Sgcg), a dystrophin-associated protein, develop muscular dystrophy and cardiomyopathy similar to their human counterparts with limb girdle muscular dystrophy. With disruption of the dystrophin complex, the muscle plasma membrane becomes leaky and muscles develop increased fibrosis.Mice from the MRL or “superhealing” strain have enhanced repair after acute injury to the skin, cornea, and heart. We now tested an admixture of the MRL genome and found that it altered the course of muscle pathology and cardiac function in a chronic disease model of skeletal and cardiac muscle. Mice lacking γ-sarcoglycan (Sgcg mice, and cardiac function was measured. Muscles were assessed for fibrosis and membrane leak using measurements of hydroxyproline and Evans blue dye. Quantitative trait locus mapping was conducted using single nucleotide polymorphisms distinct between the two parental strains.MRL/MpJ mice were bred with Sgcg model. The MRL genome was also associated with improved cardiac function with reversal of depressed fractional shortening and the left ventricular ejection fraction. We conducted a genome-wide analysis of genetic modifiers and found that a region on chromosome 2 was associated with cardiac, diaphragm muscle and abdominal muscle fibrosis.Introduction of the MRL genome reduced fibrosis but did not alter membrane leak in skeletal muscle of the These data are consistent with a model where the MRL genome acts in a dominant manner to suppress fibrosis in this chronic disease setting of heart and muscle disease. Murphy Roths Large (MRL) mice are an inbred mouse strain noted to have enhanced healing ability. This MRL strain was initially discovered because of its rapid ability to heal ear holes,2. The MThe dystrophin complex is composed of membrane-associated proteins that mediate membrane stability in heart and skeletal muscle. Mutations that disrupt expression of dystrophin or its associated proteins the sarcoglycans proteins cause progressive cardiac and skeletal muscle degeneration in humans and mouse models. At the cellular level, the loss of dystrophin or the sarcoglycans leads to a disrupted sarcolemmal membrane that is abnormally leaky. Membrane permeability results in increased intracellular calcium that triggers proteolysis and necrosis. An inflammatory response also contributes to muscle degeneration. Muscle Sgcg gene and model muscular dystrophy and relative wall thickness [(IVS thickness + PW thickness)/LVIDd] were calculated from the M-mode measurements.Twelve-week animals were evaluated by echocardiography as described,26. Inven = 1,701) informative between the parental DBA/2J and MRL/MpJ strains were genotyped in 80 SgcgMRL/D2F2-F4 Sgcg animals on the Illumina GoldenGate platform using the Mutation Mapping and Developmental Analysis Panel (MMDAP)[Single nucleotide polymorphisms  and the  (MMDAP). R packa (MMDAP),30. Signp < 0.05 was considered significant. Data from 197 Sgcg animals from an F3 intercross between the DBA/2J and 129T2/SvEmsJ backgrounds (SgcgD2/129 ) was compared to the SgcgMRL/D2cohort.For HOP and dye uptake assays, data were analyzed by one-way, unpaired ANOVA with parametric methods followed by the Tukey multiple comparison post-test , and fas allele[Sgcg null) since these mice are a model of limb girdle muscular dystrophy 2C[Sgcg null and then interbred to generate Sgcg null mice with a 50% MRL/MpJ and 50% DBA/2J contribution. In Sgcg null mice, like all dystrophin complex-associated mutations, fibrosis and collagen deposition is increased in the heart and muscle, similar to what is seen in human patients with similar mutations[Sgcg heart and muscles  when using the stringent QTLRel analysis. However, the overlapping intervals found in heart, diaphragm and abdominal muscles provide additional support that this interval modifies fibrosis.We conducted a genome-wide scan using markers that were informative in the two parental strains DBA/2J and MRL/MpJ Figure. QTLRel Ltbp4 gene[Sgcg muscular dystrophy. Ltbp4 encodes latent TGFβ-binding protein 4, and TGFβ proteins have been extensively linked to fibrosis in many disease states including muscular dystrophy[Ltbp4 allele with an additional 12 amino acids inserted in exon 12. Because the SgcgMRL/D2 cohort used here contained both the protective  and the disease-enhancing allele (Ltbp4D), we tested whether Ltbp4 genotype correlated with fibrosis in the SgcgMRL/D2 cohort. TableLtbp4 genotype in the SgcgMRL/DBA2J cohort. Therefore, Ltbp4 does not account for the suppressive effect of the MRL background, and other genetic modifiers account for this difference.The DBA/2J strain contains an insertion/deletion polymorphism within the bp4 gene that modystrophy,36. MostMutations in the dystrophin or sarcoglycan genes share a common pathological mechanism characterized by disruption of the plasma membrane of cardiomyocytes and skeletal myofibers. Membrane leak is an early step in the pathological process while fibrosis is thought to be a secondary response. Our study suggests that there are genetic pathways that target fibrosis without altering the sarcolemmal leak properties. In the heart, reduced fibrosis was correlated with functional benefit, although cardiac function may be improved from both cardiac intrinsic as well as cardiac extrinsic features. For example, improved skeletal muscle function may contribute to improved cardiac function, particularly when considering the contribution of the respiratory musculature.SgcgMRL/D2 animal that had markedly elevated fibrosis. This stands in contrast to what is normally observed in the Sgcg animals where outliers with markedly elevated fibrosis are often seen. The maximum values for fibrosis in SgcgMRL/D2 mice were all well below what was measured in SgcgD2 mice. This is consistent with multiple, dominant genetic loci imparting the improved healing capabilities, similar to what has been observed for the ear hole repair properties in the MRL strain[SgcgMRL/D2 mice, with almost complete suppression of fibrosis at 32 weeks, suggests that a persistent healing effect can arise from the MRL strain.The MRL strain typically shows the improved healing capability in its first 2–6 months of life. After 6 months of age, wild-type MRL mice develop autoimmune disorders; it is for this reason that we conducted studies using a 50% contribution of the MRL strain. Quantitative trait mapping has been used to define many different genetic regions associated with the healing properties. The autL strain. The finJag1, encoding jagged the ligand for the Notch receptor. Loss of function of Notch 3 leads to muscle hyperplasia, especially when subjected to repetitive injury[Bmp2-encoding bone morphogenetic protein 2. The BMPs mediate musculoskeletal regeneration[When considering the overlapping region on chromosome 2 from the heart, diaphragm and abdominal fibrosis data, the interval contains 49 known genes. There are additional predicted and unnamed genes and genes encoding olfactory receptors that are not considered likely candidates. Within the interval is e injury. Ex vivoe injury. Anotherneration, and givThe ability of the heart to recover after injury has been studied in the MRL strain using cryoinjury, left anterior descending ligation and ischemia reperfusion methods-10,14,15A number of mechanisms likely act in concert to achieve suppression of fibrosis and improvement of function. In skeletal muscle, where muscle stem cells robustly regenerate muscle after injury, there is indirect evidence of enhanced regeneration. Specifically, there is an increase in centrally nucleated myofibers, which is thought to reflect enhanced myoblast fusion. However, our data do not distinguish whether the MRL background exerts its effect on muscle regenerative cells, or by creating a more supportive extracellular matrix or both. MRL animals heal with embryonic characteristics with enhanced blastema formation and metabolic and gene expression features consistent with an earlier developmental state. AlteredSgcg mouse. Although membrane leak was still evident in Sgcg mice sharing a portion of the MRL genome, the reduction in fibrosis was associated with improved cardiac function. The identification of gene(s) from the MRL genome will help identify pathways important for chronic repair of myopathic processes.Herein the MRL background was shown to reduce fibrosis in a chronic model of muscular dystrophy and cardiomyopathy, the BMP: Bone morphogenetic protein; D2: (DBA/2J); DMD: Duchenne muscular dystrophy; HOP: Hydroxyproline; LTBP4: Latent TGFβ-binding protein; MRL: Murphy Roth Large; QTL: Quantitative trait loci; SNP: Single nucleotide polymorphism; TGFβ: Transforming growth factor β.The authors have no competing interests related to this work.AH conducted the phenotypic analysis of muscle. KAS conducted the genome-wide SNP analysis and phenotype analysis. GK performed the echocardiographic analysis. MH oversaw the breeding. JHC assisted with phenotypic analysis. EMM conceived the experiments, analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.Figure S1. Shown are gross images from Sgcg vs. SgcgMRL/D2 mice. Evans blue dye uptake could be readily seen in the quadriceps and diaphragm muscles and did not appear grossly altered by the presence of the MRL background. In contrast, fibrosis was visually reduced in the quadriceps and heart of SgcgMRL/D2 compared to Sgcg mice. The diaphragm muscle retained evidence of fibrosis in SgcgMRL/D2, but the white stripes of fibrosis were smaller, and intact diaphragm muscle was still evident compared to the near total replacement of diaphragm muscle in Sgcg mice. Figure S2. Shown is staining for apoptosis with TUNEL and caspase indicating no gross differences between Sgcg and SgcgMRL/D2 muscle. CD3 and MAC1 staining to examine T cell and macrophage infiltrate also did not appear grossly altered by the presence of the MRL background.Click here for file


