204 research outputs found
Genome-Wide Comparison of PU.1 and Spi-B Binding Sites in a Mouse B Lymphoma Cell Line
Background
Spi-B and PU.1 are highly related members of the E26-transformation-specific (ETS) family of transcription factors that have similar, but not identical, roles in B cell development. PU.1 and Spi-B are both expressed in B cells, and have been demonstrated to redundantly activate transcription of genes required for B cell differentiation and function. It was hypothesized that Spi-B and PU.1 occupy a similar set of regions within the genome of a B lymphoma cell line.
Results
To compare binding regions of Spi-B and PU.1, murine WEHI-279 lymphoma cells were infected with retroviral vectors encoding 3XFLAG-tagged PU.1 or Spi-B. Anti-FLAG chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) was performed. Analysis for high-stringency enriched genomic regions demonstrated that PU.1 occupied 4528 regions and Spi-B occupied 3360 regions. The majority of regions occupied by Spi-B were also occupied by PU.1. Regions bound by Spi-B and PU.1 were frequently located immediately upstream of genes associated with immune response and activation of B cells. Motif-finding revealed that both transcription factors were predominantly located at the ETS core domain (GGAA), however, other unique motifs were identified when examining regions associated with only one of the two factors. Motifs associated with unique PU.1 binding included POU2F2, while unique motifs in the Spi-B regions contained a combined ETS-IRF motif.
Conclusions
Our results suggest that complementary biological functions of PU.1 and Spi-B may be explained by their interaction with a similar set of regions in the genome of B cells. However, sites uniquely occupied by PU.1 or Spi-B provide insight into their unique functions
Regulation of B cell linker protein transcription by PU.1 and Spi-B in murine B cell acute lymphoblastic leukemia
B cell acute lymphoblastic leukemia (B-ALL) is frequently associated with mutations or chromosomal translocations of genes encoding transcription factors. Conditional deletion of genes encoding the E26-transformation-specific transcription factors, PU.1 and Spi-B, in B cells (ÎPB mice) leads to B-ALL in mice at 100% incidence rate and with a median survival of 21 wk. We hypothesized that PU.1 and Spi-B may redundantly activate transcription of genes encoding tumor suppressors in the B cell lineage. Characterization of aging ÎPB mice showed that leukemia cells expressing IL-7R were found in enlarged thymuses. IL-7R-expressing B-ALL cells grew in culture in response to IL-7 and could be maintained as cell lines. Cultured ÎPB cells expressed reduced levels of B cell linker protein (BLNK), a known tumor suppressor gene, compared with controls. The Blnk promoter contained a predicted PU.1 and/or Spi-B binding site that was required for promoter activity and occupied by PU.1 and/or Spi-B as determined by chromatin immunoprecipitation. Restoration of BLNK expression in cultured ÎPB cells opposed IL-7-dependent proliferation and induced early apoptosis. We conclude that the tumor suppressor BLNK is a target of transcriptional activation by PU.1 and Spi-B in the B cell lineage. Copyright © 2012 by The American Association of Immunologists, Inc
Biocatalytic self-assembly of tripeptide gels and emulsions
We report on the biocatalytic activation of a self-assembling (unprotected) tripeptide to stabilize oil-in-water emulsions on-demand. This is achieved by the conversion of a phosphorylated precursor into a hydrogelator using alkaline phosphatase (AP) as the trigger. The rate of conversion, controlled by the amount of enzyme used, is shown to play a key role in dictating the morphology of the nanofibrous networks produced. When these amphiphilic tripeptides are used in biphasic mixtures, nanofibers are shown to self-assemble not only at the aqueous/organic interface but also throughout the surrounding buffer, thereby stabilizing the oil-in-water droplet dispersions. The use of enzymatic activation of tripeptide emulsions gives rise to enhanced control of the emulsification process because emulsions can be stabilized on-demand by simply adding AP. In addition, control over the emulsion stabilization can be achieved by taking advantage of the kinetics of dephosphorylation and consequent formation of different stabilizing nanofibrous networks at the interface and/or in the aqueous environment. This approach can be attractive for various cosmetic, food, or biomedical applications because both tunability of the tripeptide emulsion stability and on-demand stabilization of emulsions can be achieved
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Exploratory Analysis of Mutations in Circulating Tumour DNA as Biomarkers of Treatment Response for Patients with Relapsed High-Grade Serous Ovarian Carcinoma: A Retrospective Study
Circulating tumour DNA (ctDNA) carrying tumour-specific sequence alterations may provide a minimally invasive means to dynamically assess tumour burden and response to treatment in cancer patients. Somatic mutations are a defining feature of high-grade serous ovarian carcinoma (HGSOC). We tested whether these mutations could be used as personalised markers to monitor tumour burden and early changes as a predictor of response and time to progression (TTP).
