Finally we achieved efficient tumor engraftment in recipient NOD/SCID mice, also upon inoculation of small number of cells, thus suggesting indirectly the presence of tumor initiating cells.Malignant pleural effusions (MPEs) could represent an excellent source to culture a wide variety of cancer cells from different donors. In this study, we set up culture conditions for cancer cells deriving from MPEs of several patients affected by the most frequent form of lung cancer, namely the subset of non small cell lung cancers (NSCLC) classified as Lung Adenocarcinomas (AdenoCa) which account for approximately 40% of lung cancer cases. AdenoCa malignant pleural effusions gave rise to in vitro cultures both in adherent and/or in spheroid conditions in almost all cases analyzed. We characterized in greater detail two samples which showed the most efficient propagation in vitro. In these samples we also compared gene profiles of spheroid  In recent years methodological approaches to study cancer have been deeply influenced by two major emerging concepts: a) cancer is not a single disease but rather a heterogeneous composition of multiple diseases, where we are just starting to distinguish and classify subtypes characterized by distinct genetic and epigenetic changes and by the activation of different signal transduction pathways and transcriptional programs Although evidence has been provided that this approach can be applied to obtain interesting correlations with clinical outcome in solid cancers vs non adherent spheroid cultures without applying prior enrichment for surface markers. By analyzing the global pattern of gene expression we also started to derive a signature distinctive of spheroid vs adherent AdenoCa cultures. Finally we demonstrated efficient engraftment in vivo in mice and the establishment of tumors which reproduce the histological features of the primary human tumors.Lung cancer is the leading cause of cancer mortality in the world. Current therapy is relatively ineffective and survival rate at 5-years remains only 15% for advanced disease We selected 15 cases of malignant pleural effusion obtained from patients with lung adenocarcinoma at the Department of Thoracic Surgery, University of Rome La Sapienza, Sant'Andrea Hospital. After thoracentesis, all specimens were received as fresh effusion, usually within one hour following collection, with volumes ranging from 40 to 1,500 ml. After centrifugation for 5 min at 1,500 rpm, the supernatant was decanted and the sediment resuspended in 30 ml of CytoLyte Solution . The specimen was then recentrifuged for 10 min at 1,500 rpm and the sediment resuspended in 20 ml of PreservCyt solution (Cytyc Corp.). A monolayer slide was made using the Thin Prep 2000 Processor (Cytyc Corp.). From each specimen, 5–10 slides were prepared. Of these, 1 or 2 were stained using the Papanicolaou method, and viewed independently to confirm the diagnosis and presence of cells suitable for evaluation. Tumor type was determined according to cytomorphology parameters: solitary and clusters of medium to large sized cells, hyperchromatic and eccentrical nuclei, conspicuous nucleoli and variable cytoplasmatic vacuolization. Malignant pleural fluid cells were also submitted to immunocytochemistry in order to confirm Adenocarcinoma cell origin. Diagnoses were considered ‘true positives’ if they were inagreement with the consensus cytological diagnosis.Experiments were approved by the Sant' Andrea Hospital Ethics Committee 2010 (504/10). All patients agreed to participate to the study and signed an informed consent form.To partially purify tumor cells, cells from Malignant Pleural Effusion (MPE) were subjected to discontinuous density gradient centrifugation as follows. MPE, obtained aseptically in heparinized (10 U/ml) bottles and processed within a reasonable time (4 hr), were centrifuged at 300 g for 10 min, at 4°C and cell pellet was resuspended in 30 ml of cold 1% BSA/2 mM EDTA/PBS. Cells were counted in a haemocytometer by Trypan Blue exclusion dye (Sigma-Aldrich) and then cell suspension was layered on modified Histopaque-1077 (Sigma-Aldrich) modified gradient in 50 ml conical tube . Histopaque-1077 solution of a density of about 1,065 gr/ml was obtained by diluting it with Phosphate Buffered Saline pH 7,2. Discontinuous gradient was then centrifuged at 800 g, for 30 minutes, at room temperature. During the centrifugation step the cells were separated according to their different buoyant densities: the denser fluid components such as erythrocytes and some leucocytes migrate into the lower phase through the bottom of the tube while the less dense cell fraction, including tumor cells, are enriched at the interphase. After centrifugation, cells contained in the upper gradient and in the pellet were collected and washed once with 1%BSA/2 mM EDTA/PBS. Cells from the upper gradient were then plated in “Sphere Medium” at a concentration of 100'000 cells/ml in non-tissue culture treated plates  or Ultralow binding plates (Corning). Half volume medium was replaced once a week while growth factors (bFGF and EGF) were added every other day. The pellet from density gradient centrifugation, which contains leucocytes, erythrocytes and clumps of tumor cells, was subjected to hypotonic lysis to remove erythrocytes, washed once in 10% FCS-RPMI (Invitrogen) and then plated in tissue culture treated Petri dishes .To determine their self renewal ability, sphere propagation assay were performed: adherent lung cancer cells were mechanically dissociated and plated in non-adherent culture to form P0 spheres. After 6–12 days individual spheres were formed, dissociated with Accumax and serial dilution was used to replate an average of 1 cell per well in 96-well plates to generate P1 spheres. This procedure were repeated several up to 3–4 times.6 cells analyzed for ALDH1 activity using ALDEFLUOR kit (Stem Cell Technologies) following manufacturer instruction. Briefly, cells were suspended in ALDEFLUOR assay buffer containing the fluorescent ALDH1 substrate, BODIPY-aminoacetaldehyde (BAAA), and incubated for 45 min at 37°C. The assay buffer contains also a transport inhibitor, to prevent efflux of the BAAA from the cells. BAAA passively diffuse into live cells and then is converted by intracellular ALDH1 into a negatively charged product BODIPY-aminoacetate, which is retained inside cells, labeling them with a bright fluorescent signal. After a washing step, the brightly fluorescent ALDH1-expressing cells (ALDH1br) were detected in the green fluorescence channel  of a FACSCalibur instrument (BD Biosciences). A sample of cells were stained as above with the addition of a specific ALDH1 inhibitor, diethylaminobenzaldehyde (DEAB) (Sigma), to serve as a negative control for each experiment. Because only cells with an intact cellular membrane could retain the ALDH1 reaction product, only viable ALDH1br cells were identified. Cells incubated with BAAA and DEAB were used to establish the background signal and to define the ALDH1br region. Incubation of cells with the substrate in the absence of DEAB induced a shift in BAAA fluorescence defining the ALDH1br population. Data were analyzed by FCSExpress.Cells growing in adherent or spheroid conditions, were collected, dispersed by Accumax  treatment, and 0.5–1×10H. sapiens genome databases RefSeq, ENSEMBL and GenBank. Chips were washed and scanned on the Affymetrix Complete GeneChip Instrument System, generating digitized image data (DAT) files.Single strand biotinylated cDNA was generated as follows: 100 ng of total RNA were subjected to two cycles of cDNA synthesis with the Ambion WT expression Kit (Applera). The first cycle – first strand synthesis is performed using an engineered set of random primers that exclude rRNA-matching sequences and include the T7 promoter sequences. After second-strand synthesis, the resulting cDNA is in vitro transcribed with the T7 RNA polymerase to generate a cRNA. This cRNA is subjected to a second cycle – first strand synthesis in the presence of dUTP in a fixed ratio relative to dTTP. Single strand cDNA is then purified and fragmented with a mixture of uracil DNA glycosylase and apurinic/apirimidinic endonuclease 1 (Affymetrix) in correspondence of incorporated dUTPs. DNA fragments are then terminally labeled by terminal deoxynucleotidyl transferase (Affymetrix) with biotin. The biotinylated DNA was hybridized to the Human Genechip Gene 1ST Arrays (Affymetrix), containing almost 29000 genes selected from p≤1*10−2) by the Hypergeometric test and corrected using False Discovery Rate (FDR). For the analysis of gene expression profiles in adherent cultures compared to sphere cultures, differentially expressed (DE) probe sets were filtered for fold change ≥1.5. Statistical analysis was performed using the Welch t-test, with p≤0.05. DEGs were functionally characterized using a proprietary software, Ingenuity Pathway Analysis (IPA) from Ingenuity Systems® to identify possible enriched molecular networks and canonical pathways. From the 29000 genes on the chip we obtained 1494 differentially expressed probe sets for the patient PE d/10 and 171 differentially expressed probe sets for the patient PE e/10. At this point we generated the intersection list of the two samples composed by 82 probe set. As a further filtering step, we generated a list of probes with similar behavior in the two samples by considering just those genes with a FC lower than 1.5 between the two adherent cultures. In this way we produced a curated list of 54 DE probe sets. The accession for experimental submission is MIAMExpress database number E-MEXP-3025.Data files were analyzed with AGCC  producing CEL files. Robust multichip average (RMA) normalization and data analysis were performed using GeneSpring 11.0.2 . Time analysis among the different stages of the same culture was performed using CAGED  which identifies the most probable set of clusters given a set of time series P<0.05.Using Primer3 software analysis available on the web, we selected primer sequences to optimally amplify target for qRT-PCR assay. To avoid possible amplification of contaminating genomic DNA primers were designed so that each PCR product covered at least one intron. All the primers used have been synthesized by Sigma. The expression of selected genes was examined by real-time reverse transcription-PCR (RT-PCR) using SYBR Green Detection Chemistry (Applied Biosystem). One micrograms of total RNA extracted from each of the samples were used to make complementary DNA (cDNA). Twenty nanograms of cDNA were used as template for qPCR amplification in presence of 0,2 µM specific primers, in a total volume of 25 µl. PCR reactions without cDNA samples were used as negative controls. Each reaction was done in triplicate and repeated at least twice to verify the results. SDS system software  was used to convert the fluorescent data into cycle threshold (Ct) measurements and the relative amount of specific transcript was calculated by the comparative cycle threshold method given by 4 cells subcutaneously and with 2×103 cells intra lung. Mice were housed in a highly controlled microbiological