Using multiplexed regulation of luciferase activity and GFP translocation to screen for FOXO modulators

<p>Abstract</p> <p>Background</p> <p>Independent luciferase reporter assays and fluorescent translocation assays have been successfully used in drug discovery for several molecular targets. We developed U2transLUC, an assay system in which luciferase and fluorescent read-outs can be multiplexed to provide a powerful cell-based high content screening method.</p> <p>Results</p> <p>The U2transLUC system is based on a stable cell line expressing a GFP-tagged FOXO transcription factor and a luciferase reporter gene under the control of human FOXO-responsive enhancers. The U2transLUC assay measures nuclear-cytoplasmic FOXO shuttling and FOXO-driven transcription, providing a means to analyze these two key features of FOXO regulation in the same experiment. We challenged the U2transLUC system with chemical probes with known biological activities and we were able to identify compounds with translocation and/or transactivation capacity.</p> <p>Conclusion</p> <p>Combining different biological read-outs in a single cell line offers significant advantages over conventional cell-based assays. The U2transLUC assay facilitates the maintenance and monitoring of homogeneous FOXO transcription factor expression and allows the reporter gene activity measured to be normalized with respect to cell viability. U2transLUC is suitable for high throughput screening and can identify small molecules that interfere with FOXO signaling at different levels.</p

Potential of Cu–saponite catalysts for soot combustion

H– and Na–saponite supports have been prepared by several synthesis approaches. 5% Cu/saponite catalysts have been prepared and tested for soot combustion in a NOx + O2 + N2 gas flow and with soot and catalyst mixed in loose contact mode. XRD, FT-IR, N2 adsorption and TEM characterization results revealed that the use of either surfactant or microwaves during the synthesis led to delamination of the saponite support, yielding high surface area and small crystallite size materials. The degree of delamination affected further copper oxide dispersion and soot combustion capacity of the Cu/saponite catalysts. All Cu/saponite catalysts were active for soot combustion, and the NO2-assisted mechanism seemed to prevail. The best activity was achieved with copper oxide supported on a Na–saponite prepared at pH 13 and with surfactant. This best activity was attributed to the efficient copper oxide dispersion on the high surface area delaminated saponite (603 m2 g−1) and to the presence of Na. Copper oxide reduction in H2-TPR experiments occurred at lower temperature for the Na-containing catalysts than for the H-containing counterparts, and all Cu/Na–saponite catalysts were more active for soot combustion than the corresponding Cu/H–saponite catalysts.The financial support of Generalitat Valenciana (Project PROMETEOII/2014/010), Catalan Government (2014SGR1146), Spanish Ministry of Economy and Competitiveness (Projects CTQ2012-30703 and CTQ2011-24610) and UE (FEDER funding) is acknowledged

Efficacy of bortezomib in sarcomas with high levels of MAP17 (PDZK1IP1

Sarcomas are malignant tumors accounting for a high percentage of cancer morbidity and mortality in children and young adults. Surgery and radiation therapy are the accepted treatments for most sarcomas; however, patients with metastatic disease are treated with systemic chemotherapy. Many tumors display marginal levels of chemoresponsiveness, and new treatment approaches are needed. MAP17 is a small non-glycosylated membrane protein overexpressed in carcinomas. The levels of MAP17 could be used as a prognostic marker to predict the response to bortezomib in hematological malignancies and in breast tumors. Therefore, we analyzed the expression of this oncogene in sarcomas and its relationship with clinico-pathological features, as well as tested whether it can be used as a new biomarker to predict the therapeutic response to bortezomib and new therapies for sarcomas. We found that the levels of MAP17 were related to clinical features and poor survival in a cohort of 69 patients with different sarcoma types, not being restricted to any special subtype of tumor. MAP17 expression is associated with poor overall survival (p<0.001) and worse disease-free survival (p=0.002). Cell lines with high levels of MAP17 show a better response to bortezomib in vitro. Furthermore, patient-derived xenografts (PDX) with high levels of MAP17 respond to bortezomib in vivo. Our results showed that this response is due to the lower levels of NFκB and autophagy activation. Therefore, we suggest that MAP17 is a new biomarker to predict the efficacy of bortezomib as a new therapy for sarcomas.España, Consejeria de Ciencia e innovación CTS-6844 and CTS-1848Andalucia, Consejería de Salud PI-0029-2013Andalucia, Consejería de Salud PI-0096-2014Andalucia, Consejería de Salud PI-0306-201

Effectiveness and costs of phototest in dementia and cognitive impairment screening