We performed a retrospective analysis of serial plasma samples collected during routine clinical visits from 40 patients with HGSOC undergoing heterogeneous standard of care treatment. Patient-specific assays were developed for 31 unique mutations identified in formalin-fixed paraffin-embedded tumour DNA from these patients. These assays were used to quantify ctDNA in 318 plasma samples using microfluidic digital PCR. The mutant allele fraction (TP53MAF) was compared to serum CA-125, the current gold-standard response marker for HGSOC in blood, as well as to disease volume on computed tomography scans by volumetric analysis. Changes after one cycle of treatment were compared with TTP. The median TP53MAF prior to treatment in 51 relapsed treatment courses was 8% (interquartile range [IQR] 1.2%-22%) compared to 0.7% (IQR 0.3%-2.0%) for seven untreated newly diagnosed stage IIIC/IV patients. TP53MAF correlated with volumetric measurements (Pearson = 0.59, 32 cm, ctDNA was detected at â„20 amplifiable copies per millilitre of plasma. In 49 treatment courses for relapsed disease, pre-treatment TP53MAF concentration, but not CA-125, was associated with TTP. Response to chemotherapy was seen earlier with ctDNA, with a median time to nadir of 37 d (IQR 28-54) compared with a median time to nadir of 84 d (IQR 42-116) for CA-125. In 32 relapsed treatment courses evaluable for response after one cycle of chemotherapy, a decrease in TP53MAF of >60% was an independent predictor of TTP in multivariable analysis (hazard ratio 0.22, 95% CI 0.07-0.67, = 0.008). Conversely, a decrease in TP53MAF of â€60% was associated with poor response and identified cases with TTP < 6 mo with 71% sensitivity (95% CI 42%-92%) and 88% specificity (95% CI 64%-99%). Specificity was improved when patients with recent drainage of ascites were excluded. Ascites drainage led to a reduction of TP53MAF concentration. The limitations of this study include retrospective design, small sample size, and heterogeneity of treatment within the cohort.
In this retrospective study, we demonstrated that ctDNA is correlated with volume of disease at the start of treatment in women with HGSOC and that a decrease of â€60% in TP53MAF after one cycle of chemotherapy was associated with shorter TTP. These results provide evidence that ctDNA has the potential to be a highly specific early molecular response marker in HGSOC and warrants further investigation in larger cohorts receiving uniform treatment.This work was supported by Cancer Research UK Grant numbers: A15601 (JDB), A11906 (NR), A20240 (NR), A18072 (JDB). JDB was supported by the National Institute for Health Research Cambridge Biomedical Research Centre. CAP was supported in part by the Academy of Medical Sciences, the Wellcome Trust, British Heart Foundation and Arthritis Research UK
Exoplanet phase curves: observations and theory
Phase curves are the best technique to probe the three dimensional structure
of exoplanets' atmospheres. In this chapter we first review current exoplanets
phase curve observations and the particular challenges they face. We then
describe the different physical mechanisms shaping the atmospheric phase curves
of highly irradiated tidally locked exoplanets. Finally, we discuss the
potential for future missions to further advance our understanding of these new
worlds.Comment: Fig.5 has been updated. Table 1 and corresponding figures have been
updated with new values for WASP-103b and WASP-18b. Contains a table
sumarizing phase curve observation
RADIAL VELOCITY MONITORING OFKEPLERHEARTBEAT STARS
Heartbeat stars (HB stars) are a class of eccentric binary stars with close periastron passages. The characteristic photometric HB signal evident in their light curves is produced by a combination of tidal distortion, heating, and Doppler boosting near orbital periastron. Many HB stars continue to oscillate after periastron and along the entire orbit, indicative of the tidal excitation of oscillation modes within one or both stars. These systems are among the most eccentric binaries known, and they constitute astrophysical laboratories for the study of tidal effects. We have undertaken a radial velocity (RV) monitoring campaign of Kepler HB stars in order to measure their orbits. We present our ïŹrst results here, including a sample of 22 Kepler HB systems, where for 19 of them we obtained the