environment to guarantee SPF conditions. They were maintained in IVC cages, under constant conditions of temperature (22±2°C), humidity (55%±10 UR) and light/dark cycle of 12/12 hours. The animals had free access to irradiated standard diet and water.Two-month-old male NOD/SCID mice  were used. Animals were anesthetized with ketamine and injected with 103 in size, the tumor mass was excised, collected and submitted to histological analysis. In a subset of cases tumors were minced into small pieces and transplantated into a second recipient NOD/SCID mouse. In absence of additional signs of pathology groups of mice were euthanized every two months in order to check for the presence of lung cancer metastasis. At animal sacrifice, a complete necropsy procedure was performed. Cervical, thoracic, abdominal, and pelvic organs were extracted en bloc. We assessed the development of local tumor at the injection site, measured its two largest diameters, and recorded all macroscopic tumor deposits or abnormalities in any organ, especially those in which lung cancer foci were expected . The whole block of organs was fixed with buffered formalin for 48 h, except for the lung. The lungs were excised and the tumor removed, weighed, fixed with 10% formalin in PBS. Two general pathologists analyzed histopathologically the tumor at the injection site. All the experimental procedures were approved by the Ethical Committee for the Animal Use (CESA) of the IRGS and where communicated to the Italian Ministry of Health.Cells obtained by detachment from PE d/10 and PE e/10 adherent or spheres cultures dissociated to obtain single cell suspension, were diluted in 1× PBS plus 50% matrigel before subcutaneous injection. Mice were daily monitored to avoid any sign of suffering and weekly to check for the appearance of subcutaneous tumor or weight loss due to potential tumor growth. When subcutaneous tumors volume reached at least 700 mmPE d/10 and PE e/10 were submitted to cytospins using 100000 cells per spot, centrifuged at 800 rpm for 3 minutes. The slides were allowed to dry over night and fixed with Acetone or Formaldheyde 4% for intracitoplasmatic antigens or membrane antigens, respectively. Cytospins were washed in PBS tween20 0.05%. Blocking reagent followed by primary antibody and then biotinylated anti-mouse secondary antibody were added. The primary antibody used was an anti-CK7 (DAKO Cytomatic), anti-BerEP4 (DAKO Cytomatic) anti-Vimentin (Novocastra) and ALDH 1a1 (Epitomics). After a further step of washing in PBS tween 20 0.05%, slides were treated with avidin-biotin complex for 30 min using DAKO Cytomatic LSAB+Syste-HRP detection system according to the manufacturers instructions. Staining was performed with 3,3′-diaminobenzidin and counterstaining with HE. The primary antibodies were omitted and replaced with preimmune serum in the negative control. Positive controls were adopted based on the manufacturer's instructions.Sections 3 µm thick from Xenograft mice tumor were cut onto silanized glass slides and air-dried overnight at room temperature. Sections were dewaxed in xylene and rehydrated through graded alcohol. After peroxidase inhibition, the sections were immersed in citrate buffer (pH6.0)  and irradiated twice in a microwave oven (800W) at full power for 15 min. After, the slides were incubated with the following antibodies: anti-CK7 (DAKO Cytomatic), anti-BerEP4 (DAKO Cytomatic) anti-Vimentin (Novocastra). After a further step of washing in PBS, slides were treated with avidin-biotin complex for 30 min using Dako Cytomation LSAB+Syste-HRP detection system according to the manufacturers instructions. Staining was performed with 3,3′-diaminobenzidin. and counterstaining with HE. At least 100 cells per view were counted. Cases were considered positive if at least 10% of cells were strongly stained for the antibody. The primary antibodies were omitted and replaced with preimmune serum in the negative control. Positive controls were adopted based on the manufacturer's instructions.Malignant pleural effusions were collected by thoracentesis as described in the All samples, grew in RPMI medium in the presence of 10% FBS although, as expected, a great variability from sample to sample was observed with respect to doubling time and duration of cell culture (not shown). In order to avoid long periods of adaptations in cell culture that may lead to the selection of subpopulation of cells with particular growth advantages all samples were banked in liquid nitrogen at passage 3 (p3), on average one month following MPE collection. All further studies were conducted with p4 to p6 cells, i.e. cells in the initial passages after thawing, unless otherwise specified.p4 cells all re-grew in adherence. When they were transferred into sphere forming medium (SM), i.e. DMEM/F12 medium supplemented with EGF and FGF, the majority of samples, i.e. 11 out of 15 Table 1,Figure S1). This procedure was repeated successfully at least 2 times showing, however, a gradual decrease in the frequency of sphere formation up to passages 3–4.In order to demonstrate the presence in primary cell line PE d/10 and PE e/10 of CSCs in our spheroid cultures we set up self renewal assays see  section. and br cells increased upon culturing in spheroid conditions, providing information about the presence of putative CSC in MPE primary cultures.Cancer Stem Cells (CSC) are single tumor cells which are thought to be capable to regenerate a tumor or a metastasis. The identification of CSC remains challenging and several CSC markers have been proposed. Among several, ALDH1 expression and its enzymatic activity seems to better correlate presence of CSC and aggressiveness of lung tumors vs shperoids of sample PE d/10. The amounts of the indicated transcripts, analyzed by real-time RT-PCR, were significantly increased in cell grown as spheres compared with those grown in adherence  did not reveal any substantial change (not shown). In order to better evaluate possible changes taking place over time in culture, RNA samples was extracted from adherent cultures of sample PE d/10 at passages 6, 8, 10, 13, 14 and 16 and subjected to expression profiling. We utilized CAGED an algorithm based on Bayesian Clustering by Dynamics (see Methods section), which performs Bayesian clustering on temporal gene expression data to search for the most probable set of clusters given the available data. The majority of gene clusters showed a virtually flat behaviour over time, thus confirming an overall stability of gene expression (not shown). Only one small cluster made by 69 probesets showed a moderately raising expression profile over time. (Table S1). When this cluster was functionally annotated by Gene Ontology (GO) for Biological Process (p≤1*10−2) we obtained 9 Biological Process GO categories enriched in the PE d/10. Interestingly the GO categories enriched in the PE d/10 are related to “DNA conformation changes” as reported in the supplementary (Table S2). Furthermore, we performed genome-wide microarray analysis to ask if it is possible to identify a set of genes that are differentially regulated between sphere cultures and adherent cells. We compared gene expression profiles of adherent vs sphere cultures of RNA extratcted at p6  resulting in 1494 DE probe sets for the PE d/10 adherent culture and 171 DE probe sets for the PE e/10. We then calculated the intersection list with a Venn Diagram (vs spheres between the two patients (Table S3). To discard genes whose expression doesn't vary significantly between the two adherent cultures, this DE probe set was again filtered for fold change, this time looking for genes with a negligible variation  between the two adherent cultures (Table S4). A bibliographic annotation on curated genes was performed using Ingenuity Pathway Analysis (IPA), as described in Figure S3. We finally compared the previous 82 and 54 DE gene lists obtained from MPE primary cultures with unpublished profiling data obtained comparing adherent vs sphere cultures of the stable lung cancer AdenoCa cell line NCI-H460. The results, schematically represented in the Venn Diagram  and 2×103 for orthotopic injections respectively. Results indicate that both adherent and spheroids cultures engrafted very efficiently s.c. A compendium of data obtained upon s.c. implantation is reported in Figure S2) with a variable latency time of about 10–16 weeks  obtained from lung cancer patients are a source of cancer initiating cells and may constitute an excellent model to study inter-and intra-tumoral heterogeneity vs spheroids?We have decided to focus our attention on the two samples, PE d/10 and PE e/10 that were most efficient in propagation in vitro and have started to ask the following questions: a) how is the global pattern of gene expression changing over time in culture?; b) are spheroid cultures enriched in markers commonly associated with stemness and do they differ from adherent cultures in their gene expression programs?; c) are lung AdenoCa cultures from MPEs capable of efficient engraftment in vivo and, if so, can we observe differences between adherent In response to the first question, we carried out morphological and genome wide gene expression profiling using the Affimetrix platform in sample PE e/10 over serial passages. Although we are aware of the limitations given by the small set of samples analyzed, the conclusion that can be drawn is that the overall gene expression pattern in sample PE e/10 remains stable over time. Interestingly, however, a small cluster of genes enriched for genes involved DNA packaging and maintenance undergoes a statistically significant and gradual increase from passage 6 to 16. This indirectly reinforce the concept that long term culture of primary cells may lead to the occurrence of epigenetic changes. We are currently investigating if the same trend is observed in other samples of our collection.In response to the second question, microarray analysis of samples PE d/10 and PE e/10 shows that they differ substantially in their pattern of gene expression  the rate of engraftment was similar when comparing spheroids In conclusion we have shown here that MPEs are an excellent source of tumor initiating cells in lung AdenoCa. Bulk cultures can be utilized to identify genes linked with sphere formation in vitro and enhanced tumor propagation in vivo.Figure S1Sphere assays, self renewal ability of primary lung cancer cell.(TIF)Click here for additional data file.Figure S2NOD/SCID mice subcutaneous tumor growth.(TIF)Click here for additional data file.Figure S3IPA Canonical Pathways.(TIF)Click here for additional data file.Table S1Enriched GO terms and filtered by the Hypergeometric test and corrected using False Discovery Rate (FDR).(TIF)Click here for additional data file.Table S2Cluster, containing 69 probesets, was functionally annotated by Gene Ontology (GO) for Biological Process.(TIF)Click here for additional data file.Table S369 probesets expression profile over time.(TIF)Click here for additional data file.Table S4vs spheres cell cultures.Differential expressed probe sets of 82 genes in adherent (TIF)Click here for additional data file.


