Targeting TORC1/2 Enhances Sensitivity to EGFR Inhibitors in Head and Neck Cancer Preclinical Models1

PubMed Central

Cassell, Andre; Freilino, Maria L; Lee, Jessica; Barr, Sharon; Wang, Lin; Panahandeh, Mary C; Thomas, Sufi M; Grandis, Jennifer R

2012-01-01

Head and neck squamous cell carcinoma (HNSCC) is characterized by overexpression of the epidermal growth factor receptor (EGFR) where treatments targeting EGFR have met with limited clinical success. Elucidation of the key downstream-pathways that remain activated in the setting of EGFR blockade may reveal new therapeutic targets. The present study was undertaken to test the hypothesis that inhibition of the mammalian target of rapamycin (mTOR) complex would enhance the effects of EGFR blockade in HNSCC preclinical models. Treatment of HNSCC cell lines with the newly developed TORC1/TORC2 inhibitor OSI-027/ASP4876 resulted in dose-dependent inhibition of proliferation with abrogation of phosphorylation of known downstream targets including phospho-AKT (Ser473), phospho-4E-BP1, phospho-p70s6K, and phospho-PRAS40. Furthermore, combined treatment with OSI-027 and erlotinib resulted in enhanced biochemical effects and synergistic growth inhibition in vitro. Treatment of mice bearing HNSCC xenografts with a combination of the Food and Drug Administration (FDA)-approved EGFR inhibitor cetuximab and OSI-027 demonstrated a significant reduction of tumor volumes compared with either treatment alone. These findings suggest that TORC1/TORC2 inhibition in conjunction with EGFR blockade represents a plausible therapeutic strategy for HNSCC. PMID:23226094

Pharmaceuticals and other organic chemicals in selected north-central and northwestern Arkansas streams

USGS Publications Warehouse

Haggard, B.E.; Galloway, J.M.; Green, W.R.; Meyer, M.T.

2006-01-01

Recently, our attention has focused on the low level detection of many antibiotics, pharmaceuticals, and other organic chemicals in water resources. The limited studies available suggest that urban or rural streams receiving wastewater effluent are more susceptible to contamination. The purpose of this study was to evaluate the occurrence of antibiotics, pharmaceuticals, and other organic chemicals at 18 sites on seven selected streams in Arkansas, USA, during March, April, and August 2004. Water samples were collected upstream and downstream from the influence of effluent discharges in northwestern Arkansas and at one site on a relatively undeveloped stream in north-central Arkansas. At least one antibiotic, pharmaceutical, or other organic chemical was detected at all sites, except at Spavinaw Creek near Mayesville, Arkansas. The greatest number of detections was observed at Mud Creek downstream from an effluent discharge, including 31 pharmaceuticals and other organic chemicals. The detection of these chemicals occurred in higher frequency at sites downstream from effluent discharges compared to those sites upstream from effluent discharges; total chemical concentration was also greater downstream. Wastewater effluent discharge increased the concentrations of detergent metabolites, fire retardants, fragrances and flavors, and steroids in these streams. Antibiotics and associated degradation products were only found at two streams downstream from effluent discharges. Overall, 42 of the 108 chemicals targeted in this study were found in water samples from at least one site, and the most frequently detected organic chemicals included caffeine, phenol, para-cresol, and acetyl hexamethyl tetrahydro naphthalene (AHTN). ?? ASA, CSSA, SSSA.

Strigolactone regulates shoot development through a core signalling pathway

PubMed Central

Müller, Dörte

2016-01-01

ABSTRACT Strigolactones are a recently identified class of hormone that regulate multiple aspects of plant development. The DWARF14 (D14) α/β fold protein has been identified as a strigolactone receptor, which can act through the SCFMAX2 ubiquitin ligase, but the universality of this mechanism is not clear. Multiple proteins have been suggested as targets for strigolactone signalling, including both direct proteolytic targets of SCFMAX2, and downstream targets. However, the relevance and importance of these proteins to strigolactone signalling in many cases has not been fully established. Here we assess the contribution of these targets to strigolactone signalling in adult shoot developmental responses. We find that all examined strigolactone responses are regulated by SCFMAX2 and D14, and not by other D14-like proteins. We further show that all examined strigolactone responses likely depend on degradation of SMXL proteins in the SMXL6 clade, and not on the other proposed proteolytic targets BES1 or DELLAs. Taken together, our results suggest that in the adult shoot, the dominant mode of strigolactone signalling is D14-initiated, MAX2-mediated degradation of SMXL6-related proteins. We confirm that the BRANCHED1 transcription factor and the PIN-FORMED1 auxin efflux carrier are plausible downstream targets of this pathway in the regulation of shoot branching, and show that BRC1 likely acts in parallel to PIN1. PMID:27793831

C. elegans sym-1 is a downstream target of the hunchback-like-1 developmental timing transcription factor

PubMed Central

Niwa, Ryusuke; Hada, Kazumasa; Moliyama, Kouichi; Ohniwa, Ryosuke L.; Tan, Yi-Meng; Olsson-Carter, Katherine; Chi, Woo; Reinke, Valerie; Slack, Frank J.

2010-01-01

In the nematode Caenorhabditis elegans, the let-7 microRNA (miRNA) and its family members control the timing of key developmental events in part by directly regulating expression of hunchback-like-1 (hbl-1). C. elegans hbl-1 mutants display multiple developmental timing deficiencies, including cell cycle defects during larval development. While hbl-1 is predicted to encode a transcriptional regulator, downstream targets of HBL-1 have not been fully elucidated. Here we report using microarray analysis to uncover genes downstream of HBL-1. We established a transgenic strain that overexpresses hbl-1 under the control of a heat shock promoter. Heat shock-induced hbl-1 overexpression led to retarded hypodermal structures at the adult stage, opposite to the effect seen in loss of function (lf) hbl-1 mutants. The microarray screen identified numerous potential genes that are upregulated or downregulated by HBL-1, including sym-1, which encodes a leucine-rich repeat protein with a signal sequence. We found an increase in sym-1 transcription in the heat shock-induced hbl-1 overexpression strain, while loss of hbl-1 function caused a decrease in sym-1 expression levels. Furthermore, we found that sym-1(lf) modified the hypodermal abnormalities in hbl-1 mutants. Given that SYM-1 is a protein secreted from hypodermal cells to the surrounding cuticle, we propose that the adult-specific cuticular structures may be under the temporal control of HBL-1 through regulation of sym-1 transcription. PMID:19923914

PAX3-FOXO1: Zooming in on an "undruggable" target.

PubMed

Wachtel, Marco; Schäfer, Beat W

2018-06-01

Driver oncogenes are prime targets for therapy in tumors many of which, including leukemias and sarcomas, express recurrent fusion transcription factors. One specific example for such a cancer type is alveolar rhabdomyosarcoma, which is associated in the majority of cases with the fusion protein PAX3-FOXO1. Since fusion transcription factors are challenging targets for development of small molecule inhibitors, indirect inhibitory strategies for this type of oncogenes represent a more promising approach. One can envision strategies at different molecular levels including upstream modifiers and activators, epigenetic and transcriptional co-regulators, and downstream effector targets. In this review, we will discuss the current knowledge regarding potential therapeutic targets that might contribute to indirect interference with PAX3-FOXO1 activity in alveolar rhabdomyosarcoma at the different molecular levels and extrapolate these findings to fusion transcription factors in general. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Simulating potential structural and operational changes for Detroit Dam on the North Santiam River, Oregon, for downstream temperature management

USGS Publications Warehouse

Buccola, Norman L.; Rounds, Stewart A.; Sullivan, Annett B.; Risley, John C.

2012-01-01

Detroit Dam was constructed in 1953 on the North Santiam River in western Oregon and resulted in the formation of Detroit Lake. With a full-pool storage volume of 455,100 acre-feet and a dam height of 463 feet, Detroit Lake is one of the largest and most important reservoirs in the Willamette River basin in terms of power generation, recreation, and water storage and releases. The U.S. Army Corps of Engineers operates Detroit Dam as part of a system of 13 reservoirs in the Willamette Project to meet multiple goals, which include flood-damage protection, power generation, downstream navigation, recreation, and irrigation. A distinct cycle in water temperature occurs in Detroit Lake as spring and summer heating through solar radiation creates a warm layer of water near the surface and isolates cold water below. Controlling the temperature of releases from Detroit Dam, therefore, is highly dependent on the location, characteristics, and usage of the dam's outlet structures. Prior to operational changes in 2007, Detroit Dam had a well-documented effect on downstream water temperature that was problematic for endangered salmonid fish species, releasing water that was too cold in midsummer and too warm in autumn. This unnatural seasonal temperature pattern caused problems in the timing of fish migration, spawning, and emergence. In this study, an existing calibrated 2-dimensional hydrodynamic water-quality model [CE-QUAL-W2] of Detroit Lake was used to determine how changes in dam operation or changes to the structural release points of Detroit Dam might affect downstream water temperatures under a range of historical hydrologic and meteorological conditions. The results from a subset of the Detroit Lake model scenarios then were used as forcing conditions for downstream CE-QUAL-W2 models of Big Cliff Reservoir (the small reregulating reservoir just downstream of Detroit Dam) and the North Santiam and Santiam Rivers. Many combinations of environmental, operational, and structural options were explored with the model scenarios. Multiple downstream temperature targets were used along with three sets of environmental forcing conditions representing cool/wet, normal, and hot/dry conditions. Five structural options at Detroit Dam were modeled, including the use of existing outlets, one hypothetical variable-elevation outlet such as a sliding gate, a hypothetical combination of a floating outlet and a fixed-elevation outlet, and a hypothetical combination of a floating outlet and a sliding gate. Finally, 14 sets of operational guidelines for Detroit Dam were explored to gain an understanding of the effects of imposing different downstream minimum streamflows, imposing minimum outflow rules to specific outlets, and managing the level of the lake with different timelines through the year. Selected subsets of these combinations of operational and structural scenarios were run through the downstream models of Big Cliff Reservoir and the North Santiam and Santiam Rivers to explore how hypothetical changes at Detroit Dam might provide improved temperatures for endangered salmonids downstream of the Detroit-Big Cliff Dam complex. Conclusions that can be drawn from these model scenarios include: *The water-temperature targets set by the U.S. Army Corps of Engineers for releases from Detroit Dam can be met through a combination of new dam outlets or a delayed drawdown of the lake in autumn. *Spring and summer dam operations greatly affect the available release temperatures and operational flexibility later in the autumn. Releasing warm water during midsummer tends to keep more cool water available for release in autumn. *The ability to meet downstream temperature targets during spring depends on the characteristics of the available outlets. Under existing conditions, although warm water sometimes is present at the lake surface in spring and early summer, such water may not be available for release if the lake level is either well below or well above the spillway crest. *Managing lake releases to meet downstream temperature targets depends on having outlet structures that can access both (warm) lake surface water and (cold) deeper lake water throughout the year. The existing outlets at Detroit Dam do not allow near-surface waters to be released during times when the lake surface level is below the spillway (spring and autumn). *Using the existing outlets at Detroit Dam, lake level management is important to the water temperature of releases because it controls the availability and depth of water at the spillway. When lake level is lowered below the spillway crest in late summer, the loss of access to warm water at the lake surface can result in abrupt changes to release temperatures. *Because the power-generation intakes (penstocks) are 166 feet below the full-pool lake level, imposing minimum power production requirements at Detroit Dam limits the amount of warm surface water that can be expelled from the lake in midsummer, thereby postponing and amplifying warm outflows from Detroit Lake into the autumn spawning season. *Likewise, imposing minimum power production requirements at Detroit Dam in autumn can limit the amount of cool hypolimnetic water that is released from the lake, thereby limiting cool outflows from Detroit Lake during the autumn spawning season. *Model simulations indicate that a delayed drawdown of Detroit Lake in autumn would result in better control over release temperatures in the immediate downstream vicinity of Big Cliff Dam, but the reduced outflows necessary to retain more water in the lake in late summer are more susceptible to rapid heating downstream. *Compared to the existing outlets at Detroit Dam, floating or sliding-gate outlet structures can provide greater control over release temperatures because they provide better access to warm water at the lake surface and cooler water at depth. These conclusions can be grouped into several common themes. First, optimal and flexible management and achievement of downstream temperature goals requires that releases of warm water near the surface of the lake and cold water below the thermocline are both possible with the available dam outlets during spring, summer, and autumn. This constraint can be met to some extent with existing outlets, but only if access to the spillway is extended into autumn by keeping the lake level higher than called for by the current rule curve (the typical target water-surface elevation throughout the year). If new outlets are considered, a variable-elevation outlet such as a sliding gate structure, or a floating outlet in combination with a fixed-elevation outlet at sufficient depth to access cold water, is likely to work well in terms of accessing a range of water temperatures and achieving downstream temperature targets. Furthermore, model results indicate that it is important to release warm water from near the lake surface during midsummer. If not released downstream, the warm water will build up at the top of the lake as a result of solar energy inputs and the thermocline will deepen, potentially causing warm water to reach the depth of deeper fixed-elevation outlets in autumn, particularly when the lake level is drawn down to make room for flood storage. Delaying the drawdown in autumn can help to keep the thermocline above such outlets and preserve access to cold water. Although it is important to generate hydropower at Detroit Dam, minimum power-production requirements limit the ability of dam operators to meet downstream temperature targets with existing outlet structures. The location of the power penstocks below the thermocline in spring and most of summer causes the release of more cool water during summer than is optimal. Reducing the power-production constraint allows the temperature target to be met more frequently, but at the cost of less power generation. Finally, running the Detroit Dam, Big Cliff Dam, and North Santiam and Santiam River models in series allows dam operators to evaluate how different operational strategies or combinations of new dam outlets might affect downstream temperatures for many miles of critical endangered salmonid habitat. Temperatures can change quickly in these downstream reaches as the river exchanges heat with its surroundings, and heating or cooling of 6 degrees Celsius is not unusual in the 40–50 miles downstream of Big Cliff Dam. The results published in this report supersede preliminary results published in U.S. Geological Survey Open-File Report 2011-1268 (Buccola and Rounds, 2011). Those preliminary results are still valid, but the results in this report are more current and comprehensive.

Towards β-globin gene-targeting with integrase-defective lentiviral vectors.

PubMed

Inanlou, Davoud Nouri; Yakhchali, Bagher; Khanahmad, Hossein; Gardaneh, Mossa; Movassagh, Hesam; Cohan, Reza Ahangari; Ardestani, Mehdi Shafiee; Mahdian, Reza; Zeinali, Sirous

2010-11-01

We have developed an integrase-defective lentiviral (LV) vector in combination with a gene-targeting approach for gene therapy of β-thalassemia. The β-globin gene-targeting construct has two homologous stems including sequence upstream and downstream of the β-globin gene, a β-globin gene positioned between hygromycin and neomycin resistant genes and a herpes simplex virus type 1 thymidine kinase (HSVtk) suicide gene. Utilization of integrase-defective LV as a vector for the β-globin gene increased the number of selected clones relative to non-viral methods. This method represents an important step toward the ultimate goal of a clinical gene therapy for β-thalassemia.

Elastic and inelastic scattering of 134Xe beams on C2D4 targets measured with GODDESS

NASA Astrophysics Data System (ADS)

Sims, Harrison; Cizewski, Jolie; Lapailleur, Alex; Garland, Heather; Xination, Dai; Pain, Steven; Hall, Matthew; Goddess Collaboration

2017-09-01

The GODDESS (Gammasphere-ORRUBA: Dual Detector for Experimental Structure Studies) coupling of the ORRUBA charged-particle array with Gammasphere is designed to enable high-resolution particle-gamma measurements in inverse kinematics with radioactive beams. The high resolution and coverage of GODDESS allows for multiple reaction channels to be studied simultaneously. For the stable-beam commissioning of GODDESS, the 134Xe(d,p γ)135Xe reaction was measured using a beam of 134Xe at 8 MeV/A, delivered by the ATLAS facility at Argonne National Laboratory. The beam impinged on an 800 μg/cm2 C2D4 target, and charged particles were detected in the GODDESS silicon array between 15 and 165 degrees. Coincident gamma rays were measured with Gammasphere, with 10 % efficiency at 1.3 MeV. In the detectors downstream of the target, elastically- and inelastically-scattered target ions (deuterium and carbon) were detected, populating the ground and low-lying excited states in 134Xe. An overview of GODDESS will be presented, along with the analysis of the downstream data, including the differential scattering cross sections and population of collective states in 134Xe. Work supported in part by the U.S. D.O.E. and National Science Foundation.

Cross Talk between Two Antioxidant Systems, Thioredoxin and DJ-1: Consequences for Cancer

PubMed Central

Raninga, Prahlad V.; Trapani, Giovanna Di; Tonissen, Kathryn F.

2014-01-01

Oxidative stress, which is associated with an increased concentration of reactive oxygen species (ROS), is involved in the pathogenesis of numerous diseases including cancer. In response to increased ROS levels, cellular antioxidant molecules such as thioredoxin, peroxiredoxins, glutaredoxins, DJ-1, and superoxide dismutases are upregulated to counteract the detrimental effect of ROS. However, cancer cells take advantage of upregulated antioxidant molecules for protection against ROS-induced cell damage. This review focuses on two antioxidant systems, Thioredoxin and DJ-1, which are upregulated in many human cancer types, correlating with tumour proliferation, survival, and chemo-resistance. Thus, both of these antioxidant molecules serve as potential molecular targets to treat cancer. However, targeting one of these antioxidants alone may not be an effective anti-cancer therapy. Both of these antioxidant molecules are interlinked and act on similar downstream targets such as NF-κβ, PTEN, and Nrf2 to exert cytoprotection. Inhibiting either thioredoxin or DJ-1 alone may allow the other antioxidant to activate downstream signalling cascades leading to tumour cell survival and proliferation. Targeting both thioredoxin and DJ-1 in conjunction may completely shut down the antioxidant defence system regulated by these molecules. This review focuses on the cross-talk between thioredoxin and DJ-1 and highlights the importance and consequences of targeting thioredoxin and DJ-1 together to develop an effective anti-cancer therapeutic strategy. PMID:25593990

Method of Simulating Flow-Through Area of a Pressure Regulator

NASA Technical Reports Server (NTRS)

Hass, Neal E. (Inventor); Schallhorn, Paul A. (Inventor)

2011-01-01

The flow-through area of a pressure regulator positioned in a branch of a simulated fluid flow network is generated. A target pressure is defined downstream of the pressure regulator. A projected flow-through area is generated as a non-linear function of (i) target pressure, (ii) flow-through area of the pressure regulator for a current time step and a previous time step, and (iii) pressure at the downstream location for the current time step and previous time step. A simulated flow-through area for the next time step is generated as a sum of (i) flow-through area for the current time step, and (ii) a difference between the projected flow-through area and the flow-through area for the current time step multiplied by a user-defined rate control parameter. These steps are repeated for a sequence of time steps until the pressure at the downstream location is approximately equal to the target pressure.

Edaravone alleviates Alzheimer's disease-type pathologies and cognitive deficits.

PubMed

Jiao, Shu-Sheng; Yao, Xiu-Qing; Liu, Yu-Hui; Wang, Qing-Hua; Zeng, Fan; Lu, Jian-Jun; Liu, Jia; Zhu, Chi; Shen, Lin-Lin; Liu, Cheng-Hui; Wang, Ye-Ran; Zeng, Gui-Hua; Parikh, Ankit; Chen, Jia; Liang, Chun-Rong; Xiang, Yang; Bu, Xian-Le; Deng, Juan; Li, Jing; Xu, Juan; Zeng, Yue-Qin; Xu, Xiang; Xu, Hai-Wei; Zhong, Jin-Hua; Zhou, Hua-Dong; Zhou, Xin-Fu; Wang, Yan-Jiang

2015-04-21

Alzheimer's disease (AD) is one of most devastating diseases affecting elderly people. Amyloid-β (Aβ) accumulation and the downstream pathological events such as oxidative stress play critical roles in pathogenesis of AD. Lessons from failures of current clinical trials suggest that targeting multiple key pathways of the AD pathogenesis is necessary to halt the disease progression. Here we show that Edaravone, a free radical scavenger that is marketed for acute ischemic stroke, has a potent capacity of inhibiting Aβ aggregation and attenuating Aβ-induced oxidation in vitro. When given before or after the onset of Aβ deposition via i.p. injection, Edaravone substantially reduces Aβ deposition, alleviates oxidative stress, attenuates the downstream pathologies including Tau hyperphosphorylation, glial activation, neuroinflammation, neuronal loss, synaptic dysfunction, and rescues the behavioral deficits of APPswe/PS1 mice. Oral administration of Edaravone also ameliorates the AD-like pathologies and memory deficits of the mice. These findings suggest that Edaravone holds a promise as a therapeutic agent for AD by targeting multiple key pathways of the disease pathogenesis.

TEAD1 mediates the oncogenic activities of Hippo-YAP1 signaling in osteosarcoma.

PubMed

Chai, Jiwei; Xu, Shijie; Guo, Fengbo

2017-06-24

Hippo signaling pathway is an evolutionarily conserved developmental network that governs the downstream transcriptional co-activators, YAP and TAZ, which bind to and activate the output of TEADs that responsible for cell proliferation, apoptosis, and stem cell self renewal. Emerging evidence has shown the tumor suppressor properties of Hippo signaling. However, limited knowledge is available concerning the downstream transcription factors of Hippo pathway in osteosarcoma (OS). In this study, we demonstrated that TEAD1 was the major transcription factor of Hippo signaling pathway in OS. Genetic silencing of TEAD1 suppressed multiple malignant phenotypes of OS cells including cell proliferation, apoptosis resistance, and invasive potential. Mechanistically, we showed that TEAD1 largely exerted its transcriptional control of its functional targets, PTGS2 and CYR61. Collectively, this work identifies the YAP1/TEAD1 complex as the representative dysregulated profile of Hippo signaling in OS and provides proof-of-principle that targeting TEAD1 may be a therapeutic strategy of osteosarcoma. Copyright © 2017. Published by Elsevier Inc.

Identification of three signaling molecules required for calcineurin-dependent monopolar growth induced by the DNA replication checkpoint in fission yeast.

PubMed

Kume, Kazunori; Hashimoto, Tomoyo; Suzuki, Masashi; Mizunuma, Masaki; Toda, Takashi; Hirata, Dai

2017-09-30

Cell polarity is coordinately regulated with the cell cycle. Growth polarity of the fission yeast Schizosaccharomyces pombe transits from monopolar to bipolar during G2 phase, termed NETO (new end take off). Upon perturbation of DNA replication, the checkpoint kinase Cds1/CHK2 induces NETO delay through activation of Ca 2+ /calmodulin-dependent protein phosphatase calcineurin (CN). CN in turn regulates its downstream targets including the microtubule (MT) plus-end tracking CLIP170 homologue Tip1 and the Casein kinase 1γ Cki3. However, whether and which Ca 2+ signaling molecules are involved in the NETO delay remains elusive. Here we show that 3 genes (trp1322, vcx1 and SPAC6c3.06c encoding TRP channel, antiporter and P-type ATPase, respectively) play vital roles in the NETO delay. Upon perturbation of DNA replication, these 3 genes are required for not only the NETO delay but also for the maintenance of cell viability. Trp1322 and Vcx1 act downstream of Cds1 and upstream of CN for the NETO delay, whereas SPAC6c3.06c acts downstream of CN. Consistently, Trp1322 and Vcx1, but not SPAC6c3.06c, are essential for activation of CN. Interestingly, we have found that elevated extracellular Ca 2+ per se induces a NETO delay, which depends on CN and its downstream target genes. These findings imply that Ca 2+ -CN signaling plays a central role in cell polarity control by checkpoint activation. Copyright © 2017 Elsevier Inc. All rights reserved.

JAK2V617F influences epigenomic changes in myeloproliferative neoplasms.

PubMed

Chen, Chih-Cheng; Chiu, Chia-Chen; Lee, Kuan-Der; Hsu, Chia-Chen; Chen, Hong-Chi; Huang, Tim H-M; Hsiao, Shu-Huei; Leu, Yu-Wei

2017-12-16

Negative valine (V) to phenylalanine (F) switch at the Janus kinase (JAK2) 617 codon (V617F) is the dominant driver mutation in patients with myeloproliferative neoplasms (MPNs). JAK2V617F was proved to be sufficient for cell transformation; however, independent mutations might influence the following epigenomic modifications. To assess the JAK2V617F-induced downstream epigenomic changes without interferences, we profiled the epigenomic changes in ectopically expressed JAK2V617F in Ba/F3 cells. Antibodies against phosphorylated signal transducer and activator of transcription 3 (pSTAT3) and enhancer of zeste homolog 2 (EZH2) were used for chromatin-immunoprecipitation sequencing (ChIP-seq) to detect the downstream epigenomic targets in the JAK2-STAT3 signaling pathway. To confirm the JAK2V617F-induced epigenetic changes in vivo, DNA methylation changes in the target loci in patients with MPNs were detected through methylation-specific polymerase chain reaction and were clustered against the changes within controls. We found that ectopically expressed JAK2V617F in Ba/F3 cells reduced the binding specificity; it was associated with cis-regulatory elements and recognized DNA motifs in both pSTAT3-downstream and EZH2-associated targets. Overlapping target loci between the control and JAK2V617F were <3% and 0.4%, respectively, as identified through pSTAT3 and EZH2 ChIP-seq. Furthermore, the methylation changes in the direct target loci (FOXH1, HOXC9, and SRF) were clustered independently from the control locus (L1TD1) and other mutation genes (HMGA2 and Lin28A) in the analyzed MPN samples. Therefore, JAK2V617F influences target binding in both pSTAT3 and EZH2. Without mutations in epigenetic regulators, JAK2V617F can induce downstream epigenomic modifications. Thus, epigenetic changes in JAK2 downstream targets might be trackable in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

TCF21 and the environmental sensor aryl-hydrocarbon receptor cooperate to activate a pro-inflammatory gene expression program in coronary artery smooth muscle cells

PubMed Central

Nguyen, Trieu; Iyer, Dharini; Liu, Boxiang; Wang, Ting; Sazonova, Olga; Matic, Ljubica Perisic; Maegdefessel, Lars; Quertermous, Thomas

2017-01-01

Both environmental factors and genetic loci have been associated with coronary artery disease (CAD), however gene-gene and gene-environment interactions that might identify molecular mechanisms of risk are not easily studied by human genetic approaches. We have previously identified the transcription factor TCF21 as the causal CAD gene at 6q23.2 and characterized its downstream transcriptional network that is enriched for CAD GWAS genes. Here we investigate the hypothesis that TCF21 interacts with a downstream target gene, the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that mediates the cellular response to environmental contaminants, including dioxin and polycyclic aromatic hydrocarbons (e.g., tobacco smoke). Perturbation of TCF21 expression in human coronary artery smooth muscle cells (HCASMC) revealed that TCF21 promotes expression of AHR, its heterodimerization partner ARNT, and cooperates with these factors to upregulate a number of inflammatory downstream disease related genes including IL1A, MMP1, and CYP1A1. TCF21 was shown to bind in AHR, ARNT and downstream target gene loci, and co-localization was noted for AHR-ARNT and TCF21 binding sites genome-wide in regions of HCASMC open chromatin. These regions of co-localization were found to be enriched for GWAS signals associated with cardio-metabolic as well as chronic inflammatory disease phenotypes. Finally, we show that similar to TCF21, AHR gene expression is increased in atherosclerotic lesions in mice in vivo using laser capture microdissection, and AHR protein is localized in human carotid atherosclerotic lesions where it is associated with protein kinases with a critical role in innate immune response. These data suggest that TCF21 can cooperate with AHR to activate an inflammatory gene expression program that is exacerbated by environmental stimuli, and may contribute to the overall risk for CAD. PMID:28481916

Targeting interleukin-6 for noninfectious uveitis

PubMed Central

Lin, Phoebe

2015-01-01

Interleukin-6 (IL-6) is a pleiotropic cytokine implicated in the pathogenesis of many immune-mediated disorders including several types of non-infectious uveitis. These uveitic conditions include Vogt-Koyanagi-Harada syndrome, uveitis associated with Behçet disease, and sarcoidosis. This review summarizes the role of IL-6 in immunity, highlighting its effect on Th17, Th1, and plasmablast differentiation. It reviews the downstream mediators activated in the process of IL-6 binding to its receptor complex. This review also summarizes the biologics targeting either IL-6 or the IL-6 receptor, including tocilizumab, sarilumab, sirukumab, olokizumab, clazakizumab, and siltuximab. The target, dosage, potential side effects, and potential uses of these biologics are summarized in this article based on the existing literature. In summary, anti-IL-6 therapy for non-infectious uveitis shows promise in terms of efficacy and side effect profile. PMID:26392750

New separators at the ATLAS facility

NASA Astrophysics Data System (ADS)

Back, Birger; Agfa Collaboration; Airis Team

2015-10-01

Two new separators are being built for the ATLAS facility. The Argonne Gas-Filled Analyzer (AGFA) is a novel design consisting of a single quadrupole and a multipole magnet that has both dipole and quadrupole field components. The design allows for placing Gammasphere at the target position while providing a solid angle of ~ 22 msr for capturing recoil products emitted at zero degrees. This arrangement enables studies of prompt gamma ray emission from weakly populated trans-fermium nuclei and those near the doubly-magic N = Z = 50 shell closure measured in coincidence with the recoils registered by AGFA. The Argonne In-flight Radioactive Ion Separator (AIRIS) is a magnetic chicane that will be installed immediately downstream of the last ATLAS cryostat and serve to separate radioactive ion beams generated in flight at an upstream high intensity production target. These beams will be further purified by a downstream RF sweeper and transported into a number of target stations including HELIOS, the Enge spectrograph, the FMA and Gammasphere. This talk will present the status of these two projects. This work was supported by the U.S. Department of Energy, Office of Nuclear Physics, under Contract No. DE-AC02-06CH11357.

A comprehensive review of heregulins, HER3, and HER4 as potential therapeutic targets in cancer

PubMed Central

Mota, Jose Mauricio; Collier, Katharine Ann; Barros Costa, Ricardo Lima; Taxter, Timothy; Kalyan, Aparna; Leite, Caio A.; Chae, Young Kwang; Giles, Francis J.; Carneiro, Benedito A.

2017-01-01

Heregulins (HRGs) bind to the receptors HER3 or HER4, induce receptor dimerization, and trigger downstream signaling that leads to tumor progression and resistance to targeted therapies. Increased expression of HRGs has been associated with worse clinical prognosis; therefore, attempts to block HRG-dependent tumor growth have been pursued. This manuscript summarizes the function and signaling of HRGs and review the preclinical evidence of its involvement in carcinogenesis, prognosis, and treatment resistance in several malignancies such as colorectal cancer, non-small cell lung cancer, ovarian cancer, and breast cancer. Agents in preclinical development and clinical trials of novel therapeutics targeting HRG-dependent signaling are also discussed, including anti-HER3 and -HER4 antibodies, anti-metalloproteinase agents, and HRG fusion proteins. Although several trials have indicated an acceptable safety profile, translating preclinical findings into clinical practice remains a challenge in this field, possibly due to the complexity of downstream signaling and patterns of HRG, HER3 and HER4 expression in different cancer subtypes. Improving patient selection through biomarkers and understanding the resistance mechanisms may translate into significant clinical benefits in the near future. PMID:29179520

Sex- and Tissue-specific Functions of Drosophila Doublesex Transcription Factor Target Genes

PubMed Central

Clough, Emily; Jimenez, Erin; Kim, Yoo-Ah; Whitworth, Cale; Neville, Megan C.; Hempel, Leonie; Pavlou, Hania J.; Chen, Zhen-Xia; Sturgill, David; Dale, Ryan; Smith, Harold E.; Przytycka, Teresa M.; Goodwin, Stephen F.; Van Doren, Mark; Oliver, Brian

2014-01-01

Primary sex determination “switches” evolve rapidly, but Doublesex (DSX) related transcription factors (DMRTs) act downstream of these switches to control sexual development in most animal species. Drosophila dsx encodes female- and male-specific isoforms (DSXF and DSXM), but little is known about how dsx controls sexual development, whether DSXF and DSXM bind different targets, or how DSX proteins direct different outcomes in diverse tissues. We undertook genome-wide analyses to identify DSX targets using in vivo occupancy, binding site prediction, and evolutionary conservation. We find that DSXF and DSXM bind thousands of the same targets in multiple tissues in both sexes, yet these targets have sex- and tissue-specific functions. Interestingly, DSX targets show considerable overlap with targets identified for mouse DMRT1. DSX targets include transcription factors and signaling pathway components providing for direct and indirect regulation of sex-biased expression. PMID:25535918

Synchronous detection of miRNAs, their targets and downstream proteins in transferred FFPE sections: applications in clinical and basic research.

PubMed

Zhao, Jin-yao; Liu, Chun-qing; Zhao, He-nan; Ding, Yan-Fang; Bi, Tie; Wang, Bo; Lin, Xing-chi; Guo, Gordon; Cui, Shi-ying

2012-10-01

After discovering new miRNAs, it is often difficult to determine their targets and effects on downstream protein expression. In situ hybridization (ISH) and immunohistochemistry (IHC) are two commonly used methods for clinical diagnosis and basic research. We used an optimized technique that simultaneously detects miRNAs, their binding targets and corresponding proteins on transferred serial formalin fixed paraffin embedded (FFPE) sections from patients. Combined with bioinformatics, this method was used to validate the reciprocal expression of specific miRNAs and targets that were detected by ISH, as well as the expression of downstream proteins that were detected by IHC. A complete analysis was performed using a limited number of transferred serial FFPE sections that had been stored for 1-4 years at room temperature. Some sections had even been previously stained with H&E. We identified a miRNA that regulates epithelial ovarian cancer, along with its candidate target and related downstream protein. These findings were directly validated using sub-cellular components obtained from the same patient sample. In addition, the expression of Nephrin (a podocyte marker) and Stmn1 (a recently identified marker related to glomerular development) were confirmed in transferred FFPE sections of mouse kidney. This procedure may be adapted for clinical diagnosis and basic research, providing a qualitative and efficient method to dissect the detailed spatial expression patterns of miRNA pathways in FFPE tissue, especially in cases where only a small biopsy sample can be obtained. Copyright © 2012 Elsevier Inc. All rights reserved.

Combustion mode switching with a turbocharged/supercharged engine

DOEpatents

Mond, Alan; Jiang, Li

2015-09-22

A method for switching between low- and high-dilution combustion modes in an internal combustion engine having an intake passage with an exhaust-driven turbocharger, a crankshaft-driven positive displacement supercharger downstream of the turbocharger and having variable boost controllable with a supercharger bypass valve, and a throttle valve downstream of the supercharger. The current combustion mode and mass air flow are determined. A switch to the target combustion mode is commanded when an operating condition falls within a range of predetermined operating conditions. A target mass air flow to achieve a target air-fuel ratio corresponding to the current operating condition and the target combustion mode is determined. The degree of opening of the supercharger bypass valve and the throttle valve are controlled to achieve the target mass air flow. The amount of residual exhaust gas is manipulated.

Altered decorin leads to disrupted endothelial cell function: a possible mechanism in the pathogenesis of fetal growth restriction?

PubMed

Chui, A; Murthi, P; Gunatillake, T; Brennecke, S P; Ignjatovic, V; Monagle, P T; Whitelock, J M; Said, J M

2014-08-01

Fetal growth restriction (FGR) is a key cause of adverse pregnancy outcome where maternal and fetal factors are identified as contributing to this condition. Idiopathic FGR is associated with altered vascular endothelial cell functions. Decorin (DCN) has important roles in the regulation of endothelial cell functions in vascular environments. DCN expression is reduced in FGR. The objectives were to determine the functional consequences of reduced DCN in a human microvascular endothelial cell line model (HMVEC), and to determine downstream targets of DCN and their expression in primary placental microvascular endothelial cells (PLECs) from control and FGR-affected placentae. Short-interference RNA was used to reduce DCN expression in HMVECs and the effect on proliferation, angiogenesis and thrombin generation was determined. A Growth Factor PCR Array was used to identify downstream targets of DCN. The expression of target genes in control and FGR PLECs was performed. DCN reduction decreased proliferation and angiogenesis but increased thrombin generation with no effect on apoptosis. The array identified three targets of DCN: FGF17, IL18 and MSTN. Validation of target genes confirmed decreased expression of VEGFA, MMP9, EGFR1, IGFR1 and PLGF in HMVECs and PLECs from control and FGR pregnancies. Reduction of DCN in vascular endothelial cells leads to disrupted cell functions. The targets of DCN include genes that play important roles in angiogenesis and cellular growth. Therefore, differential expression of these may contribute to the pathogenesis of FGR and disease states in other microvascular circulations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Edaravone alleviates Alzheimer’s disease-type pathologies and cognitive deficits

PubMed Central

Jiao, Shu-Sheng; Yao, Xiu-Qing; Liu, Yu-Hui; Wang, Qing-Hua; Zeng, Fan; Lu, Jian-Jun; Liu, Jia; Zhu, Chi; Shen, Lin-Lin; Liu, Cheng-Hui; Wang, Ye-Ran; Zeng, Gui-Hua; Parikh, Ankit; Chen, Jia; Liang, Chun-Rong; Xiang, Yang; Bu, Xian-Le; Deng, Juan; Li, Jing; Xu, Juan; Zeng, Yue-Qin; Xu, Xiang; Xu, Hai-Wei; Zhong, Jin-Hua; Zhou, Hua-Dong; Zhou, Xin-Fu; Wang, Yan-Jiang

2015-01-01

Alzheimer’s disease (AD) is one of most devastating diseases affecting elderly people. Amyloid-β (Aβ) accumulation and the downstream pathological events such as oxidative stress play critical roles in pathogenesis of AD. Lessons from failures of current clinical trials suggest that targeting multiple key pathways of the AD pathogenesis is necessary to halt the disease progression. Here we show that Edaravone, a free radical scavenger that is marketed for acute ischemic stroke, has a potent capacity of inhibiting Aβ aggregation and attenuating Aβ-induced oxidation in vitro. When given before or after the onset of Aβ deposition via i.p. injection, Edaravone substantially reduces Aβ deposition, alleviates oxidative stress, attenuates the downstream pathologies including Tau hyperphosphorylation, glial activation, neuroinflammation, neuronal loss, synaptic dysfunction, and rescues the behavioral deficits of APPswe/PS1 mice. Oral administration of Edaravone also ameliorates the AD-like pathologies and memory deficits of the mice. These findings suggest that Edaravone holds a promise as a therapeutic agent for AD by targeting multiple key pathways of the disease pathogenesis. PMID:25847999

Hemoglobin A1c as a Diagnostic Tool: Public Health Implications From an Actor–Network Perspective

PubMed Central

Rock, Melanie

2012-01-01

Public health arguments for collecting hemoglobin A1c (HbA1c) data, particularly in clinical settings, should be reframed to place more emphasis on nonmedical determinants of population health. We compare individual- with population-level interpretations of HbA1c titers. This comparison reveals that public health researchers need to pay close attention to diagnostic tests and their uses, including rhetorical uses. We also synthesize historical and current evidence to map out 2 possible scenarios for the future. In the first scenario, prevention efforts emphasize primary care and focus almost entirely downstream. The second scenario anticipates downstream interventions but also upstream interventions targeting environments. Our analysis adapts actor–network theory to strategic planning and forecasting in public health. PMID:22095361

Molecular mechanism of action of immune-modulatory drugs thalidomide, lenalidomide and pomalidomide in multiple myeloma

PubMed Central

Zhu, Yuan Xiao; Kortuem, K. Martin; Stewart, A. Keith

2014-01-01

Although several mechanisms have been proposed to explain the activity of thalidomide, lenalidomide and pomalidomide in multiple myeloma (MM), including demonstrable anti-angiogenic, anti-proliferative and immunomodulatory effects, the precise cellular targets and molecular mechanisms have only recently become clear. A landmark study recently identified cereblon (CRBN) as a primary target of thalidomide teratogenicity. Subsequently it was demonstrated that CRBN is also required for the anti-myeloma activity of thalidomide and related drugs, the so-called immune-modulatory drugs (IMiDs). Low CRBN expression was found to correlate with drug resistance in MM cell lines and primary MM cells. One of the downstream targets of CRBN identified is interferon regulatory factor 4 (IRF4), which is critical for myeloma cell survival and is down-regulated by IMiD treatment. CRBN is also implicated in several effects of IMiDs, such as down-regulation of tumor necrosis factor-α (TNF-α) and T cell immunomodulatory activity, demonstrating that the pleotropic actions of the IMiDs are initiated by binding to CRBN. Future dissection of CRBN downstream signaling will help to delineate the underlying mechanisms for IMiD action and eventually lead to development of new drugs with more specific anti-myeloma activities. It may also provide a biomarker to predict IMiD response and resistance. PMID:22966948

Fibroblast growth factor receptors in breast cancer: expression, downstream effects, and possible drug targets.

PubMed

Tenhagen, M; van Diest, P J; Ivanova, I A; van der Wall, E; van der Groep, P

2012-08-01

Cancer treatments are increasingly focusing on the molecular mechanisms underlying the oncogenic processes present in tumors of individual patients. Fibroblast growth factor receptors (FGFRs) are among the many molecules that are involved in oncogenesis and are currently under investigation for their potential as drug targets in breast cancer patients. These receptor tyrosine kinases play a role in several processes including proliferation, angiogenesis, and migration. Alterations in these basal processes can contribute to the development and progression of tumors. Among breast cancer patients, several subgroups have been shown to harbor genetic aberrations in FGFRs, including amplifications of FGFR1, FGFR2, and FGFR4 and mutations in FGFR2 and FGFR4. Here, we review in vitro and in vivo models that have partly elucidated the molecular implications of these different genetic aberrations, the resulting tumor characteristics, and the potential of FGFRs as therapeutic targets for breast cancer treatment.

Targeting brain metastases in ALK-rearranged non-small-cell lung cancer.

PubMed

Zhang, Isabella; Zaorsky, Nicholas G; Palmer, Joshua D; Mehra, Ranee; Lu, Bo

2015-10-01

The incidence of brain metastases has increased as a result of improved systemic control and advances in imaging. However, development of novel therapeutics with CNS activity has not advanced at the same rate. Research on molecular markers has revealed many potential targets for antineoplastic agents, and a particularly important aberration is translocation in the ALK gene, identified in non-small-cell lung cancer (NSCLC). ALK inhibitors have shown systemic efficacy against ALK-rearranged NSCLC in many clinical trials, but the effectiveness of crizotinib in CNS disease is limited by poor blood-brain barrier penetration and acquired drug resistance. In this Review, we discuss potential pathways to target ALK-rearranged brain metastases, including next generation ALK inhibitors with greater CNS penetration and mechanisms to overcome resistance. Other important mechanisms to control CNS disease include targeting pathways downstream of ALK phosphorylation, increasing the permeability of the blood-brain barrier, modifying the tumour microenvironment, and adding concurrent radiotherapy. Copyright © 2015 Elsevier Ltd. All rights reserved.

A Pan-Cancer Proteogenomic Atlas of PI3K/AKT/mTOR Pathway Alterations | Office of Cancer Genomics

Cancer.gov

Molecular alterations involving the PI3K/Akt/mTOR pathway (including mutation, copy number, protein, or RNA) were examined across 11,219 human cancers representing 32 major types. Within specific mutated genes, frequency, mutation hotspot residues, in silico predictions, and functional assays were all informative in distinguishing the subset of genetic variants more likely to have functional relevance. Multiple oncogenic pathways including PI3K/Akt/mTOR converged on similar sets of downstream transcriptional targets.

Motor Cortical Visuomotor Feedback Activity Is Initially Isolated from Downstream Targets in Output-Null Neural State Space Dimensions.

PubMed

Stavisky, Sergey D; Kao, Jonathan C; Ryu, Stephen I; Shenoy, Krishna V

2017-07-05

Neural circuits must transform new inputs into outputs without prematurely affecting downstream circuits while still maintaining other ongoing communication with these targets. We investigated how this isolation is achieved in the motor cortex when macaques received visual feedback signaling a movement perturbation. To overcome limitations in estimating the mapping from cortex to arm movements, we also conducted brain-machine interface (BMI) experiments where we could definitively identify neural firing patterns as output-null or output-potent. This revealed that perturbation-evoked responses were initially restricted to output-null patterns that cancelled out at the neural population code readout and only later entered output-potent neural dimensions. This mechanism was facilitated by the circuit's large null space and its ability to strongly modulate output-potent dimensions when generating corrective movements. These results show that the nervous system can temporarily isolate portions of a circuit's activity from its downstream targets by restricting this activity to the circuit's output-null neural dimensions. Copyright © 2017 Elsevier Inc. All rights reserved.

Perturbation biology nominates upstream-downstream drug combinations in RAF inhibitor resistant melanoma cells.

PubMed

Korkut, Anil; Wang, Weiqing; Demir, Emek; Aksoy, Bülent Arman; Jing, Xiaohong; Molinelli, Evan J; Babur, Özgün; Bemis, Debra L; Onur Sumer, Selcuk; Solit, David B; Pratilas, Christine A; Sander, Chris

2015-08-18

Resistance to targeted cancer therapies is an important clinical problem. The discovery of anti-resistance drug combinations is challenging as resistance can arise by diverse escape mechanisms. To address this challenge, we improved and applied the experimental-computational perturbation biology method. Using statistical inference, we build network models from high-throughput measurements of molecular and phenotypic responses to combinatorial targeted perturbations. The models are computationally executed to predict the effects of thousands of untested perturbations. In RAF-inhibitor resistant melanoma cells, we measured 143 proteomic/phenotypic entities under 89 perturbation conditions and predicted c-Myc as an effective therapeutic co-target with BRAF or MEK. Experiments using the BET bromodomain inhibitor JQ1 affecting the level of c-Myc protein and protein kinase inhibitors targeting the ERK pathway confirmed the prediction. In conclusion, we propose an anti-cancer strategy of co-targeting a specific upstream alteration and a general downstream point of vulnerability to prevent or overcome resistance to targeted drugs.

Integrative analysis of DNA methylation and gene expression data identifies EPAS1 as a key regulator of COPD.

PubMed

Yoo, Seungyeul; Takikawa, Sachiko; Geraghty, Patrick; Argmann, Carmen; Campbell, Joshua; Lin, Luan; Huang, Tao; Tu, Zhidong; Foronjy, Robert F; Feronjy, Robert; Spira, Avrum; Schadt, Eric E; Powell, Charles A; Zhu, Jun

2015-01-01

Chronic Obstructive Pulmonary Disease (COPD) is a complex disease. Genetic, epigenetic, and environmental factors are known to contribute to COPD risk and disease progression. Therefore we developed a systematic approach to identify key regulators of COPD that integrates genome-wide DNA methylation, gene expression, and phenotype data in lung tissue from COPD and control samples. Our integrative analysis identified 126 key regulators of COPD. We identified EPAS1 as the only key regulator whose downstream genes significantly overlapped with multiple genes sets associated with COPD disease severity. EPAS1 is distinct in comparison with other key regulators in terms of methylation profile and downstream target genes. Genes predicted to be regulated by EPAS1 were enriched for biological processes including signaling, cell communications, and system development. We confirmed that EPAS1 protein levels are lower in human COPD lung tissue compared to non-disease controls and that Epas1 gene expression is reduced in mice chronically exposed to cigarette smoke. As EPAS1 downstream genes were significantly enriched for hypoxia responsive genes in endothelial cells, we tested EPAS1 function in human endothelial cells. EPAS1 knockdown by siRNA in endothelial cells impacted genes that significantly overlapped with EPAS1 downstream genes in lung tissue including hypoxia responsive genes, and genes associated with emphysema severity. Our first integrative analysis of genome-wide DNA methylation and gene expression profiles illustrates that not only does DNA methylation play a 'causal' role in the molecular pathophysiology of COPD, but it can be leveraged to directly identify novel key mediators of this pathophysiology.

Deptor Is a Novel Target of Wnt/β-Catenin/c-Myc and Contributes to Colorectal Cancer Cell Growth.

PubMed

Wang, Qingding; Zhou, Yuning; Rychahou, Piotr; Harris, Jennifer W; Zaytseva, Yekaterina Y; Liu, Jinpeng; Wang, Chi; Weiss, Heidi L; Liu, Chunming; Lee, Eun Y; Evers, B Mark

2018-06-15

Activation of the Wnt/β-catenin signaling pathway drives colorectal cancer growth by deregulating expression of downstream target genes, including the c-myc proto-oncogene. The critical targets that mediate the functions of oncogenic c-Myc in colorectal cancer have yet to be fully elucidated. Previously, we showed that activation of PI3K/Akt/mTOR contributes to colorectal cancer growth and metastasis. Here, we show that Deptor, a suppressor of mTOR, is a direct target of Wnt/β-catenin/c-Myc signaling in colorectal cancer cells. Inhibition of Wnt/β-catenin or knockdown of c-Myc decreased, while activation of Wnt/β-catenin or overexpression of c-Myc increased the expression of Deptor. c-Myc bound the promoter of Deptor and transcriptionally regulated Deptor expression. Inhibition of Wnt/β-catenin/c-Myc signaling increased mTOR activation, and the combination of Wnt and Akt/mTOR inhibitors enhanced inhibition of colorectal cancer cell growth in vitro and in vivo Deptor expression was increased in colorectal cancer cells; knockdown of Deptor induced differentiation, decreased expression of B lymphoma Mo-MLV insertion region 1 (Bmi1), and decreased proliferation in colorectal cancer cell lines and primary human colorectal cancer cells. Importantly, our work identifies Deptor as a downstream target of the Wnt/β-catenin/c-Myc signaling pathway, acting as a tumor promoter in colorectal cancer cells. Moreover, we provide a molecular basis for the synergistic combination of Wnt and mTOR inhibitors in treating colorectal cancer with elevated c-Myc. Significance: The mTOR inhibitor DEPTOR acts as a tumor promoter and could be a potential therapeutic target in colorectal cancer. Cancer Res; 78(12); 3163-75. ©2018 AACR . ©2018 American Association for Cancer Research.

Identification and Validation of Ifit1 as an Important Innate Immune Bottleneck

DOE Office of Scientific and Technical Information (OSTI.GOV)

McDermott, Jason E.; Vartanian, Keri B.; Mitchell, Hugh D.

The innate immune system plays important roles in a number of disparate processes. Foremost, innate immunity is a first responder to invasion by pathogens and triggers early defensive responses and recruits the adaptive immune system. The innate immune system also responds to endogenous damage signals that arise from tissue injury. Recently it has been found that innate immunity plays an important role in neuroprotection against ischemic stroke through the activation of the primary innate immune receptors, Toll-like receptors (TLRs). Using several large-scale transcriptomic data sets from mouse and mouse macrophage studies we identified targets predicted to be important in controllingmore » innate immune processes initiated by TLR activation. Targets were identified as genes with high betweenness centrality, so-called bottlenecks, in networks inferred from statistical associations between gene expression patterns. A small set of putative bottlenecks were identified in each of the data sets investigated including interferon-stimulated genes (Ifit1, Ifi47, Tgtp and Oasl2) as well as genes uncharacterized in immune responses (Axud1 and Ppp1r15a). We further validated one of these targets, Ifit1, in mouse macrophages by showing that silencing it suppresses induction of predicted downstream genes by lipopolysaccharide (LPS)-mediated TLR4 activation through an unknown direct or indirect mechanism. Our study demonstrates the utility of network analysis for identification of interesting targets related to innate immune function, and highlights that Ifit1 can exert a positive regulatory effect on downstream genes.« less

VHL and Hypoxia Signaling: Beyond HIF in Cancer

PubMed Central

Zhang, Jing

2018-01-01

Von Hippel-Lindau (VHL) is an important tumor suppressor that is lost in the majority of clear cell carcinoma of renal cancer (ccRCC). Its regulatory pathway involves the activity of E3 ligase, which targets hypoxia inducible factor α (including HIF1α and HIF2α) for proteasome degradation. In recent years, emerging literature suggests that VHL also possesses other HIF-independent functions. This review will focus on VHL-mediated signaling pathways involving the latest identified substrates/binding partners, including N-Myc downstream-regulated gene 3 (NDRG3), AKT, and G9a, etc., and their physiological roles in hypoxia signaling and cancer. We will also discuss the crosstalk between VHL and NF-κB signaling. Lastly, we will review the latest findings on targeting VHL signaling in cancer. PMID:29562667

Single-step affinity purification of enzyme biotherapeutics: a platform methodology for accelerated process development.

PubMed

Brower, Kevin P; Ryakala, Venkat K; Bird, Ryan; Godawat, Rahul; Riske, Frank J; Konstantinov, Konstantin; Warikoo, Veena; Gamble, Jean

2014-01-01

Downstream sample purification for quality attribute analysis is a significant bottleneck in process development for non-antibody biologics. Multi-step chromatography process train purifications are typically required prior to many critical analytical tests. This prerequisite leads to limited throughput, long lead times to obtain purified product, and significant resource requirements. In this work, immunoaffinity purification technology has been leveraged to achieve single-step affinity purification of two different enzyme biotherapeutics (Fabrazyme® [agalsidase beta] and Enzyme 2) with polyclonal and monoclonal antibodies, respectively, as ligands. Target molecules were rapidly isolated from cell culture harvest in sufficient purity to enable analysis of critical quality attributes (CQAs). Most importantly, this is the first study that demonstrates the application of predictive analytics techniques to predict critical quality attributes of a commercial biologic. The data obtained using the affinity columns were used to generate appropriate models to predict quality attributes that would be obtained after traditional multi-step purification trains. These models empower process development decision-making with drug substance-equivalent product quality information without generation of actual drug substance. Optimization was performed to ensure maximum target recovery and minimal target protein degradation. The methodologies developed for Fabrazyme were successfully reapplied for Enzyme 2, indicating platform opportunities. The impact of the technology is significant, including reductions in time and personnel requirements, rapid product purification, and substantially increased throughput. Applications are discussed, including upstream and downstream process development support to achieve the principles of Quality by Design (QbD) as well as integration with bioprocesses as a process analytical technology (PAT). © 2014 American Institute of Chemical Engineers.

Isodeoxyelephantopin induces protective autophagy in lung cancer cells via Nrf2-p62-keap1 feedback loop

PubMed Central

Wang, Yang; Zhang, Jing; Huang, Zhi-Hao; Huang, Xiao-Hui; Zheng, Wei-Bin; Yin, Xing-Feng; Li, Yao-Lan; Li, Bin; He, Qing-Yu

2017-01-01

Isodeoxyelephantopin (ESI), isolated from Elephantopus scaber L. has been reported to exert anticancer effects. In this study, we aimed to investigate whether and how cancer cells exert protective responses against ESI treatment. Confocal fluorescence microscopy showed that ESI significantly induced autophagy flux in the lung cancer cells expressing mCherry-EGFP-LC3 reporter. Treatment of the cells with ESI increased the expression levels of the autophagy markers including LC3-II, ATG3 and Beclin1 in a dose-dependent manner. Pretreatment with autophagy inhibitor 3-methyladenine (3-MA) not only attenuated the effects of ESI on autophagy, but also enhanced the effects of ESI on cell viability and apoptosis. Mechanistically, the SILAC quantitative proteomics coupled with bioinformatics analysis revealed that the ESI-regulated proteins were mainly involved in Nrf2-mediated oxidative stress response. We found that ESI induced the nuclear translocation of Nrf2 for activating the downstream target genes including HO-1 and p62 (SQSTM1). More importantly, ESI-induced p62 could competitively bind with Keap1, and releases Nrf2 to activate downstream target gene p62 as a positive feedback loop, therefore promoting autophagy. Furthermore, knockdown of Nrf2 or p62 could abrogate the ESI-induced autophagy and significantly enhanced the anticancer effect of ESI. Taken together, we demonstrated that ESI can sustain cell survival by activating protective autophagy through Nrf2-p62-keap1 feedback loop, whereas targeting this regulatory axis combined with ESI treatment may be a promising strategy for anticancer therapy. PMID:28617433

Isodeoxyelephantopin induces protective autophagy in lung cancer cells via Nrf2-p62-keap1 feedback loop.

PubMed

Wang, Yang; Zhang, Jing; Huang, Zhi-Hao; Huang, Xiao-Hui; Zheng, Wei-Bin; Yin, Xing-Feng; Li, Yao-Lan; Li, Bin; He, Qing-Yu

2017-06-15

Isodeoxyelephantopin (ESI), isolated from Elephantopus scaber L. has been reported to exert anticancer effects. In this study, we aimed to investigate whether and how cancer cells exert protective responses against ESI treatment. Confocal fluorescence microscopy showed that ESI significantly induced autophagy flux in the lung cancer cells expressing mCherry-EGFP-LC3 reporter. Treatment of the cells with ESI increased the expression levels of the autophagy markers including LC3-II, ATG3 and Beclin1 in a dose-dependent manner. Pretreatment with autophagy inhibitor 3-methyladenine (3-MA) not only attenuated the effects of ESI on autophagy, but also enhanced the effects of ESI on cell viability and apoptosis. Mechanistically, the SILAC quantitative proteomics coupled with bioinformatics analysis revealed that the ESI-regulated proteins were mainly involved in Nrf2-mediated oxidative stress response. We found that ESI induced the nuclear translocation of Nrf2 for activating the downstream target genes including HO-1 and p62 (SQSTM1). More importantly, ESI-induced p62 could competitively bind with Keap1, and releases Nrf2 to activate downstream target gene p62 as a positive feedback loop, therefore promoting autophagy. Furthermore, knockdown of Nrf2 or p62 could abrogate the ESI-induced autophagy and significantly enhanced the anticancer effect of ESI. Taken together, we demonstrated that ESI can sustain cell survival by activating protective autophagy through Nrf2-p62-keap1 feedback loop, whereas targeting this regulatory axis combined with ESI treatment may be a promising strategy for anticancer therapy.

Hcfc1b, a zebrafish ortholog of HCFC1, regulates craniofacial development by modulating mmachc expression.

PubMed

Quintana, Anita M; Geiger, Elizabeth A; Achilly, Nate; Rosenblatt, David S; Maclean, Kenneth N; Stabler, Sally P; Artinger, Kristin B; Appel, Bruce; Shaikh, Tamim H

2014-12-01

Mutations in HCFC1 (MIM300019), have been recently associated with cblX (MIM309541), an X-linked, recessive disorder characterized by multiple congenital anomalies including craniofacial abnormalities. HCFC1 is a transcriptional co-regulator that modulates the expression of numerous downstream target genes including MMACHC, but it is not clear how these HCFC1 targets play a role in the clinical manifestations of cblX. To begin to elucidate the mechanism by which HCFC1 modulates disease phenotypes, we have carried out loss of function analyses in the developing zebrafish. Of the two HCFC1 orthologs in zebrafish, hcfc1a and hcfc1b, the loss of hcfc1b specifically results in defects in craniofacial development. Subsequent analysis revealed that hcfc1b regulates cranial neural crest cell differentiation and proliferation within the posterior pharyngeal arches. Further, the hcfc1b-mediated craniofacial abnormalities were rescued by expression of human MMACHC, a downstream target of HCFC1 that is aberrantly expressed in cblX. Furthermore, we tested distinct human HCFC1 mutations for their role in craniofacial development and demonstrated variable effects on MMACHC expression in humans and craniofacial development in zebrafish. Notably, several individuals with mutations in either HCFC1 or MMACHC have been reported to have mild to moderate facial dysmorphia. Thus, our data demonstrates that HCFC1 plays a role in craniofacial development, which is in part mediated through the regulation of MMACHC expression. Copyright © 2014 Elsevier Inc. All rights reserved.

Human myostatin negatively regulates human myoblast growth and differentiation

PubMed Central

McFarlane, Craig; Hui, Gu Zi; Amanda, Wong Zhi Wei; Lau, Hiu Yeung; Lokireddy, Sudarsanareddy; XiaoJia, Ge; Mouly, Vincent; Butler-Browne, Gillian; Gluckman, Peter D.; Sharma, Mridula

2011-01-01

Myostatin, a member of the transforming growth factor-β superfamily, has been implicated in the potent negative regulation of myogenesis in murine models. However, little is known about the mechanism(s) through which human myostatin negatively regulates human skeletal muscle growth. Using human primary myoblasts and recombinant human myostatin protein, we show here that myostatin blocks human myoblast proliferation by regulating cell cycle progression through targeted upregulation of p21. We further show that myostatin regulates myogenic differentiation through the inhibition of key myogenic regulatory factors including MyoD, via canonical Smad signaling. In addition, we have for the first time demonstrated the capability of myostatin to regulate the Notch signaling pathway during inhibition of human myoblast differentiation. Treatment with myostatin results in the upregulation of Hes1, Hes5, and Hey1 expression during differentiation; moreover, when we interfere with Notch signaling, through treatment with the γ-secretase inhibitor L-685,458, we find enhanced myotube formation despite the presence of excess myostatin. Therefore, blockade of the Notch pathway relieves myostatin repression of differentiation, and myostatin upregulates Notch downstream target genes. Immunoprecipitation studies demonstrate that myostatin treatment of myoblasts results in enhanced association of Notch1-intracellular domain with Smad3, providing an additional mechanism through which myostatin targets and represses the activity of the myogenic regulatory factor MyoD. On the basis of these results, we suggest that myostatin function and mechanism of action are very well conserved between species, and that myostatin regulation of postnatal myogenesis involves interactions with numerous downstream signaling mediators, including the Notch pathway. PMID:21508334

Human myostatin negatively regulates human myoblast growth and differentiation.

PubMed

McFarlane, Craig; Hui, Gu Zi; Amanda, Wong Zhi Wei; Lau, Hiu Yeung; Lokireddy, Sudarsanareddy; Xiaojia, Ge; Mouly, Vincent; Butler-Browne, Gillian; Gluckman, Peter D; Sharma, Mridula; Kambadur, Ravi

2011-07-01

Myostatin, a member of the transforming growth factor-β superfamily, has been implicated in the potent negative regulation of myogenesis in murine models. However, little is known about the mechanism(s) through which human myostatin negatively regulates human skeletal muscle growth. Using human primary myoblasts and recombinant human myostatin protein, we show here that myostatin blocks human myoblast proliferation by regulating cell cycle progression through targeted upregulation of p21. We further show that myostatin regulates myogenic differentiation through the inhibition of key myogenic regulatory factors including MyoD, via canonical Smad signaling. In addition, we have for the first time demonstrated the capability of myostatin to regulate the Notch signaling pathway during inhibition of human myoblast differentiation. Treatment with myostatin results in the upregulation of Hes1, Hes5, and Hey1 expression during differentiation; moreover, when we interfere with Notch signaling, through treatment with the γ-secretase inhibitor L-685,458, we find enhanced myotube formation despite the presence of excess myostatin. Therefore, blockade of the Notch pathway relieves myostatin repression of differentiation, and myostatin upregulates Notch downstream target genes. Immunoprecipitation studies demonstrate that myostatin treatment of myoblasts results in enhanced association of Notch1-intracellular domain with Smad3, providing an additional mechanism through which myostatin targets and represses the activity of the myogenic regulatory factor MyoD. On the basis of these results, we suggest that myostatin function and mechanism of action are very well conserved between species, and that myostatin regulation of postnatal myogenesis involves interactions with numerous downstream signaling mediators, including the Notch pathway.

BLISTER Regulates Polycomb-Target Genes, Represses Stress-Regulated Genes and Promotes Stress Responses in Arabidopsis thaliana.

PubMed

Kleinmanns, Julia A; Schatlowski, Nicole; Heckmann, David; Schubert, Daniel

2017-01-01

HIGHLIGHTS The PRC2 interacting protein BLISTER likely acts downstream of PRC2 to silence Polycomb target genes and is a key regulator of specific stress responses in Arabidopsis . Polycomb group (PcG) proteins are key epigenetic regulators of development. The highly conserved Polycomb repressive complex 2 (PRC2) represses thousands of target genes by trimethylating H3K27 (H3K27me3). Plant specific PcG components and functions are largely unknown, however, we previously identified the plant-specific protein BLISTER (BLI) as a PRC2 interactor. BLI regulates PcG target genes and promotes cold stress resistance. To further understand the function of BLI , we analyzed the transcriptional profile of bli-1 mutants. Approximately 40% of the up-regulated genes in bli are PcG target genes, however, bli-1 mutants did not show changes in H3K27me3 levels at all tested genes, indicating that BLI regulates PcG target genes downstream of or in parallel to PRC2. Interestingly, a significant number of BLI regulated H3K27me3 target genes is regulated by the stress hormone absciscic acid (ABA). We further reveal an overrepresentation of genes responding to abiotic stresses such as drought, high salinity, or heat stress among the up-regulated genes in bli mutants. Consistently, bli mutants showed reduced desiccation stress tolerance. We conclude that the PRC2 associated protein BLI is a key regulator of stress-responsive genes in Arabidopsis : it represses ABA-responsive PcG target genes, likely downstream of PRC2, and promotes resistance to several stresses such as cold and drought.

Novel Molecular Strategies and Targets for Opioid Drug Discovery for the Treatment of Chronic Pain

PubMed Central

Olson, Keith M.; Lei, Wei; Keresztes, Attila; LaVigne, Justin; Streicher, John M.

2017-01-01

Opioid drugs like morphine and fentanyl are the gold standard for treating moderate to severe acute and chronic pain. However, opioid drug use can be limited by serious side effects, including constipation, tolerance, respiratory suppression, and addiction. For more than 100 years, we have tried to develop opioids that decrease or eliminate these liabilities, with little success. Recent advances in understanding opioid receptor signal transduction have suggested new possibilities to activate the opioid receptors to cause analgesia, while reducing or eliminating unwanted side effects. These new approaches include designing functionally selective ligands, which activate desired signaling cascades while avoiding signaling cascades that are thought to provoke side effects. It may also be possible to directly modulate downstream signaling through the use of selective activators and inhibitors. Separate from downstream signal transduction, it has also been found that when the opioid system is stimulated, various negative feedback systems are upregulated to compensate, which can drive side effects. This has led to the development of multi-functional molecules that simultaneously activate the opioid receptor while blocking various negative feedback receptor systems including cholecystokinin and neurokinin-1. Other novel approaches include targeting heterodimers of the opioid and other receptor systems which may drive side effects, and making endogenous opioid peptides druggable, which may also reduce opioid mediated side effects. Taken together, these advances in our molecular understanding provide a path forward to break the barrier in producing an opioid with reduced or eliminated side effects, especially addiction, which may provide relief for millions of patients. PMID:28356897

Detection of Genes Regulated by Lmx1b During Limb Dorsalization

PubMed Central

Feenstra, Jennifer M.; Kanaya, Kohei; Pira, Charmaine U; Hoffman, Sarah E.; Eppey, Richard J.; Oberg, Kerby C.

2012-01-01

Lmx1b is a homeodomain transcription factor that regulates dorsal identity during limb development. Lmx1b knockout (KO) mice develop distal ventral-ventral limbs. Although induction of Lmx1b is linked to Wnt7a expression in the dorsal limb ectoderm, the downstream targets of Lmx1b that accomplish limb dorsalization are unknown. To identify genes targeted by Lmx1b, we compared gene arrays from Lmx1b KO and wildtype mouse limbs during limb dorsalization, i.e., 11.5, 12.5, and 13.5 days post coitum. We identified 54 target genes differentially expressed in all three stages. Several skeletal targets, including Emx2, Matrilin1 and Matrilin4, demonstrated a loss of scapular expression in the Lmx1b KO mice, supporting a role for Lmx1b in scapula development. Furthermore, the relative abundance of extracellular matrix-related soft tissue targets regulated by Lmx1b, such as collagens and proteoglycans, suggests a mechanism which includes changes in the extracellular matrix composition to accomplish limb dorsalization. Our study provides the most comprehensive characterization of genes regulated by Lmx1b during limb development to-date and provides targets for further investigation. PMID:22417325

Auditory cortex stimulation to suppress tinnitus: mechanisms and strategies.

PubMed

Zhang, Jinsheng

2013-01-01

Brain stimulation is an important method used to modulate neural activity and suppress tinnitus. Several auditory and non-auditory brain regions have been targeted for stimulation. This paper reviews recent progress on auditory cortex (AC) stimulation to suppress tinnitus and its underlying neural mechanisms and stimulation strategies. At the same time, the author provides his opinions and hypotheses on both animal and human models. The author also proposes a medial geniculate body (MGB)-thalamic reticular nucleus (TRN)-Gating mechanism to reflect tinnitus-related neural information coming from upstream and downstream projection structures. The upstream structures include the lower auditory brainstem and midbrain structures. The downstream structures include the AC and certain limbic centers. Both upstream and downstream information is involved in a dynamic gating mechanism in the MGB together with the TRN. When abnormal gating occurs at the thalamic level, the spilled-out information interacts with the AC to generate tinnitus. The tinnitus signals at the MGB-TRN-Gating may be modulated by different forms of stimulations including brain stimulation. Each stimulation acts as a gain modulator to control the level of tinnitus signals at the MGB-TRN-Gate. This hypothesis may explain why different types of stimulation can induce tinnitus suppression. Depending on the tinnitus etiology, MGB-TRN-Gating may be different in levels and dynamics, which cause variability in tinnitus suppression induced by different gain controllers. This may explain why the induced suppression of tinnitus by one type of stimulation varies across individual patients. Copyright © 2012. Published by Elsevier B.V.

Inhibiting 4EBP1 in Glioblastoma. | Office of Cancer Genomics

Cancer.gov

Glioblastoma is the most common and aggressive adult brain cancer. Tumors show frequent dysregulation of the PI3K-mTOR pathway. Although a number of small molecules target the PI3K-AKT-mTOR axis, their preclinical and clinical efficacy has been limited. Reasons for treatment failure include poor penetration of agents into the brain and observations that blockade of PI3K or AKT minimally affects downstream mTOR activity in glioma.

Occurrence of pharmaceuticals and other organic wastewater constituents in selected streams in northern Arkansas, 2004

USGS Publications Warehouse

Galloway, Joel M.; Haggard, Brian E.; Meyers, Michael T.; Green, W. Reed

2005-01-01

The U.S. Geological Survey, in cooperation with the University of Arkansas and the U.S. Department of Agriculture, Agricultural Research Service, collected data in 2004 to determine the occurrence of pharmaceuticals and other organic wastewater constituents, including many constituents of emerging environmental concern, in selected streams in northern Arkansas. Samples were collected in March and April 2004 from 17 sites located upstream and downstream from wastewater- treatment plant effluent discharges on 7 streams in northwestern Arkansas and at 1 stream site in a relatively undeveloped basin in north-central Arkansas. Additional samples were collected at three of the sites in August 2004. The targeted organic wastewater constituents and sample sites were selected because wastewater-treatment plant effluent discharge provides a potential point source of these constituents and analytical techniques have improved to accurately measure small amounts of these constituents in environmental samples. At least 1 of the 108 pharmaceutical or other organic wastewater constituents was detected at all sites in 2004, except at Spavinaw Creek near Maysville, Arkansas. The number of detections generally was greater at sites downstream from municipal wastewater-treatment plant effluent discharges (mean = 14) compared to sites not influenced by wastewatertreatment plants (mean = 3). Overall, 42 of the 108 constituents targeted in the collected water-quality samples were detected. The most frequently detected constituents included caffeine, phenol, para-cresol, and acetyl hexamethyl tetrahydro naphthalene.

Lateral and vertical distribution of downstream migrating juvenile sea lamprey

USGS Publications Warehouse

Sotola, V. Alex; Miehls, Scott M.; Simard, Lee G.; Marsden, J. Ellen

2018-01-01

Sea lamprey is considered an invasive and nuisance species in the Laurentian Great Lakes, Lake Champlain, and the Finger Lakes of New York and is a major focus of control efforts. Currently, management practices focus on limiting the area of infestation using barriers to block migratory adults, and lampricides to kill ammocoetes in infested tributaries. No control efforts currently target the downstream-migrating post-metamorphic life stage which could provide another management opportunity. In order to apply control methods to this life stage, a better understanding of their downstream movement patterns is needed. To quantify spatial distribution of downstream migrants, we deployed fyke and drift nets laterally and vertically across the stream channel in two tributaries of Lake Champlain. Sea lamprey was not randomly distributed across the stream width and lateral distribution showed a significant association with discharge. Results indicated that juvenile sea lamprey is most likely to be present in the thalweg and at midwater depths of the stream channel. Further, a majority of the catch occurred during high flow events, suggesting an increase in downstream movement activity when water levels are higher than base flow. Discharge and flow are strong predictors of the distribution of out-migrating sea lamprey, thus managers will need to either target capture efforts in high discharge areas of streams or develop means to guide sea lamprey away from these areas.

6-Shogaol, an active constituent of dietary ginger, induces autophagy by inhibiting the AKT/mTOR pathway in human non-small cell lung cancer A549 cells.

PubMed

Hung, Jen-Yu; Hsu, Ya-Ling; Li, Chien-Te; Ko, Ying-Chin; Ni, Wen-Chiu; Huang, Ming-Shyan; Kuo, Po-Lin

2009-10-28

This study is the first study to investigate the anticancer effect of 6-shogaol in human non-small cell lung cancer A549 cells. 6-Shogaol inhibited cell proliferation by inducing autophagic cell death, but not, predominantly, apoptosis. Pretreatment of cells with 3-methyladenine (3-MA), an autophagy inhibitor, suppressed 6-shogaol mediated antiproliferation activity, suggesting that induction of autophagy by 6-shogaol is conducive to cell death. We also found that 6-shogaol inhibited survival signaling through the AKT/mTOR signaling pathway by blocking the activation of AKT and downstream targets, including the mammalian target of rapamycin (mTOR), forkhead transcription factors (FKHR) and glycogen synthase kinase-3beta (GSK-3beta). Phosphorylation of both of mTOR's downstream targets, p70 ribosomal protein S6 kinase (p70S6 kinase) and 4E-BP1, was also diminished. Overexpression of AKT by AKT cDNA transfection decreased 6-shogaol mediated autophagic cell death, supporting inhibition of AKT beneficial to autophagy. Moreover, reduction of AKT expression by siRNA potentiated 6-shogaol's effect, also supporting inhibition of AKT beneficial to autophagy. Taken together, these findings suggest that 6-shogaol may be a promising chemopreventive agent against human non-small cell lung cancer.

Upstream water resource management to address downstream pollution concerns: A policy framework with application to the Nakdong River basin in South Korea

NASA Astrophysics Data System (ADS)

Yoon, Taeyeon; Rhodes, Charles; Shah, Farhed A.

2015-02-01

An empirical framework for assisting with water quality management is proposed that relies on open-source hydrologic data. Such data are measured periodically at fixed water stations and commonly available in time-series form. To fully exploit the data, we suggest that observations from multiple stations should be combined into a single long-panel data set, and an econometric model developed to estimate upstream management effects on downstream water quality. Selection of the model's functional form and explanatory variables would be informed by rating curves, and idiosyncrasies across and within stations handled in an error term by testing contemporary correlation, serial correlation, and heteroskedasticity. Our proposed approach is illustrated with an application to the Nakdong River basin in South Korea. Three alternative policies to achieve downstream BOD level targets are evaluated: upstream water treatment, greater dam discharge, and development of a new water source. Upstream water treatment directly cuts off incoming pollutants, thereby presenting the smallest variation in its downstream effects on BOD levels. Treatment is advantageous when reliability of water quality is a primary concern. Dam discharge is a flexible tool, and may be used strategically during a low-flow season. We consider development of a new water corridor from an extant dam as our third policy option. This turns out to be the most cost-effective way for securing lower BOD levels in the downstream target city. Even though we consider a relatively simple watershed to illustrate the usefulness of our approach, it can be adapted easily to analyze more complex upstream-downstream issues.

Proliferation of murine c-kit(pos) cardiac stem cells stimulated with IGF-1 is associated with Akt-1 mediated phosphorylation and nuclear export of FoxO3a and its effect on downstream cell cycle regulators.

PubMed

Johnson, Ann Mary; Kartha, C C

2014-04-01

Insulin-like growth factor-1 (IGF-1) is known to promote proliferation in many cell types including c-kit(pos) cardiac stem cells (CSCs). Downstream signaling pathways of IGF-1 induced CSC proliferation have not been investigated. An important downstream target of IGF-1/Akt-1 signaling is FoxO3a, a key negative regulator of cell-cycle progression. We studied the effect of IGF-1 on proliferation of c-kit(pos) murine CSCs and found that IGF-1-mediated cell proliferation is associated with FoxO3a phosphorylation and inactivation of its transcriptional activity. PI3 inhibitors LY294002 and Wortmannin abolished the effect of IGF-1 on FoxO3a phosphorylation indicating that FoxO3a phosphorylation is mediated by PI3/Akt-1 pathway. In cells with FoxO3a translocation to the cytoplasm, there is decreased expression of cell-cycle inhibitors such as p27(kip1) and p57(kip2) and increased expression of CyclinD1. Our study provides evidence that IGF-1 induced CSC proliferation could be the result of FoxO3a inactivation and its downstream effect on cell-cycle regulators.

Effects of inhibitors of vascular endothelial growth factor receptor 2 and downstream pathways of receptor tyrosine kinases involving phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin or mitogen-activated protein kinase in canine hemangiosarcoma cell lines.

PubMed

Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi

2016-07-01

Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application.

Effects of inhibitors of vascular endothelial growth factor receptor 2 and downstream pathways of receptor tyrosine kinases involving phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin or mitogen-activated protein kinase in canine hemangiosarcoma cell lines

PubMed Central

Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi

2016-01-01

Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application. PMID:27408334

Akt Regulates TNFα Synthesis Downstream of RIP1 Kinase Activation during Necroptosis

PubMed Central

McNamara, Colleen R.; Ahuja, Ruchita; Osafo-Addo, Awo D.; Barrows, Douglas; Kettenbach, Arminja; Skidan, Igor; Teng, Xin; Cuny, Gregory D.; Gerber, Scott; Degterev, Alexei

2013-01-01

Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation. PMID:23469174

Mercury Handling for the Target System for a Muon Collider

DOE Office of Scientific and Technical Information (OSTI.GOV)

Graves, Van B; Mcdonald, K; Kirk, H.

2012-01-01

The baseline target concept for a Muon Collider or Neutrino Factory is a free-stream mercury jet being impacted by an 8-GeV proton beam. The target is located within a 20-T magnetic field, which captures the generated pions that are conducted to a downstream decay channel. Both the mercury and the proton beam are introduced at slight downward angles to the magnetic axis. A pool of mercury serves as a receiving reservoir for the mercury and a dump for the unexpended proton beam. The impact energy of the remaining beam and jet are substantial, and it is required that splashes andmore » waves be controlled in order to minimize the potential for interference of pion production at the target. Design issues discussed in this paper include the nozzle, splash mitigation in the mercury pool, the mercury containment vessel, and the mercury recirculation system.« less

Anticancer molecules targeting fibroblast growth factor receptors.

PubMed

Liang, Guang; Liu, Zhiguo; Wu, Jianzhang; Cai, Yuepiao; Li, Xiaokun

2012-10-01

The fibroblast growth factor receptor (FGFR) family includes four highly conserved receptor tyrosine kinases: FGFR1-4. Upon ligand binding, FGFRs activate an array of downstream signaling pathways, such as the mitogen activated protein kinase (MAPK) and the phosphoinositide-3-kinase (PI3K)/Akt pathways. These FGFR cascades play crucial roles in tumor cell proliferation, angiogenesis, migration, and survival. The combination of knockdown studies and pharmaceutical inhibition in preclinical models demonstrates that FGFRs are attractive targets for therapeutic intervention in cancer. Multiple FGFR inhibitors with various structural skeletons have been designed, synthesized, and evaluated. Reviews on FGFRs have recently focused on FGFR signaling, pathophysiology, and functions in cancer or other diseases. In this article, we review recent advances in structure-activity relationships (SAR) of FGFR inhibitors, as well as the FGFR-targeting drug design strategies currently employed in targeting deregulated FGFRs by antibodies and small molecule inhibitors. Copyright © 2012 Elsevier Ltd. All rights reserved.

Local Circuits of V1 Layer 4B Neurons Projecting to V2 Thick Stripes Define Distinct Cell Classes and Avoid Cytochrome Oxidase Blobs

PubMed Central

Yarch, Jeff; Federer, Frederick

2017-01-01

Decades of anatomical studies on the primate primary visual cortex (V1) have led to a detailed diagram of V1 intrinsic circuitry, but this diagram lacks information about the output targets of V1 cells. Understanding how V1 local processing relates to downstream processing requires identification of neuronal populations defined by their output targets. In primates, V1 layers (L)2/3 and 4B send segregated projections to distinct cytochrome oxidase (CO) stripes in area V2: neurons in CO blob columns project to thin stripes while neurons outside blob columns project to thick and pale stripes, suggesting functional specialization of V1-to-V2 CO streams. However, the conventional diagram of V1 shows all L4B neurons, regardless of their soma location in blob or interblob columns, as projecting selectively to CO blobs in L2/3, suggesting convergence of blob/interblob information in L2/3 blobs and, possibly, some V2 stripes. However, it is unclear whether all L4B projection neurons show similar local circuitries. Using viral-mediated circuit tracing, we have identified the local circuits of L4B neurons projecting to V2 thick stripes in macaque. Consistent with previous studies, we found the somata of this L4B subpopulation to reside predominantly outside blob columns; however, unlike previous descriptions of local L4B circuits, these cells consistently projected outside CO blob columns in all layers. Thus, the local circuits of these L4B output neurons, just like their extrinsic projections to V2, preserve CO streams. Moreover, the intra-V1 laminar patterns of axonal projections identify two distinct neuron classes within this L4B subpopulation, including a rare novel neuron type, suggestive of two functionally specialized output channels. SIGNIFICANCE STATEMENT Conventional diagrams of primate primary visual cortex (V1) depict neuronal connections within and between different V1 layers, but lack information about the cells' downstream targets. This information is critical to understanding how local processing in V1 relates to downstream processing. We have identified the local circuits of a population of cells in V1 layer (L)4B that project to area V2. These cells' local circuits differ from classical descriptions of L4B circuits in both the laminar and functional compartments targeted by their axons, and identify two neuron classes. Our results demonstrate that both local intra-V1 and extrinsic V1-to-V2 connections of L4B neurons preserve CO-stream segregation, suggesting that across-stream integration occurs downstream of V1, and that output targets dictate local V1 circuitry. PMID:28077720

Perturbation biology nominates upstream–downstream drug combinations in RAF inhibitor resistant melanoma cells

PubMed Central

Korkut, Anil; Wang, Weiqing; Demir, Emek; Aksoy, Bülent Arman; Jing, Xiaohong; Molinelli, Evan J; Babur, Özgün; Bemis, Debra L; Onur Sumer, Selcuk; Solit, David B; Pratilas, Christine A; Sander, Chris

2015-01-01

Resistance to targeted cancer therapies is an important clinical problem. The discovery of anti-resistance drug combinations is challenging as resistance can arise by diverse escape mechanisms. To address this challenge, we improved and applied the experimental-computational perturbation biology method. Using statistical inference, we build network models from high-throughput measurements of molecular and phenotypic responses to combinatorial targeted perturbations. The models are computationally executed to predict the effects of thousands of untested perturbations. In RAF-inhibitor resistant melanoma cells, we measured 143 proteomic/phenotypic entities under 89 perturbation conditions and predicted c-Myc as an effective therapeutic co-target with BRAF or MEK. Experiments using the BET bromodomain inhibitor JQ1 affecting the level of c-Myc protein and protein kinase inhibitors targeting the ERK pathway confirmed the prediction. In conclusion, we propose an anti-cancer strategy of co-targeting a specific upstream alteration and a general downstream point of vulnerability to prevent or overcome resistance to targeted drugs. DOI: http://dx.doi.org/10.7554/eLife.04640.001 PMID:26284497

Integrative Analysis of DNA Methylation and Gene Expression Data Identifies EPAS1 as a Key Regulator of COPD

PubMed Central

Yoo, Seungyeul; Takikawa, Sachiko; Geraghty, Patrick; Argmann, Carmen; Campbell, Joshua; Lin, Luan; Huang, Tao; Tu, Zhidong; Feronjy, Robert; Spira, Avrum; Schadt, Eric E.; Powell, Charles A.; Zhu, Jun

2015-01-01

Chronic Obstructive Pulmonary Disease (COPD) is a complex disease. Genetic, epigenetic, and environmental factors are known to contribute to COPD risk and disease progression. Therefore we developed a systematic approach to identify key regulators of COPD that integrates genome-wide DNA methylation, gene expression, and phenotype data in lung tissue from COPD and control samples. Our integrative analysis identified 126 key regulators of COPD. We identified EPAS1 as the only key regulator whose downstream genes significantly overlapped with multiple genes sets associated with COPD disease severity. EPAS1 is distinct in comparison with other key regulators in terms of methylation profile and downstream target genes. Genes predicted to be regulated by EPAS1 were enriched for biological processes including signaling, cell communications, and system development. We confirmed that EPAS1 protein levels are lower in human COPD lung tissue compared to non-disease controls and that Epas1 gene expression is reduced in mice chronically exposed to cigarette smoke. As EPAS1 downstream genes were significantly enriched for hypoxia responsive genes in endothelial cells, we tested EPAS1 function in human endothelial cells. EPAS1 knockdown by siRNA in endothelial cells impacted genes that significantly overlapped with EPAS1 downstream genes in lung tissue including hypoxia responsive genes, and genes associated with emphysema severity. Our first integrative analysis of genome-wide DNA methylation and gene expression profiles illustrates that not only does DNA methylation play a ‘causal’ role in the molecular pathophysiology of COPD, but it can be leveraged to directly identify novel key mediators of this pathophysiology. PMID:25569234

PrimerDesign-M: A multiple-alignment based multiple-primer design tool for walking across variable genomes

DOE PAGES

Yoon, Hyejin; Leitner, Thomas

2014-12-17

Analyses of entire viral genomes or mtDNA requires comprehensive design of many primers across their genomes. In addition, simultaneous optimization of several DNA primer design criteria may improve overall experimental efficiency and downstream bioinformatic processing. To achieve these goals, we developed PrimerDesign-M. It includes several options for multiple-primer design, allowing researchers to efficiently design walking primers that cover long DNA targets, such as entire HIV-1 genomes, and that optimizes primers simultaneously informed by genetic diversity in multiple alignments and experimental design constraints given by the user. PrimerDesign-M can also design primers that include DNA barcodes and minimize primer dimerization. PrimerDesign-Mmore » finds optimal primers for highly variable DNA targets and facilitates design flexibility by suggesting alternative designs to adapt to experimental conditions.« less

WAVE2 deficiency reveals distinct roles in embryogenesis and Rac-mediated actin-based motility

PubMed Central

Yan, Catherine; Martinez-Quiles, Narcisa; Eden, Sharon; Shibata, Tomoyuki; Takeshima, Fuminao; Shinkura, Reiko; Fujiwara, Yuko; Bronson, Roderick; Snapper, Scott B.; Kirschner, Marc W.; Geha, Raif; Rosen, Fred S.; Alt, Frederick W.

2003-01-01

The Wiskott–Aldrich syndrome related protein WAVE2 is implicated in the regulation of actin-cytoskeletal reorganization downstream of the small Rho GTPase, Rac. We inactivated the WAVE2 gene by gene-targeted mutation to examine its role in murine development and in actin assembly. WAVE2-deficient embryos survived until approximately embryonic day 12.5 and displayed growth retardation and certain morphological defects, including malformations of the ventricles in the developing brain. WAVE2-deficient embryonic stem cells displayed normal proliferation, whereas WAVE2-deficient embryonic fibroblasts exhibited severe growth defects, as well as defective cell motility in response to PDGF, lamellipodium formation and Rac-mediated actin polymerization. These results imply a non-redundant role for WAVE2 in murine embryogenesis and a critical role for WAVE2 in actin-based processes downstream of Rac that are essential for cell movement. PMID:12853475

WAVE2 deficiency reveals distinct roles in embryogenesis and Rac-mediated actin-based motility.

PubMed

Yan, Catherine; Martinez-Quiles, Narcisa; Eden, Sharon; Shibata, Tomoyuki; Takeshima, Fuminao; Shinkura, Reiko; Fujiwara, Yuko; Bronson, Roderick; Snapper, Scott B; Kirschner, Marc W; Geha, Raif; Rosen, Fred S; Alt, Frederick W

2003-07-15

The Wiskott-Aldrich syndrome related protein WAVE2 is implicated in the regulation of actin-cytoskeletal reorganization downstream of the small Rho GTPase, Rac. We inactivated the WAVE2 gene by gene-targeted mutation to examine its role in murine development and in actin assembly. WAVE2-deficient embryos survived until approximately embryonic day 12.5 and displayed growth retardation and certain morphological defects, including malformations of the ventricles in the developing brain. WAVE2-deficient embryonic stem cells displayed normal proliferation, whereas WAVE2-deficient embryonic fibroblasts exhibited severe growth defects, as well as defective cell motility in response to PDGF, lamellipodium formation and Rac-mediated actin polymerization. These results imply a non-redundant role for WAVE2 in murine embryogenesis and a critical role for WAVE2 in actin-based processes downstream of Rac that are essential for cell movement.

Sestrins are differentially expressed with age in the skeletal muscle of men: A cross-sectional analysis.

PubMed

Zeng, Nina; D'Souza, Randall F; Mitchell, Cameron J; Cameron-Smith, David

2018-05-08

A gradual loss of skeletal muscle mass is a common feature of aging, leading to impaired insulin sensitivity and mobility. Sestrin1, 2, 3 are multifunctional proteins that regulate the mammalian target of rapamycin complex (mTORC1), autophagy and redox homeostasis. It is unclear how aging affects Sestrins and their downstream targets in human, therefore this study examined the basal expression of Sestrins in three age groups, young, middle-aged and older men and explored the mTORC1 pathway, autophagy markers and antioxidant regulation. Older men had less Sestrin1 and 3 protein and a different pattern of Sestrin2 electrophoretic mobility. The mRNA expression of SESN1 was highly upregulated in older men, but the discrepancy was not by microRNA expression. Although protein expressions of Sestrins were downregulated with aging, phosphorylation of AMP-dependent protein kinase (AMPKα Thr172 ) and read-outs of mTORC1 activation, ribosomal protein S6 kinase 1 (p70S6K1 Thr421/Ser424 ) and 4E-binding protein 1 (4E-BP1) mobility shift were unaltered. However, total p70S6K1 and 4E-BP1 were reduced in middle-age and older men. The mRNA expressions of autophagic markers including microtubule-associated protein 1 light chain 3 (LC3) and BCL2 interacting protein 3 (BNIP3) were upregulated in middle-age and older men. Although nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was upregulated in older men, the protein and mRNA expressions of its downstream antioxidants were either increased, decreased or unaltered. No clear relationship was observed between Sestrins and their downstream targets, yet it can be concluded that Sestrins proteins are clearly downregulated with aging. Copyright © 2017. Published by Elsevier Inc.

Applications of pathology-assisted image analysis of immunohistochemistry-based biomarkers in oncology.

PubMed

Shinde, V; Burke, K E; Chakravarty, A; Fleming, M; McDonald, A A; Berger, A; Ecsedy, J; Blakemore, S J; Tirrell, S M; Bowman, D

2014-01-01

Immunohistochemistry-based biomarkers are commonly used to understand target inhibition in key cancer pathways in preclinical models and clinical studies. Automated slide-scanning and advanced high-throughput image analysis software technologies have evolved into a routine methodology for quantitative analysis of immunohistochemistry-based biomarkers. Alongside the traditional pathology H-score based on physical slides, the pathology world is welcoming digital pathology and advanced quantitative image analysis, which have enabled tissue- and cellular-level analysis. An automated workflow was implemented that includes automated staining, slide-scanning, and image analysis methodologies to explore biomarkers involved in 2 cancer targets: Aurora A and NEDD8-activating enzyme (NAE). The 2 workflows highlight the evolution of our immunohistochemistry laboratory and the different needs and requirements of each biological assay. Skin biopsies obtained from MLN8237 (Aurora A inhibitor) phase 1 clinical trials were evaluated for mitotic and apoptotic index, while mitotic index and defects in chromosome alignment and spindles were assessed in tumor biopsies to demonstrate Aurora A inhibition. Additionally, in both preclinical xenograft models and an acute myeloid leukemia phase 1 trial of the NAE inhibitor MLN4924, development of a novel image algorithm enabled measurement of downstream pathway modulation upon NAE inhibition. In the highlighted studies, developing a biomarker strategy based on automated image analysis solutions enabled project teams to confirm target and pathway inhibition and understand downstream outcomes of target inhibition with increased throughput and quantitative accuracy. These case studies demonstrate a strategy that combines a pathologist's expertise with automated image analysis to support oncology drug discovery and development programs.

Preclinical evaluation of dasatinib alone and in combination with cabozantinib for the treatment of diffuse intrinsic pontine glioma

PubMed Central

Philippe, Cathy; Paulsson, Janna; Andreiuolo, Felipe; Guerrini-Rousseau, Léa; Cornilleau, Gaétan; Le Dret, Ludivine; Richon, Catherine; Lacroix, Ludovic; Puget, Stéphanie; Geoerger, Birgit; Vassal, Gilles; Östman, Arne; Grill, Jacques

2015-01-01

Abstract Background Platelet-derived growth factor receptor A is altered by amplification and/or mutation in diffuse intrinsic pontine glioma (DIPG). We explored in vitro on new DIPG models the efficacy of dasatinib, a multi-tyrosine kinase inhibitor targeting this receptor. Methods Gene expression profiles were generated from 41 DIPGs biopsied at diagnosis and compared with the signature associated with sensitivity/resistance to dasatinib. A panel of 12 new DIPG cell lines were established from biopsy at diagnosis, serially passaged, and characterized by gene expression analyses. Effects of dasatinib (1–10 μM) on proliferation, invasion, and cytotoxicity were determined on 4 of these cell lines using live-cell imaging and flow cytometry assays. Downstream signaling and receptor tyrosine kinases (RTKs) were assessed by western blot and phospho-RTK array. The effect of the combination with the c-Met inhibitor cabozantinib was studied on cellular growth and invasion analyzed by the Chou–Talaly method. Results DIPG primary tumors and cell lines exhibited the gene expression signature of sensitivity to dasatinib. Dasatinib reduced proliferation (half-maximal inhibitory concentration = 10–100 nM) and invasion (30%–60% reduction) at 100 nM in 4/4 cultures and induced apoptosis in 1 of 4 DIPG cell lines. Activity of downstream effectors of dasatinib targets including activin receptor 1 was strongly reduced. Since multiple RTKs were activated simultaneously in DIPG cell lines, including c-Met, which can be also amplified in DIPG, the benefit of the combination of dasatinib with cabozantinib was explored for its synergistic effects on proliferation and migration/invasion in these cell lines. Conclusion Dasatinib exhibits antitumor effects in vitro that could be increased by the combination with another RTK inhibitor targeting c-Met. PMID:25534822

Timing and Targeting of Treatment in Left Ventricular Hypertrophy.

PubMed

Nam, Deokhwa; Reineke, Erin L

2017-01-01

In most clinical cases, left ventricular hypertrophy (LVH) occurs over time from persistent cardiac stress. At the molecular level, this results in both transient and long-term changes to metabolic, sarcomeric, ion handling, and stress signaling pathways. Although this is initially an adaptive change, the mechanisms underlying LVH eventually lead to maladaptive changes including fibrosis, decreased cardiac function, and failure. Understanding the regulators of long-term changes, which are largely driven by transcriptional remodeling, is a crucial step in identifying novel therapeutic targets for preventing the downstream negative effects of LVH and treatments that could reverse or prevent it. The development of effective therapeutics, however, will require a critical understanding of what to target, how to modify important pathways, and how to identify the stage of pathology in which a specific treatment should be used.

Comparative studies on mitochondrial electron transport chain complexes of Sitophilus zeamais treated with allyl isothiocyanate and calcium phosphide.

PubMed

Zhang, Chao; Wu, Hua; Zhao, Yuan; Ma, Zhiqing; Zhang, Xing

2016-01-01

With Sitophilus zeamais as the target organism, the present study for the first time attempted to elucidate the comparative effects between allyl isothiocyanate (AITC) and calcium phosphide (Ca3P2), exposure on mitochondrial electron transport chain (ETC.) complex I & IV and their downstream effects on enzymes relevant to reactive oxygen species (ROS). In vivo, both AITC and Ca3P2 inhibited complex I and IV with similar downstream effects. In contrast with Ca3P2, the inhibition of complex I caused by AITC was dependent on time and dose. In vitro, AITC inhibited complex IV more significantly than complex I. These results indicate that mitochondrial complex IV is the primary target of AITC, and that complex I is another potential target. Copyright © 2015 Elsevier B.V. All rights reserved.

A recurrent endometrial stromal sarcoma harbors the novel fusion JAZF1-BCORL1.

PubMed

Allen, Allison J; Ali, Siraj M; Gowen, Kyle; Elvin, Julia A; Pejovic, Tanja

2017-05-01

•Genomic alterations may improve diagnostic certainty and subsequent treatment of endometrial stromal sarcoma.•Novel JAZF1-BCORL1 mutation was identified.•Targeted therapeutics to down-stream targets may improve survival benefit in these patients.

Fuel injection assembly for use in turbine engines and method of assembling same

DOEpatents

Uhm, Jong Ho; Johnson, Thomas Edward

2015-03-24

A fuel injection assembly for use in a turbine engine is provided. The fuel injection assembly includes a plurality of tube assemblies, wherein each of the tube assemblies includes an upstream portion and a downstream portion. Each tube assembly includes a plurality of tubes that extend from the upstream portion to the downstream portion or from the upstream portion through the downstream portion. At least one injection system is coupled to at least one tube assembly of the plurality of tube assemblies. The injection system includes a fluid supply member that extends from a fluid source to the downstream portion of the tube assembly. The fluid supply member includes a first end portion located in the downstream portion of the tube assembly, wherein the first end portion has at least one first opening for channeling fluid through the tube assembly to facilitate reducing a temperature therein.

IGF system targeted therapy: Therapeutic opportunities for ovarian cancer.

PubMed

Liefers-Visser, J A L; Meijering, R A M; Reyners, A K L; van der Zee, A G J; de Jong, S

2017-11-01

The insulin-like growth factor (IGF) system comprises multiple growth factor receptors, including insulin-like growth factor 1 receptor (IGF-1R), insulin receptor (IR) -A and -B. These receptors are activated upon binding to their respective growth factor ligands, IGF-I, IGF-II and insulin, and play an important role in development, maintenance, progression, survival and chemotherapeutic response of ovarian cancer. In many pre-clinical studies anti-IGF-1R/IR targeted strategies proved effective in reducing growth of ovarian cancer models. In addition, anti-IGF-1R targeted strategies potentiated the efficacy of platinum based chemotherapy. Despite the vast amount of encouraging and promising pre-clinical data, anti-IGF-1R/IR targeted strategies lacked efficacy in the clinic. The question is whether targeting the IGF-1R/IR signaling pathway still holds therapeutic potential. In this review we address the complexity of the IGF-1R/IR signaling pathway, including receptor heterodimerization within and outside the IGF system and downstream signaling. Further, we discuss the implications of this complexity on current targeted strategies and indicate therapeutic opportunities for successful targeting of the IGF-1R/IR signaling pathway in ovarian cancer. Multiple-targeted approaches circumventing bidirectional receptor tyrosine kinase (RTK) compensation and prevention of system rewiring are expected to have more therapeutic potential. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Detection of genes regulated by Lmx1b during limb dorsalization.

PubMed

Feenstra, Jennifer M; Kanaya, Kohei; Pira, Charmaine U; Hoffman, Sarah E; Eppey, Richard J; Oberg, Kerby C

2012-05-01

Lmx1b is a homeodomain transcription factor that regulates dorsal identity during limb development. Lmx1b knockout (KO) mice develop distal ventral-ventral limbs. Although induction of Lmx1b is linked to Wnt7a expression in the dorsal limb ectoderm, the downstream targets of Lmx1b that accomplish limb dorsalization are unknown. To identify genes targeted by Lmx1b, we compared gene arrays from Lmx1b KO and wild type mouse limbs during limb dorsalization, i.e., 11.5, 12.5, and 13.5 days post coitum. We identified 54 target genes that were differentially expressed in all three stages. Several skeletal targets, including Emx2, Matrilin1 and Matrilin4, demonstrated a loss of scapular expression in the Lmx1b KO mice, supporting a role for Lmx1b in scapula development. Furthermore, the relative abundance of extracellular matrix-related soft tissue targets regulated by Lmx1b, such as collagens and proteoglycans, suggests a mechanism that includes changes in the extracellular matrix composition to accomplish limb dorsalization. Our study provides the most comprehensive characterization of genes regulated by Lmx1b during limb development to-date and provides targets for further investigation. © 2012 The Authors. Development, Growth & Differentiation © 2012 Japanese Society of Developmental Biologists.

The regulation of Jmjd3 upon the expression of NF-κB downstream inflammatory genes in LPS activated vascular endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Shaoqing; Graduate School of Medicine, Nanchang University, Nanchang; Chen, Xia

Inflammatory mediators and adhesion molecules have been implicated in a variety of diseases including atherosclerosis. As both the mediator-releasing and targeted cells, vascular endothelial cells play key role in pathological processes. NF-κB signaling regulates a cluster of inflammatory factors in LPS-activated vascular endothelial cells but the underlying mechanisms remain largely unknown. Here, we investigated the epigenetic regulation of LPS upon the expression of inflammatory mediators and adhesion molecules. We found that LPS treatment promoted jmjd3 expression, enhanced Jmjd3 nuclear accumulation in human vascular endothelial cells. In addition, LPS enhanced the demethylation of H3K27me3, a specific substrate of Jmjd3. LPS treatmentmore » recruited Jmjd3 and NF-κB to the promoter region of target genes, suggesting Jmjd3 synergizes with NF-κB to activate the expression of target genes. We further found that Jmjd3 attenuated the methylation status in promoter region of target genes, culminating in target gene expression. Our findings unveil epigenetic regulations of LPS upon NF-κB pathway and identify Jmjd3 as a critical modulator of NF-κB pathway and potential therapeutic target for NF-κB related diseases including atherosclerosis.« less

Cell Density Affects the Detection of Chk1 Target Engagement by the Selective Inhibitor V158411.

PubMed

Geneste, Clara C; Massey, Andrew J

2018-02-01

Understanding drug target engagement and the relationship to downstream pharmacology is critical for drug discovery. Here we have evaluated target engagement of Chk1 by the small-molecule inhibitor V158411 using two different target engagement methods (autophosphorylation and cellular thermal shift assay [CETSA]). Target engagement measured by these methods was subsequently related to Chk1 inhibitor-dependent pharmacology. Inhibition of autophosphorylation was a robust method for measuring V158411 Chk1 target engagement. In comparison, while target engagement determined using CETSA appeared robust, the V158411 CETSA target engagement EC 50 values were 43- and 19-fold greater than the autophosphorylation IC 50 values. This difference was attributed to the higher cell density in the CETSA assay configuration. pChk1 (S296) IC 50 values determined using the CETSA assay conditions were 54- and 33-fold greater than those determined under standard conditions and were equivalent to the CETSA EC 50 values. Cellular conditions, especially cell density, influenced the target engagement of V158411 for Chk1. The effects of high cell density on apparent compound target engagement potency should be evaluated when using target engagement assays that necessitate high cell densities (such as the CETSA conditions used in this study). In such cases, the subsequent relation of these data to downstream pharmacological changes should therefore be interpreted with care.

Identification of downstream metastasis-associated target genes regulated by LSD1 in colon cancer cells.

PubMed

Chen, Jiang; Ding, Jie; Wang, Ziwei; Zhu, Jian; Wang, Xuejian; Du, Jiyi

2017-03-21

This study aims to identify downstream target genes regulated by lysine-specific demethylase 1 (LSD1) in colon cancer cells and investigate the molecular mechanisms of LSD1 influencing invasion and metastasis of colon cancer. We obtained the expression changes of downstream target genes regulated by small-interfering RNA-LSD1 and LSD1-overexpression via gene expression profiling in two human colon cancer cell lines. An Affymetrix Human Transcriptome Array 2.0 was used to identify differentially expressed genes (DEGs). We screened out LSD1-target gene associated with proliferation, metastasis, and invasion from DEGs via Gene Ontology and Pathway Studio. Subsequently, four key genes (CABYR, FOXF2, TLE4, and CDH1) were computationally predicted as metastasis-related LSD1-target genes. ChIp-PCR was applied after RT-PCR and Western blot validations to detect the occupancy of LSD1-target gene promoter-bound LSD1. A total of 3633 DEGs were significantly upregulated, and 4642 DEGs were downregulated in LSD1-silenced SW620 cells. A total of 4047 DEGs and 4240 DEGs were upregulated and downregulated in LSD1-overexpressed HT-29 cells, respectively. RT-PCR and Western blot validated the microarray analysis results. ChIP assay results demonstrated that LSD1 might be negative regulators for target genes CABYR and CDH1. The expression level of LSD1 is negatively correlated with mono- and dimethylation of histone H3 lysine4(H3K4) at LSD1- target gene promoter region. No significant mono-methylation and dimethylation of H3 lysine9 methylation was detected at the promoter region of CABYR and CDH1. LSD1- depletion contributed to the upregulation of CABYR and CDH1 through enhancing the dimethylation of H3K4 at the LSD1-target genes promoter. LSD1- overexpression mediated the downregulation of CABYR and CDH1expression through decreasing the mono- and dimethylation of H3K4 at LSD1-target gene promoter in colon cancer cells. CABYR and CDH1 might be potential LSD1-target genes in colon carcinogenesis.

EphA2 is a key effector of the MEK/ERK/RSK pathway regulating glioblastoma cell proliferation.

PubMed

Hamaoka, Yuho; Negishi, Manabu; Katoh, Hironori

2016-08-01

EphA2, a member of the Eph receptor tyrosine kinases, is frequently overexpressed in a variety of malignancies, including glioblastoma, and its expression is correlated with poor prognosis. EphA2 acts as a tumor promoter through a ligand ephrin-independent mechanism, which requires phosphorylation of EphA2 on serine 897 (S897), leading to increased cell migration and invasion. In this study, we show that ligand-independent EphA2 signaling occurs downstream of the MEK/ERK/RSK pathway and mediates epidermal growth factor (EGF)-induced cell proliferation in glioblastoma cells. Suppression of EphA2 expression by long-term exposure to ligand ephrinA1 or EphA2-targeted shRNA inhibited EGF-induced cell proliferation. Stimulation of the cells with EGF induced EphA2 S897 phosphorylation, which was suppressed by MEK and RSK inhibitors, but not by phosphatidylinositol 3-kinase (PI3K) and Akt inhibitors. The RSK inhibitor or RSK2-targeted shRNA also suppressed EGF-induced cell proliferation. Furthermore, overexpression of wild-type EphA2 promoted cell proliferation without EGF stimulation, whereas overexpression of EphA2-S897A mutant suppressed EGF- or RSK2-induced proliferation. Taken together, these results suggest that EphA2 is a key downstream target of the MEK/ERK/RSK signaling pathway in the regulation of glioblastoma cell proliferation. Copyright © 2016 Elsevier Inc. All rights reserved.

Competing targets of microRNA-608 affect anxiety and hypertension

PubMed Central

Hanin, Geula; Shenhar-Tsarfaty, Shani; Yayon, Nadav; Hoe, Yau Yin; Bennett, Estelle R.; Sklan, Ella H.; Rao, Dabeeru. C.; Rankinen, Tuomo; Bouchard, Claude; Geifman-Shochat, Susana; Shifman, Sagiv; Greenberg, David S.; Soreq, Hermona

2014-01-01

MicroRNAs (miRNAs) can repress multiple targets, but how a single de-balanced interaction affects others remained unclear. We found that changing a single miRNA–target interaction can simultaneously affect multiple other miRNA–target interactions and modify physiological phenotype. We show that miR-608 targets acetylcholinesterase (AChE) and demonstrate weakened miR-608 interaction with the rs17228616 AChE allele having a single-nucleotide polymorphism (SNP) in the 3′-untranslated region (3′UTR). In cultured cells, this weakened interaction potentiated miR-608-mediated suppression of other targets, including CDC42 and interleukin-6 (IL6). Postmortem human cortices homozygote for the minor rs17228616 allele showed AChE elevation and CDC42/IL6 decreases compared with major allele homozygotes. Additionally, minor allele heterozygote and homozygote subjects showed reduced cortisol and elevated blood pressure, predicting risk of anxiety and hypertension. Parallel suppression of the conserved brain CDC42 activity by intracerebroventricular ML141 injection caused acute anxiety in mice. We demonstrate that SNPs in miRNA-binding regions could cause expanded downstream effects changing important biological pathways. PMID:24722204

Load-induced modulation of signal transduction networks.

PubMed

Jiang, Peng; Ventura, Alejandra C; Sontag, Eduardo D; Merajver, Sofia D; Ninfa, Alexander J; Del Vecchio, Domitilla

2011-10-11

Biological signal transduction networks are commonly viewed as circuits that pass along information--in the process amplifying signals, enhancing sensitivity, or performing other signal-processing tasks--to transcriptional and other components. Here, we report on a "reverse-causality" phenomenon, which we call load-induced modulation. Through a combination of analytical and experimental tools, we discovered that signaling was modulated, in a surprising way, by downstream targets that receive the signal and, in doing so, apply what in physics is called a load. Specifically, we found that non-intuitive changes in response dynamics occurred for a covalent modification cycle when load was present. Loading altered the response time of a system, depending on whether the activity of one of the enzymes was maximal and the other was operating at its minimal rate or whether both enzymes were operating at submaximal rates. These two conditions, which we call "limit regime" and "intermediate regime," were associated with increased or decreased response times, respectively. The bandwidth, the range of frequency in which the system can process information, decreased in the presence of load, suggesting that downstream targets participate in establishing a balance between noise-filtering capabilities and a circuit's ability to process high-frequency stimulation. Nodes in a signaling network are not independent relay devices, but rather are modulated by their downstream targets.

Early clinical development of epidermal growth factor receptor targeted therapy in breast cancer.

PubMed

Matsuda, Naoko; Lim, Bora; Wang, Xiaoping; Ueno, Naoto T

2017-04-01

Epidermal growth factor receptor (EGFR) targeted treatment has been evaluated but has not shown a clear clinical benefit for breast cancer. This review article aims to consider the knowledge of the biological background of EGFR pathways in dissecting clinical studies of EGFR targeted treatment in breast cancer. Areas covered: This review focuses on the role of the EGFR pathway and the investigational drugs that target EGFR for breast cancer. Expert opinion: Recent studies have indicated that EGFR targeted therapy for breast cancer has some promising effects for patients with triple-negative breast cancer, basal-like breast cancer, and inflammatory breast cancer. However, predictive and prognostic biomarkers for EGFR targeted therapy have not been identified. The overexpression or amplification of EGFR itself may not be the true factor of induction of the canonical pathway as an oncogenic driver of breast cancer. Instead, downstream, non-canonical pathways related to EGFR may contribute to some aspects of the biological behavior of breast cancer; therefore, the blockade of the receptor could result in sufficient suppression of downstream pathways to inhibit the aggressive behavior of breast cancer. Mechanistic studies to investigate the dynamic interaction between the EGFR pathway and non-canonical pathways are warranted.

Early clinical development of epidermal growth factor receptor targeted therapy in breast cancer

PubMed Central

Matsuda, Naoko; Lim, Bora; Wang, Xiaoping; Ueno, Naoto T.

2018-01-01

Introduction Epidermal growth factor receptor (EGFR) targeted treatment has been evaluated but has not shown a clear clinical benefit for breast cancer. This review article aims to consider the knowledge of the biological background of EGFR pathways in dissecting clinical studies of EGFR targeted treatment in breast cancer. Areas covered This review focuses on the role of the EGFR pathway and the investigational drugs that target EGFR for breast cancer. Expert opinion Recent studies have indicated that EGFR targeted therapy for breast cancer has some promising effects for patients with triple-negative breast cancer, basal-like breast cancer, and inflammatory breast cancer. However, predictive and prognostic biomarkers for EGFR targeted therapy have not been identified. The overexpression or amplification of EGFR itself may not be the true factor of induction of the canonical pathway as an oncogenic driver of breast cancer. Instead, downstream, non-canonical pathways related to EGFR may contribute to some aspects of the biological behavior of breast cancer; therefore, the blockade of the receptor could result in sufficient suppression of downstream pathways to inhibit the aggressive behavior of breast cancer. Mechanistic studies to investigate the dynamic interaction between the EGFR pathway and non-canonical pathways are warranted. PMID:28271910

beta-catenin mediates insulin-like growth factor-I actions to promote cyclin D1 mRNA expression, cell proliferation and survival in oligodendroglial cultures.

PubMed

Ye, Ping; Hu, Qichen; Liu, Hedi; Yan, Yun; D'ercole, A Joseph

2010-07-01

By promoting cell proliferation, survival and maturation insulin-like growth factor (IGF)-I is essential to the normal growth and development of the central nervous system. It is clear that IGF-I actions are primarily mediated by the type I IGF receptor (IGF1R), and that phosphoinositide 3 (PI3)-Akt kinases and MAP kinases signal many of IGF-I-IGF1R actions in neural cells, including oligodendrocyte lineage cells. The precise downstream targets of these signaling pathways, however, remain to be defined. We studied oligodendroglial cells to determine whether beta-catenin, a molecule that is a downstream target of glycogen synthase kinase-3beta (GSK3beta) and plays a key role in the Wnt canonical signaling pathway, mediates IGF-I actions. We found that IGF-I increases beta-catenin protein abundance within an hour after IGF-I-induced phosphorylation of Akt and GSK3beta. Inhibiting the PI3-Akt pathway suppressed IGF-I-induced increases in beta-catenin and cyclin D1 mRNA, while suppression of GSK3beta activity simulated IGF-I actions. Knocking-down beta-catenin mRNA by RNA interference suppressed IGF-I-stimulated increases in the abundance of cyclin D1 mRNA, cell proliferation, and cell survival. Our data suggest that beta-catenin is an important downstream molecule in the PI3-Akt-GSK3beta pathway, and as such it mediates IGF-I upregulation of cyclin D1 mRNA and promotion of cell proliferation and survival in oligodendroglial cells. Copyright 2010 Wiley-Liss, Inc.

Therapeutic Approaches Targeting MYC-Driven Prostate Cancer

PubMed Central

Rebello, Richard J.; Pearson, Richard B.; Hannan, Ross D.; Furic, Luc

2017-01-01

The transcript encoding the proto-oncogene MYC is commonly overexpressed in prostate cancer (PC). MYC protein abundance is also increased in the majority of cases of advanced and metastatic castrate-resistant PC (mCRPC). Accordingly, the MYC-directed transcriptional program directly contributes to PC by upregulating the expression of a number of pro-tumorigenic factors involved in cell growth and proliferation. A key cellular process downstream of MYC activity is the regulation of ribosome biogenesis which sustains tumor growth. MYC activity also cooperates with the dysregulation of the phosphoinositol-3-kinase (PI3K)/AKT/mTOR pathway to promote PC cell survival. Recent advances in the understanding of these interactions through the use of animal models have provided significant insight into the therapeutic efficacy of targeting MYC activity by interfering with its transcriptional program, and indirectly by targeting downstream cellular events linked to MYC transformation potential. PMID:28212321

Overexpression of miRNA-497 inhibits tumor angiogenesis by targeting VEGFR2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tu, Yingfeng; Liu, Li; Zhao, Dongliang

Recent studies reported miR-497 exhibited inhibitory effects in various cancers. However, whether miR-497 is involved in inhibiting angiogenesis, which is critical for tumor growth and metastasis, is still unknown. The purpose of this study was to investigate the potential role of miR-497 in tumor angiogenesis. In this work, cell proliferation and apoptosis analyses were conducted to explore the potential function of miR-497 in HUVECs by using MTT and TUNEL assays. Western blotting (WB) was employed to validate the downstream targets of miR-497. Furthermore, in order to disclose the role of miR-497 on angiogenesis, VEGFR2-luc transgenic mice were treated with miR-497more » mimic and applied to monitor tumor angiogenesis and growth by in vivo bioluminescent imaging (BLI). The results demonstrated that overexpression of miR-497 showed inhibitory effects on VEGFR2 activation and downstream Raf/MEK/ERK signal pathways in vitro and in vivo. Moreover, overexpression of miR-497 effectively induced HUVECs apoptosis by targeting VEGFR2 and downstream PI3K/AKT signaling pathway. Furthermore, miR-497 exhibited anti-angiogenesis and anti-tumor effects in the VEGFR2-luc breast tumor model proven by BLI, WB and immunohistochemistry analysis. In summary, miR-497 inhibits tumor angiogenesis and growth via targeting VEGFR2, indicating miR-497 can be explored as a potential drug candidate for cancer therapy.« less

Overexpression of miRNA-497 inhibits tumor angiogenesis by targeting VEGFR2

DOE PAGES

Tu, Yingfeng; Liu, Li; Zhao, Dongliang; ...

2015-09-08

Recent studies reported miR-497 exhibited inhibitory effects in various cancers. However, whether miR-497 is involved in inhibiting angiogenesis, which is critical for tumor growth and metastasis, is still unknown. The purpose of this study was to investigate the potential role of miR-497 in tumor angiogenesis. In this work, cell proliferation and apoptosis analyses were conducted to explore the potential function of miR-497 in HUVECs by using MTT and TUNEL assays. Western blotting (WB) was employed to validate the downstream targets of miR-497. Furthermore, in order to disclose the role of miR-497 on angiogenesis, VEGFR2-luc transgenic mice were treated with miR-497more » mimic and applied to monitor tumor angiogenesis and growth by in vivo bioluminescent imaging (BLI). The results demonstrated that overexpression of miR-497 showed inhibitory effects on VEGFR2 activation and downstream Raf/MEK/ERK signal pathways in vitro and in vivo. Moreover, overexpression of miR-497 effectively induced HUVECs apoptosis by targeting VEGFR2 and downstream PI3K/AKT signaling pathway. Furthermore, miR-497 exhibited anti-angiogenesis and anti-tumor effects in the VEGFR2-luc breast tumor model proven by BLI, WB and immunohistochemistry analysis. In summary, miR-497 inhibits tumor angiogenesis and growth via targeting VEGFR2, indicating miR-497 can be explored as a potential drug candidate for cancer therapy.« less

Ribosomal S6 kinase (RSK) modulators: a patent review.

PubMed

Ludwik, Katarzyna A; Lannigan, Deborah A

2016-09-01

The p90 ribosomal S6 kinases (RSK) are a family of Ser/Thr protein kinases that are downstream effectors of MEK1/2-ERK1/2. Increased RSK activation is implicated in the etiology of multiple pathologies, including numerous types of cancers, cardiovascular disease, liver and lung fibrosis, and infections. The review summarizes the patent and scientific literature on small molecule modulators of RSK and their potential use as therapeutics. The patents were identified using World Intellectual Property Organization and United States Patent and Trademark Office databases. The compounds described are predominantly RSK inhibitors, but a RSK activator is also described. The majority of the inhibitors are not RSK-specific. Based on the overwhelming evidence that RSK is involved in a number of diseases that have high mortalities it seems surprising that there are no RSK modulators that have pharmacokinetic properties suitable for in vivo use. MEK1/2 inhibitors are in the clinic, but the efficacy of these compounds appears to be limited by their side effects. We hypothesize that targeting the downstream effectors of MEK1/2, like RSK, are an untapped source of drug targets and that they will generate less side effects than MEK1/2 inhibitors because they regulate fewer effectors.

Identification of Homeotic Target Genes in Drosophila Melanogaster Including Nervy, a Proto-Oncogene Homologue

PubMed Central

Feinstein, P. G.; Kornfeld, K.; Hogness, D. S.; Mann, R. S.

1995-01-01

In Drosophila, the specific morphological characteristics of each segment are determined by the homeotic genes that regulate the expression of downstream target genes. We used a subtractive hybridization procedure to isolate activated target genes of the homeotic gene Ultrabithorax (Ubx). In addition, we constructed a set of mutant genotypes that measures the regulatory contribution of individual homeotic genes to a complex target gene expression pattern. Using these mutants, we demonstrate that homeotic genes can regulate target gene expression at the start of gastrulation, suggesting a previously unknown role for the homeotic genes at this early stage. We also show that, in abdominal segments, the levels of expression for two target genes increase in response to high levels of Ubx, demonstrating that the normal down-regulation of Ubx in these segments is functional. Finally, the DNA sequence of cDNAs for one of these genes predicts a protein that is similar to a human proto-oncogene involved in acute myeloid leukemias. These results illustrate potentially general rules about the homeotic control of target gene expression and suggest that subtractive hybridization can be used to isolate interesting homeotic target genes. PMID:7498738

Cutting edge: rescue of pre-TCR but not mature TCR signaling in mice expressing membrane-targeted SLP-76.

PubMed

Bezman, Natalie A; Baker, Rebecca G; Lenox, Laurie E; Jordan, Martha S; Koretzky, Gary A

2009-05-01

SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa) organizes signaling from immunoreceptors, including the platelet collagen receptor, the pre-TCR, and the TCR, and is required for T cell development. In this study we examine a mouse in which wild-type SLP-76 is replaced with a mutant constitutively targeted to the cell membrane. Membrane-targeted SLP-76 (MTS) supports ITAM signaling in platelets and from the pre-TCR. Signaling from the mature TCR, however, is defective in MTS thymocytes, resulting in failed T cell differentiation. Defective thymic selection by MTS is not rescued by a SLP-76 mutant whose localization is restricted to the cytosol. Thus, fixed localization of SLP-76 reveals differential requirements for the subcellular localization of signaling complexes downstream of the pre-TCR vs mature TCR.

Voluntary control of intracortical oscillations for reconfiguration of network activity

PubMed Central

Corlier, Juliana; Valderrama, Mario; Navarrete, Miguel; Lehongre, Katia; Hasboun, Dominique; Adam, Claude; Belaid, Hayat; Clémenceau, Stéphane; Baulac, Michel; Charpier, Stéphane; Navarro, Vincent; Le Van Quyen, Michel

2016-01-01

Voluntary control of oscillatory activity represents a key target in the self-regulation of brain function. Using a real-time closed-loop paradigm and simultaneous macro- and micro-electrode recordings, we studied the effects of self-induced intracortical oscillatory activity (4–8 Hz) in seven neurosurgical patients. Subjects learned to robustly and specifically induce oscillations in the target frequency, confirmed by increased oscillatory event density. We have found that the session-to-session variability in performance was explained by the functional long-range decoupling of the target area suggesting a training-induced network reorganization. Downstream effects on more local activities included progressive cross-frequency-coupling with gamma oscillations (30–120 Hz), and the dynamic modulation of neuronal firing rates and spike timing, indicating an improved temporal coordination of local circuits. These findings suggest that effects of voluntary control of intracortical oscillations can be exploited to specifically target plasticity processes to reconfigure network activity, with a particular relevance for memory function or skill acquisition. PMID:27808225

Rational design and optimization of downstream processes of virus particles for biopharmaceutical applications: current advances.

PubMed

Vicente, Tiago; Mota, José P B; Peixoto, Cristina; Alves, Paula M; Carrondo, Manuel J T

2011-01-01

The advent of advanced therapies in the pharmaceutical industry has moved the spotlight into virus-like particles and viral vectors produced in cell culture holding great promise in a myriad of clinical targets, including cancer prophylaxis and treatment. Even though a couple of cases have reached the clinic, these products have yet to overcome a number of biological and technological challenges before broad utilization. Concerning the manufacturing processes, there is significant research focusing on the optimization of current cell culture systems and, more recently, on developing scalable downstream processes to generate material for pre-clinical and clinical trials. We review the current options for downstream processing of these complex biopharmaceuticals and underline current advances on knowledge-based toolboxes proposed for rational optimization of their processing. Rational tools developed to increase the yet scarce knowledge on the purification processes of complex biologicals are discussed as alternative to empirical, "black-boxed" based strategies classically used for process development. Innovative methodologies based on surface plasmon resonance, dynamic light scattering, scale-down high-throughput screening and mathematical modeling for supporting ion-exchange chromatography show great potential for a more efficient and cost-effective process design, optimization and equipment prototyping. Copyright © 2011 Elsevier Inc. All rights reserved.

Downstream processing of biopharmaceutical proteins produced in plants: the pros and cons of flocculants.

PubMed

Buyel, Johannes Felix; Fischer, Rainer

2014-01-01

All biological platforms for the manufacture of biopharmaceutical proteins produce an initially turbid extract that must be clarified to avoid fouling sensitive media such as chromatography resins. Clarification is more challenging if the feed stream contains large amounts of dispersed particles, because these rapidly clog the filter media typically used to remove suspended solids. Charged polymers (flocculants) can increase the apparent size of the dispersed particles by aggregation, facilitating the separation of solids and liquids, and thus reducing process costs. However, many different factors can affect the behavior of flocculants, including the pH and conductivity of the medium, the size and charge distribution of the particulates, and the charge density and molecular mass of the polymer. Importantly, these properties can also affect the recovery of the target protein and the overall safety profile of the process. We therefore used a design of experiments approach to establish reliable predictive models that characterize the impact of flocculants during the downstream processing of biopharmaceutical proteins. We highlight strategies for the selection of flocculants during process optimization. These strategies will contribute to the quality by design aspects of process development and facilitate the development of safe and efficient downstream processes for plant-derived pharmaceutical proteins.

Protein-intrinsic and signaling network-based sources of resistance to EGFR- and ErbB family-targeted therapies in head and neck cancer

PubMed Central

Mehra, Ranee; Serebriiskii, Ilya G.; Dunbrack, Roland L.; Robinson, Matthew K.; Burtness, Barbara; Golemis, Erica A.

2011-01-01

Agents targeting EGFR and related ErbB family proteins are valuable therapies for the treatment of many cancers. For some tumor types, including squamous cell carcinomas of the head and neck (SCCHN), antibodies targeting EGFR were the first protein-directed agents to show clinical benefit, and remain a standard component of clinical strategies for management of the disease. Nevertheless, many patients display either intrinsic or acquired resistance to these drugs; hence, major research goals are to better understand the underlying causes of resistance, and to develop new therapeutic strategies that boost the impact of EGFR/ErbB inhibitors. In this review, we first summarize current standard use of EGFR inhibitors in the context of SCCHN, and described new agents targeting EGFR currently moving through pre-clinical and clinical development. We then discuss how changes in other transmembrane receptors, including IGF1R, c-Met, and TGF-β, can confer resistance to EGFR-targeted inhibitors, and discuss new agents targeting these proteins. Moving downstream, we discuss critical EGFR-dependent effectors, including PLC-γ; PI3K and PTEN; SHC, GRB2, and RAS and the STAT proteins, as factors in resistance to EGFR-directed inhibitors and as alternative targets of therapeutic inhibition. We summarize alternative sources of resistance among cellular changes that target EGFR itself, through regulation of ligand availability, post-translational modification of EGFR, availability of EGFR partners for hetero-dimerization and control of EGFR intracellular trafficking for recycling versus degradation. Finally, we discuss new strategies to identify effective therapeutic combinations involving EGFR-targeted inhibitors, in the context of new system level data becoming available for analysis of individual tumors. PMID:21920801

Expression analysis of the N-Myc downstream-regulated gene 1 indicates that myelinating Schwann cells are the primary disease target in hereditary motor and sensory neuropathy-Lom.

PubMed

Berger, Philipp; Sirkowski, Erich E; Scherer, Steven S; Suter, Ueli

2004-11-01

Mutations in the gene encoding N-myc downstream-regulated gene-1 (NDRG1) lead to truncations of the encoded protein and are associated with an autosomal recessive demyelinating neuropathy--hereditary motor and sensory neuropathy-Lom. NDRG1 protein is highly expressed in peripheral nerve and is localized in the cytoplasm of myelinating Schwann cells, including the paranodes and Schmidt-Lanterman incisures. In contrast, sensory and motor neurons as well as their axons lack NDRG1. NDRG1 mRNA levels in developing and injured adult sciatic nerves parallel those of myelin-related genes, indicating that the expression of NDRG1 in myelinating Schwann cells is regulated by axonal interactions. Oligodendrocytes also express NDRG1, and the subtle CNS deficits of affected patients may result from a lack of NDRG1 in these cells. Our data predict that the loss of NDRG1 leads to a Schwann cell autonomous phenotype resulting in demyelination, with secondary axonal loss.

Guanosine triphosphatase activation occurs downstream of calcineurin in cardiac hypertrophy*.

PubMed

Richardson, Kenneth E; Tannous, Paul; Berenji, Kambeez; Nolan, Bridgid; Bayless, Kayla J; Davis, George E; Rothermel, Beverly A; Hill, Joseph A

2005-12-01

There is great interest in deciphering mechanisms of maladaptive remodeling in cardiac hypertrophy in the hope of affording clinical benefit. Potential targets of therapeutic intervention include the cytoplasmic phosphatase calcineurin and small guanosine triphosphate-binding proteins, such as Rac1 and RhoA, all of which have been implicated in maladaptive hypertrophy. However, little is known about the interaction-if any-between these important signaling molecules in hypertrophic heart disease. In this study, we examined the molecular interplay among these molecules, finding that Rho family guanosine triphosphatase signaling occurs either downstream of calcineurin or as a required, parallel pathway. It has been shown that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibition blocks hypertrophy, and we report here that "statin" therapy effectively suppresses small G protein activation and blunts hypertrophic growth in vitro and in vivo. Importantly, despite significant suppression of hypertrophy, clinical and hemodynamic markers remained compensated, suggesting that the hypertrophic growth induced by this pathway is not required to maintain circulatory performance.

Brucella TIR-like protein TcpB/Btp1 specifically targets the host adaptor protein MAL/TIRAP to promote infection.

PubMed

Li, Wenna; Ke, Yuehua; Wang, Yufei; Yang, Mingjuan; Gao, Junguang; Zhan, Shaoxia; Xinying, Du; Huang, Liuyu; Li, Wenfeng; Chen, Zeliang; Li, Juan

2016-08-26

Brucella spp. are known to avoid host immune recognition and weaken the immune response to infection. Brucella like accomplish this by employing two clever strategies, called the stealth strategy and hijacking strategy. The TIR domain-containing protein (TcpB/Btp1) of Brucella melitensis is thought to be involved in inhibiting host NF-κB activation by binding to adaptors downstream of Toll-like receptors. However, of the five TIR domain-containing adaptors conserved in mammals, whether MyD88 or MAL, even other three adaptors, are specifically targeted by TcpB has not been identified. Here, we confirmed the effect of TcpB on B.melitensis virulence in mice and found that TcpB selectively targets MAL. By using siRNA against MAL, we found that TcpB from B.melitensis is involved in intracellular survival and that MAL affects intracellular replication of B.melitensis. Our results confirm that TcpB specifically targets MAL/TIRAP to disrupt downstream signaling pathways and promote intra-host survival of Brucella spp. Copyright © 2016 Elsevier Inc. All rights reserved.

Fish community dynamics following dam removal in a fragmented agricultural stream

USGS Publications Warehouse

Kornis, Matthew; Weidel, Brian C.; Powers, Stephens; Diebel, Matthew W.; Cline, Timpthy; Fox, Justin; Kitchell, James F.

2014-01-01

Habitat fragmentation impedes dispersal of aquatic fauna, and barrier removal is increasingly used to increase stream network connectivity and facilitate fish dispersal. Improved understanding of fish community response to barrier removal is needed, especially in fragmented agricultural streams where numerous antiquated dams are likely destined for removal. We examined post-removal responses in two distinct fish communities formerly separated by a small aging mill dam. The dam was removed midway through the 6 year study, enabling passage for downstream fishes affiliated with a connected reservoir into previously inaccessible habitat, thus creating the potential for taxonomic homogenization between upstream and downstream communities. Both communities changed substantially post-removal. Two previously excluded species (white sucker, yellow perch) established substantial populations upstream of the former dam, contributing to a doubling of total fish biomass. Meanwhile, numerical density of pre-existing upstream fishes declined. Downstream, largemouth bass density was inversely correlated with prey fish density throughout the study, while post-removal declines in bluegill density coincided with cooler water temperature and increased suspended and benthic fine sediment. Upstream and downstream fish communities became more similar post-removal, represented by a shift in Bray-Curtis index from 14 to 41 % similarity. Our findings emphasize that barrier removal in highly fragmented stream networks can facilitate the unintended and possibly undesirable spread of species into headwater streams, including dispersal of species from remaining reservoirs. We suggest that knowledge of dispersal patterns for key piscivore and competitor species in both the target system and neighboring systems may help predict community outcomes following barrier removal.

Affinity purification mass spectrometry analysis of PD-1 uncovers SAP as a new checkpoint inhibitor.

PubMed

Peled, Michael; Tocheva, Anna S; Sandigursky, Sabina; Nayak, Shruti; Philips, Elliot A; Nichols, Kim E; Strazza, Marianne; Azoulay-Alfaguter, Inbar; Askenazi, Manor; Neel, Benjamin G; Pelzek, Adam J; Ueberheide, Beatrix; Mor, Adam

2018-01-16

Programmed cell death-1 (PD-1) is an essential inhibitory receptor in T cells. Antibodies targeting PD-1 elicit durable clinical responses in patients with multiple tumor indications. Nevertheless, a significant proportion of patients do not respond to anti-PD-1 treatment, and a better understanding of the signaling pathways downstream of PD-1 could provide biomarkers for those whose tumors respond and new therapeutic approaches for those whose tumors do not. We used affinity purification mass spectrometry to uncover multiple proteins associated with PD-1. Among these proteins, signaling lymphocytic activation molecule-associated protein (SAP) was functionally and mechanistically analyzed for its contribution to PD-1 inhibitory responses. Silencing of SAP augmented and overexpression blocked PD-1 function. T cells from patients with X-linked lymphoproliferative disease (XLP), who lack functional SAP, were hyperresponsive to PD-1 signaling, confirming its inhibitory role downstream of PD-1. Strikingly, signaling downstream of PD-1 in purified T cell subsets did not correlate with PD-1 surface expression but was inversely correlated with intracellular SAP levels. Mechanistically, SAP opposed PD-1 function by acting as a molecular shield of key tyrosine residues that are targets for the tyrosine phosphatase SHP2, which mediates PD-1 inhibitory properties. Our results identify SAP as an inhibitor of PD-1 function and SHP2 as a potential therapeutic target in patients with XLP.

RNA from the 5' end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA target site.

PubMed

Christensen, Shawn M; Ye, Junqiang; Eickbush, Thomas H

2006-11-21

Non-LTR retrotransposons insert into eukaryotic genomes by target-primed reverse transcription (TPRT), a process in which cleaved DNA targets are used to prime reverse transcription of the element's RNA transcript. Many of the steps in the integration pathway of these elements can be characterized in vitro for the R2 element because of the rigid sequence specificity of R2 for both its DNA target and its RNA template. R2 retrotransposition involves identical subunits of the R2 protein bound to different DNA sequences upstream and downstream of the insertion site. The key determinant regulating which DNA-binding conformation the protein adopts was found to be a 320-nt RNA sequence from near the 5' end of the R2 element. In the absence of this 5' RNA the R2 protein binds DNA sequences upstream of the insertion site, cleaves the first DNA strand, and conducts TPRT when RNA containing the 3' untranslated region of the R2 transcript is present. In the presence of the 320-nt 5' RNA, the R2 protein binds DNA sequences downstream of the insertion site. Cleavage of the second DNA strand by the downstream subunit does not appear to occur until after the 5' RNA is removed from this subunit. We postulate that the removal of the 5' RNA normally occurs during reverse transcription, and thus provides a critical temporal link to first- and second-strand DNA cleavage in the R2 retrotransposition reaction.

PTTG1, A novel androgen responsive gene is required for androgen-induced prostate cancer cell growth and invasion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Zheng; Jin, Bo; Jin, Yaqiong

Androgens (AR) play an important role in initiation and progression of prostate cancer. It has been shown that AR exert their effects mainly through the androgen-activated AR which binds to androgen response elements (AREs) in the regulatory regions of target genes to regulate the transcription of androgen-responsive genes, thus, identification of AR downstream target gene is critical to understand androgen function in prostate cancer. In this study, our results showed that androgen treatment of LNCaP cells induced PTTG1 expression, which was blocked by the androgen receptor antagonist, Casodex. Bioinformatics analysis and experiments using PTTG1 promoter deletion mutants showed that themore » PTTG1 promoter contains a putative androgen response element (ARE), which localizes in the −851 to −836 region of the promoter. Androgen activated androgen receptor (AR) binding to this ARE was confirmed by Chromatin immunoprecipitation (ChIP) assay. Furthermore, Knockdown of PTTG1 expression using short hairpin RNA significantly reduced androgen-induced LNCaP cell growth and invasion. In addition, we showed PTTG1 is highly expressed in metastasis prostate cancer tissue. These results suggest that PTTG1 is a novel downstream target gene of androgen receptor and take part in prostate cancer proliferation and metastasis. - Highlights: • Androgen treatment of LNCaP cells induced PTTG1 expression. • Knockdown of PTTG1 expression significantly reduced androgen-induced LNCaP cell growth and invasion. • PTTG1 is highly expressed in metastasis prostate cancer tissue. • PTTG1 is a novel downstream target gene of androgen receptor.« less

Staged electrostatic precipitator

DOEpatents

Miller, Stanley J.; Almlie, Jay C.; Zhuang, Ye

2016-03-01

A device includes a chamber having an air inlet and an air outlet. The device includes a plurality of stages including at least a first stage adjacent a second stage. The plurality of stages are disposed in the chamber and each stage has a plurality of discharge electrodes disposed in an interior region and is bounded by an upstream baffle on an end proximate the air inlet and bounded by a downstream baffle on an end proximate the air outlet. Each stage has at least one sidewall between the upstream baffle and the downstream baffle. The sidewall is configured as a collection electrode and has a plurality of apertures disposed along a length between the upstream baffle and the downstream baffle. The upstream baffle of the first stage is positioned in staggered alignment relative to the upstream baffle of the second stage and the downstream baffle of the first stage are positioned in staggered alignment relative to the downstream baffle of the second stage.

Metabolic Dysfunction Consistent with Premature Aging Results from Deletion of Pim Kinases

PubMed Central

Din, Shabana; Konstandin, Mathias H; Johnson, Bevan; Emathinger, Jacqueline; Völkers, Mirko; Toko, Haruhiro; Collins, Brett; Ormachea, Lucy; Samse, Kaitlen; Kubli, Dieter A; De La Torre, Andrea; Kraft, Andrew S; Gustafsson, Asa B; Kelly, Daniel P; Sussman, Mark A

2014-01-01

Rationale The senescent cardiac phenotype is accompanied by changes in mitochondrial function and biogenesis causing impairment in energy provision. The relationship between myocardial senescence and Pim kinases deserves attention since Pim-1 kinase is cardioprotective, in part, by preservation of mitochondrial integrity. Study of the pathological effects resulting from genetic deletion of all Pim kinase family members could provide important insight regarding cardiac mitochondrial biology and the aging phenotype. Objective Demonstrate myocardial senescence is promoted by loss of Pim leading to premature aging and aberrant mitochondrial function. Methods and Results Cardiac myocyte senescence was evident at three months of age in Pim Triple KnockOut (PTKO) mice, where all three isoforms of Pim kinase family members are genetically deleted. Cellular hypertrophic remodeling and fetal gene program activation was followed by heart failure at six months in PTKO mice. Metabolic dysfunction is an underlying cause of cardiac senescence and instigates a decline in cardiac function. Altered mitochondrial morphology is evident consequential to Pim deletion together with decreased ATP levels and increased phosphorylated AMPK, exposing an energy deficiency in PTKO mice. Expression of the genes encoding master regulators of mitochondrial biogenesis, PPARγ coactivator-1 (PGC-1) α and β were diminished in PTKO hearts, as were downstream targets included in mitochondrial energy transduction, including fatty acid oxidation. Reversal of the dysregulated metabolic phenotype was observed by overexpressing c-Myc, a downstream target of Pim kinases. Conclusion Pim kinases prevent premature cardiac aging and maintain a healthy pool of functional mitochondria leading to efficient cellular energetics. PMID:24916111

Targeting FOXM1 Improves Cytotoxicity of Paclitaxel and Cisplatinum in Platinum-Resistant Ovarian Cancer.

PubMed

Westhoff, Gina L; Chen, Yi; Teng, Nelson N H

2017-10-01

Aberrantly activated FOXM1 (forkhead box protein M1) leading to uncontrolled cell proliferation and dysregulation of FOXM1 transcription network occurs in 84% of ovarian cancer cases. It was demonstrated that thiostrepton, a thiazole antibiotic, decreases FOXM1 expression. We aimed to determine if targeting the FOXM1 pathway with thiostrepton could improve the efficacy of paclitaxel and cisplatin in human ovarian cancer ascites cells ex vivo. Human ovarian cancer cell lines and patients' ascites cells were treated with paclitaxel, cisplatin, and thiostrepton or a combination for 48 hours, and cytotoxicity was assessed. Drug combination effects were determined by calculating the combination index values using the Chou and Talalay method. Quantitative reverse transcriptase-polymerase chain reaction was performed to determine changes in FOXM1 expression and its downstream targets. Ovarian cancer cell lines and the patients' ascites cancer cells had an overexpression of FOXM1 expression levels. Targeting FOXM1 with thiostrepton decreased FOXM1 mRNA expression and its downstream targets such as CCNB1 and CDC25B, leading to cell death in both cell lines and patients' ascites cancer cells. Furthermore, addition of thiostrepton to paclitaxel and cisplatin showed synergistic effects in chemoresistant ovarian cancer patients' ascites cells ex vivo. Targeting FOXM1 may lead to novel therapeutics for chemoresistant epithelial ovarian cancer.

Coupling systematic planning and expert judgement enhances the efficiency of river restoration.

PubMed

Langhans, Simone D; Gessner, Jörn; Hermoso, Virgilio; Wolter, Christian

2016-08-01

Ineffectiveness of current river restoration practices hinders the achievement of ecological quality targets set by country-specific regulations. Recent advances in river restoration help planning efforts more systematically to reach ecological targets at the least costs. However, such approaches are often desktop-based and overlook real-world constraints. We argue that combining two techniques commonly used in the conservation arena - expert judgement and systematic planning - will deliver cost-effective restoration plans with a high potential for implementation. We tested this idea targeting the restoration of spawning habitat, i.e. gravel bars, for 11 rheophilic fish species along a river system in Germany (Havel-Spree rivers). With a group of local fish experts, we identified the location and extent of potential gravel bars along the rivers and necessary improvements to migration barriers to ensure fish passage. Restoration cost of each gravel bar included the cost of the action itself plus a fraction of the cost necessary to ensure longitudinal connectivity by upgrading or building fish passages located downstream. We set restoration targets according to the EU Water Framework Directive, i.e. relative abundance of 11 fish species in the reference community and optimised a restoration plan by prioritising a subset of restoration sites from the full set of identified sites, using the conservation planning software Marxan. Out of the 66 potential gravel bars, 36 sites which were mainly located in the downstream section of the system were selected, reflecting their cost-effectiveness given that fewer barriers needed intervention. Due to the limited overall number of sites that experts identified as being suitable for restoring spawning habitat, reaching abundance-targets was challenged. We conclude that coupling systematic river restoration planning with expert judgement produces optimised restoration plans that account for on-the-ground implementation constraints. If applied, this approach has a high potential to enhance overall efficiency of future restoration efforts. Copyright © 2016 Elsevier B.V. All rights reserved.

System and method for injecting fuel

DOEpatents

Uhm, Jong Ho; Johnson, Thomas Edward

2012-12-04

According to various embodiments, a system includes a staggered multi-nozzle assembly. The staggered multi-nozzle assembly includes a first fuel nozzle having a first axis and a first flow path extending to a first downstream end portion, wherein the first fuel nozzle has a first non-circular perimeter at the first downstream end portion. The staggered multi-nozzle assembly also includes a second fuel nozzle having a second axis and a second flow path extending to a second downstream end portion, wherein the first and second downstream end portions are axially offset from one another relative to the first and second axes. The staggered multi-nozzle assembly further includes a cap member disposed circumferentially about at least the first and second fuel nozzles to assemble the staggered multi-nozzle assembly.

c-Met in esophageal squamous cell carcinoma: an independent prognostic factor and potential therapeutic target.

PubMed

Ozawa, Yohei; Nakamura, Yasuhiro; Fujishima, Fumiyoshi; Felizola, Saulo J A; Takeda, Kenichiro; Okamoto, Hiroshi; Ito, Ken; Ishida, Hirotaka; Konno, Takuro; Kamei, Takashi; Miyata, Go; Ohuchi, Noriaki; Sasano, Hironobu

2015-06-03

c-Met is widely known as a poor prognostic factor in various human malignancies. Previous studies have suggested the involvement of c-Met and/or its ligand, hepatocyte growth factor (HGF), in esophageal squamous cell carcinoma (ESCC), but the correlation between c-Met status and clinical outcome remains unclear. Furthermore, the identification of a novel molecular therapeutic target might potentially help improve the clinical outcome of ESCC patients. The expression of c-Met and HGF was immunohistochemically assessed in 104 surgically obtained tissue specimens. The correlation between c-Met/HGF expression and patients' clinicopathological features, including survival, was evaluated. We also investigated changes in cell functions and protein expression of c-Met and its downstream signaling pathway components under treatments with HGF and/or c-Met inhibitor in ESCC cell lines. Elevated expression of c-Met was significantly correlated with tumor depth and pathological stage. Patients with high c-Met expression had significantly worse survival. In addition, multivariate analysis identified the high expression of c-Met as an independent prognostic factor. Treatment with c-Met inhibitor under HGF stimulation significantly inhibited the invasive capacity of an ESCC cell line with elevated c-Met mRNA expression. Moreover, c-Met and its downstream signaling inactivation was also detected after treatment with c-Met inhibitor. The results of our study identified c-Met expression as an independent prognostic factor in ESCC patients and demonstrated that c-Met could be a potential molecular therapeutic target for the treatment of ESCC with elevated c-Met expression.

PI3K-mediated PDGFRα signaling regulates survival and proliferation in skeletal development through p53-dependent intracellular pathways

PubMed Central

Fantauzzo, Katherine A.; Soriano, Philippe

2014-01-01

Previous studies have identified phosphatidylinositol 3-kinase (PI3K) as the main downstream effector of PDGFRα signaling during murine skeletal development. Autophosphorylation mutant knock-in embryos in which PDGFRα is unable to bind PI3K (PdgfraPI3K/PI3K) exhibit skeletal defects affecting the palatal shelves, shoulder girdle, vertebrae, and sternum. To identify proteins phosphorylated by Akt downstream from PI3K-mediated PDGFRα signaling, we immunoprecipitated Akt phosphorylation substrates from PDGF-AA-treated primary mouse embryonic palatal mesenchyme (MEPM) lysates and analyzed the peptides by nanoliquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS). Our analysis generated a list of 56 proteins, including 10 that regulate cell survival and proliferation. We demonstrate that MEPM cell survival is impaired in the presence of a PI3K inhibitor and that PdgfraPI3K/PI3K-derived MEPMs do not proliferate in response to PDGF-AA treatment. Several of the identified Akt phosphorylation targets, including Ybox1, mediate cell survival through regulation of p53. We show that Ybox1 binds both the Trp53 promoter and the p53 protein and that expression of Trp53 is significantly decreased upon PDGF-AA treatment in MEPMs. Finally, we demonstrate that introduction of a Trp53-null allele attenuates the vertebral defects found in PdgfraPI3K/PI3K neonates. Our findings identify p53 as a novel effector downstream from PI3K-engaged PDGFRα signaling that regulates survival and proliferation during skeletal development in vivo. PMID:24788519

System and method for reducing combustion dynamics and NO.sub.x in a combustor

DOEpatents

Uhm, Jong H.; Johnson, Thomas Edward

2015-11-20

A system for reducing combustion dynamics and NO.sub.x in a combustor includes a tube bundle that extends radially across at least a portion of the combustor, wherein the tube bundle comprises an upstream surface axially separated from a downstream surface. A shroud circumferentially surrounds the upstream and downstream surfaces. A plurality of tubes extends through the tube bundle from the upstream surface through the downstream surface, wherein the downstream surface is stepped to produce tubes having different lengths through the tube bundle. A method for reducing combustion dynamics and NO.sub.x in a combustor includes flowing a working fluid through a plurality of tubes radially arranged between an upstream surface and a downstream surface of an end cap that extends radially across at least a portion of the combustor, wherein the downstream surface is stepped.

Epigenetic targeting of Hedgehog pathway transcriptional output through BET bromodomain inhibition

PubMed Central

Tang, Yujie; Gholamin, Sharareh; Schubert, Simone; Willardson, Minde I.; Lee, Alex; Bandopadhayay, Pratiti; Bergthold, Guillame; Masoud, Sabran; Nguyen, Brian; Vue, Nujsaubnusi; Balansay, Brianna; Yu, Furong; Oh, Sekyung; Woo, Pamelyn; Chen, Spenser; Ponnuswami, Anitha; Monje, Michelle; Atwood, Scott X.; Whitson, Ramon J.; Mitra, Siddhartha; Cheshier, Samuel H.; Qi, Jun; Beroukhim, Rameen; Tang, Jean Y.; Wechsler-Reya, Rob; Oro, Anthony E.; Link, Brian A.; Bradner, James E.; Cho, Yoon-Jae

2014-01-01

Hedgehog signaling drives oncogenesis in several cancers and strategies targeting this pathway have been developed, most notably through inhibition of Smoothened. However, resistance to Smoothened inhibitors occurs via genetic changes of Smoothened or other downstream Hedgehog components. Here, we overcome these resistance mechanisms by modulating GLI transcription via inhibition of BET bromodomain proteins. We show the BET bromodomain protein, BRD4, regulates GLI transcription downstream of SMO and SUFU and chromatin immunoprecipitation studies reveal BRD4 directly occupies GLI1 and GLI2 promoters, with a substantial decrease in engagement of these sites upon treatment with JQ1, a small molecule inhibitor targeting BRD4. Globally, genes associated with medulloblastoma-specific GLI1 binding sites are downregulated in response to JQ1 treatment, supporting direct regulation of GLI activity by BRD4. Notably, patient- and GEMM-derived Hedgehog-driven tumors (basal cell carcinoma, medulloblastoma and atypical teratoid/rhabdoid tumor) respond to JQ1 even when harboring genetic lesions rendering them resistant to Smoothened antagonists. PMID:24973920

The Metastasis Suppressor, N-MYC Downstream-regulated Gene-1 (NDRG1), Down-regulates the ErbB Family of Receptors to Inhibit Downstream Oncogenic Signaling Pathways*

PubMed Central

Kovacevic, Zaklina; Menezes, Sharleen V.; Sahni, Sumit; Kalinowski, Danuta S.; Bae, Dong-Hun; Lane, Darius J. R.; Richardson, Des R.

2016-01-01

N-MYC downstream-regulated gene-1 (NDRG1) is a potent growth and metastasis suppressor that acts through its inhibitory effects on a wide variety of cellular signaling pathways, including the TGF-β pathway, protein kinase B (AKT)/PI3K pathway, RAS, etc. To investigate the hypothesis that its multiple effects could be regulated by a common upstream effector, the role of NDRG1 on the epidermal growth factor receptor (EGFR) and other members of the ErbB family, namely human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 3 (HER3), was examined. We demonstrate that NDRG1 markedly decreased the expression and activation of EGFR, HER2, and HER3 in response to the epidermal growth factor (EGF) ligand, while also inhibiting formation of the EGFR/HER2 and HER2/HER3 heterodimers. In addition, NDRG1 also decreased activation of the downstream MAPKK in response to EGF. Moreover, novel anti-tumor agents of the di-2-pyridylketone class of thiosemicarbazones, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, which markedly up-regulate NDRG1, were found to inhibit EGFR, HER2, and HER3 expression and phosphorylation in cancer cells. However, the mechanism involved appeared dependent on NDRG1 for di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone, but was independent of this metastasis suppressor for di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone. This observation demonstrates that small structural changes in thiosemicarbazones result in marked alterations in molecular targeting. Collectively, these results reveal a mechanism for the extensive downstream effects on cellular signaling attributed to NDRG1. Furthermore, this study identifies a novel approach for the treatment of tumors resistant to traditional EGFR inhibitors. PMID:26534963

Role of SRC-3delta4 in the Progression and Metastasis of Castration-Resistant Prostate Cancer

DTIC Science & Technology

2013-10-01

Expression of SRC-3∆4, GAPDH, and AR target genes including PSA, KLK2, IGFBP5, Cyclin A2, and UBE2C was determined by RT-qPCR analysis . Data are...Expression of AR (B), GAPDH, and TMPRSS2- ERG (C) was determined by RT-qPCR analysis . Data are presented using the comparative Ct method, in which GAPDH...input. An irrelevant region (1800 bp downstream of transcription start site) was served as a negative control. (E) and (F). ChIP analysis of SRC-3∆4’s

Type I/II cytokines, JAKs, and new strategies for treating autoimmune diseases

PubMed Central

Schwartz, Daniella M.; Bonelli, Michael; Gadina, Massimo; O’Shea, John J.

2015-01-01

Cytokines are major drivers of autoimmunity, and biologic agents targeting cytokines have revolutionized the treatment of immune-mediated diseases. Despite the effectiveness of these drugs, they do not induce complete remission in all patients, prompting the development of alternative strategies—including targeting of intracellular signal transduction pathways downstream of cytokines. Many cytokines that bind type I and type II cytokine receptors are critical regulators of immune-mediated diseases and employ the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) pathway to exert their effect. Pharmacological inhibition of JAKs block the actions of type I/II cytokines, and within the past 3 years therapeutic JAK inhibitors, or Jakinibs, have become available to rheumatologists. Jakinibs have proven effective for the treatment of rheumatoid arthritis and other inflammatory diseases. Adverse effects of these agents are largely related to their mode of action and include infections and hyperlipidemia. Jakinibs are currently being investigated for a number of new indications, and second-generation selective Jakinibs are being developed and tested. Targeting STATs could be a future avenue for the treatment of rheumatic diseases, although substantial challenges remain. Nonetheless, the ability to therapeutically target intracellular signalling pathways has already created a new paradigm for the treatment of rheumatologic disease. PMID:26633291

Approaches for targeting self-renewal pathways in cancer stem cells: implications for hematological treatments.

PubMed

Horne, Gillian A; Copland, Mhairi

2017-05-01

Self-renewal is considered a defining property of stem cells. Self-renewal is essential in embryogenesis and normal tissue repair and homeostasis. However, in cancer, self-renewal pathways, e.g. WNT, NOTCH, Hedgehog and BMP, frequently become de-regulated in stem cells, or more mature progenitor cells acquire self-renewal properties, resulting in abnormal tissue growth and tumorigenesis. Areas covered: This review considers the conserved embryonic self-renewal pathways, including WNT, NOTCH, Hedgehog and BMP. The article describes recent advances in our understanding of these pathways in leukemia and, more specifically, leukemia stem cells (LSC), how these pathways cross-talk and interact with the LSC microenvironment, and discusses the clinical implications and potential therapeutic strategies, both in preclinical and in clinical trials for hematological malignancies. Expert opinion: The conserved embryonic self-renewal pathways are frequently de-regulated in cancer stem cells (CSC), including LSCs. There is significant cross-talk between self-renewal pathways, and their downstream targets, and the microenvironment. Effective targeting of these pathways is challenging due to cross-talk, and importantly, because these pathways are important for normal stem cells as well as CSC, adverse effects on normal tissues may mean a therapeutic window cannot be identified. Nonetheless, several agents targeting these pathways are currently in clinical trials in hematological malignancies.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hughes, Michael John; McConnaughhay, Johnie Franklin

A combustor includes a tube bundle that extends radially across at least a portion of the combustor. The tube bundle includes an upstream surface axially separated from a downstream surface, and a plurality of tubes extend from the upstream surface through the downstream surface to provide fluid communication through the tube bundle. A barrier extends radially inside the tube bundle between the upstream and downstream surfaces, and a baffle extends axially inside the tube bundle between the upstream surface and the barrier.

DEPDC5 as a potential therapeutic target for epilepsy.

PubMed

Myers, Kenneth A; Scheffer, Ingrid E

2017-06-01

Dishevelled, Egl-10 and Pleckstrin (DEP) domain-containing protein 5 (DEPDC5) is a protein subunit of the GTPase-activating proteins towards Rags 1 (GATOR1) complex. GATOR1 is a recently identified modulator of mechanistic target of rapamycin (mTOR) activity. mTOR is a key regulator of cell proliferation and metabolism; disruption of the mTOR pathway is implicated in focal epilepsy, both acquired and genetic. Tuberous sclerosis is the prototypic mTOR genetic syndrome with epilepsy, however GATOR1 gene mutations have recently been shown to cause lesional and non-lesional focal epilepsy. Areas covered: This review summarizes the mTOR pathway, including regulators and downstream effectors, emphasizing recent developments in the understanding of the complex role of the GATOR1 complex. We review the epilepsy types associated with mTOR overactivity, including tuberous sclerosis, polyhydramnios megalencephaly symptomatic epilepsy, cortical dysplasia, non-lesional focal epilepsy and post-traumatic epilepsy. Currently available mTOR inhibitors are discussed, primarily rapamycin analogs and ATP competitive mTOR inhibitors. Expert opinion: DEPDC5 is an attractive therapeutic target in focal epilepsy, as effects of DEPDC5 agonists would likely be anti-epileptogenic and more selective than currently available mTOR inhibitors. Therapeutic effects might be synergistic with certain existing dietary therapies, including the ketogenic diet.

System for reducing combustion dynamics and NO.sub.x in a combustor

DOEpatents

Uhm, Jong Ho; Ziminsky, Willy Steve; Johnson, Thomas Edward; Hughes, Michael John; York, William David

2016-05-31

A combustor includes an end cap that extends radially across at least a portion of the combustor. The end cap includes an upstream surface axially separated from a downstream surface. A plurality of tubes extend from the upstream surface through the downstream surface of the end cap to provide fluid communication through the end cap. Each tube in a first set of the plurality of tubes has an inlet proximate to the upstream surface and an outlet downstream from the downstream surface. Each outlet has a first portion that extends a different axial distance from the inlet than a second portion.

Deletion of the transforming growth factor β receptor type II gene in articular chondrocytes leads to a progressive osteoarthritis-like phenotype in mice.

PubMed

Shen, Jie; Li, Jia; Wang, Baoli; Jin, Hongting; Wang, Meina; Zhang, Yejia; Yang, Yunzhi; Im, Hee-Jeong; O'Keefe, Regis; Chen, Di

2013-12-01

While transforming growth factor β (TGFβ) signaling plays a critical role in chondrocyte metabolism, the TGFβ signaling pathways and target genes involved in cartilage homeostasis and the development of osteoarthritis (OA) remain unclear. Using an in vitro cell culture method and an in vivo mouse genetic approach, we undertook this study to investigate TGFβ signaling in chondrocytes and to determine whether Mmp13 and Adamts5 are critical downstream target genes of TGFβ signaling. TGFβ receptor type II (TGFβRII)-conditional knockout (KO) (TGFβRII(Col2ER)) mice were generated by breeding TGFβRII(flox/flox) mice with Col2-CreER-transgenic mice. Histologic, histomorphometric, and gene expression analyses were performed. In vitro TGFβ signaling studies were performed using chondrogenic rat chondrosarcoma cells. To determine whether Mmp13 and Adamts5 are critical downstream target genes of TGFβ signaling, TGFβRII/matrix metalloproteinase 13 (MMP-13)- and TGFβRII/ADAMTS-5-double-KO mice were generated and analyzed. Inhibition of TGFβ signaling (deletion of the Tgfbr2 gene in chondrocytes) resulted in up-regulation of Runx2, Mmp13, and Adamts5 expression in articular cartilage tissue and progressive OA development in TGFβRII(Col2ER) mice. Deletion of the Mmp13 or Adamts5 gene significantly ameliorated the OA-like phenotype induced by the loss of TGFβ signaling. Treatment of TGFβRII(Col2ER) mice with an MMP-13 inhibitor also slowed OA progression. Mmp13 and Adamts5 are critical downstream target genes involved in the TGFβ signaling pathway during the development of OA. Copyright © 2013 by the American College of Rheumatology.

Non-coding RNAs: new biomarkers and therapeutic targets for esophageal cancer

PubMed Central

Ren, Zhipeng; Zhang, Guoliang

2017-01-01

Esophageal cancer is one of the most common gastrointestinal malignant diseases and there is still no effective treatment. The incidence of esophageal cancer in the world is relatively high and on the increase year by year. Thus, the elaboration on the carcinogenesis of esophageal cancer and the identification of new biomarkers and therapeutic targets is quite beneficial to optimizing the current therapeutic regimen for treating such deadly disease. More and more evidence has shown that non-coding RNAs play an important role in the development and progression of multiple human cancers, including esophageal cancer. microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are two functional kinds of non-coding RNAs that have been well investigated. They exert tumor suppressive or promoting effect by specifically regulating the expression of certain downstream target genes, which is tumor specific. It is also proved that miRNAs and lncRNAs level in tissue and plasma from esophageal cancer patients are closely correlated with the survival and disease progression, which could be used as a prognostic factor and therapeutic target for esophageal cancer. PMID:28388588

Non-coding RNAs: new biomarkers and therapeutic targets for esophageal cancer.

PubMed

Hou, Xiaobin; Wen, Jiaxin; Ren, Zhipeng; Zhang, Guoliang

2017-06-27

Esophageal cancer is one of the most common gastrointestinal malignant diseases and there is still no effective treatment. The incidence of esophageal cancer in the world is relatively high and on the increase year by year. Thus, the elaboration on the carcinogenesis of esophageal cancer and the identification of new biomarkers and therapeutic targets is quite beneficial to optimizing the current therapeutic regimen for treating such deadly disease. More and more evidence has shown that non-coding RNAs play an important role in the development and progression of multiple human cancers, including esophageal cancer. microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are two functional kinds of non-coding RNAs that have been well investigated. They exert tumor suppressive or promoting effect by specifically regulating the expression of certain downstream target genes, which is tumor specific. It is also proved that miRNAs and lncRNAs level in tissue and plasma from esophageal cancer patients are closely correlated with the survival and disease progression, which could be used as a prognostic factor and therapeutic target for esophageal cancer.

75 FR 31361 - Proposed Flood Elevation Determinations

Federal Register 2010, 2011, 2012, 2013, 2014

2010-06-03

... source(s) elevation ground [caret] Elevation Communities affected in meters (MSL) Effective Modified... meter. ** BFEs to be changed include the listed downstream and upstream BFEs, and include BFEs located... Sea Level, rounded to the nearest 0.1 meter. ** BFEs to be changed include the listed downstream and...

Antidepressive effects of targeting ELK-1 signal transduction.

PubMed

Apazoglou, Kallia; Farley, Séverine; Gorgievski, Victor; Belzeaux, Raoul; Lopez, Juan Pablo; Grenier, Julien; Ibrahim, El Chérif; El Khoury, Marie-Anne; Tse, Yiu C; Mongredien, Raphaele; Barbé, Alexandre; de Macedo, Carlos E A; Jaworski, Wojciech; Bochereau, Ariane; Orrico, Alejandro; Isingrini, Elsa; Guinaudie, Chloé; Mikasova, Lenka; Louis, Franck; Gautron, Sophie; Groc, Laurent; Massaad, Charbel; Yildirim, Ferah; Vialou, Vincent; Dumas, Sylvie; Marti, Fabio; Mechawar, Naguib; Morice, Elise; Wong, Tak P; Caboche, Jocelyne; Turecki, Gustavo; Giros, Bruno; Tzavara, Eleni T

2018-05-07

Depression, a devastating psychiatric disorder, is a leading cause of disability worldwide. Current antidepressants address specific symptoms of the disease, but there is vast room for improvement 1 . In this respect, new compounds that act beyond classical antidepressants to target signal transduction pathways governing synaptic plasticity and cellular resilience are highly warranted 2-4 . The extracellular signal-regulated kinase (ERK) pathway is implicated in mood regulation 5-7 , but its pleiotropic functions and lack of target specificity prohibit optimal drug development. Here, we identified the transcription factor ELK-1, an ERK downstream partner 8 , as a specific signaling module in the pathophysiology and treatment of depression that can be targeted independently of ERK. ELK1 mRNA was upregulated in postmortem hippocampal tissues from depressed suicides; in blood samples from depressed individuals, failure to reduce ELK1 expression was associated with resistance to treatment. In mice, hippocampal ELK-1 overexpression per se produced depressive behaviors; conversely, the selective inhibition of ELK-1 activation prevented depression-like molecular, plasticity and behavioral states induced by stress. Our work stresses the importance of target selectivity for a successful approach for signal-transduction-based antidepressants, singles out ELK-1 as a depression-relevant transducer downstream of ERK and brings proof-of-concept evidence for the druggability of ELK-1.

MicroRNA as therapeutic targets for treatment of depression

PubMed Central

Hansen, Katelin F; Obrietan, Karl

2013-01-01

Depression is a potentially life-threatening mental disorder affecting approximately 300 million people worldwide. Despite much effort, the molecular underpinnings of clinical depression remain poorly defined, and current treatments carry limited therapeutic efficacy and potentially burdensome side effects. Recently, small noncoding RNA molecules known as microRNA (miRNA) have gained prominence as a target for therapeutic intervention, given their capacity to regulate neuronal physiology. Further, mounting evidence suggests a prominent role for miRNA in depressive molecular signaling. Recent studies have demonstrated that dysregulation of miRNA expression occurs in animal models of depression, and in the post-mortem tissue of clinically depressed patients. Investigations into depression-associated miRNA disruption reveals dramatic effects on downstream targets, many of which are thought to contribute to depressive symptoms. Furthermore, selective serotonin reuptake inhibitors, as well as other antidepressant drugs, have the capacity to reverse aberrant depressive miRNA expression and their downstream targets. Given the powerful effects that miRNA have on the central nervous system transcriptome, and the aforementioned studies, there is a compelling rationale to begin to assess the potential contribution of miRNA to depressive etiology. Here, we review the molecular biology of miRNA, our current understanding of miRNA in relation to clinical depression, and the utility of targeting miRNA for antidepressant treatment. PMID:23935365

The challenges of modulating the 'rest and digest' system: acetylcholine receptors as drug targets.

PubMed

VanPatten, Sonya; Al-Abed, Yousef

2017-01-01

Acetylcholine, a major neurotransmitter of the parasympathetic and sympathetic nervous systems, was discovered in the early 1900s. Over the years, researchers have revealed much about its regulation, properties of its receptors and features of the downstream signaling that influence its terminal effects. The acetylcholine system, traditionally associated with neuromuscular communication, is now known to play a crucial part in modulation of the immune system and other 'rest and digest' effects. Recent research seeks to elucidate the system's role in brain functions including cognition, sleep, arousal, motivation, reward and pain. We highlight clinically approved and experimental drugs that modulate the acetylcholine receptors. The complexities in targeting the acetylcholine receptors are vast and finding future indications for drug development associated with specific acetylcholine receptors remains a challenge. Copyright © 2016 Elsevier Ltd. All rights reserved.

Manufacturing of Proteins and Antibodies: Chapter Downstream Processing Technologies : Harvest Operations.

PubMed

Turner, Richard; Joseph, Adrian; Titchener-Hooker, Nigel; Bender, Jean

2017-08-04

Cell harvesting is the separation or retention of cells and cellular debris from the supernatant containing the target molecule Selection of harvest method strongly depends on the type of cells, mode of bioreactor operation, process scale, and characteristics of the product and cell culture fluid. Most traditional harvesting methods use some form of filtration, centrifugation, or a combination of both for cell separation and/or retention. Filtration methods include normal flow depth filtration and tangential flow microfiltration. The ability to scale down predictably the selected harvest method helps to ensure successful production and is critical for conducting small-scale characterization studies for confirming parameter targets and ranges. In this chapter we describe centrifugation and depth filtration harvesting methods, share strategies for harvest optimization, present recent developments in centrifugation scale-down models, and review alternative harvesting technologies.

Molecular genetics and targeted therapeutics in biliary tract carcinoma.

PubMed

Marks, Eric I; Yee, Nelson S

2016-01-28

The primary malignancies of the biliary tract, cholangiocarcinoma and gallbladder cancer, often present at an advanced stage and are marginally sensitive to radiation and chemotherapy. Accumulating evidence indicates that molecularly targeted agents may provide new hope for improving treatment response in biliary tract carcinoma (BTC). In this article, we provide a critical review of the pathogenesis and genetic abnormalities of biliary tract neoplasms, in addition to discussing the current and emerging targeted therapeutics in BTC. Genetic studies of biliary tumors have identified the growth factors and receptors as well as their downstream signaling pathways that control the growth and survival of biliary epithelia. Target-specific monoclonal antibodies and small molecules inhibitors directed against the signaling pathways that drive BTC growth and invasion have been developed. Numerous clinical trials designed to test these agents as either monotherapy or in combination with conventional chemotherapy have been completed or are currently underway. Research focusing on understanding the molecular basis of biliary tumorigenesis will continue to identify for targeted therapy the key mutations that drive growth and invasion of biliary neoplasms. Additional strategies that have emerged for treating this malignant disease include targeting the epigenetic alterations of BTC and immunotherapy. By integrating targeted therapy with molecular profiles of biliary tumor, we hope to provide precision treatment for patients with malignant diseases of the biliary tract.

Salicylic acid suppresses jasmonic acid signaling downstream of SCFCOI1-JAZ by targeting GCC promoter motifs via transcription factor ORA59.

PubMed

Van der Does, Dieuwertje; Leon-Reyes, Antonio; Koornneef, Annemart; Van Verk, Marcel C; Rodenburg, Nicole; Pauwels, Laurens; Goossens, Alain; Körbes, Ana P; Memelink, Johan; Ritsema, Tita; Van Wees, Saskia C M; Pieterse, Corné M J

2013-02-01

Antagonism between the defense hormones salicylic acid (SA) and jasmonic acid (JA) plays a central role in the modulation of the plant immune signaling network, but the molecular mechanisms underlying this phenomenon are largely unknown. Here, we demonstrate that suppression of the JA pathway by SA functions downstream of the E3 ubiquitin-ligase Skip-Cullin-F-box complex SCF(COI1), which targets JASMONATE ZIM-domain transcriptional repressor proteins (JAZs) for proteasome-mediated degradation. In addition, neither the stability nor the JA-induced degradation of JAZs was affected by SA. In silico promoter analysis of the SA/JA crosstalk transcriptome revealed that the 1-kb promoter regions of JA-responsive genes that are suppressed by SA are significantly enriched in the JA-responsive GCC-box motifs. Using GCC:GUS lines carrying four copies of the GCC-box fused to the β-glucuronidase reporter gene, we showed that the GCC-box motif is sufficient for SA-mediated suppression of JA-responsive gene expression. Using plants overexpressing the GCC-box binding APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors ERF1 or ORA59, we found that SA strongly reduces the accumulation of ORA59 but not that of ERF1. Collectively, these data indicate that the SA pathway inhibits JA signaling downstream of the SCF(COI1)-JAZ complex by targeting GCC-box motifs in JA-responsive promoters via a negative effect on the transcriptional activator ORA59.

New insights into RAS biology reinvigorate interest in mathematical modeling of RAS signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Erickson, Keesha E.; Rukhlenko, Oleksii S.; Posner, Richard G.

RAS is the most frequently mutated gene across human cancers, but developing inhibitors of mutant RAS has proven to be challenging. Given the difficulties of targeting RAS directly, drugs that impact the other components of pathways where mutant RAS operates may potentially be effective. However, the system-level features, including different localizations of RAS isoforms, competition between downstream effectors, and interlocking feedback and feed-forward loops, must be understood to fully grasp the opportunities and limitations of inhibiting specific targets. Mathematical modeling can help us discern the system-level impacts of these features in normal and cancer cells. New technologies enable the acquisitionmore » of experimental data that will facilitate development of realistic models of oncogenic RAS behavior. In light of the wealth of empirical data accumulated over decades of study and the advancement of experimental methods for gathering new data, modelers now have the opportunity to advance progress toward realization of targeted treatment for mutant RAS-driven cancers.« less

New insights into RAS biology reinvigorate interest in mathematical modeling of RAS signaling

DOE PAGES

Erickson, Keesha E.; Rukhlenko, Oleksii S.; Posner, Richard G.; ...

2018-03-05

RAS is the most frequently mutated gene across human cancers, but developing inhibitors of mutant RAS has proven to be challenging. Given the difficulties of targeting RAS directly, drugs that impact the other components of pathways where mutant RAS operates may potentially be effective. However, the system-level features, including different localizations of RAS isoforms, competition between downstream effectors, and interlocking feedback and feed-forward loops, must be understood to fully grasp the opportunities and limitations of inhibiting specific targets. Mathematical modeling can help us discern the system-level impacts of these features in normal and cancer cells. New technologies enable the acquisitionmore » of experimental data that will facilitate development of realistic models of oncogenic RAS behavior. In light of the wealth of empirical data accumulated over decades of study and the advancement of experimental methods for gathering new data, modelers now have the opportunity to advance progress toward realization of targeted treatment for mutant RAS-driven cancers.« less

New insights into RAS biology reinvigorate interest in mathematical modeling of RAS signaling.

PubMed

Erickson, Keesha E; Rukhlenko, Oleksii S; Posner, Richard G; Hlavacek, William S; Kholodenko, Boris N

2018-03-05

RAS is the most frequently mutated gene across human cancers, but developing inhibitors of mutant RAS has proven to be challenging. Given the difficulties of targeting RAS directly, drugs that impact the other components of pathways where mutant RAS operates may potentially be effective. However, the system-level features, including different localizations of RAS isoforms, competition between downstream effectors, and interlocking feedback and feed-forward loops, must be understood to fully grasp the opportunities and limitations of inhibiting specific targets. Mathematical modeling can help us discern the system-level impacts of these features in normal and cancer cells. New technologies enable the acquisition of experimental data that will facilitate development of realistic models of oncogenic RAS behavior. In light of the wealth of empirical data accumulated over decades of study and the advancement of experimental methods for gathering new data, modelers now have the opportunity to advance progress toward realization of targeted treatment for mutant RAS-driven cancers. Copyright © 2018 Elsevier Ltd. All rights reserved.

Akt3 is a privileged first responder in isozyme-specific electrophile response.

PubMed

Long, Marcus J C; Parvez, Saba; Zhao, Yi; Surya, Sanjna L; Wang, Yiran; Zhang, Sheng; Aye, Yimon

2017-03-01

Isozyme-specific post-translational regulation fine tunes signaling events. However, redundancy in sequence or activity renders links between isozyme-specific modifications and downstream functions uncertain. Methods to study this phenomenon are underdeveloped. Here we use a redox-targeting screen to reveal that Akt3 is a first-responding isozyme sensing native electrophilic lipids. Electrophile modification of Akt3 modulated downstream pathway responses in cells and Danio rerio (zebrafish) and markedly differed from Akt2-specific oxidative regulation. Digest MS sequencing identified Akt3 C119 as the privileged cysteine that senses 4-hydroxynonenal. A C119S Akt3 mutant was hypomorphic for all downstream phenotypes shown by wild-type Akt3. This study documents isozyme-specific and chemical redox signal-personalized physiological responses.

Methylation-sensitive enrichment of minor DNA alleles using a double-strand DNA-specific nuclease.

PubMed

Liu, Yibin; Song, Chen; Ladas, Ioannis; Fitarelli-Kiehl, Mariana; Makrigiorgos, G Mike

2017-04-07

Aberrant methylation changes, often present in a minor allelic fraction in clinical samples such as plasma-circulating DNA (cfDNA), are potentially powerful prognostic and predictive biomarkers in human disease including cancer. We report on a novel, highly-multiplexed approach to facilitate analysis of clinically useful methylation changes in minor DNA populations. Methylation Specific Nuclease-assisted Minor-allele Enrichment (MS-NaME) employs a double-strand-specific DNA nuclease (DSN) to remove excess DNA with normal methylation patterns. The technique utilizes oligonucleotide-probes that direct DSN activity to multiple targets in bisulfite-treated DNA, simultaneously. Oligonucleotide probes targeting unmethylated sequences generate local double stranded regions resulting to digestion of unmethylated targets, and leaving methylated targets intact; and vice versa. Subsequent amplification of the targeted regions results in enrichment of the targeted methylated or unmethylated minority-epigenetic-alleles. We validate MS-NaME by demonstrating enrichment of RARb2, ATM, MGMT and GSTP1 promoters in multiplexed MS-NaME reactions (177-plex) using dilutions of methylated/unmethylated DNA and in DNA from clinical lung cancer samples and matched normal tissue. MS-NaME is a highly scalable single-step approach performed at the genomic DNA level in solution that combines with most downstream detection technologies including Sanger sequencing, methylation-sensitive-high-resolution melting (MS-HRM) and methylation-specific-Taqman-based-digital-PCR (digital Methylight) to boost detection of low-level aberrant methylation-changes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

SWATH2stats: An R/Bioconductor Package to Process and Convert Quantitative SWATH-MS Proteomics Data for Downstream Analysis Tools.

PubMed

Blattmann, Peter; Heusel, Moritz; Aebersold, Ruedi

2016-01-01

SWATH-MS is an acquisition and analysis technique of targeted proteomics that enables measuring several thousand proteins with high reproducibility and accuracy across many samples. OpenSWATH is popular open-source software for peptide identification and quantification from SWATH-MS data. For downstream statistical and quantitative analysis there exist different tools such as MSstats, mapDIA and aLFQ. However, the transfer of data from OpenSWATH to the downstream statistical tools is currently technically challenging. Here we introduce the R/Bioconductor package SWATH2stats, which allows convenient processing of the data into a format directly readable by the downstream analysis tools. In addition, SWATH2stats allows annotation, analyzing the variation and the reproducibility of the measurements, FDR estimation, and advanced filtering before submitting the processed data to downstream tools. These functionalities are important to quickly analyze the quality of the SWATH-MS data. Hence, SWATH2stats is a new open-source tool that summarizes several practical functionalities for analyzing, processing, and converting SWATH-MS data and thus facilitates the efficient analysis of large-scale SWATH/DIA datasets.

Cell-Specific Actions of a Human LHX3 Gene Enhancer During Pituitary and Spinal Cord Development

PubMed Central

Park, Soyoung; Mullen, Rachel D.

2013-01-01

The LIM class of homeodomain protein 3 (LHX3) transcription factor is essential for pituitary gland and nervous system development in mammals. In humans, mutations in the LHX3 gene underlie complex pediatric syndromes featuring deficits in anterior pituitary hormones and defects in the nervous system. The mechanisms that control temporal and spatial expression of the LHX3 gene are poorly understood. The proximal promoters of the human LHX3 gene are insufficient to guide expression in vivo and downstream elements including a conserved enhancer region appear to play a role in tissue-specific expression in the pituitary and nervous system. Here we characterized the activity of this downstream enhancer region in regulating gene expression at the cellular level during development. Human LHX3 enhancer-driven Cre reporter transgenic mice were generated to facilitate studies of enhancer actions. The downstream LHX3 enhancer primarily guides gene transcription in α-glycoprotein subunit -expressing cells secreting the TSHβ, LHβ, or FSHβ hormones and expressing the GATA2 and steroidogenic factor 1 transcription factors. In the developing nervous system, the enhancer serves as a targeting module active in V2a interneurons. These results demonstrate that the downstream LHX3 enhancer is important in specific endocrine and neural cell types but also indicate that additional regulatory elements are likely involved in LHX3 gene expression. Furthermore, these studies revealed significant gonadotrope cell heterogeneity during pituitary development, providing insights into the cellular physiology of this key reproductive regulatory cell. The human LHX3 enhancer-driven Cre reporter transgenic mice also provide a valuable tool for further developmental studies of cell determination and differentiation in the pituitary and nervous system. PMID:24100213

EGF stimulates the activation of EGF receptors and the selective activation of major signaling pathways during mitosis.

PubMed

Wee, Ping; Shi, Huaiping; Jiang, Jennifer; Wang, Yuluan; Wang, Zhixiang

2015-03-01

Mitosis and epidermal growth factor (EGF) receptor (EGFR) are both targets for cancer therapy. The role of EGFR signaling in mitosis has been rarely studied and poorly understood. The limited studies indicate that the activation of EGFR and downstream signaling pathways is mostly inhibited during mitosis. However, we recently showed that EGFR is phosphorylated in response to EGF stimulation in mitosis. Here we studied EGF-induced EGFR activation and the activation of major signaling pathways downstream of EGFR during mitosis. We showed that EGFR was strongly activated by EGF during mitosis as all the five major tyrosine residues including Y992, Y1045, Y1068, Y1086, and Y1173 were phosphorylated to a level similar to that in the interphase. We further showed that the activated EGFR is able to selectively activate some downstream signaling pathways while avoiding others. Activated EGFR is able to activate PI3K and AKT2, but not AKT1, which may be responsible for the observed effects of EGF against nocodazole-induced cell death. Activated EGFR is also able to activate c-Src, c-Cbl and PLC-γ1 during mitosis. However, activated EGFR is unable to activate ERK1/2 and their downstream substrates RSK and Elk-1. While it activated Ras, EGFR failed to fully activate Raf-1 in mitosis due to the lack of phosphorylation at Y341 and the lack of dephosphorylation at pS259. We conclude that contrary to the dogma, EGFR is activated by EGF during mitosis. Moreover, EGFR-mediated cell signaling is regulated differently from the interphase to specifically serve the needs of the cell in mitosis. Copyright © 2015 Elsevier Inc. All rights reserved.

A critical role for HER3 in HER2-amplified and non-amplified breast cancers: function of a kinase-dead RTK

PubMed Central

Dey, Nandini; Williams, Casey; Leyland-Jones, Brain; De, Pradip

2015-01-01

ERBB3/HER3 is the most intriguing RTK by virtue of its ability to transduce multiple cytosolic signals for the proliferation and growth of tumor cells in spite of being a “kinase dead” receptor that binds to its true ligand, heregulin. Although other members of the HER3 family like EGFR and HER2 have long been recognized to be associated with breast tumorigenesis and studied because of their predictive and prognostic value, the significance of HER3 as an irrefutable component of HER family signalosome is a relatively new development. The recent understanding of signals originating from the oncogenic partnership of HER3 with HER2 in the context of HER2 amplification/overexpression showed the critical clinical value for the treatment of HER2+BC. The downstream signaling cascade (included but not limited to the PI3K signaling) associated with signals originating from HER2:HER3 dimers play a vital role in the tumorigenesis, drug-resistance and tumor progression of HER2+BC. The upregulation of HER3 activity provides an alternate “escape route” via which tumor cells bypass either the inhibition of the HER family RTKs or the inhibition of the downstream PI3K-AKT-mTOR signaling pathway. By understanding the signaling that provides this “escape route” for these tumor cells treated with a targeted therapy (HER2 inhibitors or inhibitors of downstream PI3K-AKT-mTOR signaling pathway), we are just beginning to appreciate the prognostic value of HER3 in breast cancer. In this review, we will discuss the relevance of HER3 signaling in the context of, (1) downstream oncogenic signals and (2) therapeutic options in HER2 amplified BC. PMID:26064441

Retention of ferrofluid aggregates at the target site during magnetic drug targeting

NASA Astrophysics Data System (ADS)

Asfer, Mohammed; Saroj, Sunil Kumar; Panigrahi, Pradipta Kumar

2017-08-01

The present study reports the retention dynamics of a ferrofluid aggregate localized at the target site inside a glass capillary (500 × 500 μm2 square cross section) against a bulk flow of DI water (Re = 0.16 and 0.016) during the process of magnetic drug targeting (MDT). The dispersion dynamics of iron oxide nanoparticles (IONPs) into bulk flow for different initial size of aggregate at the target site is reported using the brightfield visualization technique. The flow field around the aggregate during the retention is evaluated using the μPIV technique. IONPs at the outer boundary experience a higher shear force as compared to the magnetic force, resulting in dispersion of IONPs into the bulk flow downstream to the aggregate. The blockage effect and the roughness of the outer boundary of the aggregate resulting from chain like clustering of IONPs contribute to the flow recirculation at the downstream region of the aggregate. The entrapment of seeding particles inside the chain like clusters of IONPs at the outer boundary of the aggregate reduces the degree of roughness resulting in a streamlined aggregate at the target site at later time. The effect of blockage, structure of the aggregate, and disturbed flow such as recirculation around the aggregate are the primary factors, which must be investigated for the effectiveness of the MDT process for in vivo applications.

Protein kinases: mechanisms and downstream targets in inflammation-mediated obesity and insulin resistance.

PubMed

Nandipati, Kalyana C; Subramanian, Saravanan; Agrawal, Devendra K

2017-02-01

Obesity-induced low-grade inflammation (metaflammation) impairs insulin receptor signaling. This has been implicated in the development of insulin resistance. Insulin signaling in the target tissues is mediated by stress kinases such as p38 mitogen-activated protein kinase, c-Jun NH2-terminal kinase, inhibitor of NF-kB kinase complex β (IKKβ), AMP-activated protein kinase, protein kinase C, Rho-associated coiled-coil containing protein kinase, and RNA-activated protein kinase. Most of these kinases phosphorylate several key regulators in glucose homeostasis. The phosphorylation of serine residues in the insulin receptor and IRS-1 molecule results in diminished enzymatic activity in the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. This has been one of the key mechanisms observed in the tissues that are implicated in insulin resistance especially in type 2 diabetes mellitus (T2-DM). Identifying the specific protein kinases involved in obesity-induced chronic inflammation may help in developing the targeted drug therapies to minimize the insulin resistance. This review is focused on the protein kinases involved in the inflammatory cascade and molecular mechanisms and their downstream targets with special reference to obesity-induced T2-DM.

Transmission blocking malaria vaccines: Assays and candidates in clinical development.

PubMed

Sauerwein, R W; Bousema, T

2015-12-22

Stimulated by recent advances in malaria control and increased funding, the elimination of malaria is now considered to be an attainable goal for an increasing number of malaria-endemic regions. This has boosted the interest in transmission-reducing interventions including vaccines that target sexual, sporogenic, and/or mosquito-stage antigens to interrupt malaria transmission (SSM-VIMT). SSM-VIMT aim to prevent human malaria infection in vaccinated communities by inhibiting parasite development within the mosquito after a blood meal taken from a gametocyte carrier. Only a handful of target antigens are in clinical development and progress has been slow over the years. Major stumbling blocks include (i) the expression of appropriately folded target proteins and their downstream purification, (ii) insufficient induction of sustained functional blocking antibody titers by candidate vaccines in humans, and (iii) validation of a number of (bio)-assays as correlate for blocking activity in the field. Here we discuss clinical manufacturing and testing of current SSM-VIMT candidates and the latest bio-assay development for clinical evaluation. New testing strategies are discussed that may accelerate the evaluation and application of SSM-VIMT. Copyright © 2015. Published by Elsevier Ltd.

Modeling Shasta Dam operations to regulate temperatures for Chinook salmon under extreme climate and climate change

NASA Astrophysics Data System (ADS)

Dai, A.; Saito, L.; Sapin, J. R.; Rajagopalan, B.; Hanna, R. B.; Kauneckis, D. L.

2014-12-01

Chinook salmon populations have declined significantly after the construction of Shasta Dam on the Sacramento River in 1945 prevented them from spawning in the cold waters upstream. In 1994, the winter-run Chinook were listed under the Endangered Species Act and 3 years later the US Bureau of Reclamation began operating a temperature control device (TCD) on the dam that allows for selective withdrawal for downstream temperature control to promote salmon spawning while also maximizing power generation. However, dam operators are responsible to other interests that depend on the reservoir for water such as agriculture, municipalities, industry, and recreation. An increase in temperatures due to climate change may place additional strain on the ability of dam operations to maintain spawning habitat for salmon downstream of the dam. We examined the capability of Shasta Dam to regulate downstream temperatures under extreme climates and climate change by using stochastically generated streamflow, stream temperature, and weather inputs with a two-dimensional CE-QUAL-W2 model under several operational options. Operation performance was evaluated using degree days and cold pool volume (volume of water below a temperature threshold). Model results indicated that a generalized operations release schedule, in which release elevations varied over the year to match downstream temperature targets, performed best overall in meeting temperature targets while preserving cold pool volume. Releasing all water out the bottom throughout the year tended to meet temperature targets at the expense of depleting the cold pool, and releasing all water out uppermost gates preserved the cold pool, but released water that was too warm during the critical spawning period. With higher air temperatures due to climate change, both degree day and cold pool volume metrics were worse than baseline conditions, which suggests that Chinook salmon may be more negatively affected under climate change.

Characterization of the SIRT family of NAD+-dependent protein deacetylases in the context of a mammalian model of hibernation, the thirteen-lined ground squirrel.

PubMed

Rouble, Andrew N; Storey, Kenneth B

2015-10-01

Hibernating mammals employ strong metabolic rate depression to survive the winter, thereby avoiding the high energy costs of maintaining a euthermic lifestyle in the face of low seasonal temperatures and limited food resources. Characteristics of this natural torpor include a significant reduction in body temperature, a shift to a lipid-based metabolism, global suppression of ATP-expensive activities, and the upregulation of selected genes that mediate biochemical reorganization and cytoprotection. Sirtuin (SIRT) proteins, an evolutionarily conserved family of NAD(+)-dependent protein deacetylases, have been shown to play important roles in the post-translational regulation of many metabolic and cytoprotective processes, suggesting a potential function for these enzymes in the control of hibernation. To assess this possibility, protein levels of the seven mammalian SIRTs (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6 and SIRT7), total SIRT activity, and the acetylation status of two downstream SIRT targets (SOD2K68 and NF-κB p65K310) were measured in skeletal muscle, liver, brown adipose and white adipose tissues of the hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus) over the course of the torpor-arousal cycle. The analysis revealed tissue-specific responses of different SIRTs at various points throughout hibernation, including a potentially interesting correlation between increased levels of SIRT3 protein, heightened total SIRT activity, and decreased acetylation of SIRT3 downstream target SOD2K68 in skeletal muscle during late torpor. These results provide evidence to suggest a possible role for the SIRT family of protein deacetylases in the regulation of the metabolic and cellular protective pathways that mediate the process of mammalian hibernation. Copyright © 2015 Elsevier Inc. All rights reserved.

Advances in treatment of achondroplasia and osteoarthritis.

PubMed

Klag, Kendra A; Horton, William A

2016-04-15

Achondroplasia (ACH) is the prototype and most common of the human chondrodysplasias. It results from gain-of-function mutations that exaggerate the signal output of the fibroblast growth factor receptor 3 (FGFR3), a receptor tyrosine kinase that negatively regulates growth plate activity and linear bone growth. Several approaches to reduce FGFR3 signaling by blocking receptor activation or inhibiting downstream signals have been proposed. Five show promise in preclinical mouse studies. Two candidate therapies target the extracellular domain of FGFR3. The first is a decoy receptor that competes for activating ligands. The second is a synthetic blocking peptide that prevents ligands from binding and activating FGFR3. Two established drugs, statins and meclozine, improve growth of ACH mice. The strongest candidate therapy employs an analog of C-type natriuretic peptide (CNP), which antagonizes the mitogen-activated-protein (MAP) kinase pathway downstream of the FGFR3 receptor and may also act independently in the growth plate. Only the CNP analog has reached clinical trials. Preliminary results of Phase 2 studies show a substantial increase in growth rate of ACH children after six months of therapy with no serious adverse effects. A challenge for drug therapy in ACH is targeting agents to the avascular growth plate. The application of gene therapy in osteoarthritis offers insights because it faces similar technical obstacles. Major advances in gene therapy include the emergence of recombinant adeno-associated virus as the vector of choice, capsid engineering to target vectors to specific tissues, and development of methods to direct vectors to articular chondrocytes. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Human Merkel cell polyomavirus small T antigen is an oncoprotein targeting the 4E-BP1 translation regulator

PubMed Central

Shuda, Masahiro; Kwun, Hyun Jin; Feng, Huichen; Chang, Yuan; Moore, Patrick S.

2011-01-01

Merkel cell polyomavirus (MCV) is the recently discovered cause of most Merkel cell carcinomas (MCCs), an aggressive form of nonmelanoma skin cancer. Although MCV is known to integrate into the tumor cell genome and to undergo mutation, the molecular mechanisms used by this virus to cause cancer are unknown. Here, we show that MCV small T (sT) antigen is expressed in most MCC tumors, where it is required for tumor cell growth. Unlike the closely related SV40 sT, MCV sT transformed rodent fibroblasts to anchorage- and contact-independent growth and promoted serum-free proliferation of human cells. These effects did not involve protein phosphatase 2A (PP2A) inhibition. MCV sT was found to act downstream in the mammalian target of rapamycin (mTOR) signaling pathway to preserve eukaryotic translation initiation factor 4E–binding protein 1 (4E-BP1) hyperphosphorylation, resulting in dysregulated cap-dependent translation. MCV sT–associated 4E-BP1 serine 65 hyperphosphorylation was resistant to mTOR complex (mTORC1) and mTORC2 inhibitors. Steady-state phosphorylation of other downstream Akt-mTOR targets, including S6K and 4E-BP2, was also increased by MCV sT. Expression of a constitutively active 4E-BP1 that could not be phosphorylated antagonized the cell transformation activity of MCV sT. Taken together, these experiments showed that 4E-BP1 inhibition is required for MCV transformation. Thus, MCV sT is an oncoprotein, and its effects on dysregulated cap-dependent translation have clinical implications for the prevention, diagnosis, and treatment of MCV-related cancers. PMID:21841310

Clinical evidence on the magnitude of change in growth pathway activity in relation to Tamoxifen resistance is required.

PubMed

Mansouri, Sepideh; Farahmand, Leila; Teymourzadeh, Azin; Majidzadeh-A, Keivan

2017-08-08

Despite prolonged disease-free survival and overall survival rates in estrogen receptor (ER)-positive patients undergoing adjuvant treatment, Tamoxifen therapy tends to fail due to eventual acquisition of resistance. Although numerous studies have emphasized the role of receptor tyrosine kinases (RTKs) in the development of Tamoxifen resistance, inadequate clinical evidence is available regarding the alteration of biomarker expression during acquired resistance, thus undermining the validity of the findings. Results of two meta-analyses investigating the effect of HER2 status on the prognosis of Tamoxifen-receiving patients have demonstrated that despite HER2-negative patients having longer disease-free survival; there is no difference in overhaul survival between the two groups. Furthermore, due to the intricate molecular interactions among estrogen receptors including ERα36, ERα66, and also RTKs, it is not surprising that RTK suppression does not restore Tamoxifen sensitivity. In considering such a complex network, we speculate that by the time HER2/EGFR is suppressed via targeted therapies, activation of ERα66 and ERα36 initiate molecular signaling pathways downstream of RTKs, thereby enhancing cell proliferation even in the presence of both Tamoxifen and RTK inhibitors. Although clinical findings regarding the molecular pathways downstream of RTKs have been thoroughly discussed in this review, further clinical studies are required in determining a consistency between preclinical and clinical findings. Discovering the best targets in preventing tumor progression requires thorough comprehension of estrogen-dependent and estrogen-independent pathways during Tamoxifen resistance development. Indeed, exploring additional clinically-proven targets would allow for better characterized treatments being available for breast cancer patients. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A Comprehensive Outline of Trastuzumab Resistance Biomarkers in HER2 Overexpressing Breast Cancer.

PubMed

Menyhárt, Otília; Santarpia, Libero; Győrffy, Balázs

2015-01-01

The introduction of trastuzumab for anti-HER2 therapy dramatically changed the clinical outcome for HER2 (ERBB2, neu) positive breast cancer patients. Today, patients eligible for trastuzumab are selected using HER2 expression/amplification status of the primary tumor. However, acquired and inherent resistance to anti-HER2 therapy in these patients poses a significant challenge, and better patient stratification will be needed to improve clinical response. Here, we provide a wide-ranging overview of potential biomarkers capable of stratifying patients regarding their response to trastuzumab. These include HER2 amplification, impaired access to the binding site (p95HER2, Δ16HER-2, MUC4), augmented signaling through other ERBB family receptors (HER1, HER3, HER4) and their ligands, activation of HER2 targets by alternate heterodimers (EphA2, IGF-1R, GDF15, MUC1*), signaling triggered by downstream members (PIK3CA, PTEN, SRC, mTOR), altered expression of cell cycle and apoptotic regulators (CDKs, p27(kip1), Bcl-2), hormone receptor status, resistance to antibody-dependent cellular cytotoxicity (FcγR), and altered miRNA expression signatures. Multigenic molecular profile analyses have revealed further genes not directly associated with classical oncogenic pathways. Although numerous biomarkers have shown promise in pre-clinical studies, many have delivered controversial results when evaluated in clinical trials. One of the keys for targeting ERBB2 will be to consider the entire ERBB family and downstream associated pathways responsible for the malignant transformation. The heterogeneity of the disease is likely to represent a significant obstacle to accurately predicting the course of resistance. The future most probably involves the incorporation of multiple biomarkers into a unified predictor enabling selection of patients for superior targeted drug administration.

Modeling Neutral Densities Downstream of a Gridded Ion Thruster

NASA Technical Reports Server (NTRS)

Soulas, George C.

2010-01-01

The details of a model for determining the neutral density downstream of a gridded ion thruster are presented. An investigation of the possible sources of neutrals emanating from and surrounding a NEXT ion thruster determined that the most significant contributors to the downstream neutral density include discharge chamber neutrals escaping through the perforated grids, neutrals escaping from the neutralizer, and vacuum facility background neutrals. For the neutral flux through the grids, near- and far-field equations are presented for rigorously determining the neutral density downstream of a cylindrical aperture. These equations are integrated into a spherically-domed convex grid geometry with a hexagonal array of apertures for determining neutral densities downstream of the ion thruster grids. The neutrals escaping from an off-center neutralizer are also modeled assuming diffuse neutral emission from the neutralizer keeper orifice. Finally, the effect of the surrounding vacuum facility neutrals is included and assumed to be constant. The model is used to predict the neutral density downstream of a NEXT ion thruster with and without neutralizer flow and a vacuum facility background pressure. The impacts of past simplifying assumptions for predicting downstream neutral densities are also examined for a NEXT ion thruster.

Effects of berberine and cinnamic acid on palmitic acid-induced intracellular triglyceride accumulation in NIT-1 pancreatic β cells.

PubMed

Zhao, Li; Jiang, Shu-Jun; Lu, Fu-Er; Xu, Li-Jun; Zou, Xin; Wang, Kai-Fu; Dong, Hui

2016-07-01

To investigate the effects of berberine (BBR) and cinnamic acid (CA), the main active components in Jiaotai Pill (, JTP), on palmitic acid (PA)-induced intracellular triglyceride (TG) accumulation in NIT-1 pancreatic β cells. Cells were incubated in culture medium containing PA (0.25 mmol/L) for 24 h. Then treatments with BBR (10 μmol/L), CA (100 μmol/L) and the combination of BBR and CA (BBR+CA) were performed respectively. Intracellular lipid accumulation was assessed by Oil Red O staining and TG content was measured by colorimetric assay. The expression of adenosine monophosphate-activated protein kinase (AMPK) protein and its downstream lipogenic and fatty acid oxidation genes, including fatty acid synthase (FAS), acetyl-coA carboxylase (ACC), phosphorylation acetyl-coA carboxylase (pACC), carnitine acyl transferase 1 (CPT-1) and sterol regulating element binding protein 1c (SREBP-1c) were determined by Western blot or real time polymerase chain reaction. PA induced an obvious lipid accumulation and a significant increase in intracellular TG content in NIT-1 cells. PA also induced a remarkable decrease in AMPK protein expression and its downstream targets such as pACC and CPT-1. Meanwhile, AMPK downstream lipogenic genes including SREBP-1c mRNA, FAS and ACC protein expressions were increased. Treatments with BBR and BBR+CA, superior to CA, significantly reversed the above genes changes in NIT-1 pancreatic β cells. However, the synergistic effect of BBR and CA on intracellular TG content was not observed in the present study. It can be concluded that in vitro, BBR and BBR+CA could inhibit PA-induced lipid accumulation by decreasing lipogenesis and increasing lipid oxidation in NIT-1 pancreatic β cells.

Modeling water quality effects of structural and operational changes to Scoggins Dam and Henry Hagg Lake, Oregon

USGS Publications Warehouse

Sullivan, Annett B.; Rounds, Stewart A.

2006-01-01

To meet water quality targets and the municipal and industrial water needs of a growing population in the Tualatin River Basin in northwestern Oregon, an expansion of Henry Hagg Lake is under consideration. Hagg Lake is the basin's primary storage reservoir and provides water during western Oregon's typically dry summers. Potential modifications include raising the dam height by 6.1 meters (20 feet), 7.6 meters (25 feet), or 12.2 meters (40 feet); installing additional outlets (possibly including a selective withdrawal tower); and adding additional inflows to provide greater reliability of filling the enlarged reservoir. One method of providing additional inflows is to route water from the upper Tualatin River through a tunnel and into Sain Creek, a tributary to the lake. Another option is to pump water from the Tualatin River (downstream of the lake) uphill and into the reservoir during the winter--the 'pump-back' option. A calibrated CE-QUAL-W2 model of Henry Hagg Lake's hydrodynamics, temperature, and water quality was used to examine the effect of these proposed changes on water quality in the lake and downstream. Most model scenarios were run with the calibrated model for 2002, a typical water year; a few scenarios were run for 2001, a drought year. More...

Expression of Biglycan in First Trimester Chorionic Villous Sampling Placental Samples and Altered Function in Telomerase-Immortalized Microvascular Endothelial Cells.

PubMed

Chui, Amy; Gunatillake, Tilini; Brennecke, Shaun P; Ignjatovic, Vera; Monagle, Paul T; Whitelock, John M; van Zanten, Dagmar E; Eijsink, Jasper; Wang, Yao; Deane, James; Borg, Anthony J; Stevenson, Janet; Erwich, Jan Jaap; Said, Joanne M; Murthi, Padma

2017-06-01

Biglycan (BGN) has reduced expression in placentae from pregnancies complicated by fetal growth restriction (FGR). We used first trimester placental samples from pregnancies with later small for gestational age (SGA) infants as a surrogate for FGR. The functional consequences of reduced BGN and the downstream targets of BGN were determined. Furthermore, the expression of targets was validated in primary placental endothelial cells isolated from FGR or control pregnancies. APPROACH AND RESULTS: BGN expression was determined using real-time polymerase chain reaction in placental tissues collected during chorionic villous sampling performed at 10 to 12 weeks' gestation from pregnancies that had known clinical outcomes, including SGA. Short-interference RNA reduced BGN expression in telomerase-immortalized microvascular endothelial cells, and the effect on proliferation, angiogenesis, and thrombin generation was determined. An angiogenesis array identified downstream targets of BGN, and their expression in control and FGR primary placental endothelial cells was validated using real-time polymerase chain reaction. Reduced BGN expression was observed in SGA placental tissues. BGN reduction decreased network formation of telomerase-immortalized microvascular endothelial cells but did not affect thrombin generation or cellular proliferation. The array identified target genes, which were further validated: angiopoetin 4 ( ANGPT4 ), platelet-derived growth factor receptor α ( PDGFRA ), tumor necrosis factor superfamily member 15 ( TNFSF15 ), angiogenin ( ANG ), serpin family C member 1 ( SERPIN1 ), angiopoietin 2 ( ANGPT2 ), and CXC motif chemokine 12 ( CXCL12 ) in telomerase-immortalized microvascular endothelial cells and primary placental endothelial cells obtained from control and FGR pregnancies. This study reports a temporal relationship between altered placental BGN expression and subsequent development of SGA. Reduction of BGN in vascular endothelial cells leads to disrupted network formation and alterations in the expression of genes involved in angiogenesis. Therefore, differential expression of these may contribute to aberrant angiogenesis in SGA pregnancies. © 2017 American Heart Association, Inc.

Microreactor and method for preparing a radiolabeled complex or a biomolecule conjugate

DOEpatents

Reichert, David E; Kenis, Paul J. A.; Wheeler, Tobias D; Desai, Amit V; Zeng, Dexing; Onal, Birce C

2015-03-17

A microreactor for preparing a radiolabeled complex or a biomolecule conjugate comprises a microchannel for fluid flow, where the microchannel comprises a mixing portion comprising one or more passive mixing elements, and a reservoir for incubating a mixed fluid. The reservoir is in fluid communication with the microchannel and is disposed downstream of the mixing portion. A method of preparing a radiolabeled complex includes flowing a radiometal solution comprising a metallic radionuclide through a downstream mixing portion of a microchannel, where the downstream mixing portion includes one or more passive mixing elements, and flowing a ligand solution comprising a bifunctional chelator through the downstream mixing portion. The ligand solution and the radiometal solution are passively mixed while in the downstream mixing portion to initiate a chelation reaction between the metallic radionuclide and the bifunctional chelator. The chelation reaction is completed to form a radiolabeled complex.

Internal baffling for fuel injector

DOEpatents

Johnson, Thomas Edward; Lacy, Benjamin; Stevenson, Christian

2014-08-05

A fuel injector includes a fuel delivery tube; a plurality of pre-mixing tubes, each pre-mixing tube comprising at least one fuel injection hole; an upstream tube support plate that supports upstream ends of the plurality of pre-mixing tubes; a downstream tube support plate that supports downstream ends of the plurality of pre-mixing tubes; an outer wall connecting the upstream tube support plate and the downstream tube support plate and defining a plenum therewith; and a baffle provided in the plenum. The baffle includes a radial portion. A fuel delivered in the upstream direction by the fuel delivery tube is directed radially outwardly in the plenum between the radial portion of the baffle and the downstream tube support plate, then in the downstream direction around an outer edge portion of the radial portion, and then radially inwardly between the radial portion and the upstream tube support plate.

Effects of wastewater effluent discharge and treatment facility upgrades on environmental and biological conditions of the upper Blue River, Johnson County, Kansas and Jackson County, Missouri, January 2003 through March 2009

USGS Publications Warehouse

Graham, Jennifer L.; Stone, Mandy L.; Rasmussen, Teresa J.; Poulton, Barry C.

2010-01-01

The Johnson County Blue River Main Wastewater Treatment Facility discharges into the upper Blue River near the border between Johnson County, Kansas and Jackson County, Missouri. During 2005 through 2007 the wastewater treatment facility underwent upgrades to increase capacity and include biological nutrient removal. The effects of wastewater effluent on environmental and biological conditions of the upper Blue River were assessed by comparing an upstream site to two sites located downstream from the wastewater treatment facility. Environmental conditions were evaluated using previously and newly collected discrete and continuous data, and were compared with an assessment of biological community composition and ecosystem function along the upstream-downstream gradient. This evaluation is useful for understanding the potential effects of wastewater effluent on water quality, biological community structure, and ecosystem function. In addition, this information can be used to help achieve National Pollution Discharge Elimination System (NPDES) wastewater effluent permit requirements after additional studies are conducted. The effects of wastewater effluent on the water-quality conditions of the upper Blue River were most evident during below-normal and normal streamflows (about 75 percent of the time), when wastewater effluent contributed more than 20 percent to total streamflow. The largest difference in water-quality conditions between the upstream and downstream sites was in nutrient concentrations. Total and inorganic nutrient concentrations at the downstream sites during below-normal and normal streamflows were 4 to 15 times larger than at the upstream site, even after upgrades to the wastewater treatment facility were completed. However, total nitrogen concentrations decreased in wastewater effluent and at the downstream site following wastewater treatment facility upgrades. Similar decreases in total phosphorus were not observed, likely because the biological phosphorus removal process was not optimized until after the study was completed. Total nitrogen and phosphorus from the wastewater treatment facility contributed a relatively small percentage (14 to 15 percent) to the annual nutrient load in the upper Blue River, but contributed substantially (as much as 75 percent) to monthly loads during seasonal low-flows in winter and summer. During 2007 and 2008, annual discharge from the wastewater treatment facility was about one-half maximum capacity, and estimated potential maximum annual loads were 1.6 to 2.4 times greater than annual loads before capacity upgrades. Even when target nutrient concentrations are met, annual nutrient loads will increase when the wastewater treatment facility is operated at full capacity. Regardless of changes in annual nutrient loads, the reduction of nutrient concentrations in the Blue River Main wastewater effluent will help prevent further degradation of the upper Blue River. The Blue River Main Wastewater Treatment Facility wastewater effluent caused changes in concentrations of several water-quality constituents that may affect biological community structure and function including larger concentrations of bioavailable nutrients (nitrate and orthophosphorus) and smaller turbidities. Streambed-sediment conditions were similar along the upstream-downstream gradient and measured constituents did not exceed probable effect concentrations. Habitat conditions declined along the upstream-downstream gradient, largely because of decreased canopy cover and riparian buffer width and increased riffle-substrate fouling. Algal biomass, primary production, and the abundance of nutrient-tolerant diatoms substantially increased downstream from the wastewater treatment facility. Likewise, the abundance of intolerant macroinvertebrate taxa and Kansas Department of Health and Environment aquatic-life-support scores, derived from macroinvertebrate data, significantly decreased downstream from the wastewater

Antihelminthic benzimidazoles are novel HIF activators that prevent oxidative neuronal death via binding to tubulin.

PubMed

Aleyasin, Hossein; Karuppagounder, Saravanan S; Kumar, Amit; Sleiman, Sama; Basso, Manuela; Ma, Thong; Siddiq, Ambreena; Chinta, Shankar J; Brochier, Camille; Langley, Brett; Haskew-Layton, Renee; Bane, Susan L; Riggins, Gregory J; Gazaryan, Irina; Starkov, Anatoly A; Andersen, Julie K; Ratan, Rajiv R

2015-01-10

Pharmacological activation of the adaptive response to hypoxia is a therapeutic strategy of growing interest for neurological conditions, including stroke, Huntington's disease, and Parkinson's disease. We screened a drug library with known safety in humans using a hippocampal neuroblast line expressing a reporter of hypoxia-inducible factor (HIF)-dependent transcription. Our screen identified more than 40 compounds with the ability to induce hypoxia response element-driven luciferase activity as well or better than deferoxamine, a canonical activator of hypoxic adaptation. Among the chemical entities identified, the antihelminthic benzimidazoles represented one pharmacophore that appeared multiple times in our screen. Secondary assays confirmed that antihelminthics stabilized the transcriptional activator HIF-1α and induced expression of a known HIF target gene, p21(cip1/waf1), in post-mitotic cortical neurons. The on-target effect of these agents in stimulating hypoxic signaling was binding to free tubulin. Moreover, antihelminthic benzimidazoles also abrogated oxidative stress-induced death in vitro, and this on-target effect also involves binding to free tubulin. These studies demonstrate that tubulin-binding drugs can activate a component of the hypoxic adaptive response, specifically the stabilization of HIF-1α and its downstream targets. Tubulin-binding drugs, including antihelminthic benzimidazoles, also abrogate oxidative neuronal death in primary neurons. Given their safety in humans and known ability to penetrate into the central nervous system, antihelminthic benzimidazoles may be considered viable candidates for treating diseases associated with oxidative neuronal death, including stroke.

Pan-SRC kinase inhibition blocks B-cell receptor oncogenic signaling in non-Hodgkin lymphoma.

PubMed

Battistello, Elena; Katanayeva, Natalya; Dheilly, Elie; Tavernari, Daniele; Donaldson, Maria C; Bonsignore, Luca; Thome, Margot; Christie, Amanda L; Murakami, Mark A; Michielin, Olivier; Ciriello, Giovanni; Zoete, Vincent; Oricchio, Elisa

2018-05-24

In diffuse large B-cell lymphoma (DLBCL), activation of the B-cell receptor (BCR) promotes multiple oncogenic signals, which are essential for tumor proliferation. Inhibition of the Bruton's tyrosine kinase (BTK), a BCR downstream target, is therapeutically effective only in a subgroup of patients with DLBCL. Here, we used lymphoma cells isolated from patients with DLBCL to measure the effects of targeted therapies on BCR signaling and to anticipate response. In lymphomas resistant to BTK inhibition, we show that blocking BTK activity enhanced tumor dependencies from alternative oncogenic signals downstream of the BCR, converging on MYC upregulation. To completely ablate the activity of the BCR, we genetically and pharmacologically repressed the activity of the SRC kinases LYN, FYN, and BLK, which are responsible for the propagation of the BCR signal. Inhibition of these kinases strongly reduced tumor growth in xenografts and cell lines derived from patients with DLBCL independent of their molecular subtype, advancing the possibility to be relevant therapeutic targets in broad and diverse groups of DLBCL patients. © 2018 by The American Society of Hematology.

Do insect repellents induce drift behaviour in aquatic non-target organisms?

PubMed

Fink, Patrick; Moelzner, Jana; Berghahn, Ruediger; von Elert, Eric

2017-01-01

Synthetic insect repellents are compounds applied to surfaces to discourage insects, mainly mosquitoes, from landing on those surfaces. As some of these repellents have repeatedly been detected in surface waters at significant concentrations, they may also exert repellent effects on aquatic non-target organisms. In running water systems, aquatic invertebrates actively enter downstream drift in order to avoid unfavourable environmental conditions. We thus tested the hypothesis that the widely used insect repellents DEET (N,N-Diethyl-m-toluamide), EBAAP (3-[N-butyl-N-acetyl]-aminopropionic acid ethyl ester) and Icaridin (1-piperidinecarboxylic acid 2-(2-hydroxyethyl)-1-methylpropyl ester) induce downstream drift behaviour in the aquatic invertebrates Gammarus pulex (Crustacea, Amphipoda) and Cloeon dipterum (Insecta, Ephemeroptera), using a laboratory-scale drift assay. We found no clear increase in the drift behaviour of both invertebrate species across a concentration gradient of eight orders of magnitude and even beyond maximum environmental concentrations for any of the three repellents. We found no evidence for a direct drift-inducing activity of insect repellents on aquatic non-target organisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

Thermo-chemotherapy Induced miR-218 upregulation inhibits the invasion of gastric cancer via targeting Gli2 and E-cadherin.

PubMed

Ruan, Qiang; Fang, Zhi-Yuan; Cui, Shu-Zhong; Zhang, Xiang-Liang; Wu, Yin-Bing; Tang, Hong-Sheng; Tu, Yi-Nuo; Ding, Yan

2015-08-01

Thermo-chemotherapy has been proven to reduce the invasion capability of cancer cells. However, the molecular mechanism underlying this anti-invasion effect is still unclear. In this study, the role of thermo-chemotherapy in the inhibition of tumor invasion was studied. The results demonstrated that expression of miR-218 was downregulated in gastric cancer tissues, which had a positive correlation with tumor invasion and metastasis. In vitro thermo-chemotherapy increased miR-218 expression in SGC7901 cells and inhibited both proliferation and invasion of cancer cells. Gli2 was identified as a downstream target of miR-218, and its expression was negatively regulated by miR-218. The thermo-chemotherapy induced miR-218 upregulation was also accompanied by increasing of E-cadherin expression. In conclusion, the present study indicates that thermo-chemotherapy can effectively decrease the invasion capability of cancer cells and increase cell-cell adhesion. miR-218 and its downstream target Gli2, as well as E-cadherin, participate in the anti-invasion process.

The Vasa homolog RDE-12 engages target mRNA and multiple Argonaute proteins to promote RNAi in C. elegans

PubMed Central

Shirayama, Masaki; Stanney, William; Gu, Weifeng; Seth, Meetu; Mello, Craig C.

2014-01-01

Summary Argonaute proteins (AGOs) are key nuclease effectors of RNA interference (RNAi) [1]. Although purified AGOs can mediate a single round of target-RNA cleavage in vitro, accessory factors are required for short-interfering (si)RNAs loading and to achieve multiple-target turnover [2, 3]. To identify AGO co-factors we immunoprecipitated the C. elegans AGO WAGO-1, which engages amplified small RNAs during RNAi [4]. These studies identified a robust association between WAGO-1 and a conserved Vasa ATPase-related protein RDE-12. rde-12 mutants are deficient in RNAi including viral suppression, and fail to produce amplified secondary siRNAs and certain endogenous siRNAs (endo-siRNAs). RDE-12 co-localizes with WAGO-1 in germline P-granules and in cytoplasmic and peri-nuclear foci in somatic cells. These findings and our genetic studies suggest that RDE-12 is first recruited to target mRNA by upstream AGOs (RDE-1 and ERGO-1) where it promotes small-RNA amplification and/or WAGO-1 loading. Downstream of these events, RDE-12 forms an RNase-resistant (target mRNA-independent) complex with WAGO-1 and may thus have additional functions in target mRNA surveillance and silencing. PMID:24684931

The Vasa Homolog RDE-12 engages target mRNA and multiple argonaute proteins to promote RNAi in C. elegans.

PubMed

Shirayama, Masaki; Stanney, William; Gu, Weifeng; Seth, Meetu; Mello, Craig C

2014-04-14

Argonaute (AGO) proteins are key nuclease effectors of RNAi. Although purified AGOs can mediate a single round of target RNA cleavage in vitro, accessory factors are required for small interfering RNA (siRNA) loading and to achieve multiple-target turnover. To identify AGO cofactors, we immunoprecipitated the C. elegans AGO WAGO-1, which engages amplified small RNAs during RNAi. These studies identified a robust association between WAGO-1 and a conserved Vasa ATPase-related protein RDE-12. rde-12 mutants are deficient in RNAi, including viral suppression, and fail to produce amplified secondary siRNAs and certain endogenous siRNAs (endo-siRNAs). RDE-12 colocalizes with WAGO-1 in germline P granules and in cytoplasmic and perinuclear foci in somatic cells. These findings and our genetic studies suggest that RDE-12 is first recruited to target mRNA by upstream AGOs (RDE-1 and ERGO-1), where it promotes small RNA amplification and/or WAGO-1 loading. Downstream of these events, RDE-12 forms an RNase-resistant (target mRNA-independent) complex with WAGO-1 and may thus have additional functions in target mRNA surveillance and silencing. Copyright © 2014 Elsevier Ltd. All rights reserved.

Modulators of Stomatal Lineage Signal Transduction Alter Membrane Contact Sites and Reveal Specialization among ERECTA Kinases.

PubMed

Ho, Chin-Min Kimmy; Paciorek, Tomasz; Abrash, Emily; Bergmann, Dominique C

2016-08-22

Signal transduction from a cell's surface to its interior requires dedicated signaling elements and a cellular environment conducive to signal propagation. Plant development, defense, and homeostasis rely on plasma membrane receptor-like kinases to perceive endogenous and environmental signals, but little is known about their immediate downstream targets and signaling modifiers. Using genetics, biochemistry, and live-cell imaging, we show that the VAP-RELATED SUPPRESSOR OF TMM (VST) family is required for ERECTA-mediated signaling in growth and cell-fate determination and reveal a role for ERECTA-LIKE2 in modulating signaling by its sister kinases. We show that VSTs are peripheral plasma membrane proteins that can form complexes with integral ER-membrane proteins, thereby potentially influencing the organization of the membrane milieu to promote efficient and differential signaling from the ERECTA-family members to their downstream intracellular targets. Copyright © 2016 Elsevier Inc. All rights reserved.

AKT in cancer: new molecular insights and advances in drug development

PubMed Central

Mundi, Prabhjot S.; Sachdev, Jasgit; McCourt, Carolyn

2016-01-01

The phosphatidylinositol‐3 kinase (PI3K)–AKT pathway is one of the most commonly dysregulated pathways in all of cancer, with somatic mutations, copy number alterations, aberrant epigenetic regulation and increased expression in a number of cancers. The carefully maintained homeostatic balance of cell division and growth on one hand, and programmed cell death on the other, is universally disturbed in tumorigenesis, and downstream effectors of the PI3K–AKT pathway play an important role in this disturbance. With a wide array of downstream effectors involved in cell survival and proliferation, the well‐characterized direct interactions of AKT make it a highly attractive yet elusive target for cancer therapy. Here, we review the salient features of this pathway, evidence of its role in promoting tumorigenesis and recent progress in the development of therapeutic agents that target AKT. PMID:27232857

Activator protein 1 promotes gemcitabine-induced apoptosis in pancreatic cancer by upregulating its downstream target Bim.

PubMed

Ren, Xiaoxia; Zhao, Wenjing; Du, Yongxing; Zhang, Taiping; You, Lei; Zhao, Yupei

2016-12-01

Gemcitabine is a commonly used chemotherapy drug in pancreatic cancer. The function of activator protein 1 (AP-1) is cell-specific, and its function depends on the expression of other complex members. In the present study, we added gemcitabine to the media of Panc-1 and SW1990 cells at clinically achieved concentrations (10 µM). Compared with constitutive c-Fos expression, c-Jun expression increased in a dose-dependent manner upon gemcitabine treatment. c-Jun overexpression increased gemcitabine-induced apoptosis through Bim activation, while cell apoptosis and Bim expression decreased following c-Jun knockdown. Furthermore, gemcitabine-induced apoptosis and Bim levels decreased when c-Jun phosphorylation was blocked by SP600125. Our findings suggest that c-Jun, which is a member of the AP-1 complex, functions in gemcitabine-induced apoptosis by regulating its downstream target Bim in pancreatic cancer cells.

Metabolite profiling of antidepressant drug action reveals novel drug targets beyond monoamine elevation.

PubMed

Webhofer, C; Gormanns, P; Tolstikov, V; Zieglgänsberger, W; Sillaber, I; Holsboer, F; Turck, C W

2011-12-13

Currently used antidepressants elevate monoamine levels in the synaptic cleft. There is good reason to assume that this is not the only source for antidepressant therapeutic activities and that secondary downstream effects may be relevant for alleviating symptoms of depression. We attempted to elucidate affected biochemical pathways downstream of monoamine reuptake inhibition by interrogating metabolomic profiles in DBA/2Ola mice after chronic paroxetine treatment. Metabolomic changes were investigated using gas chromatography-mass spectrometry profiling and group differences were analyzed by univariate and multivariate statistics. Pathways affected by antidepressant treatment were related to energy metabolism, amino acid metabolism and hormone signaling. The identified pathways reveal further antidepressant therapeutic action and represent targets for drug development efforts. A comparison of the central nervous system with blood plasma metabolite alterations identified GABA, galactose-6-phosphate and leucine as biomarker candidates for assessment of antidepressant treatment effects in the periphery.

Projection-specific visual feature encoding by layer 5 cortical subnetworks

PubMed Central

Lur, Gyorgy; Vinck, Martin A.; Tang, Lan; Cardin, Jessica A.; Higley, Michael J.

2016-01-01

Summary Primary neocortical sensory areas act as central hubs, distributing afferent information to numerous cortical and subcortical structures. However, it remains unclear whether each downstream target receives distinct versions of sensory information. We used in vivo calcium imaging combined with retrograde tracing to monitor visual response properties of three distinct subpopulations of projection neurons in primary visual cortex. While there is overlap across the groups, on average corticotectal (CT) cells exhibit lower contrast thresholds and broader tuning for orientation and spatial frequency in comparison to corticostriatal (CS) cells, while corticocortical (CC) cells have intermediate properties. Noise correlational analyses support the hypothesis that CT cells integrate information across diverse layer 5 populations, whereas CS and CC cells form more selectively interconnected groups. Overall, our findings demonstrate the existence of functional subnetworks within layer 5 that may differentially route visual information to behaviorally relevant downstream targets. PMID:26972011

Benzoxathiol derivative BOT-4-one suppresses L540 lymphoma cell survival and proliferation via inhibition of JAK3/STAT3 signaling

PubMed Central

Kim, Byung Hak; Min, Yun Sook; Choi, Jung Sook; Baeg, Gyeong-Hun; Kim, Youngsoo; Shin, Jong Wook; Kim, Tae-Yoon

2011-01-01

Persistently activated JAK/STAT3 signaling pathway plays a pivotal role in various human cancers including major carcinomas and hematologic tumors, and is implicated in cancer cell survival and proliferation. Therefore, inhibition of JAK/STAT3 signaling may be a clinical application in cancer therapy. Here, we report that 2-cyclohexylimino-6-methyl-6,7-dihydro-5H-benzo [1,3]oxathiol-4-one (BOT-4-one), a small molecule inhibitor of JAK/STAT3 signaling, induces apoptosis through inhibition of STAT3 activation. BOT-4-one suppressed cytokine (upd)-induced tyrosine phosphorylation and transcriptional activity of STAT92E, the sole Drosophila STAT homolog. Consequently, BOT-4-one significantly inhibited STAT3 tyrosine phosphorylation and expression of STAT3 downstream target gene SOCS3 in various human cancer cell lines, and its effect was more potent in JAK3-activated Hodgkin's lymphoma cell line than in JAK2-activated breast cancer and prostate cancer cell lines. In addition, BOT-4-one-treated Hodgkin's lymphoma cells showed decreased cell survival and proliferation by inducing apoptosis through down-regulation of STAT3 downstream target anti-apoptotic gene expression. These results suggest that BOT-4-one is a novel small molecule inhibitor of JAK3/STAT3 signaling and may have therapeutic potential in the treatment of human cancers harboring aberrant JAK3/STAT3 signaling, specifically Hodgkin's lymphoma. PMID:21499010

Benzoxathiol derivative BOT-4-one suppresses L540 lymphoma cell survival and proliferation via inhibition of JAK3/STAT3 signaling.

PubMed

Kim, Byung Hak; Min, Yun Sook; Choi, Jung Sook; Baeg, Gyeong Hun; Kim, Young Soo; Shin, Jong Wook; Kim, Tae Yoon; Ye, Sang Kyu

2011-05-31

Persistently activated JAK/STAT3 signaling pathway plays a pivotal role in various human cancers including major carcinomas and hematologic tumors, and is implicated in cancer cell survival and proliferation. Therefore, inhibition of JAK/STAT3 signaling may be a clinical application in cancer therapy. Here, we report that 2-cyclohexylimino-6-methyl-6,7-dihydro-5H-benzo [1,3]oxathiol-4-one (BOT-4-one), a small molecule inhibitor of JAK/STAT3 signaling, induces apoptosis through inhibition of STAT3 activation. BOT-4-one suppressed cytokine (upd)-induced tyrosine phosphorylation and transcriptional activity of STAT92E, the sole Drosophila STAT homolog. Consequently, BOT-4-one significantly inhibited STAT3 tyrosine phosphorylation and expression of STAT3 downstream target gene SOCS3 in various human cancer cell lines, and its effect was more potent in JAK3-activated Hodgkin's lymphoma cell line than in JAK2-activated breast cancer and prostate cancer cell lines. In addition, BOT-4-one-treated Hodgkin's lymphoma cells showed decreased cell survival and proliferation by inducing apoptosis through down-regulation of STAT3 downstream target anti-apoptotic gene expression. These results suggest that BOT-4-one is a novel small molecule inhibitor of JAK3/STAT3 signaling and may have therapeutic potential in the treatment of human cancers harboring aberrant JAK3/STAT3 signaling, specifically Hodgkin's lymphoma.

A positive feedback loop involving EGFR/Akt/mTORC1 and IKK/NF-κB regulates head and neck squamous cell carcinoma proliferation

PubMed Central

Li, Zhipeng; Yang, Zejia; Passaniti, Antonino; Lapidus, Rena G.; Liu, Xuefeng; Cullen, Kevin J.; Dan, Han C.

2016-01-01

The overexpression or mutation of epidermal growth factor receptor (EGFR) has been associated with a number of cancers, including head and neck squamous cell carcinoma (HNSCC). Increasing evidence indicates that both the phosphatidylinositol-3-kinase (PI3K)-Akt-mammalian target of Rapamycin (mTOR) and the nuclear factor-kappa B (NF-κB) are constitutively active and contribute to aggressive HNSCC downstream of EGFR. However, whether these two oncogenic signaling pathways exhibit molecular and functional crosstalk in HNSCC is unclear. Our results now reveal that mTORC1, not mTORC2, contributes to NF-κB activation downstream of EGFR/PI3K/Akt signaling. Mechanistically, mTORC1 enhances the inhibitor of nuclear factor kappa-B kinase (IKK) activity to accelerate NF-κB signaling. Concomitantly, activated NF-κB/IKK up-regulates EGFR expression through positive feedback regulation. Blockage of NF-κB/IKK activity by the novel IKKβ specific inhibitor, CmpdA, leads to significant inhibition of cell proliferation and induction of apoptosis. CmpdA also sensitizes intrinsic cisplatin-resistant HNSCC cells to cisplatin treatment. Our findings reveal a new mechanism by which EGFR/PI3K/Akt/mTOR signaling promotes head and neck cancer progression and underscores the need for developing a therapeutic strategy for targeting IKK/NF-κB either as a single agent or in combination with cisplatin in head and neck cancer. PMID:26895469

CTGF Mediates Smad-Dependent Transforming Growth Factor β Signaling To Regulate Mesenchymal Cell Proliferation during Palate Development

PubMed Central

Parada, Carolina; Li, Jingyuan; Iwata, Junichi; Suzuki, Akiko

2013-01-01

Transforming growth factor β (TGF-β) signaling plays crucial functions in the regulation of craniofacial development, including palatogenesis. Here, we have identified connective tissue growth factor (Ctgf) as a downstream target of the TGF-β signaling pathway in palatogenesis. The pattern of Ctgf expression in wild-type embryos suggests that it may be involved in key processes during palate development. We found that Ctgf expression is downregulated in both Wnt1-Cre; Tgfbr2fl/fl and Osr2-Cre; Smad4fl/fl palates. In Tgfbr2 mutant embryos, downregulation of Ctgf expression is associated with p38 mitogen-activated protein kinase (MAPK) overactivation, whereas loss of function of Smad4 itself leads to downregulation of Ctgf expression. We also found that CTGF regulates its own expression via TGF-β signaling. Osr2-Cre; Smad4fl/fl mice exhibit a defect in cell proliferation similar to that of Tgfbr2 mutant mice, as well as cleft palate. We detected no alteration in bone morphogenetic protein (BMP) downstream targets in Smad4 mutant palates, suggesting that the reduction in cell proliferation is due to defective transduction of TGF-β signaling via decreased Ctgf expression. Significantly, an exogenous source of CTGF was able to rescue the cell proliferation defect in both Tgfbr2 and Smad4 mutant palates. Collectively, our data suggest that CTGF regulates proliferation as a mediator of the canonical pathway of TGF-β signaling during palatogenesis. PMID:23816882

Roles of FGFR in oral carcinogenesis.

PubMed

Xie, Xiaoyan; Wang, Zhiyong; Chen, Fangman; Yuan, Yao; Wang, Jiayi; Liu, Rui; Chen, Qianming

2016-06-01

Fibroblast growth factor receptors (FGFRs) play essential roles in organ development during the embryonic period, and regulate tissue repair in adults. Accumulating evidence suggests that alterations in FGFR signalling are involved in diverse types of cancer. In this review, we focus on aberrant regulation of FGFRs in pathogenesis of oral squamous cell carcinoma (OSCC), including altered expression and subcellular location, aberrant isoform splicing and mutations. We also provide an overview of oncogenic roles of each FGFR and its downstream signalling pathways in regulating OSCC cell proliferation and metastasis. Finally, we discuss potential application of FGFRs as anti-cancer targets in the preclinical environment and in clinical practice. © 2016 John Wiley & Sons Ltd.

Articulated transition duct in turbomachine

DOEpatents

Flanagan, James Scott; McMahan, Kevin Weston; LeBegue, Jeffrey Scott; Pentecost, Ronnie Ray

2014-04-29

Turbine systems are provided. A turbine system includes a transition duct comprising an inlet, an outlet, and a duct passage extending between the inlet and the outlet and defining a longitudinal axis, a radial axis, and a tangential axis. The outlet of the transition duct is offset from the inlet along the longitudinal axis and the tangential axis. The duct passage includes an upstream portion and a downstream portion. The upstream portion extends from the inlet between an inlet end and an aft end. The downstream portion extends from the outlet between an outlet end and a head end. The turbine system further includes a joint coupling the aft end of the upstream portion and the head end of the downstream portion together. The joint is configured to allow movement of the upstream portion and the downstream portion relative to each other about or along at least one axis.

Differential IKK/NF-κB Activity is Mediated by TSC2 through mTORC1 in PTEN-null Prostate Cancer and Tuberous Sclerosis Complex Tumor Cells

PubMed Central

Gao, Yu; Gartenhaus, Ronald B.; Lapidus, Rena G.; Hussain, Arif; Zhang, Yanting; Wang, Xinghuan; Dan, Han C.

2015-01-01

The serine/threonine protein kinase Akt plays a critical role in regulating proliferation, growth and survival through phosphorylation of different downstream substrates. The mammalian target of rapamycin (mTOR) is a key target for Akt to promote tumorigenesis. It has been reported that Akt activates mTOR through phosphorylation and inhibition of the tuberous sclerosis complex (TSC) protein TSC2. Previously it was demonstrated that mTOR activates IKK/NF-κB signaling by promoting IKK activity downstream of Akt in conditions deficient of PTEN. In the current study, the mechanistic role of the tumor suppressor TSC2 was investigated in the regulation of IKK/NF-κB activity in PTEN-null prostate cancer and in TSC2 mutated tumor cells. The results demonstrate that TSC2 inhibits IKK/NF-κB activity downstream of Akt and upstream of mTORC1 in a PTEN deficient environment. However, TSC2 promotes IKK/NF-κB activity upstream of Akt and mTORC1 in TSC2 mutated tumor cells. These data indicate that TSC2 negatively or positively regulates IKK/NF-κB activity in a context-dependent manner depending on the genetic background. PMID:26374334

Effects of backpacker use, pack stock trail use, and pack stock grazing on water-quality indicators, including nutrients, E. coli, hormones, and pharmaceuticals, in Yosemite National Park, USA

USGS Publications Warehouse

Forrester, Harrison; Clow, David W.; Roche, James W.; Heyvaert, Alan C.; Battaglin, William A.

2017-01-01

We investigated how visitor-use affects water quality in wilderness in Yosemite National Park. During the summers of 2012–2014, we collected and analyzed surface-water samples for water-quality indicators, including fecal indicator bacteria Escherichia coli, nutrients (nitrogen, phosphorus, carbon), suspended sediment concentration, pharmaceuticals, and hormones. Samples were collected upstream and downstream from different types of visitor use at weekly to biweekly intervals and during summer storms. We conducted a park-wide synoptic sampling campaign during summer 2014, and sampled upstream and downstream from meadows to evaluate the mitigating effect of meadows on water quality. At pack stock stream crossings, Escherichia coli concentrations were greater downstream from crossings than upstream (median downstream increase in Escherichia coli of three colony forming units 100 mL−1), with the greatest increases occurring during storms (median downstream increase in Escherichia coli of 32 CFU 100 mL−1). At backpacker use sites, hormones, and pharmaceuticals (e.g., insect repellent) were detected at downstream sites, and Escherichia coli concentrations were greater at downstream sites (median downstream increase in Escherichia coli of 1 CFU 100 mL−1). Differences in water quality downstream vs. upstream from meadows grazed by pack stock were not detectable for most water-quality indicators, however, Escherichia coli concentrations decreased downstream, suggesting entrapment and die-off of fecal indicator bacteria in meadows. Our results indicate that under current-use levels pack stock trail use and backpacker use are associated with detectable, but relatively minor, effects on water quality, which are most pronounced during storms.

Effects of Backpacker Use, Pack Stock Trail Use, and Pack Stock Grazing on Water-Quality Indicators, Including Nutrients, E. coli, Hormones, and Pharmaceuticals, in Yosemite National Park, USA

NASA Astrophysics Data System (ADS)

Forrester, Harrison; Clow, David; Roche, James; Heyvaert, Alan; Battaglin, William

2017-09-01

We investigated how visitor-use affects water quality in wilderness in Yosemite National Park. During the summers of 2012-2014, we collected and analyzed surface-water samples for water-quality indicators, including fecal indicator bacteria Escherichia coli, nutrients (nitrogen, phosphorus, carbon), suspended sediment concentration, pharmaceuticals, and hormones. Samples were collected upstream and downstream from different types of visitor use at weekly to biweekly intervals and during summer storms. We conducted a park-wide synoptic sampling campaign during summer 2014, and sampled upstream and downstream from meadows to evaluate the mitigating effect of meadows on water quality. At pack stock stream crossings, Escherichia coli concentrations were greater downstream from crossings than upstream (median downstream increase in Escherichia coli of three colony forming units 100 mL-1), with the greatest increases occurring during storms (median downstream increase in Escherichia coli of 32 CFU 100 mL-1). At backpacker use sites, hormones, and pharmaceuticals (e.g., insect repellent) were detected at downstream sites, and Escherichia coli concentrations were greater at downstream sites (median downstream increase in Escherichia coli of 1 CFU 100 mL-1). Differences in water quality downstream vs. upstream from meadows grazed by pack stock were not detectable for most water-quality indicators, however, Escherichia coli concentrations decreased downstream, suggesting entrapment and die-off of fecal indicator bacteria in meadows. Our results indicate that under current-use levels pack stock trail use and backpacker use are associated with detectable, but relatively minor, effects on water quality, which are most pronounced during storms.

The Role of Mammalian Target of Rapamycin (mTOR) in Insulin Signaling.

PubMed

Yoon, Mee-Sup

2017-10-27

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that controls a wide spectrum of cellular processes, including cell growth, differentiation, and metabolism. mTOR forms two distinct multiprotein complexes known as mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which are characterized by the presence of raptor and rictor, respectively. mTOR controls insulin signaling by regulating several downstream components such as growth factor receptor-bound protein 10 (Grb10), insulin receptor substrate (IRS-1), F-box/WD repeat-containing protein 8 (Fbw8), and insulin like growth factor 1 receptor/insulin receptor (IGF-IR/IR). In addition, mTORC1 and mTORC2 regulate each other through a feedback loop to control cell growth. This review outlines the current understanding of mTOR regulation in insulin signaling in the context of whole body metabolism.

Targeting Wnts at the source--new mechanisms, new biomarkers, new drugs.

PubMed

Madan, Babita; Virshup, David M

2015-05-01

Wnt signaling is dysregulated in many cancers and is therefore an attractive therapeutic target. The focus of drug development has recently shifted away from downstream inhibitors of β-catenin. Active inhibitors of Wnt secretion and Wnt/receptor interactions have been developed that are now entering clinical trials. Such agents include inhibitors of Wnt secretion, as well as recombinant proteins that minimize Wnt-Frizzled interactions. These new therapies arrive together with the recent insight that cancer-specific upregulation of Wnt receptors at the cell surface regulates cellular sensitivity to Wnts. Loss-of-function mutations in RNF43 or ZNRF3 and gain-of-function chromosome translocations involving RSPO2 and RSPO3 are surprisingly common and markedly increase Wnt/β-catenin signaling in response to secreted Wnts. These mutations may be predictive biomarkers to select patients responsive to newly developed upstream Wnt inhibitors. ©2015 American Association for Cancer Research.

RhoA/Rho-kinase signaling: a therapeutic target in pulmonary hypertension.

PubMed

Barman, Scott A; Zhu, Shu; White, Richard E

2009-01-01

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by progressive elevation of pulmonary arterial pressure and vascular resistance due to pulmonary vasoconstriction and vessel remodeling as well as inflammation. Rho-kinases (ROCKs) are one of the best-described effectors of the small G-protein RhoA, and ROCKs are involved in a variety of cellular functions including muscle cell contraction, proliferation and vascular inflammation through inhibition of myosin light chain phosphatase and activation of downstream mediators. A plethora of evidence in animal models suggests that heightened RhoA/ROCK signaling is important in the pathogenesis of pulmonary hypertension by causing enhanced constriction and remodeling of the pulmonary vasculature. Both animal and clinical studies suggest that ROCK inhibitors are effective for treatment of severe PAH with minimal risk, which supports the premise that ROCKs are important therapeutic targets in pulmonary hypertension and that ROCK inhibitors are a promising new class of drugs for this devastating disease.

Regulation of Ubiquitination-Mediated Protein Degradation by Survival Kinases in Cancer

PubMed Central

Yamaguchi, Hirohito; Hsu, Jennifer L.; Hung, Mien-Chie

2011-01-01

The ubiquitin–proteasome system is essential for multiple physiological processes via selective degradation of target proteins and has been shown to plays a critical role in human cancer. Activation of oncogenic factors and inhibition of tumor suppressors have been shown to be essential for cancer development, and protein ubiquitination has been linked to the regulation of oncogenic factors and tumor suppressors. Three kinases, AKT, extracellular signal-regulated kinase, and IκB kinase, we refer to as oncokinases, are activated in multiple human cancers. We and others have identified several key downstream targets that are commonly regulated by these oncokinases, some of which are regulated directly or indirectly via ubiquitin-mediated proteasome degradation, including FOXO3, β-catenin, myeloid cell leukemia-1, and Snail. In this review, we summarize these findings from our and other groups and discuss potential future studies and applications in the clinic. PMID:22649777

Estradiol targets T cell signaling pathways in human systemic lupus.

PubMed

Walters, Emily; Rider, Virginia; Abdou, Nabih I; Greenwell, Cindy; Svojanovsky, Stan; Smith, Peter; Kimler, Bruce F

2009-12-01

The major risk factor for developing systemic lupus erythematosus (SLE) is being female. The present study utilized gene profiles of activated T cells from females with SLE and healthy controls to identify signaling pathways uniquely regulated by estradiol that could contribute to SLE pathogenesis. Selected downstream pathway genes (+/- estradiol) were measured by real time polymerase chain amplification. Estradiol uniquely upregulated six pathways in SLE T cells that control T cell function including interferon-alpha signaling. Measurement of interferon-alpha pathway target gene expression revealed significant differences (p= 0.043) in DRIP150 (+/- estradiol) in SLE T cell samples while IFIT1 expression was bimodal and correlated moderately (r= 0.55) with disease activity. The results indicate that estradiol alters signaling pathways in activated SLE T cells that control T cell function. Differential expression of transcriptional coactivators could influence estrogen-dependent gene regulation in T cell signaling and contribute to SLE onset and disease pathogenesis.

(Pro)renin Receptor Is an Amplifier of Wnt/β-Catenin Signaling in Kidney Injury and Fibrosis.

PubMed

Li, Zhen; Zhou, Lili; Wang, Yongping; Miao, Jinhua; Hong, Xue; Hou, Fan Fan; Liu, Youhua

2017-08-01

The (pro)renin receptor (PRR) is a transmembrane protein with multiple functions. However, its regulation and role in the pathogenesis of CKD remain poorly defined. Here, we report that PRR is a downstream target and an essential component of Wnt/ β -catenin signaling. In mouse models, induction of CKD by ischemia-reperfusion injury (IRI), adriamycin, or angiotensin II infusion upregulated PRR expression in kidney tubular epithelium. Immunohistochemical staining of kidney biopsy specimens also revealed induction of renal PRR in human CKD. Overexpression of either Wnt1 or β -catenin induced PRR mRNA and protein expression in vitro Notably, forced expression of PRR potentiated Wnt1-mediated β -catenin activation and augmented the expression of downstream targets such as fibronectin, plasminogen activator inhibitor 1, and α -smooth muscle actin ( α -SMA). Conversely, knockdown of PRR by siRNA abolished β -catenin activation. PRR potentiation of Wnt/ β -catenin signaling did not require renin, but required vacuolar H + ATPase activity. In the mouse model of IRI, transfection with PRR or Wnt1 expression vectors promoted β -catenin activation, aggravated kidney dysfunction, and worsened renal inflammation and fibrotic lesions. Coexpression of PRR and Wnt1 had a synergistic effect. In contrast, knockdown of PRR expression ameliorated kidney injury and fibrosis after IRI. These results indicate that PRR is both a downstream target and a crucial element in Wnt signal transmission. We conclude that PRR can promote kidney injury and fibrosis by amplifying Wnt/ β -catenin signaling. Copyright © 2017 by the American Society of Nephrology.

Burn Injury Alters Epidermal Cholinergic Mediators and Increases HMGB1 and Caspase 3 in Autologous Donor Skin and Burn Margin

PubMed Central

Holmes, Casey J.; Plichta, Jennifer K.; Gamelli, Richard L.; Radek, Katherine A.

2016-01-01

Burn wound healing complications, such as graft failure or infection, are a major source of morbidity and mortality in burn patients. The mechanisms by which local burn injury alters epidermal barrier function in autologous donor skin and surrounding burn margin are largely undefined. We hypothesized that defects in the epidermal cholinergic system may impair epidermal barrier function and innate immune responses. The objective was to identify alterations in the epidermal cholinergic pathway, and their downstream targets, associated with inflammation and cell death. We established that protein levels, but not gene expression, of the α7 nicotinic acetylcholine receptor (CHRNA7) were significantly reduced in both donor and burn margin skin. Furthermore, the gene and protein levels of an endogenous allosteric modulator of CHRNA7, secreted mammalian Ly-6/urokinase-type plasminogen activator receptor-related protein-1 (SLURP1) and acetylcholine were significantly elevated in donor and burn margin skin. As downstream proteins of inflammatory and cell death targets of nAChR activation, we found significant elevations in epidermal High Mobility Group Box Protein 1 (HMGB1) and caspase 3 in donor and burn margin skin. Lastly, we employed a novel in vitro keratinocyte burn model to establish that burn injury influences the gene expression of these cholinergic mediators and their downstream targets. These results indicate that defects in cholinergic mediators and inflammatory/apoptotic molecules in donor and burn margin skin may directly contribute to graft failure or infection in burn patients. PMID:27648692

Functional profiling of receptor tyrosine kinases and downstream signaling in human chondrosarcomas identifies pathways for rational targeted therapy.

PubMed

Zhang, Yi-Xiang; van Oosterwijk, Jolieke G; Sicinska, Ewa; Moss, Samuel; Remillard, Stephen P; van Wezel, Tom; Bühnemann, Claudia; Hassan, Andrew B; Demetri, George D; Bovée, Judith V M G; Wagner, Andrew J

2013-07-15

Chondrosarcomas are notoriously resistant to cytotoxic chemotherapeutic agents. We sought to identify critical signaling pathways that contribute to their survival and proliferation, and which may provide potential targets for rational therapeutic interventions. Activation of receptor tyrosine kinases (RTK) was surveyed using phospho-RTK arrays. S6 phosphorylation and NRAS mutational status were examined in chondrosarcoma primary tumor tissues. siRNA or small-molecule inhibitors against RTKs or downstream signaling proteins were applied to chondrosarcoma cells and changes in biochemical signaling, cell cycle, and cell viability were determined. In vivo antitumor activity of BEZ235, a phosphoinositide 3-kinase (PI3K)/mTOR inhibitor, was evaluated in a chondrosarcoma xenograft model. Several RTKs were identified as critical mediators of cell growth, but the RTK dependencies varied among cell lines. In exploration of downstream signaling pathways, strong S6 phosphorylation was found in 69% of conventional chondrosarcomas and 44% of dedifferentiated chondrosarcomas. Treatment with BEZ235 resulted in dramatic reduction in the growth of all chondrosarcoma cell lines. Tumor growth was similarly inhibited in a xenograft model of chondrosarcoma. In addition, chondrosarcoma cells with an NRAS mutation were sensitive to treatment with a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor. Functional NRAS mutations were found in 12% of conventional central chondrosarcomas. RTKs are commonly activated in chondrosarcoma, but because of their considerable heterogeneity, targeted inhibition of the PI3K/mTOR pathway represents a rational therapeutic strategy. Chondrosarcomas with NRAS mutations may benefit from treatment with MEK inhibitors.

Salicylic Acid Suppresses Jasmonic Acid Signaling Downstream of SCFCOI1-JAZ by Targeting GCC Promoter Motifs via Transcription Factor ORA59[C][W][OA

PubMed Central

Van der Does, Dieuwertje; Leon-Reyes, Antonio; Koornneef, Annemart; Van Verk, Marcel C.; Rodenburg, Nicole; Pauwels, Laurens; Goossens, Alain; Körbes, Ana P.; Memelink, Johan; Ritsema, Tita; Van Wees, Saskia C.M.; Pieterse, Corné M.J.

2013-01-01

Antagonism between the defense hormones salicylic acid (SA) and jasmonic acid (JA) plays a central role in the modulation of the plant immune signaling network, but the molecular mechanisms underlying this phenomenon are largely unknown. Here, we demonstrate that suppression of the JA pathway by SA functions downstream of the E3 ubiquitin-ligase Skip-Cullin-F-box complex SCFCOI1, which targets JASMONATE ZIM-domain transcriptional repressor proteins (JAZs) for proteasome-mediated degradation. In addition, neither the stability nor the JA-induced degradation of JAZs was affected by SA. In silico promoter analysis of the SA/JA crosstalk transcriptome revealed that the 1-kb promoter regions of JA-responsive genes that are suppressed by SA are significantly enriched in the JA-responsive GCC-box motifs. Using GCC:GUS lines carrying four copies of the GCC-box fused to the β-glucuronidase reporter gene, we showed that the GCC-box motif is sufficient for SA-mediated suppression of JA-responsive gene expression. Using plants overexpressing the GCC-box binding APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors ERF1 or ORA59, we found that SA strongly reduces the accumulation of ORA59 but not that of ERF1. Collectively, these data indicate that the SA pathway inhibits JA signaling downstream of the SCFCOI1-JAZ complex by targeting GCC-box motifs in JA-responsive promoters via a negative effect on the transcriptional activator ORA59. PMID:23435661

Transition duct with divided upstream and downstream portions

DOE Office of Scientific and Technical Information (OSTI.GOV)

McMahan, Kevin Weston; LeBegue, Jeffrey Scott; Maldonado, Jaime Javier

2015-07-14

Turbine systems are provided. In one embodiment, a turbine system includes a transition duct comprising an inlet, an outlet, and a duct passage extending between the inlet and the outlet and defining a longitudinal axis, a radial axis, and a tangential axis. The outlet of the transition duct is offset from the inlet along the longitudinal axis and the tangential axis. The duct passage includes an upstream portion extending from the inlet and a downstream portion extending from the outlet. The turbine system further includes a rib extending from an outer surface of the duct passage, the rib dividing themore » upstream portion and the downstream portion.« less

An evaluation of silver-stage American Eel conspecific chemical cueing during outmigration

USGS Publications Warehouse

Schmucker, Andrew K.; Johnson, Nicholas S.; Galbraith, Heather S.; Li, Weiming

2017-01-01

American Eel Anguilla rostrata abundance has declined in recent decades, in part because sexually maturing, silver-stage adults, outmigrating from freshwater to oceanic spawning grounds, encounter migratory blockades or perish when passing through active hydroelectric turbines. To help improve downstream passage effectiveness and increase survival rates, the role of silver-stage American Eel conspecific chemical cueing during outmigration was investigated using a new type of bioassay. Inside a laboratory flume, downstream-swimming eels were exposed to both live (putative attractant) and dead (putative repellent) conspecific washings to determine whether their trajectory of downstream movement, level of activity, or time spent inside targeted areas of the arena changed after exposure. Silver eels were not attracted to or repulsed by either odor, as none of five scoring metrics indicated a behavioral response. Results did not support the hypothesis that conspecific chemical cueing is a mechanism for downstream migration coordination or danger avoidance; however responses may not have been readily apparent in this type of assay. Fisheries managers may opt to focus future research on more feasible restoration efforts using alternate experimental designs to remedy this ecological issue.

Subsequent to suppression: Downstream comprehension consequences of noun/verb ambiguity in natural reading

PubMed Central

Stites, Mallory C.; Federmeier, Kara D.

2015-01-01

We used eye-tracking to investigate the downstream processing consequences of encountering noun/verb (NV) homographs (i.e., park) in semantically neutral but syntactically constraining contexts. Target words were followed by a prepositional phrase containing a noun that was plausible for only one meaning of the homograph. Replicating previous work, we found increased first fixation durations on NV homographs compared to unambiguous words, which persisted into the next sentence region. At the downstream noun, we found plausibility effects following ambiguous words that were correlated with the size of a reader's first fixation effect, suggesting that this effect reflects the recruitment of processing resources necessary to suppress the homograph's context-inappropriate meaning. Using these same stimuli, Lee and Federmeier (2012) found a sustained frontal negativity to the NV homographs, and, on the downstream noun, found a plausibility effect that was also positively correlated with the size of a reader's ambiguity effect. Together, these findings suggest that when only syntactic constraints are available, meaning selection recruits inhibitory mechanisms that can be measured in both first fixation slowdown and ERP ambiguity effects. PMID:25961358

VIEW OF FOSSIL CREEK DIVERSION DAM FROM DOWNSTREAM (INCLUDES 1950s ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

VIEW OF FOSSIL CREEK DIVERSION DAM FROM DOWNSTREAM (INCLUDES 1950s AUTOMATIC/REMOTE CONTROL SLUICE GATE IN UPPER CENTER OF DAM, NORTH SIDE). LOOKING NORTH-NORTHWEST - Childs-Irving Hydroelectric Project, Fossil Creek Diversion Dam, Forest Service Road 708/502, Camp Verde, Yavapai County, AZ

Airfoil

DOEpatents

Ristau, Neil; Siden, Gunnar Leif

2015-07-21

An airfoil includes a leading edge, a trailing edge downstream from the leading edge, a pressure surface between the leading and trailing edges, and a suction surface between the leading and trailing edges and opposite the pressure surface. A first convex section on the suction surface decreases in curvature downstream from the leading edge, and a throat on the suction surface is downstream from the first convex section. A second convex section is on the suction surface downstream from the throat, and a first convex segment of the second convex section increases in curvature.

Targeting the PI3K/Akt/mTOR pathway: effective combinations and clinical considerations

PubMed Central

LoPiccolo, Jaclyn; Blumenthal, Gideon M.; Bernstein, Wendy B.; Dennis, Phillip A.

2008-01-01

The PI3K/Akt/mTOR pathway is a prototypic survival pathway that is constitutively activated in many types of cancer. Mechanisms for pathway activation include loss of tumor suppressor PTEN function, amplification or mutation of PI3K, amplification or mutation of Akt, activation of growth factor receptors, and exposure to carcinogens. Once activated, signaling through Akt can be propagated to a diverse array of substrates, including mTOR, a key regulator of protein translation. This pathway is an attractive therapeutic target in cancer because it serves as a convergence point for many growth stimuli, and through its downstream substrates, controls cellular processes that contribute to the initiation and maintenance of cancer. Moreover, activation of the Akt/mTOR pathway confers resistance to many types of cancer therapy, and is a poor prognostic factor for many types of cancers. This review will provide an update on the clinical progress of various agents that target the pathway, such as the Akt inhibitors perifosine and PX-866 and mTOR inhibitors (rapamycin, CCI-779, RAD-001) and discuss strategies to combine these pathway inhibitors with conventional chemotherapy, radiotherapy, as well as newer targeted agents. We will also discuss how the complex regulation of the PI3K/Akt/mTOR pathway poses practical issues concerning the design of clinical trials, potential toxicities and criteria for patient selection. PMID:18166498

Src-dependent EGFR transactivation regulates lung inflammation via downstream signaling involving ERK1/2, PI3Kδ/Akt and NFκB induction in a murine asthma model.

PubMed

El-Hashim, Ahmed Z; Khajah, Maitham A; Renno, Waleed M; Babyson, Rhema S; Uddin, Mohib; Benter, Ibrahim F; Ezeamuzie, Charles; Akhtar, Saghir

2017-08-30

The molecular mechanisms underlying asthma pathogenesis are poorly characterized. In this study, we investigated (1) whether Src mediates epidermal growth factor receptor (EGFR) transactivation; (2) if ERK1/2, PI3Kδ/Akt and NF-κB are signaling effectors downstream of Src/EGFR activation; and (3) if upstream inhibition of Src/EGFR is more effective in downregulating the allergic inflammation than selective inhibition of downstream signaling pathways. Allergic inflammation resulted in increased phosphorylation of EGFR, Akt, ERK1/2 and IκB in the lung tissues from ovalbumin (OVA)-challenged BALB/c mice. Treatment with inhibitors of Src (SU6656) or EGFR (AG1478) reduced EGFR phosphorylation and downstream signaling which resulted in the inhibition of the OVA-induced inflammatory cell influx in bronchoalveolar lavage fluid (BALF), perivascular and peribronchial inflammation, fibrosis, goblet cell hyper/metaplasia and airway hyper-responsiveness. Treatment with pathway-selective inhibitors for ERK1/2 (PD89059) and PI3Kδ/Akt (IC-87114) respectively, or an inhibitor of NF-κB (BAY11-7085) also reduced the OVA-induced asthmatic phenotype but to a lesser extent compared to Src/EGFR inhibition. Thus, Src via EGFR transactivation and subsequent downstream activation of multiple pathways regulates the allergic airway inflammatory response. Furthermore, a broader upstream inhibition of Src/EGFR offers an attractive therapeutic alternative in the treatment of asthma relative to selectively targeting the individual downstream signaling effectors.

Proliferating cell nuclear antigen (Pcna) as a direct downstream target gene of Hoxc8

DOE Office of Scientific and Technical Information (OSTI.GOV)

Min, Hyehyun; Lee, Ji-Yeon; Bok, Jinwoong

2010-02-19

Hoxc8 is a member of Hox family transcription factors that play crucial roles in spatiotemporal body patterning during embryogenesis. Hox proteins contain a conserved 61 amino acid homeodomain, which is responsible for recognition and binding of the proteins onto Hox-specific DNA binding motifs and regulates expression of their target genes. Previously, using proteome analysis, we identified Proliferating cell nuclear antigen (Pcna) as one of the putative target genes of Hoxc8. Here, we asked whether Hoxc8 regulates Pcna expression by directly binding to the regulatory sequence of Pcna. In mouse embryos at embryonic day 11.5, the expression pattern of Pcna wasmore » similar to that of Hoxc8 along the anteroposterior body axis. Moreover, Pcna transcript levels as well as cell proliferation rate were increased by overexpression of Hoxc8 in C3H10T1/2 mouse embryonic fibroblast cells. Characterization of 2.3 kb genomic sequence upstream of Pcna coding region revealed that the upstream sequence contains several Hox core binding sequences and one Hox-Pbx binding sequence. Direct binding of Hoxc8 proteins to the Pcna regulatory sequence was verified by chromatin immunoprecipitation assay. Taken together, our data suggest that Pcna is a direct downstream target of Hoxc8.« less

Protein kinases: mechanisms and downstream targets in inflammation mediated obesity and insulin resistance

PubMed Central

Nandipati, Kalyana C; Subramanian, Saravanan; Agrawal, Devendra K

2016-01-01

Obesity induced low-grade inflammation (metaflammation) impairs insulin receptor signaling (IRS). This has been implicated in the development of insulin resistance. Insulin signaling in the target tissues is mediated by stress kinases such as p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK), inhibitor of NF-kB kinase complex beta (IKKβ), AMP activated protein kinase (AMPK), protein kinase C (PKC), Rho associated coiled-coil containing protein kinase (ROCK) and RNA-activated protein kinase (PKR), etc. Most of these kinases phosphorylate several key regulators in glucose homeostasis. The phosphorylation of serine residues in the insulin receptor (IR) and IRS-1 molecule results in diminished enzymatic activity in the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. This has been one of the key mechanisms observed in the tissues that are implicated in insulin resistance especially in Type II Diabetes Mellitus (T2-DM). Identifying the specific protein kinases involved in obesity induced chronic inflammation may help in developing the targeted drug therapies to minimize the insulin resistance. This review is focused on the protein kinases involved in the inflammatory cascade and molecular mechanisms and their downstream targets with special reference to obesity induced T2-DM. PMID:27868170

Neuroblastoma treatment in the post-genomic era.

PubMed

Esposito, Maria Rosaria; Aveic, Sanja; Seydel, Anke; Tonini, Gian Paolo

2017-02-08

Neuroblastoma is an embryonic malignancy of early childhood originating from neural crest cells and showing heterogeneous biological, morphological, genetic and clinical characteristics. The correct stratification of neuroblastoma patients within risk groups (low, intermediate, high and ultra-high) is critical for the adequate treatment of the patients.High-throughput technologies in the Omics disciplines are leading to significant insights into the molecular pathogenesis of neuroblastoma. Nonetheless, further study of Omics data is necessary to better characterise neuroblastoma tumour biology. In the present review, we report an update of compounds that are used in preclinical tests and/or in Phase I-II trials for neuroblastoma. Furthermore, we recapitulate a number of compounds targeting proteins associated to neuroblastoma: MYCN (direct and indirect inhibitors) and downstream targets, Trk, ALK and its downstream signalling pathways. In particular, for the latter, given the frequency of ALK gene deregulation in neuroblastoma patients, we discuss on second-generation ALK inhibitors in preclinical or clinical phases developed for the treatment of neuroblastoma patients resistant to crizotinib.We summarise how Omics drive clinical trials for neuroblastoma treatment and how much the research of biological targets is useful for personalised medicine. Finally, we give an overview of the most recent druggable targets selected by Omics investigation and discuss how the Omics results can provide us additional advantages for overcoming tumour drug resistance.

Molecular genetics and targeted therapeutics in biliary tract carcinoma

PubMed Central

Marks, Eric I; Yee, Nelson S

2016-01-01

The primary malignancies of the biliary tract, cholangiocarcinoma and gallbladder cancer, often present at an advanced stage and are marginally sensitive to radiation and chemotherapy. Accumulating evidence indicates that molecularly targeted agents may provide new hope for improving treatment response in biliary tract carcinoma (BTC). In this article, we provide a critical review of the pathogenesis and genetic abnormalities of biliary tract neoplasms, in addition to discussing the current and emerging targeted therapeutics in BTC. Genetic studies of biliary tumors have identified the growth factors and receptors as well as their downstream signaling pathways that control the growth and survival of biliary epithelia. Target-specific monoclonal antibodies and small molecules inhibitors directed against the signaling pathways that drive BTC growth and invasion have been developed. Numerous clinical trials designed to test these agents as either monotherapy or in combination with conventional chemotherapy have been completed or are currently underway. Research focusing on understanding the molecular basis of biliary tumorigenesis will continue to identify for targeted therapy the key mutations that drive growth and invasion of biliary neoplasms. Additional strategies that have emerged for treating this malignant disease include targeting the epigenetic alterations of BTC and immunotherapy. By integrating targeted therapy with molecular profiles of biliary tumor, we hope to provide precision treatment for patients with malignant diseases of the biliary tract. PMID:26819503

Core Promoter Functions in the Regulation of Gene Expression of Drosophila Dorsal Target Genes*

PubMed Central

Zehavi, Yonathan; Kuznetsov, Olga; Ovadia-Shochat, Avital; Juven-Gershon, Tamar

2014-01-01

Developmental processes are highly dependent on transcriptional regulation by RNA polymerase II. The RNA polymerase II core promoter is the ultimate target of a multitude of transcription factors that control transcription initiation. Core promoters consist of core promoter motifs, e.g. the initiator, TATA box, and the downstream core promoter element (DPE), which confer specific properties to the core promoter. Here, we explored the importance of core promoter functions in the dorsal-ventral developmental gene regulatory network. This network includes multiple genes that are activated by different nuclear concentrations of Dorsal, an NFκB homolog transcription factor, along the dorsal-ventral axis. We show that over two-thirds of Dorsal target genes contain DPE sequence motifs, which is significantly higher than the proportion of DPE-containing promoters in Drosophila genes. We demonstrate that multiple Dorsal target genes are evolutionarily conserved and functionally dependent on the DPE. Furthermore, we have analyzed the activation of key Dorsal target genes by Dorsal, as well as by another Rel family transcription factor, Relish, and the dependence of their activation on the DPE motif. Using hybrid enhancer-promoter constructs in Drosophila cells and embryo extracts, we have demonstrated that the core promoter composition is an important determinant of transcriptional activity of Dorsal target genes. Taken together, our results provide evidence for the importance of core promoter composition in the regulation of Dorsal target genes. PMID:24634215

System and method for reducing combustion dynamics in a combustor

DOEpatents

Uhm, Jong Ho; Ziminsky, Willy Steve; Johnson, Thomas Edward; Srinivasan, Shiva; York, William David

2016-11-29

A system for reducing combustion dynamics in a combustor includes an end cap that extends radially across the combustor and includes an upstream surface axially separated from a downstream surface. A combustion chamber is downstream of the end cap, and tubes extend from the upstream surface through the downstream surface. Each tube provides fluid communication through the end cap to the combustion chamber. The system further includes means for reducing combustion dynamics in the combustor. A method for reducing combustion dynamics in a combustor includes flowing a working fluid through tubes that extend axially through an end cap that extends radially across the combustor and obstructing at least a portion of the working fluid flowing through a first set of the tubes.

MicroRNA-99 family members suppress Homeobox A1 expression in epithelial cells.

PubMed

Chen, Dan; Chen, Zujian; Jin, Yi; Dragas, Dragan; Zhang, Leitao; Adjei, Barima S; Wang, Anxun; Dai, Yang; Zhou, Xiaofeng

2013-01-01

The miR-99 family is one of the evolutionarily most ancient microRNA families, and it plays a critical role in developmental timing and the maintenance of tissue identity. Recent studies, including reports from our group, suggested that the miR-99 family regulates various physiological processes in adult tissues, such as dermal wound healing, and a number of disease processes, including cancer. By combining 5 independent genome-wide expression profiling experiments, we identified a panel of 266 unique transcripts that were down-regulated in epithelial cells transfected with miR-99 family members. A comprehensive bioinformatics analysis using 12 different sequence-based microRNA target prediction algorithms revealed that 81 out of these 266 down-regulated transcripts are potential direct targets for the miR-99 family. Confirmation experiments and functional analyses were performed to further assess 6 selected miR-99 target genes, including mammalian Target of rapamycin (mTOR), Homeobox A1 (HOXA1), CTD small phosphatase-like (CTDSPL), N-myristoyltransferase 1 (NMT1), Transmembrane protein 30A (TMEM30A), and SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (SMARCA5). HOXA1 is a known proto-oncogene, and it also plays an important role in embryonic development. The direct targeting of the miR-99 family to two candidate binding sequences located in the HOXA1 mRNA was confirmed using a luciferase reporter gene assay and a ribonucleoprotein-immunoprecipitation (RIP-IP) assay. Ectopic transfection of miR-99 family reduced the expression of HOXA1, which, in consequence, down-regulated the expression of its downstream gene (i.e., Bcl-2) and led to reduced proliferation and cell migration, as well as enhanced apoptosis. In summary, we identified a number of high-confidence miR-99 family target genes, including proto-oncogene HOXA1, which may play an important role in regulating epithelial cell proliferation and migration during physiological disease processes, such as dermal wound healing and tumorigenesis.

TAM Receptor Tyrosine Kinases: Biologic Functions, Signaling, and Potential Therapeutic Targeting in Human Cancer

PubMed Central

Linger, Rachel M. A.; Keating, Amy K.; Earp, H. Shelton; Graham, Douglas K.

2011-01-01

Tyro-3, Axl, and Mer constitute the TAM family of receptor tyrosine kinases (RTKs) characterized by a conserved sequence within the kinase domain and adhesion molecule-like extracellular domains. This small family of RTKs regulates an intriguing mix of processes, including cell proliferation/survival, cell adhesion and migration, blood clot stabilization, and regulation of inflammatory cytokine release. Genetic or experimental alteration of TAM receptor function can contribute to a number of disease states, including coagulopathy, autoimmune disease, retinitis pigmentosa, and cancer. In this chapter, we first provide a comprehensive review of the structure, regulation, biologic functions, and down-stream signaling pathways of these receptors. In addition, we discuss recent evidence which suggests a role for TAM receptors in oncogenic mechanisms as family members are over-expressed in a spectrum of human cancers and have prognostic significance in some. Possible strategies for targeted inhibition of the TAM family in the treatment of human cancer are described. Further research will be necessary to evaluate the full clinical implications of TAM family expression and activation in cancer. PMID:18620092

Oxidized Base Damage and Single-Strand Break Repair in Mammalian Genomes: Role of Disordered Regions and Posttranslational Modifications in Early Enzymes

PubMed Central

Hegde, Muralidhar L.; Izumi, Tadahide; Mitra, Sankar

2012-01-01

Oxidative genome damage induced by reactive oxygen species includes oxidized bases, abasic (AP) sites, and single-strand breaks, all of which are repaired via the evolutionarily conserved base excision repair/single-strand break repair (BER/SSBR) pathway. BER/SSBR in mammalian cells is complex, with preferred and backup sub-pathways, and is linked to genome replication and transcription. The early BER/SSBR enzymes, namely, DNA glycosylases (DGs) and the end-processing proteins such as abasic endonuclease 1 (APE1), form complexes with downstream repair (and other noncanonical) proteins via pairwise interactions. Furthermore, a unique feature of mammalian early BER/ SSBR enzymes is the presence of a disordered terminal extension that is absent in their Escherichia coli prototypes. These nonconserved segments usually contain organelle-targeting signals, common interaction interfaces, and sites of posttranslational modifications that may be involved in regulating their repair function including lesion scanning. Finally, the linkage of BER/SSBR deficiency to cancer, aging, and human neurodegenerative diseases, and therapeutic targeting of BER/SSBR are discussed. PMID:22749145

Coordinated neuronal activity enhances corticocortical communication

PubMed Central

Zandvakili, Amin; Kohn, Adam

2015-01-01

Summary Relaying neural signals between cortical areas is central to cognition and sensory processing. The temporal coordination of activity in a source population has been suggested to determine corticocortical signaling efficacy, but others have argued that coordination is functionally irrelevant. We reasoned that if coordination significantly influenced signaling, spiking in downstream networks should be preceded by transiently elevated coordination in a source population. We developed a metric to quantify network coordination in brief epochs, and applied it to simultaneous recordings of neuronal populations in cortical areas V1 and V2 of the macaque monkey. Spiking in the input layers of V2 was preceded by brief epochs of elevated V1 coordination, but this was not the case in other layers of V2. Our results indicate that V1 coordination influences its signaling to direct downstream targets, but that coordinated V1 epochs do not propagate through multiple downstream networks as in some corticocortical signaling schemes. PMID:26291164

New targets and therapies for gastrointestinal stromal tumors.

PubMed

Wozniak, Agnieszka; Gebreyohannes, Yemarshet K; Debiec-Rychter, Maria; Schöffski, Patrick

2017-12-01

The majority of gastrointestinal stromal tumors (GIST) are driven by an abnormal receptor tyrosine kinase (RTK) signaling, occurring mainly due to somatic mutations in KIT or platelet derived growth factor receptor alpha (PDGFRA). Although the introduction of tyrosine kinase inhibitors (TKIs) has revolutionized therapy for GIST patients, with time the vast majority of them develop TKI resistance. Advances in understanding the molecular background of GIST resistance allows for the identification of new targets and the development of novel strategies to overcome or delay its occurrence. Areas covered: The focus of this review is on novel, promising therapeutic approaches to overcome heterogeneous resistance to registered TKIs. These approaches involve new TKIs, including drugs specific for a mutated form of KIT/PDGFRA, drugs with inhibitory effect against multiple RTKs, compounds targeting dysregulated downstream signaling pathways, drugs affecting KIT expression and degradation, inhibitors of cell cycle, and immunotherapeutics. Expert commentary: As the resistance to standard TKI treatment can be heterogeneous, a combinational approach for refractory GIST could be beneficial. Moreover, the understanding of the molecular background of resistant disease would allow development of a more personalized approach for these patients and their response to targeted therapy could be monitored closely using 'liquid biopsy'.

Characterization and Placement of Wetlands for Integrated Conservation Practice Planning

EPA Science Inventory

Constructed wetlands have been recognized as an efficient and cost-effective conservation practice to protect water quality through reducing the transport of sediments and nutrients from upstream croplands to downstream water bodies. The challenge resides in targeting the strateg...

MicroRNA regulation of viral immunity, latency, and carcinogenesis of selected tumor viruses and HIV.

PubMed

Wang, Ling; Li, Guangyu; Yao, Zhi Q; Moorman, Jonathan P; Ning, Shunbin

2015-09-01

MicroRNAs (miRNAs) function as key regulators in immune responses and cancer development. In the contexts of infection with oncogenic viruses, miRNAs are engaged in viral persistence, latency establishment and maintenance, and oncogenesis. In this review, we summarize the potential roles and mechanisms of viral and cellular miRNAs in the host-pathogen interactions during infection with selected tumor viruses and HIV, which include (i) repressing viral replication and facilitating latency establishment by targeting viral transcripts, (ii) evading innate and adaptive immune responses via toll-like receptors, RIG-I-like receptors, T-cell receptor, and B-cell receptor pathways by targeting signaling molecules such as TRAF6, IRAK1, IKKε, and MyD88, as well as downstream targets including regulatory cytokines such as tumor necrosis factor α, interferon γ, interleukin 10, and transforming growth factor β, (iii) antagonizing intrinsic and extrinsic apoptosis pathways by targeting pro-apoptotic or anti-apoptotic gene transcripts such as the Bcl-2 family and caspase-3, (iv) modulating cell proliferation and survival through regulation of the Wnt, PI3K/Akt, Erk/MAPK, and Jak/STAT signaling pathways, as well as the signaling pathways triggered by viral oncoproteins such as Epstein-Barr Virus LMP1, by targeting Wnt-inhibiting factor 1, SHIP, pTEN, and SOCSs, and (v) regulating cell cycle progression by targeting cell cycle inhibitors such as p21/WAF1 and p27/KIP1. Further elucidation of the interaction between miRNAs and these key biological events will facilitate our understanding of the pathogenesis of viral latency and oncogenesis and may lead to the identification of miRNAs as novel targets for developing new therapeutic or preventive interventions. Copyright © 2015 John Wiley & Sons, Ltd.

Targeted therapy in advanced gastric carcinoma: the future is beginning.

PubMed

Schinzari, G; Cassano, A; Orlandi, A; Basso, M; Barone, C

2014-01-01

Gastric cancer represents one of the most common cancer worldwide. Unfortunately, the majority of patients present in advanced stage and outcome still remains poor with high mortality rate despite decreasing incidence and new diagnostic and therapeutic strategies. Although utility of classical chemotherapy agents has been widely explored, advances have been slow and the efficacy of these agents has reached a plateau of median overall survival not higher than 12 months. Therefore, researchers focused their attention on better understanding molecular biology of carcinogenesis and deeper knowledge of the cancer cell phenotype, as well on development of rationally designed drugs that would target specific molecular aberrancies in signal transduction pathways. These targets include cell surface receptors, circulating growth and angiogenic factors and other molecules involved in downstream intracellular signaling pathways, including receptor tyrosine kinases. However, therapeutic advances in gastric cancer are not so encouraging when compared to other solid organ malignancies such as breast and colorectal cancer. This article reviews the role of targeted agents in gastric cancer as single-agent therapy or in combination regimens, including their rational and emerging mechanism of action, current and emerging data. We focused our attention mainly on published phase III studies, therefore cornerstone clinical trials with trastuzumab and bevacizumab have been largely discussed. Phase III studies presented in important international meetings are also reviewed as well phase II published studies and promising new therapies investigated in preclinical or phase I studies. Today, in first-line treatment only trastuzumab has shown significantly increased survival in combination with chemotherapy, whereas ramucirumab as single agent resulted effective in progressing patients, but - despite several disappointing results - these are the proof of principle that targeting the proper molecular aberration is the best way for implementing outcome of therapy.

The Ste12-like transcription factor MaSte12 is involved in pathogenicity by regulating the appressorium formation in the entomopathogenic fungus, Metarhizium acridum.

PubMed

Wei, Qinglv; Du, Yanru; Jin, Kai; Xia, Yuxian

2017-12-01

Homeodomain transcription factor Ste12 is a key target activated by the pathogenic mitogen-activated-protein kinase pathway, and the activated Ste12p protein regulates downstream gene expression levels to modulate phenotypes. However, the functions of Ste12-like genes in entomopathogenic fungi remain poorly understood and little is known about the downstream genes regulated by Ste12. In this study, we characterized the functions of a Ste12 orthologue in Metarhizium acridum, MaSte12, and identified its downstream target genes. The deletion mutant (ΔMaSte12) is defective in conidial germination but not in hyphal growth, conidiation, or stress tolerance. Bioassays showed that ΔMaSte12 had a dramatically decreased virulence in topical inoculations, but no significant difference was found in intrahemolymph injections when the penetration process was bypassed. The mature appressorium formation rate of ΔMaSte12 was less than 10% on locust wings, with the majority hyphae forming appressorium-like, curved but no swollen structures. Digital gene expression profiling revealed that some genes involved in cell wall synthesis and remodeling, appressorium development, and insect cuticle penetration were downregulated in ΔMaSte12. Thus, MaSte12 has critical roles in the pathogenicity of the entomopathogenic fungus M. acridum, and our study provides some explanations for the impairment of fungal virulence in ΔMaSte12. In addition, virulence is very important for fungal biocontrol agents to control insect pests effectively. This study demonstrated that MaSte12 is involved in fungal virulence but not conidial yield or fungal stress tolerance in M. acridum. Thus, MaSte12 and its downstream genes may be candidates for enhancing fungal virulence to improve mycoinsecticides.

A Topical Mitochondria-Targeted Redox Cycling Nitroxide Mitigates Oxidative Stress Induced Skin Damage

PubMed Central

Brand, Rhonda M.; Epperly, Michael W.; Stottlemyer, J. Mark; Skoda, Erin M.; Gao, Xiang; Li, Song; Huq, Saiful; Wipf, Peter; Kagan, Valerian E.; Greenberger, Joel S.; Falo, Louis D.

2017-01-01

Skin is the largest human organ and provides a first line of defense that includes physical, chemical, and immune mechanisms to combat environmental stress. Radiation is a prevalent environmental stressor. Radiation induced skin damage ranges from photoaging and cutaneous carcinogenesis from UV exposure, to treatment-limiting radiation dermatitis associated with radiotherapy, to cutaneous radiation syndrome, a frequently fatal consequence of exposures from nuclear accidents. The major mechanism of skin injury common to these exposures is radiation induced oxidative stress. Efforts to prevent or mitigate radiation damage have included development of antioxidants capable of reducing reactive oxygen species (ROS). Mitochondria are particularly susceptible to oxidative stress, and mitochondrial dependent apoptosis plays a major role in radiation induced tissue damage. We reasoned that targeting a redox cycling nitroxide to mitochondria could prevent ROS accumulation, limiting downstream oxidative damage and preserving mitochondrial function. Here we show that in both mouse and human skin, topical application of a mitochondrial targeted antioxidant prevents and mitigates radiation induced skin damage characterized by clinical dermatitis, loss of barrier function, inflammation, and fibrosis. Further, damage mitigation is associated with reduced apoptosis, preservation of the skin’s antioxidant capacity, and reduction of irreversible DNA and protein oxidation associated with oxidative stress. PMID:27794421

Inhibitory and combinatorial effect of diphyllin, a v-ATPase blocker, on influenza viruses.

PubMed

Chen, Hui-Wen; Cheng, Jenna Xiao; Liu, Ming-Tsan; King, Kevin; Peng, Ju-Yi; Zhang, Xin-Quan; Wang, Ching-Ho; Shresta, Sujan; Schooley, Robert T; Liu, Yu-Tsueng

2013-09-01

An influenza pandemic poses a serious threat to humans and animals. Conventional treatments against influenza include two classes of pathogen-targeting antivirals: M2 ion channel blockers (such as amantadine) and neuraminidase inhibitors (such as oseltamivir). Examination of the mechanism of influenza viral infection has shown that endosomal acidification plays a major role in facilitating the fusion between viral and endosomal membranes. This pathway has led to investigations on vacuolar ATPase (v-ATPase) activity, whose role as a regulating factor on influenza virus replication has been verified in extensive genome-wide screenings. Blocking v-ATPase activity thus presents the opportunity to interfere with influenza viral infection by preventing the pH-dependent membrane fusion between endosomes and virions. This study aims to apply diphyllin, a natural compound shown to be as a novel v-ATPase inhibitor, as a potential antiviral for various influenza virus strains using cell-based assays. The results show that diphyllin alters cellular susceptibility to influenza viruses through the inhibition of endosomal acidification, thus interfering with downstream virus replication, including that of known drug-resistant strains. In addition, combinatorial treatment of the host-targeting diphyllin with pathogen-targeting therapeutics (oseltamivir and amantadine) demonstrates enhanced antiviral effects and cell protection in vitro. Copyright © 2013 Elsevier B.V. All rights reserved.

Purification of target proteins from intracellular inclusions mediated by intein cleavable polyhydroxyalkanoate synthase fusions.

PubMed

Du, Jinping; Rehm, Bernd H A

2017-11-02

Recombinant protein production and purification from Escherichia coli is often accompanied with expensive and complicated procedures, especially for therapeutic proteins. Here it was demonstrated that, by using an intein cleavable polyhydroxyalkanoate synthase fusion, recombinant proteins can be first produced and sequestered on a natural resin, the polyhydroxyalkanoate (PHA) inclusions, then separated from contaminating host proteins via simple PHA bead isolation steps, and finally purified by specific release into the soluble fraction induced by a pH reduction. By translationally fusing a target protein to PHA synthase using a self-cleaving intein as linker, intracellular production of PHA beads was achieved. Upon isolation of respective PHA beads the soluble pure target protein was released by a simple pH shift to 6. The utility of this approach was exemplified by producing six target proteins, including Aequorea victoria green fluorescent protein (GFP), Mycobacterium tuberculosis vaccine candidate Rv1626, the immunoglobulin G (IgG) binding ZZ domain of protein A derived from Staphylococcus aureus, human tumor necrosis factor alpha (TNFα), human granulocyte colony-stimulating factor (G-CSF), and human interferon alpha 2b (IFNα2b). Here a new method for production and purification of a tag-less protein was developed through intein cleavable polyhydroxyalkanoate synthase fusion. Pure target protein could be easily obtained without laborious downstream processing.

MicroRNA-320a suppresses in GBM patients and modulates glioma cell functions by targeting IGF-1R.

PubMed

Guo, Tianzhu; Feng, Ying; Liu, Qingyang; Yang, Xue; Jiang, Tao; Chen, Yan; Zhang, Quangeng

2014-11-01

Glioblastoma (GBM) is the most aggressive and malignant glioma. Currently, a few modern surgical and medical therapeutic strategies are applied for GBM, but the prognosis of GBM patients remains poor, and the average median survival time is only 14.6 months. In this study, we for the first time found that the levels of miR-320a were decreased in both GBM patients and glioma cells. In GBM patients, elevated miR-320a expression was associated with better prognosis. In addition, insulin-like growth factor-1 receptor (IGF-1R) was identified as a key direct target of miR-320a. Overexpression of miR-320a led to the inhibition of cell proliferation, migration, invasion, as well as tumorigenesis by targeting IGF-1R, and thus regulated the signaling pathways downstream, including PI3K/AKT and MAPK/ERK. In tumor orthotopic xenograft experiment, the tumor growth was depressed and survival time of mice model was prolonged when miR-320a was overexpressed. Therefore, our results suggested that miR-320a could suppress tumor development and growth by targeting IGF-1R, and miR-320a might serve as a new effective target for anti-cancer therapy strategies.

M$^3$: A New Muon Missing Momentum Experiment to Probe $$(g-2)_{\\mu}$$ and Dark Matter at Fermilab

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kahn, Yonatan; Krnjaic, Gordan; Tran, Nhan

New light, weakly-coupled particles are commonly invoked to address the persistentmore » $$\\sim 4\\sigma$$ anomaly in $$(g-2)_\\mu$$ and serve as mediators between dark and visible matter. If such particles couple predominantly to heavier generations and decay invisibly, much of their best-motivated parameter space is inaccessible with existing experimental techniques. In this paper, we present a new fixed-target, missing-momentum search strategy to probe invisibly decaying particles that couple preferentially to muons. In our setup, a relativistic muon beam impinges on a thick active target. The signal consists of events in which a muon loses a large fraction of its incident momentum inside the target without initiating any detectable electromagnetic or hadronic activity in downstream veto systems. We propose a two-phase experiment, M$^3$ (Muon Missing Momentum), based at Fermilab. Phase 1 with $$\\sim 10^{10}$$ muons on target can test the remaining parameter space for which light invisibly-decaying particles can resolve the $$(g-2)_\\mu$$ anomaly, while Phase 2 with $$\\sim 10^{13}$$ muons on target can test much of the predictive parameter space over which sub-GeV dark matter achieves freeze-out via muon-philic forces, including gauged $$U(1)_{L_\\mu - L_\\tau}$$.« less

ZNF750 is a p63 Target Gene that Induces KLF4 to Drive Terminal Epidermal Differentiation

PubMed Central

Sen, George L.; Boxer, Lisa D.; Webster, Dan E.; Bussat, Rose T.; Qu, Kun; Zarnegar, Brian J.; Johnston, Danielle; Siprashvili, Zurab; Khavari, Paul A.

2012-01-01

SUMMARY Disrupted epidermal differentiation characterizes numerous diseases that impact >25% of the population. In a search for dominant mediators of differentiation, we defined a requirement for ZNF750 in terminal epidermal differentiation. ZNF750 controlled genes mutated in numerous human skin diseases, including FLG, LOR, LCE3B, ALOXE3, and SPINK5. ZNF750 induced progenitor differentiation via an evolutionarily conserved C2H2 zinc finger motif. The epidermal master regulator, p63, bound the ZNF750 promoter and was necessary for its induction. ZNF750 restored differentiation to p63-deficient tissue, suggesting it acts downstream of p63. A search for functionally important ZNF750 targets via analysis of ZNF750-regulated genes identified KLF4, a transcription factor that activates late epidermal differentiation. ZNF750 binds to KLF4 at multiple sites flanking the transcriptional start site and controls its expression. ZNF750 thus directly links a tissue-specifying factor, p63, to an effector of terminal differentiation, KLF4, and represents a potential future target for disorders of this process. PMID:22364861

New Insights into Protein Kinase B/Akt Signaling: Role of Localized Akt Activation and Compartment-Specific Target Proteins for the Cellular Radiation Response.