Caecm33 double mutant showed reduced biofilm formation and causes less damage to gingival mucosa tissues. This was confirmed by the reduced level of necrotic cells and Bax/Bcl2 gene expression as apoptotic markers. In contrast, parental and Caecm33 mutant strains decreased basement membrane protein production (laminin 5 and type IV collagen). We thus propose that ECM33 gene/protein represents a novel target for the prevention and treatment of infections caused by Candida.In this study we demonstrated that Δ Candida species are the most frequent cause of life-threatening invasive fungal infections in the immunocompromised host  reduction assay [Candida cells served as controls. To confirm biofilm formation, the Candida-seeded SE discs were transferred to microscope slides, stained with calcofluor-white (0.05% (vol/vol)) and examined under a fluorescence microscope at λmax = 432 nm.The first step was to prepare the substrate material (silicone elastomer) for biofilm formation. Silicone elastomer (SE) sheets were obtained from Invotec International . Following the manufacturer's instructions, the catheter material sheet was cleaned by scrubbing it thoroughly with a clean, soft-bristled brush in a hot water/hand soap solution, rinsed with distilled water, and autoclaved. Flat circular discs 15 mm in diameter were obtained by cutting through the catheter sheets with a cork borer, as previously described . The steon assay . Discs cC. albicans pathogenesis.Gingival mucosa samples were biopsied from healthy donors following their informed consent and approval by the University Lavals ethics committee. Gingival fibroblasts and epithelial cells were subsequently extracted . FollowiC. albicans (104 cells/cm2) strains for 24 h. Noninfected EHOM was used as the control (Ctrl). Following infection and culture, the biopsies were collected, fixed with 4% paraformaldehyde solution, embedded in paraffin, and stained with hematoxylin-eosin for analysis.EHOM was first grown in a serum-free, antifungal-free DMEH medium for 48 h and then placed in contact with one of the C. albicans strains, multiple specimens were collected from each infected EHOM and stained with hematoxylin-eosin. Each slide was assigned a number, and measurements were made independently by two different observers. Each blind observer measured the thickness of C. albicans biofilms at regular intervals using a calibrated image analysis system , as we previously reported [P value of <0.05 was considered statistically significant (n = 4).To quantitatively assess the differences in biofilm formation by the selected reported . Ten meaCandida strain. LDH activity was calculated as the percetage of total LDH activity release = (ITabsorbance − NCabsorbance)/(PCabsorbance − NCabsorbance) × 100. Differences between groups were determined by an ANOVA Student t-test and was considered significant at a P value of <0.05 (n = 4).The release of lactate dehydrogenase (LDH) from the EHOM into the surrounding medium was monitored as a measure of tissue damage/cell necrosis. LDH activity release into the medium of the infected tissue (IT) was measured at 24 h using a Cytox96 kit  according to the manufacturer's instructions. A positive control (PC) was obtained by culturing the cells for one hour in the presence of 1% Triton X-100, while a negative control (NC) was obtained by culturing cells not exposed to any Candida strains, total RNA was extracted from each EHOM and used to comparatively analyze Bax and Bcl2 gene expression by qRT-PCR [Candida-infected EHOM was used to prepare protein cell lysates which were then subjected to SDS polyacrylamide gel electrophoresis and electroblotting onto PVDF membranes [Following EHOM infection with the  qRT-PCR  and GAPDembranes . The memCandida strains, the EHOM protein lysates were used to assess laminin 5 and type IV collagen production by western blotting using mouse antilaminin 5 (1 : 200) or mouse antitype IV collagen (1 : 1000). Each membrane was washed in TBS-T then incubated with the appropriate secondary horseradish peroxidase-labeled antibody (1 : 1000) for 1 h at 23°C. Protein detection was performed as described above.Following contact for 24 h with one of the Caecm33 mutant tended to form less biofilms than did the isogenic wild-type strain  on the outer layer of the epithelium  decreased compared to that observed with the CAF2-infected tissue  down regulated in the infected tissues. It is important to note that all of the tested Candida strains significantly decreased laminin 5 and type IV collagen levels in the infected EHOM (ECM33 gene mutation did not prevent the laminin 5 and type IV collagen decreases.Western blot results show elevated basal levels of laminin 5 and type IV collagen proteins in the noninfected EHOM , whereasted EHOM  comparedC. albicans adhesion to various surfaces, including mucosal tissue and catheter disks, is the first step in biofilm formation and host infection [Candida adhesion to and biofilm formation on catheter discs were modulated by ECM33 gene, and that the disruption of this gene led to decreased biofilm formation, which supports previously reported data with medical devices [nfection , 33. Our devices . Thus ad devices .C. albicans adhesion to mammalian cells and tissue is under the control of various genes, such as ECM33. Using an EHOM model, we demonstrated that Caecm33 mutant was unable to damage tissue structure, compared to the CAF2 strains. This inability to cause tissue damage may be linked to a defect in the ability of Caecm33 mutant to form hyphae. Indeed, it was reported that Caecm33 mutants displayed filamentation defects in both liquid and solid media [ECM33 mutant to damage the EHOM tissue may also be due to cell wall architectural changes that hamper interactions with host cell/tissue. Caecm33 mutants have in fact been shown to exhibit significant cell changes, including an abnormally electron-dense outer mannoprotein layer [id media . The inain layer . This arCaecm33 mutants were also less capable of promoting cell death/apoptosis by reducing LDH release and Bax gene expression. These findings support the reduced tissue disorganization when placed in contact with the Caecm33 mutants. Recent studies suggest that the decreased host cell damage caused by Caecm33 mutants is likely due in part to the reduced endocytosis of these strains [Caecm33 mutant strains may also secrete less lytic enzymes, such as secreted aspartyl proteases and phospholipases [Caecm33 mutant strains. strains . Caecm33olipases , thus coECM33-positive gene may also modulate some physiological mechanisms such as BM protein production. Indeed, following contact with the CAF2 and Caecm33 mutant strains, both laminin 5 and type IV collagen levels decreased significantly. By decreasing BM protein production, Candida may thus deregulate epithelial and connective tissue structural interactions, thereby reducing their capacity to prevent its invasion [Candida invasion requires adhesion to BM proteins as well as their degradation [Candida is capable of degrading these proteins, this does not necessarily mean that Caecm33 mutant will, in turn, downregulate laminin 5 and type IV collagen protein production. The deletion of single or double Caecm33 alleles led to the same effect as that produced by the CAF2 strains on BM protein expression. This suggests that ECM33 mutation does not reduce the ability of Candida to decrease BM protein synthesis by gingival cells. Further research will be conducted to shed light on this possible mechanism.EHOM disorganization following contact with the invasion . It has radation . AlthougCaecm33 mutant was not able to damage tissue structure and promote cell apoptosis. We also demonstrated that biofilm formation was reduced by Caecm33 mutants compared to parental strains. Finally, we showed that parental and Caecm33 mutant strains downregulated laminin 5 and type IV collagen production. Overall data thus suggest that ECM33 gene plays an active role in Candida-host interactions and is responsible for tissue damage that ultimately leads to candidiasis.In conclusion, we used an engineered human oral mucosa model to demonstrate that


Gene expression profiling of human breast tumors has uncovered several molecular signatures that can divide breast cancer patients into good and poor outcome groups. However, these signatures typically comprise many genes (~50-100), and the prognostic tests associated with identifying these signatures in patient tumor specimens require complicated methods, which are not routinely available in most hospital pathology laboratories, thus limiting their use. Hence, there is a need for more practical methods to predict patient survival.We modified a feature selection algorithm and used survival analysis to derive a 2-gene signature that accurately predicts breast cancer patient survival.KIAA0191 expression in the context of E2F1 expression levels. This approach led to highly accurate survival predictions in a large cohort of breast cancer patients using only a 2-gene signature.We developed a tree based decision method that segregated patients into various risk groups using E2F1 and KIAA0191 expression that is relevant to the pathogenesis of breast cancer. Furthermore, our findings raise the prospect that the practicality of patient prognosis methods may be improved by reducing the number of genes required for analysis. Indeed, our E2F1/KIAA0191 2-gene signature would be highly amenable for an immunohistochemistry based test, which is commonly used in hospital laboratories.Our observations suggest a possible relationship between  Traditionally, a variety of clinical and histopathological characteristics have been employed to make predictions regarding the potential clinical outcomes of breast cancer patients. However, the advent of gene expression profiling technologies has enabled the use of molecular signatures to provide improved predictions of clinical outcome over traditional methods -5. TheseBecause the expression of proliferation associated genes has been shown to group breast cancer patients into good and poor risk groups , we sougKIAA0191 transcripts can be used in conjunction with those of E2F1 to more accurately predict breast cancer patient survival than does E2F1 expression alone. KIAA0191, commonly known as TUT4 or ZCCHC11, encodes a canonical poly (A) polymerase, whose function involves the polyadenylation of pre-mRNA in the nucleus [. KIAA0191 has been shown to work in concert with Lin28 to suppress microRNA biogenesis through uridylation of pre-microRNA. Importantly, KIAA0191 function has not been previously linked to E2F1. Here we demonstrate that the expression of KIAA0191 transcripts alone is not related to breast cancer patient survival. However, in the context of average to high expression of E2F1 transcript levels, high KIAA0191 expression was linked to poor breast cancer patient prognosis, whereas low KIAA0191 expression was linked to good outcome for these patients. Interestingly, our study identified a potentially novel functional relationship between