Keplerian orbit and for 3 other systems we did not detect a statistically signiïŹcant RV variability. Results presented here are based on 218 spectra obtained with the Keck/HIRES spectrograph during the 2015 Kepler observing season, and they have allowed us to obtain the largest sample of HB stars with orbits measured using a single instrument, which roughly doubles the number of HB stars with an RV measured orbit. The 19 systems measured here have orbital periods from 7 to 90 days and eccentricities from 0.2 to 0.9. We show that HB stars draw the upper envelope of the eccentricityâperiod distribution. Therefore, HB stars likely represent a population of stars currently undergoing high eccentricity migration via tidal orbital circularization, and they will allow for new tests
of high eccentricity migration theories
Germline whole exome sequencing and large-scale replication identifies FANCM as a likely high grade serous ovarian cancer susceptibility gene
We analyzed whole exome sequencing data in germline DNA from 412 high grade serous ovarian cancer (HGSOC) cases from The Cancer Genome Atlas Project and identified 5,517 genes harboring a predicted deleterious germline coding mutation in at least one HGSOC case. Gene-set enrichment analysis showed enrichment for genes involved in DNA repair (p = 1.8x10(-3)). Twelve DNA repair genes - APEX1, APLF, ATX, EME1, FANCL, FANCM, MAD2L2, PARP2, PARP3, POLN, RAD54L and SMUG1 - were prioritized for targeted sequencing in up to 3,107 HGSOC cases, 1,491 cases of other epithelial ovarian cancer (EOC) subtypes and 3,368 unaffected controls of European origin. We estimated mutation prevalence for each gene and tested for associations with disease risk. Mutations were identified in both cases and controls in all genes except MAD2L2, where we found no evidence of mutations in controls. In FANCM we observed a higher mutation frequency in HGSOC cases compared to controls (29/3,107 cases, 0.96 percent; 13/3,368 controls, 0.38 percent; P = 0.008) with little evidence for association with other subtypes (6/1,491, 0.40 percent; P = 0.82). The relative risk of HGSOC associated with deleterious FANCM mutations was estimated to be 2.5 (95% CI 1.3 - 5.0; P = 0.006). In summary, whole exome sequencing of EOC cases with large-scale replication in case-control studies has identified FANCM as a likely novel susceptibility gene for HGSOC, with mutations associated with a moderate increase in risk. These data may have clinical implications for risk prediction and prevention approaches for high-grade serous ovarian cancer in the future and a significant impact on reducing disease mortality
Germline whole exome sequencing and large-scale replication identifies FANCM as a likely high grade serous ovarian cancer susceptibility gene.
We analyzed whole exome sequencing data in germline DNA from 412 high grade serous ovarian cancer (HGSOC) cases from The Cancer Genome Atlas Project and identified 5,517 genes harboring a predicted deleterious germline coding mutation in at least one HGSOC case. Gene-set enrichment analysis showed enrichment for genes involved in DNA repair (p = 1.8Ă10-3). Twelve DNA repair genes - APEX1, APLF, ATX, EME1, FANCL, FANCM, MAD2L2, PARP2, PARP3, POLN, RAD54L and SMUG1 - were prioritized for targeted sequencing in up to 3,107 HGSOC cases, 1,491 cases of other epithelial ovarian cancer (EOC) subtypes and 3,368 unaffected controls of European origin. We estimated mutation prevalence for each gene and tested for associations with disease risk. Mutations were identified in both cases and controls in all genes except MAD2L2, where we found no evidence of mutations in controls. In FANCM we observed a higher mutation frequency in HGSOC cases compared to controls (29/3,107 cases, 0.96 percent; 13/3,368 controls, 0.38 percent; P=0.008) with little evidence for association with other subtypes (6/1,491, 0.40 percent; P=0.82). The relative risk of HGSOC associated with deleterious FANCM mutations was estimated to be 2.5 (95% CI 1.3 - 5.0; P=0.006). In summary, whole exome sequencing of EOC cases with large-scale replication in case-control studies has identified FANCM as a likely novel susceptibility gene for HGSOC, with mutations associated with a moderate increase in risk. These data may have clinical implications for risk prediction and prevention approaches for high-grade serous ovarian cancer in the future and a significant impact on reducing disease mortality
