each flat. In total, 4388 points were imported into ArcMap and inverse-distance weighted (IDW) to interpolate elevations for all exposed sand flat surfaces. IDW data were used to calculate surface area exposed for each flat at successive tidal heights .Benthic macroinvertebrates were sampled on 12 November 2008, approximately at the temporal mid-point of our shorebird censusing. We assumed benthic invertebrate densities did not exhibit dramatic variation from mid-October to mid-December, a period without major recruitment pulses e.g., , major s2 in surface area and 10 cm deep, was taken at each of the three microhabitat levels at each tidal stage on Semi-Enclosed Flat and at Broad Flat. Additionally, a full set of 3 replicate samples from each microhabitat was taken at Shell Flat; because this flat was much smaller than the others, fewer replicates were taken here in order to maintain comparable spacing between samples. Tidal Creek Flat was sampled at late ebb and low tides with 8 replicate samples per microhabitat level per tidal stage. A set of 7 replicate samples per level were obtained from Island Flat at low tide. The 10-cm depth was selected to capture benthic organisms that would be within probing range of the whimbrel (Numenius phaeopus), and to capture vertically-moving organisms that would periodically be available to surface-feeding shorebirds. Our sampling protocol was inclusive of all species of macroinvertebrates found in this depth range in order to match our focus on the entire community of shorebirds, which includes multiple feeding guilds. This depth was sufficient to capture most if not all organisms that serve as shorebird prey on intertidal sand flats of North Carolina inlets. The only deep-burrowing vermiform invertebrates in this biogeographic area  were rare, judging from virtual absence of fecal casting evidence on the surface . The only local deep-burrowing bivalve, the adult razor clam, Tagelus plebeius, can be recognized by its evident dual siphon holes at the surface Mercenaria mercenaria and Chione cancellata) are all suspension feeders so, unlike some deposit feeders, their siphons are not extended and thus vulnerable when the intertidal habitat is exposed at low tide.At each tidal stage, up to three microhabitats were distinguishable on each tidal flat, based on relative elevation and apparent water content: “saturated” , “glossy” (a film of surface water visible on the sediment), and “subtidal” (<3 cm of water cover). As sampling at later tidal stages focused on newly exposed areas, a “saturated” zone at late ebb tide was lower in elevation than a “subtidal” sample taken at mid ebb tide . BecauseThe 199 samples were returned to the laboratory on ice and sieved immediately on 0.5-mm mesh, with contents preserved using 10% buffered formalin with Rose Bengal stain. After sorting and enumerating by species, genus, or occasionally a higher taxonomic level, organisms were placed in 35% ethanol for ≤60 d until they could be dried at 60°C to constant mass (∼48 hours) and weighed. For each sample, dry mass was computed for the four major taxonomic groups: polychaetes; crustaceans ; bivalves; and gastropods.P-values but rely on them less than on F ratios, R statistics, and t statistics to assess potential biological patterns and provide evidence for rejection of null hypotheses.One-way Model I ANOVAs were used to test each major group for differences in abundance, and separately in biomass, among flats at each tidal level. Both response variables were log (x+1)-transformed to meet statistical assumptions of normality and homogeneity of variance. To determine whether benthic community composition differed among flats, we performed one-way ANOSIMs , and percent composition was computed for each sample. Grain-size group means were compared (1) among flats and (2) among tidal elevations within flats using one-way Model I ANOVAs.To test for among-flat differences in grain-size distribution, we performed a one-way ANOSIM based on a Euclidean distance resemblance matrix with flat as the independent variable and grain-size distributions from individual samples as replicates. To remove collinearity, the size classes “coarse/very coarse sand” and “gravel” were excluded from the analysis because they were highly negatively correlated with “fine sand” (−0.898). We further constructed a similarity matrix from mean grain-size distributions for each flat/tidal stage combination and used it as the basis for an n-MDS ordination (Euclidean distance) and hierarchical cluster analysis.−1(range 0–32 km hr−1) and air temperatures between 8 and 20°C (mean 14°C). During the first week of observations, several wooden stakes were inserted in Broad Flat in order to facilitate tidal height comparisons across dates. Shorebird surveys were conducted by walking the length of the sand spit along the vegetation line  ordination using a Bray-Curtis similarity matrix was constructed from the means of each flat/tidal stage combination along with a hierarchical clustering (PRIMER v.6.1.11) of these means.2 to contrast foraging guild distributions, and computed Simpson’s D to compare shorebird community diversity among tidal stages.Significant multivariate PERMANOVAs were followed by univariate ANOVAs and Tukey-Kramer HSD post hoc contrasts to compare foraging densities at each tidal stage. Again, Shell Flat was excluded. In addition, we used χThe relationship between sediment grain-size distribution and benthic community structure was assessed using the BEST procedure in PRIMER6 The BEST procedure was also used to assess the spatiotemporal relationship between tidal flat sediment grain-size composition and the shorebird community. To match sediments with shorebird samples, the process of compositing sediment samples used in the sediment-benthos BEST analysis described above was repeated until a one-to-one sample correspondence was reached, providing each shorebird community sample with a matching sediment profile for a given flat at a given tidal stage. Shorebird observations from December were excluded because spring tides during that half month of sampling changed the distributions of birds on the flats relative to sediment sample locations, resulting in a poor spatial match. The composite shorebird density data were log(x+1)-transformed, and their Bray-Curtis similarity matrix served as the fixed matrix for the BEST analysis.S = 0.94) confirmed that benthic community patterns were preserved within the subset of most frequent species. We performed a BEST analysis using the fixed shorebird Bray-Curtis similarity matrix and the active benthic Bray-Curtis similarity matrix from the reduced species set. As with the sediment dataset in the previous analysis, benthic samples were composited until a one-to-one sample correspondence was reached between benthic and shorebird samples. In this way, each shorebird community sample was matched with the benthic community composition of a given flat at a given tidal stage.The relationship between benthic macrofaunal and shorebird communities was assessed using the BEST procedure as well. This analysis was performed using the shorebird density and benthic abundance datasets, with the similarity matrix from the shorebird dataset serving as the fixed matrix. To reduce numbers of benthic species (67), we included only those found in five percent or more of the total samples . To ensuA final BEST analysis drew upon all three datasets. The composited sediment and benthic macrofauna datasets were combined on a single spreadsheet to form an active matrix that supplied explanatory variables from both datasets at the same time. Once again, the Bray-Curtis similarity matrix from the shorebird dataset served as the fixed matrix for the BEST analysis.Marine Corps Base Camp Lejeune (MCBCL) Environmental Management Division approved this research, and MCBCL Range Control granted us access to the study site.Nereis spp. (20%), Capitella capitata (17%), Haploscoloplos robustus (16%), Heteromastus filiformis (14%), Paraonis sp. (8%), or Aricidea fragilis (5%). Crustaceans consisted mostly of amphipods (94%), but also contained a few other small crustaceans including decapods , and isopods. Bivalves were mainly Donax variabilis, Gemma gemma, or Mercenaria mercenaria, and the most abundant gastropods were Nassarius obsoletus and Littorina irrorata.Almost all (98%) invertebrates sampled belonged to one of four major taxonomic groups: polychaetes (53%), crustaceans (34%), bivalves (6%), or gastropods (5%) . Individual pairwise comparisons of flats  revealed2,18 = 8.60, P<0.01) and a trend of increasing very fine sand. Shell Flat showed a marked coarsening of sediments from mid ebb to late ebb and low tide with increases in coarse/very coarse sand  and decreases from mid to late ebb and low tidal levels in fine sand .Each separate sediment grain size class revealed highly significant differences in percentage composition among tidal flats . The flat most clearly distinguishable from all others was Shell Flat, which had the coarsest sediments by far . Tidal CResults of analysis of similarity conducted on complete grain-size distributions of each tidal flat revealed a significant difference among the flats . Subsequent n-MDS ordination of sediment grain size distributions by flat and tidal stage demonstrated that Shell Flat’s late ebb tide and low tide sediment composition differed considerably from all other flats and tidal stages , while tShorebird community composition differed among the flats at each tidal stage, but exhibited no significant difference across the sampling dates . All pos2 (8) = 118.5; Tidal Creek: χ2 (8) = 75.4; Island: χ2 (8) = 61.5; Shell χ2 (8) = 27.4; P<0.001 for all). Unlike Broad Flat, Semi-Enclosed Flat had a large proportion (78%) of visual foragers, while Tidal Creek Flat was used almost exclusively (91%) by tactile foragers. Though Island Flat was not dominated by a particular foraging guild, it had a higher percentage of mixed foragers (35%) than the other flats. Shell Flat was rarely used by shorebirds, but the few birds that did use it were all visual foragers (3 species).Similarly, an examination of flat use by shorebird foraging guild showed clear differences among flats . When obUnivariate analyses of average shorebird density revealed many significant differences among flats . Semi-EnCalidris alpina), which was most abundant at that tidal stage  and black-bellied plover , tended to increase in abundance from mid ebb to low tide  was most abundant at mid ebb tide, and rarely foraged on the flats at later tidal stages (Arenaria interpres), piping plovers (Charadrius melodus), willets , and yellowlegs (Tringa spp.) also contributed to observed species diversity. Though diversity decreased at late ebb tide with the influx of the numerically dominant dunlin , their low tide decline, combined with the increase in abundance of other common species  of only 0.119 with “silt/clay” and “very fine sand” size classes. However, the BEST analysis between sediments and composite benthic samples  yielded a much higher optimized correlation . The BEST analysis between composited sediment size distributions for each tidal elevation of each flat and the corresponding shorebird community composition based on average density at each tidal elevation of every flat had an optimized correlation of ρs = 0.575 (p<0.01) with 2 variables: “medium sand” and “fine sand” . The BEST analysis comparing the composited samples of the 14-species subset of most frequent benthic invertebrates at each tidal elevation of every flat to the shorebird community composition based on abundances at each level of each flat optimized with 1 variable (crustaceans) at ρs = 0.561 (p<0.01). Finally, when all three composited datasets were used concurrently , the BEST analysis optimized at ρs = 0.711 (p<0.01) with 4 variables ).A BEST analysis between sediment size classes and benthic macrofaunal community composition in the glossy zone  produced a Spearman correlation coefficient , explaining a large portion of the pattern in shorebird foraging habitat use. Adding information on sediment grain-size distributions to the benthic invertebrate community dataset and forming a composite of predictor variables revealed that inclusion of grain size information further improved by about 27% the correlation with the distribution pattern of feeding shorebirds (ρs = 0.71). One might have expected the granulometry of the flats to have had its influence on where shorebirds feed indirectly via the strong impacts of grain size composition on the benthic invertebrates themselves. Indeed, grain size and benthic macrofauna exhibited a correlation coefficient of 0.69, indicating that much of the spatial variance in benthic invertebrate community composition could be explained by local changes in sediment size composition. Yet, the further direct explanatory value of adding in sediment grain size information to benthic invertebrate composition in explaining shorebird habitat use suggests the operation of direct effects of sediment size beyond indirect effects operating through determination of prey types and abundances  in our analyses because their inclusion could compromise the interpretability of our results: any observed increase in variance explained by an included landscape variable might actually be driven by any number of additional unmeasured or co-varying variables.It is possible that bird behavior also contributed to the unexplained variability in shorebird spatiotemporal distributions although the specific mechanisms are not clear. Agonistic behaviors did not play a major role in structuring patterns of patch use as few negative interactions were observed, consistent with species-specific literature e.g., , which rPrey abundance and availability (as mediated by sediment characteristics) explain much of the variation in foraging patterns of the shorebird community, a conclusion mirroring ones arising previously from studies of habitat selection by single species. Our multivariate analyses confirmed this relationship for guilds representing different foraging modes and revealed that no single patch type is ideal for all guilds that comprise the entire shorebird community. Habitat heterogeneity  is necessary to support the full spectrum of shorebirds with different prey preferences and foraging modes. Consequently, inlets containing extensive areas of heterogeneous intertidal sand and mud flats are important to sustaining functional diversity of shorebird communities.Results of our study have important implications for shorebird conservation and coastal management. The sediments that form intertidal sand flats near inlets are part of a dynamic ocean beach and inlet sand-sharing system with a finite sand resource. When humans intervene in this sand-sharing system by erecting groins and jetties intended to capture and retain sand in front of a specific shoreline property to protect against erosion, such interventions disrupt the natural flow of sand and commonly result in downdrift sand deficits Table S1Total intertidal area (hectares) of each tidal flat at the three tidal stages of observation, as a function of tidal amplitude.(DOCX)Click here for additional data file.Table S2Benthic macrofaunal species found in at least five percent of samples.(DOCX)Click here for additional data file.Table S3−2) by flat.Phylum Polychaeta densities (organisms m(DOCX)Click here for additional data file.Table S4−2) by flat.Phylum Arthropoda densities (organisms m(DOCX)Click here for additional data file.Table S5−2) by flat.Phylum Mollusca densities (organisms m(DOCX)Click here for additional data file.Table S6−2) of organisms from additional phyla.Flat-specific densities (organisms m(DOCX)Click here for additional data file.Table S7Results of one-way ANOVAs comparing benthic invertebrate abundance among tidal flats at each tidal stage, and post hoc contrasts using Tukey-Kramer HSD.(DOCX)Click here for additional data file.Table S8Results of one-way ANOVAs comparing benthic invertebrate biomass among tidal flats at each tidal stage, and post hoc contrasts using Tukey-Kramer HSD.(DOCX)Click here for additional data file.Table S9Results of post hoc pairwise comparisions of flats following Analysis of Similarity (ANOSIM) on benthic invertebrate community composition dataset of abundances by species .(DOCX)Click here for additional data file.Table S10Results of Tukey-Kramer HSD post hoc comparisons of all possible pairs of flats based on percentage composition of each size class of sediments.(DOCX)Click here for additional data file.


Despite the continually increasing rates of adverse perinatal outcomes across the globe, the molecular mechanisms that underlie adverse perinatal outcomes are not completely understood. Clinical studies report that 10% of pregnant women will experience a urinary tract infection (UTI) and there is an association of UTIs with adverse perinatal outcomes. We introduced bacterial cystitis into successfully outbred female mice at gestational day 14 to follow pregnancy outcomes and immunological responses to determine the mechanisms that underlie UTI-mediated adverse outcomes. Outbred fetuses from mothers experiencing localized cystitis displayed intrauterine growth restriction (20–80%) as early as 48 hours post-infection and throughout the remainder of normal gestation. Robust infiltration of cellular innate immune effectors was observed in the uteroplacental tissue following introduction of UTI despite absence of viable bacteria. The magnitude of serum proinflammatory cytokines is elevated in the maternal serum during UTI. This study demonstrates that a localized infection can dramatically impact the immunological status as well as the function of non-infected distal organs and tissues. This model can be used as a platform to determine the mechanism(s) by which proinflammatory changes occur between non-contiguous genitourinary organs Adverse perinatal outcomes include both prematurity (birth prior to 37 weeks) and/or low birthweight (weight below 2500 grams). There is a discernible etiology in only 50% of adverse perinatal outcomes Pregnancy is a unique situation in which the fetus carries both maternal (“self”) and paternal (“non-self”) antigens. While the immune system normally functions to attack non-self stimuli, successful gestation requires the maternal immune response to “ignore” paternal antigens produced by the fetus . While mechanisms of tolerance between mother and fetus are not completely understood, it is clear that dendritic cells (DCs), T cells and natural killerd (NK) cells play crucial roles at the maternal-fetal interface and in maternal tissues to ensure that pregnancy proceeds successfully Maternal infections contribute to nearly 40% of all adverse perinatal outcomes UTIs affect almost 50% of all women (10% of pregnant women) and manifest in a variety of clinical presentations  Escherichia coli (UPEC) that has been extensively studied to determine the molecular details of UTI pathogenesis  for mating with inbred CH3/HeN males (MHC haplotype k) to produce outbred fetuses see . The genThe genitourinary tissues, fetus and blood from pregnant mice were cultured for viable UPEC to determine the tissue distribution of UPEC following introduction into the urinary tract. No bacteria were detected in any of the tissues analyzed from sham-treated mice. Viable bacteria were present throughout pregnancy and parturition in the bladders of mothers that received UPEC . The bac+, Gr-1+hi, Ly6C+, F4/80−) and macrophages    were ele F4/80+) . At 96 het al. suggested a functional role for phagocytes during and after the natural course of parturition. We observed a difference in the magnitude of PMN and macrophage infiltration between the uteroplacental tissue and the non-pregnant uterus (data not shown), suggesting that pregnancy presents a unique situation, which may promote proinflammatory immune responses in the presence of infection. Interestingly, our results indicate that inflammation-associated cellular effectors are present in tissues that lack UPEC colonization,  suggesting that either soluble bacterial factors or transient host-derived mediators result in such an inflammatory environment.We next evaluated whether the induction of localized cystitis in the bladder would elicit a cellular immune response in adjacent genitourinary tissues. Renal tissue demonstrates a similar cellular pro-inflammatory infiltrate as observed in the bladder , despite+, MHC II+med-hi) in the bladders of infected mice 48 and 96 hours post-infection   which are important for maintenance of fetal tolerance, remained unchanged in uteroplacental tissues at all of the post-infection time points analyzed (The maturation state of DCs dictates the success or failure of outbred pregnancies ctively) . The preanalyzed . The simAs serum levels of pro-inflammatory cytokines are elevated during UTI In order to identify early changes in the placenta that lead to fetal demise, we compared transcriptional changes in the presence and absence of experimental UTI. Placenta and uterus were harvested on gestational day 16 (48 hours post UTI) from mice from the sham cohort and mice from that received experimental UTI on gestational day 14. The tissue samples were processed for extraction of mRNA and subjected to Agilent microarray analysis . There wet al. presented a mouse model of pyelonephritis-induced preterm birth and low birth weights while investigating the virulence of the Dr adhesion et al. provides additional insight into the effects of intrauterine infection on fetal development. In our system, the offspring of mothers that experienced experimental UTI displayed up to 80% decrease in fetal weight when compared to non-infected mothers. This phenomenon occurs as early as 48 hours post introduction of cystitis and continues throughout gestation. An important feature of our model of adverse perinatal outcome model is that localized cystitis induced delayed infiltration of PMNs and macrophages in distal organs. We demonstrated that bladders from mice with experimental UTI had an expected increase of professional phagocytes as a result of localized infection; however, inflammatory cells were also observed in the kidney, even in the absence of viable bacteria. Furthermore, a robust cellular inflammatory response was observed in the uteroplacental tissue of those mice with experimental UTI when no viable bacteria were recovered (the same tissue sample tested for both bacteria and cellular infiltrate). In fact, there was a correlation between PMN infiltration and diminished fetal weight gain  and Th17 cytokines (IL-17) are significantly elevated at delivery. Furthermore, IL-6 and TGF-β are key factors in the development of Th17 cells. Similar systemic inflammation along with an imbalance of Th1 and Th2 cells in the uterus is a dominant component implicated in the pathogenesis of pre-eclampsia In conclusion, our murine model of maternal non-disseminated UTI-induced adverse perinatal outcomes provides a platform to further elucidate the inter-organ cross talk that occurs during localized infections.4–6 week old C57Bl/6 female mice were obtained from Jackson Lab, . Females were outbred with C3H/HeN males less than 1 year of age purchased from Harlan Labs . Forty-eight hours prior to the overnight mating sessions, males and females were exchanged in the cages to allow the mice to become familiar with their future mate's scent in an attempt to increase the rate of conception. To synchronize impregnation and maintain precise infection and delivery dates, 2 females were housed with 1 male  overnight for less than 16 hours. Therefore, successful pregnancies were determined by maternal weight gain following cohousing. Female mice that did not become pregnant as determined by every other day weight measurements were returned to the mating rotation every 2–3 weeks until conception occurred. Females were deemed pregnant if they had gained at least 2 grams over a 1-week period. The distinct histocompatibility complex classes chosen will invoke fetal tolerance to prevent fetal rejection. Our mating combination resulted in the typical number of fetuses with a gestational length of 20 days . With th8/mL UTI89/pANT4 or sterile phosphate buffered saline (PBS) as previously described UTI89/pANT4 Following isoflurane anesthesia per IACUC protocols, renal arteries of the pregnant mice were severed and blood was aspirated into tubes containing EDTA. Bladder, kidneys, and uteroplacental tissues were minced into small pieces with scissors and were digested for 25–30 minutes at 37°C under slight agitation. Bladders and kidneys were digested in 0.5 mg/ml collagenase and 100 µg/ml DNase I in RPMI 1640 medium  containing 0.5% heat-inactivated fetal calf serum  and 20 mM HEPES as previously described Cell suspensions were washed in FACS buffer and filtered through a 70 µm nylon filter  to remove cellular debris. One mL of ACK lysing buffer  was added to the kidney, spleen, and bone marrow to lyse red blood cells. A discontinuous 20, 40, and 80% Percoll gradient was prepared as described Each organ sample, including blood, was plated on LB plates containing ampicillin sulfate . Serial dilutions of each organ were plated in consecutive rows on LB agar and LB agar containing ampicillin to enumerate bacterial burden. Undiluted blood samples were also plated. Colony counts were then recorded for any growth and labeled as none detected if no growth was witnessed. The number of colonies was indistinguishable on LB agar and LB agar containing ampicillin.Maternal serum was analyzed for various cytokine levels using a Th1/Th2 and a Mouse Inflammation Cytokines Bead Arrays . Samples taken at the time of harvest from severed renal vessels were spun for 10 minutes at 10,000 rpm. Serum was then collected and frozen at −80°C for batch testing. Fifty microliters of serum was used for each sample tested. If more than 50 µl was available, samples were run in duplicate. We then used an LSR II cytometer to measure the following: IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-17, IFN-γ, and TNF-α. Software included in the kits was used for conversion into pg/ml.Samples of the placenta and the uterus were taken on gestational day 16 from 4 mice. Two mice received experimental UTI or shame inoculation on gestational day 14. Tissue samples were homogenized in 1 ml TRIZOL® Reagent per 50–100 mg of tissue using power homogenizer Polytron. The homogenized samples were incubated for 5 minutes at room temperature and then 0.2 ml of chloroform per 1 ml of Trizol were added to the tube. Total RNA was isolated from the homogenized samples with TRIZOL reagent according to the manufacturer's recommendations.Two-color gene expression analysis was performed in-house by the Biomedical Genomics Core. In outline, 500 ng of total RNA was amplified and labeled with either Cy3  or Cy5  using the Low Linear Amplification Kit . This labeling reaction produced 7–10.0 µg of Cy3-labeled cRNA (anti-sense), by first converting mRNA primed with an oligo (d)T-T7 primer into dsDNA with MMLV-RT and then amplifying the sample using T7 RNA Polymerase in the presence of Cy3-CTP. After purification, 825 ng of each the test and control cRNA was fragmented and co-hybridized to the Whole Mouse Genome Oligo Microarray  array for 17 hr. at 65°C. This array consists of 44,000 60-mer oligonucleotides, representing 21,609 known genes represented by 33,661 transcripts.Microarray slides were washed and then scanned with an Agilent G2505C Microarray Scanner. Images were analyzed with Feature Extraction 10.7  in two color gene expression mode. Median foreground intensities were obtained for each spot and imported into the mathematical software package “R”. The intensities were corrected for the scanner offset but not further background corrected. The dataset was filtered to remove positive control elements. Using the negative controls on the arrays, the background threshold was determined and all values less than this value were set to the threshold value. Finally, the data were global loess normalized using the LIMMA microarray processing package in “R” . Fold change values were then calculated for each element on the array and averaged for the replicate samples. Genes with a fold change >2 fold up or down were considered differentially expressed. All microarray data is MIAME compliant and the raw transcriptional data has been deposited into the MIAME compliant database, GEO, accession number GSE32028.Exact values (symbols) or median values (graphs) are represented in each figure. Statistical significance was determined using two-tailed non-parametric Mann-Whitney U test .Figure S1Maternal weight gain during UTI. Mothers were weighed daily following impregnation to validate pregnancy. On gestational day 14, mothers received PBS (black lines) or experimental UTI (gray lines) and were weighed at least every other day to assess the effect of UTI on maternal weight gain. The rate of gain (slope of the line) are not different between the cohorts. Only three representative mothers from the sham cohort and 7 of the UTI cohort are depicted for clarity.(TIF)Click here for additional data file.Figure S2Gating strategy for identification of infiltrating myeloid cells. Representative graphs indicate the markers and the populations that were selected for analysis.(TIF)Click here for additional data file.Figure S3Correlation of PMN infiltration with IUGR. The magnitude of PMN infiltration into the uteroplacental tissues was inversely correlated with the weight of each offspring at 96 hours in pregnant mice that received experimental UTI (gray) or sham infection (black). Each data point represents the weight of a single fetus with the magnitude of the PMN infiltration into the uteroplacental tissues.(TIF)Click here for additional data file.Figure S4Correlation of IL-6 with IUGR. The magnitude of IL-6 serum levels was inversely correlated with the weight of each offspring at 96 hours in pregnant mice that received experimental UTI (gray) or sham infection (black). Each data point represents the weight of a single fetus with the magnitude of the maternal circulatory IL-levels.(TIF)Click here for additional data file.


Most of current strategies for antiviral therapeutics target the virus specifically and directly, but an alternative approach to drug discovery might be to enhance the immune response to a broad range of viruses. Based on clinical observation in humans and successful genetic strategies in experimental models, we reasoned that an improved interferon (IFN) signaling system might better protect against viral infection. Here we aimed to identify small molecular weight compounds that might mimic this beneficial effect and improve antiviral defense. Accordingly, we developed a cell-based high-throughput screening (HTS) assay to identify small molecules that enhance the IFN signaling pathway components. The assay is based on a phenotypic screen for increased IFN-stimulated response element (ISRE) activity in a fully automated and robust format (Z′>0.7). Application of this assay system to a library of 2240 compounds  led to the identification of 64 compounds with significant ISRE activity. From these, we chose the anthracycline antibiotic, idarubicin, for further validation and mechanism based on activity in the sub-µM range. We found that idarubicin action to increase ISRE activity was manifest by other members of this drug class and was independent of cytotoxic or topoisomerase inhibitory effects as well as endogenous IFN signaling or production. We also observed that this compound conferred a consequent increase in IFN-stimulated gene (ISG) expression and a significant antiviral effect using a similar dose-range in a cell-culture system inoculated with encephalomyocarditis virus (EMCV). The antiviral effect was also found at compound concentrations below the ones observed for cytotoxicity. Taken together, our results provide proof of concept for using activators of components of the IFN signaling pathway to improve IFN efficacy and antiviral immune defense as well as a validated HTS approach to identify small molecules that might achieve this therapeutic benefit. There has been significant progress in the development of vaccines and therapeutics against viruses, but there are still major gaps in medical therapy for some of the most common types of viral infections. For these types of infections, vaccines can still be ineffective due to new and emergent strains and can exhibit significant off-target effects In the present study, we aimed to mimic the beneficial actions of STAT1 modification with a small molecule that also enhances the activity of the IFN signaling pathway. We describe here the development of a high-throughput screening (HTS) system for novel small molecular weight compounds  that might increase ISG expression and antiviral activity. To develop this screening system, we generated cell lines that stably express the human interferon-stimulated response element (ISRE) driving a luciferase reporter gene. The ISRE gene promoter element is responsible for type I IFN signaling that mediates host defense against a wide range of viruses Based on the observation that STAT1-CC-expressing cells show increased activity of the endogenous ISRE promoter element To achieve assay automation and miniaturization, the ISRE activity assay was first automated in 96-well plates and then reformatted for 384-well plates. In the 384-well format, the assay exhibited a near-maximal signal at 8000 cells per well and consistent well-to-well and plate-to-plate reproducibility . In 96- We used the automated ISRE-activity assay to perform a screen of a 2240 chemical compound library. This library consisted of 2160 compounds from the Johns Hopkins Clinical Compound Library (JHCCL) of FDA approved or approvable drugs After raw data were normalized, scaled to z-scores, and summarized, we found that 321 data points  had an ISRE activity z-score ≥2 . This daEach of the 64 primary hits was subjected to primary validation for ISRE activity over a broader range of concentrations of drug and IFN-β (0–15 U/ml). Among the primary and confirmed screening hits, idarubicin hydrochloride ranked highest in potency for enhancing ISRE activity . During the ISRE validation, we found that idarubicin caused a concentration-dependent increase in ISRE activity over a range of IFN-β treatment concentrations, with highly significant effects as low as 25 nM idarubicin in combination with 15 U/ml IFN-β .The structure for idarubicin shows characteristic features of an anthracycline antibiotic unrelated to any other antiviral compound in clinical use. To further validate the effect of idarubicin on ISRE activity, we tested three other anthracyclines  with very similar chemical structures to idarubicin. Each of these compounds also showed a capacity to significantly increase ISRE activity . In addiWe also found a cytotoxic effect of idarubicin that is consistent with previous observations 50 for idarubicin decreased as the concentration of IFN-β increased  . For exancreased . These fncreased . These rncreased . TogetheOAS1), particularly with IFN-β treatment (GBP1) and three other ISGs   and U3A-STAT1 cells were treated with a range of concentrations of idarubicin and IFN-β and then harvested for gene expression using quantitative real-time PCR assay. We found that idarubicin increased the expression of the antiviral gene 2′,5′-oligoadenylate synthetase 1  levels and virus-induced cytopathic effect. We selected EMCV since it was previously found to be sensitive to STAT1-CC-dependent improvement in IFN signaling The present study was undertaken to discover antiviral therapeutics that broadly increase host defense. We focused on the IFN system that is central to the antiviral response, although we recognized that other labs have pursued this target with limited success in the past. Some of these previous investigators have used administration of IFN itself to increase the antiviral response, but for this therapeutic goal and others, the side effects of IFN administration have proven to be rate-limiting Our specific approach was based on previous success with the use of a modified STAT1 signaling pathway. In this work, we demonstrated that a designer form of STAT1 (designated STAT1-CC based on double-cysteine substitutions) was able to enhance IFN signaling and better control viral replication Considering these factors and observations in the STAT1-CC model system, we designed a cell-based luciferase reporter assay for measuring type I-dependent ISRE activity. This assay proved to yield excellent signal-to-background and Ź factors, specificity for IFN-β treatment over IFN-γ treatment, and suitability for automation and screening. Furthermore, because the construct design uses the Click Beetle Green luciferase, the assay can be paired with other luciferase reporter genes to develop dual color assays to report activity of other signaling pathways, including the type II IFN-γ activated sequence (GAS) promoter activity that mediates defense against intracellular bacteria. A related approach was used to screen for small molecules that increase GAS activity for anti-proliferative and pro-apoptotic effects in cancer cells Our primary screen identified idarubicin on the basis of its capacity to significantly increase ISRE activity. Subsequent validation assays demonstrated that idarubicin facilitates STAT1-dependent ISG expression and STAT1-directed control of viral replication and cytopathic effect. While others previously reported the antiviral properties of anthracyclines some time ago, no mechanism of their antiviral action was elucidated In a further analysis of drug mechanism, our study demonstrates that the antiviral activity of idarubicin and other closely related anthracyclines is derived from enhancing the activity of the type I IFN signaling pathway. Our data further show that the enhancing effect of idarubicin is based on ISRE activation and ISG expression independent of IFN production or IFN–IFN-receptor interaction, since the effect of idarubicin is unchanged by IFN-receptor blockade. These findings suggest that idarubicin activation of the ISRE is due to an action in the IFN signaling pathway distal to ligand-receptor binding, e.g., at the level of receptor-associated JAK kinases or further downstream at the formation, transport, binding, and/or assembly of the ISRE transcriptional complex. In that regard, anthracyclines are known to inhibit DNA and RNA synthesis by intercalating between base pairs of the DNA/RNA strand to prevent replication, but whether this mechanism can affect ISRE or other gene promoter elements still needs to be defined. The present screening approach overcomes the uncertainty in molecular mechanism by using a phenotypic (rather than a target-based) screening approach and thereby captures compounds that increase the activity of the IFN signaling pathway by either established or undefined mechanisms.In sum, we describe and validate a phenotypic screening strategy to identify small molecules that enhance the activity of the type I IFN signaling pathway and consequently improve antiviral host defense. This approach is designed to lead to discovery of drugs with activity against a broad range of viruses for clinical application as well as experimental tool compounds to further understand IFN-dependent immune mechanisms. Current approaches to defining the basis for IFN signal transduction, particularly in vivo, often rely on complex transgenic and gene targeting approaches. Thus, the use of small molecule enhancers (SMEs) of the IFN signaling pathway may provide much greater flexibility and ease of application to achieve transient adjustment of IFN-related actions and consequent scientific and clinical benefit. Our approach should thereby prove useful to discover drugs with activity against a broad range of viruses as well as effectiveness in other conditions  where the efficacy of IFN treatment might benefit from enhancing the IFN signaling pathway.IFN-β and IFN-γ were obtained from PBL Interferon source , diluted and aliquoted according of manufacturer's recommendation, and stored at −80°C. The Johns Hopkins Clinical Compound Library (JHCCL) was obtained from Dr. David Sullivan at the Johns Hopkins University To construct the pISRE-CBG99 vector, we first generated a 5x-repeat of the ISRE sequence and a TATAA box (5xISRE-TATAA) in the pUCMinusMCS vector from Blue Heron Biotechnology  and then cloned this sequence into the Chroma-Luc pCBG99-Basic reporter vector from Promega  and ligated into Xma1 and Nco1 sites using T4 DNA ligase from Life Technologies . The DNA sequence of the resultant pISRE-CBG99 vector was confirmed by carrying out BigDye Terminator v3.1 sequencing reactions (Life Technologies) on an ABI capillary sequencer. This vector and the pPUR selection vector from Clontech  were co-transfected at a 9∶1 ratio into 2fTGH or HEK293T cells to increase the likelihood that cells tolerating puromycin selection (0.5 μg/ml) contained one or more copies of pISRE-CBG99 in addition to pPUR. The 2fTGH cells To optimize the luciferase light reaction, we tested a series of flash and glow luminescent substrate systems in both lysed and live 2fTGH-ISRE-CBG99 and HEK293T-ISRE-CBG99 cells, including D-luciferin , the Chroma-Glo Luciferase assay system from Promega and the steadylite plus reporter gene assay system from Perkin Elmer  under a range of incubation conditions. The steadylite plus system was selected based on kinetic profile. Effects of IFN-β concentration and treatment time were assessed with all other variables constant.The assay was automated in a 96-well format with a customized and fully integrated robotic system. The system equipment included: a Caliper Sciclone ALH 3000 workstation (Perkin Elmer) and a EL406 washer (BioTek) for liquid handling, an automated Liconic incubator (Thermo Scientific) for cold storage of plates, an automated Cytomat incubator (Thermo Scientific) for cell culture environment, a separate hotel for storage of plates at room temperature, a Synergy 4 plate reader, a Flexiseal plate heat sealer , a Caliper Twister II, and a Beckman Sagian Orca robotic arm on a linear rail . Construction allowed for transfer of plates, reagents, and plasticware between all instruments, so that there was no need for any manual interference during screening assays. This entire system was enclosed in a custom-made laminar flow hood to allow for HTS screening capability under BSL2 sterile conditions. After the system demonstrated satisfactory performance in a 96-well format, the assay was miniaturized to a 384-well format and re-tested for reproducibility and stability under IFN-β and vehicle (1% DMSO) treatment conditions.To achieve simultaneous treatment of cells with IFN-β and various compound concentrations and to avoid reagent degradation over time, the screen was run in a modular manner with a precise timeline . The first step included production of plates with appropriate concentrations of compound and IFN-β and then storage at 4°C. A separate plate was made for each of the four compound concentrations . The Twister II, Sciclone, Orca, and Liconic cold storage incubator handled this step. For the second step, cells were plated at 8000 cells per well in 384-well assay plates (n = 56). This step was accomplished in seven batches (8 assay plates per batch) using the Sciclone. A uniform suspension of cells was maintained by intermittent mixing on the Sciclone deck between cell plating. Simultaneously, the compound stock plates were sealed using the Flexiseal and stacked back into a Twister II rack for storage. For the third step, cells were allowed to grow for 11 h and then were treated with compound and IFN solutions. This step required that a plate containing cells be brought from the Cytomat incubator to the Sciclone deck in concert with a set of compound/IFN dilutions plates from the cold storage incubator. Cell treatments were timed so that each assay plate would be incubated for 10.3 h before the final step of performing the luciferase assay. For this last step, robotics were programmed so that each assay plate developed the luciferase light reaction for 40 min at 25°C in the plate hotel and then was delivered to the Synergy 4 plate reader for determination of luminescence. For this assay, the BioTek EL406 washer was used for aspiration of media and dispersion of substrate. In entirety, the screen took 41.6 h to complete.The raw data from the HTS assay were subjected to statistical analysis using cellHTS2 50), this data was fit to a four-parameter concentration-response curve as described previously Hits from the primary screen were validated using the ISRE activity-luciferase reporter assay over a broad range of compound concentrations (0.01–25 µM) in the absence or presence of IFN-β . To determine drug potency, as defined by half-maximal effective concentration (ECA resazurin (Alamar Blue) metabolism assay was used to assess cell toxicity during compound treatment OAS1 and GBP1) and normalizer gene ornithine decarboxylase antizyme (OAZ1). The normalizer gene OAZ1 was selected and validated for cell samples treated with and without IFN-β. In brief, candidate normalizer genes were selected from a combination of invariant genes selected from previous microarray data Expression of ISG's was assessed with real-time quantitative PCR assay for the corresponding mRNA level. For these experiments, U3A and U3A-STAT1 cells were first treated with the programmed combination of compound and IFN-β for 12 h and then were washed twice with cold Dulbecco's PBS followed by lysis with Cells-to-cDNA II lysis buffer (Life Technologies) and treatment with DNase according to the manufacturer's instructions. The U3A cells were obtained from G. Stark (Cleveland Clinic) and complemented with STAT1 to generate U3A-STAT1 cells as described previously Cells were cultured overnight and then treated with compound and IFN-β for 6 h. Thereafter, cells were washed and then were inoculated with EMCV  for 1 h at MOI 1 as described previously Figure S1Miniaturization and automation of the ISRE activity assay. (A) Effect of cell density on ISRE activity as a function fo IFN-β concentration in 384-well plates. B) Well-to-well reproducibility for the assay performed in 384-well automated format using 8000 cells per well.(EPS)Click here for additional data file.Figure S2Scheme for the HTS automation protocol. Each box represents one of the sequential steps in the screening process. Timing and time allotment for each step is also indicated.(EPS)Click here for additional data file.