Small interfering RNA targeting HIF-1α reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro

Background: : Hypoxia inducible factor-1 has been identified as a potential target to overcome hypoxia-induced radioresistance The aim of the present study was to investigate whether selective HIF-1 inhibition via small interfering RNA (siRNA) targeting hypoxia-inducible factor 1α (HIF-1α) affects hypoxia-induced radioresistance in HT 1080 human fibrosarcoma cells. Material and Methods: : HIF-1α expression in HT 1080 human fibrosarcoma cells in vitro was silenced using HIF-1α siRNA sequence primers. Quantitative real-time polymerase chain reaction assay was performed to quantify the mRNA expression of HIF-1α. HIF-1α protein levels were studied by Western blotting at 20% (air) or after 12 hours at 0.1% O2 (hypoxia). Cells were assayed for clonogenic survival after irradiation with 2, 5, or 10 Gy, under normoxic or hypoxic conditions in the presence of HIF-1α-targeted or control siRNA sequences. A modified oxygen enhancement ratio (OER´) was calculated as the ratio of the doses to achieve the same survival at 0.1% O2 as at ambient oxygen tensions. OER´ was obtained at cell survival levels of 50%, 37%, and 10%. Results: : HIF-1α-targeted siRNA enhanced radiation treatment efficacy under severely hypoxic conditions compared to tumor cells treated with scrambled control siRNA. OER was reduced on all survival levels after treatment with HIF-1α-targeted siRNA, suggesting that inhibition of HIF-1 activation by using HIF-1α-targeted siRNA increases radiosensitivity of hypoxic tumor cells in vitro. Conclusion: : Inhibition of HIF-1 activation by using HIF-1α-targeted siRNA clearly acts synergistically with radiotherapy and increase radiosensitivity of hypoxic cells in vitr

Inhibition of N-Myc down regulated gene 1 in in vitro cultured human glioblastoma cells

AIM: To study short dsRNA oligonucleotides (siRNA) as a potent tool for artificially modulating gene expression of N-Myc down regulated gene 1 (NDRG1) gene induced under different physiological conditions (Normoxia and hypoxia) modulating NDRG1 transcription, mRNA stability and translation. METHODS: A cell line established from a patient with glioblastoma multiforme. Plasmid DNA for transfections was prepared with the Endofree Plasmid Maxi kit. From plates containing 5 x 10(7) cells, nuclear extracts were prepared according to previous protocols. The pSUPER-NDRG1 vectors were designed, two sequences were selected from the human NDRG1 cDNA (5'-GCATTATTGGCATGGGAAC-3' and 5'-ATGCAGAGTAACGTGGAAG-3'. reverse transcription polymerase chain reaction was performed using primers designed using published information on -actin and hypoxia-inducible factor (HIF)-1 mRNA sequences in GenBank. NDRG1 mRNA and protein level expression results under different conditions of hypoxia or reoxygenation were compared to aerobic control conditions using the Mann-Whitney U test. Reoxygenation values were also compared to the NDRG1 levels after 24 h of hypoxia (P < 0.05 was considered significant). RESULTS: siRNA- and iodoacetate (IAA)-mediated downregulation of NDRG1 mRNA and protein expression in vitro in human glioblastoma cell lines showed a nearly complete inhibition of NDRG1 expression when compared to the results obtained due to the inhibitory role of glycolysis inhibitor IAA. Hypoxia responsive elements bound by nuclear HIF-1 in human glioblastoma cells in vitro under different oxygenation conditions and the clearly enhanced binding of nuclear extracts from glioblastoma cell samples exposed to extreme hypoxic conditions confirmed the HIF-1 Western blotting results. CONCLUSION: NDRG1 represents an additional diagnostic marker for brain tumor detection, due to the role of hypoxia in regulating this gene, and it can represent a potential target for tumor treatment in human glioblastoma. The siRNA method can represent an elegant alternative to modulate the expression of the hypoxia induced NDRG1 gene and can help to monitor the development of the cancer disease treatment outcome through monitoring the expression of this gene in the patients undergoing the different therapeutic treatment alternatives available nowadays