Trabecular meshwork and Schlemm's canal are the tissues appointed to modulate the aqueous humour outflow from the anterior chamber. The impairment of their functions drives to an intraocular pressure increase. The selective laser trabeculoplasty is a laser therapy of the trabecular meshwork able to decrease intraocular pressure. The exact response mechanism to this treatment has not been clearly delineated yet. The herein presented study is aimed at studying the gene expression changes induced in trabecular meshwork cells by selective laser trabeculoplasty (SLT) in order to better understand the mechanisms subtending its efficacy.th and 6th hour from treatment. On the whole, selective laser trabeculoplasty modulates in trabecular meshwork the expression of genes involved in cell motility, intercellular connections, extracellular matrix production, protein repair, DNA repair, membrane repair, reactive oxygen species production, glutamate toxicity, antioxidant activities, and inflammation.Primary human trabecular meshwork cells cultured in fibroblast medium underwent selective laser trabeculoplasty treatment. RNA was extracted from a pool of cells 30 minutes after treatment while the remaining cells were further cultured and RNA was extracted respectively 2 and 6 hours after treatment. Control cells stored in incubator in absence of SLT treatment were used as reference samples. Gene expression was evaluated by hybridization on miRNA-microarray and laser scanner analysis. Scanning electron microscopic examination was performed on 2 Trabecular meshwork samples after SLT at 4SLT did not induce any phenotypic alteration in TM samples. TM is a complex tissue possessing a great variety of function pivotal for the active regulation of aqueous humour outflow from the anterior chamber. SLT is able to modulate these functions at the postgenomic molecular level without inducing damage either at molecular or phenotypic levels. Glaucoma is a neurodegenerative multi-factorial disease affecting different target tissues: the lateral geniculate nucleus and the visual cortex in the central nervous system, The first “mechanical theory” stating that the laser application results in the local shrinkage of the meshwork, opening the intertrabecular spaces between the laser sites SLT applied on rabbit eyes increases in aqueous humour lipid peroxidase and free oxygen radicals levels The study adhered to the tenets of the Declaration of Helsinki and was approved by the Ethical Board of the Ophthalmologic Division.SLT did not induce any phenotypic alteration in TM samples collected from corneal donors as evaluated by electron scanning microscopy either 3 h and 6 h after treatment . By compSLT treatment induced a time-related change in the gene expression profile of TM cells. As evaluated by scatter plot such a change was well detectable starting since two hours after SLT treatment and persisted up to 6 hours , 2 hours (T2) and 6 hours (T6) .No effect was observed after 30 min since SLT treatment. Conversely, after 2 hours the number of genes changing their expression more than 2-fold as compared to controls accounted for 513, further rising up to 3,774 after 6 hours.per se cell damage. This finding is in line with the lack of any alteration in TM after 24 h since SLT treatment as ob served by electron microscopy .Complete microarray data are available at GEO database (GEO number GSE29697) (Our work provides evidence at postgenomic level that TM actively modulates the aqueous humor outflow through a variety of molecular mechanisms that can be modulated by SLT.Of the 67 genes modulated by SLT 7 (12%), encoding for activities involved in cell motility and contraction were upregulated by SLT indicating that this treatment enhance the contracting capacity of TM cells. The contractile function, cell shape, and cell adhesion properties of TM and SC cells have been implicated widely in modulation of aqueous humor outflow through the conventional pathway The above reported results could confirm the hypothesis of the “mechanical theory” of Wise and Witter Indeed, 15 out of the 67 genes (22%) modulated by SLT encoded for activities involved in maintaining TM tissue integrity. Among these, genes involved in adherence to basal membrane and intercellular connection and in extracellular matrix removal resulted up regulated. Conversely, SLT downregulated gene involved in the production of extracellular matrix. These mechanisms contributes to the maintenance of TM cellularity counteracting its decrease. It was demonstrated that a relative increase in the extracellular matrix/cells ratio is crucial for POAG progression ++ homeostasis were modulated by SLT. This finding probably reflects the SLT effect on mitochondrion (see below), which is the main intracellular Ca++ depot. Intracellular Ca++ release is a pivotal step of the intrinsic apoptotic pathway. Thus this situation may be interpreted as a decreased trend towards apoptosis.Furthermore 8 out of 67 genes whose expression was affected by SLT treatment are cell membrane components. Genes encoding for activities involved in intracellular Ca2+ stress. The dysfunction in calcium regulation by these cells may contribute to the failure of this tissue to control IOP.A study + uptake. On the whole these genes contribute to tissue integrity by stimulating cell growth and volume maintenance. SLC3A2, involved in the synthesis of nitric oxide, was upregulated. This indicates that SLT activates mechanisms contributing to the maintenance of TM integrity such as blood perfusion improvement, being nitric oxide able to increase vessel diameter and perfusion rate.SLT treatment increased the expression of genes encoding for cell growth factor, aminoacids, and Na8 out of the 67 (12%) SLT-modulated genes were involved in mitochondrial functions. 7 of these genes, involved in ion transport and lipid peroxidation, were downregulated. This trend indicates a silencing of mitochondrial activities producing intracellular reactive oxygen species. However this situation does not result in energy shortage. In fact a gene (ATP5J) involved in ATP synthesis was upregulated.6 genes involved in energy production (9% of 67), 3 genes, involved in glucose supply and anaerobic glycolysis, were upregulated. In parallel, 3 of these genes, involved in ATP consumption, were downregulated. These finding indicate that TM cells as a consequence of SLT exposure decrease energy consumption and increase energy production by anaerobic mechanisms. This balance results in the reduction of endogenous production of reactive oxygen species.Furthermore, among 8 out of 67 genes (10%) affected by SLT treatment are involved in response to oxidative stress and production of antioxidant activities. Upregulated antioxidant activities include glutathione, cyestine rich proteins, bilirubin metabolites, mitochondrial cytochrome. Two genes involved in removal of retinol derivatives were downregulated, suggesting that retinol metabolites too contribute to the increasing of the antioxidant status.The role of reactive oxygen species in the pathogenicity of glaucoma is supported by increasing evidences Moreover an increased trend to DNA repair was observed. In fact among the 5 (7% of 67) genes involved in DNA repair and cell cycle, 2 involved in base repair and 1 inhibiting cell cycle were upregulated, while 3 inducing cell cycle progression were downregulated. This figure indicates a trend to cell cycle rate decrease paralleled by increased DNA repair.Luna et al. All the 7 genes (10% out of 67) involved in inflammation and recruitment of inflammatory cells were downregulated after SLT treatment. This situation results in an immunosuppressive effect bearing relevance because inflammation has been proposed as a possible pathogenic step of POAG. These findings are in line with the ability of light to induce immunosuppression by modulating gene expression in exposed tissues 3 out of the 67 genes modulated by SLT (4%) are involved in glutamate metabolism. This confirm a possible interaction between glutamate metabolism and the IOP in the optic nerve head where, in rabbits, the elevation of the IOP causes an increase in the glutamate levels The expression of 9 genes (13%) characterizing neural tissue, as inferred from Swissprotein annotation, was well detectable expressed in TM cells and modulated by SLT treatment. These genes are identified by ‘*’ in The similarity between TM and neural tissue is important for glaucoma pathogenesis because they can share common pathogenic mechanisms. The herein reported results provide evidence that glutamate cytotoxic effects representing a major pathogenic element for nerve tissues targeted by glaucoma, are also important for TM homeostasis and may be modulated in TM by SLT treatment. A trend to decreasing TM cell to the adverse glutamate effects was detected. In fact, SLT decreased the expression of gene encoding for the NMDA glutamate receptors, which mediate glutamate induced cytotoxicity. In parallel, two genes encoding for enzymes involved in glutamate catabolism were upregulated.in vivo is not clear, it is thought that a high IOP, can extend the trabecular beams, obtaining an increased exposure of endothelial cells in AH by increasing the surface of filtration On the whole SLT induces in TM cell motility, increased intercellular connections, decreased extracellular matrix production, protein repair, DNA repair, membrane repair, decreased reactive oxygen species production, decreased glutamate toxicity, increased antioxidant activities, and decreased inflammation. Nevertheless it has to be taken into account that the analysis of gene expression can give a broad quantity of data but not all of them will be actually effective in cell. As a matter of that Alvarado et al. For instance, Bone morphogenetic proteins that controls multiple functions in a variety of cells are present in human optic nerve head (ONH) tissues, isolated ONH astrocytes, and Lamina Cribrosa cells In conclusion SLT significantly increased the tonographic outflow facility and decreased IOP in patients with primary open angle glaucoma and ocular hypertension For their absolute interest are those related to stress oxidative which confirm its pathogenetic importance http://www.sciencellonline.com for details] were cultured in fibroblast medium , 2% fetal bovine serum , fibroblast growth supplement  and penicillin/streptomycin solution  using poly-L-lysine-coated flasks having a 6 mm diameter. Cell were growth to semi confluence accounting for approximately 1×105 cell per well. When reaching semi-confluence cells underwent SLT treatment.Primary human TM cell line [HTM, ScienCell, San Diego, California: cat. n. 6590, see TM cells underwent SLT under similar conditions usually adopted for human subjects using a SLT laser system . Specific treatment conditions were Q-swithched frequency doubled with Nd:YAG laser at 532 nm.Wavelength, energy settings at 0.5 mJ single pulse, pulse duration 3 nsec, and treatment spot size of 400 micron.After treatment cells were immediately restored in incubator and then collected by scraping after 30 min (T0.5), 2 hours (T2), and 6 hours (T6). Control cells stored in incubator in absence of SLT treatment were used as reference samples.th and 6th hour from treatment  were performed on 2 Trabecular meshwork samples after SLT at 4reatment . Immediahttp://www.micro-array.nl/libraries.html. Because of the poor amount of available cell extracted RNA underwent reverse transcription and pre-plateau amplification as previously reported The expression of 18,401 human genes was tested in SLT-treated cells by cDNA microarray. Custom microarray as made available from the Microarray Department-University of Amsterdam, were used. The whole list of spotted genes is available at the website http://www.mged.org/Workgroups/MIAME/miame.htmlAll data is MIAME compliant and that the raw data has been deposited in a MIAME compliant database , as detailed on the MGED Society website Purified RNA underwent reverse transcription and amplification using quantitative real-time PCR (qPCR) prior to probe synthesis for array hybridization. A particular retrotranscription protocol  aimed at the introduction of a target sequence at 3′ and 5′ of the cDNA library was applied as follows: RNA was incubated with a mix of a target sequence-oligo(dT)-linked primer and a target sequence-oligo(dG)-linked at 72°C for 5 minutes then a master-mix solution was added containing reverse transcriptase and dNTPs mix. Samples were then incubated at 42°C for 90 minutes. Reaction was terminated by adding ethylenediaminetetraacetic acid, mixture diluted in phosphate buffer and synthesized cDNA purified by column chromatography using a commercially available purification kit . A PCR mix, including the specific primer for the target sequences and the fluorescent tracers SYBR GREEN, was added to the purified cDNA. The PCR reaction was performed at 95°C ×1 minute, 50 amplification cycles at 95°C ×15 s, 65°C ×30 s and 68°C ×3 minutes. The amplification curve was observed in real time by recording fluorescence and the pre-plateau amplification cycle was identified for each sample. A second set of sample was amplified to the identified pre-plateau cycle using the same conditions without the fluorescent tracer. This procedure was applied to all samples in order to standardize the amplification process thus allowing reliable comparisons of microarray data among different samples. PCR reactions were performed in a thermocycler equipped with microvial rotating support .2 ug of amplified cDNA, as purified by column chromatography, were converted in aminomodified probes performing a three cycle PCR using a PCR mix containing N-18 random primers, aminomodified dNTPs and Taq polymerase. The reaction was performed at 95°C ×5minutes, 94°C ×1 minute, 25°C ×90 s, 50°C ×10 minutes and 68°C ×5 minutes. Synthesized amino-modified oligo-nucleotides were purified by column chromatography and alcohol precipitation then labelled with fluorescent tracers Cy3 or Cy5 by incubation at room temperature in the dark for 90 minutes. Fluorescent oligonucleotides were precipitated by cold ethanol and sodium acetate, and then purified by column chromatography. The efficacy of the procedure was checked by spectrophotometric analysis measuring abosorbance at 550 (Cy3) and 650 (Cy5). Standardized amounts of labelled probes were hybridized on glass cDNA microarrays.Probes were lyophilized, diluted in 4 ul of EDTA 10 mM and incubated at 95°C for 10 min. Hybridization solution (18 ul) was added and labelled probe mixed together to a final volume of 44 ul. Mixture was then transferred onto microarrays which were then covered with a coverslip and hybridized overnight at 50°C in a Hybridization Cassette . After 16 hours the microarrays were washed twice in a low stringency wash buffer  and twice in a high stringency wash buffer . Microarray were dried in centrifuge and signal acquired in a laser scanner .Data analysis was performed subtracting for each microarray the local spot background from raw spot intensity, log transformation, normalization per chip and per array . Each gene was spotted in quadruplicate on the used microarray. Accordingly, results represent the mean among 4 data. Data generated for each mRNA were compared among the various experimental groups by volcano-plot analysis taking into account as thresholds of two-fold variation and statistical significance P<0.05 as evaluated by ANOVA after Bonferroni multiple testing correction. Global mRNA expression profiles were compared by hierarchical cluster analysis (HC) and bidimensional principal component analysis of variance (PCA).www.genecards.org) and Swissprotein database (http://expasy.org/sprot/).Gene function was inferred from annotation reported in gene databank  and Weizmann Institute (http://www.genecards.org/) databases. Predictor genes were identified by statistical support vector machine analysis, which is a supervised learning methods that predicts, for each given input, which of possible classes the input is a member of identifying the most relevant genes affecting this classification”.“Biological function of genes whose expression was modulate by SLT was inferred from data available on Swissprot (Table S1Gene whose expression in TM cells was modulated by SLT treatment (control vs. SLT). 89 genes.(DOC)Click here for additional data file.


