The data shows results acquired in a large cohort of 5668 ethnic Arabs involved in a common variants association study for coronary artery disease (CAD) and myocardial infarction (MI) using the Affymetrix Axiom Genotyping platform   Specifications tableValue of the data•Genomic distribution of risk variants for CAD/MI in ethnic Arabs.•Comparative analysis of the genomic distribution of the associated loci for CAD/MI between the Arab population and other ethnic groups.•Regional association plots demonstrate loci on 2q33, 8q13, 9p31 for CAD and on 21q22.11 for MI.•Principal component analysis comparison with 11 other MApMAp3 populations shows variations and clustering of ethnic populations.•The Saudi Arab cohort show similarities with the Caucasian populations.1The summary puts together the clinical features of the studied cases versus controls, the genomic distribution of the implicated variants and the principal component analysis of the data, as well as comparison of the Saudi population with other ethnic groups, Fig. 3.The figure shows the loci on loci 2q33, 8q13, 9p31 associated with coronary artery disease and locus 21q22.11.associated with and myocardial infarction.The figure shows the first and second principal component plot for the Saudi Arab samples (4431 samples: 2165 cases and 2266 controls) with eleven other HapMap3 populations .The figure displays the first and second principal component plot of the Saudi Arab samples (4431 samples: 2165 cases and 2266 controls) without Hapmap3 populations.2The discovery study involved 5668 Saudi Arabs who were subjected to genotyping using Affymetrix Axiom Genome-Wide ASI Array (Asian population). Genotyping data were generated using the Axiom GT1 algorithm and an IBS/IBD analysis in PLINK


A large number of studies have reported the effectiveness of psychotropic agents targeting 5-HT2A and D2 receptors in these disorders. In addition to the individual functions of these receptors, the interaction between the two neurotransmitter systems has been studied in the living brain. However, little is known about their regional relationship in individual human brains. We investigated regional relationships between 5-HT2A and D2 receptors using positron emission tomography (PET) and a bicluster analysis of the correlation matrix of individual variation in the two receptor densities to identify groups of distinctive regional correlations between the two receptors.Serotonin 2A  was calculated from PET data for 76 cerebral cortical regions. A correlation matrix was calculated between the binding potentials of [18F]altanserin and [11C]FLB 457 for those regions. The regional relationships were investigated using a bicluster analysis of the correlation matrix with an iterative signature algorithm.Seven healthy volunteers underwent PET scans with [2A and D2 receptors, with the former in regions related to sensorimotor integration  and the latter in most cortical regions. The second cluster identified another distinct profile of correlation coefficients between 5-HT2A receptors in the bilateral hippocampi and D2 receptors in most cortical regions.We identified two clusters of regions. The first cluster identified a distinct profile of correlation coefficients between 5-HT2A and D2 receptors in sensorimotor integration and hippocampal function. A bicluster analysis of the correlation matrix of these neuroreceptors may be beneficial in understanding molecular networks in the human brain.The observation of two distinct clusters in the correlation matrix suggests regional interactions between 5-HT Psychotropic agents targeting 5-HT2A and D2 receptors are used in the treatment of these disorders FLB 457. The scan protocol consisted of 35 frames and lasted 90 minutes. The injected dose and specific activity were 235 ± 4.8 MBq and 220 ± 68 GBq/μmol at the time of injection, respectively. For evaluation of density of serotonin 5-HT2A receptors, a 90-minute dynamic PET scan was performed after an injection of [18F]altanserin. The scan protocol consisted of 33 frames and lasted 90 minutes. The injected dose and specific activity were 191 ± 5.2 MBq and 153 ± 73 GBq/μmol at the time of injection, respectively. Each PET scan was preceded by a transmission scan for attenuation correction using a 137Cs source. A head holder was used to minimize head movement. The two scans were performed on separate days .Each subject underwent two PET scans to visualize serotonin 5-HT18F]altanserin PET, arterial blood samples were obtained manually 33 times after the injection of the radioligand to obtain an arterial input function. Each blood sample was centrifuged to obtain plasma and blood cell fractions, and the concentrations of radioactivity in whole blood and plasma were measured. The fractions of the parent compound and its radiometabolites in plasma were determined using high-performance liquid chromatography from 6 samples for each subject.For the FLB 457 altanserin and BPND of [11C]FLB 457 for the 76 regions (the two parameters are referred to as BP hereafter). The relationships between the distributions of the 5-HT2A and D2 receptors were investigated using unsupervised biclustering of regions on the correlation matrix with an iterative signature algorithm (ISA) FLB 457 changes slowly with aging (10% decrease per decade) [BP of [18F]altanserin also changes slowly with aging (less than 10% decrease per decade) altanserin for 5-HT2A receptors and [11C]FLB 457 for D2 receptors in cerebral cortical regions.An excel file contains (XLSX)Click here for additional data file.S2 Filer values) between individual binding potential values of [18F]altanserin for 5-HT2A receptors (columns) and [11C]FLB 457 for D2 receptors (rows) in 76 regions.An excel file contains Spearman’s correlation coefficients ((XLSX)Click here for additional data file.


Inhibition of VEGF signaling by the stress-induced matricellular protein TSP1 plays a role in modulating the angiogenic response to VEGF in both health and disease. TSP1 binding to CD47 inhibits VEGFR2 activation. The full implications of this inhibitory interaction are unknown. We developed a detailed rule-based computational model to inquire if TSP1-CD47 signaling through VEGF had downstream effects upon ERK1/2 and calcium. Our Simulations suggest that enhanced degradation of VEGFR2 initiated by the binding of TSP1 to CD47 is sufficient to explain the inhibition of VEGFR2 phosphorylation, calcium elevation, and ERK1/2 activation downstream of VEGF. A complementary mechanism involving the recruitment of phosphatases to the VEGFR2 complex with consequent increase in the rate of receptor dephosphorylation may augment the inhibition of the VEGF signal. The model was then utilized to simulate the effect of inhibiting external TSP1 or the depletion of CD47 as potential therapeutic strategies in restoring VEGF signaling. Results suggest that depleting CD47 is a more efficient strategy in inhibiting the effects of TSP1/CD47 on VEGF signaling. Our results highlight the utility of  VEGF is crucial in normal angiogenesis during embryonic vascular development  by TSP1  given in the Supplementary Material Section. We also included the SBML file associated with the model.Table Binding of PLCγ to pVEGFR2 and subsequent phosphorylation and dissociation of PLCγ from the receptor is described by a Michaelis-Menten type reaction as follows:pVEGFR2 denotes all the species containing phosphorylated VEGFR2 which is determined by BioNetGen. Using this approach lowers the number of reactions generated by the rules and prevents combinatorial explosion in the model.Ras activation by S1P is modeled as a Michaelis-Menten type reaction as follows:S1PRas and Km, S1PRas determining the strength of Ras activation by S1P.with parameters kCurrent through the CRAC channels is modeled according to the following equation. This model is a simplified version of the CRAC component of the mathematical model developed by Schmeitz et al.  in T-celThe steady-state current (Equation 4) as a function of ER calcium concentration is described by an empirically determined Hill function . The set of ordinary differential equations (ODEs) describing the reaction network was numerically integrated using SUNDIAL numerical solver suite  algorithm described in Marino et al. . The parTo accurately capture receptor dynamics and signaling to downstream targets, we opted to use a rule-based modeling approach where molecular details of the species and the corresponding rules for the interactions are implemented in a programing environment such as BioNetGen   via the IP3 sensitive receptors and calcium regulation involving plasma membrane and SERCA pumps. CRAC channels are also a prominent hallmark of the calcium cycling module Figure  see met. The com−1 vs. 1.24 × 10−3 s−1). This reduction in recycling rate is sufficient to explain the rapid decay of total cellular receptor levels in the absence of NRP1. In fact, it is the combined effects of internalization, recycling, and degradation that determine the rapid decay of VEGFR2 cellular level . Samples traces for four different degradation rates are also shown in Figure We simulate the direct effect of TSP1 by assuming that CD47 binding to TSP1 leads to accelerated VEGFR2 degradation. It is assumed that TSP1/CD47/VEGFR2 undergo internalization together and either recycle back to the membrane or degrade with a higher degradation rate . In cells devoid of C47, VEGFR2 signaling is not affected by TSP1. This implies that TSP1/CD47 interaction is necessary for VEGFR2 degradation. For the simulations here, cells are exposed to 2 nM TSP1 for 10 min, followed by 40 min exposure to 50 ng/ml VEGF. This is similar to the experimental protocol in Kaur et al. . Accordi2 (7000 receptors per cell) to 2.2 receptors/μm2 (3100 receptors per cell). Figure 2 does not affect TSP1 inhibition of VEGFR2 signaling.The effect of VEGFR degradation and CD47 surface levels on ERK1/2 activation is investigated in Figure Overall, the results here demonstrate that the enhanced degradation of VEGFR2 by TSP1/CD47 interaction is a viable mechanism to explain the global shutdown of VEGFR2 signaling. The dependence on TSP1 concentration is also consistent with the experimental data indicating the viability of the enhanced degradation mechanism.We next consider the hypothesis that TSP1 might insert some of its inhibitory effects on VEGFR2 by recruiting phosphatases to the receptor complex. For the simulations here the cells are exposed to 2 nM TSP1 for 10 min, followed by the addition of VEGF for 40 min. It is assumed that the increase in dephosphorylation rate is the sole mechanism. As shown in Figure Next, we consider a different layer of TSP1 effects involving the experimentally suggested alteration of calcium signaling by TSP1  is modeled in a simple manner by lowering the concentration of TSP1 and monitoring the recovery of calcium and pERK1/2 signals. We consider a mixed mechanism involving enhanced VEGFR2 degradation (10-fold) and increased VEGFR2 dephosphorylation (51-fold). These values are consistent with the two-parameter scan in Figures The effect of depleting CD47 as a therapeutic strategy is summarized in Figures The simulations demonstrate that CD47 depletion may be a more effective strategy than targeting TSP1 in restoring VEGF signaling to calcium and ERK1/2. CD47 depletion is particularly effective in restoring ERK1/2 activation downstream of VEGFR2.in silico approach to computationally investigate potential mechanisms of inhibition. The model incorporated detailed molecular mechanisms using a rule-based model for receptor-receptor interaction and a coarse-grained model for signaling to calcium and ERK1/2 from the activated receptors. The inclusion of detailed calcium cycling module in the model allowed us to study the role of TSP1 in inhibiting calcium signaling and the potential crosstalk with ERK1/2 activation. Fluorescene resonance energy transfer based (FRET-based) experiments in endothelial cells had shown the preassociation of VEGFR2 with CD47 in endothelial and T cells . Indeed, only 18% depletion of CD47 was needed to restore ERK1/2 activation signal compared with 72% TSP1 inhibition. The results highlight the important role of efficient and sustained therapeutic agent delivery to affected tissues to ensure above-threshold inhibition of TSP1 and CD47. They further suggest that CD47 depletion should be considered as an initial approach in pro-angiogenic therapeutic intervention. It is plausible that different endothelial cell types may exhibit differential inhibition thresholds to TSP1 and CD47 which would further necessitate the need for sustained delivery of anti-TSP1 and anti-CD47 agents.Simulations here also proposed the possibility of inhibiting TSP1 or CD47 as potential interventions for restoring VEGF-mediated angiogenic response. The model showed that sequestering TSP1 effectively normalized calcium signaling and ERK1/2 activation, albeit requiring at least 80% inhibition. This high threshold for inhibition might preclude the Further complicating anti-TSP1 therapy, platelets contain significant pre-formed TSP1 that are a depot source of protein. Thus, targeting CD47 is an alternative strategy expected to be more efficient at preserving pro-angiogenic signals. Additionally, VEGF can also signal via NO while CD47 can interact with cell membrane SIRPα  and has equity interest in the same and in Tioma Therapeutics . The other 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.