PubMed

Szymonowicz, Klaudia; Oeck, Sebastian; Malewicz, Nathalie M; Jendrossek, Verena

2018-03-18

Genetic alterations driving aberrant activation of the survival kinase Protein Kinase B (Akt) are observed with high frequency during malignant transformation and cancer progression. Oncogenic gene mutations coding for the upstream regulators or Akt, e.g., growth factor receptors, RAS and phosphatidylinositol-3-kinase (PI3K), or for one of the three Akt isoforms as well as loss of the tumor suppressor Phosphatase and Tensin Homolog on Chromosome Ten (PTEN) lead to constitutive activation of Akt. By activating Akt, these genetic alterations not only promote growth, proliferation and malignant behavior of cancer cells by phosphorylation of various downstream signaling molecules and signaling nodes but can also contribute to chemo- and radioresistance in many types of tumors. Here we review current knowledge on the mechanisms dictating Akt's activation and target selection including the involvement of miRNAs and with focus on compartmentalization of the signaling network. Moreover, we discuss recent advances in the cross-talk with DNA damage response highlighting nuclear Akt target proteins with potential involvement in the regulation of DNA double strand break repair.

Gas turbine premixer with internal cooling

DOEpatents

York, William David; Johnson, Thomas Edward; Lacy, Benjamin Paul; Stevenson, Christian Xavier

2012-12-18

A system that includes a turbine fuel nozzle comprising an air-fuel premixer. The air-fuel premixed includes a swirl vane configured to swirl fuel and air in a downstream direction, wherein the swirl vane comprises an internal coolant path from a downstream end portion in an upstream direction through a substantial length of the swirl vane.

The role of headwater streams in downstream water quality

USGS Publications Warehouse

Alexander, R.B.; Boyer, E.W.; Smith, R.A.; Schwarz, G.E.; Moore, R.B.

2007-01-01

Knowledge of headwater influences on the water-quality and flow conditions of downstream waters is essential to water-resource management at all governmental levels; this includes recent court decisions on the jurisdiction of the Federal Clean Water Act (CWA) over upland areas that contribute to larger downstream water bodies. We review current watershed research and use a water-quality model to investigate headwater influences on downstream receiving waters. Our evaluations demonstrate the intrinsic connections of headwaters to landscape processes and downstream waters through their influence on the supply, transport, and fate of water and solutes in watersheds. Hydrological processes in headwater catchments control the recharge of subsurface water stores, flow paths, and residence times of water throughout landscapes. The dynamic coupling of hydrological and biogeochemical processes in upland streams further controls the chemical form, timing, and longitudinal distances of solute transport to downstream waters. We apply the spatially explicit, mass-balance watershed model SPARROW to consider transport and transformations of water and nutrients throughout stream networks in the northeastern United States. We simulate fluxes of nitrogen, a primary nutrient that is a water-quality concern for acidification of streams and lakes and eutrophication of coastal waters, and refine the model structure to include literature observations of nitrogen removal in streams and lakes. We quantify nitrogen transport from headwaters to downstream navigable waters, where headwaters are defined within the model as first-order, perennial streams that include flow and nitrogen contributions from smaller, intermittent and ephemeral streams. We find that first-order headwaters contribute approximately 70% of the mean-annual water volume and 65% of the nitrogen flux in second-order streams. Their contributions to mean water volume and nitrogen flux decline only marginally to about 55% and 40% in fourth- and higher-order rivers that include navigable waters and their tributaries. These results underscore the profound influence that headwater areas have on shaping downstream water quantity and water quality. The results have relevance to water-resource management and regulatory decisions and potentially broaden understanding of the spatial extent of Federal CWA jurisdiction in U.S. waters. ?? 2007 American Water Resources Association.

The Role of Headwater Streams in Downstream Water Quality1

PubMed Central

Alexander, Richard B; Boyer, Elizabeth W; Smith, Richard A; Schwarz, Gregory E; Moore, Richard B

2007-01-01

Knowledge of headwater influences on the water-quality and flow conditions of downstream waters is essential to water-resource management at all governmental levels; this includes recent court decisions on the jurisdiction of the Federal Clean Water Act (CWA) over upland areas that contribute to larger downstream water bodies. We review current watershed research and use a water-quality model to investigate headwater influences on downstream receiving waters. Our evaluations demonstrate the intrinsic connections of headwaters to landscape processes and downstream waters through their influence on the supply, transport, and fate of water and solutes in watersheds. Hydrological processes in headwater catchments control the recharge of subsurface water stores, flow paths, and residence times of water throughout landscapes. The dynamic coupling of hydrological and biogeochemical processes in upland streams further controls the chemical form, timing, and longitudinal distances of solute transport to downstream waters. We apply the spatially explicit, mass-balance watershed model SPARROW to consider transport and transformations of water and nutrients throughout stream networks in the northeastern United States. We simulate fluxes of nitrogen, a primary nutrient that is a water-quality concern for acidification of streams and lakes and eutrophication of coastal waters, and refine the model structure to include literature observations of nitrogen removal in streams and lakes. We quantify nitrogen transport from headwaters to downstream navigable waters, where headwaters are defined within the model as first-order, perennial streams that include flow and nitrogen contributions from smaller, intermittent and ephemeral streams. We find that first-order headwaters contribute approximately 70% of the mean-annual water volume and 65% of the nitrogen flux in second-order streams. Their contributions to mean water volume and nitrogen flux decline only marginally to about 55% and 40% in fourth- and higher-order rivers that include navigable waters and their tributaries. These results underscore the profound influence that headwater areas have on shaping downstream water quantity and water quality. The results have relevance to water-resource management and regulatory decisions and potentially broaden understanding of the spatial extent of Federal CWA jurisdiction in U.S. waters. PMID:22457565

Cognitive Dysfunctions in Intellectual Disabilities: The Contributions of the Ras-MAPK and PI3K-AKT-mTOR Pathways.

PubMed

Borrie, Sarah C; Brems, Hilde; Legius, Eric; Bagni, Claudia

2017-08-31

The Ras-MAPK and PI3K-AKT-mTOR signaling cascades were originally identified as cancer regulatory pathways but have now been demonstrated to be critical for synaptic plasticity and behavior. Neurodevelopmental disorders arising from mutations in these pathways exhibit related neurological phenotypes, including cognitive dysfunction, autism, and intellectual disability. The downstream targets of these pathways include regulation of transcription and protein synthesis. Other disorders that affect protein translation include fragile X syndrome (an important cause of syndromal autism), and other translational regulators are now also linked to autism. Here, we review how mechanisms of synaptic plasticity have been revealed by studies of mouse models for Ras-MAPK, PI3K-AKT-mTOR, and translation regulatory pathway disorders. We discuss the face validity of these mouse models and review current progress in clinical trials directed at ameliorating cognitive and behavioral symptoms.

2',3-dihydroxy-5-methoxybiphenyl suppresses fMLP-induced superoxide anion production and cathepsin G release by targeting the β-subunit of G-protein in human neutrophils.

PubMed

Liao, Hsiang-Ruei; Chen, Ih-Sheng; Liu, Fu-Chao; Lin, Shinn-Zhi; Tseng, Ching-Ping

2018-06-15

This study investigates the effect and the underlying mechanism of 2',3-dihydroxy-5-methoxybiphenyl (RIR-2), a lignan extracted from the roots of Rhaphiolepis indica (L.) Lindl. ex Ker var. tashiroi Hayata ex Matsum. & Hayata (Rosaceae), on N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced respiratory burst and cathepsin G in human neutrophils. Signaling pathways regulated by RIR-2 which modulated fMLP-induced respiratory burst were evaluated by an interaction between β subunit of G-protein (Gβ) with downstream signaling induced by fMLP and by immunoblotting analysis of the downstream targets of Gβ-protein. RIR-2 inhibited fMLP-induced superoxide anion production (IC 50 :2.57 ± 0.22 μM), cathepsin G release (IC 50 :18.72 ± 3.76 μM) and migration in a concentration dependent manner. RIR-2 specifically suppresses fMLP-induced Src family kinases phosphorylation by inhibiting the interaction between Gβ-protein with Src kinases without inhibiting Src kinases activities, therefore, RIR-2 attenuated the downstream targets of Src kinase, such as phosphorylation of Raf/ERK, AKT, P38, PLCγ2, PKC and translocation Tec, p47 ph ° x and P40 ph ° x from the cytosol to the inner leaflet of the plasma membrane. Furthermore, RIR-2 attenuated fMLP-induced intracellular calcium mobilization by inhibiting the interaction between Gβ-protein with PLCβ2. RIR-2 was not a competitive or allosteric antagonist of fMLP. On the contrary, phorbol 12-myristate 13-acetate (PMA)-induced phosphorylation of Src, AKT, P38, PKC and membrane localization of p47 ph ° x and P40 ph ° x remained unaffected. RIR-2 specifically modulates fMLP-mediated neutrophil superoxide anion production and cathepsin G release by inhibiting the interaction between Gβ-protein with downstream signaling which subsequently interferes with the activation of intracellular calcium, PLCγ2, AKT, p38, PKC, ERK, p47 ph ° x and p40 phox . Copyright © 2018 Elsevier B.V. All rights reserved.

Antihelminthic Benzimidazoles Are Novel HIF Activators That Prevent Oxidative Neuronal Death via Binding to Tubulin

PubMed Central

Aleyasin, Hossein; Karuppagounder, Saravanan S.; Kumar, Amit; Sleiman, Sama; Basso, Manuela; Ma, Thong; Siddiq, Ambreena; Chinta, Shankar J.; Brochier, Camille; Langley, Brett; Haskew-Layton, Renee; Bane, Susan L.; Riggins, Gregory J.; Gazaryan, Irina; Starkov, Anatoly A.; Andersen, Julie K.

2015-01-01

Abstract Aims: Pharmacological activation of the adaptive response to hypoxia is a therapeutic strategy of growing interest for neurological conditions, including stroke, Huntington's disease, and Parkinson's disease. We screened a drug library with known safety in humans using a hippocampal neuroblast line expressing a reporter of hypoxia-inducible factor (HIF)-dependent transcription. Results: Our screen identified more than 40 compounds with the ability to induce hypoxia response element-driven luciferase activity as well or better than deferoxamine, a canonical activator of hypoxic adaptation. Among the chemical entities identified, the antihelminthic benzimidazoles represented one pharmacophore that appeared multiple times in our screen. Secondary assays confirmed that antihelminthics stabilized the transcriptional activator HIF-1α and induced expression of a known HIF target gene, p21cip1/waf1, in post-mitotic cortical neurons. The on-target effect of these agents in stimulating hypoxic signaling was binding to free tubulin. Moreover, antihelminthic benzimidazoles also abrogated oxidative stress-induced death in vitro, and this on-target effect also involves binding to free tubulin. Innovation and Conclusions: These studies demonstrate that tubulin-binding drugs can activate a component of the hypoxic adaptive response, specifically the stabilization of HIF-1α and its downstream targets. Tubulin-binding drugs, including antihelminthic benzimidazoles, also abrogate oxidative neuronal death in primary neurons. Given their safety in humans and known ability to penetrate into the central nervous system, antihelminthic benzimidazoles may be considered viable candidates for treating diseases associated with oxidative neuronal death, including stroke. Antioxid. Redox Signal. 22, 121–134. PMID:24766300

PAR1 deletions downstream of SHOX are the most frequent defect in a Spanish cohort of Léri-Weill dyschondrosteosis (LWD) probands.

PubMed

Benito-Sanz, Sara; del Blanco, Darya Gorbenko; Aza-Carmona, Miriam; Magano, Luis F; Lapunzina, Pablo; Argente, Jesús; Campos-Barros, Angel; Heath, Karen E

2006-10-01

Léri-Weill dyschondrosteosis (LWD) is a skeletal dysplasia characterized by disproportionate short stature and Madelung deformity. Mutations or deletions of the SHOX gene have been previously identified as the main cause of LWD. We recently identified the existence of a second class of pseudoautosomal region 1 (PAR1) deletions which do not include SHOX, implicated in the etiopathogenesis of LWD. The deletions map at least 30-250 kb downstream of SHOX, are variable in size and clearly cosegregate with the LWD phenotype. In order to determine the frequency of this new type of deletions in the Spanish population we analyzed the distribution of PAR1 defects, including the screening of SHOX deletions, mutations, and PAR1 deletions downstream of SHOX, in a total of 26 LWD probands by a combination of MLPA, microsatellite analysis, SNP genotyping, dHPLC, and DNA sequencing. A molecular defect was identified in 16/26 LWD patients (61.5%): 10 PAR1 deletions downstream of SHOX, four SHOX encompassing deletions, and two SHOX mutations. No apparent phenotypic differences were observed between patients with SHOX defects and those with PAR1 deletions downstream of SHOX. In the examined cohort of Spanish LWD probands, PAR1 deletions downstream of SHOX represent the highest proportion of identified mutations (38%) compared to SHOX deletions (15%) and mutations (8%). As a consequence of our findings, the screening of this region should be included in the routine genetic testing of LWD. Also, LWD patients who tested negative for SHOX defects should be re-evaluated for PAR1 deletions downstream of SHOX.

Armored long non-coding RNA MEG3 targeting EGFR based on recombinant MS2 bacteriophage virus-like particles against hepatocellular carcinoma.

PubMed

Chang, Le; Wang, Guojing; Jia, Tingting; Zhang, Lei; Li, Yulong; Han, Yanxi; Zhang, Kuo; Lin, Guigao; Zhang, Rui; Li, Jinming; Wang, Lunan

2016-04-26

Hepatocellular carcinoma (HCC) is one of the most frequently diagnosed cancers worldwide. However, the treatment of patients with HCC is particularly challenging. Long non-coding RNA maternally expressed gene 3 (MEG3) has been identified as a potential suppressor of several types of tumors, but the delivery of long RNA remains problematic, limiting its applications. In the present study, we designed a novel delivery system based on MS2 virus-like particles (VLPs) crosslinked with GE11 polypeptide. This vector was found to be fast, effective and safe for the targeted delivery of lncRNA MEG3 RNA to the epidermal growth factor receptor (EGFR)-positive HCC cell lines without the activation of EGFR downstream pathways, and significantly attenuated both in vitro and in vivo tumor cell growth. Our study also revealed that the targeted delivery was mainly dependent on clathrin-mediated endocytosis and MEG3 RNA suppresses tumor growth mainly via increasing the expression of p53 and its downstream gene GDF15, but decreasing the expression of MDM2. Thus, this vector is promising as a novel delivery system and may facilitate a new approach to lncRNA based cancer therapy.

Understanding the origin of non-immune cell-mediated weakness in the idiopathic inflammatory myopathies - potential role of ER stress pathways.

PubMed

Lightfoot, Adam P; Nagaraju, Kanneboyina; McArdle, Anne; Cooper, Robert G

2015-11-01

Discussion of endoplasmic reticulum (ER) stress pathway activation in idiopathic inflammatory myopathies (IIM), and downstream mechanisms causative of muscle weakness. In IIM, ER stress is an important pathogenic process, but how it causes muscle dysfunction is unknown. We discuss relevant pathways modified in response to ER stress in IIM: reactive oxygen species (ROS) generation and mitochondrial dysfunction, and muscle cytokine (myokine) generation. First, ER stress pathway activation can induce changes in mitochondrial bioenergetics and ROS production. ROS can oxidize cellular components, causing muscle contractile dysfunction and energy deficits. Novel compounds targeting ROS generation and/or mitochondrial dysfunction can improve muscle function in several myopathologies. Second, recent research has demonstrated that skeletal muscle produces multiple myokines. It is suggested that these play a role in causing muscle weakness. Myokines are capable of immune cell recruitment, thus contributing to perturbed muscle function. A characterization of myokines in IIM would clarify their pathogenic role, and so identify new therapeutic targets. ER stress pathway activation is clearly of etiological relevance in IIM. Research to better understand mechanisms of weakness downstream of ER stress is now required, and which may discover new therapeutic targets for nonimmune cell-mediated weakness.

The role of calcium signalling in the chondrogenic response of mesenchymal stem cells to hydrostatic pressure.

PubMed

Steward, A J; Kelly, D J; Wagner, D R

2014-10-28

The object of this study was to elucidate the role of Ca++ signalling in the chondrogenic response of mesenchymal stem cells (MSCs) to hydrostatic pressure (HP). MSCs were seeded into agarose hydrogels, subjected to HP or kept in free swelling conditions, and were cultured either with or without pharmacological inhibitors of Ca++ mobility and downstream targets. Chelating free Ca++, inhibiting voltage-gated calcium channels, and depleting intracellular calcium storessuppressed the beneficial effect of HP on chondrogenesis, indicating that Ca++ mobility may play an important role in the mechanotransduction of HP. However, inhibition of stretch-activated calcium channels in the current experiment yielded similar results to the control group, suggesting that mechanotransduction of HP is distinct from loads that generate cell deformations. Inhibition of the downstream targets calmodulin, calmodulin kinase II, and calcineurin all knocked down the effect of HP on chondrogenesis, implicating these targets in MSCs response to HP. All of the pharmacological inhibitors that abolished the chondrogenic response to HP also maintained a punctate vimentin organisation in the presence of HP, as opposed to the mechanoresponsive groups where the vimentin structure became more diffuse. These results suggest that Ca++ signalling may transduce HP via vimentin adaptation to loading.

Phosphoproteomics reveals ALK promote cell progress via RAS/ JNK pathway in neuroblastoma.

PubMed

Chen, Kai; Lv, Fan; Xu, Guofeng; Zhang, Min; Wu, Yeming; Wu, Zhixiang

2016-11-15

Emerging evidence suggests receptor tyrosine kinase ALK as a promising therapeutic target in neuroblastoma. However, clinical trials reveal that a limited proportion of ALK-positive neuroblastoma patients experience clinical benefits from Crizotinib, a clinically approved specific inhibitor of ALK. The precise molecular mechanisms of aberrant ALK activity in neuroblastoma remain elusive, limiting the clinical application of ALK as a therapeutic target in neuroblastoma. Here, we describe a deep quantitative phosphoproteomic approach in which Crizotinib-treated neuroblastoma cell lines bearing aberrant ALK are used to investigate downstream regulated phosphoproteins. We identified more than 19,500-and quantitatively analyzed approximately 10,000-phosphorylation sites from each cell line, ultimately detecting 450-790 significantly-regulated phosphorylation sites. Multiple layers of bioinformatic analysis of the significantly-regulated phosphoproteins identified RAS/JNK as a downstream signaling pathway of ALK, independent of the ALK variant present. Further experiments demonstrated that ALK/JNK signaling could be inactivated by either ALK- or JNK-specific inhibitors, resulting in cell growth inhibition by induction of cell cycle arrest and cell apoptosis. Our study broadly defines the phosphoproteome in response to ALK inhibition and provides a resource for further clinical investigation of ALK as therapeutic target for the treatment of neuroblastoma.

Targeting BTK for the treatment of FLT3-ITD mutated acute myeloid leukemia.

PubMed

Pillinger, Genevra; Abdul-Aziz, Amina; Zaitseva, Lyubov; Lawes, Matthew; MacEwan, David J; Bowles, Kristian M; Rushworth, Stuart A

2015-08-21

Approximately 20% of patients with acute myeloid leukaemia (AML) have a mutation in FMS-like-tyrosine-kinase-3 (FLT3). FLT3 is a trans-membrane receptor with a tyrosine kinase domain which, when activated, initiates a cascade of phosphorylated proteins including the SRC family of kinases. Recently our group and others have shown that pharmacologic inhibition and genetic knockdown of Bruton's tyrosine kinase (BTK) blocks AML blast proliferation, leukaemic cell adhesion to bone marrow stromal cells as well as migration of AML blasts. The anti-proliferative effects of BTK inhibition in human AML are mediated via inhibition of downstream NF-κB pro-survival signalling however the upstream drivers of BTK activation in human AML have yet to be fully characterised. Here we place the FLT3-ITD upstream of BTK in AML and show that the BTK inhibitor ibrutinib inhibits the survival and proliferation of FLT3-ITD primary AML blasts and AML cell lines. Furthermore ibrutinib inhibits the activation of downstream kinases including MAPK, AKT and STAT5. In addition we show that BTK RNAi inhibits proliferation of FLT3-ITD AML cells. Finally we report that ibrutinib reverses the cyto-protective role of BMSC on FLT3-ITD AML survival. These results argue for the evaluation of ibrutinib in patients with FLT3-ITD mutated AML.

Dynamic Black-Level Correction and Artifact Flagging in the Kepler Data Pipeline

NASA Technical Reports Server (NTRS)

Clarke, B. D.; Kolodziejczak, J. J.; Caldwell, D. A.

2013-01-01

Instrument-induced artifacts in the raw Kepler pixel data include time-varying crosstalk from the fine guidance sensor (FGS) clock signals, manifestations of drifting moiré pattern as locally correlated nonstationary noise and rolling bands in the images which find their way into the calibrated pixel time series and ultimately into the calibrated target flux time series. Using a combination of raw science pixel data, full frame images, reverse-clocked pixel data and ancillary temperature data the Keplerpipeline models and removes the FGS crosstalk artifacts by dynamically adjusting the black level correction. By examining the residuals to the model fits, the pipeline detects and flags spatial regions and time intervals of strong time-varying blacklevel (rolling bands ) on a per row per cadence basis. These flags are made available to downstream users of the data since the uncorrected rolling band artifacts could complicate processing or lead to misinterpretation of instrument behavior as stellar. This model fitting and artifact flagging is performed within the new stand-alone pipeline model called Dynablack. We discuss the implementation of Dynablack in the Kepler data pipeline and present results regarding the improvement in calibrated pixels and the expected improvement in cotrending performances as a result of including FGS corrections in the calibration. We also discuss the effectiveness of the rolling band flagging for downstream users and illustrate with some affected light curves.

La-related Protein 1 (LARP1) Represses Terminal Oligopyrimidine (TOP) mRNA Translation Downstream of mTOR Complex 1 (mTORC1).

PubMed

Fonseca, Bruno D; Zakaria, Chadi; Jia, Jian-Jun; Graber, Tyson E; Svitkin, Yuri; Tahmasebi, Soroush; Healy, Danielle; Hoang, Huy-Dung; Jensen, Jacob M; Diao, Ilo T; Lussier, Alexandre; Dajadian, Christopher; Padmanabhan, Niranjan; Wang, Walter; Matta-Camacho, Edna; Hearnden, Jaclyn; Smith, Ewan M; Tsukumo, Yoshinori; Yanagiya, Akiko; Morita, Masahiro; Petroulakis, Emmanuel; González, Jose L; Hernández, Greco; Alain, Tommy; Damgaard, Christian K

2015-06-26

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of protein synthesis. The best studied targets of mTORC1 in translation are the eukaryotic initiation factor-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). In this study, we identify the La-related protein 1 (LARP1) as a key novel target of mTORC1 with a fundamental role in terminal oligopyrimidine (TOP) mRNA translation. Recent genome-wide studies indicate that TOP and TOP-like mRNAs compose a large portion of the mTORC1 translatome, but the mechanism by which mTORC1 controls TOP mRNA translation is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5'TOP motif to repress TOP mRNA translation; and (iv) LARP1 competes with the eukaryotic initiation factor (eIF) 4G for TOP mRNA binding. Importantly, from a drug resistance standpoint, our data also show that reducing LARP1 protein levels by RNA interference attenuates the inhibitory effect of rapamycin, Torin1, and amino acid deprivation on TOP mRNA translation. Collectively, our findings demonstrate that LARP1 functions as an important repressor of TOP mRNA translation downstream of mTORC1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Nuclear cereblon modulates transcriptional activity of Ikaros and regulates its downstream target, enkephalin, in human neuroblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wada, Takeyoshi; Asahi, Toru; Research Organization for Nano & Life Innovation, Waseda University #03C309, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480

2016-08-26

The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as amore » thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin. - Highlights: • We found that CRBN is a nucleocytoplasmic shutting protein and identified the key domain for nucleocytoplasmic shuttling. • CRBN associates with the transcription factor Ikaros via the N-terminal domain. • CRBN modulates Ikaros-mediated transcriptional regulation and its downstream target, enkephalin.« less

Evolving therapeutic strategies for Duchenne muscular dystrophy: targeting downstream events.

PubMed

Tidball, James G; Wehling-Henricks, Michelle

2004-12-01

Duchenne muscular dystrophy (DMD) is a progressive, lethal, muscle wasting disease that affects 1 of 3500 boys born worldwide. The disease results from mutation of the dystrophin gene that encodes a cytoskeletal protein associated with the muscle cell membrane. Although gene therapy will likely provide the cure for DMD, it remains on the distant horizon, emphasizing the need for more rapid development of palliative treatments that build on improved understanding of the complex pathology of dystrophin deficiency. In this review, we have focused on therapeutic strategies that target downstream events in the pathologic progression of DMD. Much of this work has been developed initially using the dystrophin-deficient mdx mouse to explore basic features of the pathophysiology of dystrophin deficiency and to test potential therapeutic interventions to slow, reverse, or compensate for functional losses that occur in muscular dystrophy. In some cases, the initial findings in the mdx model have led to clinical treatments for DMD boys that have produced improvements in muscle function and quality of life. Many of these investigations have concerned interventions that can affect protein balance in muscle, by inhibiting specific proteases implicated in the DMD pathology, or by providing anabolic factors or depleting catabolic factors that can contribute to muscle wasting. Other investigations have exploited the use of anti-inflammatory agents that can reduce the contribution of leukocytes to promoting secondary damage to dystrophic muscle. A third general strategy is designed to increase the regenerative capacity of dystrophic muscle and thereby help retain functional muscle mass. Each of these general approaches to slowing the pathology of dystrophin deficiency has yielded encouragement and suggests that targeting downstream events in dystrophinopathy can yield worthwhile, functional improvements in DMD.

La-related Protein 1 (LARP1) Represses Terminal Oligopyrimidine (TOP) mRNA Translation Downstream of mTOR Complex 1 (mTORC1)*

PubMed Central

Fonseca, Bruno D.; Zakaria, Chadi; Jia, Jian-Jun; Graber, Tyson E.; Svitkin, Yuri; Tahmasebi, Soroush; Healy, Danielle; Hoang, Huy-Dung; Jensen, Jacob M.; Diao, Ilo T.; Lussier, Alexandre; Dajadian, Christopher; Padmanabhan, Niranjan; Wang, Walter; Matta-Camacho, Edna; Hearnden, Jaclyn; Smith, Ewan M.; Tsukumo, Yoshinori; Yanagiya, Akiko; Morita, Masahiro; Petroulakis, Emmanuel; González, Jose L.; Hernández, Greco; Alain, Tommy; Damgaard, Christian K.

2015-01-01

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of protein synthesis. The best studied targets of mTORC1 in translation are the eukaryotic initiation factor-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). In this study, we identify the La-related protein 1 (LARP1) as a key novel target of mTORC1 with a fundamental role in terminal oligopyrimidine (TOP) mRNA translation. Recent genome-wide studies indicate that TOP and TOP-like mRNAs compose a large portion of the mTORC1 translatome, but the mechanism by which mTORC1 controls TOP mRNA translation is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5′TOP motif to repress TOP mRNA translation; and (iv) LARP1 competes with the eukaryotic initiation factor (eIF) 4G for TOP mRNA binding. Importantly, from a drug resistance standpoint, our data also show that reducing LARP1 protein levels by RNA interference attenuates the inhibitory effect of rapamycin, Torin1, and amino acid deprivation on TOP mRNA translation. Collectively, our findings demonstrate that LARP1 functions as an important repressor of TOP mRNA translation downstream of mTORC1. PMID:25940091

MicroRNA-29 regulates myocardial microvascular endothelial cells proliferation and migration in association with IGF1 in type 2 diabetes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Zhenjie; Jiang, Runxia; Yue, Qingcai

Background: In our study, we investigated the expression and function of microRNA-29 in myocardial microvascular endothelial cells (MMEVC) in type 2 diabetic Goto-Kakizaki (GK) rats. Methods: MiR-29 gene expression was compared, by qRT-PCR between diabetic GK rat MMEVC and non-diabetic Wistar rat MMEVC. MiR-29 was downregulated in GK MMEVC and its effect on angiogenic properties of proliferation and migration was examined. Potential downstream target gene of miR-29, insulin growth factor 1 (IGF1), was assessed by dual-luciferase reporter assay, qRT-PCR and western blot in GK MMEVC. IGF1 was also downregulated by siRNA in miR-29-downregulated GK MMEVC. Its effect on miR-29-associated angiogenicmore » regulation on MMEVC proliferation and migration was further investigated. Results: MiR-29 was substantially upregulated in GK MMEVC than in Wistar MMEVC. Transfection of synthetic miR-29 inhibitor successfully downregulate endogenous miR-29 in GK MMEVC, and subsequently promoted angiogenesis by increasing cell proliferation and migration. IGF1 was confirmed to be downstream target gene of miR-29 in GK MMEVC, with its gene and protein expressions both upregulated in miR-29-downregualted GK MMEVC. Conversely, siRNA-mediated IGF1 downregulation reversed the pro-angiogenic effect of miR-29 downregulation in GK MMEVC, as it decreased cell proliferation and migration. Conclusion: Our study suggests that miR-29 downregulation, through its inverse regulation on downstream target of IGF1 gene, is a pro-angiogenic factor in MMEVC in type 2 diabetic rats.« less

Current and Future Trials of Targeted Therapies in Cutaneous Melanoma

PubMed Central

Madhunapantula, SubbaRao V.; Robertson, Gavin P.; Drabick, Joseph J.

2013-01-01

In order to effectively treat melanoma, targeted inhibition of key mechanistic events regulating melanoma development such as cell proliferation, survival, angiogenesis and invasion or metastasis needs to be accomplished. The Mitogen Activated Protein Kinase (MAPK) pathway has been identified as a key player in melanoma development making this cascade an important therapeutic target. However, identification of the ideal pathway member to therapeutically target for maximal clinical benefit remains a challenge. In normal cells, the MAPK pathway relays extracellular signals from the cell membrane to the nucleus via a cascade of phosphorylation events, which promote cancer development. Dysregulation of the MAPK pathway occurs frequently in many human cancers including melanoma. Mutations in the B-RAF and RAS genes, genetic or epigenetic modifications are the key aberrations observed in this signaling cascade. Constitutive activation of this pathway causes oncogenic transformation of cells by promoting cell proliferation, invasion, metastasis, migration, survival and angiogenesis. This review provides an overview of (a) key members of MAPK signaling regulating melanoma development; (b) key proteins which can serve as biomarkers to assess disease progression; (c) the clinical efficacy of various pharmacological agents targeting MAPK pathway; (d) current clinical trials evaluating downstream targets of the MAPK pathway; (e) issues associated with pharmacological agents such as drug resistance, induction of cancers; and finally (e) various strategies overcoming drug resistance. PMID:23288642

Upregulation of microRNA-137 expression by Slug promotes tumor invasion and metastasis of non-small cell lung cancer cells through suppression of TFAP2C.

PubMed

Chang, Tzu-Hua; Tsai, Meng-Feng; Gow, Chien-Hung; Wu, Shang-Gin; Liu, Yi-Nan; Chang, Yih-Leong; Yu, Sung-Liang; Tsai, Hsing-Chen; Lin, Shih-Wen; Chen, Yen-Wei; Kuo, Po-Yen; Yang, Pan-Chyr; Shih, Jin-Yuan

2017-08-28

The epithelial-mesenchymal transition (EMT) regulator, Slug, plays multifaceted roles in controlling lung cancer progression, but its downstream targets and mechanisms in promoting lung cancer progression have not been well defined. In particular, the miRNAs downstream of Slug in non-small cell lung cancer (NSCLC) remain undetermined. Here, we report that miR-137 is downstream of the EMT regulator, Slug, in lung cancer cells. Slug binds directly to the E-box of the miR-137 promoter and up-regulates its expression in lung cancer cells. Knockdown of miR-137 abolished Slug-induced cancer invasion and migration, whereas upregulation of miR-137 was found to trigger lung cancer cell invasion and progression by direct suppressing TFAP2C (transcription factor AP-2 gamma). Clinical data showed that lung adenocarcinoma patients with low-level expression of Slug and miR-137 but high-level expression of TFAP2C experienced significantly better survival. miR-137 is a Slug-induced miRNA that relays the pro-metastatic effects of Slug by targeting TFAP2C. Our findings add new components to the Slug-mediated regulatory network in lung cancer, and suggest that Slug, miR-137, and TFAP2C may be useful prognostic markers in lung adenocarcinoma. Copyright © 2017 Elsevier B.V. All rights reserved.

Brachyury downstream notochord differentiation in the ascidian embryo

PubMed Central

Takahashi, Hiroki; Hotta, Kohji; Erives, Albert; Di Gregorio, Anna; Zeller, Robert W.; Levine, Michael; Satoh, Nori

1999-01-01

The ascidian tadpole represents the most simplified chordate body plan. It contains a notochord composed of just 40 cells, but as in vertebrates Brachyury is essential for notochord differentiation. Here, we show that the misexpression of the Brachyury gene (Ci-Bra) of Ciona intestinalis is sufficient to transform endoderm into notochord. Subtractive hybridization screens were conducted to identify potential Brachyury target genes that are induced upon Ci-Bra misexpression. Of 501 independent cDNA clones that were surveyed, 38 were specifically expressed in notochord cells. These potential Ci-Bra downstream genes appear to encode a broad spectrum of divergent proteins associated with notochord formation. PMID:10385620

miRNA-133 augments coelomocyte phagocytosis in bacteria-challenged Apostichopus japonicus via targeting the TLR component of IRAK-1 in vitro and in vivo

PubMed Central

Lu, Meng; Zhang, Peng-Juan; Li, Cheng-Hua; Lv, Zhi-Meng; Zhang, Wei-Wei; Jin, Chun-Hua

2015-01-01

In this study, we explored the potential roles of miRNA-133 in regulating TLR pathways in the sea cucumber Apostichopus japonicus. Target screening of RNA-Seq data successfully identified interleukin-1 receptor-associated kinase (AjIRAK−1) as a putative target of miR-133. This result was further validated by negative expression profiles in Vibrio splendidus-challenged coelomocytes and lipopolysaccharide (LPS)-exposed cell cultures. HEK-293T cells transfected with a dual-luciferase reporter fused to the 3′UTR of wild-type or mutant AjIRAK-1 exhibited a 52.9% reduction in luciferase activity (p < 0.01) compared to controls. Co-infection with a miR-133 mimics or a specific siRNA targeting AjIRAK-1 significantly repressed the mRNA and protein expression levels of AjIRAK-1 and its downstream molecules, such as AjTRAF6 and Ajp105, in primary coelomocytes. In contrast, a miR-133 inhibitor significantly increased the expression of these TLR pathway members. The injection of miR-133 agomir or AjIRAK-1 siRNA into sea cucumbers not only decreased the expression of AjIRAK-1 and its downstream molecules but also significantly increased V. splendidus coelomocyte phagocytosis. All of the present data provide direct evidence that miR-133 is involved in TLR cascade modulation through AjIRAK-1 targeting to promote V. splendidus coelomocyte phagocytosis in these non-model invertebrates. PMID:26223836

miRNA-133 augments coelomocyte phagocytosis in bacteria-challenged Apostichopus japonicus via targeting the TLR component of IRAK-1 in vitro and in vivo.

PubMed

Lu, Meng; Zhang, Peng-Juan; Li, Cheng-Hua; Lv, Zhi-Meng; Zhang, Wei-Wei; Jin, Chun-Hua

2015-07-30

In this study, we explored the potential roles of miRNA-133 in regulating TLR pathways in the sea cucumber Apostichopus japonicus. Target screening of RNA-Seq data successfully identified interleukin-1 receptor-associated kinase (AjIRAK-1) as a putative target of miR-133. This result was further validated by negative expression profiles in Vibrio splendidus-challenged coelomocytes and lipopolysaccharide (LPS)-exposed cell cultures. HEK-293T cells transfected with a dual-luciferase reporter fused to the 3'UTR of wild-type or mutant AjIRAK-1 exhibited a 52.9% reduction in luciferase activity (p < 0.01) compared to controls. Co-infection with a miR-133 mimics or a specific siRNA targeting AjIRAK-1 significantly repressed the mRNA and protein expression levels of AjIRAK-1 and its downstream molecules, such as AjTRAF6 and Ajp105, in primary coelomocytes. In contrast, a miR-133 inhibitor significantly increased the expression of these TLR pathway members. The injection of miR-133 agomir or AjIRAK-1 siRNA into sea cucumbers not only decreased the expression of AjIRAK-1 and its downstream molecules but also significantly increased V. splendidus coelomocyte phagocytosis. All of the present data provide direct evidence that miR-133 is involved in TLR cascade modulation through AjIRAK-1 targeting to promote V. splendidus coelomocyte phagocytosis in these non-model invertebrates.

Impact of surfactants on the target recognition of Fab-conjugated PLGA nanoparticles.

PubMed

Kennedy, Patrick J; Perreira, Ines; Ferreira, Daniel; Nestor, Marika; Oliveira, Carla; Granja, Pedro L; Sarmento, Bruno

2018-06-01

Targeted drug delivery with nanoparticles (NPs) requires proper surface ligand presentation and availability. Surfactants are often used as stabilizers in the production of targeted NPs. Here, we evaluated the impact of surfactants on ligand functionalization and downstream molecular recognition. Our model system consisted of fluorescent poly(lactic-co-glycolic acid) (PLGA) NPs that were nanoprecipitated in one of a small panel of commonly-used surfactants followed by equivalent washes and conjugation of an engineered Fab antibody fragment. Size, polydispersity index and zeta potential were determined by dynamic light scattering and laser Doppler anemometry, and Fab presence on the NPs was assessed by enzyme-linked immunosorbent assay. Most importantly, Fab-decorated NP binding to the cell surface receptor was monitored by fluorescence-activated cell sorting. 2% polyvinyl alcohol, 1% sodium cholate, 0.5% Pluronic F127 (F127) and 2% Tween-80 were initially tested. Of the four surfactants tested, PLGA NPs in 0.5% F127 and 2% Tween-80 had the highest cell binding. These two surfactants were then retested in two different concentrations, 0.5% and 2%. The Fab-decorated PLGA NPs in 2% F127 had the highest cell binding. This study highlights the impact of common surfactants and their concentrations on the downstream targeting of ligand-decorated NPs. Similar principles should be applied in the development of future targeted nanosystems where surfactants are employed. Copyright © 2018 Elsevier B.V. All rights reserved.

Pharmacogenetics of cystic fibrosis treatment.

PubMed

Carter, Suzanne C; McKone, Edward F

2016-08-01

Cystic fibrosis (CF) is genetic autosomal recessive disease caused by reduced or absent function of CFTR protein. Treatments for patients with CF have primarily focused on the downstream end-organ consequences of defective CFTR. Since the discovery of the CFTR gene that causes CF in 1989 there have been tremendous advances in our understanding of the genetics and pathophysiology of CF. This has recently led to the development of new CFTR mutation-specific targeted therapies for select patients with CF. This review will discuss the characteristics of the CFTR gene, the CFTR mutations that cause CF and the new mutation specific pharmacological treatments including gene therapy that are contributing to the dawning of a new era in cystic fibrosis care.

cAMP signaling in skeletal muscle adaptation: hypertrophy, metabolism, and regeneration

PubMed Central

Stewart, Randi

2012-01-01

Among organ systems, skeletal muscle is perhaps the most structurally specialized. The remarkable subcellular architecture of this tissue allows it to empower movement with instructions from motor neurons. Despite this high degree of specialization, skeletal muscle also has intrinsic signaling mechanisms that allow adaptation to long-term changes in demand and regeneration after acute damage. The second messenger adenosine 3′,5′-monophosphate (cAMP) not only elicits acute changes within myofibers during exercise but also contributes to myofiber size and metabolic phenotype in the long term. Strikingly, sustained activation of cAMP signaling leads to pronounced hypertrophic responses in skeletal myofibers through largely elusive molecular mechanisms. These pathways can promote hypertrophy and combat atrophy in animal models of disorders including muscular dystrophy, age-related atrophy, denervation injury, disuse atrophy, cancer cachexia, and sepsis. cAMP also participates in muscle development and regeneration mediated by muscle precursor cells; thus, downstream signaling pathways may potentially be harnessed to promote muscle regeneration in patients with acute damage or muscular dystrophy. In this review, we summarize studies implicating cAMP signaling in skeletal muscle adaptation. We also highlight ligands that induce cAMP signaling and downstream effectors that are promising pharmacological targets. PMID:22354781

Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

PubMed Central

Lee-Sherick, Alisa B.; Zhang, Weihe; Menachof, Kelly K.; Hill, Amanda A.; Rinella, Sean; Kirkpatrick, Gregory; Page, Lauren S.; Stashko, Michael A.; Jordan, Craig T.; Wei, Qi; Liu, Jing; Zhang, Dehui; DeRyckere, Deborah; Wang, Xiaodong; Frye, Stephen; Earp, H. Shelton; Graham, Douglas K.

2015-01-01

Mer and Flt3 receptor tyrosine kinases have been implicated as therapeutic targets in acute myeloid leukemia (AML). In this manuscript we describe UNC1666, a novel ATP-competitive small molecule tyrosine kinase inhibitor, which potently diminishes Mer and Flt3 phosphorylation in AML. Treatment with UNC1666 mediated biochemical and functional effects in AML cell lines expressing Mer or Flt3 internal tandem duplication (ITD), including decreased phosphorylation of Mer, Flt3 and downstream effectors Stat, Akt and Erk, induction of apoptosis in up to 98% of cells, and reduction of colony formation by greater than 90%, compared to treatment with vehicle. These effects were dose-dependent, with inhibition of downstream signaling and functional effects correlating with the degree of Mer or Flt3 kinase inhibition. Treatment of primary AML patient samples expressing Mer and/or Flt3-ITD with UNC1666 also inhibited Mer and Flt3 intracellular signaling, induced apoptosis, and inhibited colony formation. In summary, UNC1666 is a novel potent small molecule tyrosine kinase inhibitor that decreases oncogenic signaling and myeloblast survival, thereby validating dual Mer/Flt3 inhibition as an attractive treatment strategy for AML. PMID:25762638

Noncanonical control of C. elegans germline apoptosis by the insulin/IGF-1 and Ras/MAPK signaling pathways.

PubMed

Perrin, A J; Gunda, M; Yu, B; Yen, K; Ito, S; Forster, S; Tissenbaum, H A; Derry, W B

2013-01-01

The insulin/IGF-1 pathway controls a number of physiological processes in the nematode worm Caenorhabditis elegans, including development, aging and stress response. We previously found that the Akt/PKB ortholog AKT-1 dampens the apoptotic response to genotoxic stress in the germline by negatively regulating the p53-like transcription factor CEP-1. Here, we report unexpected rearrangements to the insulin/IGF-1 pathway, whereby the insulin-like receptor DAF-2 and 3-phosphoinositide-dependent protein kinase PDK-1 oppose AKT-1 to promote DNA damage-induced apoptosis. While DNA damage does not affect phosphorylation at the PDK-1 site Thr350/Thr308 of AKT-1, it increased phosphorylation at Ser517/Ser473. Although ablation of daf-2 or pdk-1 completely suppressed akt-1-dependent apoptosis, the transcriptional activation of CEP-1 was unaffected, suggesting that daf-2 and pdk-1 act independently or downstream of cep-1 and akt-1. Ablation of the akt-1 paralog akt-2 or the downstream target of the insulin/IGF-1 pathway daf-16 (a FOXO transcription factor) restored sensitivity to damage-induced apoptosis in daf-2 and pdk-1 mutants. In addition, daf-2 and pdk-1 mutants have reduced levels of phospho-MPK-1/ERK in their germ cells, indicating that the insulin/IGF-1 pathway promotes Ras signaling in the germline. Ablation of the Ras effector gla-3, a negative regulator of mpk-1, restored sensitivity to apoptosis in daf-2 mutants, suggesting that gla-3 acts downstream of daf-2. In addition, the hypersensitivity of let-60/Ras gain-of-function mutants to damage-induced apoptosis was suppressed to wild-type levels by ablation of daf-2. Thus, insulin/IGF-1 signaling selectively engages AKT-2/DAF-16 to promote DNA damage-induced germ cell apoptosis downstream of CEP-1 through the Ras pathway.

Transcriptomic-based effects monitoring for endocrine active chemicals: Assessing relative contribution of treated wastewater to downstream pollution

EPA Science Inventory

The present study investigated whether combining of targeted analytical chemistry methods with unsupervised, data-rich methodologies (i.e. transcriptomics) can be utilized to evaluate relative contributions of wastewater treatment plant (WWTP) effluents to biological effects. The...

Characterization and placement of wetlands for integrated watershed conservation practice planning

USDA-ARS?s Scientific Manuscript database

Constructed wetlands have been recognized as an efficient and cost-effective conservation practice to protect water quality through reducing the transport of sediments and nutrients from upstream croplands to downstream water bodies. The challenge resides in targeting the strategic location of wetla...

Rous Sarcoma Virus RNA Stability Element Inhibits Deadenylation of mRNAs with Long 3′UTRs

PubMed Central

Balagopal, Vidya; Beemon, Karen L.

2017-01-01

All retroviruses use their full-length primary transcript as the major mRNA for Group-specific antigen (Gag) capsid proteins. This results in a long 3′ untranslated region (UTR) downstream of the termination codon. In the case of Rous sarcoma virus (RSV), there is a 7 kb 3′UTR downstream of the gag terminator, containing the pol, env, and src genes. mRNAs containing long 3′UTRs, like those with premature termination codons, are frequently recognized by the cellular nonsense-mediated mRNA decay (NMD) machinery and targeted for degradation. To prevent this, RSV has evolved an RNA stability element (RSE) in the RNA immediately downstream of the gag termination codon. This 400-nt RNA sequence stabilizes premature termination codons (PTCs) in gag. It also stabilizes globin mRNAs with long 3′UTRs, when placed downstream of the termination codon. It is not clear how the RSE stabilizes the mRNA and prevents decay. We show here that the presence of RSE inhibits deadenylation severely. In addition, the RSE also impairs decapping (DCP2) and 5′-3′ exonucleolytic (XRN1) function in knockdown experiments in human cells. PMID:28763028

The essential and downstream common proteins of amyotrophic lateral sclerosis: A protein-protein interaction network analysis.

PubMed

Mao, Yimin; Kuo, Su-Wei; Chen, Le; Heckman, C J; Jiang, M C

2017-01-01

Amyotrophic Lateral Sclerosis (ALS) is a devastative neurodegenerative disease characterized by selective loss of motoneurons. While several breakthroughs have been made in identifying ALS genetic defects, the detailed molecular mechanisms are still unclear. These genetic defects involve in numerous biological processes, which converge to a common destiny: motoneuron degeneration. In addition, the common comorbid Frontotemporal Dementia (FTD) further complicates the investigation of ALS etiology. In this study, we aimed to explore the protein-protein interaction network built on known ALS-causative genes to identify essential proteins and common downstream proteins between classical ALS and ALS+FTD (classical ALS + ALS/FTD) groups. The results suggest that classical ALS and ALS+FTD share similar essential protein set (VCP, FUS, TDP-43 and hnRNPA1) but have distinctive functional enrichment profiles. Thus, disruptions to these essential proteins might cause motoneuron susceptible to cellular stresses and eventually vulnerable to proteinopathies. Moreover, we identified a common downstream protein, ubiquitin-C, extensively interconnected with ALS-causative proteins (22 out of 24) which was not linked to ALS previously. Our in silico approach provides the computational background for identifying ALS therapeutic targets, and points out the potential downstream common ground of ALS-causative mutations.

Atox1 Contains Positive Residues That Mediate Membrane Association and Aid Subsequent Copper Loading

PubMed Central

Flores, Adrian G.; Unger, Vinzenz M.

2013-01-01

Copper chaperones bind intracellular copper and ensure proper trafficking to downstream targets via protein-protein interactions. In contrast to the mechanisms of copper binding and transfer to downstream targets, the mechanisms of initial copper loading of the chaperones are largely unknown. Here we demonstrate that antioxidant protein 1 (Atox1 in human cells), the principal cellular copper chaperone responsible for delivery of copper to the secretory pathway, possesses the ability to interact with negatively charged lipid headgroups via distinct surface lysine residues. Moreover, loss of these residues lowers the efficiency of copper loading of Atox1 in vivo, suggesting that the membrane may play a scaffolding role in copper distribution to Atox1. These findings complement the recent discovery that the membrane also facilitates copper loading of the copper chaperone for superoxide dismutase 1 and provide further support for the emerging paradigm that the membrane bilayer plays a central role in cellular copper acquisition and distribution. PMID:24036897

Atox1 contains positive residues that mediate membrane association and aid subsequent copper loading.

PubMed

Flores, Adrian G; Unger, Vinzenz M

2013-12-01

Copper chaperones bind intracellular copper and ensure proper trafficking to downstream targets via protein-protein interactions. In contrast to the mechanisms of copper binding and transfer to downstream targets, the mechanisms of initial copper loading of the chaperones are largely unknown. Here, we demonstrate that antioxidant protein 1 (Atox1 in human cells), the principal cellular copper chaperone responsible for delivery of copper to the secretory pathway, possesses the ability to interact with negatively charged lipid headgroups via distinct surface lysine residues. Moreover, loss of these residues lowers the efficiency of copper loading of Atox1 in vivo, suggesting that the membrane may play a scaffolding role in copper distribution to Atox1. These findings complement the recent discovery that the membrane also facilitates copper loading of the copper chaperone for superoxide dismutase 1 and provide further support for the emerging paradigm that the membrane bilayer plays a central role in cellular copper acquisition and distribution.

Peptide amphiphile nanofiber hydrogel delivery of sonic hedgehog protein to the cavernous nerve to promote regeneration and prevent erectile dysfunction.

PubMed

Choe, Shawn; Bond, Christopher W; Harrington, Daniel A; Stupp, Samuel I; McVary, Kevin T; Podlasek, Carol A

2017-01-01

Erectile dysfunction (ED) has high impact on quality of life in prostatectomy, diabetic and aging patients. An underlying mechanism is cavernous nerve (CN) injury, which causes ED in up to 80% of prostatectomy patients. We examine how sonic hedgehog (SHH) treatment with innovative peptide amphiphile nanofiber hydrogels (PA), promotes CN regeneration after injury. SHH and its receptors patched (PTCH1) and smoothened (SMO) are localized in PG neurons and glia. SMO undergoes anterograde transport to signal to downstream targets. With crush injury, PG neurons degenerate and undergo apoptosis. SHH protein decreases, SMO localization changes to the neuronal cell surface, and anterograde transport stops. With SHH treatment SHH is taken up at the injury site and undergoes retrograde transport to PG neurons, allowing SMO transport to occur, and neurons remain intact. SHH treatment prevents neuronal degeneration, maintains neuronal, glial and downstream target signaling, and is significant as a regenerative therapy. Published by Elsevier Inc.

HAND2 Targets Define a Network of Transcriptional Regulators that Compartmentalize the Early Limb Bud Mesenchyme

DOE PAGES

Osterwalder, Marco; Speziale, Dario; Shoukry, Malak; ...

2014-11-10

The genetic networks that govern vertebrate development are well studied, but how the interactions of trans-acting factors with cis-regulatory modules (CRMs) are integrated into spatiotemporal regulation of gene expression is not clear. The transcriptional regulator HAND2 is required during limb, heart, and branchial arch development. Here, we identify the genomic regions enriched in HAND2 chromatin complexes from mouse embryos and limb buds. Then we analyze the HAND2 target CRMs in the genomic landscapes encoding transcriptional regulators required in early limb buds. HAND2 controls the expression of genes functioning in the proximal limb bud and orchestrates the establishment of anterior andmore » posterior polarity of the nascent limb bud mesenchyme by impacting Gli3 and Tbx3 expression. TBX3 is required downstream of HAND2 to refine the posterior Gli3 expression boundary. In conclusion, our analysis uncovers the transcriptional circuits that function in establishing distinct mesenchymal compartments downstream of HAND2 and upstream of SHH signaling.« less

FANCD2 functions as a critical factor downstream of MiTF to maintain the proliferation and survival of melanoma cells.

PubMed

Bourseguin, Julie; Bonet, Caroline; Renaud, Emilie; Pandiani, Charlotte; Boncompagni, Marina; Giuliano, Sandy; Pawlikowska, Patrycja; Karmous-Benailly, Houda; Ballotti, Robert; Rosselli, Filippo; Bertolotto, Corine

2016-11-09

Proteins involved in genetic stability maintenance and safeguarding DNA replication act not only against cancer initiation but could also play a major role in sustaining cancer progression. Here, we report that the FANC pathway is highly expressed in metastatic melanoma harboring the oncogenic microphthalmia-associated transcription factor (MiTF). We show that MiTF downregulation in melanoma cells lowers the expression of several FANC genes and proteins. Moreover, we observe that, similarly to the consequence of MiTF downregulation, FANC pathway silencing alters proliferation, migration and senescence of human melanoma cells. We demonstrate that the FANC pathway acts downstream MiTF and establish the existence of an epistatic relationship between MiTF and the FANC pathway. Our findings point to a central role of the FANC pathway in cellular and chromosomal resistance to both DNA damage and targeted therapies in melanoma cells. Thus, the FANC pathway is a promising new therapeutic target in melanoma treatment.

FANCD2 functions as a critical factor downstream of MiTF to maintain the proliferation and survival of melanoma cells

PubMed Central

Bourseguin, Julie; Bonet, Caroline; Renaud, Emilie; Pandiani, Charlotte; Boncompagni, Marina; Giuliano, Sandy; Pawlikowska, Patrycja; Karmous-Benailly, Houda; Ballotti, Robert; Rosselli, Filippo; Bertolotto, Corine

2016-01-01

Proteins involved in genetic stability maintenance and safeguarding DNA replication act not only against cancer initiation but could also play a major role in sustaining cancer progression. Here, we report that the FANC pathway is highly expressed in metastatic melanoma harboring the oncogenic microphthalmia-associated transcription factor (MiTF). We show that MiTF downregulation in melanoma cells lowers the expression of several FANC genes and proteins. Moreover, we observe that, similarly to the consequence of MiTF downregulation, FANC pathway silencing alters proliferation, migration and senescence of human melanoma cells. We demonstrate that the FANC pathway acts downstream MiTF and establish the existence of an epistatic relationship between MiTF and the FANC pathway. Our findings point to a central role of the FANC pathway in cellular and chromosomal resistance to both DNA damage and targeted therapies in melanoma cells. Thus, the FANC pathway is a promising new therapeutic target in melanoma treatment. PMID:27827420

Fuel injection assembly for use in turbine engines and method of assembling same

DOEpatents

Berry, Jonathan Dwight; Johnson, Thomas Edward; York, William David; Uhm, Jong Ho

2015-12-15

A fuel injection assembly for use in a turbine engine is provided. The fuel injection assembly includes an end cover, an endcap assembly, a fluid supply chamber, and a plurality of tube assemblies positioned at the endcap assembly. Each of the tube assemblies includes housing having a fuel plenum and a cooling fluid plenum. The cooling fluid plenum is positioned downstream from the fuel plenum and separated from the fuel plenum by an intermediate wall. The plurality of tube assemblies also include a plurality of tubes that extends through the housing. Each of the plurality of tubes is coupled in flow communication with the fluid supply chamber and a combustion chamber positioned downstream from the tube assembly. The plurality of tube assemblies further includes an aft plate at a downstream end of the cooling fluid plenum. The plate includes at least one aperture.

Silencing of Pokemon enhances caspase-dependent apoptosis via fas- and mitochondria-mediated pathways in hepatocellular carcinoma cells.

PubMed

Zhang, Yu-Qin; Xiao, Chuan-Xing; Lin, Bi-Yun; Shi, Ying; Liu, Yun-Peng; Liu, Jing-Jing; Guleng, Bayasi; Ren, Jian-Lin

2013-01-01

The role of Pokemon (POK erythroid myeloid ontogenic actor), a recently identified POK transcription factor with proto-oncogenic activity, in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC) and promotes HCC cell proliferation and migration via an AKT- and ERK- dependent manner. In the present study, we used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induced apoptosis was significantly increased in cells with silenced Pokemon. Western blots showed that p53 expression and phosphorylation were significantly increased in Pokemon defective cells, thereby initiating the mitochondria-mediated and death receptor-mediated apoptotic pathways. In the mitochondria-mediated pathway, expression of pro-apoptotic Bcl-2 family members (including Bad, Bid, Bim and Puma) as well as AIF was increased and decreasing the mitochondrial membrane potential resulted in cytochrome C released from mitochondrial in HepG2 si-Pokemon cells. In addition, upon oxaliplatin treatment of Pokemon-silenced cells, the FAS receptor, FADD and their downstream targets caspase-10 and caspase-8 were activated, causing increased release of caspase-8 active fragments p18 and p10. Increased activated caspase-8-mediated cleavage and activation of downstream effector caspases such as caspase-9 and caspase-3 was observed in HepG2 si-Pokemon cells as compared to control. Therefore, Pokemon might serve as an important mediator of crosstalk between intrinsic and extrinsic apoptotic pathways in HCC cells. Moreover, our findings suggest that Pokemon could be an attractive therapeutic target gene for human cancer therapy.