MicroRNAs (miRNAs) are an extensive family of small regulatory RNAs that function by binding to complementary mRNAs, primarily in the 3′ untranslated region (3′UTRs), and then inhibiting their expression by reducing mRNA translation and/or stability Upon infection of a cell, viruses encounter a wide range of miRNA species, generally more than 50 different miRNAs per cell, and these miRNAs vary greatly between tissues. For example, miR-122 is expressed at very high levels in hepatocytes, but is absent from almost all other cells, while miR-1 is primarily expressed in muscle tissue and miR-128 in neuronal cells Analyses of the interactions of viruses with cellular miRNAs have revealed that viruses can influence cellular miRNA biogenesis and effector mechanisms in several different ways. Viruses can clearly benefit from miRNA expression. For example, almost all herpesviruses that have been examined express substantial numbers of miRNAs, and these can facilitate viral replication and/or regulate viral entry or exit from latency While certain viruses can clearly benefit from cellular miRNAs, it has been unclear how viruses avoid inhibition of viral mRNA function by cellular miRNAs. Indeed, several reports demonstrating inhibition of viruses by cellular miRNAs have been published Viruses block miRNA function. This appears rare, as virus-infected cells generally contain normal levels of miRNAs, and most viruses can be inhibited by specific small interfering RNAs (siRNAs), which function indistinguishably from miRNAs in mammalian cells Viruses evolve to avoid 3′UTR targets complementary to cellular miRNAs. Because full complementarity to the seed is generally critical for miRNA inhibition, single nucleotide mutations should block inhibition Viruses evolve very short 3′UTRs. RISC recruitment to open reading frames (ORFs) does not effectively inhibit mRNA function, most probably because translating ribosomes sweep bound RISCs off the mRNA Viruses evolve structured 3′UTRs. Some viruses, especially retroviruses, alphaviruses, and coronaviruses, contain extensive 3′UTRs in at least some viral mRNA species. For example, the HIV-1 mRNA that encodes Gag and Gag-Pol has a 3′UTR that is several thousand nucleotides in length. Similarly, the coronavirus mRNA encoding the viral ORF1a and ORF1b proteins has a 3′UTR more than 10,000 nucleotides in length. How do these very long 3′UTRs avoid functioning as targets for multiple miRNAs? One possibility is that these 3′UTRs have evolved high levels of RNA secondary structure, which would be predicted to globally restrict binding by miRNA-programmed RISCs Relatively little is known about the secondary structure of viral RNAs, although some data suggest that high levels of secondary structure are a common feature + T cells) or in a non-physiological target cell (HeLa cells) has been examined using a technology called photo-activatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP). The PAR-CLIP technique involves pulsing cells with the highly photoactivatable uridine analog 4-thiouridine and then crosslinking endogenous RNAs to bound proteins by irradiation at 365 nm Recently, the ability of the HIV-1 genome to bind to endogenous cellular miRNAs in relevant target cells (CD4Analysis of RISC binding to the HIV-1 genome indeed identified several binding sites that were occupied by RISCs programmed by endogenous cellular miRNAs and some of these could be shown, by indicator assays, to confer a modest repression of mRNA function While a more complete understanding of the interaction of viruses with cellular miRNAs must await a more detailed dissection of the effect of endogenous miRNAs on a wide range of viral species, current data suggest that viruses have likely evolved a number of strategies to avoid inhibition by these ubiquitous cellular regulatory RNAs. Whether the perturbation of these avoidance strategies has the potential to lead to the development of reagents that are useful in disease prevention, such as novel forms of attenuated viral vaccines, remains to be determined.


Both were shown to be radiosensitive with respect to clonogenic survival curves of bone marrow stromal cells in culture and severity of oral cavity mucositis during single fraction or fractionated radiotherapy. Oral administration of the antioxidant GS-nitroxide, JP4-039, provided significant radioprotection, and also ameliorated distant bone marrow suppression  in Fancd2−/− mice. These data suggest that radiation protection by targeting the mitochondria may be of therapeutic benefit even in the setting of defects in the DNA repair process for irradiation-induced DNA double strand breaks.Radiation oncologists have observed variation in normal tissue responses between patients in many instances with no apparent explanation. The association of clinical tissue radiosensitivity with specific genetic repair defects  has been well established, but there are unexplained differences between patients in the general population with respect to the intensity and rapidity of appearance of normal tissue toxicity including radiation dermatitis, oral cavity mucositis, esophagitis, as well as differences in response of normal tissues to standard analgesic or other palliative measures. Strategies for the use of clinical radioprotectors have included modalities designed to either prevent and/or palliate the consequences of radiosensitivity. Most prominently, modification of total dose, fraction size, or total time of treatment delivery has been necessary in many patients, but such modifications may reduce the likelihood of local control and/or radiocurability. As a model system in which to study potential radioprotection by mitochondrial-targeted antioxidant small molecules, we have studied cell lines and tissues from Fanconi anemia (Fancd2 The relative susceptibility of individual patients to acute and chronic side effects of therapeutic ionizing irradiation has been the subject of intense controversy . RadiatiDespite uniform approaches toward reducing normal tissue toxicity, there remain continual reports of variation between patients with respect to the likelihood of developing rapid normal tissue damage effects, symptomatology, and potential requirement for radiotherapy treatment breaks, reduced total dose, or reduced fraction size. The mechanism of the radiosensitivity of tissues in specific patients is not always available. Specific DNA repair defects have been associated with radiosensitivity. These include the genetic defects in ataxia telangiectasia, Bloom’s syndrome, Wegner’s syndrome, and Fanconi anemia –4. HowevFancd2−/−) mice were bred from the mating of Fancd2+/− heterozygote pairs and were derived from either the C57BL/6 background . Non-adherent cells were removed weekly and assayed for total cells produced, as well as those cells capable of producing colonies in semi-solid medium in secondary culture. Colonies were scored at day 7 and day 14. The results of these studies showed significant decrease in the capacity of cultures to produce hematopoietic cells over 22 weeks . Non-adherent cells from the 4-week harvest of long-term marrow cultures were carried in medium supplemented with IL-3 and clonal cell lines established. These are termed hematopoietic cells and are IL-3-dependent multilineage colony forming cells capable of forming neutrophils, macrophages, erythroid cells, megakaryocytes, and monocytes. Briefly, the adherent cell layer from 4-week-old continuous marrow cultures was trypsinized and cells were passaged weekly in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics according to published methods. After 10 weeks of passage, cell lines were cloned by limiting dilution technique in 96 well plates and using Poisson statistics. Single cell-derived clonal lines were passaged weekly according to published methods.Bone marrow stromal cell lines from several mouse strains were derived from the adherent layer of long-term bone marrow cultures . Brieflyin vitro.Interleukin-3 (IL-3)-dependent hematopoietic progenitor cell lines were derived from the non-adherent cells of 4-week-old long-term bone marrow cultures and were passaged in Iscove’s medium supplemented with 10% fetal bovine serum and 10 μM recombinant murine IL-3 , accordiBone marrow stromal cell line irradiation survival curves were carried out with cells irradiated to doses between 0 and 800 cGy using a cesium gamma cell irradiator. Cells were plated in six well tissue culture plates and colonies on the adherent layer consisting of >50 cells per colony were scored at day 7. IL-3-dependent hematopoietic progenitor cell lines were irradiated in suspension culture to doses described above, but then plated into semi-solid medium culture containing 0.8% methylcellulose containing Iscove’s medium supplemented with 10 μM IL-3. Non-adherent cell-derived colonies in agar secondary culture of size greater than 50 cells were scored at day 7 , 12.p-values <0.05 were considered statistically significant. All the radiation survival curve studies were done in triplicate and the results calculated based on the average of three experiments  was measured using a commercial kit . This assay measures the antioxidant capacity of cells based on the ability of cellular antioxidants to reduce Cu++ to Cu+, which reacts with bathocuproine to form a color complex absorbing at 480–490 nm. The antioxidant capacity was compared to a standard curve generator using Trolox units and all data was, therefore, expressed as millimolar or equivalents of Trolox units . At least five sections per animal, and at least 20 animals per experiment were analyzed. Over 1000 high power microscopic fields were scored for percent ulceration, and the results presented as the percent ulceration , 10. Thein vitro radiation protection and mitigation experiments, the compound was administered at 10 μM, and JP4-039 was added either one hour before irradiation or immediately after irradiation and maintained in the culture plates for 7 days up to the time of scoring the colony assay.The small molecule GS-nitroxide, JP4-039 , 13, andin vivo experiments, JP4-039 was suspended in a novel emulsion (F15) containing Tween detergent, which facilitates localization of the compound in the locally applied tissue  were derived from both the C57BL/6J and the FVB/N background. The advantage of having Fancd2−/− mice from two different genetic background mouse strains added robustness to the data for any experiments purporting to show radiosensitivity of normal tissues. As shown in Table Fancd2+/+ littermates, Fancd2−/− mice of both strains were markedly radiosensitive. In particular, bone marrow stromal cells and hematopoietic progenitor cells show distinctly different phenotypes with respect to radiation sensitivity  mouse model dose response curves were carried out delivering doses ranging from 24 to 30 Gy. In both model systems and both background strains, there was uniform radiosensitivity of the Fancd2−/− genotype. Table Fancd2−/− mice, including experiments using single fraction, fractionated irradiation, and dose response curves. Of interest, the day 2 scoring after irradiation did not allow a significant variation between mouse genotypes; however, by day 5, ulceration was well-established and was more significant in the Fancd2−/− mice   Table . The nit−/− genotype. In this figure, cells in culture were incubated with JP4-039 labeled with the BODIPY fluorochrome ] oral cavity tissue and amelioration of that radiosensitivity in mice receiving JP4-039/F15, even in the presence of orthotopic tumors.In experiments treating orthotopic tumors derived from the TC1 squamous cell carcinoma cell line (derived from C57BL/6J mice), single fraction or fractionated irradiation of mice with palpable tumors was successful in in vitro was not altered by JP4-039 administration prior to or after irradiation. Given that JP4-039 has been demonstrated to localize to the mitochondria  has