KIR3DS1 gene was associated with increased susceptibility for ocular toxoplasmosis. The activating KIR together with their HLA ligands (KIR3DS1-Bw4-80Ile and KIR2DS1+/C2++ KIR3DS1+/Bw4-80Ile+) were associated with increased susceptibility for ocular toxoplasmosis and its clinical manifestations. KIR-HLA inhibitory pairs -KIR2DL3/2DL3-C1/C1 and KIR2DL3/2DL3-C1- were associated with decreased susceptibility for ocular toxoplasmosis and its clinical forms, while the KIR3DS1−/KIR3DL1+/Bw4-80Ile+ combination was associated as a protective factor against the development of ocular toxoplasmosis and, in particular, against recurrent manifestations. Our data demonstrate that activating and inhibitory KIR genes may influence the development of ocular toxoplasmosis.The objective of this study was to investigate the influence of the genes encoding the KIR receptors and their HLA ligands in the susceptibility of ocular toxoplasmosis. A total of 297 patients serologically-diagnosed with toxoplasmosis were selected and stratified according to the presence (n = 148) or absence (n = 149) of ocular scars/lesions due to toxoplasmosis. The group of patients with scars/lesions was further subdivided into two groups according to the type of ocular manifestation observed: primary (n = 120) or recurrent (n = 28). Genotyping was performed by PCR-SSOP. Statistical analyses were conducted using the Chi-square test, and odds ratio with a 95% confidence interval was also calculated to evaluate the risk association. The activating  Toxoplasma gondii infection2T. gondiiOcular Toxoplasmosis, the most common form of posterior uveitis, results from The damage to ocular tissues led to the proposal of phenomena that may be related to pathogenic mechanisms of ocular toxoplasmosis, including autoimmune mechanisms15dim cytotoxic cells and a decrease in immunoregulatory NK CD56bright cells have been identified in children congenitally infected with T. gondii who have active eye lesions. Furthermore, subsets of NK cells and CD8+ T cells play a crucial role as biomarkers of cicatricial lesion of the eyein vitro during T. gondii infections, due to an increased production of interferon-gamma (IFN-γ) in patients with congenital ocular toxoplasmosisIncreases in the numbers of circulating proinflammatory monocytes and NK CD56KIR genes are responsible for coding the KIR receptors of NK cells. These genes comprise a family of 15 genes located on chromosome 19q13.4 characterized as inhibitors  or activators , and two pseudogenes (KIR2DP1 and -3DP1). Moreover, based on the content of the genes, KIR genotypes are divided into two groups designated AA and BX (BB and AB) that differ in the number and type of KIR genesThe effector function of NK cells is regulated by a set of receptors named killer immunoglobulin-like receptors (KIR) expressed on the cell surface that recognize human leukocyte antigen (HLA) class I molecules of target cells9KIR genes have been described as risk or protective factors in different types of non-toxoplasmic uveitis and inflammatory ocular diseases. These diseases include Behcet’s uveitis1315KIR genes are also associated with many other infectious diseases1718202122232425T. gondii infectionKIR genes that encode the immune receptors of NK cells and can trigger local inflammation in the eye has not been elucidated in ocular toxoplasmosis yet. The objective of this study was to investigate the influence of the genes encoding the KIR receptors and their HLA ligands in the resistance or susceptibility to the development of ocular toxoplasmosis.NK cells have great importance in the control of P < 0.0001, t = 7.00), with the subgroup of patients with primary manifestations of ocular toxoplasmosis  and with the subgroup of patients with the recurrent form of the disease . A higher mean age was also observed for the subgroup of patients with primary manifestations than those who had recurrent manifestations .The characteristics of the study population with respect to age, gender, clinical diagnosis and serological profile are shown in KIR2DL2/3 and KIR3DL1/S1 were in Hardy–Weinberg equilibrium (P > 0.05) in this study population. However, KIR3DL1/S1 for the patient group that developed ocular toxoplasmosis was not in Hardy-Weinberg equilibrium (P = 0.03). KIR framework genes, KIR2DL4, KIR3DL2, KIR3DL3 and KIR3DP1, as expected were present in all samples, which are important internal controls to check the quality of genotyping.The distribution of genotype frequencies of KIR gene frequencies and AA and BX genotype frequencies are shown in P = 0.003, Pc = 0.04) was observed for the KIR3DS1 activating gene. There was also a positive association between KIR3DS1 and recurrent manifestations of disease  when this patient group was compared to patients without ocular toxoplasmosis, although it was lost after applying the Bonferroni correction. On the other hand, the KIR2DS2 activating gene was associated with decreased susceptibility for ocular toxoplasmosis , but the significance was also lost after applying the Bonferroni correction. No significant difference was observed in the AA and BX genotype frequencies between all groups investigated in this study.The distribution of P > 0.05) in all groups studied. The frequencies of the HLA class I ligands of KIR  were analyzed and were similar between groups , patients with primary manifestations  and patients with recurrent manifestations  than patients without ocular toxoplasmosis.Data on the distribution of KIR genes with their HLA class I ligands are listed in KIR2DL3 inhibitory allele in the homozygous state and presence of its ligands whether homozygous or not (KIR2DL3/2DL3-C1/C1 and KIR2DL3/2DL3-C1) was associated with resistance to ocular toxoplasmosis when patients without ocular toxoplasmosis were compared with those with ocular toxoplasmosis , primary manifestations  and recurrent manifestations  (The ctively) .P = 0.01; Pc = 0.04) and patients with primary manifestations and without ocular toxoplasmosis  when only two pairs of activating ligands were present ( present . Signifi present .+/C2++ KIR3DS1+/Bw4–80Ile+) is responsible for increasing the risk of developing ocular toxoplasmosis . The combination is also responsible for its primary  and recurrent clinical forms  compared to patients without ocular toxoplasmosis  for the association mentioned above was investigated. The combination involving KIR2DS1 and KIR3DS1 in the presence of their respective ligands  and recurrent manifestations of the disease  was observed for the KIR3DS1−/KIR3DL1+/Bw4–80Ile+ combination (KIR2DL1 and the Bw4-80Ile ligand in the absence of KIR3DS1) when compared to patients without ocular toxoplasmosis. This correlation was also observed when patients with recurrent manifestations were compared to patients with primary manifestations .The correlation between the distribution of activating and inhibitory KIR and their respective HLA ligands was analyzed . A decreT. gondii infection. To the best of our knowledge, this is the first study of KIR genes and HLA ligands in the immunopathology of ocular toxoplasmosis.Possible causes of ocular manifestations of toxoplasmosis have not been fully elucidated, but it is believed that factors related both to the parasite and the host contribute to the development of this disease4T. gondii infection can occur at any time of life, and although most cases of ocular toxoplasmosis occur due to infections acquired after birth, a significant number of patients can acquire the disease congenitally and the resulting scars trend to be persistent30The difference in the mean ages of the patient groups of this study was carefully discussed previouslyKIR genes, after Bonferroni correction only the KIR3DS1 activating gene was associated with increased risk of developing ocular toxoplasmosis with the other associations being lost. The Bonferroni correction decreases the chance of a significant difference by chance alone, making the data more robustKIR3DS1 with ocular toxoplasmosis can be explained by the absence of KIR3DL1, because 3DS1 and 3DL1 segregate as alleles of a single locus. Thus, the presence of KIR3DS1, and consequently the absence of KIR3DL1, can create an increased potential for the activation of NK cells owing to decreases in the ratio of inhibitory/activating receptors. Previous studies have shown involvement of the KIR3DS1/L1 alleles in various types of non-toxoplasmic uveitis and inflammatory eye diseases triggered by autoimmune factors. Levinson et al.KIR3DS1 gene in their haplotype have an increased risk of developing Vogt-Koyanagi-Harada syndrome, while the presence of KIR3DL1 was associated to protection against the development of the disease. A similar result was observed by Moon et al.KIR3DS1 was associated as a risk factor and KIR3DL1 was associated as a protective factor.Regarding the distribution of KIR3DL1/S1 for the patient group that developed the ocular toxoplasmosis were not in Hardy-Weinberg equilibrium. However, some authors claim that this equilibrium should only be investigated in the control group, because it represents the general population34KIR3DS1 might be changing the distribution of these alleles in individuals with ocular toxoplasmosis, resulting in a deviation from the Hardy-Weinberg equilibrium. Considering that numerous precautions were adopted to prevent bias in this study, we can safely say that the 3DS1 gene exerts a real influence on the development of ocular toxoplasmosis, even though KIR3DL1/S1 were not in Hardy-Weinberg equilibrium.In this study, KIR genes in immune response is highly dependent on the HLA molecules expressed on the target cell surface. Therefore, KIR receptors influence susceptibility for or protection against certain illnesses by means of a balance in activation and inhibition signals that regulate the NK cell effector function937The function of KIR genes were analyzed in the presence of their respective ligands (KIR-HLA), the KIR3DS1-Bw4-80Ile pair was associated with the development of ocular toxoplasmosis irrespective of the type of clinical manifestation (primary or recurrent); while KIR2DL3/2DL3-C1/C1 and KIR2DL3/2DL3-C1 were associated with protection against the development of ocular toxoplasmosis and its clinical manifestations. In accord with our results, other studies found an association involving the KIR3DS1-Bw4-80Ile pair in other human diseases404142KIR2DL3 and KIR2DL2 are considered alleles, as are KIR3DL1 and KIR3DS1. Although the KIR2DL2-C1 interaction is stronger than the KIR2DL3-C1 interaction, the inhibitory