Profiling the immune landscape in mucinous ovarian carcinoma
Objective: Mucinous ovarian carcinoma (MOC) is a rare histotype of ovarian cancer, with low response rates to standard chemotherapy, and very poor survival for patients diagnosed at advanced stage. There is a limited understanding of the MOC immune landscape, and consequently whether immune checkpoint inhibitors could be considered for a subset of patients. Methods: We performed multicolor immunohistochemistry (IHC) and immunofluorescence (IF) on tissue microarrays in a cohort of 126 MOC patients. Cell densities were calculated in the epithelial and stromal components for tumor-associated macrophages (CD68+/PD-L1+, CD68+/PD-L1-), T cells (CD3+/CD8-, CD3+/CD8+), putative T-regulatory cells (Tregs, FOXP3+), B cells (CD20+/CD79A+), plasma cells (CD20-/CD79a+), and PD-L1+ and PD-1+ cells, and compared these values with clinical factors. Univariate and multivariable Cox Proportional Hazards assessed overall survival. Unsupervised k-means clustering identified patient subsets with common patterns of immune cell infiltration. Results: Mean densities of PD1+ cells, PD-L1- macrophages, CD4+ and CD8+ T cells, and FOXP3+ Tregs were higher in the stroma compared to the epithelium. Tumors from advanced (Stage III/IV) MOC had greater epithelial infiltration of PD-L1- macrophages, and fewer PD-L1+ macrophages compared with Stage I/II cancers (p = 0.004 and p = 0.014 respectively). Patients with high epithelial density of FOXP3+ cells, CD8+/FOXP3+ cells, or PD-L1- macrophages, had poorer survival, and high epithelial CD79a + plasma cells conferred better survival, all upon univariate analysis only. Clustering showed that most MOC (86%) had an immune depleted (cold) phenotype, with only a small proportion (11/76,14%) considered immune inflamed (hot) based on T cell and PD-L1 infiltrates. Conclusion: In summary, MOCs are mostly immunogenically âcoldâ, suggesting they may have limited response to current immunotherapies
Population-based targeted sequencing of 54 candidate genes identifies PALB2 as a susceptibility gene for high-grade serous ovarian cancer
PURPOSE: The known epithelial ovarian cancer (EOC) susceptibility genes account for less than 50% of the heritable risk of ovarian cancer suggesting that other susceptibility genes exist. The aim of this study was to evaluate the contribution to ovarian cancer susceptibility of rare deleterious germline variants in a set of candidate genes. METHODS: We sequenced the coding region of 54 candidate genes in 6385 invasive EOC cases and 6115 controls of broad European ancestry. Genes with an increased frequency of putative deleterious variants in cases versus controls were further examined in an independent set of 14â135 EOC cases and 28â655 controls from the Ovarian Cancer Association Consortium and the UK Biobank. For each gene, we estimated the EOC risks and evaluated associations between germline variant status and clinical characteristics. RESULTS: The ORs associated for high-grade serous ovarian cancer were 3.01 for PALB2 (95%âCI 1.59 to 5.68; p=0.00068), 1.99 for POLK (95%âCI 1.15 to 3.43; p=0.014) and 4.07 for SLX4 (95%âCI 1.34 to 12.4; p=0.013). Deleterious mutations in FBXO10 were associated with a reduced risk of disease (OR 0.27, 95%âCI 0.07 to 1.00, p=0.049). However, based on the Bayes false discovery probability, only the association for PALB2 in high-grade serous ovarian cancer is likely to represent a true positive. CONCLUSIONS: We have found strong evidence that carriers of PALB2 deleterious mutations are at increased risk of high-grade serous ovarian cancer. Whether the magnitude of risk is sufficiently high to warrant the inclusion of PALB2 in cancer gene panels for ovarian cancer risk testing is unclear; much larger sample sizes will be needed to provide sufficiently precise estimates for clinical counselling
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