E2F1 and KIAA0191, which may be of clinical relevance to breast cancer patients.Using an algorithm we published recently , we founhttp://microarray-pubs.stanford.edu/wound_NKI/explore.html) for our analyses. We also downloaded a matrix containing clinical data for the patients that provided samples for the microarray profiles used in the present study from the same location. We created a master data matrix by combining the gene expression profiles with indices for survival and metastasis for each patient. Patients included within this cohort had either stage I or II breast cancer and were less than 53 years of age. The prevalence of lymph-node positive and lymph-node negative disease was approximately 50% for each, respectively.We used data from the Stanford microarray repository  was calculated using the Hmisc package in R .http://www.random.org).Randomly selected genes were obtained by using a random number generator  generally were expressed at low levels in tumors where high-average E2F1 expression was not associated with poor patient survival. The mostly highly ranked candidate among the 295 patient cohort was KIAA0191, which is also commonly known as TUT4 or ZCCHC11 [Because eviously  to find KIAA0191 expression alone was related to patient survival, we divided the patient cohort into KIAA0191 high and low expressing groups, as described above. This led to 154 patients being selected for the KIAA0191 high expression group and 141 patients being selected for the low expression group .To learn whether p Figure . We compKIAA0191 expression was only predictive of patient survival in the context of E2F1 transcript levels, we devised a tree-based decision strategy to integrate KIAA0191 expression into our model of using E2F1 transcript abundance to separate breast cancer patients into good and poor prognosis groups  and BUB1, which like E2F1, are independently prognostic (data not shown) and linked to cell proliferation [AURKA or BUB1 transcript levels for that of E2F1, resulted in highly similar risk grouping as observed with E2F1 expression , however future studies will need to replicate these findings using independent gene expression data sets.An advantage of our 2-gene signature over currently available prognostic signatures is that it may be suitable for development as an immunohistochemical based test. As mentioned previously, immunohistochemical based tests are faster, cheaper, and have greater availability to patients, than the currently available mRNA based tests. Furthermore, antibodies that `recognize E2F1 and KIAA0191 are commercially available, and several protocols exist for the quantification of protein expression using immunohistochemistry . HoweverE2F1 expression was most related to poor patient outcome when that patient's tumor also expressed high levels of KIAA0191. When KIAA0191 was not expressed at high levels, the relationship between high E2F1 transcript levels and poor outcome was significantly reduced. In line with these observations, average levels of E2F1 expression were associated with poor patient outcome when KIAA0191 was highly expressed, and good patient outcome when KIAA0191 was expressed at low levels. Indeed, we took advantage of this relationship to generate a 2-gene based decision tree, which made highly accurate predictions about patient outcome, while only taking into account the expression of 2 genes.A major implication of this study is that it is important to understand the context in which a gene's expression is most highly related to patient survival. For example, we observed that high We envision that the identification of gene signatures, which are highly predictive, but consist of relatively few genes (2-3 genes), would allow the use of immunohistochemical or immunofluorescent based assays that are commonly used in hospital-based pathology laboratories to readily guide the use of chemotherapeutics in breast cancer patients. Importantly, immunohistochemical or immunofluorescent testing does not require long distance transfer of tumor samples to molecular profiling facilities (as is the case for MammaPrint™ and Oncotype DX) and thus would provide a less time-consuming and less costly means of providing prognostic information to breast cancer patients.The authors have filed a provisional patent on the use of 2-gene signatures for breast cancer patient prognosis.RMH, conception of project and performed research; RMH, and JAH, interpretation of data and writing of manuscript. All authors have read and approved the final manuscript.


Drosophila lymph gland. Mechanisms that modulate and integrate these signals are poorly understood. Here we report that mutation in a conserved endocytic protein Asrij affects signal transmission and causes aberrant lymph gland hematopoiesis. Mammalian Asrij (Ociad1) is expressed in stem cells of the blood vascular system and is implicated in several cancers. We found that Drosophila Asrij is a pan-hemocyte marker and localizes to a subset of endocytic vesicles. Loss of asrij causes hyperproliferation of lymph gland lobes coupled with increased hemocyte differentiation, thereby depleting the pool of quiescent hemocyte precursors. This co-relates with fewer Col+ cells in the hematopoietic stem cell niche of asrij mutants. Asrij null mutants also show excess specification of crystal cells that express the RUNX factor Lozenge (Lz), a target of Notch signaling. Asrij mutant lymph glands show increased N in sorting endosomes suggesting aberrant trafficking. In vitro assays also show impaired traffic of fluorescent probes in asrij null hemocytes. Taken together our data suggest a role for Asrij in causing increased Notch signaling thereby affecting hemocyte differentiation. Thus, conserved endocytic functions may control blood cell progenitor quiescence and differentiation.Several signaling pathways control blood cell (hemocyte) development in the  Drosophila hematopoiesis Drosophila hematopoiesis.The conservation of mechanisms as well as ontogeny of blood development over the course of evolution is well established asrij expression in Drosophila hemocytes asrij expression is also seen in Drosophila prohemocytes and is independent of the prohemocyte marker Serpent (Srp) Ovarian Carcinoma Immunoreactive Antigen domain 1 (Ociad1). Mouse Asrij localizes to endocytic vesicles Drosophila yeast two hybrid screen asrij/ociad1 has not been reported. However, mis-regulation of ociad1 is associated with several hematological neoplasms asrij in hematopoiesis, we undertook a functional analysis of Asrij in Drosophila.We previously reported Drosophila lymph gland is the best studied site of hematopoiesis. Lymph gland hemocytes are released only at metamorphosis By the third instar the lymph gland has additional secondary and tertiary lobes residing between segments T3 to A3 asrij leads to a dramatic increase in the number of lymph gland lobes. Asrij blocks hemocyte precursor differentiation and controls hemocyte number. We present a detailed analysis of the hematopoietic defects associated with asrij mutants. We also show that Asrij modulates Notch signaling and discuss the importance of endosomal trafficking in hematopoiesis. These results provide definitive genetic evidence that loss of asrij promotes aberrant cell proliferation and differentiation in vivo and will help enhance our understanding of pathways affected in hematopoietic disorders.In this report, we use genetic analyses to reveal an important role for endocytic proteins in hematopoiesis. We show that Asrij is expressed in embryonic and lymph gland hemocytes. A null mutation in Drosophila stocks were maintained under standard rearing conditions at 25°C. Canton-S was used as the wild type reference strain. Respective UAS or GAL4 parent stocks or w1118 were used as controls where appropriate. P element stock KG08017 (Bloomington # 14935) was used to generate excision lines of asrij by following standard procedure (see asrij cDNA (BDGP clone ID AT12418) was cloned in pPUAST vector Dmasrij-RNAi transgenic flies were obtained from National Institute of Genetics, Japan. Other stocks used in this study were HemolectinGAL4 (Bloomington # 6395), e33cGAL4  and lzGAL4UASGFP (Bloomington # 6314), domelessGal4;UAS-mCD8GFP .dure see . For expE. coli. Antisera were checked for specificity to the immunogen by Western blot analysis . cDNA was prepared using Superscript enzyme (Invitrogen Bioservices) and used as a template for PCR amplification. qRT-PCR was performed using SYBR green chemistry in a Rotor Gene 3000 (Corbett Life Science 3000) and analyzed with the accompanying software. Primer sequences used for RT-PCR and qRT-PCR are provided in Circulating hemocyte counts were obtained as described before asrij mRNA expression in embryonic hemocytes asrij RNA by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and of protein by Western blot analysis and immunolocalization at different developmental stages of Drosophila melanogaster. We analyzed asrij mRNA expression at the whole animal level and found that it is present at all developmental stages  (asrij knockdown and over-expression constructs under the control of the UAS-GAL4 system (asrij RNA (asrij knockdown cells had greatly reduced Asrij expression (≤50%) in all hemocyte classes (data not shown).The larval lymph gland is a good model for studying conserved mechanisms in definitive hematopoiesis cessive) –F as wel4 system . The nulsrij RNA  and protsrij RNA –F. asrij9arj/9arj embryonic lymph glands (asrij null mutant larvae (9/arj9arj) showed higher number of secondary lymph gland lobes which expanded into posterior segments up to A4 or A5 . Staining for Odd expression showed that h glands ' were coh glands '. 2–4 pah glands . All asrA4 or A5  as compaA4 or A5 . The muthenotype . This ph drivers  (n>50).  