signal generated by the absence of KIR2DL2 (KIR2DL3/2DL3-C1/C1 and KIR2DL3/2DL3-C1) appears to be sufficient to inhibit the effector function of NK cells and protect against elevated inflammation and tissue damage. It has been shown that abnormal expressions of inhibitory receptors of NK cells, including the KIR2DL3 receptor, may be associated with the development of Behcet’s diseaseIn this study, where the +/C2++ KIR3DS1+/Bw4-80Ile+, although the KIR2DS1 and KIR3DS1 genes are not in linkage disequilibrium as observed among patients with ocular toxoplasmosis  and patients without ocular toxoplasmosis . The combination of one pair (inhibitory) in the absence of the other pair (activating) was analyzed. It was possible to observe that the KIR3DS1−/KIR3DL1+/Bw4-80Ile+ combination decreases the risk of developing ocular toxoplasmosis and its recurrent clinical forms.It is important to highlight the results observed for the number of pairs of KIR-HLA ligands and the correlation between the distribution of activating KIR and their respective HLA ligands. A higher frequency of only two pairs of ligands was observed in patients with ocular toxoplasmosis and in patients with primary manifestations compared to patients without ocular toxoplasmosis. The combination responsible for this association was KIR2DS1et al.These results may suggest that the activating function mediated by KIR2DS1 plus KIR3DS1 and their respective HLA ligands is, in fact, an important factor interfering in ocular toxoplasmosis, since such a combination may affect the balance of inhibiting/activating signals. NK cells are activated when there is an increase of activation signals, even if there is a combination of strong or weak inhibitory signalsT. gondii infection. IL-12 stimulates NK cells to produce IFN-γ and to promote the development of Th-1 cells that produce IFN-γ, a cytokine involved in the activation of macrophages, the main phagocytes in chronic inflammation47T. gondii infection promotes the production of factors such as IFN-γ that suppress the immune privilege of this organ50NK, CD4 and CD8 T cells, and type 1 cytokines, such as IFN-γ and IL-2, play a protective role in T. gondii infection, and the mechanisms involved may be associated with both the pathogenesis and protective effects that control tissue damage. It has been shown that increases in the frequency of circulating NK cells and proinflammatory monocytes in children infected by T. gondii, particularly in those with active ocular lesions, are indicative of a strong and persistent proinflammatory response. Moreover, subsets of NK cells and CD8+ T cells act as biomarkers for cicatricial lesions of the eye51The immune response can determine the development of eye injuries resulting from T. gondii and NK cytotoxicity assays to better understanding the role of NK cells and the expression of KIR in the immunopathogenesis of ocular toxoplasmosis.The current study investigated the KIR-HLA ligand as a risk factor in ocular toxoplasmosis and these results may improve to understanding of the immunopathogenic mechanism involving NK cells in ocular manifestations related to toxoplasmosis. However, others studies should be performed such as histological analyses of the ocular tissue affected by KIR3DS1, KIR3DS1-Bw4-80Ile and KIR3DS1+/Bw4-80Ile++ KIR2DS1+/C2+) and inhibitory signals as protective factors .In conclusion, the results of this study show that activating and inhibitory KIR in the presence of their respective HLA ligands may have influence on the development of ocular toxoplasmosis and its clinical form in this population. In particular this is seen with the strong presence of activating signals as risk factors This study was approved by the Research Ethics Committee of the Medicine School in São José do Rio Preto (#1980/2009) and all individuals who agreed to participate signed informed consent forms. The experiments were carried out in accordance with the approved relevant guidelines and regulations.A total of 297 unrelated patients from the Retinopathy Outpatient Service of Hospital de Base of the Medicine School in São José do Rio Preto (HB-FUNFARME) and Medical Outpatient Clinic (AME) in São José do Rio Preto participated in this study.The study subjects have been described previouslyT. gondii and epidemiological data. Besides, although patients self-reported themselves as European descent, mixed African and European descent, and African descent, due to high miscegenation of the Brazilian population they were defined as a population of mixed ethnicityAll individuals who participated in this study were monitored and evaluated in respect to clinical symptoms, serology for In this study, in order to avoid bias in the results, all patients were selected after clinical examination using the same criteria. Additionally, the probability of variations in the allele frequencies due to ethnic background was minimized by matching patients with ocular toxoplasmosis and patients without ocular toxoplasmosis from similar ethnic backgrounds. Furthermore, gender and residence in the same geographical areas were carefully matched during group selection.KIR genes with statistical power of more than 90% and it was chosen according to frequency of KIR genes recorded in Allele*Frequencies database  observed in a population located in the southeast region of Brazil and defined as a population of mixed ethnicity.The number of patients enrolled is sufficient to demonstrate whether there is an association between ocular toxoplasmosis and The inclusion criteria of patients with ocular toxoplasmosis were positive laboratory diagnosis of toxoplasmosis, the presence of ocular scars/lesions due to toxoplasmosis and live in municipalities in the northwest region of the State of Sao Paulo . The inclusion criteria of patients without ocular toxoplasmosis were positive laboratory diagnosis of toxoplasmosis but without ocular scars/lesions due to toxoplasmosis and living in the same geographical region as the patients with ocular toxoplasmosis. All patients were clinically evaluated by two experienced physicians.The exclusion criteria were: patients with other infectious and parasitic diseases, patients with any type of mental disability, patients with blood dyscrasia and using oral anticoagulants and related patients.Anti-T. gondii antibodies were detected by immunosorbent assay (ELISA) according to the manufacturer’s instructions . The microplates were read using Epoch™ equipment using the Gen5™ 2.0 software . The samples were tested in duplicate and in cases of indeterminate results, the samples were retested in duplicate. Positive and negative controls were included in all reactions.Blood samples were collected into tubes without anticoagulant to obtain serum. All patients were clinically assessed using an indirect binocular ophthalmoscope . The evaluation of visual acuity followed the logMAR Early Treatment Diabetic Retinopathy Study chart (ETDRS) criteriaT. gondii. This is therefore a presumptive diagnosis.As no invasive test was performed, the ocular toxoplasmosis diagnostic criteria used were the same as in the clinical practice: injury identified by ophthalmoscopy associated with positive serology for ® DNA Blood Mini Kit, QIAGEN, the Netherlands) following the manufacturer’s instructions. All DNA samples were subjected to an evaluation of concentration and purity using the ratio of the absorbance at optical densities (OD) of 260 and 280 nm with Epoch™ equipment . KIR and HLA-A, -B and -C were genotyped according to manufacturer’s instructions by Polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSOP) protocols with Luminex® technology . This technique uses PCR-amplified DNA with specific biotinylated primers. The amplified product is hybridized by complementary DNA probes conjugated to fluorescently coded microspheres, with detection using R-Phycoerythrin-conjugated Streptavidin (SAPE). The data were interpreted using a computer program .Blood samples were also collected into tubes containing EDTA anticoagulant for DNA extraction. DNA of all patients was extracted using the commercial kit for silica column extraction . HLA-Bw4 molecules were divided into two groups based on whether isoleucine or threonine was present at position 80 (Bw4-80Ile and Bw4-80Thr). KIR3DS1 binds to Bw4-80Ile molecules. HLA-KIR ligand specificities were considered according to Carr http://www.allelefrequencies.net). Individual genotypes were determined to be AA when the genes KIR2DL1, KIR2DL3, KIR2DL4, KIR2DS4, KIR3DL1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1 were present. The presence of one or more of the following genes: KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5 and KIR3DS1 characterized the BX genotype.Two types of KIR genotypes have been described based on the content of the genes: AA and BX (BB and AB) . Odds ratio (OR) with a 95% confidence interval (95% CI) was also calculated to evaluate the risk association. The mean ages were compared using the t-test. Differences with P-values < 0.05 corrected by the Bonferroni inequality method for multiple comparisons (Pc) were considered statistically significant. A Hardy-Weinberg equilibrium fit was performed by calculating expected genotype frequencies and comparing that with the observed values for KIR2DL2/3, KIR3DL1/S1, and the HLA alleles using ARLEQUIN software, version 3.1. (http://cmpg.unibe.ch/software/arlequin3).KIR, HLA and KIR-HLA frequencies were obtained by direct counting. The comparisons of the frequencies of HLA ligands, KIR genes, KIR AA and BX genotypes and KIR with or without ligands between groups of patients were performed with the Chi-square test with Yates’ correction or, when necessary, Fisher’s exact test using the program Graph Pad Instat .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Lineage tracing of human B cells reveals the in vivo landscape of human antibody class switching. Published 2, August 2016In the originally published article, the primer sequences used for PCR amplification of immunoglobulin heavy chain genes (IGH) which bind in the constant region were reported incorrectly in Table 5. The correct oligonucleotide sequences are now reported in the corrected Table 5.In the 'Library preparation' section of 'Materials and methods', the text “a pooled set of five isotype-specific IGH constant region primers that contain 8 random nts (Table 5)” has been changed to “a pooled set of ten isotype-specific IGH constant region primers that contain 8 or 12 random nts (Table 5)”.The article has been corrected accordingly.


The following statistics were accidentally reversed: "The prevalence in rural areas is nearly double that of urban areas for both early marriage (39% versus 22%) and early childbearing (42% versus 21%)." This sentence should read: "The prevalence in rural areas is nearly double that of urban areas for both early marriage (42% versus 21%) and early childbearing (39% versus 22%)."2)The statistic on the prevalence of sexual initiation in the following sentence was incorrect: "For instance, the median age of sexual debut is 17.7 years, approximately 1 year prior to marriage, and the prevalence of sexual initiation among 15–19 year olds is 27%. This sentence should read": For instance, the median age of sexual debut is 17.7 years, approximately 1 year prior to marriage, and the prevalence of sexual initiation among 15–19 year olds is 49%.”After publication of the article , it has


Interestingly, orthotopic tumours arising from transplantation of 4T1 murine mammary tumour cells exhibit both phosphorylated Stat3 and mCLCA5 expression. However, we demonstrate that expression is highly compartmentalized to distinct subpopulations of cells, and that Stat3 retains a suppressive effect on mCLCA5 expression in 4T1 tumour cells. These findings enhance our understanding of the regulation of CLCA channel expression both in vitro and in vivo, and in particular, demonstrate that expression of mCLCA1 and mCLCA2 during involution is profoundly dependent upon Stat3, whereas the relationship between mCLCA5 and Stat3 activity is reciprocal and restricted to different subpopulations of cells.Mammary gland regression at the cessation of lactation (involution) is an exquisitely orchestrated process of cell death and tissue remodelling in which Stat3 signalling has an essential role. The involution microenvironment of the mammary gland is considered to be pro-tumourigenic and a proportion of cases of pregnancy-associated breast cancer are suggested to originate in tandem with involution. However, the apparent paradox that STAT3 is required for cell death in normal mammary gland, but is associated with breast cancer cell survival, has not been resolved. Herein, we investigate Stat3-mediated regulation of expression of members of the calcium-activated chloride channel regulator (CLCA) family of proteins during involution and mammary carcinogenesis. Using the conditionally immortal mammary epithelial cell line KIM-2, together with mice exhibiting mammary epithelial cell-specific deletion of Stat3 during lactation, we demonstrate that expression of mCLCA1 and mCLCA2 is elevated in concert with activation of Stat3. By contrast, murine CLCA5 (mCLCA5), the murine orthologue of human CLCA2, is significantly upregulated at 24, 72 and 96 h of involution in Stat3 knockout mice, suggesting a reciprocal regulation of these proteins by Stat3  Although the mechanism by which they activate other channel proteins to trigger movement of chloride ions across membranes is not clear, there is some evidence that they can be secreted, and thereby potentially activate chloride channels.16 apoptosis, tumourigenesis and mucus cell differentiation.17 Of particular interest, both CLCA2 and CLCA4 have been shown to inhibit proliferation of breast cancer cells when ectopically expressed in vitro,18 whereas normal breast tissue exhibits high levels of CLCA2 in both acini and small ducts.19 Murine CLCA5 (mCLCA5) is highly homologous to human CLCA2 (hCLCA2) and is considered to be its orthologue.20 Widely expressed in the cytoplasmic granules of granular layer keratinocytes in stratified squamous epithelia,21 mCLCA5 is downregulated in some murine mammary tumour cell lines, including 4T1 cells, where re-expression inhibits proliferation.22 Thus, hCLCA2 and its murine orthologue have been proposed as tumour suppressor genes in breast cancer.24 However, other investigators have demonstrated an upregulation of hCLCA2 in triple-negative breast cancer patients with a poor prognosis,25 perhaps suggesting breast cancer subtype specific roles, and exemplifying the need for further characterization of this poorly understood protein family.CLCA proteins have been associated with diverse functions including cell adhesion,mClca1 and mClca2 that share ~96% complementary DNA sequence identity, and encode mCLCA1 and mCLCA2, respectively.26 These proteins are reciprocally expressed during mammary gland development with mCLCA2 being expressed at high levels during lactation and involution, whereas mCLCA1 expression is suppressed during involution.27 This pattern suggests different functional roles for these highly related genes.Other murine genes include Given the pro-tumourigenic potential of the involution mammary microenvironment, the critical role of Stat3 in orchestrating regression and the intriguing regulation of mCLCA proteins, we hypothesized that mammary epithelial expression of Stat3 would influence the expression of mCLCA proteins.28 Differentiation was confirmed by robust upregulation in expression of the milk protein β-casein , whereas expression of mCLCA1 is markedly upregulated between 12 and 48 h of involution (first phase), and subsequently maintained from 48 h onwards  microarray data that have been previously published,Stat3fl/fl; BLG-Cre; hereafter referred to as Stat3 KO) and compared these with age-matched controls lacking Cre expression (control). Using qRT-PCR, we compared expression of mCLCA1, mCLCA2 and mCLCA5 during lactation and involution, utilizing samples collected at 42 h involution, close to the onset of the irreversible phase (unpublished data).To further interrogate a possible relationship between Stat3 activity and CLCA protein expression, we utilized mice with a mammary epithelial cell-specific conditional deletion of Stat3 , we identified at least two different cell populations at the edge of the tumour . Strikin35 is likely to be critical to the invasive nature of the neoplastic cells at the tumour margin.We therefore suggest that in individual neoplastic cells, the negative regulation of mCLCA5 by pStat3 remains functional. Although both mCLCA5 and pStat3 are expressed predominantly at the invasive edge of the tumour, minimal co-localization of nuclear Stat3 and cytoplasmic mCLCA5 is observed. It seems likely that mCLCA5 expression may be predominantly in the tumour cells, whereas pStat3 nuclear localization is seen in both the tumour cells and immune and stromal compartments at the invasive front. Thus, activity of pStat3, a known breast cancer oncogene,36 and it is tempting to speculatively attribute such as role to mCLCA5 in this context at the invasive edge of the tumour  Stat3. This suggests that Stat3 retains a suppressive effect on mCLCA5 expression in 4T1 tumour cells. It is in accordance with the findings of others, and therefore tempting to speculate, that downregulation of mCLCA5 in the presence of cells expressing Stat3 activity reflects an invasive phenotype in these cells and potential epithelial–mesenchymal transition.37 However, in this context it is important to note the compelling data demonstrating that re-expression of mCLCA5 in 4T1 cells inhibits proliferation22 and that loss of hCLCA2 expression in human MCF10A cells augments proliferation of the cells,24 whereas re-expression elicits a reduction in tumour cell growth.19 In the in vivo 4T1 murine model described in our study, cellular behaviour may be influenced by the presence of subpopulations of neoplastic cells in which either Stat3 activity suppresses mCLCA5 expression, or mCLCA5 is expressed in the absence of Stat3 activity. The influence of the immune cell compartment also needs to be considered particularly given that the 4T1 model involves introduction of syngeneic tumour cells into immune-competent mice. It is possible that mCLCA5 secretion is stimulated in host cells, such as stromal components or immune cells, in an attempt to negatively regulate tumour growth. Currently, the role of mCLCA5 at the invasive front of the tumour is unclear. Future work will require development of inducible models of mCLCA5 expression and/or Stat3 activity in vivo in different cell types to understand the relationship between the key oncogene Stat3 and the intriguing factor mCLCA5.However, our data also raise the question of the role of the observed mCLCA5 expression at the invasive front. One interpretation is that mCLCA5 expression at the invasive edge may be beneficial to tumour growth with mCLCA5 acting in a non-cell autonomous manner to augment tumour cell proliferation.Although in normal mammary gland, Stat3 regulates cell death, breast cancer cells frequently become addicted to Stat3 and require Stat3 activity for survival. Our data demonstrate that Stat3 is a negative regulator of mCLCA5 during mammary gland involution, and that in tumourigenesis in an immune-competent murine model, mCLCA5 expression is suppressed in cells with high levels of nuclear Stat3. Stat3 appears to be a cell autonomous negative regulator of this protein.28 Human LIF (a generous gift from Dr Jennifer Nichols) or recombinant mouse OSM  were used at a final concentration of 10 or 20 ng/ml (LIF), or 25 ng/ ml (OSM). For imaging, KIM-2 cells were differentiated in 35 mm diameter glass bottom dishes . Murine 4T1 cells were purchased from ATCC  and were maintained according to ATCC protocols.Maintenance and differentiation of KIM-2 cells and instigation of hormone withdrawal, were as previously described.38 The primer sequences utilized are detailed in Standard protocols were followed.38 Antibodies employed were: anti-phospho-Stat3 , anti-Stat3 , anti-GAPDH  , and anti-alpha-tubulin .Standard protocols were followed.Stat3fl/fl; BLG-Cre mice40 were utilized and involution induced as previously described.9 All animals were treated according to the local ethical committee and the UK Home Office guidelines.μl containing 1 × 105 4T1 cells with matrigel  was injected into the right- or left-fourth mammary gland fat pad of ~12-week-old virgin Balb/c mice under anaesthesia and analgesia.41 Tumour development was monitored, mice were killed and tumours were harvested at day 25 post injection. Tumour tissue  was collected for RNA and protein extraction. Tissue for histological sectioning was dissected. Formalin fixation, histological sectioning and staining followed routine protocols.A final volume of 16 KIM-2 cells in glass bottom dishes were fixed for 10 min in ice cold methanol and immunofluorescence staining for E-cadherin  was carried out following a standard protocol.Paraffin embedded tumour specimens were prepared as three micron sections on positively charged slides . Immunohistochemical staining for mCLCA5 , vimentin  and murine pStat3   was carried out using a routine protocol employing an automated immunohistochemistry system .Immunofluoresence staining for E-cadherin , mCLCA5 , vimentin  and murine total Stat3   was carried out following de-paraffinization and antigen retrieval for 20 min at 90 °C using Dako Envision Flex Target Antigen Retrieval Solution High pH in a PT Link and Pre-Treatment Module for Tissue Specimens (both Dako). For Fc receptor blocking prior to staining for murine total Stat3 , anti-mouse CD16/CD32 clone 93 was utilized . Isotype- and species-matched immunoglobulins were used as a negative control.