Silencing of Pokemon Enhances Caspase-Dependent Apoptosis via Fas- and Mitochondria-Mediated Pathways in Hepatocellular Carcinoma Cells

PubMed Central

Lin, Bi-Yun; Shi, Ying; Liu, Yun-Peng; Liu, Jing-Jing; Guleng, Bayasi; Ren, Jian-Lin

2013-01-01

The role of Pokemon (POK erythroid myeloid ontogenic actor), a recently identified POK transcription factor with proto-oncogenic activity, in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC) and promotes HCC cell proliferation and migration via an AKT- and ERK- dependent manner. In the present study, we used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induced apoptosis was significantly increased in cells with silenced Pokemon. Western blots showed that p53 expression and phosphorylation were significantly increased in Pokemon defective cells, thereby initiating the mitochondria-mediated and death receptor-mediated apoptotic pathways. In the mitochondria-mediated pathway, expression of pro-apoptotic Bcl-2 family members (including Bad, Bid, Bim and Puma) as well as AIF was increased and decreasing the mitochondrial membrane potential resulted in cytochrome C released from mitochondrial in HepG2 si-Pokemon cells. In addition, upon oxaliplatin treatment of Pokemon-silenced cells, the FAS receptor, FADD and their downstream targets caspase-10 and caspase-8 were activated, causing increased release of caspase-8 active fragments p18 and p10. Increased activated caspase-8-mediated cleavage and activation of downstream effector caspases such as caspase-9 and caspase-3 was observed in HepG2 si-Pokemon cells as compared to control. Therefore, Pokemon might serve as an important mediator of crosstalk between intrinsic and extrinsic apoptotic pathways in HCC cells. Moreover, our findings suggest that Pokemon could be an attractive therapeutic target gene for human cancer therapy. PMID:23874836

Timing of androgen receptor disruption and estrogen exposure underlies a spectrum of congenital penile anomalies

PubMed Central

Armfield, Brooke A.; Cohn, Martin J.

2015-01-01

Congenital penile anomalies (CPAs) are among the most common human birth defects. Reports of CPAs, which include hypospadias, chordee, micropenis, and ambiguous genitalia, have risen sharply in recent decades, but the causes of these malformations are rarely identified. Both genetic anomalies and environmental factors, such as antiandrogenic and estrogenic endocrine disrupting chemicals (EDCs), are suspected to cause CPAs; however, little is known about the temporal window(s) of sensitivity to EDCs, or the tissue-specific roles and downstream targets of the androgen receptor (AR) in external genitalia. Here, we show that the full spectrum of CPAs can be produced by disrupting AR at different developmental stages and in specific cell types in the mouse genital tubercle. Inactivation of AR during a narrow window of prenatal development results in hypospadias and chordee, whereas earlier disruptions cause ambiguous genitalia and later disruptions cause micropenis. The neonatal phase of penile development is controlled by the balance of AR to estrogen receptor α (ERα) activity; either inhibition of androgen or augmentation of estrogen signaling can induce micropenis. AR and ERα have opposite effects on cell division, apoptosis, and regulation of Hedgehog, fibroblast growth factor, bone morphogenetic protein, and Wnt signaling in the genital tubercle. We identify Indian hedgehog (Ihh) as a novel downstream target of AR in external genitalia and show that conditional deletion of Ihh inhibits penile masculinization. These studies reveal previously unidentified cellular and molecular mechanisms by which antiandrogenic and estrogenic signals induce penile malformations and demonstrate that the timing of endocrine disruption can determine the type of CPA. PMID:26598695

Peripheral noxious stimulation reduces withdrawal threshold to mechanical stimuli after spinal cord injury: Role of tumor necrosis factor alpha and apoptosis

PubMed Central

Woller, Sarah A.; Huie, J. Russell; Hartman, John J.; Hook, Michelle A.; Miranda, Rajesh C.; Huang, Yung-Jen; Ferguson, Adam R.; Grau, James W.

2014-01-01

We previously showed that peripheral noxious input after spinal cord injury (SCI) inhibits beneficial spinal plasticity and impairs recovery of locomotor and bladder functions. These observations suggest that noxious input may similarly affect the development and maintenance of chronic neuropathic pain, an important consequence of SCI. In adult rats with a moderate contusion SCI, we investigated the effect of noxious tail stimulation, administered one day after SCI, on mechanical withdrawal responses to von Frey stimuli from 1 to 28 days, post-treatment. In addition, because the pro-inflammatory cytokine tumor necrosis factor α (TNFα) is implicated in numerous injury-induced processes including pain hypersensitivity, we assessed the temporal and spatial expression of TNFα, TNF receptors, and several downstream signaling targets after stimulation. Our results showed that unlike sham surgery or SCI only, nociceptive stimulation following SCI induced mechanical sensitivity by 24 hours. These behavioral changes were accompanied by increased expression of TNFα. Cellular assessments of downstream targets of TNFα revealed that nociceptive stimulation increased the expression of caspase 8 and the active subunit (12 kDa) of caspase 3 at a time point consistent with the onset of mechanical allodynia, indicative of active apoptosis. In addition, immunohistochemical analysis revealed distinct morphological signs of apoptosis in neurons and microglia at 24 hours post-stimulation. Interestingly, expression of the inflammatory mediator NFκB was unaltered by nociceptive stimulation. These results suggest that noxious input caudal to the level of SCI can increase the onset and expression of behavioral responses indicative of pain, potentially involving TNFα signaling. PMID:25180012

Insulin-mediated signaling promotes proliferation and survival of glioblastoma through Akt activation

PubMed Central

Gong, Yuanying; Ma, Yufang; Sinyuk, Maksim; Loganathan, Sudan; Thompson, Reid C.; Sarkaria, Jann N.; Chen, Wenbiao; Lathia, Justin D.; Mobley, Bret C.; Clark, Stephen W.; Wang, Jialiang

2016-01-01

Background Metabolic complications such as obesity, hyperglycemia, and type 2 diabetes are associated with poor outcomes in patients with glioblastoma. To control peritumoral edema, use of chronic high-dose steroids in glioblastoma patients is common, which can result in de novo diabetic symptoms. These metabolic complications may affect tumors via profound mechanisms, including activation of insulin receptor (InsR) and the related insulin-like growth factor 1 receptor (IGF1R) in malignant cells. Methods In the present study, we assessed expression of InsR in glioblastoma surgical specimens and glioblastoma response to insulin at physiologically relevant concentrations. We further determined whether genetic or pharmacological targeting of InsR affected oncogenic functions of glioblastoma in vitro and in vivo. Results We showed that InsR was commonly expressed in glioblastoma surgical specimens and xenograft tumor lines, with mitogenic isoform-A predominating. Insulin at physiologically relevant concentrations promoted glioblastoma cell growth and survival, potentially via Akt activation. Depletion of InsR impaired cellular functions and repressed orthotopic tumor growth. The absence of InsR compromised downstream Akt activity, but yet stimulated IGF1R expression. Targeting both InsR and IGF1R with dual kinase inhibitors resulted in effective blockade of downstream signaling, loss of cell viability, and repression of xenograft tumor growth. Conclusions Taken together, our work suggests that glioblastoma is sensitive to the mitogenic functions of insulin, thus significant insulin exposure imposes risks to glioblastoma patients. Additionally, dual inhibition of InsR and IGF1R exhibits promise for treating glioblastoma. PMID:26136493

Toxicity assessment of sediments collected upstream and downstream from the White Dam in Clarke County, Georgia

USGS Publications Warehouse

Lasier, Peter J.

2018-06-06

The White Dam in Clarke County, Georgia, has been proposed for breaching. Efforts to determine potential risks to downstream biota included assessments of sediment collected in the vicinity of the dam. Sediments collected from sites upstream and downstream from the dam were evaluated for toxicity in 42-day exposures using the freshwater amphipod Hyalella azteca. Endpoints of the study were survival, growth, and reproduction of H. azteca. Results indicated no significant differences between the collected sediments and the water-only treatment used for comparison of the test endpoints. Therefore, based on the laboratory experiments in this study, sediment migration downstream from a breach of the Dam may not pose a toxicity risk to downstream biota.

Combination of a proteomics approach and reengineering of meso scale network models for prediction of mode-of-action for tyrosine kinase inhibitors.

PubMed

Balabanov, Stefan; Wilhelm, Thomas; Venz, Simone; Keller, Gunhild; Scharf, Christian; Pospisil, Heike; Braig, Melanie; Barett, Christine; Bokemeyer, Carsten; Walther, Reinhard; Brümmendorf, Tim H; Schuppert, Andreas

2013-01-01

In drug discovery, the characterisation of the precise modes of action (MoA) and of unwanted off-target effects of novel molecularly targeted compounds is of highest relevance. Recent approaches for identification of MoA have employed various techniques for modeling of well defined signaling pathways including structural information, changes in phenotypic behavior of cells and gene expression patterns after drug treatment. However, efficient approaches focusing on proteome wide data for the identification of MoA including interference with mutations are underrepresented. As mutations are key drivers of drug resistance in molecularly targeted tumor therapies, efficient analysis and modeling of downstream effects of mutations on drug MoA is a key to efficient development of improved targeted anti-cancer drugs. Here we present a combination of a global proteome analysis, reengineering of network models and integration of apoptosis data used to infer the mode-of-action of various tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) cell lines expressing wild type as well as TKI resistance conferring mutants of BCR-ABL. The inferred network models provide a tool to predict the main MoA of drugs as well as to grouping of drugs with known similar kinase inhibitory activity patterns in comparison to drugs with an additional MoA. We believe that our direct network reconstruction approach, demonstrated on proteomics data, can provide a complementary method to the established network reconstruction approaches for the preclinical modeling of the MoA of various types of targeted drugs in cancer treatment. Hence it may contribute to the more precise prediction of clinically relevant on- and off-target effects of TKIs.

Combination of a Proteomics Approach and Reengineering of Meso Scale Network Models for Prediction of Mode-of-Action for Tyrosine Kinase Inhibitors

PubMed Central

Balabanov, Stefan; Wilhelm, Thomas; Venz, Simone; Keller, Gunhild; Scharf, Christian; Pospisil, Heike; Braig, Melanie; Barett, Christine; Bokemeyer, Carsten; Walther, Reinhard

2013-01-01

In drug discovery, the characterisation of the precise modes of action (MoA) and of unwanted off-target effects of novel molecularly targeted compounds is of highest relevance. Recent approaches for identification of MoA have employed various techniques for modeling of well defined signaling pathways including structural information, changes in phenotypic behavior of cells and gene expression patterns after drug treatment. However, efficient approaches focusing on proteome wide data for the identification of MoA including interference with mutations are underrepresented. As mutations are key drivers of drug resistance in molecularly targeted tumor therapies, efficient analysis and modeling of downstream effects of mutations on drug MoA is a key to efficient development of improved targeted anti-cancer drugs. Here we present a combination of a global proteome analysis, reengineering of network models and integration of apoptosis data used to infer the mode-of-action of various tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) cell lines expressing wild type as well as TKI resistance conferring mutants of BCR-ABL. The inferred network models provide a tool to predict the main MoA of drugs as well as to grouping of drugs with known similar kinase inhibitory activity patterns in comparison to drugs with an additional MoA. We believe that our direct network reconstruction approach, demonstrated on proteomics data, can provide a complementary method to the established network reconstruction approaches for the preclinical modeling of the MoA of various types of targeted drugs in cancer treatment. Hence it may contribute to the more precise prediction of clinically relevant on- and off-target effects of TKIs. PMID:23326482

XCR1 promotes cell growth and migration and is correlated with bone metastasis in non-small cell lung cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Ting; Han, Shuai; Wu, Zhipeng

Bone metastasis occurs in approximately 30–40% patients with advanced non-small cell lung cancer (NSCLC), but the mechanism underlying this bone metastasis remains poorly understood. The chemokine super family is believed to play an important role in tumor metastasis in lung cancer. The chemokine receptor XCR1 has been identified to promote cell proliferation and migration in oral cancer and ovarian carcinoma, but the role of XCR1 in lung cancer has not been reported. In this study, we demonstrated for the first time that XCR1 was overexpressed in lung cancer bone metastasis as compared with that in patients with primary lung cancer.more » In addition, the XCR1 ligand XCL1 promoted the proliferation and migration of lung cancer cells markedly, and knockdown of XCR1 by siRNA abolished the effect of XCL1 in cell proliferation and migration. Furthermore, we identified JAK2/STAT3 as a novel downstream pathway of XCR1, while XCL1/XCR1 increased the mRNA level of the downstream of JAK2/STAT3 including PIM1, JunB, TTP, MMP2 and MMP9. These results indicate that XCR1 is a new potential therapeutic target for the treatment of lung cancer bone metastasis. - Highlights: • XCR1 is overexpressed in bone metastasis compared with primary NSCLC. • XCR1 activation by XCL1 promotes lung cancer cell proliferation and migration. • JAK2/STAT3 is a novel potential downstream pathway of XCR1.« less

Drosophila CHIP protects against mitochondrial dysfunction by acting downstream of Pink1 in parallel with Parkin.

PubMed

Chen, Jia; Xue, Jin; Ruan, Jingsong; Zhao, Juan; Tang, Beisha; Duan, Ranhui

2017-12-01

Mitochondrial kinase PTEN-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase Parkin function in a common pathway to regulate mitochondrial homeostasis contributing to the pathogenesis of Parkinson disease. The carboxyl terminus of Hsc70-interacting protein (CHIP) acts as a heat shock protein 70/heat shock protein 90 cochaperone to mediate protein folding or as an E3 ubiquitin ligase to target proteins for degradation. In this study, overexpression of Drosophila CHIP suppressed a range of Pink1 mutant phenotypes in flies, including abnormal wing posture, thoracic indentation, locomotion defects, muscle degeneration, and loss of dopaminergic neurons. Mitochondrial defects of Pink1 mutant, such as excessive fusion, reduced ATP content, and crista disorganization, were rescued by CHIP but not its ligase-dead mutants. Similar phenotypes and mitochondrial impairment were ameliorated in Parkin mutant flies by wild-type CHIP. Inactivation of CHIP with null fly mutants resulted in mitochondrial defects, such as reduced thoracic ATP content at 3 d old, decreased thoracic mitochondrial DNA content, and defective mitochondrial morphology at 60 d old. CHIP mutants did not exacerbate the phenotypes of Pink1 mutant flies but markedly shortened the life span of Parkin mutant flies. These results indicate that CHIP is involved in mitochondrial integrity and may act downstream of Pink1 in parallel with Parkin.-Chen, J., Xue, J., Ruan, J., Zhao, J., Tang, B., Duan, R. Drosophila CHIP protects against mitochondrial dysfunction by acting downstream of Pink1 in parallel with Parkin. © FASEB.

Analysis of water use strategies of the desert riparian forest plant community in inland rivers of two arid regions in northwestern China

NASA Astrophysics Data System (ADS)

Chen, Y. N.; Li, W. H.; Zhou, H. H.; Chen, Y. P.; Hao, X. M.; Fu, A. H.; Ma, J. X.

2014-10-01

Studies of the water use of the desert riparian forest plant community in arid regions and analyses of the response and adaptive strategies of plants to environmental stress are of great significance to the formulation of effective ecological conservation and restoration strategies. Taking two inland rivers in the arid regions of northwestern China, downstream of the Tarim River and Heihe River Basin as the research target regions, this paper explored the stem water potential, sap flow, root hydraulic lift, and characteristics of plant water sources of the major constructive species in the desert riparian forest, Populus euphratica and Tamarix ramosissima. Specifically, this was accomplished by combining the monitoring of field physiological and ecological indicators, and the analysis of laboratory tests. Then, the water use differences of species in different ecological environments and their ecological significance were analyzed. This study indicated that: (1) in terms of water sources, Populus euphratica and Tamarix ramosissima mainly used deep subsoil water and underground water, but the plant root system in the downstream of the Tarim River was more diversified than that in the downstream of the Heihe River in water absorption, (2) in terms of water distribution, Populus euphratica root possessed hydraulic lift capacity, but Populus euphratica root in the downstream of the Tarim River presented stronger hydraulic lift capacity and more significant ecological effect of water redistribution, (3) in terms of water transport, the plants in the downstream of the Heihe River can adapt to the environment through the current limiting of branch xylem, while plants in the downstream of the Tarim River substantially increased the survival probability of the whole plant by sacrificing weak branches and improving the water acquisition capacity of dominant branches; and (4) in terms of water dissipation, the water use and consumption of Populus euphratica at night exhibited no significant difference, but the water use and consumption of Populus euphratica in the downstream of the Tarim River in the day was significantly higher than that in the downstream of the Heihe River, and the essential reason for this is the groundwater depth. The ecology in the downstream of the Heihe River has been in balance in the maintenance and development stage, while desert riparian forest plants in the downstream of the Tarim River are still in severe arid stress.

Influence of Wastewater Discharge on the Metabolic Potential of the Microbial Community in River Sediments.

PubMed

Li, Dong; Sharp, Jonathan O; Drewes, Jörg E

2016-01-01

To reveal the variation of microbial community functions during water filtration process in river sediments, which has been utilized widely in natural water treatment systems, this study investigates the influence of municipal wastewater discharge to streams on the phylotype and metabolic potential of the microbiome in upstream and particularly various depths of downstream river sediments. Cluster analyses based on both microbial phylogenetic and functional data collectively revealed that shallow upstream sediments grouped with those from deeper subsurface downstream regions. These sediment samples were distinct from those found in shallow downstream sediments. Functional genes associated with carbohydrate, xenobiotic, and certain amino acid metabolisms were overrepresented in upstream and deep downstream samples. In contrast, the more immediate contact with wastewater discharge in shallow downstream samples resulted in an increase in the relative abundance of genes associated with nitrogen, sulfur, purine and pyrimidine metabolisms, as well as restriction-modification systems. More diverse bacterial phyla were associated with upstream and deep downstream sediments, mainly including Actinobacteria, Planctomycetes, and Firmicutes. In contrast, in shallow downstream sediments, genera affiliated with Betaproteobacteria and Gammaproteobacteria were enriched with putative functions that included ammonia and sulfur oxidation, polyphosphate accumulation, and methylotrophic bacteria. Collectively, these results highlight the enhanced capabilities of microbial communities residing in deeper stream sediments for the transformation of water contaminants and thus provide a foundation for better design of natural water treatment systems to further improve the removal of contaminants.

Slp-76 is a critical determinant of NK-cell mediated recognition of missing-self targets.

PubMed

Lampe, Kristin; Endale, Mehari; Cashman, Siobhan; Fang, Hao; Mattner, Jochen; Hildeman, David; Hoebe, Kasper

2015-07-01

Absence of MHC class I expression is an important mechanism by which NK cells recognize a variety of target cells, yet the pathways underlying "missing-self" recognition, including the involvement of activating receptors, remain poorly understood. Using ethyl-N-nitrosourea mutagenesis in mice, we identified a germline mutant, designated Ace, with a marked defect in NK cell mediated recognition and elimination of "missing-self" targets. The causative mutation was linked to chromosome 11 and identified as a missense mutation (Thr428Ile) in the SH2 domain of Slp-76-a critical adapter molecule downstream of ITAM-containing surface receptors. The Slp-76 Ace mutation behaved as a hypomorphic allele-while no major defects were observed in conventional T-cell development/function, a marked defect in NK cell mediated elimination of β2-microglobulin (β2M) deficient target cells was observed. Further studies revealed Slp-76 to control NK-cell receptor expression and maturation; however, activation of Slp-76(ace/ace) NK cells through ITAM-containing NK-cell receptors or allogeneic/tumor target cells appeared largely unaffected. Imagestream analysis of the NK-β2M(-/-) target cell synapse revealed a specific defect in actin recruitment to the conjugate synapse in Slp-76(ace/ace) NK cells. Overall these studies establish Slp-76 as a critical determinant of NK-cell development and NK cell mediated elimination of missing-self target cells in mice. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Slp-76 is a critical determinant of NK cell-mediated recognition of missing-self targets

PubMed Central

Lampe, Kristin; Endale, Mehari; Cashman, Siobhan; Fang, Hao; Mattner, Jochen; Hildeman, David; Hoebe, Kasper

2015-01-01

Absence of MHC class I expression is an important mechanism by which NK cells recognize a variety of target cells, yet the pathways underlying “missing-self” recognition, including the involvement of activating receptors, remain poorly understood. Using ENU mutagenesis in mice, we identified a germline mutant, designated Ace, with a marked defect in NK cell-mediated recognition and elimination of “missing-self” targets. The causative mutation was linked to chromosome 11 and identified as a missense mutation [Thr428Ile] in the SH2 domain of Slp-76—a critical adapter molecule downstream of ITAM-containing surface receptors. The Slp-76 Ace mutation behaved as a hypomorphic allele—while no major defects were observed in conventional T cell development/function, a marked defect in NK cell-mediated elimination of β2-Microglobulin (β2M)-deficient target cells was observed. Further studies revealed Slp-76 to control NK cell receptor expression and maturation, however, activation of Slp-76ace/ace NK cells through ITAM-containing NK cell receptors or allogeneic/tumor target cells appeared largely unaffected. Imagestream analysis of the NK-β2M−/− target cell synapse, revealed a specific defect in actin recruitment to the conjugate synapse in Slp-76ace/ace NK cells. Overall these studies establish Slp-76 as a critical determinant of NK cell development and NK cell-mediated elimination of missing-self target cells. PMID:25929249

Targeting BTK through microRNA in chronic lymphocytic leukemia

PubMed Central

Bottoni, Arianna; Rizzotto, Lara; Lai, Tzung-Huei; Liu, Chaomei; Smith, Lisa L.; Mantel, Rose; Reiff, Sean; El-Gamal, Dalia; Larkin, Karilyn; Johnson, Amy J.; Lapalombella, Rosa; Lehman, Amy; Plunkett, William; Byrd, John C.; Blachly, James S.; Woyach, Jennifer A.

2016-01-01

Bruton’s tyrosine kinase (BTK) is a critical mediator of survival in B-cell neoplasms. Although BTK inhibitors have transformed therapy in chronic lymphocytic leukemia (CLL), patients with high-risk genetics are at risk for relapse and have a poor prognosis. Identification of novel therapeutic strategies for this group of patients is an urgent unmet clinical need, and therapies that target BTK via alternative mechanisms may fill this niche. Herein, we identify a set of microRNAs (miRs) that target BTK in primary CLL cells and show that the histone deacetylase (HDAC) repressor complex is recruited to these miR promoters to silence their expression. Targeting the HDACs by using either RNA interference against HDAC1 in CLL or a small molecule inhibitor (HDACi) in CLL and mantle cell lymphoma restored the expression of the BTK-targeting miRs with loss of BTK protein and downstream signaling and consequent cell death. We have also made the novel and clinically relevant discovery that inhibition of HDAC induces the BTK-targeting miRs in ibrutinib-sensitive and resistant CLL to effectively reduce both wild-type and C481S-mutant BTK. This finding identifies a novel strategy that may be promising as a therapeutic modality to eliminate the C481S-mutant BTK clone that drives resistance to ibrutinib and provides the rationale for a combination strategy that includes ibrutinib to dually target BTK to suppress its prosurvival signaling. PMID:27756747

Targeting BTK through microRNA in chronic lymphocytic leukemia.

PubMed

Bottoni, Arianna; Rizzotto, Lara; Lai, Tzung-Huei; Liu, Chaomei; Smith, Lisa L; Mantel, Rose; Reiff, Sean; El-Gamal, Dalia; Larkin, Karilyn; Johnson, Amy J; Lapalombella, Rosa; Lehman, Amy; Plunkett, William; Byrd, John C; Blachly, James S; Woyach, Jennifer A; Sampath, Deepa

2016-12-29

Bruton's tyrosine kinase (BTK) is a critical mediator of survival in B-cell neoplasms. Although BTK inhibitors have transformed therapy in chronic lymphocytic leukemia (CLL), patients with high-risk genetics are at risk for relapse and have a poor prognosis. Identification of novel therapeutic strategies for this group of patients is an urgent unmet clinical need, and therapies that target BTK via alternative mechanisms may fill this niche. Herein, we identify a set of microRNAs (miRs) that target BTK in primary CLL cells and show that the histone deacetylase (HDAC) repressor complex is recruited to these miR promoters to silence their expression. Targeting the HDACs by using either RNA interference against HDAC1 in CLL or a small molecule inhibitor (HDACi) in CLL and mantle cell lymphoma restored the expression of the BTK-targeting miRs with loss of BTK protein and downstream signaling and consequent cell death. We have also made the novel and clinically relevant discovery that inhibition of HDAC induces the BTK-targeting miRs in ibrutinib-sensitive and resistant CLL to effectively reduce both wild-type and C481S-mutant BTK. This finding identifies a novel strategy that may be promising as a therapeutic modality to eliminate the C481S-mutant BTK clone that drives resistance to ibrutinib and provides the rationale for a combination strategy that includes ibrutinib to dually target BTK to suppress its prosurvival signaling. © 2016 by The American Society of Hematology.

System and method for reducing combustion dynamics in a combustor

DOEpatents

Uhm, Jong Ho; Johnson, Thomas Edward; Zuo, Baifang; York, William David

2013-08-20

A system for reducing combustion dynamics in a combustor includes an end cap having an upstream surface axially separated from a downstream surface, and tube bundles extend through the end cap. A diluent supply in fluid communication with the end cap provides diluent flow to the end cap. Diluent distributors circumferentially arranged inside at least one tube bundle extend downstream from the downstream surface and provide fluid communication for the diluent flow through the end cap. A method for reducing combustion dynamics in a combustor includes flowing fuel through tube bundles that extend axially through an end cap, flowing a diluent through diluent distributors into a combustion chamber, wherein the diluent distributors are circumferentially arranged inside at least one tube bundle and each diluent distributor extends downstream from the end cap, and forming a diluent barrier in the combustion chamber between at least one pair of adjacent tube bundles.

Enhancement of the anti-tumor activity of FGFR1 inhibition in squamous cell lung cancer by targeting downstream signaling involved in glucose metabolism

PubMed Central

Fumarola, Claudia; Cretella, Daniele; La Monica, Silvia; Bonelli, Mara A.; Alfieri, Roberta; Caffarra, Cristina; Quaini, Federico; Madeddu, Denise; Falco, Angela; Cavazzoni, Andrea; Digiacomo, Graziana; Mazzaschi, Giulia; Vivo, Valentina; Barocelli, Elisabetta; Tiseo, Marcello; Petronini, Pier Giorgio; Ardizzoni, Andrea

2017-01-01

Fibroblast Growth Factor Receptor (FGFR) signaling is a complex pathway which controls several processes, including cell proliferation, survival, migration, and metabolism. FGFR1 signaling is frequently deregulated via amplification/over-expression in NSCLC of squamous histotype (SQCLC), however its inhibition has not been successfully translated in clinical setting. We determined whether targeting downstream signaling implicated in FGFR1 effects on glucose metabolism potentiates the anti-tumor activity of FGFR1 inhibition in SQCLC. In FGFR1 amplified/over-expressing SQCLC cell lines, FGF2-mediated stimulation of FGFR1 under serum-deprivation activated both MAPK and AKT/mTOR pathways and increased glucose uptake, glycolysis, and lactate production, through AKT/mTOR-dependent HIF-1α accumulation and up-regulation of GLUT-1 glucose transporter. These effects were hindered by PD173074 and NVP-BGJ398, selective FGFR inhibitors, as well as by dovitinib, a multi-kinase inhibitor. Glucose metabolism was hampered by the FGFR inhibitors also under hypoxic conditions, with consequent inhibition of cell proliferation and viability. In presence of serum, glucose metabolism was impaired only in cell models in which FGFR1 inhibition was associated with AKT/mTOR down-regulation. When the activation of the AKT/mTOR pathway persisted despite FGFR1 down-regulation, the efficacy of NVP-BGJ398 could be significantly improved by the combination with NVP-BEZ235 or other inhibitors of this signaling cascade, both in vitro and in xenotransplanted nude mice. Collectively our results indicate that inhibition of FGFR1 signaling impacts on cancer cell growth also by affecting glucose energy metabolism. In addition, this study strongly suggests that the therapeutic efficacy of FGFR1 targeting molecules in SQCLC may be implemented by combined treatments tackling on glucose metabolism. PMID:29190880

Twist1 Transcriptional Targets in the Developing Atrio-Ventricular Canal of the Mouse

PubMed Central

Vrljicak, Pavle; Cullum, Rebecca; Xu, Eric; Chang, Alex C. Y.; Wederell, Elizabeth D.; Bilenky, Mikhail; Jones, Steven J. M.; Marra, Marco A.; Karsan, Aly; Hoodless, Pamela A.

2012-01-01

Malformations of the cardiovascular system are the most common type of birth defect in humans, frequently affecting the formation of valves and septa. During heart valve and septa formation, cells from the atrio-ventricular canal (AVC) and outflow tract (OFT) regions of the heart undergo an epithelial-to-mesenchymal transformation (EMT) and invade the underlying extracellular matrix to give rise to endocardial cushions. Subsequent maturation of newly formed mesenchyme cells leads to thin stress-resistant leaflets. TWIST1 is a basic helix-loop-helix transcription factor expressed in newly formed mesenchyme cells of the AVC and OFT that has been shown to play roles in cell survival, cell proliferation and differentiation. However, the downstream targets of TWIST1 during heart valve formation remain unclear. To identify genes important for heart valve development downstream of TWIST1, we performed global gene expression profiling of AVC, OFT, atria and ventricles of the embryonic day 10.5 mouse heart by tag-sequencing (Tag-seq). Using this resource we identified a novel set of 939 genes, including 123 regulators of transcription, enriched in the valve forming regions of the heart. We compared these genes to a Tag-seq library from the Twist1 null developing valves revealing significant gene expression changes. These changes were consistent with a role of TWIST1 in controlling differentiation of mesenchymal cells following their transformation from endothelium in the mouse. To study the role of TWIST1 at the DNA level we performed chromatin immunoprecipitation and identified novel direct targets of TWIST1 in the developing heart valves. Our findings support a role for TWIST1 in the differentiation of AVC mesenchyme post-EMT in the mouse, and suggest that TWIST1 can exert its function by direct DNA binding to activate valve specific gene expression. PMID:22815831

Supply Chain Modeling for Fluorspar and Hydroflouric Acid and Implications for Further Analyses

DTIC Science & Technology

2015-04-01

Critical Materials, Volume 1 Chapter 2. Fluorspar-HF Supply Chain 4 Foreign Supply Other usesUS Supply Fluorspar Mining HF Production Downstream...analysis are listed across the top: Fluorspar Mining , HF Production, and (pro- duction of) Downstream Products (using HF). • U.S. supply is represented by...material flows from fluorspar mining , to HF production, to downstream fluorine-containing products. – Black lines are material flows included in the supply

Pharmacology of cognitive enhancers for exposure-based therapy of fear, anxiety and trauma-related disorders

PubMed Central

Singewald, N.; Schmuckermair, C.; Whittle, N.; Holmes, A.; Ressler, K.J.

2015-01-01

Pathological fear and anxiety are highly debilitating and, despite considerable advances in psychotherapy and pharmacotherapy they remain insufficiently treated in many patients with PTSD, phobias, panic and other anxiety disorders. Increasing preclinical and clinical evidence indicates that pharmacological treatments including cognitive enhancers, when given as adjuncts to psychotherapeutic approaches [cognitive behavioral therapy including extinction-based exposure therapy] enhance treatment efficacy, while using anxiolytics such as benzodiazepines as adjuncts can undermine long-term treatment success. The purpose of this review is to outline the literature showing how pharmacological interventions targeting neurotransmitter systems including serotonin, dopamine, noradrenaline, histamine, glutamate, GABA, cannabinoids, neuropeptides (oxytocin, neuropeptides Y and S, opioids) and other targets (neurotrophins BDNF and FGF2, glucocorticoids, L-type-calcium channels, epigenetic modifications) as well as their downstream signaling pathways, can augment fear extinction and strengthen extinction memory persistently in preclinical models. Particularly promising approaches are discussed in regard to their effects on specific aspects of fear extinction namely, acquisition, consolidation and retrieval, including long-term protection from return of fear (relapse) phenomena like spontaneous recovery, reinstatement and renewal of fear. We also highlight the promising translational value of the preclinial research and the clinical potential of targeting certain neurochemical systems with, for example d-cycloserine, yohimbine, cortisol, and L-DOPA. The current body of research reveals important new insights into the neurobiology and neurochemistry of fear extinction and holds significant promise for pharmacologically-augmented psychotherapy as an improved approach to treat trauma and anxiety-related disorders in a more efficient and persistent way promoting enhanced symptom remission and recovery. PMID:25550231

MicroRNA expression and protein acetylation pattern in respiratory and limb muscles of Parp-1(-/-) and Parp-2(-/-) mice with lung cancer cachexia.

PubMed

Chacon-Cabrera, Alba; Fermoselle, Clara; Salmela, Ida; Yelamos, Jose; Barreiro, Esther

2015-12-01

Current treatment options for cachexia, which impairs disease prognosis, are limited. Muscle-enriched microRNAs and protein acetylation are involved in muscle wasting including lung cancer (LC) cachexia. Poly(ADP-ribose) polymerases (PARP) are involved in muscle metabolism. We hypothesized that muscle-enriched microRNA, protein hyperacetylation, and expression levels of myogenic transcription factors (MTFs) and downstream targets, muscle loss and function improve in LC cachectic Parp-1(−/−) and Parp-2(−/−) mice. Body and muscle weights, grip strength, muscle phenotype, muscle-enriched microRNAs (miR-1, -133, -206, and -486), protein acetylation, acetylated levels of FoxO1, FoxO3, and PGC-1α, histone deacetylases (HDACs) including SIRT1, MTFs, and downstream targets (α-actin, PGC-1α, and creatine kinase) were evaluated in diaphragm and gastrocnemius of LC (LP07 adenocarcinoma) wild type (WT), Parp-1(−/−) and Parp-2−/− mice. Compared to WT cachectic animals, in both respiratory and limb muscles of Parp-1(−/−) and Parp-2(−/−) cachectic mice: downregulation of muscle-specific microRNAs was counterbalanced especially in gastrocnemius of Parp-1(−/−) mice; increased protein acetylation was attenuated (improvement in HDAC3, SIRT-1, and acetylated FoxO3 levels in both muscles, acetylated FoxO1 levels in the diaphragm); reduced MTFs and creatine kinase levels were mitigated; body and muscle weights, strength, and muscle fiber sizes improved, while tumor weight and growth decreased. These molecular findings may explain the improvements seen in body and muscle weights, limb muscle force and fiber sizes in both Parp-1(−/−) and Parp-2(−/−) cachectic mice. PARP-1 and -2 play a role in cancer-induced cachexia, thus selective pharmacological inhibition of PARP-1 and -2 may be of interest in clinical settings. Copyright © 2015 Elsevier B.V. All rights reserved.

Chromatin-Specific Regulation of Mammalian rDNA Transcription by Clustered TTF-I Binding Sites

PubMed Central

Diermeier, Sarah D.; Németh, Attila; Rehli, Michael; Grummt, Ingrid; Längst, Gernot

2013-01-01

Enhancers and promoters often contain multiple binding sites for the same transcription factor, suggesting that homotypic clustering of binding sites may serve a role in transcription regulation. Here we show that clustering of binding sites for the transcription termination factor TTF-I downstream of the pre-rRNA coding region specifies transcription termination, increases the efficiency of transcription initiation and affects the three-dimensional structure of rRNA genes. On chromatin templates, but not on free rDNA, clustered binding sites promote cooperative binding of TTF-I, loading TTF-I to the downstream terminators before it binds to the rDNA promoter. Interaction of TTF-I with target sites upstream and downstream of the rDNA transcription unit connects these distal DNA elements by forming a chromatin loop between the rDNA promoter and the terminators. The results imply that clustered binding sites increase the binding affinity of transcription factors in chromatin, thus influencing the timing and strength of DNA-dependent processes. PMID:24068958

Msx genes are important apoptosis effectors downstream of the Shh/Gli3 pathway in the limb.

PubMed

Lallemand, Yvan; Bensoussan, Vardina; Cloment, Cécile Saint; Robert, Benoît

2009-07-15

In tetrapods, the anteroposterior (AP) patterning of the limb is under the control of the antagonistic activities of the secreted factor Sonic hedgehog (Shh) and Gli3R, the truncated repressor form of the transcription factor Gli3. In this report, we show that Msx1 and Msx2 are targets and downstream effectors of Gli3R. Consequently, in Shh null mutants, Msx genes are overexpressed and, furthermore, partially responsible for the limb phenotype. This is exemplified by the fact that reducing Msx activity in Shh mutants partially restores a normal limb development. Finally, we show that the main action of the Msx genes, in both normal and Shh(-/-) limb development, is to control cell death in the mesenchyme. We propose that, in the limb, Msx genes act downstream of the Shh/Gli3 pathway by transducing BMP signaling and that, in the absence of Shh signaling, their deregulation contributes to the extensive apoptosis that impairs limb development.

Consumption‐Based Conservation Targeting: Linking Biodiversity Loss to Upstream Demand through a Global Wildlife Footprint

PubMed Central

Berlow, Eric; Conlisk, Erin; Erb, Karlheinz; Iha, Katsunori; Martinez, Neo; Newman, Erica A.; Plutzar, Christoph; Smith, Adam B.; Harte, John

2016-01-01

Abstract Although most conservation efforts address the direct, local causes of biodiversity loss, effective long‐term conservation will require complementary efforts to reduce the upstream economic pressures, such as demands for food and forest products, which ultimately drive these downstream losses. Here, we present a wildlife footprint analysis that links global losses of wild birds to consumer purchases across 57 economic sectors in 129 regions. The United States, India, China, and Brazil have the largest regional wildlife footprints, while per‐person footprints are highest in Mongolia, Australia, Botswana, and the United Arab Emirates. A US$100 purchase of bovine meat or rice products occupies approximately 0.1 km2 of wild bird ranges, displacing 1–2 individual birds, for 1 year. Globally significant importer regions, including Japan, the United Kingdom, Germany, Italy, and France, have large footprints that drive wildlife losses elsewhere in the world and represent important targets for consumption‐focused conservation attention. PMID:29104616

Chemical Genetics Reveals an RGS/G-Protein Role in the Action of a Compound

PubMed Central

Fitzgerald, Kevin; Tertyshnikova, Svetlana; Moore, Lisa; Bjerke, Lynn; Burley, Ben; Cao, Jian; Carroll, Pamela; Choy, Robert; Doberstein, Steve; Dubaquie, Yves; Franke, Yvonne; Kopczynski, Jenny; Korswagen, Hendrik; Krystek, Stanley R; Lodge, Nicholas J; Plasterk, Ronald; Starrett, John; Stouch, Terry; Thalody, George; Wayne, Honey; van der Linden, Alexander; Zhang, Yongmei; Walker, Stephen G; Cockett, Mark; Wardwell-Swanson, Judi; Ross-Macdonald, Petra; Kindt, Rachel M

2006-01-01

We report here on a chemical genetic screen designed to address the mechanism of action of a small molecule. Small molecules that were active in models of urinary incontinence were tested on the nematode Caenorhabditis elegans, and the resulting phenotypes were used as readouts in a genetic screen to identify possible molecular targets. The mutations giving resistance to compound were found to affect members of the RGS protein/G-protein complex. Studies in mammalian systems confirmed that the small molecules inhibit muscarinic G-protein coupled receptor (GPCR) signaling involving G-αq (G-protein alpha subunit). Our studies suggest that the small molecules act at the level of the RGS/G-αq signaling complex, and define new mutations in both RGS and G-αq, including a unique hypo-adapation allele of G-αq. These findings suggest that therapeutics targeted to downstream components of GPCR signaling may be effective for treatment of diseases involving inappropriate receptor activation. PMID:16683034

Scientific evidence and rationale for the development of curcumin and resveratrol as nutraceutricals for joint health.

PubMed

Mobasheri, Ali; Henrotin, Yves; Biesalski, Hans-Konrad; Shakibaei, Mehdi

2012-01-01

Interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) are key cytokines that drive the production of inflammatory mediators and matrix-degrading enzymes in osteoarthritis (OA). These proinflammatory cytokines bind to their respective cell surface receptors and activate inflammatory signaling pathways culminating with the activation of nuclear factor κB (NF-κB), a transcription factor that can be triggered by a host of stress-related stimuli including, excessive mechanical stress and ECM degradation products. Once activated, NF-κB regulates the expression of many cytokines, chemokines, adhesion molecules, inflammatory mediators, and several matrix-degrading enzymes. Therefore, proinflammatory cytokines, their cell surface receptors, NF-κB and downstream signaling pathways are therapeutic targets in OA. This paper critically reviews the recent literature and outlines the potential prophylactic properties of plant-derived phytochemicals such as curcumin and resveratrol for targeting NF-κB signaling and inflammation in OA to determine whether these phytochemicals can be used as functional foods.

RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators

PubMed Central

Redfern, Andrew D.; Colley, Shane M.; Beveridge, Dianne J.; Ikeda, Naoya; Epis, Michael R.; Li, Xia; Foulds, Charles E.; Stuart, Lisa M.; Barker, Andrew; Russell, Victoria J.; Ramsay, Kerry; Kobelke, Simon J.; Li, Xiaotao; Hatchell, Esme C.; Payne, Christine; Giles, Keith M.; Messineo, Adriana; Gatignol, Anne; Lanz, Rainer B.; O’Malley, Bert W.; Leedman, Peter J.

2013-01-01

The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing. PMID:23550157

Influenza A virus targets a cGAS-independent STING pathway that controls enveloped RNA viruses.

PubMed

Holm, Christian K; Rahbek, Stine H; Gad, Hans Henrik; Bak, Rasmus O; Jakobsen, Martin R; Jiang, Zhaozaho; Hansen, Anne Louise; Jensen, Simon K; Sun, Chenglong; Thomsen, Martin K; Laustsen, Anders; Nielsen, Camilla G; Severinsen, Kasper; Xiong, Yingluo; Burdette, Dara L; Hornung, Veit; Lebbink, Robert Jan; Duch, Mogens; Fitzgerald, Katherine A; Bahrami, Shervin; Mikkelsen, Jakob Giehm; Hartmann, Rune; Paludan, Søren R

2016-02-19

Stimulator of interferon genes (STING) is known be involved in control of DNA viruses but has an unexplored role in control of RNA viruses. During infection with DNA viruses STING is activated downstream of cGAMP synthase (cGAS) to induce type I interferon. Here we identify a STING-dependent, cGAS-independent pathway important for full interferon production and antiviral control of enveloped RNA viruses, including influenza A virus (IAV). Further, IAV interacts with STING through its conserved hemagglutinin fusion peptide (FP). Interestingly, FP antagonizes interferon production induced by membrane fusion or IAV but not by cGAMP or DNA. Similar to the enveloped RNA viruses, membrane fusion stimulates interferon production in a STING-dependent but cGAS-independent manner. Abolishment of this pathway led to reduced interferon production and impaired control of enveloped RNA viruses. Thus, enveloped RNA viruses stimulate a cGAS-independent STING pathway, which is targeted by IAV.

The PDZ domain of the guanine nucleotide exchange factor PDZGEF directs binding to phosphatidic acid during brush border formation.

PubMed

Consonni, Sarah V; Brouwer, Patricia M; van Slobbe, Eleonora S; Bos, Johannes L

2014-01-01

PDZGEF is a guanine nucleotide exchange factor for the small G protein Rap. It was recently found that PDZGEF contributes to establishment of intestinal epithelial polarity downstream of the kinase Lkb1. By binding to phosphatidic acid enriched at the apical membrane, PDZGEF locally activates Rap2a resulting in induction of brush border formation via a pathway that includes the polarity players TNIK, Mst4 and Ezrin. Here we show that the PDZ domain of PDZGEF is essential and sufficient for targeting PDZGEF to the apical membrane of polarized intestinal epithelial cells. Inhibition of PLD and consequently production of phosphatidic acid inhibitis targeting of PDZGEF to the plasma membrane. Furthermore, localization requires specific positively charged residues within the PDZ domain. We conclude that local accumulation of PDZGEF at the apical membrane during establishment of epithelial polarity is mediated by electrostatic interactions between positively charged side chains in the PDZ domain and negatively charged phosphatidic acid.

The PDZ Domain of the Guanine Nucleotide Exchange Factor PDZGEF Directs Binding to Phosphatidic Acid during Brush Border Formation

PubMed Central

Consonni, Sarah V.; Brouwer, Patricia M.; van Slobbe, Eleonora S.; Bos, Johannes L.

2014-01-01

PDZGEF is a guanine nucleotide exchange factor for the small G protein Rap. It was recently found that PDZGEF contributes to establishment of intestinal epithelial polarity downstream of the kinase Lkb1. By binding to phosphatidic acid enriched at the apical membrane, PDZGEF locally activates Rap2a resulting in induction of brush border formation via a pathway that includes the polarity players TNIK, Mst4 and Ezrin. Here we show that the PDZ domain of PDZGEF is essential and sufficient for targeting PDZGEF to the apical membrane of polarized intestinal epithelial cells. Inhibition of PLD and consequently production of phosphatidic acid inhibitis targeting of PDZGEF to the plasma membrane. Furthermore, localization requires specific positively charged residues within the PDZ domain. We conclude that local accumulation of PDZGEF at the apical membrane during establishment of epithelial polarity is mediated by electrostatic interactions between positively charged side chains in the PDZ domain and negatively charged phosphatidic acid. PMID:24858808

RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators.

PubMed

Redfern, Andrew D; Colley, Shane M; Beveridge, Dianne J; Ikeda, Naoya; Epis, Michael R; Li, Xia; Foulds, Charles E; Stuart, Lisa M; Barker, Andrew; Russell, Victoria J; Ramsay, Kerry; Kobelke, Simon J; Li, Xiaotao; Hatchell, Esme C; Payne, Christine; Giles, Keith M; Messineo, Adriana; Gatignol, Anne; Lanz, Rainer B; O'Malley, Bert W; Leedman, Peter J

2013-04-16

The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing.

Stereotyped responses of Drosophila peptidergic neuronal ensemble depend on downstream neuromodulators

PubMed Central

Mena, Wilson; Diegelmann, Sören; Wegener, Christian; Ewer, John

2016-01-01

Neuropeptides play a key role in the regulation of behaviors and physiological responses including alertness, social recognition, and hunger, yet, their mechanism of action is poorly understood. Here, we focus on the endocrine control ecdysis behavior, which is used by arthropods to shed their cuticle at the end of every molt. Ecdysis is triggered by ETH (Ecdysis triggering hormone), and we show that the response of peptidergic neurons that produce CCAP (crustacean cardioactive peptide), which are key targets of ETH and control the onset of ecdysis behavior, depends fundamentally on the actions of neuropeptides produced by other direct targets of ETH and released in a broad paracrine manner within the CNS; by autocrine influences from the CCAP neurons themselves; and by inhibitory actions mediated by GABA. Our findings provide insights into how this critical insect behavior is controlled and general principles for understanding how neuropeptides organize neuronal activity and behaviors. DOI: http://dx.doi.org/10.7554/eLife.19686.001 PMID:27976997

Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.

PubMed

Kayagaki, Nobuhiko; Stowe, Irma B; Lee, Bettina L; O'Rourke, Karen; Anderson, Keith; Warming, Søren; Cuellar, Trinna; Haley, Benjamin; Roose-Girma, Merone; Phung, Qui T; Liu, Peter S; Lill, Jennie R; Li, Hong; Wu, Jiansheng; Kummerfeld, Sarah; Zhang, Juan; Lee, Wyne P; Snipas, Scott J; Salvesen, Guy S; Morris, Lucy X; Fitzgerald, Linda; Zhang, Yafei; Bertram, Edward M; Goodnow, Christopher C; Dixit, Vishva M

2015-10-29

Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1β processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1β maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd(-/-) mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1β secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd(-/-) mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.

acn-1, a C. elegans homologue of ACE, genetically interacts with the let-7 microRNA and other heterochronic genes.

PubMed

Metheetrairut, Chanatip; Ahuja, Yuri; Slack, Frank J

2017-10-02

The heterochronic pathway in C. elegans controls the relative timing of cell fate decisions during post-embryonic development. It includes a network of microRNAs (miRNAs), such as let-7, and protein-coding genes, such as the stemness factors, LIN-28 and LIN-41. Here we identified the acn-1 gene, a homologue of mammalian angiotensin-converting enzyme (ACE), as a new suppressor of the stem cell developmental defects of let-7 mutants. Since acn-1 null mutants die during early larval development, we used RNAi to characterize the role of acn-1 in C. elegans seam cell development, and determined its interaction with heterochronic factors, including let-7 and its downstream interactors - lin-41, hbl-1, and apl-1. We demonstrate that although RNAi knockdown of acn-1 is insufficient to cause heterochronic defects on its own, loss of acn-1 suppresses the retarded phenotypes of let-7 mutants and enhances the precocious phenotypes of hbl-1, though not lin-41, mutants. Conversely, the pattern of acn-1 expression, which oscillates during larval development, is disrupted by lin-41 mutants but not by hbl-1 mutants. Finally, we show that acn-1(RNAi) enhances the let-7-suppressing phenotypes caused by loss of apl-1, a homologue of the Alzheimer's disease-causing amyloid precursor protein (APP), while significantly disrupting the expression of apl-1 during the L4 larval stage. In conclusion, acn-1 interacts with heterochronic genes and appears to function downstream of let-7 and its target genes, including lin-41 and apl-1.

Diversity in migratory patterns among Neotropical fishes in a highly regulated river basin.

PubMed

Makrakis, M C; Miranda, L E; Makrakis, S; Fontes Júnior, H M; Morlis, W G; Dias, J H P; Garcia, J O

2012-07-01

Migratory behaviour of selected fish species is described in the Paraná River, Brazil-Argentina-Paraguay, to search for patterns relevant to tropical regulated river systems. In a 10 year mark-recapture study, spanning a 1425 km section of the river, 32 867 fishes composed of 18 species were released and 1083 fishes were recaptured. The fishes recaptured were at liberty an average 166 days (maximum 1548 days) and travelled an average 35 km (range 0-625 km). Cluster analysis applied to variables descriptive of movement behaviour identified four general movement patterns. Cluster 1 included species that moved long distances (mean 164 km) upstream (54%) and downstream (40%) the mainstem river and showed high incidence (27%) of passage through dams; cluster 2 also exhibited high rate of movement along the mainstem (49% upstream, 13% downstream), but moved small distances (mean 10 km); cluster 3 included the most fishes moving laterally into tributaries (45%) or not moving at all (25%), but little downstream movement (8%); fishes in cluster 4 exhibited little upstream movement (13%) and farthest downstream movements (mean 41 km). Whereas species could be numerically clustered with statistical models, a species ordination showed ample spread, suggesting that species exhibit diverse movement patterns that cannot be easily classified into just a few classes. The cluster and ordination procedures also showed that adults and juveniles of the same species exhibit similar movement patterns. Conventional concepts about Neotropical migratory fishes portray them as travelling long distances upstream. The present results broaden these concepts suggesting that migratory movements are more diverse, could be long, short or at times absent, upriver, downriver or lateral, and the diversity of movements can vary within and among species. The intense lateral migrations exhibited by a diversity of species, especially to and from large tributaries (above reservoirs) and reservoir tributaries, illustrate the importance of these habitats for the fish species life cycle. Considering that the Paraná River is highly impounded, special attention should be given to the few remaining low-impact habitats as they continue to be targets of hydropower development that will probably intensify the effects on migratory fish stocks. © 2012 The Authors. Journal of Fish Biology © 2012 The Fisheries Society of the British Isles.

Dimpled/grooved face on a fuel injection nozzle body for flame stabilization and related method

DOEpatents

Uhm, Jong Ho; Johnson, Thomas Edward; Kim, Kwanwoo; Zuo, Baifang

2013-08-20

A fuel injection head for a fuel nozzle used in a gas turbine combustor includes a substantially hollow body formed with an upstream end face, a downstream end face and a peripheral wall extending therebetween. A plurality of pre-mix tubes or passages extend axially through the hollow body with inlets at the upstream end face and outlets at the downstream end face. An exterior surface of the downstream end face is formed with three-dimensional surface features that increase a total surface area of the exterior surface as compared to a substantially flat, planar downstream end face.

Emergent Rules for Codon Choice Elucidated by Editing Rare Arginine Codons in Escherichia coli

DTIC Science & Technology

2016-09-20

alternative codons are more likely to be viable. To evaluate synonymous and nonsynonymous alternatives to essential AGRs further, we imple- mented a CRISPR ... Crispr -assisted MAGE). First, we designed oligos that changed not only the target AGR codon to NNN but also made several synonymous changes at least 50...nt downstream that would disrupt a 20-bp CRISPR target lo- cus. MAGE was used to replace each AGR with NNN in parallel, and CRISPR /cas9 was used to

Dana-Farber Cancer Institute: Identification of Therapeutic Targets Across Cancer Types | Office of Cancer Genomics

Cancer.gov

The Dana Farber Cancer Institute CTD2 Center focuses on the use of high-throughput genetic and bioinformatic approaches to identify and credential oncogenes and co-dependencies in cancers. This Center aims to provide the cancer research community with information that will facilitate the prioritization of targets based on both genomic and functional evidence, inform the most appropriate genetic context for downstream mechanistic and validation studies, and enable the translation of this information into therapeutics and diagnostics.

Does Targeted, Disease-Specific Public Research Funding Influence Pharmaceutical Innovation?

ERIC Educational Resources Information Center

Blume-Kohout, Margaret E.

2012-01-01

Public funding for biomedical research is often justified as a means to encourage development of more (and better) treatments for disease. However, few studies have investigated the relationship between these expenditures and downstream pharmaceutical innovation. In particular, although recent analyses have shown a clear contribution of federally…

Yorkie and Scalloped: partners in growth activation.

PubMed

Bandura, Jennifer L; Edgar, Bruce A

2008-03-01

The Hippo (Hpo) signaling pathway limits organ growth in organisms from Drosophila to mammals by suppressing the activity of the transcriptional coactivator Yorkie (Yki)/YAP. The TEAD/TEF factor Scalloped (Sd) has been identified as the first known transcription factor to partner with Yki as a downstream target of Hpo signaling.

Conservative site-specific and single-copy transgenesis in human LINE-1 elements

PubMed Central

Vijaya Chandra, Shree Harsha; Makhija, Harshyaa; Peter, Sabrina; Myint Wai, Cho Mar; Li, Jinming; Zhu, Jindong; Ren, Zhonglu; D'Alcontres, Martina Stagno; Siau, Jia Wei; Chee, Sharon; Ghadessy, Farid John; Dröge, Peter

2016-01-01

Genome engineering of human cells plays an important role in biotechnology and molecular medicine. In particular, insertions of functional multi-transgene cassettes into suitable endogenous sequences will lead to novel applications. Although several tools have been exploited in this context, safety issues such as cytotoxicity, insertional mutagenesis and off-target cleavage together with limitations in cargo size/expression often compromise utility. Phage λ integrase (Int) is a transgenesis tool that mediates conservative site-specific integration of 48 kb DNA into a safe harbor site of the bacterial genome. Here, we show that an Int variant precisely recombines large episomes into a sequence, termed attH4X, found in 1000 human Long INterspersed Elements-1 (LINE-1). We demonstrate single-copy transgenesis through attH4X-targeting in various cell lines including hESCs, with the flexibility of selecting clones according to transgene performance and downstream applications. This is exemplified with pluripotency reporter cassettes and constitutively expressed payloads that remain functional in LINE1-targeted hESCs and differentiated progenies. Furthermore, LINE-1 targeting does not induce DNA damage-response or chromosomal aberrations, and neither global nor localized endogenous gene expression is substantially affected. Hence, this simple transgene addition tool should become particularly useful for applications that require engineering of the human genome with multi-transgenes. PMID:26673710

Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins.

PubMed

Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka

2016-04-01

The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for various biological applications, including genome editing. We developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR to isolate target genomic regions from cells for their biochemical characterization. In this study, we developed 'in vitro enChIP' using recombinant CRISPR ribonucleoproteins (RNPs) to isolate target genomic regions. in vitro enChIP has the great advantage over conventional enChIP of not requiring expression of CRISPR complexes in cells. We first showed that in vitro enChIP using recombinant CRISPR RNPs can be used to isolate target DNA from mixtures of purified DNA in a sequence-specific manner. In addition, we showed that this technology can be used to efficiently isolate target genomic regions, while retaining their intracellular molecular interactions, with negligible contamination from irrelevant genomic regions. Thus, in vitro enChIP technology is of potential use for sequence-specific isolation of DNA, as well as for identification of molecules interacting with genomic regions of interest in vivo in combination with downstream analysis. © 2016 The Authors. Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Depletion of polycistronic transcripts using short interfering RNAs: cDNA synthesis method affects levels of non-targeted genes determined by quantitative PCR.