embryos  or seconaffected . Furtheraffected .asrij null mutant as compared to wild type. However the mutant secondary lobes showed a dramatic increase in mitotic foci , which is an indicator of mitosis. There was no significant change in the number of mitotic foci in the primary lobe of tic foci , indicatasrij mutants  as compared to controls . (+ cells in the mutant (20±5.7) as compared to control (38.4±11.2) suggested a role for asrij in maintenance of Col+ cells  - expressing cells. The primary role of the PSC in the third instar larva is to act as a hematopoietic stem cell niche whereby it signals to maintain hemocyte precursors in the medullary zone. During metamorphosis or upon immune challenge the precursors differentiate  H3P see  suggestiasrij mutant a large number of P1+ plasmatocytes were seen all over the primary lobe -mediated signaling through the Notch (N) pathway, results in specification of Lzasrij mutant lymph gland cells showed a change in NICD localization. Co-staining for the membrane-localized lymph gland marker Pvr showed that while NICD was localized primarily to the membrane in control cells, asrij mutant cells showed diffuse NICD staining  is used as an indicator of molecular movement within the cell during endocytosis Asrij null hemocytes showed greatly reduced uptake of the probe  are lethal (Inamdar 2003), asrij null mutant is homozygous viable. Just as mutations in human asrij are associated with cancers Drosophila asrij loss-of-function mutants also cause hyperproliferation and premature differentiation of precursors, indicating that the mutant phenotype is the result of perturbation in conserved gene function. Our results indicate that Asrij interacts with multiple signaling pathways and will be an important tool in the analysis of hematopoiesis.We have used 9arj mutant phenotype is the supernumerary posterior lymph gland lobes. The origin of the secondary lymph gland lobes is not understood and no precursors are detected in the embryo asrij mutants we see a bona fide primary lobe and excess secondary lobes arising in the larva. This suggests the presence of previously undetected larval lymph gland precursors whose proliferation was suppressed by Asrij. Alternatively, asrij may suppress specification of posterior lymph gland progenitors in larval development. In addition, asrij may control proliferation of circulating hemocytes as we see increase in their number. Similar phenotypes were reported for other mutants that show overgrowth in mutant lymph glands and increase in circulating hemocytes Asrij mutants provide an excellent tool to elucidate events in hematopoiesis and interrogate signaling pathways implicated in proliferation of lymph gland lobes.A remarkable feature of the asrij controls proliferation by integrating with Notch signaling. Previous studies report the effect of N signaling on the primary lobe or circulating hemocytes asrij leads to increased Notch signaling and hence hyperproliferation in secondary lobes. However, additional signals required to maintain quiescence may be absent and hence there is increased differentiation to Lz+ crystal cells, which is a read out for Notch signaling. On the other hand, plasmatocyte differentiation is not seen in asrij mutant secondary lobes. This suggests that pathways that promote plasmatocyte differentiation are not active in the posterior or not controlled by Asrij.The intriguing question remains of how Asrij can promote both proliferation and differentiation in posterior lobes and differentiation in the primary lobe. Multiple signals in the anterior lobe are integrated in time and space to maintain the PSC and control precursor differentiation. These may provide mechanisms independent of or complementary to Asrij function in the control of proliferation. Such details are not available for the secondary lobes, which are believed to harbor a homogenous population of quiescent precursors. Loss of Asrij leads to hyperplastic effects in the secondary lobes. Hyperplasia is also a phenotype associated with excess N signaling. We propose that asrij knockdown or overexpression flies. Experiments using either GAL4 driver gave similar phenotypes to those using the null mutant (9/arj9arj) or the 9/Dfarj flies. These results validate that the phenotypes seen are primarily due to the effect on asrij. Though HmlGal4 is reported to drive expression only in the primary lobe and hemocytes, multiple experiments that we have done clearly show that expression using HmlGAL4 affects function in the secondary lymph gland lobes. This could be either due to previously unreported low level or leaky activity of the driver in posterior lobes or due to a non-autonomous effect of manipulating asrij activity in primary lobes and in circulating hemocytes. The interaction between hemocytes in lymph gland posterior lobes and in hemolymph merits further investigation.As none of the hemocyte-specific GAL4 drivers is reported to function only in the lymph gland primary and secondary lobes, we used the best available drivers e33cGAL4  asrij mutants show disturbed pericardial cell arrangement, the heartbeat of 9arj homozygous larvae is normal (data not shown) indicating no functional effect on cardiac rhythm. This is in agreement with earlier reports that pericardial cells are not required for normal cardiac function As Asrij is involved in vesicular traffic, it may affect multiple signaling pathways and possibly have non-autonomous or secondary effects. Though + cells in asrij mutant, no appreciable reduction in Antp+ cell number was seen, suggesting that Asrij may affect maintenance of Col+ cells, which needs to be investigated. The reduced Col+ PSC in asrij mutants could affect MZ quiescence. Cells in the MZ are compact, bounded by extracellular matrix (ECM) and maintained in a slow-cycling quiescent state by signals from the PSC Asrij null lymph glands have loosely packed cells with greatly reduced expression of the Wg target DE-cad . Hematopoiesis in Drosophila is governed by a transcription factor cascade initiated by the GATA factor Srp. Hemocyte division, density and possibly viability are controlled by the Toll/Cactus and JAK/STAT pathways which also activate immunity genes In addition to interaction of Asrij with ARF1 + cells. Up-regulation of Notch has been implicated in human blood cell disorders such as, T cell acute lymphoblastic leukemia lethal giant discs (lgd) that affect protein sorting in the late endosomes or MVB result in Notch gain- of- function phenotypes asrij mutant is ligand dependent or independent. Asrij mutants provide an excellent tool to understand the mechanism involved in precocious N signaling leading to blood cell disorders. Further asrij mutants are viable and can be used in studying post-embryonic Notch signaling in various contexts.Notch processing is quite complex and several tissue- specific components are implicated in its activation Drosophila at multiple levels leading to increased hematopoiesis by enhancing precursor proliferation and differentiation. Our results indicate a role for Asrij in PSC maintenance, which in turn affects precursor quiescence. In addition, a more direct role for Asrij is implicated in crystal cell specification via control of NICD traffic.The importance of signaling proteins, receptors and transcriptional targets of the N and Wg pathways for cancer development is well established. In contrast, data regarding endocytic molecules that traffic the pathway components and modulate their activity is limited. Here we show that loss of the endocytic protein Asrij affects Drosophila. The early onset of Asrij expression during development suggests that it could be a key player in vertebrate hematopoiesis as well. By virtue of its ability to control cellular traffic, Asrij may control cell adhesion, proliferation and differentiation, which makes it difficult to tease out the exact mechanism of its action. Understanding how asrij controls the balance between stem cell number and committed precursors may aid in disease correction and regenerative medicine. Together, our findings indicate that endocytosis is a key modulator of lymph gland hematopoiesis and provide in vivo demonstration that genetic loss of endocytic components can lead to accelerated hematopoietic development and facilitate premature differentiation.Our study demonstrates the value of a comparative approach in identifying functions of conserved mammalian genes in Figure S1A–B) Antigen-antibody competition assay to validate the specificity of Asrij antibody. Western blot showing specificity of Asrij (A) antibody. Lanes:  Blot probed with antibody preincubated with 25 or 50 µg of corresponding antigen.  blot probed with antibody without preincubation with antigen. (B–C) Asrij expression (green) by immunostaining with anti-Asrij antibodies could not be detected in several other tissues examined including wing disc (B), fat body (C). Nuclei stained with DAPI (blue). Panels to the extreme right are merged images. Scale bar:  50 µm.((TIF)Click here for additional data file.Figure S2Conservation and subcellular localisation of Asrij in Drosophila melanogaster. (A) Schematic representing conservation in OCIA domain of Asrij. The N half of the Asrij protein including predicted helices are conserved in Drosophila, mouse and human. (B–E) Subcellular localization of Asrij. Immufluorescence analysis of hemocytes stained for expression of Asrij  and subcellular marker proteins  such as (B) Rab5, (C) Rab11, (D) dArl8 and (E) GM130. Nuclei are stained with DAPI (blue). Panels to the extreme right in each set are merged images. Scale bar: (B–E): 5 µm.(TIF)Click here for additional data file.Figure S3Southern blot analysis confirms insertion in 9arj mutant. (A) Schematic showing the details of the asrij null mutant. (B) Southern blot of Hind III digested genomic DNA from asrij excision lines probed with 32P-labelled cDNA. Lanes. 1: CS, 2: BL14935, 3: arj9/arj9 and 4: Marker. A 3.2 kbp band of expected size is seen in wild type whereas arj9/arj9 mutant has 2 bands of 2.4 kbp and 1.3 kbp due to 550 bp remnant of P element sequence. Analysis of asrij (C) transcript expression by RT-PCR and (D) protein expression by immunoblot with anti-Asrij antibody. Genotypes are as indicated above the lanes. (E–F) Immunofluorescence analysis of Asrij (green) expression in hemocytes of wild type (E) and 9arj/9arj mutant (F). Hemocytes are identified by the expression of the pan hemocyte marker Hemese (red). Nuclei marked by DAPI (blue). Scale bar:  5 µm.(TIF)Click here for additional data file.Figure S4Dextran uptake is reduced in Asrij null hemocytes. (A) Total cell associated fluorescence of internalized FITC Dextran 5 min after starting the incubation of wild type (CS), asrij null (9/arj9arj) and rescue  hemocytes (P = 0.002). (B–C) Representative images of wild type (B) and 9arj/9arj mutant (C) hemocytes showing the uptake of FITC Dextran. Cell boundary is marked by a white line. Scale bar:  5 µm.(TIF)Click here for additional data file.Table S1List of primers used for RT-PCR and qRT-PCR.(DOC)Click here for additional data file.Text S1(DOC)Click here for additional data file.