<p>Abstract</p> <p>Background</p> <p>To assess and compare the effectiveness and costs of Phototest, Mini Mental State Examination (MMSE), and Memory Impairment Screen (MIS) to screen for dementia (DEM) and cognitive impairment (CI).</p> <p>Methods</p> <p>A phase III study was conducted over one year in consecutive patients with suspicion of CI or DEM at four Primary Care (PC) centers. After undergoing all screening tests at the PC center, participants were extensively evaluated by researchers blinded to screening test results in a Cognitive-Behavioral Neurology Unit (CBNU). The gold standard diagnosis was established by consensus of expert neurologists. Effectiveness was assessed by the proportion of correct diagnoses (diagnostic accuracy [DA]) and by the kappa index of concordance between test results and gold standard diagnoses. Costs were based on public prices and hospital accounts.</p> <p>Results</p> <p>The study included 140 subjects (48 with DEM, 37 with CI without DEM, and 55 without CI). The MIS could not be applied to 23 illiterate subjects (16.4%). For DEM, the maximum effectiveness of the MMSE was obtained with different cutoff points as a function of educational level [k = 0.31 (95% Confidence interval [95%CI], 0.19-0.43), DA = 0.60 (95%CI, 0.52-0.68)], and that of the MIS with a cutoff of 3/4 [k = 0.63 (95%CI, 0.48-0.78), DA = 0.83 (95%CI, 0.80-0.92)]. Effectiveness of the Phototest [k = 0.71 (95%CI, 0.59-0.83), DA = 0.87 (95%CI, 0.80-0.92)] was similar to that of the MIS and higher than that of the MMSE. Costs were higher with MMSE (275.9 ± 193.3€ [mean ± sd euros]) than with Phototest (208.2 ± 196.8€) or MIS (201.3 ± 193.4€), whose costs did not significantly differ. For CI, the effectiveness did not significantly differ between MIS [k = 0.59 (95%CI, 0.45-0.74), DA = 0.79 (95%CI, 0.64-0.97)] and Phototest [k = 0.58 (95%CI, 0.45-0.74), DA = 0.78 (95%CI, 0.64-0.95)] and was lowest for the MMSE [k = 0.27 (95%CI, 0.09-0.45), DA = 0.69 (95%CI, 0.56-0.84)]. Costs were higher for MMSE (393.4 ± 121.8€) than for Phototest (287.0 ± 197.4€) or MIS (300.1 ± 165.6€), whose costs did not significantly differ.</p> <p>Conclusion</p> <p>MMSE is not an effective instrument in our setting. For both DEM and CI, the Phototest and MIS are more effective and less costly, with no difference between them. However, MIS could not be applied to the appreciable percentage of our population who were illiterate.</p

Quality of life of patients with metastatic pancreatic adenocarcinoma initiating first-line chemotherapy in routine practice

Background: Despite advances in surgery, radiotherapy, and chemotherapy, pancreatic adenocarcinoma often progresses rapidly and causes death. The physical decline of these patients is expected to impact their quality of life (QoL). Therefore, in addition to objective measures of effectiveness, the evaluation of health-related QoL should be considered a matter of major concern when assessing therapy outcomes. Methods: Observational, prospective, multicenter study including patients with metastatic pancreatic adenocarcinoma who started first-line chemotherapy in 12 Spanish centers. Treatment and clinical characteristics were recorded at baseline. Patients' health-related quality of life, ECOG, and Karnofsky index were measured at baseline, at Days 15 and 30, and every four weeks up to 6months of chemotherapy. Health-related quality of life was measured using the EORTC-QLQ-C30 and EQ-5D questionnaires. Other endpoints included overall survival and progression-free survival. Results: The study sample included 116 patients (median age of 65years). Mean (SD) scores for the QLQ-C30 global health status scale showed a significant increasing trend throughout the treatment (p=0.005). Patients with either a Karnofsky index of 70-80 or ECOG 2 showed greater improvement in the QLQ-C30 global health status score than the corresponding groups with better performance status (p 50 at baseline had significantly greater overall survival and progression-free survival (p=0.005 and p=0.021, respectively). No significant associations were observed regarding the EQ-5D score. Conclusions: Most metastatic pancreatic adenocarcinoma patients receiving first-line chemotherapy showed an increase in health-related quality of life scores throughout the treatment. Patients with lower performance status and health-related quality of life at baseline tended to greater improvement. The EORTC QLQ-C30 scale allowed us to measure the health-related quality of life of metastatic pancreatic adenocarcinoma patients receiving first-line chemotherapy

A Dual-Color Fluorescence-Based Platform to Identify Selective Inhibitors of Akt Signaling