Effects of HIF-1 inhibition by chetomin on hypoxia-related transcription and radiosensitivity in HT 1080 human fibrosarcoma cells

<p>Abstract</p> <p>Background</p> <p>Hypoxia-inducible factor-1 (HIF-1) overexpression has been linked to tumor progression and poor prognosis. We investigated whether targeting of HIF-1 using chetomin, a disrupter of the interaction of HIF-1 with the transcriptional coactivator p300, influences the radiosensitivity of hypoxic HT 1080 human fibrosarcoma cells.</p> <p>Methods</p> <p>Optimal dose of chetomin was determined by EGFP-HRE gene reporter assay in stably transfected HT 1080 cells. Cells were assayed for expression of the hypoxia-inducible genes carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) by RT-PCR and for clonogenic survival after irradiation with 2, 5 or 10 Gy, under normoxic or hypoxic (0.1% O₂, 12 h) conditions in the presence or absence of chetomin (150 nM, 12 h, pre-treatment of 4 h).</p> <p>Results</p> <p>Chetomin treatment significantly reduced CA9 and VEGF mRNA expression in hypoxic cells to 44.4 ± 7.2% and 39.6 ± 16.0%, respectively, of untreated hypoxic controls. Chetomin clearly reduced the modified oxygen enhancement ratio (OER') compared to untreated cells, from 2.02 to 1.27, from 1.86 to 1.22 and from 1.49 to 1.06 at the 50%, 37% and 10% clonogenic survival levels, respectively.</p> <p>Conclusion</p> <p>HIF-1 inhibition by chetomin effectively reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells <it>in vitro</it>.</p

The classification of Information and Communication Technology Investment in Financial Accounting

Financial accounting is well known in its responsibility for book keeping the organisational expenditure and the preparation of the financial statements. ICT investment has become important to investors and not reporting these investments on financial statement leads to misevaluation of the organisation market value. Moreover, the misclassification of ICT investment has been indicated, yet not investigated in the past researches. The unreported ICT investment and the misclassification of ICT investment could affect the measurement of ICT investment at firm level. By analysing the content of the financial statement for 86 firms listing in Australian Stock Exchange, this study explains how ICT investments were being classified with the other investment in financial reports from 2006 to 2010. Differentiating between ICT asset and expense is an initial step into the understanding about the classification of ICT investment in financial accounting. The accounting standards requires the capitalisation conditions including future economic benefit, controllability, identifiability, existence, and reliability measurement to be justified for the expenditure before it can be capitalised as asset. The study use fuzzy set qualitative and comparative analysis (fsQCA) to analyse the information collected from the experts in the accounting fields. Base on fsQCA analysis, the study is able to shows that the factors considered by the organisation to differentiate ICT asset from ICT expense is beyond the requirement in definition of asset stated in the International Accounting Standards and the Australian Accounting Standards