MLH1 and MSH2. Patients inheriting this predisposition are susceptible to colorectal, endometrial and other extracolonic tumors. It has recently been shown that germline deletions of the last few exons of the EPCAM gene are involved in the etiology of Lynch syndrome. Such constitutional mutations lead to subsequent epigenetic silencing of a neighbouring gene, here, MSH2, causing Lynch syndrome. Thus, deletions of the last few exons of EPCAM constitute a distinct class of mutations associated with HNPCC. Worldwide, several investigators have reported families with EPCAM 3’end deletions. The risk of colorectal cancer in carriers of EPCAM deletions is comparable to situations when patients are MSH2 mutation carriers, and is associated with high expression levels of EPCAM in colorectal cancer stem cells. A lower risk of endometrial cancer was also reported. Until now the standard diagnostic tests for Lynch syndrome have contained analyses such as immunohistochemistry and tests for microsatellite instability of mismatch repair genes. The identification of EPCAM deletions or larger EPCAM-MSH2 deletions should be included in routine mutation screening, as this has implications for cancer predisposition.Lynch syndrome  is a frequent, autosomal, dominantly-inherited cancer predisposition syndrome caused by various germline alterations that affect DNA mismatch repair genes, mainly  MSH2, MLH1, MSH6, but also PMS2) .Kempers and colleagues (2011) in their studies established different cancer risks associated with carriers . They reEPCAM and MSH2 protein expression is a feature highly specific for cancers from EPCAM deletion carriers, suggesting EPCAM immunohistochemistry as a potential analysis tool for the identification of Lynch syndrome patients with EPCAM germline deletions [EPCAM protein expression was retained in some cancers from EPCAM mutation carriers. Investigators have not determined the relationship between EPCAM protein expression status in cancers and localization of an EPCAM germline deletion [MSH2 inactivation) determined EPCAM expression in tumor cells.  revealed that concomitant lack of eletions . Howeverdeletion . This isCAM gene .EPCAM gene  encodes a carcinoma-associated antigen which is a glycosylated member of a family that includes at least two type I membrane proteins [The proteins . It is lEPCAM is located in the basolateral membrane but in cancer tissues this protein is homogeneously distributed on the cell surface. EPCAM is not only implicated in mediating epithelial-specific intercellular adhesion, but also in intracellular signaling, migration, proliferation and differentiation. The extracellular part of EPCAM contains an epidermal growth factor-like domain and a presumed thyroglobulin domain. Activation of EPCAM signaling is mediated by intra-membrane proteolysis through which the extracellular domain is shed and the intracellular domain (EpICD) is released into the cytoplasm  the performance of EPCAM immunohistochemistry before MLPA (Multiplex Ligation-dependent Probe Amplification) analysis [analysis .EPCAM which did not affect the open reading frame of MSH2, in a cohort of LS-suspected patients [EPCAM and inactivation of MSH2 was independently reported by two groups. Kovacs et al. (2009) detected four different deletions encompassing the last few exons of EPCAM, in five families with one or more tumors exhibiting microsatellite instability and loss of the MSH2 protein [EPCAM 3′ end deletions in four Dutch families with colorectal cancer, showing high microsatellite instability and loss of the MSH2 protein, but in which no mutations in MSH2 were found. Additionally, in these families the deletion co-segregated with the occurrence of MSH2-deficient tumors and, in addition, was found to lead to transcriptional read-through into the MSH2 locus [MSH2 promoter present on the same allele as the 3′ end EPCAM deletion. Sequence analysis defined the deletion breakpoints, with a deletion of 4909 bp denoted 859-1462_*1999del from EPCAM cDNA. Haplotype analysis suggested that this mutation originated from a common founder. All six high-MSI tumors from these families showed methylation of the MSH2 promoter by methylation-specific PCR and subsequent bisulfite sequencing  first reported deletions encompassing the 3’end of patients . Five ye protein . At the H2 locus . What’s 005 firstMSH2 promoter methylation and identified a heterozygous deletion of 22.8 kb  that segregated with the disease [EPCAM gene to approximately 2.4 kb upstream of MSH2, encompassing the 3’ end of EPCAM and leaving the MSH2 promoter intact. The same authors identified the same mutation in another Chinese family, where there was no evidence for a founder mutation. Analyses such as RT-PCR and methylation-specific PCR of tissue samples from affected individuals showed that methylation of MSH2 was limited to EPCAM-expressing cells  also analyzed the family reported by Chan et al. (2006) with heritable 009 also EPCAM germline deletions [EPCAM germline deletions. Therefore they hypothesized that a type of second somatic hit, leading to MSH2 inactivation during tumor development, determines EPCAM expression in the tumor cells. In four out of six tumors, investigators detected lack of EPCAM expression accompanied by biallelic deletions affecting the EPCAM gene. In contrast, monoallelic retention of the EPCAM gene was observed in the remaining two tumors with retained EPCAM protein expression. These results indicate that EPCAM expression in tumors from EPCAM deletion carriers depends on the localization of a second somatic hit that inactivates MSH2. These data demonstrate the lack of EPCAM protein expression observed in tumors from a combination of a germline mutation and second somatic hit. They also show that heterozygous EPCAM germline deletions are not necessarily associated with loss of EPCAM expression in tumor tissue. The detection of a somatic mutational event causing MSH2 inactivation in one of the EPCAM positive tumors, explains why some tumors from EPCAM deletion carriers show loss of MSH2, but retained EPCAM expression [Huth et al. (2012) reported that lack of EPCAM expression occurs in many, but not all, tumors from Lynch syndrome patients with eletions . The difpression .EPCAM deletions appeared to vary between populations and was found to represent at least 1-3% of the explained Lynch syndrome families. Detailed analysis of the EPCAM deletions revealed their range of variability as well as their Alu-repeat-mediated origin as a likely mechanism for these rearrangements [EPCAM deletion breakpoints characterized by various authors were located within repetitive Alu elements  found only in primates and comprise about 10.5% of the human genome [Alu-mediated homologous recombination involve a cross-over at regions of shared sequence identity between two parental Alu elements located in cis in the same orientation, with loss of the loop of intervening genomic sequence during the exchange. These deletions are identifiable by signatory tracts of perfect Alu-derived sequence identity, overlapping or adjacent to the deletion breakpoints, such that the deletion junction cannot be mapped to a precise nucleotide. Perez-Cabornero and colleagues (2011) mapped each of the deletion breakpoints to within short stretches of fused Alu sequences that shared close homology to their respective parental Alu elements [Alu elements in the causation of Lynch syndrome.n genome . These an genome . Deletioelements . This isEPCAM deletions were found to be present in various populations from different geographic origins [MSH2 inactivating mutations. All Lynch syndrome-associated tumors from EPCAM deletion carriers that were available for testing showed hypermethylation of the MSH2 promoter [Alu repeat-mediated recombination events [Alu repeats spread across this locus [Worldwide,  origins ,28 , EPCAM gene [EPCAM founder deletion, thus far observed by Ligtenberg et al. in seven Dutch families, was found to be present in nine out of ten additional families from The Netherlands, but in none of the families from other geographic origins, thus confirming its founder nature (Table EPCAM deletions: c.858+2568_*4596del (found in three families) and c.858+2488_*7469del . Furthermore, they describe the EPCAM c.858+2568_*4596del mutation as the first reported EPCAM founder mutation in Spain , Switzerland (deletion 3 in Table n = 2), and the United States (deletion 6 in Table n = 2) or from multiple origins (deletions 5 and 10 in Table n = 3) [In addition, six EPCAM is based on matching the Amsterdam or Bethesda criteria and is associated with an MSI phenotype or loss of MMR protein expression in tumors.Mutational screening of carriers of 3’end deletions of EPCAM deletion carriers and MSH2 mutation carriers [EPCAM immunohistochemistry can yield inconspicuous results in a subset of tumors from EPCAM deletion carriers, namely if a second somatic MSH2 inactivating hit does not affect the EPCAM gene [EPCAM immunohistochemistry may detect EPCAM deletions already at a precancerous stage [Immunohistochemical analysis of MMR protein expression is a hallmark of Lynch syndrome diagnostics, but it cannot distinguish between carriers . The depCAM gene . Moreoveus stage .EPCAM gene and MSH2 promoter methylation are detected by using multiplex ligation-dependent probe amplification (MLPA) analysis containing probes for a specific region [EPCAM gene in four out of six analyzed tumors. In the remaining two tumors, no biallelic EPCAM deletions were observed, and the allelic profile obtained for the EPCAM gene region was identical in DNA isolated from tumor tissue and matched blood samples. All tumors showing biallelic deletions in the EPCAM gene region were negative for EPCAM protein expression, while EPCAM protein expression was retained in the tumors which retained a one normal EPCAM allele. These scientists also proved that the MLPA technique is applicable for the detection of heterozygous and homozygous deletions in DNA isolated from paraffin-embedded tumor tissue (when for example no blood is available) [Germline rearrangements in the c region ,19,32. Hailable) .EPCAM gene, Ligtenberg and Kuiper (2009) used long range PCR and real-time quantitative RT-PCR. For long range PCR across the deletion, in Dutch families, a TAKARA PCR kit was used with primers on either side of the deletion. Further, they specified the deletion by direct-sequencing, using a forward primer in combination with an internal reverse primer. For the Chinese families they performed multiple long range PCRs, which yielded an aberrant amplicon suggestive of a large deletion mutation [As a additional method for detecting specified deletions in the mutation . To detemutation ,21.MLH1, MSH2, MSH6, PMS2, EPCAM, APC and MUTYH, including single nucleotide variants (SNVs), small insertions and deletions, and large copy number variants. These scientists focused on defining the sensitivity of heterozygous variant detection because pathogenic mutations in Lynch and polyposis syndromes are almost always heterozygous, except in the MUTYH gene. There was 100% reproducibility of mutation detection between independent runs [Alu or other repetitive DNA elements [Recently, Pritchard and his team from the USA (2012) notified a comprehensive and cost-effective test called “ColoSeq” that detects all classes of mutations in Lynch and polyposis syndrome genes, using solution-based targeted capture and next-generation sequencing . Due to ent runs . The Colelements .EPCAM deletion mapping in routine diagnostics on suspected Lynch syndrome families should be considered. Some studies suggest that the frequency of EPCAM deletions as a cause of Lynch syndrome is up to 30% in patients with MSH2–negative tumors (from IHC results) or approximately 20% of LS patients without a mutation in MMR genes [Based on previous worldwide results, there is a strong suggestion that implementation of MR genes ,22.EPCAM deletions in the Lynch syndrome, as it is a more frequent cause of LS than mutations in PMS2 or MSH6[This underlines the importance of 2 or MSH6.EPCAM gene as a second hit in tumors from EPCAM deletion carriers suggests that the localization of somatic events inactivating mismatch repair genes in Lynch syndrome is not random, but related to the underlying germline mutation [The frequent occurrence of somatic deletions affecting the mutation .EPCAM 3’end deletions are a recurrent cause of Lynch syndrome, and detection should be implemented in routine Lynch syndrome diagnostics.In conclusion, Authors declare that they have no competing interests.Conception and design: KT; drafting the manuscript: KT; critical revision for important intelectual content: GK; final approval: KT, GK, JL. All authors read and approved the final manuscript.


Formica selysi. We showed that workers from both single- and multiple-queen colonies discriminated against foreign eggs from single-queen colonies, whereas they surprisingly accepted foreign eggs from multiple-queen colonies. Chemical analyses then demonstrated that social origins of eggs and workers could be discriminated on the basis of their chemical profiles, a signal generally involved in nestmate discrimination. These findings provide the first evidence in social insects that social origins of eggs interfere with nestmate discrimination and are encoded by chemical signatures.The ability of group members to discriminate against foreigners is a keystone in the evolution of sociality. In social insects, colony social structure (number of queens) is generally thought to influence abilities of resident workers to discriminate between nestmates and non-nestmates. However, whether social origin of introduced individuals has an effect on their acceptance in conspecific colonies remains poorly explored. Using egg-acceptance bioassays, we tested the influence of social origin of queen-laid eggs on their acceptance by foreign workers in the ant  The evolution of sociality requires recognition mechanisms allowing group members to direct cooperative or aggressive behaviours towards the correct individuals. In particular, the ability to discriminate against non-group members is important as it may help to prevent colony invasion, to avoid exploitation of group resources by outsiders and to limit erosion of relatedness between group members In social insects, nestmate recognition is generally mediated by the blend of chemical compounds (CC) present on the cuticle of individuals Camponotus floridanus, queens and queen-laid eggs presenting CC associated with high fertility rates elicit fewer aggression from foreign conspecific workers Odours of intruders may also influence nestmate discrimination through distinct pathways. One way is to present a relatively small global-amount of CC, so that resident workers are not able to compare intruders' odours to colony-profile Solenopsis invicta, workers from multiple-queen ( =  polygyne) colonies tolerated foreign conspecific queens only if these queens came from polygyne colonies Messor barbatus, workers from single-queen ( =  monogyne) colonies accepted more foreign conspecific workers from polygyne than monogyne colonies Formica selysi, resident workers were less aggressive towards foreign workers originating from colonies with similar than alternative social forms S. invicta queens and M. barbatus workers present subtle differences of chemical profiles when originating from monogyne and polygyne colonies F. selysi workers reflect their social origin remained however untested so far.Signals associated with the social origin of intruders are also known to influence discrimination against foreign queens and workers. In the fire ant F. selysi, recent works suggest that social origin of foreign eggs could interfere with their elimination rate. In particular, monogyne workers rejected eggs from foreign monogyne colonies, while polygyne workers accepted eggs from foreign polygyne colonies Contrary to adult intruders, the influence of social origin of foreign eggs  on their elimination by resident workers has never been explored in social insects. The elimination of foreign eggs can be important to maintain colony integrity, as subsequent brood from infiltrated queens may dilute relatedness among nestmates, monopolise colony resources, parasitize host colonies and hence can decrease inclusive fitness of resident workers and induce colony collapse F. selysi (i) to test the influence of social origin of eggs on their acceptance by foreign workers, and (ii) to investigate the association between chemical profiles and social origins of workers and eggs. Using egg-acceptance bioassays, we first investigated whether monogyne and polygyne workers discriminated between eggs from their own colony, foreign monogyne colony and foreign polygyne colony. Then, we determined whether social origin of eggs and workers could be discriminated on the basis of their chemical profiles by testing qualitative and quantitative differences between chemical profiles of individuals (i.e. eggs and workers) sampled in 23 monogyne and 23 polygyne field colonies.Our present study was conducted in the ant F. selysi is located between Sierre and Susten along the river Rhône in central Valais, Switzerland . In this population, monogyne and polygyne colonies live in close proximity with no sign of genetic differentiation or mating incompatibilities between social forms The study population of et al.ad libitum  as an injection internal standard. A 2 µl sample of the extract was injected on a Agilent 7890 gas chromatograph fitted with a HP-5MS fused silica capillary column  linked to a mass analyzer (Agilent 5975 mass spectrometer). The injector was used in splitless mode with a splitless time of 2 min. Injector temperature was held constant at 250°C. An oven program that began at 70°C (1 min) and was ramped at 20°C/min to 140°C, then 3°C/min to 230°C, 2°C/min to 260°C and 3°C/min to 300°C (10 min). Carrier gas was Helium at a flow rate of 1 ml/min. Electron impact positive ions at 70 eV were recorded in the scanning mode (mass range scanned 40–550 amu). The mass spectra were interpreted by fragmentation analysis and comparison to previous publications n-alkane standards  were compared to published data. MSD Chemstation Agilent Technologies software was used to calculate the retention time and total area of each peak for subsequent analysis.Chemical analyses were made on the hexane extracts described above, which were previously evaporated and reconstituted in 100 µl of hexane with 10 ng/µl of eicosane (p>0.05), and pairwise comparisons between egg origins tested using post-hoc Tukey HSD tests. Because eggs or workers originating from the same field colony were sometimes used twice in the analyses , we included the colony of origin of eggs and workers as random factors in all the analyses.The survival of eggs was tested using mixed-effect models (GLMMs) within groups of monogyne and polygyne workers. In these analyses, the origin of eggs  was used as a fixed factor and the arcsine-transformed proportion of eggs still alive after 24 hours  entered as response variable. The normality of residuals were tested using Shapiro Wilcoxon tests . The significance of each DA was evaluated (i) by testing difference between groups using Wilks' Lamba tests and (ii) through the percentage of correct assignment of eggs or workers to their social origin, which was given by statistical models and cross-validations (Leave-one-out method). The structure coefficients (i.e. correlations between discriminating variables and discriminant functions) were used to assess the importance of each peak in discriminating social origin of eggs and workers. According to Mardia's criterion U-tests. To avoid limitations inherent to analyses of compositional data, the area of each peak was transformed according to Aitchison formula i,jZ is the transformed area of peak i for colony j, Y is the area of the peak i for colony j; and j)g was added to all peak areas Because a sample size of at least three times the number of variable is recommended for multivariate analyses t-tests. For each sampled colony, the total absolute quantity of extracted CC was calculated using the formula iTot is the total absolute quantity of CC extracted from colony i, ijAbs is the GC-MS area of the peak j for colony i, ISq is the quantity of internal standard introduced in the sample (here 20 ng) and iISa is the GC-MS area of the internal standard in the colony i.Differences in the total amount of CC extracted from groups of monogyne and polygyne eggs or workers were investigated using F2,30.96 = 5.19, p = 0.011; polygyne workers: F2,17.94 = 13.75, p<0.001). Overall, foreign eggs originating from monogyne colonies survived significantly less than nestmate eggs  or foreign eggs from polygyne colonies . By contrast, there was no significant difference between the survival rates of nestmate eggs and