The data presented in this article are related to the research article entitled “Cyr61/CCN1 is involved in the pathogenesis of psoriasis vulgaris via promoting IL-8 production by keratinocytes in a JNK/NF-κB pathway”   Specifications TableValue of the data•This data characterizes the distribution of Cyr61 expression in skin lesion of patients with psoriasis vulgaris.•These data could be used for developing improved strategy in the treatment of psoriasis.1in vitro and anti-Cyr61mAb in vivo   on the shaved back and the right ear, representing a daily dose of 3.125 mg of the active compound. Control mice were treated similarly with vaseline . Mice received intraperitoneal injections of 200 μg/day of either anti-Cyr61 mAb 093G9 or control IgG1  2 days after IMQ treatment. After16 days later mice were sacrificed and skin samples were collected and inspected National Natural Science Foundation of China , Excellent Young Doctor Foundation of Shanghai Ninth People׳s Hospital (201608), Education Ministry Research Fund for the Doctoral Program (20130073110003), Science and Technology Commission of Shanghai Municipality (13JC1402300) and Shanghai Cultivate Outstanding Young Teachers in Colleges and Universities Scientific Research Fund (JDY09062).This work was funded by


Insufficient licensing of DNA replication origins has been shown to result in genome instability, stem cell deficiency, and cancers. However, it is unclear whether the DNA damage resulting from deficient replication licensing occurs generally or if specific sites are preferentially affected. To map locations of ongoing DNA damage in vivo, the DNAs present in red blood cell micronuclei were sequenced. Many micronuclei are the product of DNA breaks that leave acentromeric remnants that failed to segregate during mitosis and should reflect the locations of breaks. To validate the approach we show that micronuclear sequences identify known common fragile sites under conditions that induce breaks at these locations (hydroxyurea). In MCM2 deficient mice a different set of preferred breakage sites is identified that includes the tumor suppressor gene Tcf3, which is known to contribute to T-lymphocytic leukemias that arise in these mice, and the 45S rRNA gene repeats. Many RBC micronuclei result from double strand DNA breaks that give rise to acentromeric chromosomal fragments that fail to incorporate into nuclei during mitosis and consequently remain in the cell following enucleation. Here, RBC micronuclear DNA is sequenced (Mic-Seq) to define the locations of breaks genome-wide and this assay is used to study ongoing genome instability resulting from insufficient DNA replication origin licensing. Using a mouse model, we show that there is increased instability at discrete sites across the genome, which include genes that are recurrently deleted in the T-lymphocytic leukemias that eventually arise in these mice. Mic-Seq may provide an effective means of predicting locations that are susceptible to genetic damage and these predictions may have prognostic value. Licensing of DNA replication origins begins early during the G1-phase of the cell cycle when ORC and CDC6 recruit CDT1-MCM2-7 to the chromatin [reviewed in Chaos3/Chaos3) females succumb to mammary adenocarcinomas when carried on the C3H genetic background on which it was isolated [IRES-CreERT2/ IRES-CreERT2) mice exhibit near to 100% penetrance of T-lymphocytic leukemia (TLL) within 5 months on the 129Sv background on which they were constructed [The cancers that arise in MCM deficient mice can be highly specific to a particular genetic background. For example, >80% of hypomorphic MCM4  with a preponderance of deletions averaging 400–500 kbp in size –10. Seveexamined . Tcf3 is in mice . Importaent mice .Mcm2-7, which occurs in many tumors [Tcf3 gene is also the site of elevated ongoing genome instability even prior to tumor initiation in MCM2 deficient mice on the 129Sv genetic background. The increased instability at this site predicts the high rate of genetic lesions affecting the Tcf3 gene and consequent high rate of TLL in these mice. In addition to loss of Tcf3, MCM2 deficient mice exhibit genetic lesions within the 45S ribosomal RNA gene repeats clusters on Chrs 12, 16, 18 and 19 consistent with the observed nucleolar associated DNA damage foci in aging HSCs. The present study demonstrates that the consequences of reduced MCM expression on local genome instability is reflected in micronuclear DNA sequences allowing prediction of specific genetic lesions in the etiology of cancer and during aging.Replication licensing may become limiting due to genetic deletion of y tumors , oncogeny tumors  and duriy tumors . It is ny tumors . In the To map sites of ongoing genomic instability across the genome we take advantage of the fact that in the hematopoietic system of mammals DNA remnants resulting from genetic damage events during differentiation of hematopoietic stem cells (HSCs) to erythrocytes are retained in the cells as micronuclei following enucleation . To isolSequence tag density in the WBC and GRN fractions of wt mice are largely uniform both between and across individual chromosomes. In contrast, in the MN fraction, although the minimal sequence tag density is similar between chromosomes, there is a significant deviation from the average sequence tag density as a function of position across each chromosome. This, in part, reflects the frequency with which different portions of each chromosome are present in micronuclei. Similar patterns are seen in the MN fractions of two different wt mice where the correlation between experimental repeats is 0.988. Several parameters are defined to describe the observed changes quantitatively . First, Functions describing β and ρ for wt animals were established using Chr7 and Chr11 since, by inspection, there are few localized increases in sequence tag density on these chromosomes. Both β and ρ are non-linear where the best fit for each is a quadratic function  panel a.To identify localized regions of the genome exhibiting elevated instability, γ values were estimated by determining the rate of the sequence tag coverage change within smoothed and normalized 20 kbp windows genome wide. Although the overall contribution of β and ρ to differential representation of centromere distal and, to a lesser extent proximal ends of whole chromosomes is significant over entire chromosomes, over shorter 20 kb intervals these effects are minimal and the slope of the sequence tag density largely reflects the localized effect γ e.g. , γ trackPrior studies have shown that agents that inhibit replication fork progression lead to chromosome breakage at specific locations across the genome referred to as common fragile sites. Characteristics of these sites have been defined where breakage typically occurs at large, late replicating, transcriptionally active genes . To deteThe micronuclear fractions from HU treated mice show a more rapid increase in sequence tag density as a function of distance from the centromere relative to wt mice and the value estimated for β (again using Chr7 and Chr11) is increased ~5 fold  panel a.Wwox, Inspection of the micronuclear sequence data from HU treated mice reveals sharp increases in sequence tag coverage distal to discrete locations suggesting that specific sites are disproportionately affected by HU treatment  protein 2 deficient mice . MCM2 deComparison of the sequence tag densities derived from micronuclei of wt and MCM2 deficient 129Sv mice shows that the additional breaks resulting from MCM2 deficiency suppress ρ values and modestly increase β values relative to wt cells  panel a.http://www.ncbi.nlm.nih.gov/gene/19791). These are typically composed of 30–40 repeats of an approximately 45 kb repeating unit at each location . One poin situ hybridization (FISH) plus spectral karyotyping (SKY) to localize a probe for the 45S ribosomal gene  to specific chromosomes. Results from these studies  and mapping breaks in banded metaphase chromosomes. Numerous studies have characterized sites that are frequently affected in human and mouse cells and led to identification of a set of locations termed common fragile sites that are affected under conditions of chemically induced replication stress in a high proportion of individuals. These sites have been extensively characterized and are significantly associated with the presence of large, transcriptionally active, and late replicating genes over 300 kbp in size . The frein vivo. Many micronuclear sequences result from the presence of double strand DNA breaks that lead to failure of acentromeric portions of chromosomes to segregate to the nucleus during mitosis. Further we take advantage of the fact that micronuclei are retained in maturing RBCs following enucleation in mammals. In contrast to defining fragile sites cytogenetically, harvesting micronuclei from RBCs allows recovery of tens of millions chromosomal remnants each of which defines a breakpoint that can be queried at nucleotide level resolution by high throughput sequencing. Although there is a concern that erythroblasts (just before enucleation) may not reflect the DNA repair and checkpoint responses typical of other cells, application of the Mic-Seq method to define fragile sites in mice treated with hydryoxyurea shows that the method identifies 5 of the 8 molecularly characterized fragile sites previously defined in mouse lymphocytes [Wwox and Immp2 genes. Further sites detected in this study support that the majority of the most sensitive HU induced fragile sites occur within subdomains of the transcribed regions of large (>300 kbp), transcriptionally active, genes similar to prior studies and consistent with the possibility of interference between the transcription and replication machinery under conditions of DNA polymerase inhibition [Here we have used the representation of different genomic regions in micronuclear DNA sequences to infer the frequency of chromosome breaks in erythroid cells phocytes  includinhibition –16, 20.Similar to HU treatment, reduced replication licensing results in genome instability, increased DSBs, and chromosomal deletions and rearrangements –5, 9. ThShort nascent strand analysis has shown that origin usage does not decline uniformly across the genome, but rather specific locations lose function preferentially, in MCM2 deficient MEFs . These lTcf3 is sufficient to drive TLL formation [Even within early replicating regions of the genome chromosomal breaks detected by Mic-Seq show only a modest correlation with locations where SNS analysis demonstrates that origin usage is most affected. Nonetheless, it is important that many of the locations showing recurrent deletions in the TLLs that arise in MCM2 deficient mice  are siteormation  and the The strongest signals observed by Mic-Seq in MCM2 deficient mice are associated with chromosomes carrying nucleoli and implicate a high rate of DSBs within rDNA clusters as the location of much of the DNA damage, and the source of a large proportion of the additional micronuclei, found in MCM2 deficient mice. This observation confirms and extends prior studies showing that, as mice age, MCM levels are suppressed in HSCs and, coincident with the loss of MCM expression, nucleolar associated γ-H2AX foci accumulate . The preThese results demonstrate that experimental reduction of MCM proteins in young asymptomatic mice is sufficient to cause a profile of genome instability that predicts at least a subset of chromosomal locations where genetic damage is found in both cancers and during aging. The observation that, unlike HU, many locations where MCM2 deficiency causes instability can already be detected in young wt mice suggests that even under normal conditions the distribution of licensed DNA replication origins contributes to base line levels of chromosomal instability. Although replication stress is widely recognized as a potent cause of genomic instability , 25, theAnimal husbandry programs and protocol reviews are in compliance with NIH, USDA, and New York State Standards. Mice were maintained in facilities covered under NIH assurance #A-3143-01, certified by New York State for the use of living animals, and the USDA APIHS registration as research facility #21–124. The studies were approved by the Roswell Park Cancer Institute Animal Care and Use Committee under Protocols 817M and 876M.IRES-CreERT2/IRES-CreERT2 (MCM2 deficient) mice were used in studies addressing the effects of MCM2 deficiency. For studies addressing the effects of hydroxyurea (HU), 3 month old wild type 129Sv mice were administered HU continuously in the drinking water at the concentrations indicated in the text. Blood samples were taken by retro-orbital bleed or cardiac puncture.Five to six week old wild type 129Sv and Mcm2 For flow cytometric analysis of micronuclei  blood saCombined SKY/FISH was performed on wt or MCM2 deficient mouse embryonic fibroblast (passage 3) by the Roswell Park Cancer Institute SKY/FISH core facility. The rDNA probe for FISH analysis was prepared using Nick Translation Reagent Kit 07J00-001 (Abbott Molecular Inc.) Green-dUTP 02N32-050 (Abbott Molecular Inc.) to fluorescently label a 7109 bp EcoRI fragment from human genomic ribosomal gene DNA containing a portion of the 18S ribosomal RNA gene, the intergenic spacer, the 5.8S ribosomal RNA gene and a portion of the 28S ribosomal RNA gene.Between 400–500 μl of whole blood was washed with 10 ml of phosphate buffered saline (PBS) 3 times and re-suspended in 3 ml PBS. The sample was then layered over 2 ml of Lymphocyte Separation Medium  in a 15 ml centrifuge tube and spun for 15 min at 800 RPM. Lymphocytes (WBCs) at the PBS-media interphases were collected and placed in a 15 ml tube and washed 3 times with 10 ml PBS prior to pelleting for DNA isolation. Separation media was removed from the red blood cell (RBC)/granulocyte (GRN) pellet in the original tube and cells were washed 3 times with 10 ml PBS and pelleted. The cell pellet was then resuspended in 4 ml of RLF lyse buffer and incubated at room temperature for 5 minutes prior to spinning at 800 RPM for 5 minutes. The supernatant was collected as the RBC micronuclear fraction (MN). The pellet is the granulocyte fraction and was washed with 10 ml PBS and pelleted for DNA isolation. WBC and GRN pellets were re-suspended in 2 ml 1X lysis buffer with 1 ug/ml proteinase K and the RBC fraction was brought to 1X lysis buffer and 1 μg/ml proteinase K using 4X lysis buffer. Samples were incubated at 37°C overnight and extracted with an equal volume of phenol-chloroform-isoamyl alcohol. Nucleic acid was precipitated with isopropanol, washed with 70% ethanol and re-suspended in 100 ul TE buffer. Samples were treated with 2000 units ribonuclease T1 (Invitrogen) and 3 ug ribonuclease A (Invitrogen) for 30 minutes at 37°C followed by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation of the DNA.Libraries were prepared for DNA sequencing using Wafergen prep X kits and TruSeq Index tags . Paired end 100 nt sequences were generated using an Illumina HiSeq2500. A summary of numbers of mapped sequence tags generated for each sequencing library is given in Sequence mapping and coverage generation: Sequences were aligned to the reference mouse genome (mm9) using BWA [sing BWA  with defThe Pearson correlation coefficient is 0.975 between MCM2 deficient samples 1 and 2, 0.938 between HU treated samples 1 and 2, and 0.969, 0.978, and 0.988 between wt samples 1 and 2, 2 and 3 and 1 and 3 respectively.  