been documented in cells and tissues from both mouse strains. These results provide strong evidence that an intrinsic DNA repair defect, documented in Fancd2−/− mice, can be overcome with respect to therapeutic radiation by administration of a mitochondrial-targeted radioprotectant. These data further suggest that the initiation of radiation-induced apoptosis, which occurs in the nucleus with DNA double strand breaks, may not necessarily be fatal if the downstream effects of this initiation can be countered. The mitochondria have been shown to be a critical target for radiation protection given the strong association of mitochondrial-mediated apoptosis with ionizing irradiation  mice demonstrated suppression of bone marrow colony forming progenitor cells at a greater magnitude after head and neck irradiation, than did heterozygote littermates or Fancd2+/+ control mice. That the suppression of bone marrow colony forming cells was also ameliorated by administration of head and neck localized JP4-039/F15 suggests that the humoral mediators, through the circulation, which caused the “abscopal” or “bystander” effect, may be more readily identified in these interesting Fancd2−/− mice (Studies with −/− mice , 32, 33.Fancd2−/− mice should be helpful in defining many radiobiologic parameters associated with tissue, organ, and organ system responses to therapeutic fractionated irradiation in an intrinsically radiosensitive microenvironment. Furthermore, radiobiologic studies in tumors bearing Fancd2−/− and FancG−/− mice may lead to the clinical translation of JP4-039/F15 as a potential radioprotectant during radiotherapy of Fanconi anemia patients, or other patients with genetic defects in DNA repair.Further studies with The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The zebrafish has emerged as a useful model system for the analysis of vertebrate organ development and function, and it is suitable for the identification of organotoxic or disease-modulating compounds on a larger scale. However, to fully exploit its potential in high content screening applications, dedicated protocols are required allowing the consistent visualization of inner organs such as the embryonic kidney. To this end, we developed a high content screening compatible pipeline for the automated imaging of standardized views of the developing pronephros in zebrafish larvae. Using a custom designed tool, cavities were generated in agarose coated microtiter plates allowing for accurate positioning and orientation of zebrafish larvae. This enabled the subsequent automated acquisition of stable and consistent dorsal views of pronephric kidneys. The established pipeline was applied in a pilot screen for the analysis of the impact of potentially nephrotoxic drugs on zebrafish pronephros development in the Tg(wt1b:EGFP) transgenic line in which the developing pronephros is highlighted by GFP expression. The consistent image data that was acquired allowed for quantification of gross morphological pronephric phenotypes, revealing concentration dependent effects of several compounds on nephrogenesis. In addition, applicability of the imaging pipeline was further confirmed in a morpholino based model for cilia-associated human genetic disorders associated with different intraflagellar transport genes. The developed tools and pipeline can be used to study various aspects in zebrafish kidney research, and can be readily adapted for the analysis of other organ systems.The analysis of kidney malformation caused by environmental influences during nephrogenesis or by hereditary nephropathies requires animal models allowing the  Adverse environmental conditions and genetic influences can have a major impact on organogenesis. According to statistical surveys, 22-50% of all pregnant women take drugs within the first trimenon of gestation, often whilst they are unaware of their pregnancy . Howeverin vivo imaging of kidney development in a spatiotemporal context is not feasible in rodents, necessitating animal models that are experimentally more accessible. Furthermore, dedicated imaging techniques are required which enable the in vivo visualization of developing organs and tissues on a larger scale.Despite rodent studies and observational reports in humans, detailed data on potential harmful side effects on nephrogenesis is still missing for many drugs and chemical compounds ,5. Additin vitro and in vivo model systems [in vivo assays. Moreover, the high degree of anatomical and physiological homology to higher vertebrates renders it a relevant model for biomedical research [The development of high content screening (HCS) technologies has had a major impact on biomedical and pharmaceutical research, as these platforms are suitable for a wide range of large-scale investigations using  systems . Due to research . Thus, tresearch 910–11.research 13–14.Despite its widespread usage in large scale experiments, it remains challenging to fully exploit the advantages of this model system in HCS experiments. Zebrafish embryos are largely incompatible with standard HCS protocols developed for other model systems, one of the reasons being that their relatively large size and complex three-dimensional shape often lead to random positioning and orientation of embryos within wells of microtiter plates. This especially hampers reproducibility and detailed visualization of cell or tissue morphology, as well as developmental processes. Whilst several studies and recent technological advances have tried to address these challenges, they often require sophisticated technical setups  or are iin vivo model system with automated imaging technologies could serve as a tool for the large scale analysis of kidney phenotypes. However, to our current knowledge, a screening platform compatible with in vivo imaging of zebrafish larval kidneys has not been described yet.In the zebrafish larva, the functional pronephros comprises only 2 nephrons with fused glomeruli located ventrally to the dorsal aorta. Interestingly, despite tremendous differences in nephron number, the composition of a single nephron shows great homology at the cellular and molecular level between human and zebrafish ,18. AddiTg(wt1b:EGFP) transgenic line in which the developing pronephros is highlighted by GFP expression [in vivo data was cross-validated by histological analysis. In addition, we demonstrate that the established microscopy platform can also be utilized for genetic disease models.Here, we delineate the development of an automated HCS compatible imaging pipeline designed for live imaging of zebrafish kidneys in chemical screening scenarios. Using a custom designed orientation tool, embryos could be accurately positioned in wells of microtiter plates allowing consistent imaging of dorsal views of the pronephros. Subsequent automated imaging was performed on a standard widefield screening microscope and a data handling and visualization pipeline was developed. A pilotscreen for morphological kidney abnormalities was performed using a subset of potentially nephrotoxic drugs applied to larvae of the pression . The obtAll zebrafish husbandry and experimental procedures were performed in accordance with the German animal protection standards and were approved by the Government of Baden-Württemberg, Regierungspräsidium Karlsruhe, Germany (Aktenzeichen 35-9185.64).Tg(wt1b:EGFP) transgenic line [Adult zebrafish of the nic line  were mainic line . Eggs wenic line . Embryosnic line . Prior tA subset of certain drug classes was chosen for which an adverse effect on the developing kidney had been described in animal and/or human studies . To evalift172 gene, and ift80 morpholino has been previously published [5’-AGGTGTATGTGGAACCTGTGATAAG-3’, ift172 Exon2 splice donor blocking MO: 5’-ACGAGATGAGAGCTTACTTTTGGGT-3’, ift172 Exon1 splice blocking MO: 5’-ACGCTGTCAATATTTTACCTGAGGC-3’, Standard control (ctr) oligonucleotide: 5’-CCTCTTACCTCAGTTACAATTTATA-3’.Antisense splice blocking morpholino oligonucleotides  were designed against the exon1-intron1 and exon2 –intron2 boundary of zebrafish ublished . Morpholublished 25–26. AThe tool is made from brass and consists of a base plate with 96 perpendicular pins arranged in a certain way to match the positions of the wells of a microtiter plate. The tool was produced by CNC milling . In a first step, the base plate with the dimensions of 140 mm x 100 mm x 21 mm was milled by a four flute end mill (20 mm). Secondly, the array of 96 pins with 11 mm height and rectangular footprint (5.9 mm x 1 mm) was created by partly thinning the base plate down to 9 mm between the pins . Finally, the ends of the 96 pins were tapered to a conical tip with 60° by utilizing a standard engraving tool. 70 µl of 1% agarose in fish water was added to each well of a 96 well microtiter plate  using a multi-channel pipette. The agarose coated well plates were pre-cooled at room temperature for one minute. To generate grooves the brass tool was inserted, while adjusting the penetration depth of pins using spacers. After solidification the tool was carefully removed and plates were optionally stored in plastic bags at 4°C. Embryos were transferred in 100 µl fish water containing PTU and tricaine (see above) and manually arrayed and oriented under a stereomicroscope. To aid in positioning of regions of interest within the grid-based and fixed field views of the Scan^R system, embryos were positioned in such a way that all yolk sacs were approximately at the same position within cavities using features of the well plate as guidelines. Embryos were imaged on a standard Scan^R high-content screening microscope   as previMacro S1). In brief, maximum projection images were duplicated, automatically thresholded and the resulting binary images were eroded. The position of kidneys was detected by measuring the center of mass. The corresponding coordinates were restored on original images, a bounding box was defined and images were cropped accordingly. Cropped images were loaded into a stack and overview images were generated using the ‘Make Montage’ function of Fiji. Data handling, generation of multilayer tiffs and generation of maximum projections were carried out using custom written Perl scripts and Fiji  macros aTg(wt1b:EGFP) zebrafish. For description of glomerular changes, the distance between the glomeruli was determined and glomeruli were classified as either normal or showing glomerular malformation judged by the glomerular area and structure. Heatmaps were generated using matrix2png [Overall morphology was scored on a Leica MZ10 F stereomicroscope . Lethality and pericardial/yolk sac edema were rated for each experiment. Blinded analysis of tubular and glomerular phenotypes was performed by SS and JHW on maximum projections of deconvolved z-stacks. Using ImageJ, 2 parameters were manually measured for tubular structures: i) maximum distance between the tubules and ii) angle between neck segment and proximal convoluted tubule  being vitrix2png .Larvae were fixed in 4% paraformaldehyde overnight at 4°C. Samples were dehydrated through an ethanol series and processed for embedding in Paraffin . 3 μm sections were cut using a Leica RM 2165 microtome . Sections were deparaffinized in xylene and rehydrated through graded washes of ethanol in water before staining with hematoxylin and eosin. The stained sections were imaged with a Leica DMI4000 B microscope equipped with a Leica DFC320 digital camera.Data were evaluated using IBM® SPSS® Statistics Version 21. For lethality and edema rates and glomerular fusion and malformation, statistical analysis was performed by Chi-square test. Datasets of low sample sizes were additionally tested using Fisher’s exact test. For tubular angle and distance, means among treatment groups were compared using ANOVA with Bonferroni correction for multiple comparisons as a post-hoc test. Significance was defined as p<0.05. The visualization of bilateral symmetric organs of zebrafish embryos usually demands dorsal or ventral views. However, consistent large scale imaging of zebrafish embryos remains challenging in automated screening experiments, as stable and reproducible positioning is complicated by the size and complex three-dimensional shape of embryos. To enable a simplified handling and precise positioning of zebrafish larvae and to achieve consistent visualization of tissues in high content screening scenarios, we have developed a tool to create agarose molds in a standard microtiter plate. The tool allows the preparation of agarose coated 96 well plates in a single replication step. The tool consists of a base plate with 96 rectangular pins, whose positions exactly match the centers of wells of standard and commercially available 96 well microtiter plates 1A, B. TWe recently demonstrated an alternative protocol for automated dorsal imaging of oriented larvae using a silicone tool to generate an array of 96 agarose molds . HoweverTg(wt1b:EGFP) stable transgenic line were raised to the desired developmental stage (48 or 72 hpf) and then transferred into microtiter plates containing agarose cavities as described above  transgenic line. Glomerular alterations were subdivided into (i.) glomerular malformation indicated by abnormal or reduced glomerular shape and area, and (ii.) incomplete glomerular fusion representing aberrant pronephros development. Tubular parameters were classified into (i.) the angle between the tubular neck segment and the proximal convoluted tubule and (ii.) variations in the maximum distance between the 2 tubular systems  constitutes the bidirectional transport of protein complexes along axonemal microtubules. IFT plays an essential role in the assembly and function of cilia and flagella by contributing to cell motility, sensory perception and cilium-based signaling ,52. IFT8ystrophy , a congeystrophy , no humarmations , indicaturvature . ift80 and ift172 were designed as described in the Methods section. By using the standard positioning tool as described above, automated imaging of dorsally positioned morpholino-injected Tg(wt1b:EGFP) zebrafish (3 dpf) was performed in 96 well plates (ift80 morphants [Tg(wt1b:EGFP) zebrafish confirmed the formation of large pronephric cysts  and tubular (lower panels) sections at 48 hours post fertilization. (A) control, (B) penicillin (20 mM), (C) ampicillin (40 mM), (D) gentamicin (40 mM), (E) kanamycin (40 mM), (F) acetaminophen (40 mM), (G) captopril (40 mM), (H) losartan (10 mM) and (I) indomethacin (0.75 mM) treatment.(TIF)Click here for additional data file.Figure S2Cross-sections of pronephric regions after morpholino injections. Shown are glomerular (upper panels) and tubular (lower panels) sections at 72 hours post fertilization. (A) control-MO, (B) ift80-MO and (C) ift172-MO injection.(TIF)Click here for additional data file.Table S1Lethality and edema formation in zebrafish larvae following drug treatment.(DOCX)Click here for additional data file.Table S2Glomerular alterations in zebrafish larvae following drug treatment.(DOCX)Click here for additional data file.Table S3Tubular angle and distance in zebrafish larvae following drug treatment.(DOCX)Click here for additional data file.Macro S1Fiji macro to crop images and generate overview images.(ZIP)Click here for additional data file.


Mmp10 as a gene that is highly induced in tumor-initiating lung bronchioalveolar stem cells (BASCs) upon activation of oncogenic Kras in a mouse model of lung adenocarcinoma. However, the potential role of Mmp10 in lung tumorigenesis has not been addressed. Here, we demonstrate that Mmp10 is overexpressed in lung tumors induced by either the smoke carcinogen urethane or oncogenic Kras. In addition, we report a significant reduction in lung tumor number and size after urethane exposure or genetic activation of oncogenic Kras in Mmp10 null (−/−Mmp10) mice. This inhibitory effect is reflected in a defect in the ability of Mmp10-deficient BASCs to expand and undergo transformation in response to urethane or oncogenic Kras in vivo and in vitro, demonstrating a role for Mmp10 in the tumor-initiating activity of Kras-transformed lung stem cells. To determine the potential relevance of MMP10 in human cancer we analyzed Mmp10 expression in publicly-available gene expression profiles of human cancers. Our analysis reveals that MMP10 is highly overexpressed in human lung tumors. Gene set enhancement analysis (GSEA) demonstrates that elevated MMP10 expression correlates with both cancer stem cell and tumor metastasis genomic signatures in human lung cancer. Finally, Mmp10 is elevated in many human tumor types suggesting a widespread role for Mmp10 in human malignancy. We conclude that Mmp10 plays an important role in lung tumor initiation via maintenance of a highly tumorigenic, cancer-initiating, stem-like cell population, and that Mmp10 expression is associated with stem-like, highly metastatic genotypes in human lung cancers. These results indicate that Mmp10 may represent a novel therapeutic approach to target lung cancer stem cells.Matrix metalloproteinase 10  is a member of a large family of structurally related matrix metalloproteinases, many of which have been implicated in tumor progression, invasion and metastasis. We recently identified  Non-small cell lung cancer (NSCLC) is the most common cause of cancer death in the United States Emerging evidence supports the existence of rare subpopulations of cancer cells with stem-like characteristics in vitroKras-mediated lung cancer initiation, lung cancer stem cell expansion, and metastasis. Our data indicate that Mmp10 is an attractive therapeutic target for CSCs.We recently demonstrated that Mmp10 is required for the transformed growth and invasion of human NSCLC cells Mmp10 is involved in lung tumor formation, mice were treated with the smoke carcinogen urethane to induce lung adenocarcinoma tumors using well-established protocols −−/ mice were exposed to urethane, these mice developed significantly fewer , whereas less than 1% of the tumors in LA2/Mmp10−/−Kras mice were grade 3 , putative regional stem cells that reside at the terminal bronchioles adjacent to the alveolar space Mmp10-deficiency affects the expansion of BASCs in response to urethane and oncogenic Kras in vivo. We identified and quantitated BASCs in paraffin sections of mouse lungs by dual immunofluorescence staining for surfactant protein C (SPC) and the Clara cell-specific protein (CCSP) as described previously Kras caused an expansion of BASCs at the terminal bronchioles  when compared with control Ntg mice  Kras allele can be activated by Cre-mediated recombination LSL-Kras, and −/−LSL-Kras/Mmp-10 mice and characterized for enrichment of BASCs using dual immunofluorescence and flow cytometry. Our BASC preparations consisted of >86% SPC/CCSP double positive cells when analyzed by flow cytometry confirming isolation of a highly enriched BASC population (LSL-Kras mice with adenovirus expressing Cre-recombinase (AdCre) to activate the oncogenic Kras allele led to a significant increase in Mmp10 mRNA abundance, whereas AdCre-treated BASCs from −/−LSL-Kras/Mmp10 mice expressed no detectable Mmp10 mRNA as expected  into two equal sized groups of 30 samples comprised of the tumors with the highest (High) and lowest (Low) Mmp10 RNA expression, respectively. Statistical analysis confirmed these two groups of samples express significantly different levels of Mmp10 mRNA  cancer stem cell signatures were enriched in the high Mmp10 samples, and that 14 signatures were significantly enriched with a p-value <0.05 and FDR <25% (We previously demonstrated that Mmp10 is overexpressed in human NSCLC and is important for transformed growth and invasion of human NSCLC cells data not shown). We also performed an unbiased analysis using every gene set in the MSig database. In this analysis, the top stem cell signatures remained significantly enriched  and Sca1- cells exhibit tumor-initiating activity in Kras mice, demonstrating that BASCs are not the sole source of tumor-initiating cells in this model Kras mutations, often develop at the bronchio-alveolar duct junction and display either airway or alveolar differentiation, or both The matrix metalloproteinases (MMPs) have long been implicated in tumor progression and metastasis. We recently demonstrated that Mmp10 is overexpressed in NSCLC and is a critical target of oncogenic Mmp10 in lung CSCs is cell autonomous. Both the LA2Kras and urethane tumor models show Mmp10 staining in tumor cells, with little to no staining in tumor associated-stroma or morphological normal lung epithelium. More importantly, the tumor inhibitory effects of the genetic loss of Mmp10 are reflected in a defect in oncogenic expansion of BASCs in vivo and in vitro. Thus, while many MMPs produced by the tumor microenvironment play prominent roles in the invasive and metastatic properties of lung tumor cells, our data demonstrate that Mmp10 specifically functions to support the autonomous growth of CSCs. However, our data do not exclude a contributory role for Mmp10 produced by and/or exerting its effects upon the tumor microenvironment. However, our studies do provide new insight into a largely unappreciated role for Mmp10 in the regulation of CSC behavior. Interestingly, Mmps have been implicated in regulation of tumor cell growth through cleavage and activation of cell surface proteins involved in cell growth regulation such as Notch Our data provide evidence that the role of Cancer stem cells (CSCs) are defined by their ability to clonally expand, initiate tumors, maintain tumor progression and participate in tumor metastasis. The phenotype of these cells is associated with a genotype related to that of embryonic stem cells. Our finding that Mmp10 expression is associated with CSC genotypes in human lung tumors provides compelling circumstantial evidence that Mmp10 plays a critical role in maintenance of CSCs within human lung tumors. In this regard, it is interesting to note that Mmp10 expression has been observed to be elevated in tumor-initiating stem-like cells isolated from human small cell lung cancer cell lines in vitroThe leading cause of cancer-related deaths in lung cancer patients is metastatic dissemination. CSCs are thought to be the cells within a tumor that have the capability of metastasizing to distant sites. Our finding that Mmp10 is highly expressed in lung cancer-initiating BASCs, and is associated with the CSC genotype in human lung tumors suggests that Mmp10 may promote both CSC maintenance and metastatic potential through its role in CSC proliferation and metastatic behavior. Our finding that Mmp10 is elevated in human CSCs and that Mmp10 is highly expressed at the interface between mouse lung tumors and the surrounding tissue, suggesting a role for Mmp10 in tumor invasion, and are consistent with our previous funding that Mmp10 is required for invasion of human NSCLC cells Mmp10 (−/−Mmp10) mice were obtained from the National Cancer Institute Mutant Mouse Regional Resource Center (MMRRC). The mice were generated on a mixed 129/C57BL/6J background and harbor a targeted disruption of exons 1–3 of the mouse Mmp10 gene. The mice were bred onto a pure C57BL/6J background through 10 generations. Genotyping was conducted by PCR using primers recommended by MMRRC. −/−Mmp10 mice and non-transgenic littermates were injected intraperitoneally with urethane at 1 mg/kg body weight weekly for six weeks to induce lung tumors. Control mice were injected with saline. Mice were analyzed twelve weeks after the first injection for the presence of pulmonary lesions. LA2Kras mice, generated as previously described −/−Mmp10 mice to generate bitransgenic LA2/Mmp10−/−Kras mice. LA2Kras and LA2/Mmp10−/−Kras mice were harvested at the time points indicated to assessed tumor number, tumor size, tumor burden and pathological classification by a