Error fields associated with positions determined by these methods range from hundreds of metres to hundreds of kilometres. However, low spatial accuracy of tracks masks important details, such as foraging patterns. Here we use a fast-acquisition global positioning system (Fastloc GPS) tag with remote data retrieval to track long-term movements, in near real time and position accuracy of <70 m, of the world's largest bony fish, the ocean sunfish Mola mola. Search-like movements occurred over at least three distinct spatial scales. At fine scales, sunfish spent longer in highly localised areas with faster, straighter excursions between them. These ‘stopovers’ during long-distance movement appear consistent with finding and exploiting food patches. This demonstrates the feasibility of GPS tagging to provide tracks of unparalleled accuracy for monitoring movements of large pelagic fish, and with nearly four times as many locations obtained by the GPS tag than by a conventional Argos transmitter. The results signal the potential of GPS-tagged pelagic fish that surface regularly to be detectors of resource ‘hotspots’ in the blue ocean and provides a new capability for understanding large pelagic fish behaviour and habitat use that is relevant to ocean management and species conservation.Satellite tracking of large pelagic fish provides insights on free-ranging behaviour, distributions and population structuring. Up to now, such fish have been tracked remotely using two principal methods: direct positioning of transmitters by Argos polar-orbiting satellites, and satellite relay of tag-derived light-level data for  Determining the movements of individual free-ranging animals is important for a number of reasons such as assessing patterns of habitat utilisation, prey search strategies and defining critical conservation areas. Technological developments have transformed our ability to track a broad range of animals post hoc track reconstruction The movements and behaviour of large pelagic fish such as sharks and tuna have been tracked over large spatio-temporal scales by remote means using two main approaches: (i) direct, near real time positioning of animal-attached platform terminal transmitters (PTTs) by Argos polar-orbiting satellites, and (ii) satellite relay of tag-derived light-level data for Mola mola, and which sets the scene for a new era in fish biotelemetry.In this paper we describe the first long-term GPS tracking of a large pelagic fish that surfaces relatively frequently Mola mola  captured in a tuna pound net off southern Portugal Three ocean sunfish −1, then by reference to the number of satellites acquired to resolve each location. Because Fastloc GPS location estimates vary, with a higher number of satellites generally yielding more accurate locations, we removed positions with <5 satellites  Two time series of locations were retrieved remotely from tags towed by sunfish. The first comprised standard Argos locations determined from the Doppler shift in the receiver-uplink signal frequency as the satellite passes overhead  http://www.medspiration.org/), sea surface height (SSH) and geostrophic currents  (direction and speed vectors) . To examine changes in movement path tortuosity as a function of spatial scale, the first passage times (FPT) were calculated using custom-written software . FPT is defined as the time required for an animal to cross a circle of given radius r: rt is the FPT for a circle of radius rThe time series of filtered GPS locations were each analysed in ArcGIS to determine distances and times between consecutive positions, thus giving over-the-ground speed estimates. Paths were then mapped on time-referenced, remotely-sensed images of sea surface temperature (SST)  was obtained for sunfish 2 presumably because this individual spent more time shallower than 1.5 m compared with the two other tagged sunfish. For the longest track (S3), 346 GPS locations were obtained, with 67.1% of GPS positions fixed from between 5 and 8 satellites, compared with only 91 conventional Argos PTT locations of lower spatial accuracy for S3.A total of 612 Fastloc GPS locations were retrieved remotely from tags via Argos, with 3.0, 44.2 and 3.8 locations d−1 compared with movement rates of 10.0 and 19.6 km d−1 for sunfish 1 and 2 respectively.Sunfish 1 and 2, both 0.6 m total length, were tracked in May 2008 for 15 and 5 days covering estimated total distances of 150.5 and 98.1 km respectively, prior to the tag becoming detached from fish 1, and a cessation of Argos uplinks from the tag attached to fish 2 . Sunfishvice versa  resulted in clustering of consecutive GPS locations, but a faster, directed movement (>90 km d−1) between 28 and 29 December relocated the fish to a location 2.8 km away (−1) before exhibiting slower speeds on 1–3 January, resulting in clustering of locations over these days, before a faster, directed movement on 4 January and slowing again on 5 January  GPS tracking of a large pelagic fish and shows the enormous potential for this technique, where GPS-quality location data is retrieved remotely via conventional Argos satellites. By freeing researchers from the restriction of working on estuarine or nearshore species in order to physically recover tags to download GPS acquisition data Mola mola, showing that a large (153 kg) individual swam slower (mean and maximum speeds) than two smaller individuals of 48 and 59 kg body mass. In the context of our GPS tracking study, it is conceivable that if relatively slower swimming speeds were exhibited by the larger sunfish (S3), they may have contributed to longer tag retention time through reduction in drag-associated forces acting on the tow body at the point of attachment.The tags we deployed on the two smaller sunfish produced much shorter tracks than anticipated. The tag attached to sunfish 1 became detached after 15 days, whereafter it remained transmitting at the surface for a further week before the batteries were exhausted. It is possible that the relatively large size of the tag and the increased drag from a towed float caused this problem. The cessation of transmissions from the tag attached to sunfish 2 after 5 days may have resulted from tag failure or from the fish dying and sinking to the sea bottom. If it was the death of the fish then we might have expected the tag to re-surface and transmit when it finally broke free from the sunfish carcass (due to scavenger activity). As this did not occur it seems likely that the short tracking time was due to tag failure. Regardless of that contention, it is possible that attaching the tag to a larger sunfish (i.e. S3) resulted in longer term tracking. Watanabe and Sato th Century contradictory observations about M. mola swimming abilities were presented, with them being described as active swimmers in one study M. mola are not passive drifters but active swimmers with movement rates within the range observed for pelagic sharks and other pelagic fishes In the early part of the 20n = 78 locations, 24 Dec 08 – 8 Jan 09). A similar intermittent pattern of intensive and extensive movement has been observed in filter-feeding basking sharks feeding on patchy zooplankton in shelf waters th and 20th January 2009 (see Self-similar patterns of relatively sharp transitions between area restricted movements and faster, directed movements were apparent at three distinct scales. Particularly at the fine scale, sunfish 3 exhibited pronounced slowing of movement rate over periods of 1–3 days during which movements were spatially constrained with often <500 m between locations during a day; see 2009 see . Ocean sThe GPS technique we demonstrate for sunfish presents the capability to resolve much finer scale behaviours, such as within and between-patch foraging, than are possible with other techniques presently available for fish tracking. In addition, the technique captures large-scale movements over long periods of time. This suggests that GPS-tracked large pelagic fish could be useful resource detectors of pelagic prey patches or biodiversity ‘hotspots’ in the blue ocean, where satellite remote sensing of ocean colour cannot be used routinely to determine enhanced secondary and tertiary productivity over the appropriate spatio-temporal scales to develop ‘prey fields’


Lyssaviruses are RNA viruses with single-strand, negative-sense genomes responsible for rabies-like diseases in mammals. To date, genomic and evolutionary studies have most often utilized partial genome sequences, particularly of the nucleoprotein and glycoprotein genes, with little consideration of genome-scale evolution. Herein, we report the first genomic and evolutionary analysis using complete genome sequences of all recognised lyssavirus genotypes, including 14 new complete genomes of field isolates from 6 genotypes and one genotype that is completely sequenced for the first time. In doing so we significantly increase the extent of genome sequence data available for these important viruses. Our analysis of these genome sequence data reveals that all lyssaviruses have the same genomic organization. A phylogenetic analysis reveals strong geographical structuring, with the greatest genetic diversity in Africa, and an independent origin for the two known genotypes that infect European bats. We also suggest that multiple genotypes may exist within the diversity of viruses currently classified as ‘Lagos Bat’. In sum, we show that rigorous phylogenetic techniques based on full length genome sequence provide the best discriminatory power for genotype classification within the lyssaviruses. RhabdoviridaeLyssaviruses (LYSSAV) are RNA viruses with single-stranded, negative-sense genomes of the family Despite the importance of LYSSAV for human and wildlife populations, the number of complete genome sequences of field isolates of LYSSAV is sparse, with only eight currently available for limited type species Total RNA  was isolhttp://tree.bio.ed.ac.uk/). GenBank accession numbers for the sequences newly acquired here are designated EU293108-EU293121.Long-range PCR products were obtained using ExTaq (Takara) and specific primers  using ma4 model of nucleotide substitution inferred by ModelTest Phylogenetic analysis of LYSSAV genomes was based on a multiple alignment of concatenated coding region sequences (12105 nt). A maximum likelihood (ML) phylogenetic analysis of these data was undertaken using PAUP* In total we determined 14 new complete genome sequences of field isolates representing six  of the seven genotypes of LYSSAV, with complete genome from GT4 obtained for the first time. These genomes were combined with eight genomes described previously . Eight field isolates of viruses isolated from humans, canids and bats were chosen as representative of the diversity of GT1. Two vaccine strains (SAD-B19 and PV) were included in all sequence comparisons but not in the phylogenetic analysis. Our study also represent the first analysis of the intrinsic genetic diversity of GT2, GT3, GT4, GT5 and GT6 based on full length genomes.N) to synonymous substitutions (dS) per site (dN/dS), estimated using the maximum likelihood SLAC  method N/dS reflects major differences in selective constraint among proteins. This trend was also reflected in previous analyses of full length genomes of vaccine strains All genomes have the same structural organization although their lengths varied between 11918 nt. (GT7) and 12016 nt. (GT2) . The preOur study represents the largest analysis of the 3′ and 5′ UTR of the lyssavirus genomes undertaken to date. The 3′ UTR comprises 70 nt and includes the leader regions potentially transcribed into the leader RNA. The 5′UTR region comprises 86–145 nt and contains the trailer regions of size 68–69 nt. Both the 3′ and 5′ UTR have conserved signals that play a role to modulate replication and transcription  [27]. OuThere have been several attempts to estimate the evolutionary relationships among lyssaviruses, with most utilizing only one or two genes Notably, our study represents the first analysis of the genetic diversity of four complete genomes of GT2 and GT4, both of which are African in origin. While little variation is seen within GT4, the degree of divergence among the two GT2 isolates (Lagos bat virus – LBV) is striking  and greater than that seen within any other genotype. Hence, although 0406SEN and 8619NGA are related according to the arbitrary classification system based on nucleotide identity between N coding regions (80.3% between 0406SEN and 8619NGA compared to a cut-off of 80%) Finally, we suggest that the phylogenetic methods used here – based on a realistic model of nucleotide substitution, a robust phylogenetic method, and rigorous bootstrap resampling – represent a more powerful method of lyssavirus classification than those based on pairwise genetic diversity alone, particularly as they account for any lineage-specific rate variation that will compromise all distance-based approaches used to date. This method has also been proposed for HIV to try to standardize viral classification Table S1List of primers(0.06 MB PDF)Click here for additional data file.Table S2Transcription and termination signals for all lyssavirus genotypes.(0.05 MB PDF)Click here for additional data file.Figure S1Comparison of the 5′ and the reverse complementary 3′ genomic termini of the antigenomic (+) sense RNA of lyssaviruses. Identical nucleotides are indicated by a vertical line. A, 23 lyssaviruses representing the 7 genotypes. B, consensus sequences. Only regions corresponding to 3′ and 5′ UTR sequences are shown. TTS: transcription termination signal.(0.27 MB DOC)Click here for additional data file.


Cardiovascular development is vital for embryonic survival and growth. Early gestation embryo loss or malformation has been linked to yolk sac vasculopathy and congenital heart defects (CHDs). However, the molecular pathways that underlie these structural defects in humans remain largely unknown hindering the development of molecular-based diagnostic tools and novel therapies.ex vivo model of heart development. Additionally, a successful application of a cluster of protein biomarkers  as a prenatal screen for CHDs was confirmed in a study of human amniotic fluid (AF) samples from women carrying normal fetuses and those with CHDs.Murine embryos were exposed to high glucose, a condition known to induce cardiovascular defects in both animal models and humans. We further employed a mass spectrometry-based proteomics approach to identify proteins differentially expressed in embryos with defects from those with normal cardiovascular development. The proteins detected by mass spectrometry  were validated by Western blotting and immunoflorescent staining of the yolk sac and heart. The proteins within the proteomic dataset clustered to adhesion/migration, differentiation, transport, and insulin signaling pathways. A functional role for several proteins  was demonstrated in an The novel finding that WNT16, ST14 and Pcsk1 protein levels increase in fetuses with CHDs suggests that these proteins may play a role in the etiology of human CHDs. The information gained through this bed-side to bench translational approach contributes to a more complete understanding of the protein pathways dysregulated during cardiovascular development and provides novel avenues for diagnostic and therapeutic interventions, beneficial to fetuses at risk for CHDs. The foundations of the primitive heart form between gastrulation and early organogenesis, as critical specification, differentiation, and morphogenic events occur. During this period, the embryo is highly susceptible to environmental insults and genetic lesions that perturb cardiovascular development. These disturbances lead to congenital heart defects (CHDs), a significant cause of spontaneous abortions (20%) and infant mortality  Clinical strategies to diagnose cardiac structural anomalies rely on imaging the visible structural defect. Indications for fetal echocardiography include risk factors based on family history, exposures, or other fetal findings The development of such interventions has been hindered by our incomplete knowledge of human cardiac development. A number of genes that drive cardiovascular development have been described in mouse, zebrafish, and chicken model systems, although only a handful of genes such as PTPN11, TBX5 GATA4, and Nkx2.5 have been definitively identified in the etiology of human CHDs Identification of proteins involved in specific cardiac developmental events using whole embryonic hearts is difficult because it requires distinguishing and deconvoluting candidate proteins expressed concurrently during endocardial cushion morphogenesis, cardiomyocyte differentiation, looping and/or chamber specification. Therefore, our strategy utilized yolk sac vascular tissue rather than cardiac tissue for proteomic studies. Our hypothesis was that proteins involved in cardiac development and CHDs could be isolated and characterized from the vascular tissue dataset for the following reasons. Yolk sac tissue at the primary capillary plexus stage provides a relatively simple, homogeneous tissue consisting of few cell types in which the primary developmental event is endothelial differentiation and assembly into a mature vascular bed. In the heart, endothelial cell differentiation via EMT is a key event in endocardial cushion morphogenesis. Additionally, trabeculation and coronary system development both also rely on endothelial transdifferentiation or endothelial cell reorganization and assembly. Thus, during development of both the yolk sac and heart, endothelial biology plays a central role. The overlapping developmental pathways in the vasculature and heart have been revealed by the generation of numerous knockout mice that have described both vascular and cardiac phenotypes. This pattern may be a reflection of a common endothelial/cardiac progenitor , non-cell autonomous interactions between endothelial and cardiac progenitor lines which are intermingling during migration through the splanchnic mesoderm, or the intimate juxtaposition of differentiated endothelial cells and cardiomyocytes in the embryonic heart ex vivo cardiac culture model revealed novel roles for Collagen IV, NOGO-A/B, WNT16 and ADAM15 in heart development. Together with the immunofluorescence data, the functional analysis data confirmed that our approach did identify proteins relevant to cardiac development. In our dataset, molecules that may be useful as biomarkers in human AF were targeted, in particular secreted proteins and protein ectodomains. The successful application of a cluster of proteins  as biomarkers of CHDs was confirmed in a study of human AF samples from women carrying normal fetuses and those with CHDs. This study adds new information to our understanding of the molecular pathways underlying normal and abnormal cardiovascular development, paving the way for the development of novel prenatal screens and treatments for human fetuses with CHDs.From our vascular proteomic dataset, we assembled a list of differentially expressed proteins which were validated by Western blot. The data accrued through these studies highlight the importance of adhesion/migration, differentiation, transport, and insulin/insulin like growth factor (IGF) pathways in cardiovascular development. Immunofluorescence microscopy studies demonstrated the presence of many of these proteins  in the embryonic heart. Subsequent functional analysis in an CD1 mice (Charles River) were maintained under standard conditions. Timed-matings were detected by the presence of a vaginal plug. NOGO-A/B mice were provided by Steve Strittmatter . The Yale University Animal Care and Use Committee approved all animal protocols and experimentation was performed in accordance with National Institute of Health regulations.Conceptuses were harvested at 7.5 dpc and cultured for 48 hours in pooled rat serum supplemented with 20 µm of the nitric oxide donor NOC-18 bis-ethanamine) , 10 pg/ml rVEGF (Chemicon), 20 mM L-glucose (control) and/or 20 mM D-glucose. The developmental stage was determined using Downs and Davies' criteria for staging morphological landmarks of the primitive streak, neural plate and head fold Atrioventricular canals were isolated from E9.0 embryos and placed onto a 1 mg/ml Type I Rat Tail collagen gel (BD Biosciences). The explants were allowed to adhere prior to the addition of media  Individual 8.5 dpc yolk sacs were lysed in RapiGest SF (Waters), digested with trypsin (Princeton Separations), and desalted using C-18 columns (VivaScience). LC-MS and LC-MS-MS analysis were performed on a Q-TOF API-US quadrupole time of flight tandem mass spectrometer (Waters) equipped alternatively with electrospray and nonospray sources. Peptides were eluted into the source following chromatography on a capillary HPLC CapLC system equipped with a dual wave length UV detector (Waters). For both standard ESI (flow set at 10 uL/min) and nonoflow (flow set at 100 nL/min) ionization methods a linear 30 min gradient of increasing acetonitrile concentration was applied to fractionate and elute the samples. The mass spectrometer's source voltage for this application was set at 3.5 kV with scan time duration of 1 s/scan and spectra were acquired using both survey and parent scan MS methods with data dependent acquisition (DDA) setting defined by MassLynx software instrument interface. Following acquisition, data was preprocessed automatically by MassLynx and the resulting MS-MS spectra was submitted to MZmine for post-acquisition processing  to generate the peak intensity matrix Conceptuses were harvested from timed-pregnant mice at the indicated time points, frozen in OCT medium and cryo-sectioned (10 µm). Immunoreactivity was visualized with Alexa Fluor 594 or 488 secondary antibodies (Invitrogen). Nuclei were stained with DAPI. Digital images were captured on an Olympus IX71 Inverted Microscope.Yolk sacs were harvested from culture conditions at the indicated time points and lysed in RIPA Buffer (Upstate) supplemented with 1% SDS, Complete Protease Inhibitor Cocktail (Roche) and Phosphatase Inhibitor Cocktails . The samples were homogenized and soluble extracts obtained by centrifugation. Protein concentrations were determined by BCA Assay (Bio-Rad) and 25 µg was electrophoresed on SDS-PAGE gels (Bio-Rad). Luminescence was performed using the Western Lightening Chemiluminescence Reagent (PerkinElmer) and densitometry using Quantity One Software (Bio-Rad).Primary antibodies included NOGO-A , PECAM (J.A.M), Laminin 1 (J.A.M.), α smooth muscle cell actin (Sigma), ADAM15 ectodomain (R&D Systems), WNT16 (BD Pharmigen), SCVT (Slc23a1) , Collagen IV , PIASγ (Abcam), TRPC5 (Chemicon), KIF21B (Upstate), Erk2 (Santa Cruz), NOGO-A/B (Santa Cruz), Enolase 1 , and the following from Novus: Fbxo42, Jumonji, Pcsk1, ST14, CHST3 and ZCWCC1 (Morca2a).Yolk sacs were lysed in 50 mM Tris/1% Triton-X and homogenized with a motorized pestle. 25 µg of protein was mixed with non-reducing Laemmli buffer and loaded onto a 10% Zymogram Gel (Bio-Rad) for electrophoresis. The gels were washed in 2.5% Triton X-100, followed by water, and incubated for 48–72 h at 37°C in a 50 mM Tris-HCl buffer, pH 8.0, containing 5 mM calcium chloride or 10 mM EDTA (negative control for MMP activity). Gels were stained in 50% methanol, 10% acetic acid containing 0.1% Coomassie Blue R250 and imaged on a BioRad Chemidoc XRS.Via ultrasound-guided amniocentesis, 16–22 week gestation amniotic fluids (AFs) were obtained from healthy women (average age 27.7±6.6) carrying fetuses with (n = 17) and without (n = 16) cardiac defects from 2004–2007 at Yale New Haven Hospital. The Human Investigation Committee of Yale University approved our study protocol, and written informed consent for research purpose was obtained from all participants before the procedure. The purpose of the amniocentesis was clinical: to rule out chromosomal abnormalities. Criteria for the “diseased group” were heart defect visualized on at least two ultrasound exams. In cases resulting in live newborns, the defects were confirmed postnatally. In the remaining cases, the pregnancy course was terminated and the defect was confirmed in elected autopsies. CHDs included ventricular septal defects, valvular stenosis, complete atrioventricular septal defects, bilateral ventriculomegaly, hypoplastic ventricle, tetralogy of Fallot, and transposition of the great arteries. Criteria for the “non-diseased group” were AF samples from women undergoing genetic testing who had a normal course of pregnancy including term delivery. All fetuses in the non-diseased group were euploid and had normal heart ultrasound examinations during pregnancy and a normal physical exam at birth.Frozen AFs were centrifuged and aliquoted prior to assay. Western Blot was performed as described above on 25 µg of AF. A “standard” control sample was chosen to run on each gel. Coomassie staining of gels confirmed equal protein load. Films were scanned and analyzed in Quantity One as follows. Bands were selected for quantification using the automated volume contour tool. Band densities were calculated by computer algorithm as the volume, the sum of pixel intensities multiplied by pixel area, adjusted by global background subtraction. Blotting and analysis was performed by a blinded observer.Univariate logistic regression was performed using the design packet in R (GNU S). The AF measurements were log-transformed using log2 and the regression coefficient and odds ratio (OR) were calculated. The OR can be interpreted as the increased risk of developing a cardiac defect per doubling of the AF measurement. The mean classification error was calculated using 10 by 10 cross-validation. An attempt to perform multiple regression analysis did result in inflated regression coefficient estimates due to strong multicollinearity. After normalization and centering of all biomarker data, Principle Component Analysis (PCA), a clustering tool that reduces multidimensional data sets to identify new meaningful underlying variables, was performed using the SVD function in Matlab.ex vivo embryo cultures results in vasculopathy in the yolk sac and inhibition of EMT in the cardiac endocardium One premise of our study is that proteomic profiling of hyperglycemia exposed vascular tissue will enrich for factors involved in endothelial differentiation or dysfunction. Hyperglycemia was used as an “inducer” because increased vascular and cardiac defects are observed in the offspring of diabetic rodents (streptozotocin-induced) and the infants of diabetic women Under normoglycemic conditions, control embryos exhibit normal organogenesis and vascular development of the yolk sac  while hy165 or a nitric oxide donor supplementation reverses the morphological, functional and biochemical changes induced by hyperglycemia in the cardiovascular system We hypothesized that the hyperglycemic insult may be used to enrich for factors involved in endothelial differentiation/dysfunction. Due to the global biological effects of hyperglycemia, we expected a large number of proteins to exhibit expression changes, but only a subset to be relevant to endothelial differentiation/dysfunction. Therefore two additional groups, “rescue conditions”, were included in the experimental design. Our previous studies demonstrated that exogenous rVEGF-A165, or hyperglycemia plus nitric oxide donor. Yolk sacs were harvested at 24 hours, at the end of the primary capillary plexus stage and beginning of the vascular remodeling stage, and processed individually for mass spectrometry. LC-MS detected 39,120 peaks which were subsequently analyzed using MZmine software www.matrixscience.com) and NCBInr Database.Groups of ten 7.5 dpc conceptuses were cultured in normoglycemia, hyperglycemia, hyperglycemia plus rVEGF-A2 ratio (of treatment group to control) against −log10 p value (2 (fold change)>1] but returned to control levels by both rVEGF and nitric oxide donor [inclusion criteria: p>0.05 or p<0.05 and log2 (fold change)<1]. Significant peptide peaks were ranked by fold change and those greater than or equal to 2 [log2 (fold change)≥1] were included. Of the 143 peptide peaks, approximately 75% were upregulated. The majority of peptide peaks display a 2–4 fold change [log2 (fold change)≥1–2]. Though these changes appear minor, in developmental contexts slight changes in growth factors or signaling pathways disrupt cardiovascular development. For instance, a one fold increase or half fold decrease in VEGF leads to lethal cardiac defects To globally visualize the dataset, volcano plots were generated depicting each of the 12,908 peptide peaks as a point and plotting the log p value . The redCandidate proteins with the highest Mowse score Several proteins in our dataset mapped to the Gene Ontology term “Proteolysis”. These proteins function in varied biological processes involving proteolytic activity such as cell-cell adhesion, migration/invasion, matrix remodeling, growth factor release/activation, receptor shedding and cytokine signaling. The candidate proteases detected, included ADAM15, MMP2, Suppressor of tumorigenicity 14 (ST14) and Proprotein convertase 1 (Pcsk1).A number of candidate proteins that directly affect differentiation and embryonic development were discovered in our dataset, including Hairy and enhancer of split 6 (HES6), Notch1, NOGO-A, Jumonji, and WNT16. Several other candidate proteins identified in our screen mapped to the Gene Ontology term “Transport” including Transient receptor potential protein (TRPC5), a calcium channel, Sodium/vitamin C transporter (SVCT or Slc23a1), a sodium/ascorbate cotransporter, and ATP binding proteins . Other proteins of note found in our database include Fbxo42 (part of the ubiquitin ligase complex), Protein inhibitor of activated STAT4 (PIASγ) (a SUMO ligase), Kinesin-like protein KIF21b (a microtubule binding motor protein), Enolase 1 (an enzyme in the glycolysis pathway) and Microrchidia 2a (Morc2a or ZCWCC1).Several candidate factors in our dataset have been correlated with disease states including the Ets family member Elf5, which is upregulated in breast cancer In order to assess the biological significance of the dysregulation of these candidate proteins and validate our MS-MS results , WesternWe discovered a number of unnamed and predicted proteins with significant dysregulation. Continued curation of the public databases will reveal the identities and functions of unnamed proteins identified from cDNA initiatives; however, as little information is currently available on such proteins these were not further pursued. Many proteins were identified from multiple different significant peptide peaks (i.e. different fragments within the same protein). The consolidated list consisting of 44 proteins is presented in To validate our assumption that proteins relevant to cardiac development may be isolated from vascular tissue, proteins from the validated candidate list were tested by immunofluorescence microscopy for their presence in the endocardial cushion at E9.5 (a stage of cardiac development when EMT is actively occurring). This is a time and place during cardiogenesis that is widely recognized to be a “hot spot” of cardiac anomalies.Laminin 1 was strongly present at the interface of the myocardial and mesenchymal zones of the cushion, consistent with basement membrane localization . TRPC5 aex vivo atrioventricular canal assay, that has yielded many insights into the molecular events that underlie endocardial cushion development in vivo valve development and provides a system amenable to experimental manipulation Proteins were selected for further study in a functional assay, the Normal cultures (IgG treatment) exhibited the characteristic phenotype of EMT, displaying elongated, invasive mesenchymal cells . A pilot study was initiated to determine if the expression of these proteins vary in the AF of a cohort of healthy, non-diabetic pregnant women. AF was obtained from routine amniocentesis of women carrying normal fetuses (n = 17) and women carrying fetuses with CHDs (n = 16) (population details in CHST3 was significantly (p<0.01) increased in the AF of women carrying fetuses with CHDs (mean = 380) compared to controls (mean = 176) by Student's t-test , but not significant by univariate logistic regression  ; Table 2The biomarkers that showed significant association with CHDs in univariate logistic regression were WNT 16, Pcsk1 and ST14 . WNT16 wex vivo model of vasculopathy, we have identified proteins and pathways that are disrupted during cardiovascular development and lead to the observed pathological phenotype. Dysregulation of this set of proteins in a “normal” fetus may signal that the fetus is congenitally at risk or encountering an environmental stressor. Several of these protein targets  provided the basis for a novel prenatal screen for the detection of CHDs, suggesting dysregulation of WNT16, ST14 and Pcsk1 may play a role in the etiology of human CHDs. Several forms of CHDs are known to have a genetic basis such as microdeletions (22q11) in DiGeorge Syndrome and single gene mutations (JAG1) in Alagille Syndrome. It would be of interest to determine if individuals with CHDs carry genetic mutations in WNT16, ST14 or Pcsk1. At the protein level, these markers are overexpressed therefore if sequence variants are found mutations would likely affect protein function and/or activity. In the case of ST14 and Pcsk1, both of which are enzymes, a hypomorphic or hypermorphic mutation in these genes has the potential to affect multiple substrates and hence several cellular processes.Applying a proteomic approach to embryonic vascular tissue generated from an Changes in cellular processes not only reflect the molecular origins of disease but also provide avenues for molecular therapies to ameliorate the defect. To this end, we identified several proteins, previously unknown to have a role in cardiovascular development, with diverse functions that cluster to adhesion/migration, differentiation, transport, IGF signaling pathways . AlthougPreviously, we demonstrated that yolk sac vasculopathy is associated with increased nitric oxide, however the nitric oxide targets remained elusive On the other hand, positive regulation of cardiac EMT relies on Wnt/β-catenin signaling however the specific Wnts involved are largely unknown with the exception of Wnt9a in the avian heart Tbx20 in the avian endocardial cushion resulted in decreased chondroitin sulfate proteoglycans and increased mesenchyme Proteoglycans and extracellular matrix are important players in cell adhesion, migration, proliferation, and angiogenesis, processes important in both development and disease. CHST3 catalyzes the transfer of sulfate to position 6 of the N-acetylgalactosamine  residue of chondroitin. Human endothelial cells express CHST3 mRNA while endothelial cells overlying the porcine mitral valve produce chondroitin sulphate An additional factor involved in adhesion/migration identified in our study is the protease ADAM15. Multiple individual ADAM isoform KO mice display cardiac defects, including Adam 9, 17, and 19 In addition to proteins involved in differentiation and adhesion/migration, several proteins involved in the IGF/insulin pathway were identified in our study. Early in embryogenesis the embryo does not produce insulin nor does maternal insulin cross the placental barrier. However, embryos express high levels of IGF receptors and early growth is mediated via IGF signaling through Akt. Factors that regulate IGF receptor engagement and its downstream signaling cascades were identified both on the extracellular face, including the proteases Pcsk1 and ST14, and the intracellular face, including ZFP106 (or son of insulin receptor mutant) and HES6. Pcsk1 is a secreted protease that cleaves precursors including neuropeptides, renin, somatostatin, and insulin/IGF Another molecule in the IGF signaling pathway, ST14, a transmembrane serine protease, has been implicated in metastatic cancer, which utilizes a molecular program reminiscent of embryonic EMT Cell movements, growth and differentiation depend on environmental homeostasis; of particular importance during development is redox balance. According to current knowledge, SVCT transports ascorbate into cells and thereby participates in metabolic processes, matrix synthesis and antioxidant defense in vivo streptozocin-induced diabetic model in mice, we detected differences in specific Laminin chains, ADAM15 ectodomain, and MMP2 activity in maternal sera from mice carrying a high proportion of fetuses with heart defects compared to those with a high proportion of fetuses with normal hearts (unpublished data). These data suggest that protein fragments are released into the maternal circulation in sufficient quantities that allow detection early in pregnancy . Experiments are currently underway to evaluate the levels of WNT16, ST14, and Pcsk1 in maternal sera from murine models and humans.Molecular-based diagnostic tests that detect fetuses “at risk” provide clinicians with information to guide clinical interventions aimed at improving outcome. First-trimester biochemical screening is becoming a powerful tool, allowing clinicians to efficiently triage patients The creation of new biochemical diagnostic tools to assay proteins such as Pcsk1, ST14, and WNT16 may give insights into the well-being of the fetus by sensing an inability to combat an environmental insult, disruption in growth, or damage due to reactive oxygen species. Affordable, rapid, and accurate molecular-based diagnosis of potential problems in fetal cardiovascular development early in pregnancy will provide clinicians with information to guide clinical interventions and pave the way toward eradicating one of the major global causes of infant mortality in the world. In the future, dietary supplementation or molecular therapies based on Pcsk1, ST14 and WNT16 or their targets may ameliorate the deficiencies that ultimately lead to the formation of a CHD in the first trimester. Therefore isolating the specific substrate targets of ST14 and Pcsk1 that disrupt cardiac development and investigating the transcriptional or post-transcriptional regulatory mechanisms that lead to overexpression of ST14 and Pcsk1 will aid in deciphering the role of these proteins in the etiology of CHDs. Further, continued mining of the proteomic dataset coupled with analysis of novel factors in physiologically relevant models will continue to yield fruitful avenues for investigation into the biology of cardiovascular development and pathogenic mechanisms of CHDs in the hope of advancing prevention, diagnosis and treatment.Table S1(2.75 MB XLS)Click here for additional data file.


While it is accepted that a majority of invasive breast cancer progresses from a ductal carcinoma in situ (DCIS) precursor stage, very little is known about the factors that promote survival of DCIS neoplastic cells within the hypoxic, nutrient deprived intraductal microenvironment.Ex vivo organoid culture of fresh human DCIS lesions, without enzymatic treatment or sorting, induced the emergence of neoplastic epithelial cells exhibiting the following characteristics: a) spontaneous generation of hundreds of spheroids and duct-like 3-D structures in culture within 2–4 weeks; b) tumorigenicity in NOD/SCID mice; c) cytogenetically abnormal  compared to the normal karyotype of the non-neoplastic cells in the source patient's breast tissue; d) in vitro migration and invasion of autologous breast stroma; and e) up-regulation of signal pathways linked to, and components of, cellular autophagy. Multiple autophagy markers were present in the patient's original DCIS lesion and the mouse xenograft. We tested whether autophagy was necessary for survival of cytogenetically abnormal DCIS cells. The lysosomotropic inhibitor (chloroquine phosphate) of autophagy completely suppressed the generation of DCIS spheroids/3-D structures, suppressed ex vivo invasion of autologous stroma, induced apoptosis, suppressed autophagy associated proteins including Atg5, AKT/PI3 Kinase and mTOR, eliminated cytogenetically abnormal spheroid forming cells from the organ culture, and abrogated xenograft tumor formation.We examined the hypothesis that fresh human DCIS lesions contain pre-existing carcinoma precursor cells. We characterized these cells by full genome molecular cytogenetics (Illumina HumanCytoSNP profile), and signal pathway profiling , and demonstrated that autophagy is required for survival and anchorage independent growth of the cytogenetically abnormal tumorigenic DCIS cells. Cytogenetically abnormal spheroid forming, tumorigenic, and invasive neoplastic epithelial cells pre-exist in human DCIS and require cellular autophagy for survival. While the transition from in situ to invasive cancer is central to the origin of the malignant phenotype, very little is known about the time of onset, and the triggering mechanism, that switches in situ neoplastic lesions to overt invasive carcinoma in the human breast. Ductal Carcinoma In Situ (DCIS), the most common type of non-invasive breast cancer in women, is defined as a proliferation of neoplastic epithelial cells within the duct that is normally surrounded by myoepithelial cells and an intact basement membrane et al employing the ‘MINO’  mouse model of DCIS concluded that malignant aggressiveness is pre-programmed in the pre-cancer stem cell It has been previously hypothesized that breast cancer progression is a multi-step process involving a continuum of changes from the normal phenotype to hyperplastic lesions, carcinomas in situ, invasive carcinoma, and finally to metastatic disease In the present study we studied the cellular processes that promote the survival of the invasive precursor cells that exist within the human breast intraductal niche. Living human DCIS ducts were explanted into organoid culture, in serum free conditions, to expose the putative DCIS neoplastic cells within the duct. We characterized the biologic phenotype of these emerging cells using xenograft transplantation, and propagation in organ culture and evaluated the survival mechanisms employed by these cells. Cytogenetically abnormal DCIS lesion derived epithelial cells were identified by their phenotype of anchorage independent growth as 3-D spheroids and duct like structures, invasion of autologous stroma, and tumorigenicity in SCID NOD mice. We find that autophagy is a principal, and perhaps necessary, survival pathway in DCIS.To date, the macroautophagy survival pathway (autophagy) has not been implicated in the survival of malignant precursor cells within human DCIS lesions. Autophagy (self-eating) is a dynamic, catabolic process of organelle digestion that generates ATP during periods of nutrient limitation The existence of tumorigenic cells within fresh human DCIS duct segments was first tested using xenotransplantation In order to identify and characterize the tumorigenic neoplastic cells within the DCIS tissue that were responsible for the tumorigenic phenotype observed in the mouse xenografts, we cultured fresh living human DCIS ductal fragments to generate DCIS neoplastic cell outgrowths. The outgrowths spontaneously generated 3-D structures including spheroids and duct-like structures . The numin vitro organoid cultivation successfully propagated DCIS derived epithelial cells with anchorage independent growth, defined as upward growing and expanding spheroids, and lobulated, duct-like 3-D formations with pseudo lumens, in serum free medium supplemented with EGF and insulin (Organoid culture was used to characterize the DCIS neoplastic cells that were implicated in tumorigenesis by the xenograft experiments. Migratory proliferative cells that were positive for human specific EpCAM were observed to migrate out of the DCIS duct organoids grown in culture for as little as 2 weeks (within 2–4 weeks). Continued  insulin .DCIS cultured neoplastic epithelial cells migrated over the surface of autologous stroma and formed multilayered colonies with clear epithelial morphology . InvasivThe genotype of the isolated DCIS spheroid forming cells was evaluated and compared to the non-neoplastic tissue from the same patient. The spheroid forming DCIS epithelial cells were found to be cytogenetically abnormal compared to the normal karyotype of the anchorage dependent monolayer cells cultured from the same patient's breast tissue lesion. Full genotypic data output included allele calls from “tagged” single nucleotide polymorphism (SNP) sites and signal intensity values from non-polymorphic sites to determine DNA copy number values . Molecular cytogenetic profiles demonstrated cytogenetic alterations in the isolated DCIS spheroids (3–5 spheroids per prep) and isolated pseudoductular structures. The spheroid abnormal karyotype signature included loss of copy number on chromosome 5, 6, 8, and 13, and gain of copy number on chromosomes 1, 5, and 17. Abnormalities were present in all DCIS cell spheroids and pseudoductular isolates  and arosWithin the organoid cultures we sought to characterize the functional signal pathways that may be involved in the phenotype of these cells. It would not be possible to measure a large number of protein signal pathway endpoints and post-translational modifications by conventional flow cytometry following enzymatic dissociation, even within a hundred spheroids. Consequently we used Reverse Phase Protein Microarray (RPMA) analysis of 59 cell signaling kinase endpoints, representing stem cell markers, autophagy, adhesion, invasion, and pro-survival pathways . RPMA teComparison of the spheroids to the flat, single layer epithelial cells in the same culture revealed a set of activated signaling pathways consistent with a progenitor-type classification. Autophagy markers (Atg5 and LC3B) were elevated in the spheroids in comparison to the epithelial and cuboidal monolayer cells. p38 MAPK Thr180/Tyr182 and SMAD2 Ser465/467, cell signaling proteins associated with survival and stress, were elevated in the spheroids in comparison to the epithelial and cuboidal monolayers. The spheroids exhibited progenitor cell characteristics as evidenced by up-regulation of stem cell markers (CD44), down-regulation of cell adhesion markers (E-Cadherin), up-regulation of invasion related metalloproteinases (MMP14), and up-regulation of COX-2 .To test whether these differences in cell signaling proteins were a stable characteristic of the observed phenotype, we conducted an independent verification analysis. RPMA was performed on a different set of harvested cultured spheroids, epithelial cells, and stromal fibroblasts from the same patient derived cultures that were propagated over several months . Autophain vivo, DCIS cultured organoids, and murine human DCIS xenografts. Intermediate and high-grade DCIS lesions were positive by immunohistochemistry for autophagy pathway proteins Atg5, Beclin-1 and LC3B, which are involved in the nucleation of autophagosomes , which disrupts autophagy, markedly suppressed xenograft tumor formation. Zero of seven DCIS cultured cell strains treated with CQ for greater than 4 days formed tumors, whereas 21 of 27 untreated cultured DCIS cell transplants yielded xenograft tumors . Treatment of organoids or propagated DCIS epithelial cells with CQ markedly suppressed outgrowth, spheroid formation , and indWe examined the cytogenetics of CQ treated organ cultures. The surviving cells after CQ treatment were found to be cytogenetically normal  & S6. ThThe untreated DCIS neoplastic epthelial cells grew in a multilayered colony on autologous stroma, formed spheroids on the surface of the colony  and invaCQ treatment virtually abolished spheroid and 3-D growth. As shown in in vitro anchorage independent, neoplastic epithelial cells that generate 3-D structures including spheroids and duct-like structures. Neoplastic cells with this phenotype can emerge from a high proportion of replicate DCIS lesion samples from the same patient, and can be serially propagated for at least one year. No anchorage independent cells arose from tissue containing histologically verified normal appearing glands and adipose tissue. The anchorage independent epithelial cells were observed to arise from all grades of DCIS including ADH  generation of neoplasms in NOD SCID mice, b) anchorage independent 3-D structures that increase in size and frequency over time, which can be subcultured for at least one year , c) abnoIt is unlikely that the neoplastic cell strains propagated from fresh human DCIS ducts are isolates of micro-invasion areas or invasive cancer in the original histopathology.The patient source material was verified histologically to be devoid of microinvasion or invasive cancer before and after DCIS lesion tissue procurement. The verification was by an independent pathologist with no knowledge of the research findings for the individual specimen. The DCIS lesion source material was evaluated by IHC for type IV collagen and was found to contain an intact basement membrane . If the in vitro culture. The early emergence of chromosomally abnormal cells may be the drivers of the dominant neoplastic phenotype observed during DCIS culture outgrowth.The propagated spheroid forming cells, generated from fresh human DCIS tissue, were cytogenetically abnormal and were associated with xenograft tumor formation. We hypothesize that these cytogenetically abnormal cells pre-exist in the fresh human DCIS duct. This hypothesis was supported by the results of the Illumina 300,000 cytoSNP beadchip comparing the molecular cytogenetics profile of the organoid, the epithelial and cuboidal cultured monolayers, and the spheroids and 3-D structures grown in the same mixed culture. It is well established that the transition from normal to neoplastic growth involves deregulation at various cellular levels including gene-specific mutational events, alterations in signal transduction and growth control pathways, and dysfunctional DNA synthesis and repair mechanisms resulting in the generation of chromosomal abnormalities. At the chromosomal level, it is now accepted that some degree of copy number variation (CNV) and copy-neutral loss-of-heterozygosity (LOH) exists in the normal karyotype Transformed cells containing regions of excessive CNV can change the dosage of key regulatory genes and protein output; while de novo regions of LOH can potentially expose harmful recessive alleles. Of note was the strikingly similar loss on chromosome 6 in the spheroid forming cells of three different patients. This region on chromosome 6 narrowly encompasses the SUPT3H gene . Little is a known about the encoded 399 amino acid protein. It is thought to be a transcription factor, participating in the STAGA complex (SPT3-TAF9-GCN5-acetylase) involved in p53 and c-Myc regulation A strong rationale links autophagy to the survival and invasion of pre-malignant breast cancer. The first link is hypoxia and nutrient stress The data presented herein, strongly support the conclusion that autophagy plays a necessary role in the DCIS cell malignant phenotype:a) autophagy is up-regulated in the in vivo DCIS lesion as shown by immunohistochemistry; b) autophagy is up regulated in the cultured DCIS spheroids and 3-D structures, as shown by immunohistochemistry and immunofluorescence; c) autophagy is up-regulated in mouse xenografts as shown by immunohistochemistry; d) autophagy signal proteins are up-regulated in cultured DCIS spheroids, with validation after long term culture, by reverse phase protein microarray measurement; e) disruption of autophagy by chloroquine phosphate (CQ) completely abrogated xenograft tumor formation; f) CQ completely blocked growth and invasion of DCIS cells on autologous stroma; g) CQ markedly suppressed DCIS spheroid formation and outgrowth in culture for independent experiments; and h) CQ completely eliminated cytogenetically abnormal cells from the DCIS cultured cells. Based on these established mechanistic roles, autophagy constitutes a novel target for treating DCIS and arresting DCIS transition to overt invasion.Autophagy is an established strategy for a cell to avoid apoptosis in the face of oxidative, hypoxic and nutrient deprivation stress. Our results indicate that autophagy is a requirement for the survival and the malignant phenotype (tumorigenicity and invasion) of cytogenetically abnormal cultured human DCIS cells. It is possible that the observed genetic abnormalities directly or indirectly induced the up-regulation of autophagy and thereby promoted survival of the DCIS cells. It is also possible that autophagy was not driven directly by the genetic changes. Instead the malignant precursor cells up-regulated autophagy as a necessary means to generate anchorage independent 3-D structures such as spheroids and pseudo ducts. In either case, the end result is the same: Autophagy is required for the observed phenotype.in vitro in tamoxifen resistant breast carcinoma cells by blocking autophagy dependent cell survival Chloroquine phosphate, which suppressed or abolished the anchorage independent, cytogenetically abnormal anchorage independent neoplastic DCIS cells, is an orally administered small molecule inhibitor which blocks the autophagy pathway by accumulating in autophagosomes and inhibiting autophagosomal formation/function et alThese data provide additional evidence that the local ductal tissue microenvironment influences the progression of in situ carcinomas. Breast stromal cells Even though the cytogenetically abnormal DCIS epithelial cells we have derived appear to have the characteristics of progenitor cells by RPMA analysis , our metex vivo model to study the biology of DCIS. Moreover, the yield, and properties, of progenitor-like cells from samples of human DCIS lesions following in vivo treatment of DCIS can be used to screen the effectiveness of an in vivo therapy. Finally, autophagy constitutes a new treatment target for DCIS.In conclusion, fresh human DCIS organoid culture reveals cytogenetically abnormal, neoplastic, anchorage independent cells that require autophagy for survival. Invasive neoplastic cells may pre-exist within the human breast DCIS duct but are apparently held in check by the ductal niche, and can be coaxed to emerge in organ culture when the duct is cut open. Blockade of the autophagy pathway abolishes the propagation, invasion, and 3-D growth of the DCIS neoplastic cells. The reproducible derivation of cytogenetically abnormal cells with fresh human DCIS lesions provides a new This study was conducted according to the principles expressed in the Declaration of Helsinki, with written informed patient consent. The study was approved by the institutional review boards of Inova Fairfax Hospital and George Mason University. Animal care and use was approved by Institutional Animal Care and Use Committee of George Mason University and the US Department of Defense according to (a) DOD Directive 3216.1, “The Use of Laboratory Animals in DOD Programs”, (b) US Army Regulation 40–33, “The Care and Use of Laboratory Animals in DOD Programs”, and (c) Animal Welfare Regulations .Fresh, sterile breast DCIS tissue was obtained from patients undergoing standard of care surgery for suspected or biopsy confirmed neoplasia at Inova Fairfax Hospital, Falls Church, VA . Written2, containing one or more discernable duct segments with associated stroma. Dissected breast ductal tissue was grown in serum free DMEM/F12 medium supplemented with human recombinant EGF , insulin , streptomycin sulfate  and gentamicin sulfate , with or without 0.36% (v/v) murine Engelbreth-Holm-Swarm (EHS) derived, growth factor reduced, basement membrane extract  at 37°C in a humidified 5.0% CO2 atmosphere. Medium was replaced three times per week. Organoids were submerged in a minimum volume of medium (just enough to cover the duct fragments) to maximize gas exchange. Non-adherent organoids were removed from the culture flask. Cultures were maintained continuously for up to one year. Periodically, organoids were removed, under microscopic visualization, for propagation into new culture flasks or phenotypic and molecular analysis.The tissue was dissected into organoids approximately 3 mmAutophagy was inhibited in organoid cultures by treating cultures with chloroquine diphosphate (CQ)  in DMEM/F12 medium as described above. CQ-containing medium was replaced three times per week for a period of 6 months. Comparable untreated control cultures were maintained in identical medium lacking chloroquine with similar media changes.Prkdcscid/NCrHsd; Harlan or The Jackson Laboratory) with a 17β Estradiol pellet . The DCIS organoids, prior to transplantation into the murine mammary fat pad, were incubated in DMEM/F12 medium supplemented with EGF, insulin, streptomycin, gentamicin, or estrogen (Sigma) for 6 to 18 hours. The study was conducted under a contract with Biocon Inc., Rockville, MD.Human DCIS tumor tissue, propagated organoid tissue, or spheroids generated from primary DCIS organoid cultures were transplanted transdermally into the mammary fat pad of female NOD/SCID mice  twice weekly. Mice exhibiting evidence of tumor growth were sacrificed as necessary or after 120 days. Complete necropsies were performed for evidence of metastasis. Tumor tissue, when present, was either excised for immunohistochemical analysis or injected into a different NOD SCID mouse for propagation of the tumor.Formalin fixed murine tissue or DCIS organoids were processed and paraffin embedded by commercial laboratories . Formalin fixed paraffin embedded (FFPE) tissue sections (5 µm or 1 µm thickness) mounted on positively charged glass slides were baked at 56°C for 20 min., deparaffinized in xylene and rehydrated in a series of graded alcohols  with a final rinse in wash buffer . Immunostaining was performed on a Dako Autostainer with an Envision+HRP staining kit (Dako) per manufacturer's instructions . StainedSpheroids were aspirated directly from the culture flask under direct microscopic visualization, mounted on positively charged glass microscope slides, fixed in 16% paraformaldehyde (Fisher Scientific), and stored dessicated at 4°C. FFPE murine xenograft tissue sections were deparaffinized in xylene, and rehydrated in graded alcohols. Spheroids and FFPE sections were incubated at room temperature with anti-human specific epithelial antigen conjugated to FITC  , or mouse immunoglobulin IgG1 as a negative control (Dako). Slides were rinsed in borate buffer pH 8, then nuclear counterstained with ProLong Gold+DAPI (Invitrogen). Images were captured with a Nikon Eclipse C1si confocal microscope in different channels for EpCAM-FITC  and DAPI  using the 20× objective.LysoTracker Red  and nuclear counterstain Hoechst 33258 pentahydrate  were added to DMEM/F12 culture medium as described above and incubated with live human DCIS organoid cell cultures for 0.5 hour. Medium containing dye was removed and replaced with fresh medium. Images were captured with either a Nikon Eclipse C1si confocal or a Nikon Eclipse TE200 microscope in different channels for LysoTracker Red  and Hoechst 33258 (pseudo-colored blue) using either the 10× or 20× objective.Cellular outgrowths were removed from the culture flask by scraping or aspiration with a pipette and were spun briefly to pellet the cells. Medium was removed by aspiration and the cell pellet was subjected to lysis with a 10% (v/v) solution of Tris(2-carboxyethyl)phosphine  in Tissue Protein Extraction Reagent /Tris-glycine 2X SDS buffer (Invitrogen). Cell lysates were stored at –80°C prior to microarray construction. Cellular lysates were printed on glass backed nitrocellulose array slides  using an Aushon 2470 arrayer  equipped with 350 µm pins as previously described Total protein per microarray spot was determined with a Sypro Ruby protein stain (Invitrogen/Molecular Probes) per manufacturer's directions and imaged with a CCD camera .Cellular outgrowths were removed from the culture flask by scraping or aspiration with a pipette and were spun briefly to pellet the cells. Culture medium was removed by aspiration and the cell pellet was immediately frozen on dry ice and stored at –80°C prior to nucleic acid extraction. Nucleic acid preparations derived from human breast tissue and/or cell culture out growths were tested using quantitative PCR (qPCR), PicoGreen (Invitrogen) staining and fluorometry . Microarray-based genomic analysis was performed using CytoSNP-12 beadchips  and analyzed on an Illumina BeadStation 500 GX laser scanner The DNA extraction and purification was performed using a DNA purification column . Approximately 200 ng of DNA at a concentration of 50 ng/µL was amplified, fragmented, precipitated, re-suspended, and hybridized to the Illumina CytoSNP-12 beadchips. After single-base extension, sample DNA was stained and the chip was washed, dried, and scanned for the resulting 300,000 SNP calls and copy number values.Raw fluorescence data was converted to genotypic data using the Illumina GenomeStudio software program. Data analysis was performed using the Illumina KaryoStudio software program that converts genotypic and signal intensity data into a “molecular karyotype” showing B allele frequency, Log R ratio, LOH score and Copy Number Score. Log R ratio, which is the log (base 2) ratio of the normalized R value for the particular SNP divided by the expected normalized R value, was employed. The red line in the log R plot indicates a smoothing series with a 200 kb moving average window. Thus, a Log R Ratio\2 was considered to represent a true amplification and Log R Ratio\-1.5 was considered to represent a probable homozygous deletion. Additionally, B allele frequency data was used to identify regions of copy-neutral and hemizygous LOH.Standard deviation (SD) or standard error of the mean (SEM) was calculated for small group comparisons. The Student t-test, two tailed with Welch's correction, was used to calculate the p-value, and to determine the statistical difference of epithelial outgrowth area before and after CQ treatment and spheroid generation. P values <0.05 were considered significant . Wilcoxon rank sum was used to determine the differences between CQ treated and untreated groups for the reverse phase protein arrays . A p = 0.1 was considered different for small sample sizes.Figure S1www.cellsignal.com).Macroautophagy cell signaling pathway. Autophagy  is a catabolic process that can either maintain cellular homeostasis or result in cell death. Intracellular signaling kinases such as AKT, PI3 Kinase, ERK, Bcl-2, and mTOR regulate autophagy. Reverse phase protein microarrays (RPMA) were employed in the present study to evaluate the activation (phosphorylation) of signal pathway proteins that are associated with autophagy. Pathway diagram reproduced courtesy of Cell Signaling Technology, Inc. ((0.93 MB TIF)Click here for additional data file.Figure S2Human DCIS xenograft tumor histology. H&E staining of mouse xenograft tumor derived from fresh human DCIS cells. (A) Human DCIS explant derived mouse tumor (20x). (B) Xenograft tumor is comprised of pleomorphic epithelial cells with prominent nucleoli and partial ductal/glandular differentiation directly abutting or invading surrounding stroma (40x).(7.27 MB TIF)Click here for additional data file.Figure S3Molecular karyotype of chromosome 6 from human cultured DCIS cells shows a deletion p21.1/12.3. The upper panels show the log R ratio plots from 3 different patients . These data represent DNA ploidy, or copy number, for the displayed chromosomal region with the red line indicating the statistical average value. A log R ratio of 0.0 equals a DNA copy number of 2 (diploid). Deflection downward of the red line indicates loss of DNA copy number. Each blue dot represents the log R ratio value for each SNP. The shaded regions represent segments of DNA deviating from a copy number of 2 as determined by the Illumina GenomeStudio 2.0 software. The software uses both quantitative fluorescence intensity and qualitative genotypic data for determining copy number values. The color code is as follows: orange indicates a region of 1 copy; red, 0 copies; blue, 3 copies; purple, 4 or more copies; and green, copy-neutral LOH (2 copies). The center panel shows the chromosomal ideogram indicating cytological bands with the centromere in red. The small window shows the region expanded in the figure and the nucleotide positions for this region are shown below the ideogram. The lower panel shows the cytogenetic bands and genetic map for genes located in the expanded region. Note that the region of the deletion for these 3 patients (orange) corresponds to the transcript for SUPT3H.(0.18 MB TIF)Click here for additional data file.Figure S4Molecular karyotype of chromosome 5 from chloroquine treated or untreated cultured human DCIS cells. The upper panel shows log 2 ratio plots of 2 different samples from the same patient . In the upper panel, the top plot shows the log R ratio from chloroquine treated human DCIS cell cultures showing normal ploidy, while the lower plot shows a number of extended regions of gain and loss of content on chromosome 5. The color code is as follows: orange indicates a region of 1 copy; red, 0 copies; blue, 3 copies; purple, 4 or more copies; and green, copy-neutral LOH (2 copies). Blue and purple regions show an increase of copy number extending from nucleotide position ∼31 Mb to ∼43 Mb  affecting the dosage of numerous genes. Additional regions of copy number gain are present distally, including subtelomeric regions. Extended regions of copy number loss are indicated in orange (one copy) and red (0 copy). The lower panel shows the cytogenetic banding pattern and the corresponding nucleotide positions beginning with the p-telomere.(0.22 MB TIF)Click here for additional data file.Figure S5Allele frequency of chromosome 17 q-arm for spheroids from DCIS organoid culture. Molecular karyotype of chromosome 17 q arm. The upper panel shows log R ratio and B allele frequency plots of genomic DNA from an organoid sample (case 09-148 spheroids/3-D structure). The top plot shows an upper deflection of the red averaging line indicating a gain in copy number (blue) spanning ∼14 Mb of chromosomal content. This is the largest of multiple gain of copy number regions on the q-arm in this neoplastic sample (data not shown). The bottom plot shows the B allele frequency data for this same region. Within the blue shaded region, the B allele frequency data for heterogeneous SNPs  is split into 2 lines at values above and below 0.5, indicating the presence of 3 copies of DNA in this region, consistent with the log 2 ratio data shown above. The bottom panel shows the chromosome 17 ideogram with the expanded region outlined below. The gain of DNA copy number extends from q22 to q24.3.(0.15 MB TIF)Click here for additional data file.Figure S6Molecular karyotype of chromosome 6 from chloroquine treated or untreated cultured human DCIS cells. Molecular karyotype of chromosome 6 from chloroquine treated cultured DCIS epithelial monolayer devoid of spheroids and untreated spheroids for case 09-148. Molecular karyotype of chromosome 6; p21.1/p12.3: The upper panel shows the log R ratio plots from 2 different samples from the same patient. In the upper panel, the top plot shows the log R ratio from cell cultures treated with chloroquine phosphate showing normal ploidy, while the lower plot shows a hemizygous deletion (orange) of the SUPT3H locus in cultures displaying spheroid and 3-D structures. This data represents DNA ploidy, or copy number, for the displayed chromosomal region with the red line indicating the statistical average value. A log R ratio of 0.0 equals a DNA copy number of 2 (diploid). Deflection downward of the red line indicates loss of DNA copy number. Each blue dot represents the log R ratio value for each SNP. The shaded regions represent segments of DNA deviating from a copy number of 2 as determined by the Illumina GenomeStudio 2.0 software. The software uses both quantitative fluorescence intensity and qualitative genotypic data for determining copy number values. The color code is as follows: orange indicates a region of 1 copy; red, 0 copies; blue, 3 copies; purple, 4 or more copies; and green, copy-neutral LOH (2 copies). The center panel shows the chromosomal ideogram indicating cytological bands with the centromere in red. The small window shows the region expanded in the figure and the nucleotide positions for this region are shown below the ideogram. The lower panel shows the cytogenetic bands and genetic map for genes located in the expanded region.(0.58 MB TIF)Click here for additional data file.Figure S7Signal pathway alterations induced by chloroquine treatment of cultured DCIS spheroids. Complete set of cell signaling kinases measured by reverse phase protein microarray for the subset shown in (0.46 MB TIF)Click here for additional data file.Figure S8Basement membrane remains intact in DCIS cultured organoids. Collagen type IV immunohistochemistry demonstrates intact basement membrane surrounding breast ducts (brown staining). (A) Case 08-352 FFPE primary breast tissue at time of procurement. Scale bar 100 µm, magnification 10×. (B) The basement membrane remains intact surrounding the duct that is within the tissue fragment and surviving after the breast organoid was growing in culture for 12 weeks .(2.39 MB TIF)Click here for additional data file.Table S1Validated primary antibodies for Reverse Phase Protein Microarray analysis.(0.03 MB XLS)Click here for additional data file.Table S2Verification of DCIS progenitor cell phenotype: Reverse Phase Protein Microarray analysis of DCIS cultured epithelial cells, spheroids and stromal fibroblasts.(0.04 MB XLS)Click here for additional data file.Table S3Primary antibodies and antigen retrieval conditions for immunohistochemistry.(0.03 MB DOC)Click here for additional data file.