Based on the extremely high linear correlations, data was pooled for samples from the same experimental condition to reach better accuracy and allow the use of smaller window sizes in subsequent analyses. A window based approach is employed to infer the parameters in the model by counting read pairs in overlapping windows. Proper paired reads with mapping scores greater than 20 are assigned to a window if the middle point of the left-end read is within the window. A 40 kb window was applied where adjacent windows are overlapped by 20 kb and for pooled data, a 20 kb window with a 10 kb overlap is used.Window count data are first normalized against the GRN fraction where deviation in sequence coverage is expected to result from GC bias, other sequencing biases and strain specific bias as the reference genome is not 129Sv. The GRN fraction from the HU untreated control was used as it shows similar bias with MN fractions and windows with read counts outside of three standard deviations are excluded. The data are further normalized to remove any effect related to sequencing depth using scaling factors determined by α estimates. The number of read pairs for each sample is related to parameter estimates as more reads often have better coverage/higher α. α values are estimated using the minimum median value of 1000 consecutive data points, this estimate ignores β and ρ effects and is an over-estimate. Then the median ratio is selected as a base line for data scaling after excluding chromosomes with strong α changes, which include chr 6, 12, 15, 16, 18, 19, and X. The normalization will make any biological effects comparable as they are scaled to the same level. For the pooled data, the following factors are applied: MCM2 deficient (1.742), HU treated (1.000) and wt (2.037).γ plots: To extract γ values, pooled coverage data was windowed and smoothed monotonically. Monotonic smoothing is used to suppress the effect of local changes in sequence tag coverage due to GC bias or other effects that are unrelated to the cumulative changes in coverage expected for γ. For chromosomes exhibiting significant levels of ρ (Chr1 and Chr2 in wt samples), a monotonically decreasing smoothing line is fitted before the minimum median value point while after that point an increasing smoothing line is fitted. R package scam [age scam  is used 2 + ρ(Chr length—x)2 where deviation from this relationship is defined as γ. Finally, the distance effects of γ values related to telomeres were normalized by multiplying the corresponding distance and the γ peaks do not show any relationship with genomic locations after normalization.γ plots were further normalized by removing β and ρ effects estimated using chr7 and 11. To estimate β and ρ, a quadratic function with 2 quadratic terms for beta and rho separately is fitted using non-linear weighted least-squares method provided by nls R function. Chr7 and chr11 are used for the inference as, by inspection, they have smallest γ effects. To further estimate the accuracy of the estimation, bootstraps of 50% data points without replacement with 1000 repeats were carried out to estimate standard deviation and 95% confidence interval. The effect of α, β and ρ on sequence tag coverage at various positions across a chromosome is then described as α + βxTo establish cutoff values for normalized γ peaks, the γ values (with β and ρ effect removed) on chr11 and chr7 are used to estimate the null distribution and the histograms are shown in Major satellite (MaSat) and minor satellite (MiSat) sequences  were conhttps://www.ncbi.nlm.nih.gov/sra/?term=SRP091564) under accession number SRP091564.Sequence data from this study have been submitted to the NCBI short reads archive (SRA),  and GRN (panel b) blood fractions of the 6 week old wt 129Sv mouse shown in (TIF)Click here for additional data file.S2 FigWild type 129Sv mice were treated with varying concentrations of HU (n = 3 for each condition) as indicated and assayed for RBC MN frequency at 1 week or 3 weeks of treatment in panel (a) and by CBCs at three week of treatment in panel (b). Panel (c) is a list of genes over 300 kbp in length that contain the largest γ peak regions in HU treated mice. These include sites 1–3 and 5 (indicated in parentheses following the gene name) as marked in (TIF)Click here for additional data file.S3 FigPanels A and B show 20X images of blood smears from 6 week old129Sv wt (panel a) and MCM2 deficient (panel b) mice stained with acridine orange (green). Arrows indicate micronuclei. Panel c shows RBC micronuclear frequency determined by flow cytometry for wt ) and MCM2 deficient  6 week old129Sv mice (error bars indicate s.d.). Panel d shows SKY/FISH analysis to identify chromosomes hybridizing to a 45S rRNA gene probe sequence where signal is seen as green . Chromosomes 6, 12, 15, 16, 18, and 19 are marked by arrows as indicated. Panel (e) shows the number of chromatids exhibiting 45S rRNA gene signal and panel (f) shows the average signal strength per chromatid  in 13 metaphase spreads (52 chromatids) for each chromosome (error bars indicate s.d.). Panel (g) shows the ratio of sequence tags mapping to the major (MaSat) relative to the minor (MiSat) satellites for wt and MCM2 deficient granulocyte (GRN), micronuclear (MN) and white blood cell (WBC) fractions as indicated.(TIF)Click here for additional data file.S4 FigPanels a-f show MEFs from wt (a-c) and MCM2 deficient (d-f) embryos stained for nucleolin , γH2AX  and counter stained with DAPI (blue). Panels c and f are overlays of panels a/b and d/e respectively and the proportion of nucleoli containing γH2AX foci in wt n = 122, 345) verses MCM2 deficient  cells are quantified for two experiments in panel g. Panels h and i compare sequence tag density over the 45S rRNA gene using data from total genomic DNA from thymus to estimate the ability to map sequences across the repeat (h) or short nascent strands prepared from wt (blue) or MCM2 deficient (red) MEFs by nascent strand capture and release (i) extracted from data in [2, 345 ve(TIF)Click here for additional data file.S5 FigPanel a shows β and ρ values estimated from chr7 using pooled wt, MCM2 deficient and HU treated samples. To further estimate the accuracy of the estimation, bootstraps of 50% data points without replacement with 1000 repeats were carried out to estimate standard deviation and 95% confidence interval as shown in the table. Panel b shows normalized α values for individual wt (3), MCM2 deficient (2) and HU treated (2) samples. Panel c shows a plot of 45S ribosomal RNA gene (rDNA) sequence tag coverage (y-axis) against major satellite (MaSat) coverage for individual wt (3), MCM2 deficient (2) and HU treated (2) samples from both GRN fractions (G) and MN fractions (R). The shaded area indicates the 95% confidence interval excluding the MCM2 deficient MN samples.(TIF)Click here for additional data file.S6 FigChr7 and Chr11 were chosen to estimate experimental variance in γ values since by inspection they exhibit few local γ peaks. In panel a, γ value distributions are shown for each chromosome for hydroxyurea treated (HUEX), MCM2 deficient (Mcm2) and wild type (wt) mice as indicated. Panel b shows the standard deviations estimated from each of the distributions shown in panel a.(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.S3 Table(TIF)Click here for additional data file.


The prognostic variability of thyroid carcinomas has led to the search for accurate biomarkers at the molecular level. Follicular thyroid carcinoma (FTC) is a typical example of differentiated thyroid carcinomas (DTC) in which challenges are faced in the differential diagnosis.We used high-throughput paired-end RNA sequencing technology to study four cases of FTC with different degree of capsular invasion: two minimally invasive (mFTC) and two widely invasive FTC (wFTC). We searched by genes differentially expressed between mFTC and wFTC, in an attempt to find biomarkers of thyroid cancer diagnosis and/or progression. Selected biomarkers were validated by real-time quantitative PCR in 137 frozen thyroid samples and in an independent dataset (TCGA), evaluating the diagnostic and the prognostic performance of the candidate biomarkers.C1QL1, LCN2, CRABP1 and CILP were differentially expressed in DTC in comparison with normal thyroid tissues. LCN2 and CRABP1 were also differentially expressed in DTC when compared with follicular thyroid adenoma. Additionally, overexpression of LCN2 and C1QL1 were found to be independent predictors of extrathyroidal extension in DTC.We identified 17 genes significantly differentially expressed between mFTC and wFTC. CRABP1 and the overexpression of LCN2 may be useful diagnostic biomarkers in thyroid tumours with questionable malignity, and the overexpression of LCN2 and C1QL1 may be useful for prognostic purposes.We conclude that the underexpression of The online version of this article (10.1186/s12885-017-3948-3) contains supplementary material, which is available to authorized users. Thyroid cancer is the most frequent type of endocrine cancer with an incidence of 12 cases per 100,000 individuals . PapillaBRAF mutations have been shown to be useful for predicting recurrence and/or disease persistence , 20 FTA, and 19 normal samples of adjacent/contralateral thyroid tissues from patients with DTC from this series. The histology of all DTC was revised by two pathologists (CE and MSS) and the final classification was made according to the WHO criteria  > 1 was considered as gain of expression, and fold change [log2 (2-ΔΔCT)] ≤ 1 was established as normal gene expression. For CRABP1 and CILP genes, fold change [log2 (2-ΔΔCT)] < −1 was considered as loss of expression, and fold change [log2 (2-ΔΔCT)] ≥ −1 was established as normal gene expression. The diagnostic performance (sensitivity and specificity) of those cut-off values are presented in Table The relative quantification of target genes was determined using the comparative CT method 2systems) . This prsystems .C1QL1, LCN2, CRABP1 and CILP genes. Gene expression values for each sample are normalized read counts, estimated by using RSEM software  and LCN2  expression were significantly associated with the extrathyroidal extension of the tumour. In the multivariate model, gain of C1QL1  and LCN2  expression were independent predictive factors for extrathyroidal extension in DTC. Additionally, gain of C1QL1 expression was observed in the larger (> 4 cm) DTC (OR = 4.60), but the difference was only borderline in terms of statistical significance (P = 0.056). No associations were found between CRABP1 and CILP expression and prognostic factors of thyroid cancer.In the univariate analysis, gain of the The clinical behaviour of thyroid cancer is still difficult to predict and clinical and histopathological prognostic factors remain the key elements for diagnosis and prognosis of the patients . Due to C1QL1, LCN2, CRABP1 and CILP genes were selected for further validations in a larger series of DTC, using FTAs and a pool of normal thyroid tissues for comparison.In total, 17 genes were found to be differentially expressed in mFTC and wFTC. After customized filtering and validation of expression values by qPCR, CRABP1 was differentially expressed between DTC, FTA and normal thyroid tissue. Expression of CRABP1 was significantly lower in FTC, FVPTC and PTC and in all ten thyroid cancer cell lines than in normal thyroid and FTA. In the ROC analysis, AUC for the CRABP1 had the highest value (0.902), in the comparison of DTC versus normal thyroid. Gene expression from thyroid samples in TCGA reinforced CRABP1 as biomarker in DTC.Remarkably, CRABP1 (cellular retinoic acid binding protein1) encodes a specific binding protein for a vitamin A family member and is thought to play an important role in retinoic acid-mediated differentiation and proliferation processes. Loss of CRABP1 expression in thyroid cancer was shown in previous studies ]. Our results indicate that LCN2 expression may also serve as a useful biomarker in DTC, namely for the FNAB diagnosis of thyroid nodules.e stress . Consist cancers . Our datehaviour . LCN2 haC1QL1 was found differentially expressed between DTC and normal thyroid tissue. We observed a clear cut gain of C1QL1 expression in FTC, FVPTC and PTC. The relative low number of FTC of the present series can justify the lack of statistical significance verified in the comparison of FTC with normal thyroid tissue regarding C1QL1 expression. Gain of C1QL1 expression was significantly associated with the presence of extrathyroidal extension in DTC. In PTC, tumours with gain of C1QL1 expression were significantly larger than tumours without C1QL1 overexpression. Since extrathyroidal extension and tumour size are major prognostic factors of thyroid cancer it may be advance that gain of expression of C1QL1 will probably be a good indicator of clinical evolution. Notably, gain of C1QL1 expression was significantly higher in wFTC than in mFTC, reinforcing the assumption that gain of C1QL1 expression is related with thyroid cancer progression. Although we found an association of C1QL1 overexpression and lymphocytic thyroiditis in DTC, further statistical analyses revealed no significant differences of C1QL1 expression between the subtypes of DTC with lymphocytic thyroiditis and without lymphocytic thyroiditis (P = 0.316). Gene expression from thyroid samples in TCGA reinforced C1QL1 as biomarker in DTC.C1QL1 is member of a subfamily of small secreted proteins of unknown function -C1q-like that are expressed almost exclusively in brain, and produced in differential patterns by specific types of neurons  as input. For evaluation of the combined biomarker panel the sum of log2 (2-ΔΔCT) expression values from genes with gain (C1QL1 and LCN2) and loss (CRABP1 and CILP) in DTC were used. AUC, area under the curve; CI, confidence interval. (DOCX 14 kb)Receiver Operating Characteristics (ROC) curve analysis. ROC curves for individual biomarkers were generated using log2  of C1QL1 (a), LCN2 (b), CRABP1 (c) and CILP (d) in differentiated thyroid cancer (DTC) and normal thyroid (NT) tissues available in The Cancer Genome Atlas (TCGA). Each dot represents the gene expression value of each sample. The lines represent the averages. Statistical significance values: *, P < 0.001. (JPEG 518 kb)Gene expression of Additional file 12: Table S6.Clinicopathological and genetic data of the FTC classified by classes based on gene expression. (DOCX 18 kb)Additional file 13: Table S7.Clinicopathological and genetic data of the FVPTC classified by classes based on gene expression. (DOCX 17 kb)Additional file 14: Table S8.Clinicopathological and genetic data of the PTC classified by classes based on gene expression. (DOCX 17 kb)


EPB41L3 in 148 anal and perianal biopsies to determine whether high levels of methylation would be associated with anal intraepithelial neoplasia (AIN). The most prevalent HPV type was HPV16, detected in 54% of the 30 benign biopsies, 33% of the 43 low-grade AIN (lgAIN), 82% of the 59 high grade AIN (hgAIN) and 4 of the 5 anal cancers. A methylation score was developed (0.561*HPV16me+0.439*EPB41L3) which had increasing values with severity of disease: the mean was 8.1% in benign, 13.2% in lgAIN, 22.3% in hgAIN and 49.3% in cancers (p < 0.0001). The methylation score as a triage classifier at a cut-off of 8.8 gave a sensitivity of 90.6% , specificity of 50.7%  and area under the curve of 0.82 (95% CI: 0.75–0.89) for separating hgAIN and cancer from benign and lgAIN biopsies. We conclude that methylation of HPV16 and EPB41L3 show highly significant association with increasing severity of AIN and cancer and may be useful as biomarkers in anal disease.We studied DNA methylation patterns of human papillomavirus (HPV) and tumor suppressor gene  Human papillomavirus (HPV) infects a majority of people worldwide. Infection can occur at any age and can either be transient  or could be persistent and last for many decades . High riAnal cancer has been growing in incidence in the past few decades, especially in women and also in men who have sex with men (MSM). Furthermore, anal cancer incidence is higher in HIV-positive MSM with approximately 100 cases compared to 25 cases per 100,000 in HIV-negative MSM  and onlyAnal cytology sampling is problematic because anal folds may hide lesions and this has resulted in recommendations for more frequent sampling to compensate for poor sensitivity . Normal,a priori hypothesis that high levels of methylation at genomic positions associated with hgCIN would also be associated with hgAIN. Here, we focus on the methylation of host gene EPB41L3 and the high risk viral types: HPV16, HPV18, HPV31 and HPV33. EPB41L3 (Erythrocyte Membrane Protein Band 4.1 like 3) is a tumor suppressor gene that inhibits cell proliferation, promotes apoptosis and has been found to be highly methylated in many cancers such as lung, cervix, ovarian and breast [DNA methylation testing of HPV and human genes has been validated as an accurate method for detection of CIN2 and CIN3 –22. Leved breast –28.There were 30 biopsies with <AIN, 43 lgAIN, 59 hgAIN and 5 cancers among the anal samples and 11 biopsies of high-grade perianal lesions (Table About a third 47/148) of samples were infected with multiple HPV types. 33% of anal biopsies with either <AIN, or lgAIN histopathology were not infected by hrHPV types while 53% and 33%, respectively, were HPV16 positive , but no significant differences in the likelihood ratios were found for methylation of HPV18, HPV31 and HPV33 and these latter markers were dropped from further analysis . The bivariable logistic regression using EPB41L3 and HPV16me was highly significant , there was a highly significant trend of increased methylation with disease progression . Figure EPB41L3, HPV16me and the DNAme score.The DNAme levels of p < 0.0001) for EPB41L3, 0.781  for HPV16me and 0.821  for the DNAme score. Figure The ROC curves comparing the methylation levels in the <AIN and lgAIN samples to the hgAIN and cancer cases Figure  had an ASensitivity and specificity of the DNAme score to detect hgAIN and cancers and the proportion of positive samples identified by the DNAme score is shown in Table EPB41L3 against HPV16me with the relationship having a weak Spearman correlation. We also investigated whether the DNAme score would be able to correctly identify the 11 high-grade perianal samples  of HPV16. The AUC of the methylation score for separating <AIN and lgAIN from hgAIN and cancer was 0.82  for DNA methylation, similar to what has been generally observed for cervical cancer. The DNAme score we developed is a multi-biomarker panel composed of three CpG sites within the EPB41L3. These data demonstrate that DNA methylation provides significant diagnostic information for detecting hgAIN in addition to that provided by HPV16 genotyping and suggests that a methylation test may be used to detect essentially all the cancers. The methylation score in HPV16 negative people did not show a significant discriminating effect. However, we cannot be sure of the lack of value of methylation information in the HPV16 negative samples because the analysis was underpowered for this endpoint. These results indicate the need for larger studies and a search for more genes that could provide additional triage information in people infected with hrHPV types other than HPV16.It is notable that the triage information for hgAIN and cancer provided by the methylation score was significantly higher only in HPV16 positive people ; howeverA limitation of the study is that we used FFPE biopsies. Our results need to be replicated in an adequately powered study of exfoliated anal cells because in routine practice hrHPV positive patients would have methylation tests performed on exfoliated cells collected by a swab or similar device. This would allow efficient triage to HRA, thus reducing costs, anxiety and possible over-treatment of low risk people. Our study had incomplete information on HIV status and the small size of the HIV-negative subset likely produced some ascertainment bias. Another limitation of our study is the use of patients (mostly MSM) recruited from two sites specializing in anal HPV-related disease in London. It remains to be seen if our results can be duplicated in other settings.We conclude that high levels of DNA methylation are associated with hgAIN and anal cancer. This finding should be further explored to better understand the biological mechanisms and the value of DNA methylation testing as a molecular triage of hrHPV positive individuals for high-resolution anoscopy screening.A set of anal and perianal biopsy specimens were obtained from 148 patients  of whom 94 were HIV positive, 40 were HIV negative and 14 had not been tested for their HIV status . The forIn patients undergoing HRA, biopsies were obtained from areas of clinical interest that exhibited acetowhite changes (regardless of vascular changes) on 5% acetic acid application. Most of the biopsies showed morphological changes varying from slight to severe which distinguished them from the surrounding non-acetowhite normal epithelium. The main clinical endpoint for all comparisons was the histology result. The biopsies were graded histopathologically using the AIN terminology  which isWe used H&E sections annotated by MS as a guide to dissect areas of interest on the corresponding four unstained 5 μm sections, as previously described . If moreThe samples were tested using the PapType High Risk HPV Detection and Genotyping kit  according to the manufacturer's instructions. The kit is able to detect 13 high-risk HPV types  a possible high risk type (HPV66) and two low-risk types (HPV6 and HPV11). The PapType test was performed with 10 μL of DNA in a final reaction volume of 20 μL with the addition of 2% Tween 20. The PCR reaction amplifies a variable region of the L1 gene of the HPV genome. A fragment of the human cardiac myosin light chain gene (MLC-1) was co-amplified in the same reaction vessel as a quality and quantity control. For simplicity, we categorized the samples into three HPV genotype groups independently of whether they were singly or multiply infected: those infected by (1) HPV16, (2) by any other high-risk types (including HPV66) and (3) those HPV negative or infected only by a low-risk type (lrHPV). We did not combine HPV18 with HPV16 due to obvious differences in viral disease characteristics as noted in earlier studies .EPB41L3 and viral late genomic regions of HPV16  and HPV18 , HPV31  and HPV33  as previously described [EPB41L3 (HPV assays) in a 25 μl volume with final concentration of reagents of 1× for Coral Load and PyroMark mix, 0.2 μM of PCR primers. PCR cycling conditions were 15 minutes at 94°C, followed by 45 cycles of 94°C, 54°C, 72°C each for 30 seconds and a final extension at 72°C for 10 minutes. The PCR products were pyrosequenced using a PyroMark™Q96 ID (Qiagen) instrument as previously described [Bisulfite conversions on 20 μl of DNA extracts were done using EZ DNA Methylation Kit  following the manufacturer's instructions. DNA methylation (DNAme) was measured by pyrosequencing for human biomarker escribed . All pyrEPB41L3 and HPV positive samples were imputed using a median regression with age as a predictor and DNA methylation as an outcome. All statistical analyses were performed on the imputed data set [EPB41L3, 22 for HPV16L1, 31 for HPV16L2, 5 for HPV18, 11 for HPV31 and none for HPV33. Of the 11 perianal samples, 4 had missing values for HPV16L1, 1 for HPV16L2 and 3 for HPV18. No value was missing for EPB41L3, HPV31 nor HPV33 , a variable called HPV16me was created by taking the geometric mean of DNAme levels of HPV16L1 and HPV16L2.Statistical analyses for the anal and perianal lesions were not combined. Spearman correlations were calculated between the markers. Since methylation values of HPV16L1 and HPV16L2 regions were correlated  with 95% confidence intervals were estimated and reported. Performance of the markers were assessed univariably by the LR χ2 test and the area under the curve (AUC) with 95% confidence intervals (CI) using the Delong method [Univariable and bivariable logistic models were fitted for statistically significant genes with the outcome measures 0 = <AIN and lgAIN, and 1 = hgAIN and cancer. The likelihood ratio (LR) χg method . SensitiEPB41L3 and HPV16me was computed as a linear predictor of the fitted bivariable logistic regression. Confidence intervals for difference in sensitivities and specificities between the DNAme score at different cut-offs and HPV16 genotyping was computed using 2000 stratified bootstrap replicates as recommended by Carpenter and Bithell [EPB41L3, HPV16me and the DNAme score to test for significant changes of methylation between the groups .A combined DNAme score of  Bithell . Cuzick Multiple imputations analyses were performed to test whether the single imputation led to any bias . We usedp-values were two-sided with significance set at α < 0.05. Analyses were undertaken using R statistical software version 3.3.1 [All on 3.3.1 .


To the editor: We greatly appreciate the editorial by Penttinen and Friede summarising the data regarding recent observations in the United States (US) of decreased effectiveness of the influenza A(H1N1)pdm09 strains  included in live attenuated influenza vaccines (LAIV) [luenza AHN1pdm09 sBased on evidence presently available to us, we believe that reduced replicative fitness of the A/California and A/Bolivia (H1N1)pdm09 LAIV strains is the most probable root cause for the reduced vaccine effectiveness (VE). From 2010/11 through 2013/14, LAIV VE in children aged 2–17 years against matched A(H3N2) and B strains has been comparable to that observed with IIV . LaboratIn the context of reduced replicative fitness, vaccine–virus interference may have contributed to the observed reduced VE. However, vaccine–virus interference specific to the quadrivalent formulation appears to be an unlikely root cause of the reduced VE with LAIV. LAIV3 demonstrated reduced VE against A(H1N1)pdm09 in 2010/11 in the US . AdditioBecause rates of vaccine coverage in the US have historically been higher compared with European countries, questions have been raised regarding the role of prior vaccination in the reduced AH1N1)pdm09 effectiveness N1pdm09 e-10. No sIn 2013/14, with LAIV4 containing the A/California strain, a statistically significant correlation was observed between reduced LAIV VE against (H1N1)pdm09 viruses and higher outdoor temperatures during LAIV lot unloading at US distributors . In laboWe have initiated a multifaceted scientific investigation into the causes of the recently observed reduced effectiveness of LAIV, with the goal of identifying a more effective A(H1N1)pdm09 LAIV strain for potential inclusion in the 2017/18 LAIV formulation. All potential hypotheses continue to be evaluated. We welcome the input and support of the multiple stakeholders involved, including national public health agencies, the World Health Organization, and additional external scientific experts, as we work together to ensure that VE of LAIV is improved in future influenza seasons.


After 3 days as a Category 3 and 4 hurricane in Haiti and Bahamas, Hurricane Matthew moved along the coast of the southeastern United States during October 6−8, 2016  . DrowninDespite public health warnings to avoid flood waters, among all 23 hurricane-related drownings, 18 deaths (78%) occurred in motor vehicles . As little as 6 inches of water might result in loss of control of a vehicle, and 2 feet of water can carry most cars away (


The BRCA1-associated protein 1 (BAP1) is an example of a critical player in determining the outcome of the interaction of Genes 3 Original studies by Carbone’s group identified the association of germline mutations in BAP1 with early onset of benign melanocytic skin tumours and a later high incidence of malignant mesothelioma together with uveal and cutaneous melanoma.6 The study conducted on two unrelated US families, referred to as ‘L’ and ‘W’ for their origins from Louisiana and Wisconsin respectively, implicated the 3p21 chromosomal region with an unexpectedly high incidence of malignant mesothelioma following minimal exposure to asbestos.2 Sequencing of this locus led to the identification of heterozygous mutations in BAP1, uncovering a novel familial autosomal dominant cancer syndrome. Indeed, biallelic somatic mutations in BAP1 are also frequently observed in human cancers.8 Nearly all cancer types have been reported in carriers of BAP1 germline mutations, but the high incidence of tumours associated with environmental stressors, such as mesothelioma (with asbestos), melanoma and skin tumours (with UV), highlights BAP1 as a critical player in the interaction of genes with environment.BAP1 is an ubiquitin C-terminal hydrolase belonging to a larger group of deubiquitylating enzymes.3 Nuclear BAP1 indeed deubiquitylates BRCA1‐associated RING domain 1 (BARD1), which in a heterodimer with BRCA1, forms the E3 ubiquitin ligase that regulates the DNA damage response (DDR).10 Loss of BAP1 resembles BRCA1 deficiency, resulting in hypersensitivity to ionising radiation.3 However, an important unexplored aspect of BAP1 is the significant proportion of the protein that can be sequestered in the cytoplasm by multi-monoubiquitylation of its nuclear localisation signal. In this study Bononi et al. uncovered a cytoplasmic function of BAP1, implicating a novel mechanism for the contribution of BAP1 loss to environmental carcinogenesis.Despite the critical relevance of BAP1 for cancer predisposition, the mechanistic insights underlining its function have so far been elusive. Originally identified in a yeast two‐hybrid screen as an interactor of the RING finger domain of the tumour suppressor BRCA1, BAP1 was eventually disproved as a BRCA1 deubiquitylating enzyme.+/− and BAP1+/+ fibroblasts derived from carriers and control members of families ‘W’ and ‘L’. BAP1+/− fibroblasts express 50% BAP1 protein levels as compared to control fibroblasts, and represent an excellent cellular model to experimentally recapitulate BAP1 deficiency in pre-carcinogenic conditions. In the work published in Nature1 the authors showed in subcellular fractionation experiments a significant accumulation of BAP1 in the cytoplasm and more specifically in the endoplasmic reticulum (ER). A reduced level of BAP1 in BAP1+/− fibroblasts mainly affected the ER fraction of the protein, suggesting a potential dysfunction of the ER in the mutant cells.1 As BAP1+/− cells appeared hyposensitive to a wide range of pro-apoptotic drugs and inducers, the authors speculated that BAP1 may contribute to control of the pro-apoptotic Ca2+ release from the ER. BAP1+/− fibroblasts indeed displayed a reduced level of Ca2+ release from the ER, which was restored by re-expression of chimeric BAP1 mutants selectively targeting ER and cytoplasm, but not the nucleus. Thus, Carbone et al. reported an unanticipated cytoplasmic function of BAP1 that could explain its critical role in determining the biological outcome of its interaction with the environmental stressors.1The authors employed an elegant experimental approach, using BAP12+ flux to the cytoplasm. The data clearly indicated that BAP1 physically interacts with the type-3 inositol-1,4,5-trisphosphate-receptor (IP3R3), and BAP1 expression level is directly correlated with IP3R3 in wild-type and mutant fibroblasts. Deeper biochemical analysis indeed demonstrated that BAP1 enzymatically controls IP3R3 ubiquitination and thus positively influences its stability. Loss of BAP1 therefore results in reduced expression of IP3R3 with correspondingly reduced cytoplasmic Ca2+ release, which in turn determines resistance to apoptotic inducers. The authors also observed an increase of cytoplasmic Ca2+ release and apoptosis in crocidolite asbestos-treated primary human mesothelial cells. Remarkably, depletion of BAP1 or IP3R3 promoted crocidolite asbestos/TNF-α-induced transformation of primary mesothelial cells, and re-expression of IP3R3 in BAP1-depleted cells partially reverted the phenotype.1 However, experiments with subcellular specific chimeric mutants of BAP1 indicated that both nuclear and cytoplasmic BAP1 contributed to the phenotype. This latter data opens the question regarding the specific contribution of the balance between cytoplasmic and nuclear BAP1 to cell death and genomic stability upon interaction with environmental stressors.As a ubiquitin hydrolase, BAP1 exerts an enzymatic activity which directly influences stability and activation of its substrates. Investigating the cytoplasmic function of BAP1, the authors focused on the possible interaction between BAP1 and ER channels that control CaCell Death & Differentiation2 by the same groups reports an intriguing observation in BAP1+/− individuals, which might shed light on the molecular basis of the transformation potential of these cells.2 By metabolic profiling of fibroblasts and plasma of carriers and control members from ‘W’ and ‘L’ families, the authors identified a clear alteration in cellular metabolism adopted by the mutant cells. BAP1+/− fibroblasts displayed a typical ‘Warburg effect’, as their cellular metabolism largely relied on aerobic glycolysis, increased lactate production and reduced mitochondrial respiration.2 Metabolic profiling of plasma from carrier individuals also displayed a similar distribution of the metabolites, and their profile was predictive of the genotype with 100% accuracy. The originality of these data relies on the observation of metabolic rewiring typical of transformed cells in a premalignant cellular setting, thus suggesting that that ‘Warburg effect’ might facilitate cancer transformation rather than be an adaptive process occurring in malignancy. Whether the observed metabolic change is associated with alteration of the Ca2+ flux and ER/mitochondria function of BAP1+/− cells will require further investigation and will probably highlight additional layers of complexity on the contribution of BAP1 deficiency to carcinogen hypersensitivity.A second study published in +/− carrying individuals and suggest new relevant questions, related to BAP1-dependent cancer susceptibility, and to more general gene/environment interactions -environmental signals can trigger BAP1 subcellular localisation and consequentially balance its contribution to genomic stability and cell death. Also, it will be important to assess the contribution of BAP1 somatic mutations to the tumorigenic process, and whether their incidence correlates with a selective mutational landscape. In addition, the contribution of premalignant metabolic rewiring to tumour transformation will stimulate a broad interest in the field. This aspect might also have wider implications for the genes−environment interaction, as it might imply that dietary regimes could influence metabolism and cellular transformation in specific predisposed conditions such as BAP1 mutations. Finally, these studies further highlight the importance of the integration of multidisciplinary approaches to dissect the interactions between genetic and environmental factors, to translate this information and stratify patients for prevention and treatment, and to provide novel biomarkers that predict risk of exposure.In conclusion, these studies have clarified novel important molecular aspects associated with the cancer predisposition of BAP1ractions . It is n