A candidate super-Earth planet orbiting near the snow line of Barnard’s star

Barnard’s star is a red dwarf, and has the largest proper motion (apparent motion across the sky) of all known stars. At a distance of 1.8 parsecs, it is the closest single star to the Sun; only the three stars in the α Centauri system are closer. Barnard’s star is also among the least magnetically active red dwarfs known and has an estimated age older than the Solar System. Its properties make it a prime target for planetary searches; various techniques with different sensitivity limits have been used previously, including radial-velocity imaging, astrometry and direct imaging, but all ultimately led to negative or null results. Here we combine numerous measurements from high-precision radial-velocity instruments, revealing the presence of a low-amplitude periodic signal with a period of 233 days. Independent photometric and spectroscopic monitoring, as well as an analysis of instrumental systematic effects, suggest that this signal is best explained as arising from a planetary companion. The candidate planet around Barnard’s star is a cold super-Earth, with a minimum mass of 3.2 times that of Earth, orbiting near its snow line (the minimum distance from the star at which volatile compounds could condense). The combination of all radial-velocity datasets spanning 20 years of measurements additionally reveals a long-term modulation that could arise from a stellar magnetic-activity cycle or from a more distant planetary object. Because of its proximity to the Sun, the candidate planet has a maximum angular separation of 220 milliarcseconds from Barnard’s star, making it an excellent target for direct imaging and astrometric observations in the future. © 2018, Springer Nature Limited.The results are based on observations made with the CARMENES instrument at the 3.5-m telescope of the Centro Astronomico Hispano-Aleman de Calar Alto (CAHA, Almeria, Spain), funded by the German Max-Planck-Gesellschaft (MPG), the Spanish Consejo Superior de Investigaciones Cientificas (CSIC), the European Union and the CARMENES Consortium members; the 90-cm telescope at the Sierra Nevada Observatory (Granada, Spain) and the 40-cm robotic telescope at the SPACEOBS observatory (San Pedro de Atacama, Chile), both operated by the Instituto de Astrofisica de Andalucia (IAA); and the 80-cm Joan Oro Telescope (TJO) of the Montsec Astronomical Observatory (OAdM), owned by the Generalitat de Catalunya and operated by the Institute of Space Studies of Catalonia (IEEC). This research was supported by the following institutions, grants and fellowships: Spanish MINECO ESP2016-80435-C2-1-R, ESP2016-80435-C2-2-R, AYA2016-79425-C3-1-P, AYA2016-79245-C3-2-P, AYA2016-79425-C3-3-P, AYA2015-69350-C3-2-P, ESP2014-54362-P, AYA2014-56359-P, RYC-2013-14875; Generalitat de Catalunya/CERCA programme; Fondo Europeo de Desarrollo Regional (FEDER); German Science Foundation (DFG) Research Unit FOR2544, project JE 701/3-1; STFC Consolidated Grants ST/P000584/1, ST/P000592/1, ST/M001008/1; NSF AST-0307493; Queen Mary University of London Scholarship; Perren foundation grant; CONICYT-FONDECYT 1161218, 3180405; Swiss National Science Foundation (SNSF); Koshland Foundation and McDonald-Leapman grant; and NASA Hubble Fellowship grant HST-HF2-51399.001. J.T. is a Hubble Fellow

Beetroot supplementation improves the physiological responses to incline walking

The final publication is available at Springer via http://dx.doi.org/10.1007/s00421-018-3843-xPurpose: We investigated the effects of an acute 24-h nitrate-rich beetroot juice supplement (BR) on the energy cost, exercise efficiency and blood pressure responses to intermittent walking at different gradients. Methods: In a double-blind, cross-over design, eight participants were provided with a total of 350 ml of nitrate-rich (~20.5 mmol nitrate) BR or placebo (PLA) across 24-h before completing intermittent walking at 3 km/h on treadmill at gradients of 1%, 5%, 10%, 15% and 20%. Results: Resting mean arterial pressure (MAP) was ~4.1% lower after BR (93 vs. 89 mmHg; P = 0.001), as well as during exercise (102 vs. 99 mmHg; P = 0.011) and recovery (97 vs. 94 mmHg; P = 0.001). Exercising (1227 vs. 1129 ml/min P < 0.001) and end-stage (1404 vs. 1249 ml/min; P = 0.002) oxygen uptake (O2) was lower in BR compared to PLA, which was accompanied by an average reduction in phase II ̇O2 amplitude (1067 vs. 940 ml/min; P = 0.025). Similarly, recovery O2 (509 vs. 458 ml/min; P = 0.001) was lower in BR. Whole-blood potassium concentration increased from pre-post exercise in PLA (4.1 ± 0.3 vs. 4.5 ± 0.3 mmol/L; P = 0.013) but not BR (4.1 ± 0.31 vs. 4.3 ± 0.2 mmol/L; P = 0.188). Conclusions: Energy cost of exercise, recovery of O2, MAP and blood markers were ameliorated after BR. Previously reported mechanisms explain these findings, which are more noticeable during less efficient walking at steep gradients (15-20%). These findings have practical implications for hill-walkers

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival