Identification of Importin 8 (IPO8) as the most accurate reference gene for the clinicopathological analysis of lung specimens

<p>Abstract</p> <p>Background</p> <p>The accurate normalization of differentially expressed genes in lung cancer is essential for the identification of novel therapeutic targets and biomarkers by real time RT-PCR and microarrays. Although classical "housekeeping" genes, such as GAPDH, HPRT1, and beta-actin have been widely used in the past, their accuracy as reference genes for lung tissues has not been proven.</p> <p>Results</p> <p>We have conducted a thorough analysis of a panel of 16 candidate reference genes for lung specimens and lung cell lines. Gene expression was measured by quantitative real time RT-PCR and expression stability was analyzed with the softwares <it>GeNorm </it>and <it>NormFinder</it>, mean of |ΔCt| (= |Ct Normal-Ct tumor|) ± SEM, and correlation coefficients among genes. Systematic comparison between candidates led us to the identification of a subset of suitable reference genes for clinical samples: IPO8, ACTB, POLR2A, 18S, and PPIA. Further analysis showed that IPO8 had a very low mean of |ΔCt| (0.70 ± 0.09), with no statistically significant differences between normal and malignant samples and with excellent expression stability.</p> <p>Conclusion</p> <p>Our data show that IPO8 is the most accurate reference gene for clinical lung specimens. In addition, we demonstrate that the commonly used genes GAPDH and HPRT1 are inappropriate to normalize data derived from lung biopsies, although they are suitable as reference genes for lung cell lines. We thus propose IPO8 as a novel reference gene for lung cancer samples.</p

Demonstrating Immersive Media Delivery on 5G Broadcast and Multicast Testing Networks

This work presents eight demonstrators and one showcase developed within the 5G-Xcast project. They experimentally demonstrate and validate key technical enablers for the future of media delivery, associated with multicast and broadcast communication capabilities in 5th Generation (5G). In 5G-Xcast, three existing testbeds: IRT in Munich (Germany), 5GIC in Surrey (UK), and TUAS in Turku (Finland), have been developed into 5G broadcast and multicast testing networks, which enables us to demonstrate our vision of a converged 5G infrastructure with fixed and mobile accesses and terrestrial broadcast, delivering immersive audio-visual media content. Built upon the improved testing networks, the demonstrators and showcase developed in 5G-Xcast show the impact of the technology developed in the project. Our demonstrations predominantly cover use cases belonging to two verticals: Media & Entertainment and Public Warning, which are future 5G scenarios relevant to multicast and broadcast delivery. In this paper, we present the development of these demonstrators, the showcase, and the testbeds. We also provide key findings from the experiments and demonstrations, which not only validate the technical solutions developed in the project, but also illustrate the potential technical impact of these solutions for broadcasters, content providers, operators, and other industries interested in the future immersive media delivery.Comment: 16 pages, 22 figures, IEEE Trans. Broadcastin

Abstract PO-017 : radiotherapy in combination with the brain penetrant ATM inhibitor AZD1390 does not exacerbate radiation toxicity of neural stem cells in vitro or in vivo

While radiotherapy (RT) is fundamental for the treatment of brain tumors, irradiation of the brain frequently causes devastating effects on cognitive function and quality of life. DNA damage within neural stem cells (NSC) is a key factor in the pathogenesis of radiation-induced cognitive dysfunction. The ataxia telangiectasia mutated (ATM) kinase is a central protein in the DNA damage response and a critical determinant of tumor cell survival after radiation. ATM inhibition potently radiosensitizes preclinical models of GBM in vitro and in vivo. A novel, brain penetrant ATM inhibitor AZD1390, which is predicted to achieve brain tumor concentrations in the range of 1-5nM, is currently in early phase clinical evaluation in combination with RT. In marked contrast to observations in tumor models, genetic knockdown of ATM has radioprotective effects on NSC in vitro; the proposed mechanism is via suppression of p53 mediated apoptosis. The purpose of this study was to investigate the impact of AZD1390 on survival responses and mode of death in NSCs exposed to RT in vitro and in vivo. NSCs were derived from the telencephalon of E13 mouse embryos. Cells were treated with AZD1390 (0.1-10nM) 1 hour prior to ionizing radiation (IR; 0-5 Gy). Mode and timing of cell death was interrogated using IncuCyte live cell analysis to measure proliferation, cytotoxicity and apoptosis up to 72 hours post-IR. Cell viability and neurosphere formation assays were also used to measure radiation sensitivity in vitro. C57BL/6 mice received 20Gy hemibrain irradiation +/- 7-day treatment with AZD1390 (10mg/kg). Immunohistochemistry for Ki67 and Sox2 was used to assess effects on NSC in the subventricular zone (SVZ) 50 days post-irradiation. In vitro AZD1390 (1-10nM) inhibited ATM kinase function within 1 hour, evidenced by abrogation of KAP1 and p53 phosphorylation. NSCs primarily undergo apoptosis in response to IR. AZD1390 at 1 and 3nM significantly reduced apoptosis in irradiated NSCs (ratios of annexin V area under the curve 1.95 and 2 respectively); 10 nM had no effect on this parameter. Proliferation rates and cell viability after radiation were preserved at all drug concentrations. AZD1390 at 1nM did not modulate radiation effects on neurosphere formation whereas at 10nM a radiosensitizing effect was observed (ratio of SF[3Gy]=0.25). In vivo, IR decreased the number of Ki67 positive proliferating cells (92% reduction) and Sox2-positive cells (24% reduction) in the SVZ after 50 days; these effects were not exacerbated by addition of AZD1390. Acute effects (24 hours post-IR) are under investigation. We demonstrate in vitro that AZD1390 has radioprotective effects on NSCs at clinically achievable concentrations. In vivo, treatment with AZD1390 did not enhance the effects of radiation on NSCs in the SVZ. In the context of its profound radiosensitizing effects on GBM models, the absence of radiosensitization of NSCs both in vitro and in vivo strengthens the rationale for evaluating AZD1390 in combination with RT in GBM patients. Citation Format: Rodrigo Guttierez-Quintana, David J. Walker, Mark R. Jackson, Natividad Gomez-Roman, Sandeep Chahal, Stephen T. Durant, Anthony J. Chalmers. Radiotherapy in combination with the brain penetrant ATM inhibitor AZD1390 does not exacerbate radiation toxicity of neural stem cells in vitro or in vivo

Development of a novel splice array platform and its application in the identification of alternative splice variants in lung cancer

<p>Abstract</p> <p>Background</p> <p>Microarrays strategies, which allow for the characterization of thousands of alternative splice forms in a single test, can be applied to identify differential alternative splicing events. In this study, a novel splice array approach was developed, including the design of a high-density oligonucleotide array, a labeling procedure, and an algorithm to identify splice events.</p> <p>Results</p> <p>The array consisted of exon probes and thermodynamically balanced junction probes. Suboptimal probes were tagged and considered in the final analysis. An unbiased labeling protocol was developed using random primers. The algorithm used to distinguish changes in expression from changes in splicing was calibrated using internal non-spliced control sequences. The performance of this splice array was validated with artificial constructs for <it>CDC6</it>, <it>VEGF</it>, and <it>PCBP4 </it>isoforms. The platform was then applied to the analysis of differential splice forms in lung cancer samples compared to matched normal lung tissue. Overexpression of splice isoforms was identified for genes encoding <it>CEACAM1</it>, <it>FHL-1</it>, <it>MLPH</it>, and <it>SUSD2. </it>None of these splicing isoforms had been previously associated with lung cancer.</p> <p>Conclusions</p> <p>This methodology enables the detection of alternative splicing events in complex biological samples, providing a powerful tool to identify novel diagnostic and prognostic biomarkers for cancer and other pathologies.</p

Combined inhibition of the Fanconi anaemia (FA) pathway and ATR promotes R-loop generation and profound radiosensitisation in glioblastoma

Glioblastoma is a deadly cancer in which treatment resistance is mediated through extensive intratumoural heterogeneity including difficult-to-treat glioblastoma stem cell (GSC) subpopulations. GSC eradication represents an attractive therapeutic goal, but these cells possess upregulated DNA damage response (DDR) processes, resulting in a chemo- and radioresistant phenotype. However, recent studies have demonstrated that elevated replication stress in GSCs may partially explain DDR upregulation and resistance, thus highlighting a potential therapeutically exploitable vulnerability. ATR and the FA-pathway are both fundamental to cellular DNA replication stress responses and maintaining replication fork stability. Since we have previously shown the FA-pathway is inactive in normal brain, but is re-activated in glioblastoma with potential to provide a cancer-specific foundation for combination DDR therapies, we explored the therapeutic potential of simultaneous inhibition of the FA-pathway (FAPi) and ATR (ATRi), in addition to other FA-pathway-based DDR inhibitor (DDRi) combinations. We find that compared with single agent treatments, combined inhibition of the FA-pathway and ATR in both 2D and 3D GSC ex vivo models promotes a substantial increase in conflicts between DNA replication and transcription (R-loops) which is further exacerbated by ionising radiation (IR). Molecular analyses of DNA damage indicate that FAPi+ATRi increases peak DNA damage post-IR treatments, with sustained elevation of DNA damage even at 24 hours post-treatment. In conclusion, simultaneously targeting the FA-pathway and ATR represents an appealing therapeutic strategy for glioblastoma. This approach promotes substantial R-loop generation, likely through exacerbating constitutively high levels of DNA replication stress previously observed in GSCs, with deleterious effects in these treatment resistant cells. Our findings underline the value of developing clinical FA pathway inhibitors and also support the application of current ATR inhibitors to molecularly-selected subsets of glioblastoma, namely, those with defects in one of 22 currently known FA-pathway genes which include BRCA1/FANCS and BRCA2/FANCD1

Solution of an industrially relevant coupled magneto–mechanical problem set on an axisymmetric domain

Eddy currents are generated when low frequency magnetic fields interact with conducting components and this, in turn, generates Lorentz forces, which can cause these metallic components to deform and vibrate. An important application of this magneto–mechanical coupling is in coil design for magnetic resonance imaging scanners, where such vibrations can have unwanted effects such as ghosting of images, reduction of the life span of devices and discomfort for the patient. This work is aimed at developing an accurate computational tool for better understanding these deformations by considering a benchmark problem proposed by Siemens plc (Kruip, personal communication, 2013) on an idealised axisymmetric geometry. We present a new fixed point algorithm and develop new weak variational statements, which use a stress tensor approach for force calculation and permit discretisation using H1 conforming hp-version finite elements. Numerical results are included, which show the importance of high order finite elements for predicting the eddy currents and the associated coupling in the resonance region

Antibody-Based HIV-1 Vaccines: Recent Developments and Future Directions: A summary report from a Global HIV Vaccine Enterprise Working Group

The authors discuss humoral immune responses to HIV and approaches to designing vaccines that induce viral neutralizing and other potentially protective antibodies

Identifying synergistic regulation involving c-Myc and sp1 in human tissues

Combinatorial gene regulation largely contributes to phenotypic versatility in higher eukaryotes. Genome-wide chromatin immuno-precipitation (ChIP) combined with expression profiling can dissect regulatory circuits around transcriptional regulators. Here, we integrate tiling array measurements of DNA-binding sites for c-Myc, sp1, TFIID and modified histones with a tissue expression atlas to establish the functional correspondence between physical binding, promoter activity and transcriptional regulation. For this we develop SLM, a methodology to map c-Myc and sp1-binding sites and then classify sites as sp1-only, c-Myc-only or dual. Dual sites show several distinct features compared to the single regulator sites: specifically, they exhibit overall higher degree of conservation between human and rodents, stronger correlation with TFIID-bound promoters, and preference for permissive chromatin state. By applying regression models to an expression atlas we identified a functionally distinct signature for strong dual c-Myc/sp1 sites. Namely, the correlation with c-Myc expression in promoters harboring dual-sites is increased for stronger sp1 sites by strong sp1 binding and the effect is largest in proliferating tissues. Our approach shows how integrated functional analyses can uncover tissue-specific and combinatorial regulatory dependencies in mammals

Towards the high-accuracy determination of the 238U fission cross section at the threshold region at CERN - N-TOF

The 238U fission cross section is an international standard beyond 2 MeV where the fission plateau starts. However, due to its importance in fission reactors, this cross-section should be very accurately known also in the threshold region below 2 MeV. The 238U fission cross section has been measured relative to the 235U fission cross section at CERN - n-TOF with different detection systems. These datasets have been collected and suitably combined to increase the counting statistics in the threshold region from about 300 keV up to 3 MeV. The results are compared with other experimental data, evaluated libraries, and the IAEA standards

Surgical management of pulmonary inflammatory pseudotumors: A single center experience

<p>Abstract</p> <p>Background</p> <p>The pulmonary inflammatory pseudotumor (PIP) is a rare disease. It is still debated whether it represents an inflammatory lesion characterized by uncontrolled cell growth or a true neoplasm. PIP is characterized by a cellular polymorphism.</p> <p>Methods</p> <p>We retrospectively analyzed 8 patients with PIP treated by surgery between 2001 and 2009. Preoperative thoracic computed tomography (CT) scan was performed in all cases. All patients underwent preoperative bronchoscopy with washing and brushing and/or transbronchial biopsy and preoperative cytology examination</p> <p>Results</p> <p>There were 5 men and 3 women, aged between 38 and 69 years (mean of 58 years). 3 patients (37%) were asymptomatic. The others had symptoms characterized by chest pain, shortness of breath and persistent cough or hemoptysis. 5 patients had neutrophilic leucocytosis. CT scan demonstrated solitary nodules (maximum diameter <3 cm) in 5 patients (62%) and lung masses (maximum diameter >3 cm) in 3 patients (37%). In 2 patients there were signs of pleural infiltration. Distant lesions were excluded in all cases. A preoperative histology examination failed to reach a definitive diagnosis in all patients. At surgery, we performed two lobectomies, one segmentectomy and five wedge resections, these being performed with videothoracoscopy (VATS), except for one patient where open surgery was used. Complete tumor resection was obtained in all patients. According to the Matsubara classification, there were 2 cases of organizing pneumonia, 5 cases of fibrous histiocytoma and one case of lymphoplasmacytoma. All patients were discharged alive from hospital between 4 and 7 days after surgery. At follow-up CT scan performed annually (range 11 to 112 months) (mean 58 months), there were no residual lesions, neither local nor distant recurrences.</p> <p>Conclusions</p> <p>PIP is a rare disease. Many synonyms have been used for this disease, usually in relation to the most represented cell type. The true incidence is unclear. Preoperative diagnosis is difficult to reach, despite performing a bronchoscopy or a transparietal needle aspiration. Different classifications have been proposed for PIP. Either medical, radiation or surgical therapy has been used for PIP. Whenever possible, surgery should be considered the standard treatment. Complete surgical resection is advocated to prevent recurrence.</p