foreign polygyne eggs . A lower intrinsic viability of monogyne than polygyne eggs is unlikely to explain these results, as there was no significant difference between the survival rates of both types of eggs introduced with nestmate workers .The social origin of foreign eggs significantly influenced their survival rate in both groups of monogyne and polygyne workers (nC23), pentacosane (nC25) and heptacosane (nC27) or 9-monoenes  . Peaks 1 to 9 are very volatile compounds and are usually associated with the Dufour gland F. selysi were found in large quantities like aldehydes, alcohols and acid esters Chemical profiles of eggs and workers contained qualitative differences. Thirty-two peaks are exclusively present in workers and one is present in eggs only . ChemicaAfter Bonferonni correction, the relative amount of 7 out of 67 peaks extracted from eggs and 6 out of 98 peaks extracted from workers were significantly different between monogyne and polygyne colonies  and S1. Wilks' lambda = 0.117, F1,30 = 15.05, p<0.0001; eggs: Wilks' lambda = 0.225, F1,30 = 6.90, p<0.0001). Moreover, 97.8% of workers and eggs were correctly assigned to their social origins by statistical models, and respectively 91.3% and 84.8% by cross-validation method. According to Mardia's criterion, five and 14 out of the 15 selected peaks significantly contributed in discriminating social origin of workers and eggs, respectively , and significantly smaller in polygyne than monogyne workers . Despite these differences, the social origins of both eggs and workers were not associated with the presence/absence of specific CC .The total amount of extracted CC was significantly larger in polygyne than monogyne eggs (polygyne: 388.2±20.2 ng (mean ± SE); monogyne: 335.3±14.7 ng; cific CC . Also, tF. selysi workers discriminated against foreign monogyne eggs, whereas they did not discriminate between nestmate and foreign polygyne ones. Chemical analyses confirmed that discrimination between eggs from alternative social origins could be based on quantitative differences in their chemical profiles. Finally, we showed that chemical profiles of F. selysi workers reflected their social origin, a result in accordance with studies in M. barbatus and S. invictaUnderstanding which proximate factors influence the ability of group members to discriminate against foreigners is fundamental to gain a better understanding of the evolution and maintenance of complex social systems. Our egg-acceptance bioassays provide the first evidence in social insects that social origin of queen-laid eggs is associated with cues that interfere with nestmate discrimination. In particular, all F. selysi workers towards foreign conspecific workers, where the level of aggression decreases with matches between social origins A somewhat surprising result of our bioassays was that both monogyne and polygyne workers rejected foreign monogyne eggs, whereas they accepted foreign polygyne ones. First, the similar behavioral response of monogyne and polygyne workers towards foreign eggs stands in contrast with the distinct behavioral response of both types of Ultimately, the risk of egg elimination by foreign workers is unlikely to have specifically selected for the socially-related cues interfering with nestmate discrimination of eggs, as in this species, mated queens introduced in foreign colonies are generally killed before any egg production C. floridanus, wherein workers accepted foreign conspecific queens only if they presented signals associated with high fertility F. selysi.Proximately, the influence of social origin of eggs on their tolerance by foreign workers indicates either that (i) eggs lack reliable colony-recognition cues when they are produced in polygyne colonies, or that (ii) signals associated with polygyne origins prevent workers to perceive cues of colony-membership. The first hypothesis seems less likely, since the global-amount of extracted CC was larger in polygyne than monogyne eggs and the chemical profiles of eggs presented some differences across polygyne colonies (Mean variance in the relative amount of each peak across polygyne colonies (± SE) = 0.439±0.049). By contrast, a signal interfering with nestmate discrimination against queens has already been described in the monogyne ant F. selysi (this study), F. selysi, the genetic background of monogyne and polygyne colonies is unlikely to produce specific chemical signatures, as no sign of genetic differentiation or mating incompatibilities have been found between social forms in the studied populations Results from behavioural tests of nestmate discrimination towards eggs and workers imply that resident workers detect signals associated with the social origin of conspecific intruders in F. selysi remain, however, open for further investigations.Studies assessing or manipulating the availability of informations are a necessary instrument in unravelling the diversity of outcomes of social evolution and adaptive behaviour in general Table S1Chemical compounds extracted from eggs and workers in F. selysi. Relative amount (%) of all the chemical compounds extracted from groups of workers or eggs originating from monogyne (Mo) and polygyne (Po) colonies. Retention time (RT) is the mean retention time of CC across all extractions. The 15 peaks with the largest variation of relative amount between Mo and Po colonies were included in the discriminant function analyses for workers (W) and eggs (E). P-values remaining significant after Bonferonni corrections are indicated in bold . Unknown compounds with identical numbers correspond to the same family of compounds. Main compounds contained in complex peaks are represented in bold.(DOC)Click here for additional data file.


To compare diabetes risk assessment tools in estimating risk of developing type 2 diabetes (T2DM) and to evaluate cardiometabolic risk profiles in a middle-aged Irish population.Future risk of developing T2DM was estimated using 7 risk scores, including clinical measures with or without anthropometric, biological and lifestyle data, in the cross-sectional Mitchelstown cohort of 2,047 middle-aged men and women. Cardiometabolic phenotypes including markers of glucose metabolism, inflammatory and lipid profiles were determined.Estimates of subjects at risk for developing T2DM varied considerably according to the risk assessment tool used (0.3% to 20%), with higher proportions of males at risk . Extrapolated to the Irish population of similar age, the overall number of adults at high risk of developing T2DM ranges from 3,378 to 236,632. Numbers of non-optimal metabolic features were generally greater among those at high risk of developing T2DM. However, cardiometabolic profile characterisation revealed that only those classified at high risk by the Griffin (UK Cambridge) score displayed a more pro-inflammatory, obese, hypertensive, dysglycaemic and insulin resistant metabolic phenotype.Most diabetes risk scores examined offer limited ability to identify subjects with metabolic abnormalities and at risk of developing T2DM. Our results highlight the need to validate diabetes risk scoring tools for each population studied and the potential for developing an Irish diabetes risk score, which may help to promote self awareness and identify high risk individuals and diabetes hot spots for targeted public health interventions. Type 2 diabetes mellitus (T2DM) is a common metabolic condition associated with increased morbidity and mortality, largely due to increased cardiovascular risk 1c (HbA1c) on estimated diabetes risk.Various diabetes risk assessment tools have been developed in numerous populations, either for self-assessment relying on readily available health information; others need to be completed by the physician and require clinical and/or biological data th birthday during the cohort recruitment, were included. Ethics committee approval conforming to the Declaration of Helsinki was obtained from the Clinical Research Ethics Committee of University College Cork. All participants provided written informed consent. Individuals with doctor diagnosed diabetes or FPG ≥7.0 mmol/L were excluded, resulting in 1,862 non-diabetic individuals at risk of developing T2DM who were included in the current study. For comparative purposes HbA1c was also used to define T2DM (HbA1c ≥6.5% and/or treatment for diabetes), resulting in 1,823 non-diabetic individuals for analyses. Participants with missing data for FPG, HbA1c or diabetes treatment were excluded. All collected source data are maintained and stored at the study research office, in the Department of Epidemiology and Public Health, University College Cork. Specific proposals for future collaboration for which data would be made available would be welcomed. Further information can be found on the Centre for Diet and Health Research website, http://www.ucc.ie/en/hrbc/projects/cluster3 or by contacting the corresponding author.The Cork and Kerry Diabetes and Heart Disease Study (Phase II) was a single centre, cross-sectional study conducted between 2010 and 2011 All participants attended the clinic in the morning after an overnight fast (minimum 8 h). Fasting blood samples were taken on arrival. Participants completed a General Health Questionnaire (GHQ), a Food Frequency Questionnaire (FFQ) and the International Physical Activity Questionnaire (IPAQ). Data on age, gender, family history, medication/medical history and lifestyle factors was gathered through a self-completed GHQ. Participants answered questions regards personal and family diabetes diagnosis/treatment and personal hypertension diagnosis/treatment. Corticosteroid use was also used in the current analysis. Smoking status was defined as never, former and current smokers. Alcohol consumption included questions regards past and current intake to define drinkers, never or former drinkers. Diet was assessed using a modified version of the EPIC FFQ, validated for use in the Irish population, which was previously used in the Cork and Kerry Phase 1 study 1c was measured using an automated high-performance liquid chromatography analyser (Tosoh HLC-723 (G7), Tosoh Europe N.V, Tessenderlo, Belgium). Complement component c3 (C3) was determined by immunoturbidimetric assay . Serum insulin, C reactive protein (CRP), tumour necrosis factors aTNF-Plasma and serum were prepared from fasting blood samples. FPG concentrations were determined using a glucose hexokinase assay and serum high density lipoprotein (HDL) cholesterol, low density lipoprotein cholesterol, triglycerides and uric acid levels were analysed using enzymatic colorimetric tests  on an Olympus 5400 automatic analyser  by Cork University Hospital Biochemistry Laboratory. HbAet al., et al., et al., et al., et al., based on the Framingham Offspring Study Seven diabetes risk scores were assessed including the 9-year risk score based on the French DESIR study by Balkau 10 transformation as appropriate. Differences between groups were analyzed by independent t-tests or Mann Whitney U tests. Non-optimal cardiometabolic risk features were determined using the NCEP ATP III metabolic syndrome (MetS) criteria th percentile cut-offs were used for HOMA and for generating a composite inflammatory score based on C3, CRP, TNF-www.cso.ie) to ascertain the number of non-diabetic subjects in Ireland. Next the number of at risk subjects was estimated for each risk score by applying the age-group and gender-specific estimates obtained in the Mitchelstown cohort to the corresponding diabetes-free population. Correlations and pair-wise comparison of agreement between risk scores were assessed by Spearman correlation coefficient and Cohen’s kappa, respectively. Statistical analyses were carried out using SPSS version 18.0 for Windows .Proportions of individuals at high risk of developing T2DM were calculated according to each diabetes risk score and expressed as percentages and 95% confidence intervals (CI). Cardiometabolic and inflammatory markers were assessed for normality of distribution, and skewed variables were normalised by log1c cut-offs, respectively) were used in the analyses. The proportion of individuals in the Mitchelstown cohort at high risk for developing T2DM is presented in 1c was used to exclude T2DM. In contrast higher proportions of at risk subjects were identified using the Kahn Enhanced score, whereas identical percentage values were obtained using the Griffin score. Examination of estimated risk according to age group across each risk score consistently showed lowest risk in the 45–54 year age group  included in the analyses are presented in ge group . Greatesge group , the oveP<0.001) and Kahn Basic risk scores (P<0.005). Closer examination of individual inflammatory profiles, clinical characteristics, anthropometric measurements and markers of lipid and glucose homeostasis according to diabetes risk classification are presented in Greater numbers of non-optimal metabolic features were generally observed among individuals classified as at high risk of developing T2DM, with the exception of the Wilson score . Signifip<0.001). Comparison of the clinical risk scores  was disappointing  were obtained for the Griffin and Kahn scores (Basic and Enhanced), which all include age. Alcohol intake was only included in the Kahn Enhanced risk score, which pooled non-drinkers and former drinkers into a single group, thereby not taking the U-shaped association between alcohol intake and T2DM risk into account. The Griffin score was based on a Caucasian UK population aged 40–79 years, whereas the Kahn US population (45–64 years of age) included 22.8% black participants. Different scoring was applied according to race, which would not impact on our findings, but the accuracy of the Kahn scores in predicting diabetes risk for people older than 64 years of age has not been confirmed. Indeed comparison of risk estimates from each score according to age group revealed lowest risk in the 45–54 year old age group for all scores. It is thought that over the next 25 years the greatest increase in T2DM in developed countries will be observed in the over 65 year old age group Lifestyle and pharmacological interventions can delay or prevent the development of T2DM While the predictive ability of these diabetes risk scores cannot be assessed at present the planned longitudinal follow-up of the Mitchelstown cohort will enable their predictive and discriminative value to be ascertained. Nevertheless we examined the predictive value of these scores in the Cork and Kerry Phase I Study, which was initiated in 1998 (n = 1018) and re-screened in 2008 (n = 359) et al., analysed the validity of 3 diabetes risk score models in predicting risk in a multi-ethnic cohort Limited comparisons of diabetes risk scores in combination with cardiometabolic profiling exist. A Swiss comparative study examining the same diabetes risk scores, except for the Schulze score, also reported wide variation in predicted risk estimates, with the lowest risk identified by the Wilson and Balkau scores and the greatest risk by the FINDRISC and Griffin scores Our study has several strengths including a high participation rate (67%), inclusion of questionnaires to assess dietary and lifestyle behaviours, detailed family and medical histories, extensive biochemical profiling and collection of anthropometric measurements which allowed us to generate diabetes risk scores using a range of anthropometric, clinical, biological and/or lifestyle factors and also to compare cardiometabolic profiles of at risk individuals according to each score which has not been achieved in comparative studies to date In conclusion, we demonstrate wide variation in the estimates of middle-aged people at risk for developing T2DM according to each risk score used suggesting that these risk assessment tools require validation for each population under consideration. This data highlights the need to develop an Irish diabetes risk score which at an individual level (if designed for self-assessment by a lay person) could promote self awareness of risk factors and modifiable risk behaviours and at a national level  could identify diabetes hot spots for targeted public health interventions. Early identification of high risk individuals could allow earlier diagnosis and personalised and/or public health targeted interventions, thus attenuating the development of diabetes and associated cardiometabolic complications. For example, risk stratification using a two step approach consisting of a preliminary assessment based on a risk score followed by more in depth biological and clinical measurements may offer a more cost effective strategy to identify high risk individuals.Table S1Characteristics of each study used to develop diabetes risk scores.(DOCX)Click here for additional data file.Table S2Characteristics of each model for predicting incident diabetes risk.(DOCX)Click here for additional data file.Table S3Characteristics of the non-diabetic Mitchelstown cohort assessed by the diabetes risk scoring tools.(DOCX)Click here for additional data file.Table S4Spearman correlation coefficients between risk scores.(DOCX)Click here for additional data file.Table S5Comparison of gender and age distributions between the Mitchelstown cohort and the 2011 Irish population.(DOCX)Click here for additional data file.Table S6Comparison of diabetes risk estimates and classification of incident cases in the Cork and Kerry Phase I studies (1998 and 2008).(DOCX)Click here for additional data file.


Cdc42 knockdown also inhibited generation of mammospheres in vitro and tumours in vivo, demonstrating the additional importance of this pathway in tumour initiating cell (TIC) function. These findings provide a new regulatory pathway that is subverted in cancer cells, a novel means of attacking TIC and non-TIC aspects of BLBCs, a lead molecule (ML141) that confers sensitivity to low µM TMX in vitro and in vivo and also appear to be novel in enhancing sensitivity to a non-canonical mode of action of an established therapeutic agent.We found that basal-like breast cancer (BLBC) cells use Cdc42 to inhibit function of the redox/Fyn/c-Cbl (RFC) pathway, which normally functions to convert small increases in oxidative status into enhanced degradation of c-Cbl target proteins. Restoration of RFC pathway function by genetic or pharmacological Cdc42 inhibition enabled harnessing of pro-oxidant effects of low µM tamoxifen (TMX) concentrations – concentrations utilized in trials on multiple tumour types – to suppress division and induce death of BLBC cells  Two of the most critical goals in cancer research are to identify means of increasing the utility of existing therapeutic strategies and of discovering new vulnerabilities of cancer cells. Such goals are most effectively achieved by the discovery of molecular mechanisms that play a role in both resistance to existing therapies and in other critical properties of cancer cells.Tamoxifen (TMX) offers an exceptionally attractive target for such efforts, both because of the extensive experience with this agent and because of its two distinct modes of action. At mid-nanomolar (nM) exposure levels, TMX inhibits the division of oestrogen-dependent luminal breast cancer cells. Induction of cell death, however, requires exposure to micromolar (µM) concentrations of TMX. Such µM TMX concentrations are clinically relevant even with standard exposures to TMX  20 days after mice were xenografted with a parallel comparison with a sham control group. To examine the effects of the combination of TMX + ML141, tumours were first established for 24 days prior to initiation of drug treatment. Animals were then treated with TMX  and/or ML141  for 14 days. Six mice for each transplant were injected were euthanized when the tumours were approximately 2 cm in the largest diameter according to regulations for use of vertebrate animals in research. All procedures were approved by the University of Rochester Committee on Animal Resources.Breast cancer cell lines expressing firefly luciferase were suspended in 20 µL PBS and injected orthotopically into mammary fat pads of female NOD/SCID mice. Tumourigenicities of breast cancer cell lines were assessed in NOD/SCID mice by implantation with serially diluted numbers of cells treated with Cdc42, c-Cbl or scrambled shRNA constructs. Effects of systemic TMX administration on tumour growth in Cdc42 depleted breast cancer cells were examined by injection of 10d-luciferin. Normalized photon flux represents the ratio of the photon flux detected after inoculation to that detected before inoculation.Tumour size was monitored using IVIS 100 Imaging station after intraperitoneal injection of 150 µg/g body weight HYC, YMY and BMS conducted all experiments and all were involved in data interpretation. MN supervised the research and data interpretation. The manuscript was written by HYC, BMS and MN.