Background: Inhibition of Akt signaling is considered one of the most promising therapeutic strategies for many cancers. However, rational target-orientated approaches to cell based drug screens for anti-cancer agents have historically been compromised by the notorious absence of suitable control cells. Methodology/Principal Findings: In order to address this fundamental problem, we have developed BaFiso, a live-cell screening platform to identify specific inhibitors of this pathway. BaFiso relies on the co-culture of isogenic cell lines that have been engineered to sustain interleukin-3 independent survival of the parental Ba/F3 cells, and that are individually tagged with different fluorescent proteins. Whilst in the first of these two lines cell survival in the absence of IL-3 is dependent on the expression of activated Akt, the cells expressing constitutively-activated Stat5 signaling display IL-3 independent growth and survival in an Akt-independent manner. Small molecules can then be screened in these lines to identify inhibitors that rescue IL-3 dependence. Conclusions/Significance: BaFiso measures differential cell survival using multiparametric live cell imaging and permits selective inhibitors of Akt signaling to be identified. BaFiso is a platform technology suitable for the identification of smal

Proton Pump Inhibitors and the Risk of Early Aseptic Loosening in Hip and Knee Arthroplasty

Introduction: The use of proton pump inhibitors (PPIs) has been associated with a higher risk of osteoporotic fractures and non-unions rates. However, the relation between the use of PPIs and the development of aseptic loosening in arthroplasty procedures has not been studied. The objective of this study is to analyze the relation between the use of PPIs, and the risk of early aseptic loosening in total knee arthroplasty (TKA) and total hip arthroplasty (THA). Materials and methods: A nested case-control study was conducted on patients who were subjected THA or TKA in our center between 2010 and 2014. Cases were patients subjected to revision surgery due to early aseptic loosening during the study period. Cases were matched with controls who did not require any type of revision surgery by type of joint replacement (THA/TKA), gender, age (+/− 2 years), and follow-up time (±6 months). Odds Ratios were adjusted to potential confounders. Results: The crude and adjusted ORs (95% CI) of undergoing revision surgery for aseptic loosening following primary total knee arthroplasty or total hip arthroplasty, were 6.25 (2.04–19.23) and 6.10 (1.71–21.73), respectively, for any use PPIs compared with non-users. Crude and adjusted ORs, were 11.6 (2.93–45.88) and 17.1 (2.41–121.66), respectively, for patients with a Proportion of Days Covered (PDC) for PPIs <.5 (Table 2). In addition, the crude and adjusted ORs of undergoing revision surgery, were 5.05 (1.59–16.02) and 5.01 (1.36–18.44), respectively, for patients with a PDC for PPIs ≥.5. Discussion: These results suggest that PPIs should be used with caution in patients with TKA and THA, and that the use of these drugs should not be prolonged unless there was a justifiable indication. Conclusions: The use of PPIs and was associated with a higher risk of early aseptic loosening in patients subjected to THA and TKA

Evidence for Color Dichotomy in the Primordial Neptunian Trojan Population

In the current model of early Solar System evolution, the stable members of the Jovian and Neptunian Trojan populations were captured into resonance from the leftover reservoir of planetesimals during the outward migration of the giant planets. As a result, both Jovian and Neptunian Trojans share a common origin with the primordial disk population, whose other surviving members constitute today's trans-Neptunian object (TNO) populations. The cold classical TNOs are ultra-red, while the dynamically excited "hot" population of TNOs contains a mixture of ultra-red and blue objects. In contrast, Jovian and Neptunian Trojans are observed to be blue. While the absence of ultra-red Jovian Trojans can be readily explained by the sublimation of volatile material from their surfaces due to the high flux of solar radiation at 5AU, the lack of ultra-red Neptunian Trojans presents both a puzzle and a challenge to formation models. In this work we report the discovery by the Dark Energy Survey (DES) of two new dynamically stable L4 Neptunian Trojans,2013 VX30 and 2014 UU240, both with inclinations i >30 degrees, making them the highest-inclination known stable Neptunian Trojans. We have measured the colors of these and three other dynamically stable Neptunian Trojans previously observed by DES, and find that 2013 VX30 is ultra-red, the first such Neptunian Trojan in its class. As such, 2013 VX30 may be a "missing link" between the Trojan and TNO populations. Using a simulation of the DES TNO detection efficiency, we find that there are 162 +/- 73 Trojans with Hr < 10 at the L4 Lagrange point of Neptune. Moreover, the blue-to-red Neptunian Trojan population ratio should be higher than 17:1. Based on this result, we discuss the possible origin of the ultra-red Neptunian Trojan population and its implications for the formation history of Neptunian Trojans