board-certified pathologist (A.K.). −/−Mmp10 and non-transgenic mice served as negative controls. All animal experiments were approved by the Institutional Animal Care and Use Committee of Mayo Clinic and were conducted under approved IACUC protocol# A30308. Mouse tissues were prepared for histology and immunohistochemistry as previously described Nullizygous LSL-Kras mice −/−Mmp10 mice to generate bitransgenic −/−LSL-Kras/Mmp10 mice. Lung epithelial cells were isolated from Ntg, −/−Mmp10, LSL-Kras, and −/−LSL-Kras/Mmp10 mice and BASC isolation, Cre-recombinase treatment and ex vivo culture were carried out as described previously BASCs were quantified in formalin fixed, paraffin embedded mouse lung tissues as described previously Total RNA was extracted from BASC cells using the RNeasy Plus Mini Kit . QPCR reagents for mouse Mmp10 mRNA were purchased from Applied Biosystems . QPCR was carried out using an Applied Biosystems 7900 thermal cycler, and data was analyzed using the SDS 2.3 software package. Data were normalized to 18S RNA.BASCs were incubated for 1 hour at 4°C with Alexa Fluor 488-conjugated CD133  and Alexa Fluor 647 conjugated Notch4  antibodies or respective isotype controls. Cells were incubated for 1 hour at 4°C with CCSP  and SPC  antibodies followed by a 30 minute incubation with Alexa Fluor 488- and Alexa Fluor 647 conjugated secondary antibodies . Flow cytometry was performed on an Accuri C6 flow cytometer and analyzed using CFlow Plus software .t test and one-way ANOVA statistical analyses were done using SigmaStat 3.5. A P value of less than 0.05 was considered statistically significant.Differences in the number and distribution of BASCs in the terminal bronchioles were assessed using the Cochrane-Armitage test using StatsDirect 2.6.1. Differences in tumor grade were assessed using the Mann-Whitney U test in Stats-Direct 2.6.1. The Student's www.nextbio.com) The correlation between Mmp10 and various types of human cancer was determined using the NextBio data mining framework  are comprised of gene expression measurements from NSCLC tumors. http://www.broadinstitute.org/gsea/msigdb/index.jsp) Gene Set Enrichment Analysis (GSEA) was carried out on all of the lung cancer gene expression datasets described above


Cancer, much like most human disease, is routinely studied by utilizing model organisms. Of these model organisms, mice are often dominant. However, our assumptions of functional equivalence fail to consider the opportunity for divergence conferred by ~180 Million Years (MY) of independent evolution between these species. For a given set of human disease related genes, it is therefore important to determine if functional equivalency has been retained between species. In this study we test the hypothesis that cancer associated genes have different patterns of substitution akin to adaptive evolution in different mammal lineages.Our analysis of the current literature and colon cancer databases identified 22 genes exhibiting colon cancer associated germline mutations. We identified orthologs for these 22 genes across a set of high coverage (>6X) vertebrate genomes. Analysis of these orthologous datasets revealed significant levels of positive selection. Evidence of lineage-specific positive selection was identified in 14 genes in both ancestral and extant lineages. Lineage-specific positive selection was detected in the ancestral Euarchontoglires and Hominidae lineages for STK11, in the ancestral primate lineage for CDH1, in the ancestral Murinae lineage for both SDHC and MSH6 genes and the ancestral Muridae lineage for TSC1.Identifying positive selection in the Primate, Hominidae, Muridae and Murinae lineages suggests an ancestral functional shift in these genes between the rodent and primate lineages. Analyses such as this, combining evolutionary theory and predictions - along with medically relevant data, can thus provide us with important clues for modeling human diseases. TDP1 gene, required in Topo1-DNA complex repair, protein sequence similarity of 81%. A point mutation from an adenine to a guanine at position 1478 in human TDP1 is linked with a disorder known as SCAN1 that results in cerebellar atrophy and peripheral neuropathy. However, this mutation in mouse does not result in the same condition/phenotype [+/− women having a 50% risk of developing breast cancer, while BRCA+/− mice do not exhibit increased susceptibility to cancer [Mouse models are currently used to research many human cancers including colon cancer. On a genome wide scale, mouse protein sequences share 78.5% sequence identity with human counterparts . With suenotype . SpecifidN) to synonymous substitutions per synonymous site (dS) is known as ω, and indicates the selective pressure at work in that sequence. If ω > 1 it signifies positive selective pressure, ω = 1 signifies neutral evolution, while ω < 1 indicates purifying selective pressure. Previous work assessed the level of positive selection present in mammal genomes and estimated 5%-9% of genes in mammals are under positive selection under a Bayesian framework, and thus provides us with a reference or expected level of positive selection for our analysis [Positive selection is the retention and spread of advantageous mutations throughout a population and has long been considered synonymous with protein functional shift. There are a number of driving forces for positive selection including external mechanisms such as adaptation to different ecological niches and response to disease and internal mechanisms such as co-evolution and compensatory mutations , all of nalysis ,8.Here we have applied a Maximum Likelihood method based on codon models of evolution to assess the selective pressures across our dataset . These mWe have taken colon cancer as an example for our study given the large amount of mutation and epigenetic data available for this form of cancer . LineageColorectal cancer (CRC) is the third most commonly diagnosed cancer in males and second in females and we have focused on this in our study . CRC ariHereditary Non-Polyposis Colorectal Cancer (HNPCC) is a hereditary predisposition for the development of colorectal cancer, and accounts for 3% of all colon cancer cases . The 22 MLH1 (mutL homologue 1) is a mismatch repair gene and is commonly associated with HNPCC. Missense mutations in MLH1 occur in the C-terminal domain, which is responsible for constitutive dimerization with the mismatch repair endonuclease PMS2 . StudiesTo assess if there is evidence for protein functional shift from the patterns of substitution in colon cancer associated genes we have carried out selective pressure analyses using codon models of lineage-specific rate heterogeneity.The colon cancer gene dataset used in this study consists of 22 genes taken from the Cancer Gene Census at the Sanger Institute . All 22 The coding DNA sequences of the single gene orthologs were translated and the resulting amino acid sequences were aligned using the default parameters in ClustalW 2.0.12 ,31. We mIt has been shown through computer simulations that sequence length has an impact on the power to infer positive selection . Power wRecombination events can result in the incorrect detection of positive selection. To reduce the detection of potential false positives from our analyses, we have implemented GENECONV (version 1.81a) . GENECONSelective pressure analyses were performed using Codeml from PAML version 4.4 ,37. BecaSeven site-heterogeneous models were used, we have retained conventional annotations for these models: Model 1a , Model 2a (Selection), Model 3 Discrete (k = 2), Model 3 Discrete (k = 3), Model 7, Model 8 and Model 8a. Two lineage-specific heterogeneous models were used: Model A and Model A Null. These models have been applied similarly elsewhere .χ2 distribution with degrees of freedom equal to the number of additional free parameters in the alternative model compared to the null model. Because of this, the critical value of the test statistic can be determined from standard statistical tables. If the p-value of the test statistic exceeds that critical value , then the null model can be rejected. For example, if the test statistic of an LRT comparing Model 1a  with Model 2a (Selection) is greater than the critical value determined from the χ2 distribution, Model 1 a can be rejected. If ω1 > 1 under Model 2a, positive selection may be inferred. Additional file The goodness-of-fit of the different models was assessed statistically using a likelihood ratio test (LRT). The LRT compares the log-likelihoods of a null model with the alternative model. For hierarchically nested models, the test statistic of an LRT approximates the In cases where positive selection is inferred, the posterior probability of a site belonging to the positively selected class is estimated using two calculations: Naïve Empirical Bayes (NEB) or Bayes Empirical Bayes (BEB). If both BEB and NEB are predicted, we will preferentially use the BEB results as these have been shown to be more robust .In-house software was designed to prepare all files for analysis and to process all output. PAML output files were parsed for parameter estimates and log likelihood values, and LRTs were performed , Northern and Western European (C), and African Yoruba (Y) populations. The “integrated haplotype score” or iHS, described first in [Selective pressures within the present day human population were analyzed for those genes with evidence of lineage-specific positive selection in the human ancestral lineages. The online tool SNP@Evolutionirst in , was empirst in . A derivStarting with a dataset of 22 genes, we identified single gene orthologs across 21 complete vertebrate genomes. Ortholog identification resulted in families with between 15 and 21 taxa, and alignment lengths of between 507 and 9,189 base pairs thus satisfying the criteria described in the materials and methods section. The test for recombination on all 22 genes is summarized in Additional file To assess the selective pressure variation, we performed both site- and lineage-specific selective pressure analyses and subsequently assessed the statistical significance of all results via LRT analyses to ascertain the codon evolutionary model of best fit. In those cases where the ω value vastly exceeds 1, we have simply denoted them as ω > > 1 throughout the manuscript, as there is no biological significance for these extremely large ω values (the precise numbers are shown in the Tables throughout). The lineage-specific analyses are more pertinent to the main focus of the paper, i.e. - the identification of species-specific patterns of substitution in these colon cancer associated genes. Therefore the lineage-specific results have been described in detail in the following section. Site-specific results are briefly summarized on a gene-by-gene basis. All positively selected sites were assessed using functional information from the Uniprot database . The modLineage-specific models of codon evolution were assessed at multiple phylogenetic depths, (i) the extant lineages within the Euarchontoglires clade, and (ii), all ancestral lineages leading from the Euarchontoglires to modern mouse and human were also tested independently as depicted in Figure In the following section, we have analyzed the positively selected sites for those genes with evidence of lineage-specific positive selection in the context of their potential functional relevance for those genes. This was carried out for all genes where functional sites and/or domains have been elucidated. All sites described were calculated via Bayes Empirical Bayes (BEB) analysis (unless otherwise specified). In all cases we are assessing the potential functional importance of residues based on their sequence position. There are instances where we identify stretches of protein sequence under positive selection - there is a possibility that these sites may have very different functions despite their sequence position. For a total 16 of the 22 genes there were partial or complete 3D structures available. However, many of the positively selected sites identified were located in regions that were not yet fully resolved at the structural level, and so only the 3D model for STK11 is given. The most ancestral branch tested was the Euarchontoglires ancestral branch, i.e. the ancestor of the Primate, Rodent and Glires clades as depicted in Figure The branch leading from the Euarchontoglires ancestor towards the primates was analyzed, we have termed this branch the ancestral Primate branch as depicted in Figure The next branch in the primate clade is that leading to modern great apes, i.e. Hominidae, as depicted in Figure A second gene showing evidence of positive selection in the Hominidae ancestral branch is the VHL dataset consisting of 18 taxa. The VHL gene encodes Von Hippel-Lindaue tumour suppressor protein. Mutations occurring in this gene can result in von Hippel-Lindau disease (VHDL) - a dominantly inherited familial cancer syndrome . VHL exhAnalysis of modern non-human primates also identified positive selection in a number of genes. In VHL positive selection was detected in the Chimpanzee lineage where 1.2% of sites have ω > > 1, and also in the Marmoset lineage where 5.5% sites have ω > > 1. Sites under positive selection were compared against human Swiss-Prot entry (P40337), however the region (1–60) was only represented by 11/18 species in the alignment and therefore we do not have sufficient confidence in these positions to explore these sites in more detail.The MSH6 gene dataset contained 19 taxa and showed evidence of positive selection in both the Gorilla and Marmoset lineages each displaying 3.2% of sites with ω > > 1. Gorilla and Marmoset extant lineages were compared against human P52701) Swiss-Prot entry. No relevant functional information could be extracted from positively selected sites in Gorilla, however 2/45 positively selected sites in Marmoset fall in close proximity to cancer variants. In marmoset, positively selected site 803 PP = 0.551) coincides with Human colorectal cancer variants D803G [ coincide701 SwissMSH2 alignment consists of 18 taxa. The function of the MSH2 protein is in post-replicative DNA mismatch repair system (MMR). Mutations in MSH2 result in hereditary non-polyposis colorectal cancer type 1 (HNPCC1) . LineageTuberous sclerosis 2 protein (TSC2) interacts with TSC1 protein and mutations in this gene can cause tuberous sclerosis type 2 . The aliATM acts as a DNA checkpoint sensor by activating checkpoint signaling upon double strand breaks . The aliThe extant Orangutan lineage also showed evidence of positive selection in the TSC1 gene for 1.2% of its alignment (ω > > 1). Positively selected sites were compared against human Swiss-Prot sequence (Q92574) and mouse Swiss-Prot sequence (Q9EP53) however there was insufficient information to extrapolate the potential functional impact of these sites.Homo sapiens, i.e. the primate and Hominidae lineages , were further analyzed to determine if there is evidence for ongoing positive directional selection in modern day human populations. The integrated haplotype score, iHS [Genes displaying evidence of positive selection in lineages leading to re, iHS , was calThe ancestral Muridae branch marks the most recent common ancestor of modern mouse, rat and guinea pig species and is depicted in Figure in vitro functional assaying given their uniqueness to the rodent clade and their retention in all modern rodents tested.The ancestral Murinae branch defines the most recent common ancestor of mouse and rat. In total there were two genes identified as being under positive selection in the Murinae lineage. The first is the MSH6 gene that acts as a DNA mismatch repair protein and is a component of the post–replicative DNA mismatch repair system . MSH6 alThe second gene with evidence of positive selection on the ancestral Murinae lineage is the SDHC  gene. The SDHC function is to act as a membrane-anchoring subunit for the SDH protein. Defects in this protein are reported in paragangliomas and gastric stromal sarcomas . The datThe SDHC gene again showed evidence of positive selection, this time in the extant Rabbit lineage with 35.59% of sites under positive selection (ω = 3.59). 15/51 positively selected sites were identified as occurring within 10 amino acid positions of the metal binding site 127, also mentioned in the ancestral Murinae analysis. While there are extremely high levels of positive selection identified in the rabbit lineage, no other relevant functional information could be gleaned from the databases at this point.The MUTYH alignment consisted of 21 taxa and showed evidence of lineage-specific positive selection in 1.4% of sites in the extant Rabbit lineage (ω > > 1). Positively selected sites were compared to human (Q9UIF7) and mouse (Q99P21) Swiss-Prot entries, however no relevant functional information could be extrapolated. Radical substitutions occurred in all 5 BEB sites in the extant Rabbit lineage, three of which are at positions 485–487 in the Nudix hydrolase domain.The MLH1 gene codes for a critical protein involved with the post-replicative DNA mismatch repair system. Defects in this gene result in hereditary non-polyposis colorectal cancer type 2 (HNPCC2) . The aliFinally, the BHD gene showed evidence of positive selection in the extant Rabbit lineage. The function of the BHD gene is still largely unknown, however it is thought that it may be a tumour suppressor and it may be involved in colorectal tumorigenesis . The aliMADH4 is the co-activator and mediator of signal transduction by TGF-beta. Defects in MADH4 result in pancreatic, colorectal, juvenile polyposis syndrome, juvenile intestinal polyposis and primary pulmonary hypertension ,70. The NF1 is thought to be a regulator of RAS activity . DefectsTSC1 also shows evidence of positive selection in the extant guinea pig lineage where 1.2% of the sites have ω > > 1. As before, the positively selected sites were compared against human Swiss-Prot sequence (Q92574) and mouse Swiss-Prot sequence (Q9EP53) however there was insufficient information to extrapolate potential functional impacts of these sites.The site-specific results may be beneficial to those working on rational mutagenesis and/or the identification of functionally important regions in these colon cancer associated genes and so these results have been summarized. We have identified five genes that have signatures of site-specific positive selection, namely: CDH1, MUTYH, PMS1, PMS2 and TP53, representing ~23% of the dataset. For each of these five genes, the model of best fit was the site-heterogeneous model “model 8”, see Table Defects in the CDH1 member of the Cadherin family are linked to hereditary diffuse gastric cancer ,28. The The MUTYH dataset consisted of 21 taxa and site-specific analysis identified 18 sites under positive selection (ω = 2.44), representing 2.8% of the MUTYH protein (Table PMS1 (postmeiotic segregation increased 1) encodes a DNA mismatch repair protein and this dataset consists of 20 taxa. Defects in PMS1 are reported to cause hereditary non-polyposis colorectal cancer type 3 (HNPCC3) . AnalysiMismatch repair endonuclease PMS2 (postmeiotic segregation increased 2) is a component of the post-replicative DNA mismatch repair system . DefectsTP53 (cellular tumor antigen p53) acts as a tumor suppressor by inducing apoptosis or arresting growth depending on the physiological circumstances and cell type . The TP5The results we have presented are indicative of selective pressures acting in a lineage-specific manner. The positively selected sites we have identified in this study frequently reside in regions of functional importance, such as glycosylation sites, protease cleavage sites, and sites known to interact with proteins involved in DNA damage repair pathways. Also of note, positively selected residues are frequently located at, or in close proximity to, known cancer associated sites although the statistical significance of these coincidences cannot be concluded with such a small sample sizes. Larger sample sizes and more complete functional information will be hugely beneficial in resolving whether these positively selected residues are most likely positioned to or at variants associated with cancer.Drosophila species indicate that positive selection is pervasive in this species with positive selection present in ~25% of the genes [In using the mouse as a model organism for colon cancer, we are making an assumption that the orthologs in both species are functioning in precisely the same way despite ~ 180 MY of independent evolution. We found no evidence of functional divergence in the extant human and mouse lineages for the genes analyzed. However, upon testing the lineages leading from the MRCA of mouse and human, i.e. Euarchontoglires, positive selection has occurred on certain ancestral branches and in specific extant lineages. In the ancestral lineages of primates, rodents and glires there is evidence of positive selection in 6 of the 22 genes tested  of the species tested vary enormously, with estimations for modern human populations in the range of Ne = 7,500 to 3,100 [Ne = 58,000 to 25,000 [Ne may also contribute to detection of false positives. We have also detected weak evidence for ongoing selective pressure in the human genome on the STK11 and CDH1, but these signals of selection may be artifacts of the very small effective population size of modern humans. Smaller Ne values are associated with increased fixation of slightly deleterious substitutions and subsequent elevated ω values [Ne[[Overall, these results indicate that the function of certain proteins associated with colon cancer display distinct lineage-specific patterns of substitution indicative of positive selection in the ancestral human and mouse lineages. There are a number of selective pressures on any given protein that can contribute to patterns of substitution that are “roteins . Compensroteins . The effo 3,100 , while e 25,000  and this values . Such slues [Ne[ will aiddN: Nonsynonymous substitutions per nonsynonymous site; dS: Synonymous substitutions per synonymous sites; LRT: Likelihood ratio test; ML: Maximum likelihood; MY: Millions of years; Ne: Effective population size; NEB: Naïve empirical bayes; PP: Posterior probability.BEB: Bayes empirical bayes; The authors declare no conflict of interest.CCM and KS carried out all data assembly. KS, CCM and AEW carried out all homolog identification and MSAs. CCM carried out all data quality and phylogeny analyses. CCM, KS, AEW, and TAW carried out all selective pressure analyses and designed the necessary software. ML carried out all structural analyses. All authors participated in drafting the manuscript. MJO'C conceived of the study, its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.Details of the data used in the analysis, the 21 species and their genome coverage. Orthologs that were not found by the Ensembl genome browser are labeled in black, orthologs identified are shown in white.Click here for fileComplete set of all multiple sequence alignments used in the analysis. The data is presented on a gene-by-gene basis in nexus format. Click here for fileLikelihood ratio tests performed and their associated significance values. Click here for fileFull set of models, associated likelihood scores and parameter estimates for all genes in the colon cancer gene dataset. This information is given alphabetically on a gene-by-gene basis. All estimated parameters, Likelihood values and BEB or NEB sites are listed. Click here for fileFull set of recombination test results on a per gene and per species basis. The value highlighted in yellow for TP53 represents a region where recombination was detected with reasonable confidence that also coincided with a positively selected residue . Click here for file