A patient with chemical corneal burn presented two months after the acute episode of chemical injury to his right eye (OD) and was diagnosed with severe limbal stem cell deficiency and with vascularized corneal opacity OD. Limbal cell transplantation and penetrating keratoplasty (PKP) was performed. The autologous and allograft limbal tissue included peripheral cornea, limbus and conjunctiva obtained from contralateral eye and cadaveric eye bank cornea. Central corneal button was used for a PKP with 32 intermittent sutures. One year after the procedure, the corneal surface remains clear with a best corrected visual acuity of 6/12 (-2.00 DS / -2.75 DC-/ 150°. Eighteen sutures are still in place; no vascularization has extended beyond the host graft junction. Ocular surface is wetting well with no filamentary keratitis.Combined autologous and allograft limbal cell transplant can be performed for severe deficiency of corneal stem cells in a patient with chemical corneal burn. Chemical burns of the eye are one of the leading causes of visual disability. Chemical burns, particularly alkali burns lead to gross limbal stem cell deficiency. Limbal dThis case report describes a patient with alkali burn induced severe stem cell deficiency and vascularized corneal opacity, treated with combined autologous and allograft limbal cell transplantation and penetrating keratoplasty.A 24-year-old patient sustained alkali burn two month prior to his presentation to our clinic. He had been managed with topical antibiotics, lubricants and steroid. Amnion grafting had been done to protect and heal the corneal surface. At presentation, he complained of severe pain, redness, watering and poor vision in the right eye (OD). There was no significant past history of ocular or systemic disease.Ophthalmic examination revealed an unaided vision of 6/60 OD with no further improvement with refraction. There was narrowing of the palpebral aperture with fornix cicatrization. Slit lamp examination revealed bulbar and ciliary congestion, 360° superficial and deep corneal vascularization with vessels extending 4–5 mm from the limbus into the center of the cornea Figures  and 2. TPre-operatively, anesthetic clearance was obtained and informed consent was taken from the patient and all surgical risks were explained to the patient including graft rejection.The surgical technique included the following steps:The donor tissue consisting of corneal-limbal-conjunctival explant , 2–clock hours each (11–1 o′clock and 5–7 o′clock hours), were harvested from the contralateral eye and the eye bank cornea.An 8.5 mm donor corneal graft was obtained from the same donor eye bank cornea.Limbal stem cell harvest: A double-edged calibrated diamond knife was set to 125 µm, and used to make a circumferential corneal incision parallel to the limbus, and two radial incisions were then made extending from either ends of the limbal tissue to beyond the limbus under the conjunctiva. Three millimeters of the conjunctiva, attached to the corneal and limbal explants along the limbal border, was then excised using a Barron Hess burg disposable donor punch to obtain an 8.5 mm donor corneal button. The peripheral corneal and conjunctiva tissue from the eye bank cornea was used to develop a 3 × 5 mm corneal–limbal–conjunctival graft tissue using the degraded diamond knife and lamellar dissector.Preparation of recipient bed: The conjunctiva at the limbus was incised 360° and hemostasis was achieved using bipolar cautery. Abnormal corneal epithelium and the superficial fibro-vascular scar tissue were removed using a blunt dissector. The diamond knife was set at 75 microns and a bed corresponding in dimension to the donor explants was fashioned at the recipient site(s). The donor limbal graft was sutured onto the recipient eye with two interrupted 10-0 monofilament nylon sutures at the corneal margin and two along the scleral edge of the explants. The conjunctiva of the recipient eye was approximated to the donor conjunctiva with 8-0 Vicryl sutures, taking a bite into the episclera. An 8 mm Barron Hessburg punch was used to make a corneal punch at the recipient site and anterior chamber was entered using a #15-blade. The corneal button was removed using corneal scissors. The 8.5 mm donor cornea was then sutured to the recipient bed using 32 interrupted 10-0 monofilament nylon sutures.Postoperatively, topical prednisolone 1% eye drops four times; moxifloxacillin eye drops four times, preservative free topical lubricants and ointment TobraDex at night was commenced.Oral prednisolone (1 mg/kg body weight) was administered for 1 week and tapered over 6 weeks. Follow-up checks were performed on day 1, 2, 3, 4, 10 and 14, and then after 1 month, and monthly for 1 year. Sutures were removed 4 months postoperatively, whenever they got loose and collected mucus on them, unaided and best-corrected vision, slit lamp examination, IOP measurement using the Tonopen and dilated fundus examination was performed during each visit.No intraoperative complications occurred. Postoperatively, the limbal graft showed epithelial outgrowths within the first two days and almost 90% corneal surface was completely epithelialized within two weeks . There wVarious techniques of limbal transplantation have been reported. All these procedures remove the host′s altered corneal epithelium and provide a new source of epithelium for the diseased ocular surface. From the donor tissue obtained from the fellow eye (auto graft) and/or cThis study describes a patient who sustained alkali burn OD, developed severe limbal ischemia and corneal vascularization with repeated corneal epithelial defect and underwent a combination of auto and allograft limbal stem cell transplant along with penetrating keratoplasty (PKP). In this case, the clinical features were suggestive of limbal stem cell ischemia; however, an impression cytology for Periodic Acid Schiff stain (PAS) for conjunctival goblet cells and immunohistochemistry analysis for cytokines CK 19 and CK 3 confirms limbal cell ischemia.‐7 SuperfIn conclusion, the use of autologous and allograft limbal transplantation with corneal, scleral and conjunctival carriers was useful for ocular surface reconstruction and graft survival in-patient with chemical burn and severe limbal stem cell loss.Adequate immunosuppression was required in the initial period to prevent graft rejection.


A common complication associated with diabetes is painful or painless diabetic peripheral neuropathy (DPN). The mechanisms and determinants responsible for these peripheral neuropathies are poorly understood. Using both streptozotocin (STZ)-induced and transgene-mediated murine models of type 1 diabetes (T1D), we demonstrate that Transient Receptor Potential Vanilloid 1 (TRPV1) expression varies with the neuropathic phenotype. We have found that both STZ- and transgene-mediated T1D are associated with two distinct phases of thermal pain sensitivity that parallel changes in TRPV1 as determined by paw withdrawal latency (PWL). An early phase of hyperalgesia and a late phase of hypoalgesia are evident. TRPV1-mediated whole cell currents are larger and smaller in dorsal root ganglion (DRG) neurons collected from hyperalgesic and hypoalgesic mice. Resiniferatoxin (RTX) binding, a measure of TRPV1 expression is increased and decreased in DRG and paw skin of hyperalgesic and hypoalgesic mice, respectively. Immunohistochemical labeling of spinal cord lamina I and II, dorsal root ganglion (DRG), and paw skin from hyperalgesic and hypoalgesic mice reveal increased and decreased TRPV1 expression, respectively. A role for TRPV1 in thermal DPN is further suggested by the failure of STZ treatment to influence thermal nociception in TRPV1 deficient mice. These findings demonstrate that altered TRPV1 expression and function contribute to diabetes-induced changes in thermal perception. Diabetic peripheral neuropathy (DPN) is a common chronic complication of diabetes mellitus . Symptom2+ permeant non-selective cation channel expressed predominantly by unmyelinated C-fibers and thinly myelinated Aδ fibers and plays a major role in inflammatory thermal sensation -RTX binding assays in DRG obtained from STZ treated diabetic or transgenic diabetic or vehicle injected non-diabetic mice  and decreased in hypoalgesic mice . The DRG obtained from hypoalgesic transgenic diabetic mice showed lower  [3H]-RTX binding as compared to control mice).Changes in capsaicin-induced currents may result from altered receptor sensitivity as well as expression. Therefore, to confirm that the TRPV1 protein content is different, TRPV1 levels were quantitated by using [ice Fig.  Compared3H]-RTX binding assays  and lower in hypoalgesic mice  as compared to non-diabetic control mice (188.8 ± 26.5 fmol/mg tissue). The paw skin of hypoalgesic transgenic diabetic mice showed lower [3H]-RTX binding  as compared to paw skin harvested from control nondiabetic mice. Thus, changes in TRPV1 protein expression at both the peripheral nerve terminals and DRGs may contribute to the abnormal thermal sensitivity observed in diabetic mice. As the Kd of this radio ligand in most tissues is ~0.1 nM, it is estimated that > 90% of the available receptors are labeled with this concentration [3H]-RTX reflects an increase in the total TRPV1 levels.Alterations in DRG TRPV1 expression may be secondary to changes that occur in the periphery. To determine the state of TRPV1 protein expression in the periphery, we quantitated TRPV1 levels in paw skin using -RTX binding results and further examine TRPV1 expression, immunohistochemistry was performed on spinal cord, DRG and paw skin tissue harvested from STZ treated or transgenic diabetic hyperalgesic, hypoalgesic mice and age matched non-diabetic mice (Ins-HA.D2). TRPV1 is expressed in the central terminals of DRG neurons, which synapse with the second order dorsal horn neurons at laminae I and II of the spinal cord  can manifest as focal, autonomic, or peripheral sensory neuropathy . AlteredSTZ-induced diabetic mice exhibited two phases of altered thermal pain sensitivity, an initial transient phase of hyperalgesia followed by a phase of hypoalgesia. Similarly, when subjected to the hot plate test, transgenic diabetic mice exhibited two phases of altered thermal pain sensitivity. However, the transition to different phases was not uniform in these animals. Careful examination of thermal sensitivities of individual mice over the course of the study allowed for classification of three unique phenotypes. One group exhibited thermal sensitivities similar to those observed with STZ treated mice. Thus, phenotype 1 was characterized by a period of hyperalgesia followed by a sustained phase of hypoalgesia. A second group of diabetic TCR-SFE/Ins-HA mice exhibited thermal hypoalgesia at approximately the same age as observed for phenotype 1 mice but lacked an early hyperalgesic phase (phenotype 2). The third group of animals became slightly hyperalgesic and remained hyperalgesic during the course of the study (phenotype 3). It is unclear why these unique thermal sensitivity phenotypes are observed among syngenic mice with uniformly dysregulated glucose control. Together these behavioral studies show altered heat sensation, a finding in diabetic neuropathy, is present in our animal models ,49.We tested the hypothesis that the altered pain sensation is due to changes in TRPV1 because the altered temperature sensitivity corresponds to the temperature range that activates TRPV1. We provide four lines of evidence to support the involvement of TRPV1. First, thermal sensitivity was not altered among STZ-induced diabetic TRPV1 deficient mice demonstrating the thermal sensitivity in diabetic mice is dependent on TRPV1. However, a significantly higher PWL was observed in TRPV1 knock-out mice as compared to wild type mice with similar back ground strain suggesting that TRPV1 knock-out mice has deficiency in acute thermal pain sensation Fig. .Second, TRPV1 mediated capsaicin induced currents in DRG neurons collected from mice exhibiting hyperalgesia or hypoalgesia were significantly larger or smaller, respectively, compared to currents obtained from euglycemic control mice. The change could be as a result of enhanced expression or sensitivity. The increase was observed in saturating concentrations of capsaicin confirming increased TRPV1 protein levels rather than as a result of increased sensitivity due to phosphorylation of TRPV1 .Third, RTX binding assays demonstrated that TRPV1 expression parallels thermal sensitivity. DRG neurons from hyperalgesic mice bound more RTX and hypoalgesic mice bound less RTX than DRGs from age-matched euglycemic control mice. It is conceivable that peripheral TRPV1 expression might influence expression in DRG; therefore, we determined TRPV1 levels from the paw skin. Peripheral TRPV1 expression mirrored that observed in DRG neurons. Thus, changes in TPRV1 expression parallel the thermal sensitivity observed among diabetic mice and are seen both at peripheral tissue and in DRG.Fourth, we used immunohistochemical localization of TRPV1 in DH of the spinal cord, DRG neurons and the paw skin to confirm changes in TRPV1 expression detected in RTX binding assays and to determine if increased RTX binding was the result of ectopic TRPV1 expression (on neurons other than C and Aδ fibers). In the DH, there was no obvious co-localization of TRPV1 with the neuronal marker NeuN, suggesting that the distribution of TRPV1 in DH is predominantly in fibers projecting from DRG rather than in the cell body of DH neurons. We observed a significant increase in expression of TRPV1 in DH, DRG and paw skin from hyperalgesic diabetic mice as compared to age-matched control mice. Significant decreases were also observed among tissues harvested from hypoalgesic diabetic mice. Furthermore, the number of neurons expressing TRPV1 is altered in tissues harvested from hypoalgesic mice, but the total number of neurons remains unchanged. This is important to address because it has been suggested that there may be neuronal loss in diabetes leading to altered thermal sensitivity . We founIn diabetes, the link between abnormal thermal sensitivity and hyperglycemia resulting from insulin deficiency is not clear. Here, we have shown that TRPV1 is a major player, but questions still exist with respect to how TRPV1 function and expression are modulated in diabetes. Previously, we showed that insulin and IGF-1, acting via the IGF-1 receptor, potentiates TRPV1 current by activating PKC . Based oWe have recently showed Increased sensitivity of TRPV1 in STZ-induced diabetic mice has been shown that increased thermal sensitivity results from sensitization of TRPV1, possibly through PKC ,41,54. ITRPV1 is present in areas that are not exposed to sufficient changes in temperature to cause channel activation and is widely believed to have functions other than temperature sensation. TRPV1 is detected throughout the neuroaxis by RT-PCR  and iden


Several chronic respiratory diseases exhibit hyperactive immune responses in the lung: abundant inflammatory mediators; infiltrating neutrophils, macrophages, lymphocytes and other immune cells; and increased level of proteases. Such diseases include cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) and severe/neutrophilic asthma. Paradoxically, patients with these diseases are also susceptible to detrimental bacterial infection and colonization. In this paper, we seek to explain how a positive feedback mechanism via IL-8 could lead to desensitization of epithelial cells to pathogen recognition thus perpetuating bacterial colonization and chronic disease states in the lung. Such insight was obtained from mathematical modeling of the IRAK/TRAF6 signaling module, and is consistent with existing clinical evidence. The potential implications for targeted treatment regimes for these persistent respiratory diseases are explored. Human epithelial cells are the first line of defense against external pathogens and particulates. The innate immune responses mediated by these cells are important for host defense against bacteria and viruses The IRAK4/IRAK1 complex is recruited to activated receptors by stimuli such as cytokines, bacteria, or viruses through the interaction of IRAK4 with MyD88 and additional partners. It is phosphorylated at multiple residues to produce activated IRAK1 proteins. The activated IRAK1 then forms a complex with TRAF6 to activate downstream MAPK and NF-kappaB cascades and subsequently induce the production of chemokines (for example IL-8), cytokines and antibacterial peptides. The level of activated IRAK1 proteins is controlled by the rapid ubiquitin-targeted degradation of phosphorylated IRAK1. This key signaling component in innate immunity will be referred as the core IRAK/TRAF6 module in this paper .IL-8, a major chemoattractant for neutrophil recruitment, is produced by epithelial cells upon IRAK/TRAF6 activation. A positive feedback loop from IL-8 signals to NF-kappaB though the IRAK module has been reported A detailed schematic model of the TLR and IL-1 signaling cascades is available and the robustness of the IRAK signaling has been discussed previously Intrigued by the reported IL-8 positive feedback loop to IRAK signaling by Monna, et al Depending on the initial cellular states at the time of stimulation, this signaling module would respond to IRAK4 activation by external stimuli  with different outcomes in time series analysis. Given a pulse-like stimulus from baseline (at 0), the cells can either induce a response in IL-8 level that returns to the initial state of 0 (The parameters that impact the state switches between “resolved” (State 0) and “self-reinforced” (State 1) of final IL-8 levels are examined through both sensitivity analyses (data not shown) and parameter scans  using MaThere are many possible intracellular or extracellular factors that could impact these parameters to create a heightened innate immune response: viral/bacterial infections An interesting result arose when we introduced a spike of external stimulus as an input for the IRAK/TRAF6 module at State 1  than by non-CF cells . This is consistent with the observation that activity of NF-kappaB . Higher rates of production of IL-8 were observed in cells from smokers with COPD, and persistent inflammation is associated with COPD long after smoke cessation. A higher number of severe asthma patients, compared with mild asthma patients, exhibit a disease phenotype of persistent high IL-8 level, higher neutrophil counts, and higher bacteria endotoxins in their induced sputum In summary, these disparate chronic respiratory diseases have a common theme in their disease manifestation: increased IL-8 levels, increase neutrophils, and ineffective host defense to certain pathogens. These observations suggest this model is operative in these diseases.To mount a defense against pathogens or respond to other damaging or noxious stimuli, the hosts' innate immune systems need to achieve allostases; in other words, maintain sufficient innate response yet not become hyper-responsive or desensitized to microbial recognition. For the species to survive these pathological or physiological insults, there is substantial evolutionary and population diversity for these sensitive parameters in and around the IRAK/TRAF6 module. Our model predicts that the qualitative diversity in these parameters affect the host's responsiveness towards environmental pathogens Genetic variation in signaling intermediates, frequency or severity of bacterial/viral infections, exposure to tobacco smoke, aging, or the presence or absence of alternative cytokine profiles are all possible susceptibility factors that can contribute to the changes in the identified sensitive parameters in the IRAK/TRAF6 signaling module. In cystic fibrosis, COPD and neutrophilic asthma, chronic inflammation and damage to the mucosal epithelial barrier could predispose patients to bacteria infection . The firStaphylococcus aureus signal though IRAK-dependent TLR2 Pseudomonas aeruginosa is recognized through IRAK-dependent TLR5 signaling S. aureus and P. aeruginosa) as are IRAK4-deficient children with the non-responsive IRAK/TRAF6 signaling Paradoxically, our model also predicts that constitutively expressed IL-8 could desensitize the TLR-IRAK-TRAF6 signaling. Innate immune responses to Our exploration of the IRAK/TRAF6 signaling module has been focused primarily on specific cell types, specifically epithelial cells and recruited neutrophils. However, there is obviously substantially more complexity in the extracellular signals and cell types that affect the epithelial environment, including monocytes, dendritic cells, mast cells, macrophages, and various lymphocytes P. aeruginosa in cystic fibrosis patients. Such a result can be explained by this model. Blocking neutrophil elastase would reduce the feedback from IL-8 to the IRAK/TRAF6 module by reducing the protease cleaved CXCR1 fragments, which signal to epithelial TLR2 to activate the IRAK/TRAF6 module. This model also helps to explain why impaired bacterial killing only impacts the clearance of certain bacteria in cystic fibrosis. In addition to topical treatments, our model also predicts that well-dosed systemic administration of drugs that reduce chemokine production , could increase bacteria clearance and rescue from these particular disease states. Given the broad range of inflammatory diseases potentially associated with this mechanism, these results have important therapeutic implications in the treatment of chronic respiratory diseases including cystic fibrosis, COPD and neutrophilic asthma.The underlying mechanisms of the aforementioned diseases suggest that their treatment should be distinguished from diseases with similar symptoms but with different mechanisms. In chronic respiratory diseases characterized by both chronic neutrophilic inflammation and bacteria infection, this model suggests that epithelial cells may be trapped in a self-reinforcing positive feedback loop. Endogenous counter-regulatory pathways could be insufficient to resolve pro-inflammatory signaling. There would therefore be a need for external therapeutic interventions that break such a cycle. For example, Hartl et al www.sbml.org) package. The initial concentrations of the model species were mostly estimated. The initial parameters were either estimated or constrained using literature or experimental data −1 and the Km defined in µM−1. The state presented in the attached model is prone to the switch between State 0 and State 1.The IRAK/TRAF6 model was constructed in SBML  using JDTime series analyses of the model were performed in either SBW JDesigner or Matlab Simbiology. Sensitivities of parameters in regards to IL-8 level  in the systems were identified using the MatLab SimBiology module with the Sundial solver. Parameter scans on all the individual parameters were performed using the Jarnac simulation service in the SBW package. Those sensitive parameters that exhibited a change from/to 0 in IL-8 levels in the scanned ranges were then simulated close to the switching range in the Matlab Simbiology parameter scan. In addition to the parameters identified in th, 2008.Text mining of diseases associated with IL-8 constitutive levels or expression were performed using MedScan 1.0 (available from Ariadne 5.0) looking for either co-occurance or Ariadne-defined relationships. Only those diseases with at least 2 relevant references associated with IL-8 were selected. The published network was generated based on the first 10000 PubMed abstracts downloaded with the search terms “Interleukin-8” AND (“constitutive” OR “level” OR “expression”) on September 18Model S1The IRAK/TRAF6 model in the SBML format. Quantitative  enzyme kinetics data and literature knowledge on structure of the signaling pathways were incorporated to construct a parameterized IRAK/TRAF6 model (0.01 MB ZIP)Click here for additional data file.