PubMed

Hanning, Jennifer E; Groves, Ian J; Pett, Mark R; Coleman, Nicholas

2013-05-21

Short interfering RNAs (siRNAs) are often used to deplete viral polycistronic transcripts, such as those encoded by human papillomavirus (HPV). There are conflicting data in the literature concerning how siRNAs targeting one HPV gene can affect levels of other genes in the polycistronic transcripts. We hypothesised that the conflict might be partly explained by the method of cDNA synthesis used prior to transcript quantification. We treated HPV16-positive cervical keratinocytes with siRNAs targeting the HPV16 E7 gene and used quantitative PCR to compare transcript levels of E7 with those of E6 and E2, viral genes located upstream and downstream of the target site respectively. We compared our findings from cDNA generated using oligo-dT primers alone with those from cDNA generated using a combination of random hexamer and oligo-dT primers. Our data show that when polycistronic transcripts are targeted by siRNAs, there is a period when untranslatable cleaved mRNA upstream of the siRNA binding site remains detectable by PCR, if cDNA is generated using random hexamer primers. Such false indications of mRNA abundance are avoided using oligo-dT primers. The period corresponds to the time taken for siRNA activity and degradation of the cleaved transcripts. Genes downstream of the siRNA binding site are detectable during this interval, regardless of how the cDNA is generated. These data emphasise the importance of the cDNA synthesis method used when measuring transcript abundance following siRNA depletion of polycistronic transcripts. They provide a partial explanation for erroneous reports suggesting that siRNAs targeting HPV E7 can have gene-specific effects.

Depletion of polycistronic transcripts using short interfering RNAs: cDNA synthesis method affects levels of non-targeted genes determined by quantitative PCR

PubMed Central

2013-01-01

Background Short interfering RNAs (siRNAs) are often used to deplete viral polycistronic transcripts, such as those encoded by human papillomavirus (HPV). There are conflicting data in the literature concerning how siRNAs targeting one HPV gene can affect levels of other genes in the polycistronic transcripts. We hypothesised that the conflict might be partly explained by the method of cDNA synthesis used prior to transcript quantification. Findings We treated HPV16-positive cervical keratinocytes with siRNAs targeting the HPV16 E7 gene and used quantitative PCR to compare transcript levels of E7 with those of E6 and E2, viral genes located upstream and downstream of the target site respectively. We compared our findings from cDNA generated using oligo-dT primers alone with those from cDNA generated using a combination of random hexamer and oligo-dT primers. Our data show that when polycistronic transcripts are targeted by siRNAs, there is a period when untranslatable cleaved mRNA upstream of the siRNA binding site remains detectable by PCR, if cDNA is generated using random hexamer primers. Such false indications of mRNA abundance are avoided using oligo-dT primers. The period corresponds to the time taken for siRNA activity and degradation of the cleaved transcripts. Genes downstream of the siRNA binding site are detectable during this interval, regardless of how the cDNA is generated. Conclusions These data emphasise the importance of the cDNA synthesis method used when measuring transcript abundance following siRNA depletion of polycistronic transcripts. They provide a partial explanation for erroneous reports suggesting that siRNAs targeting HPV E7 can have gene-specific effects. PMID:23693071

A New Strategy to Control and Eradicate "Undruggable" Oncogenic K-RAS-Driven Pancreatic Cancer: Molecular Insights and Core Principles Learned from Developmental and Evolutionary Biology.

PubMed

Van Sciver, Robert E; Lee, Michael P; Lee, Caroline Dasom; Lafever, Alex C; Svyatova, Elizaveta; Kanda, Kevin; Colliver, Amber L; Siewertsz van Reesema, Lauren L; Tang-Tan, Angela M; Zheleva, Vasilena; Bwayi, Monicah N; Bian, Minglei; Schmidt, Rebecca L; Matrisian, Lynn M; Petersen, Gloria M; Tang, Amy H

2018-05-14

Oncogenic K-RAS mutations are found in virtually all pancreatic cancers, making K-RAS one of the most targeted oncoproteins for drug development in cancer therapies. Despite intense research efforts over the past three decades, oncogenic K-RAS has remained largely "undruggable". Rather than targeting an upstream component of the RAS signaling pathway (i.e., EGFR/HER2) and/or the midstream effector kinases (i.e., RAF/MEK/ERK/PI3K/mTOR), we propose an alternative strategy to control oncogenic K-RAS signal by targeting its most downstream signaling module, Seven-In-Absentia Homolog (SIAH). SIAH E3 ligase controls the signal output of oncogenic K-RAS hyperactivation that drives unchecked cell proliferation, uncontrolled tumor growth, and rapid cancer cell dissemination in human pancreatic cancer. Therefore, SIAH is an ideal therapeutic target as it is an extraordinarily conserved downstream signaling gatekeeper indispensable for proper RAS signaling. Guided by molecular insights and core principles obtained from developmental and evolutionary biology, we propose an anti-SIAH-centered anti-K-RAS strategy as a logical and alternative anticancer strategy to dampen uncontrolled K-RAS hyperactivation and halt tumor growth and metastasis in pancreatic cancer. The clinical utility of developing SIAH as both a tumor-specific and therapy-responsive biomarker, as well as a viable anti-K-RAS drug target, is logically simple and conceptually innovative. SIAH clearly constitutes a major tumor vulnerability and K-RAS signaling bottleneck in pancreatic ductal adenocarcinoma (PDAC). Given the high degree of evolutionary conservation in the K-RAS/SIAH signaling pathway, an anti-SIAH-based anti-PDAC therapy will synergize with covalent K-RAS inhibitors and direct K-RAS targeted initiatives to control and eradicate pancreatic cancer in the future.

Potential signaling pathways as therapeutic targets for overcoming chemoresistance in mucinous ovarian cancer

PubMed Central

Niiro, Emiko; Morioka, Sachiko; Iwai, Kana; Yamada, Yuki; Ogawa, Kenji; Kawahara, Naoki; Kobayashi, Hiroshi

2018-01-01

Cases of mucinous ovarian cancer are predominantly resistant to chemotherapies. The present review summarizes current knowledge of the therapeutic potential of targeting the Wingless (WNT) pathway, with particular emphasis on preclinical and clinical studies, for improving the chemoresistance and treatment of mucinous ovarian cancer. A review was conducted of English language literature published between January 2000 and October 2017 that concerned potential signaling pathways associated with the chemoresistance of mucinous ovarian cancer. The literature indicated that aberrant activation of growth factor and WNT signaling pathways is specifically observed in mucinous ovarian cancer. An evolutionarily conserved signaling cascade system including epidermal growth factor/RAS/RAF/mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase, phosphoinositide 3-kinase/Akt and WNT signaling regulates a variety of cellular functions; their crosstalk mutually enhances signaling activity and induces chemoresistance. Novel antagonists, modulators and inhibitors have been developed for targeting the components of the WNT signaling pathway, namely Frizzled, low-density lipoprotein receptor-related protein 5/6, Dishevelled, casein kinase 1, AXIN, glycogen synthase kinase 3β and β-catenin. Targeted inhibition of WNT signaling represents a rational and promising novel approach to overcome chemoresistance, and several WNT inhibitors are being evaluated in preclinical studies. In conclusion, the WNT receptors and their downstream components may serve as novel therapeutic targets for overcoming chemoresistance in mucinous ovarian cancer. PMID:29564122

Effects of high salinity wastewater discharges on unionid mussels in the Allegheny River, Pennsylvania

USGS Publications Warehouse

Kathleen Patnode,; Hittle, Elizabeth A.; Robert Anderson,; Lora Zimmerman,; Fulton, John W.

2015-01-01

We examined the effect of high salinity wastewater (brine) from oil and natural gas drilling on freshwater mussels in the Allegheny River, Pennsylvania, during 2012. Mussel cages (N = 5 per site) were deployed at two sites upstream and four sites downstream of a brine treatment facility on the Allegheny River. Each cage contained 20 juvenile northern riffleshell mussels Epioblasma torulosa rangiana). Continuous specific conductance and temperature data were recorded by water quality probes deployed at each site. To measure the amount of mixing throughout the entire study area, specific conductance surveys were completed two times during low-flow conditions along transects from bank to bank that targeted upstream (reference) reaches, a municipal wastewater treatment plant discharge upstream of the brine-facility discharge, the brine facility, and downstream reaches. Specific conductance data indicated that high specific conductance water from the brine facility (4,000–12,000 µS/cm; mean 7,846) compared to the reference reach (103–188 µS/cm; mean 151) is carried along the left descending bank of the river and that dilution of the discharge via mixing does not occur until 0.5 mi (805 m) downstream. Juvenile northern riffleshell mussel survival was severely impaired within the high specific conductance zone (2 and 34% at and downstream of the brine facility, respectively) and at the municipal wastewater treatment plant (21%) compared to background (84%). We surveyed native mussels (family Unionidae) at 10 transects: 3 upstream, 3 within, and 4 downstream of the high specific conductance zone. Unionid mussel abundance and diversity were lower for all transects within and downstream of the high conductivity zone compared to upstream. The results of this study clearly demonstrate in situ toxicity to juvenile northern riffleshell mussels, a federally endangered species, and to the native unionid mussel assemblage located downstream of a brine discharge to the Allegheny River.

Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma

PubMed Central

Venkatesh, Humsa S.; Tam, Lydia T.; Woo, Pamelyn J.; Lennon, James; Nagaraja, Surya; Gillespie, Shawn M.; Ni, Jing; Duveau, Damien Y.; Morris, Patrick J.; Zhao, Jean J.; Thomas, Craig J.; Monje, Michelle

2017-01-01

Summary High-grade gliomas (HGG) are a devastating group of cancers, representing the leading cause of brain tumor-related death in both children and adults. Therapies aimed at mechanisms intrinsic to the glioma cell have translated to only limited success; effective therapeutic strategies will need to also target elements of the tumor microenvironment that promote glioma progression. We recently demonstrated that neuronal activity robustly promotes the growth of a range of molecularly and clinically distinct HGG types, including adult glioblastoma (GBM), anaplastic oligodendroglioma, pediatric GBM, and diffuse intrinsic pontine glioma (DIPG)1. An important mechanism mediating this neural regulation of brain cancer is activity-dependent cleavage and secretion of the synaptic molecule neuroligin-3 (NLGN3), which promotes glioma proliferation through the PI3K-mTOR pathway1. However, neuroligin-3 necessity to glioma growth, proteolytic mechanism of secretion and further molecular consequences in glioma remain to be clarified. Here, we demonstrate a striking dependence of HGG growth on microenvironmental neuroligin-3, elucidate signaling cascades downstream of neuroligin-3 binding in glioma and determine a therapeutically targetable mechanism of secretion. Patient-derived orthotopic xenografts of pediatric GBM, DIPG and adult GBM fail to grow in Nlgn3 knockout mice. Neuroligin-3 stimulates numerous oncogenic pathways, including early focal adhesion kinase activation upstream of PI3K-mTOR, and induces transcriptional changes including upregulation of numerous synapse-related genes in glioma cells. Neuroligin-3 is cleaved from both neurons and oligodendrocyte precursor cells via the ADAM10 sheddase. ADAM10 inhibitors prevent release of neuroligin-3 into the tumor microenvironment and robustly block HGG xenograft growth. This work defines a promising strategy for targeting neuroligin-3 secretion, which could prove transformative for HGG therapy. PMID:28959975

Molecular basis for the action of a dietary flavonoid revealed by the comprehensive identification of apigenin human targets

PubMed Central

Arango, Daniel; Morohashi, Kengo; Yilmaz, Alper; Kuramochi, Kouji; Parihar, Arti; Brahimaj, Bledi; Grotewold, Erich; Doseff, Andrea I.

2013-01-01

Flavonoids constitute the largest class of dietary phytochemicals, adding essential health value to our diet, and are emerging as key nutraceuticals. Cellular targets for dietary phytochemicals remain largely unknown, posing significant challenges for the regulation of dietary supplements and the understanding of how nutraceuticals provide health value. Here, we describe the identification of human cellular targets of apigenin, a flavonoid abundantly present in fruits and vegetables, using an innovative high-throughput approach that combines phage display with second generation sequencing. The 160 identified high-confidence candidate apigenin targets are significantly enriched in three main functional categories: GTPase activation, membrane transport, and mRNA metabolism/alternative splicing. This last category includes the heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2), a factor involved in splicing regulation, mRNA stability, and mRNA transport. Apigenin binds to the C-terminal glycine-rich domain of hnRNPA2, preventing hnRNPA2 from forming homodimers, and therefore, it perturbs the alternative splicing of several human hnRNPA2 targets. Our results provide a framework to understand how dietary phytochemicals exert their actions by binding to many functionally diverse cellular targets. In turn, some of them may modulate the activity of a large number of downstream genes, which is exemplified here by the effects of apigenin on the alternative splicing activity of hnRNPA2. Hence, in contrast to small-molecule pharmaceuticals designed for defined target specificity, dietary phytochemicals affect a large number of cellular targets with varied affinities that, combined, result in their recognized health benefits. PMID:23697369

Bruton tyrosine kinase represents a promising therapeutic target for treatment of chronic lymphocytic leukemia and is effectively targeted by PCI-32765

PubMed Central

Herman, Sarah E. M.; Gordon, Amber L.; Hertlein, Erin; Ramanunni, Asha; Zhang, Xiaoli; Jaglowski, Samantha; Flynn, Joseph; Jones, Jeffrey; Blum, Kristie A.; Buggy, Joseph J.; Hamdy, Ahmed

2011-01-01

B-cell receptor (BCR) signaling is aberrantly activated in chronic lymphocytic leukemia (CLL). Bruton tyrosine kinase (BTK) is essential to BCR signaling and in knockout mouse models its mutation has a relatively B cell–specific phenotype. Herein, we demonstrate that BTK protein and mRNA are significantly over expressed in CLL compared with normal B cells. Although BTK is not always constitutively active in CLL cells, BCR or CD40 signaling is accompanied by effective activation of this pathway. Using the irreversible BTK inhibitor PCI-32765, we demonstrate modest apoptosis in CLL cells that is greater than that observed in normal B cells. No influence of PCI-32765 on T-cell survival is observed. Treatment of CD40 or BCR activated CLL cells with PCI-32765 results in inhibition of BTK tyrosine phosphorylation and also effectively abrogates downstream survival pathways activated by this kinase including ERK1/2, PI3K, and NF-κB. In addition, PCI-32765 inhibits activation-induced proliferation of CLL cells in vitro, and effectively blocks survival signals provided externally to CLL cells from the microenvironment including soluble factors (CD40L, BAFF, IL-6, IL-4, and TNF-α), fibronectin engagement, and stromal cell contact. Based on these collective data, future efforts targeting BTK with the irreversible inhibitor PCI-32765 in clinical trials of CLL patients is warranted. PMID:21422473

Yorkie Facilitates Organ Growth and Metamorphosis in Bombyx.

PubMed

Liu, Shumin; Zhang, Panli; Song, Hong-Sheng; Qi, Hai-Sheng; Wei, Zhao-Jun; Zhang, Guozheng; Zhan, Shuai; Liu, Zhihong; Li, Sheng

2016-01-01

The Hippo pathway, which was identified from genetic screens in the fruit fly, Drosophila melanogaster, has a major size-control function in animals. All key components of the Hippo pathway, including the transcriptional coactivator Yorkie that is the most critical substrate and downstream effector of the Hippo kinase cassette, are found in the silkworm, Bombyx mori. As revealed by microarray and quantitative real-time PCR, expression of Hippo pathway genes is particularly enriched in several mitotic tissues, including the ovary, testis, and wing disc. Developmental profiles of Hippo pathway genes are generally similar (with the exception of Yorkie) within each organ, but vary greatly in different tissues showing nearly opposing expression patterns in the wing disc and the posterior silk gland (PSG) on day 2 of the prepupal stage. Importantly, the reduction of Yorkie expression by RNAi downregulated Yorkie target genes in the ovary, decreased egg number, and delayed larval-pupal-adult metamorphosis. In contrast, baculovirus-mediated Yorkie(CA) overexpression upregulated Yorkie target genes in the PSG, increased PSG size, and accelerated larval-pupal metamorphosis. Together the results show that Yorkie potentially facilitates organ growth and metamorphosis, and suggest that the evolutionarily conserved Hippo pathway is critical for size control, particularly for PSG growth, in the silkworm.

Magnetic molecularly imprinted polymer for the isolation and detection of biotin and biotinylated biomolecules.

PubMed

Ben Aissa, A; Herrera-Chacon, A; Pupin, R R; Sotomayor, M D P T; Pividori, M I

2017-02-15

Magnetic separation based on biologically-modified magnetic particles is a preconcentration procedure commonly integrated in magneto actuated platforms for the detection of a huge range of targets. However, the main drawback of this material is the low stability and high cost. In this work, a novel hybrid molecularly-imprinted polymer with magnetic properties is presented with affinity towards biotin and biotinylated biomolecules. During the synthesis of the magneto core-shell particles, biotin was used as a template. The characterization of this material by microscopy techniques including SEM, TEM and confocal microscopy is presented. The application of the magnetic-MIPs for the detection of biotin and biotinylated DNA in magneto-actuated platforms is also described for the first time. The magnetic-MIP showed a significant immobilization capacity of biotinylated molecules, giving rise to a cheaper and a robust method (it is not required to be stored at 4°C) with high binding capacity for the separation and purification under magnetic actuation of a wide range of biotinylated molecules, and their downstream application including determination of their specific targets. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of wastewater treatment facility closure on endocrine disrupting chemicals in a Coastal Plain stream

USGS Publications Warehouse

Bradley, Paul M.; Journey, Celeste A.; Clark, Jimmy M.

2016-01-01

Wastewater treatment facility (WWTF) closures are rare environmental remediation events; offering unique insight into contaminant persistence, long-term wastewater impacts, and ecosystem recovery processes. The U.S. Geological Survey assessed the fate of select endocrine disrupting chemicals (EDC) in surface water and streambed sediment one year before and one year after closure of a long-term WWTF located within the Spirit Creek watershed at Fort Gordon, Georgia. Sample sites included a WWTF-effluent control located upstream from the outfall, three downstream effluent-impacted sites located between the outfall and Spirit Lake, and one downstream from the lake's outfall. Prior to closure, the 2.2-km stream segment downstream from the WWTF outfall was characterized by EDC concentrations significantly higher (α = 0.05) than at the control site; indicating substantial downstream transport and limited in-stream attenuation of EDC, including pharmaceuticals, estrogens, alkylphenol ethoxylate (APE) metabolites, and organophosphate flame retardants (OPFR). Wastewater-derived pharmaceutical, APE metabolites, and OPFR compounds were also detected in the outflow of Spirit Lake, indicating the potential for EDC transport to aquatic ecosystems downstream of Fort Gordon under effluent discharge conditions. After the WWTF closure, no significant differences in concentrations or numbers of detected EDC compounds were observed between control and downstream locations. The results indicated EDC pseudo-persistence under preclosure, continuous supply conditions, with rapid attenuation following WWTF closure. Low concentrations of EDC at the control site throughout the study and comparable concentrations in downstream locations after WWTF closure indicated additional, continuing, upstream contaminant sources within the Spirit Creek watershed. 

miR-223/Hsp70/JNK/JUN/miR-223 feedback loop modulates the chemoresistance of osteosarcoma to cisplatin.

PubMed

Tang, Qi; Yuan, Qi; Li, Hui; Wang, Wanchun; Xie, Guangrong; Zhu, Kewei; Li, Ding

2018-03-11

Osteosarcoma (OS) is a primary bone malignancy with a five-year survival rate of 60%; the chemoresistance of OS still remains a huge challenge. Heat shock protein 70 (Hsp70), a member of HSP family, is overexpressed in OS cell lines and involved in the resistance of OS cell lines. In addition, miRNAs have been involved in the carcinogenesis and chemoresistance of OS; of them, miR-223 has been reported to be underexpressed and serve as a tumor suppressor in OS through targeting Hsp90B1, also a member of HSP family. Herein, online tools predicted that Hsp70 might be a direct target of miR-223. In the present study, miR-223 expression was down-regulated in OS tissues and cell lines; miR-223 overexpression enhanced the cellular effects of cisplatin (CDDP) on OS cell lines. Through binding to the HSPA1A 3'UTR, miR-223 could regulate Hsp70 protein levels and downstream JNK/JUN signaling pathway, thus modulating OS cell apoptosis through Hsp70 under CDDP stress. Finally, JUN, a downstream transcription factor of JNK signaling, could bind to the promoter region of miR-223 to promote its transcription. In summary, miR-223, Hsp70 and downstream JNK/JUN formed a feedback loop to modulate the chemoresistance of OS to CDDP. Copyright © 2018 Elsevier Inc. All rights reserved.

Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors

PubMed Central

Karlsson, Hannah; Svensson, Emma; Gigg, Camilla; Jarvius, Malin; Olsson-Strömberg, Ulla; Savoldo, Barbara; Dotti, Gianpietro; Loskog, Angelica

2015-01-01

CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs. PMID:26700307

Preclinical efficacy of maternal embryonic leucine-zipper kinase (MELK) inhibition in acute myeloid leukemia.

PubMed

Alachkar, Houda; Mutonga, Martin B G; Metzeler, Klaus H; Fulton, Noreen; Malnassy, Gregory; Herold, Tobias; Spiekermann, Karsten; Bohlander, Stefan K; Hiddemann, Wolfgang; Matsuo, Yo; Stock, Wendy; Nakamura, Yusuke

2014-12-15

Maternal embryonic leucine-zipper kinase (MELK), which was reported to be frequently up-regulated in various types of solid cancer, plays critical roles in formation and maintenance of cancer stem cells. However, little is known about the relevance of this kinase in hematologic malignancies. Here we report characterization of possible roles of MELK in acute myeloid leukemia (AML). MELK is expressed in AML cell lines and AML blasts with higher levels in less differentiated cells. MELK is frequently upregulated in AML with complex karyotypes and is associated with worse clinical outcome. MELK knockdown resulted in growth inhibition and apoptosis of leukemic cells. Hence, we investigated the potent anti-leukemia activity of OTS167, a small molecule MELK kinase inhibitor, in AML, and found that the compound induced cell differentiation and apoptosis as well as decreased migration of AML cells. MELK expression was positively correlated with the expression of FOXM1 as well as its downstream target genes. Furthermore, MELK inhibition resulted in downregulation of FOXM1 activity and the expression of its downstream targets. Taken together, and given that OTS167 is undergoing a phase I clinical trial in solid cancer, our study warrants clinical evaluation of this compound as a novel targeted therapy for AML patients.

Preclinical efficacy of maternal embryonic leucine-zipper kinase (MELK) inhibition in acute myeloid leukemia

PubMed Central

Alachkar, Houda; Mutonga, Martin B.G.; Metzeler, Klaus H.; Fulton, Noreen; Malnassy, Gregory; Herold, Tobias; Spiekermann, Karsten; Bohlander, Stefan K.; Hiddemann, Wolfgang; Matsuo, Yo; Stock, Wendy; Nakamura, Yusuke

2014-01-01

Maternal embryonic leucine-zipper kinase (MELK), which was reported to be frequently up-regulated in various types of solid cancer, plays critical roles in formation and maintenance of cancer stem cells. However, little is known about the relevance of this kinase in hematologic malignancies. Here we report characterization of possible roles of MELK in acute myeloid leukemia (AML). MELK is expressed in AML cell lines and AML blasts with higher levels in less differentiated cells. MELK is frequently upregulated in AML with complex karyotypes and is associated with worse clinical outcome. MELK knockdown resulted in growth inhibition and apoptosis of leukemic cells. Hence, we investigated the potent anti-leukemia activity of OTS167, a small molecule MELK kinase inhibitor, in AML, and found that the compound induced cell differentiation and apoptosis as well as decreased migration of AML cells. MELK expression was positively correlated with the expression of FOXM1 as well as its downstream target genes. Furthermore, MELK inhibition resulted in downregulation of FOXM1 activity and the expression of its downstream targets. Taken together, and given that OTS167 is undergoing a phase I clinical trial in solid cancer, our study warrants clinical evaluation of this compound as a novel targeted therapy for AML patients. PMID:25365263

Changes in RNA polymerase II progression influence somatic hypermutation of Ig-related genes by AID

PubMed Central

Kodgire, Prashant; Mukkawar, Priyanka; Ratnam, Sarayu; Martin, Terence E.

2013-01-01

Somatic hypermutation (SHM) of Ig genes is initiated by the activation-induced cytidine deaminase (AID), and requires target gene transcription. We previously proposed that AID may associate with the RNA polymerase II (Pol). Here, to determine aspects of the transcription process required for SHM, we knocked-in a transcription terminator into an Ig gene variable region in DT40 chicken B cell line. We found that the human β-globin terminator was an efficient inhibitor of downstream transcription in these cells. The terminator reduced mutations downstream of the poly(A) signal, suggesting that the process of transcription is essential for efficient SHM and that AID has better access to its target when Pol is in the elongating rather than terminating mode. Mutations upstream of the poly(A) site were almost doubled in the active terminator clones compared with an inactivated terminator, and this region showed more single-stranded DNA, indicating that Pol pausing assists SHM. Moreover, the nontranscribed DNA strand was the preferred SHM target upstream of the active terminator. Pol pausing during poly(A) site recognition may facilitate persistence of negative supercoils, exposing the coding single strand and possibly allowing the nascent RNA intermittent reannealing with the template strand, for prolonged access of AID. PMID:23752228

Searching for Dark Photons with the SeaQuest Spectrometer

NASA Astrophysics Data System (ADS)

Uemura, Sho; SeaQuest Collaboration

2017-09-01

The existence of a dark sector, containing families of particles that do not couple directly to the Standard Model, is motivated as a possible model for dark matter. A ``dark photon'' - a massive vector boson that couples weakly to electric charge - is a common component of dark sector models. The SeaQuest spectrometer at Fermilab is designed to detect dimuon pairs produced by the interaction of a 120 GeV proton beam with a rotating set of thin fixed targets. An iron-filled magnet downstream of the target, 5 meters in length, serves as a beam dump. The SeaQuest spectrometer is sensitive to dark photons that are mostly produced in the beam dump and decay to dimuons, and a SeaQuest search for dark sector particles was approved as Fermilab experiment E1067. As part of E1067, a displaced-vertex trigger was built, installed and commissioned this year. This trigger uses two planes of extruded scintillators to identify dimuons originating far downstream of the target, and is sensitive to dark photons that travel deep inside the beam dump before decaying to dimuons. This trigger will be used to take data parasitically with the primary SeaQuest physics program. In this talk I will present the displaced-vertex trigger and its performance, and projected sensitivity from future running.

mTOR up-regulation of PFKFB3 is essential for acute myeloid leukemia cell survival

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feng, Yonghuai; Institute of Hematology, Peking University, Beijing; Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Beijing

Although mTOR (mammalian target of rapamycin) activation is frequently observed in acute myeloid leukemia (AML) patients, the precise function and the downstream targets of mTOR are poorly understood. Here we revealed that PFKFB3, but not PFKFB1, PFKFB2 nor PFKFB4 was a novel downstream substrate of mTOR signaling pathway as PFKFB3 level was augmented after knocking down TSC2 in THP1 and OCI-AML3 cells. Importantly, PFKFB3 silencing suppressed glycolysis and cell proliferation of TSC2 silencing OCI-AML3 cells and activated apoptosis pathway. These results suggested that mTOR up-regulation of PFKFB3 was essential for AML cells survival. Mechanistically, Rapamycin treatment or Raptor knockdown reducedmore » the expression of PFKFB3 in TSC2 knockdown cells, while Rictor silencing did not have such effect. Furthermore, we also revealed that mTORC1 up-regulated PFKFB3 was dependent on hypoxia-inducible factor 1α (HIF1α), a positive regulator of glycolysis. Moreover, PFKFB3 inhibitor PFK15 and rapamycin synergistically blunted the AML cell proliferation. Taken together, PFKFB3 was a promising drug target in AML patients harboring mTOR hyper-activation.« less

Elevated YAP and its downstream targets CCN1 and CCN2 in basal cell carcinoma: impact on keratinocyte proliferation and stromal cell activation.

PubMed

Quan, Taihao; Xu, Yiru; Qin, Zhaoping; Robichaud, Patrick; Betcher, Stephanie; Calderone, Ken; He, Tianyuan; Johnson, Timothy M; Voorhees, John J; Fisher, Gary J

2014-04-01

Yes-associated protein (YAP) is a transcriptional co-activator of hippo signaling pathway, which plays an important role in organ size control and tumorigenesis. Here we report that YAP and its downstream transcriptional targets CCN1 and CCN2 are markedly elevated in keratinocytes in human skin basal cell carcinoma tumor islands. In human keratinocytes, knockdown of YAP significantly reduced expression of CCN1 and CCN2, and repressed proliferation and survival. This inhibition of proliferation and survival was rescued by restoration of CCN1 expression, but not by CCN2 expression. In basal cell carcinoma stroma, CCN2-regulated genes type I collagen, fibronectin, and α-smooth muscle actin were highly expressed. Furthermore, atomic force microscopy revealed increased tissue stiffness in basal cell carcinoma stroma compared to normal dermis. These data provide evidence that up-regulation of YAP in basal cell carcinoma impacts both aberrant keratinocyte proliferation, via CCN1, and tumor stroma cell activation and stroma remodeling, via CCN2. Targeting YAP and/or CCN1 and CCN2 may provide clinical benefit in basal cell carcinoma. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Quantification of DNA cleavage specificity in Hi-C experiments.

PubMed

Meluzzi, Dario; Arya, Gaurav

2016-01-08

Hi-C experiments produce large numbers of DNA sequence read pairs that are typically analyzed to deduce genomewide interactions between arbitrary loci. A key step in these experiments is the cleavage of cross-linked chromatin with a restriction endonuclease. Although this cleavage should happen specifically at the enzyme's recognition sequence, an unknown proportion of cleavage events may involve other sequences, owing to the enzyme's star activity or to random DNA breakage. A quantitative estimation of these non-specific cleavages may enable simulating realistic Hi-C read pairs for validation of downstream analyses, monitoring the reproducibility of experimental conditions and investigating biophysical properties that correlate with DNA cleavage patterns. Here we describe a computational method for analyzing Hi-C read pairs to estimate the fractions of cleavages at different possible targets. The method relies on expressing an observed local target distribution downstream of aligned reads as a linear combination of known conditional local target distributions. We validated this method using Hi-C read pairs obtained by computer simulation. Application of the method to experimental Hi-C datasets from murine cells revealed interesting similarities and differences in patterns of cleavage across the various experiments considered. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Evidence for transcriptional interference in a dual-luciferase reporter system.

PubMed

Wu, Guo-Qing; Wang, Xiao; Zhou, Hong-Ying; Chai, Ke-Qun; Xue, Qian; Zheng, Ai-Hong; Zhu, Xiu-Ming; Xiao, Jian-Yong; Ying, Xu-Hua; Wang, Fu-Wei; Rui, Tao; Xu, Li-Yun; Zhang, Yong-Kui; Liao, Yi-Ji; Xie, Dan; Lu, Li-Qin; Huang, Dong-Sheng

2015-12-01

The dual-luciferase reporter assay is widely used for microRNA target identification and the functional validation of predicted targets. To determine whether curcumin regulates expression of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) by targeting its 3'untranslated region (3'UTR), two luciferase reporter systems containing exactly the same sequence of the EZH2 3'UTR were used to perform dual-luciferase reporter assays. Surprisingly, there were certain discrepancies between the luciferase activities derived from these two reporter constructs. We normalized luciferase activity to an internal control to determine the amount of the reporter construct successfully transfected into cells, induced a transcriptional block with flavopiridol, quantified renilla luciferase mRNA levels, and compared the absolute luciferase activity among the different groups. The results suggested that curcumin promoted the transcription of the luciferase genes located downstream of the simian vacuolating virus 40 (SV40) early enhancer/promoter, but not those located downstream of the human cytomegalovirus (CMV) immediate-early or the herpes simplex virus thymidine kinase (HSV-TK) promoters. These results explain the discrepancies between the two luciferase reporter systems. The current study underscores the importance of taking caution when interpreting the results of dual-luciferase reporter assays and provides strategies to overcome the potential pitfall accompanying dual-luciferase reporter systems.

Evidence for transcriptional interference in a dual-luciferase reporter system

PubMed Central

Wu, Guo-Qing; Wang, Xiao; Zhou, Hong-Ying; Chai, Ke-Qun; Xue, Qian; Zheng, Ai-Hong; Zhu, Xiu-Ming; Xiao, Jian-Yong; Ying, Xu-Hua; Wang, Fu-Wei; Rui, Tao; Xu, Li-Yun; Zhang, Yong-Kui; Liao, Yi-Ji; Xie, Dan; Lu, Li-Qin; Huang, Dong-Sheng

2015-01-01

The dual-luciferase reporter assay is widely used for microRNA target identification and the functional validation of predicted targets. To determine whether curcumin regulates expression of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) by targeting its 3′untranslated region (3′UTR), two luciferase reporter systems containing exactly the same sequence of the EZH2 3′UTR were used to perform dual-luciferase reporter assays. Surprisingly, there were certain discrepancies between the luciferase activities derived from these two reporter constructs. We normalized luciferase activity to an internal control to determine the amount of the reporter construct successfully transfected into cells, induced a transcriptional block with flavopiridol, quantified renilla luciferase mRNA levels, and compared the absolute luciferase activity among the different groups. The results suggested that curcumin promoted the transcription of the luciferase genes located downstream of the simian vacuolating virus 40 (SV40) early enhancer/promoter, but not those located downstream of the human cytomegalovirus (CMV) immediate-early or the herpes simplex virus thymidine kinase (HSV-TK) promoters. These results explain the discrepancies between the two luciferase reporter systems. The current study underscores the importance of taking caution when interpreting the results of dual-luciferase reporter assays and provides strategies to overcome the potential pitfall accompanying dual-luciferase reporter systems. PMID:26620302

Assessing downstream flood impacts due to a potential GLOF from Imja Lake in Nepal

NASA Astrophysics Data System (ADS)

Somos-Valenzuela, M. A.; McKinney, D. C.; Byers, A. C.; Rounce, D. R.; Portocarrero, C.; Lamsal, D.

2014-11-01

Glacial-dominated areas pose unique challenges to downstream communities in adapting to recent and continuing global climate change, including increased threats of glacial lake outburst floods (GLOFs) that can increase risk due to flooding of downstream communities and cause substantial impacts on regional social, environmental and economic systems. The Imja glacial lake in Nepal, with potential to generate a GLOF, was studied using a two-dimensional debris flow inundation model in order to evaluate the effectiveness of proposed measures to reduce possible flooding impacts to downstream communities by lowering the lake level. The results indicate that only minor flood impact reduction is achieved in the downstream community of Dingboche with modest (~3 m) lake lowering. Lowering the lake by 10 m shows a significant reduction in inundated area. However, lowering the lake by 20 m almost eliminates all flood impact at Dingboche. Further downstream at Phakding, the impact of the GLOF is significant and similar reductions in inundation are likely as a result of lake lowering.

An electrophysiological investigation of reinforcement effects in attention deficit/hyperactivity disorder: Dissociating cue sensitivity from down-stream effects on target engagement and performance.

PubMed

Chronaki, Georgia; Soltesz, Fruzsina; Benikos, Nicholas; Sonuga-Barke, Edmund J S

2017-12-01

Neural hypo-sensitivity to cues predicting positive reinforcement has been observed in ADHD using the Monetary Incentive Delay (MID) task. Here we report the first study using an electrophysiological analogue of this task to distinguish between (i) cue related anticipation of reinforcement and downstream effects on (ii) target engagement and (iii) performance in a clinical sample of adolescents with ADHD and controls. Thirty-one controls and 32 adolescents with ADHD aged 10-16 years performed the electrophysiological (e)-MID task - in which preparatory cues signal whether a response to an upcoming target will be reinforced or not - under three conditions; positive reinforcement, negative reinforcement (response cost) and no consequence (neutral). We extracted values for both cue-related potentials known to be, both, associated with response preparation and modulated by reinforcement (Cue P3 and Cue CNV) and target-related potentials (target P3) and compared these between ADHD and controls. ADHD and controls did not differ on cue-related components on neutral trials. Against expectation, adolescents with ADHD displayed Cue P3 and Cue CNV reinforcement-related enhancement (versus neutral trials) compared to controls. ADHD individuals displayed smaller target P3 amplitudes and slower and more variable performance - but effects were not modulated by reinforcement contingencies. When age, IQ and conduct problems were controlled effects were marginally significant but the pattern of results did not change. ADHD was associated with hypersensitivity to positive (and marginally negative) reinforcement reflected on components often thought to be associated with response preparation - however these did not translate into improved attention to targets. In the case of ADHD, upregulated CNV may be a specific marker of hyper-arousal rather than an enhancement of anticipatory attention to upcoming targets. Future studies should examine the effects of age, IQ and conduct problems on reinforcement sensitivity in ADHD. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Chemokine receptor binding and signal transduction in native cells of the central nervous system.

PubMed

Davis, Christopher N; Chen, Shuzhen; Boehme, Stefen A; Bacon, Kevin B; Harrison, Jeffrey K

2003-04-01

Chemokine receptors belong to the superfamily of seven-transmembrane-spanning, G-protein-coupled receptors, and their expression by central nervous system cells is clearly documented. As this gene family has become the target of novel therapeutic development, the analysis of these receptors requires radioligand binding techniques as well as methods that entail assessing receptor stimulation of signal transduction pathways. Herein, we describe specific protocols for measuring radiolabeled chemokine binding to their cognate receptors on cultured glial cells as well as to receptors expressed in heterologous cell systems. Multiple downstream signaling pathways, including intracellular calcium influx and receptor-dependent kinase activation, are associated with chemokine receptor stimulation. Protocols for measuring these signaling events in chemokine-receptor-expressing cells are also presented.

Insights from human genetic studies of lung and organ fibrosis.

PubMed

Garcia, Christine Kim

2018-01-02

Genetic investigations of fibrotic diseases, including those of late onset, often yield unanticipated insights into disease pathogenesis. This Review focuses on pathways underlying lung fibrosis that are generalizable to other organs. Herein, we discuss genetic variants subdivided into those that shorten telomeres, activate the DNA damage response, change resident protein expression or function, or affect organelle activity. Genetic studies provide a window into the downstream cascade of maladaptive responses and pathways that lead to tissue fibrosis. In addition, these studies reveal interactions between genetic variants, environmental factors, and age that influence the phenotypic spectrum of disease. The discovery of forces counterbalancing inherited risk alleles identifies potential therapeutic targets, thus providing hope for future prevention or reversal of fibrosis.

Rho proteins of plants--functional cycle and regulation of cytoskeletal dynamics.

PubMed

Mucha, Elena; Fricke, Inka; Schaefer, Antje; Wittinghofer, Alfred; Berken, Antje

2011-11-01

Rho-related ROP proteins are molecular switches that essentially regulate a wide variety of processes. Of central interest is their influence on the plant cytoskeleton by which they affect vital processes like cell division, growth, morphogenesis, and pathogen defense. ROPs switch between GTP- and GDP-bound conformations by strictly regulated nucleotide exchange and GTP-hydrolysis, and only the active GTP-form interacts with downstream effectors to ultimately provoke a biological response. However, the mode of action of the engaged regulators and effectors as well as their upstream and downstream interaction partners have long been largely unknown. As opposed to analogous systems in animals and fungi, plants use specific GTPase activating proteins (RopGAPs) with a unique domain composition and novel guanine nucleotide exchange factors (RopGEFs) with a probable link to cell surface receptors. Moreover, plants comprise novel effector molecules and adapters connecting ROPs to mostly unknown downstream targets on the route to the cytoskeleton. This review aims to summarize recent knowledge on the molecular mechanisms and reaction cascades involved in ROP dependent cytoskeletal rearrangements, addressing the structure and function of the unusual RopGAPs, RopGEFs and effectors, and the upstream and downstream pathways linking ROPs to cell receptor-like kinases, actin filaments, and microtubules. Copyright © 2010 Elsevier GmbH. All rights reserved.

Tissue engineering and microRNAs: future perspectives in regenerative medicine.

PubMed

Gori, Manuele; Trombetta, Marcella; Santini, Daniele; Rainer, Alberto

2015-01-01

Tissue engineering is a growing area of biomedical research, holding great promise for a broad range of potential applications in the field of regenerative medicine. In recent decades, multiple tissue engineering strategies have been adopted to mimic and improve specific biological functions of tissues and organs, including biomimetic materials, drug-releasing scaffolds, stem cells, and dynamic culture systems. MicroRNAs (miRNAs), noncoding small RNAs that negatively regulate the expression of downstream target mRNAs, are considered a novel class of molecular targets and therapeutics that may play an important role in tissue engineering. Herein, we highlight the latest achievements in regenerative medicine, focusing on the role of miRNAs as key modulators of gene expression, stem cell self-renewal, proliferation and differentiation, and eventually in driving cell fate decisions. Finally, we will discuss the contribution of miRNAs in regulating the rearrangement of the tissue microenvironment and angiogenesis, and the range of strategies for miRNA delivery into target cells and tissues. Manipulation of miRNAs is an alternative approach and an attractive strategy for controlling several aspects of tissue engineering, although some issues concerning their in vivo effects and optimal delivery methods still remain uncovered.

Analysis of DNA methylation in FFPE tissues using the MethyLight technology.

PubMed

Dallol, Ashraf; Al-Ali, Waleed; Al-Shaibani, Amina; Al-Mulla, Fahd

2011-01-01

Novel biomarkers are sought after by mining DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues. Such tissues offer the great advantage of often having complete clinical data (including survival), as well as the tissues are amenable for laser microdissection targeting specific tissue areas. Downstream analysis of such DNA includes mutational screens and methylation profiling. Screening for mutations by sequencing requires a significant amount of DNA for PCR and cycle sequencing. This is self-inhibitory if the gene screened has a large number of exons. Profiling DNA methylation using the MethyLight technology circumvents this problem and allows for the mining of several biomarkers from DNA extracted from a single microscope slide of the tissue of interest. We describe in this chapter a detailed protocol for MethyLight and its use in the determination of CpG Island Methylator Phenotype status in FFPE colorectal cancer samples.

Homeodomain-Interacting Protein Kinase-2: A Critical Regulator of the DNA Damage Response and the Epigenome

PubMed Central

Kuwano, Yuki; Nishida, Kensei; Akaike, Yoko; Kurokawa, Ken; Nishikawa, Tatsuya; Masuda, Kiyoshi; Rokutan, Kazuhito

2016-01-01

Homeodomain-interacting protein kinase 2 (HIPK2) is a serine/threonine kinase that phosphorylates and activates the apoptotic program through interaction with diverse downstream targets including tumor suppressor p53. HIPK2 is activated by genotoxic stimuli and modulates cell fate following DNA damage. The DNA damage response (DDR) is triggered by DNA lesions or chromatin alterations. The DDR regulates DNA repair, cell cycle checkpoint activation, and apoptosis to restore genome integrity and cellular homeostasis. Maintenance of the DDR is essential to prevent development of diseases caused by genomic instability, including cancer, defects of development, and neurodegenerative disorders. Recent studies reveal a novel HIPK2-mediated pathway for DDR through interaction with chromatin remodeling factor homeodomain protein 1γ. In this review, we will highlight the molecular mechanisms of HIPK2 and show its functions as a crucial DDR regulator. PMID:27689990

Trans-acting epigenetic effects of chromosomal aneuploidies: lessons from Down syndrome and mouse models

PubMed Central

Do, Catherine; Xing, Zhuo; Yu, Y Eugene; Tycko, Benjamin

2017-01-01

An important line of postgenomic research seeks to understand how genetic factors can influence epigenetic patterning. Here we review epigenetic effects of chromosomal aneuploidies, focusing on findings in Down syndrome (DS, trisomy 21). Recent work in human DS and mouse models has shown that the extra chromosome 21 acts in trans to produce epigenetic changes, including differential CpG methylation (DS-DM), in specific sets of downstream target genes, mostly on other chromosomes. Mechanistic hypotheses emerging from these data include roles of chromosome 21-linked methylation pathway genes (DNMT3L and others) and transcription factor genes (RUNX1, OLIG2, GABPA, ERG and ETS2) in shaping the patterns of DS-DM. The findings may have broader implications for trans-acting epigenetic effects of chromosomal and subchromosomal aneuploidies in other human developmental and neuropsychiatric disorders, and in cancers. PMID:27911079

RGS16 inhibits breast cancer cell growth by mitigating phosphatidylinositol 3-kinase signaling.

PubMed

Liang, Genqing; Bansal, Geetanjali; Xie, Zhihui; Druey, Kirk M

2009-08-07

Aberrant activity of the phosphatidylinositol 3-kinase (PI3K) pathway supports growth of many tumors including those of breast, lung, and prostate. Resistance of breast cancer cells to targeted chemotherapies including tyrosine kinase inhibitors (TKI) has been linked to persistent PI3K activity, which may in part be due to increased membrane expression of epidermal growth factor (EGF) receptors (HER2 and HER3). Recently we found that proteins of the RGS (regulator of G protein signaling) family suppress PI3K activity downstream of the receptor by sequestering its p85alpha subunit from signaling complexes. Because a substantial percentage of breast tumors have RGS16 mutations and reduced RGS16 protein expression, we investigated the link between regulation of PI3K activity by RGS16 and breast cancer cell growth. RGS16 overexpression in MCF7 breast cancer cells inhibited EGF-induced proliferation and Akt phosphorylation, whereas shRNA-mediated extinction of RGS16 augmented cell growth and resistance to TKI treatment. Exposure to TKI also reduced RGS16 expression in MCF7 and BT474 cell lines. RGS16 bound the amino-terminal SH2 and inter-SH2 domains of p85alpha and inhibited its interaction with the EGF receptor-associated adapter protein Gab1. These results suggest that the loss of RGS16 in some breast tumors enhances PI3K signaling elicited by growth factors and thereby promotes proliferation and TKI evasion downstream of HER activation.

Proceedings of a workshop on American Eel passage technologies

USGS Publications Warehouse

Haro, Alexander J.

2013-01-01

Recent concerns regarding a decline in recruitment of American eels (Anguilla rostrata) have prompted efforts to restore this species to historic habitats by providing passage for both upstream migrant juveniles and downstream migrant adults at riverine barriers, including low-head and hydroelectric dams (Castonguay et al. 1994, Haro et al. 2000). These efforts include development of management plans and stock assessment reviews in both the US and Canada (COSEWIC 2006, Canadian Eel Working Group 2009, DFO 2010, MacGregor et al. 2010, ASMFC 2000, ASMFC 2006, ASMFC 2008, Williams and Threader 2007), which target improvement of upstream and downstream passage for eels, as well as identification and prioritization of research needs for development of new and more effective passage technologies for American eels. Traditional upstream fish passage structures, such as fishways and fish lifts, are often ineffective passing juvenile eels, and specialized passage structures for this species are needed. Although designs for such passage structures are available and diverse (Knights and White 1998, Porcher 2002, FAO/DVWK 2002, Solomon and Beach 2004a,b, Environment Agency UK 2011), many biologists, managers, and engineers are unfamiliar with eel pass design and operation, or unaware of the technical options available for upstream eel passage, Better coordination is needed to account for eel passage requirements during restoration efforts for other diadromous fish species. Also, appropriately siting eel passes at hydropower projects is critical, and siting can be difficult and complex due to physical restrictions in access to points of natural concentrations of eels, dynamic hydraulics of tailrace areas, and presence of significant competing flows from turbine outfalls or spill. As a result, some constructed eel passes are sited poorly and may pass only a fraction of the number of eels attempting to pass the barrier. When sited and constructed appropriately, however, eel passes can effectively pass thousands of individuals in a season (Appendix D). technologies for preventing impingement and entrainment mortality and injury of downstream migrant eels at hydropower projects are not well developed. Traditional downstream fish passage mitigative techniques originally developed for salmonids and other species are frequently ineffective passing eels (Richkus and Dixon 2003, EPRI 2001, Bruijs and Durif 2009). Large hydropower projects, with high project flows or intake openings that cannot be fitted with racks or screens with openings small enough to exclude eels, pose significant passage problems for this species, and turbine impingement and entrainment mortality of eels can be as high as 100%. Spill mortality and injury may also be significant for eels, given their tendency to move during high flow events when projects typically spill large amounts of flow. Delays in migration of eels that have difficulty locating and utilizing bypass entrances can also be significant. Therefore, downstream passage technologies are at a much more nebulous state of development than upstream passage technologies, and require further evaluation and improvement before rigorous design guidelines can be established. There have been few studies conducted to evaluate effectiveness of current mitigative measures for both upstream and downstream passage of eels. Research is needed to determine eel migratory timing, behavior, and appropriate mitigation technologies for specific sites and eel life history stages. Both upstream and downstream eel passage structures can be difficult to evaluate in terms of performance, and examples of how evaluation and monitoring can be accomplished were reviewed at the workshop.

The Advantages of Targeted Protein Degradation Over Inhibition: An RTK Case Study.

PubMed

Burslem, George M; Smith, Blake E; Lai, Ashton C; Jaime-Figueroa, Saul; McQuaid, Daniel C; Bondeson, Daniel P; Toure, Momar; Dong, Hanqing; Qian, Yimin; Wang, Jing; Crew, Andrew P; Hines, John; Crews, Craig M

2018-01-18

Proteolysis targeting chimera (PROTAC) technology has emerged over the last two decades as a powerful tool for targeted degradation of endogenous proteins. Herein we describe the development of PROTACs for receptor tyrosine kinases, a protein family yet to be targeted for induced protein degradation. The use of VHL-recruiting PROTACs against this protein family reveals several advantages of degradation over inhibition alone: direct comparisons of fully functional, target-degrading PROTACs with target-inhibiting variants that contain an inactivated E3 ligase-recruiting ligand show that degradation leads to more potent inhibition of cell proliferation and a more durable and sustained downstream signaling response, and thus addresses the kinome rewiring challenge seen with many receptor tyrosine kinase inhibitors. Combined, these findings demonstrate the ability to target receptor tyrosine kinases for degradation using the PROTAC technology and outline the advantages of this degradation-based approach. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development and validation of a clinical trial patient stratification assay that interrogates 27 mutation sites in MAPK pathway genes.

PubMed

Chang, Ken C N; Galuska, Stefan; Weiner, Russell; Marton, Matthew J

2013-01-01

Somatic mutations identified on genes related to the cancer-developing signaling pathways have drawn attention in the field of personalized medicine in recent years. Treatments developed to target a specific signaling pathway may not be effective when tumor activating mutations occur downstream of the target and bypass the targeted mechanism. For instance, mutations detected in KRAS/BRAF/NRAS genes can lead to EGFR-independent intracellular signaling pathway activation. Most patients with these mutations do not respond well to anti-EGFR treatment. In an effort to detect various mutations in FFPE tissue samples among multiple solid tumor types for patient stratification many mutation assays were evaluated. Since there were more than 30 specific mutations among three targeted RAS/RAF oncogenes that could activate MAPK pathway genes, a custom designed Single Nucleotide Primer Extension (SNPE) multiplexing mutation assay was developed and analytically validated as a clinical trial assay. Throughout the process of developing and validating the assay we overcame many technical challenges which include: the designing of PCR primers for FFPE tumor tissue samples versus normal blood samples, designing of probes for detecting consecutive nucleotide double mutations, the kinetics and thermodynamics aspects of probes competition among themselves and against target PCR templates, as well as validating an assay when positive control tumor tissue or cell lines with specific mutations are not available. We used Next Generation sequencing to resolve discordant calls between the SNPE mutation assay and Sanger sequencing. We also applied a triplicate rule to reduce potential false positives and false negatives, and proposed special considerations including pre-define a cut-off percentage for detecting very low mutant copies in the wild-type DNA background.

System for tuning a combustor of a gas turbine

DOEpatents

Hughes, Michael John

2016-12-27

A system for tuning a combustor of a gas turbine includes a flow sleeve having an annular main body. The main body includes an upstream end, a downstream end, an inner surface and an outer surface. A cooling channel extends along the inner surface of the main body. The cooling channel extends at least partially between the downstream end and the upstream end of the main body.

Sound suppression mixer

NASA Technical Reports Server (NTRS)

Brown, William H. (Inventor)

1994-01-01

A gas turbine engine flow mixer includes at least one chute having first and second spaced apart sidewalls joined together at a leading edge, with the sidewalls having first and second trailing edges defining therebetween a chute outlet. The first trailing edge is spaced longitudinally downstream from the second trailing edge for defining a septum in the first sidewall extending downstream from the second trailing edge. The septum includes a plurality of noise attenuating apertures.

A Hox regulatory network establishes motor neuron pool identity and target-muscle connectivity.

PubMed

Dasen, Jeremy S; Tice, Bonnie C; Brenner-Morton, Susan; Jessell, Thomas M

2005-11-04

Spinal motor neurons acquire specialized "pool" identities that determine their ability to form selective connections with target muscles in the limb, but the molecular basis of this striking example of neuronal specificity has remained unclear. We show here that a Hox transcriptional regulatory network specifies motor neuron pool identity and connectivity. Two interdependent sets of Hox regulatory interactions operate within motor neurons, one assigning rostrocaudal motor pool position and a second directing motor pool diversity at a single segmental level. This Hox regulatory network directs the downstream transcriptional identity of motor neuron pools and defines the pattern of target-muscle connectivity.

Drug targeting of oncogenic pathways in melanoma.

PubMed

Fecher, Leslie A; Amaravadi, Ravi K; Schuchter, Lynn M; Flaherty, Keith T

2009-06-01

Melanoma continues to be one of the most aggressive and morbid malignancies once metastatic. Overall survival for advanced unresectable melanoma has not changed over the past several decades. However, the presence of some long-term survivors of metastatic melanoma highlights the heterogeneity of this disease and the potential for improved outcomes. Current research is uncovering the molecular and genetic scaffolding of normal and aberrant cell function. The known oncogenic pathways in melanoma and the attempts to develop therapy for them are discussed. The targeting of certain cellular processes, downstream of the common genetic alterations, for which the issues of target and drug validation are somewhat distinct, are also highlighted.

RSK2 is a new Pim2 target with pro-survival functions in FLT3-ITD-positive acute myeloid leukemia.

PubMed

Hospital, M-A; Jacquel, A; Mazed, F; Saland, E; Larrue, C; Mondesir, J; Birsen, R; Green, A S; Lambert, M; Sujobert, P; Gautier, E-F; Salnot, V; Le Gall, M; Decroocq, J; Poulain, L; Jacque, N; Fontenay, M; Kosmider, O; Récher, C; Auberger, P; Mayeux, P; Bouscary, D; Sarry, J-E; Tamburini, J

2018-03-01

Acute myeloid leukemia (AML) with the FLT3 internal tandem duplication (FLT3-ITD AML) accounts for 20-30% of AML cases. This subtype usually responds poorly to conventional therapies, and might become resistant to FLT3 tyrosine kinase inhibitors (TKIs) due to molecular bypass mechanisms. New therapeutic strategies focusing on resistance mechanisms are therefore urgently needed. Pim kinases are FLT3-ITD oncogenic targets that have been implicated in FLT3 TKI resistance. However, their precise biological function downstream of FLT3-ITD requires further investigation. We performed high-throughput transcriptomic and proteomic analyses in Pim2-depleted FLT3-ITD AML cells and found that Pim2 predominantly controlled apoptosis through Bax expression and mitochondria disruption. We identified ribosomal protein S6 kinase A3 (RSK2), a 90 kDa serine/threonine kinase involved in the mitogen-activated protein kinase cascade encoded by the RPS6KA3 gene, as a novel Pim2 target. Ectopic expression of an RPS6KA3 allele rescued the viability of Pim2-depleted cells, supporting the involvement of RSK2 in AML cell survival downstream of Pim2. Finally, we showed that RPS6KA3 knockdown reduced the propagation of human AML cells in vivo in mice. Our results point to RSK2 as a novel Pim2 target with translational therapeutic potential in FLT3-ITD AML.

GLI activation by atypical protein kinase C ι/λ regulates the growth of basal cell carcinomas.

PubMed

Atwood, Scott X; Li, Mischa; Lee, Alex; Tang, Jean Y; Oro, Anthony E

2013-02-28

Growth of basal cell carcinomas (BCCs) requires high levels of hedgehog (HH) signalling through the transcription factor GLI. Although inhibitors of membrane protein smoothened (SMO) effectively suppress HH signalling, early tumour resistance illustrates the need for additional downstream targets for therapy. Here we identify atypical protein kinase C ι/λ (aPKC-ι/λ) as a novel GLI regulator in mammals. aPKC-ι/λ and its polarity signalling partners co-localize at the centrosome and form a complex with missing-in-metastasis (MIM), a scaffolding protein that potentiates HH signalling. Genetic or pharmacological loss of aPKC-ι/λ function blocks HH signalling and proliferation of BCC cells. Prkci is a HH target gene that forms a positive feedback loop with GLI and exists at increased levels in BCCs. Genome-wide transcriptional profiling shows that aPKC-ι/λ and SMO control the expression of similar genes in tumour cells. aPKC-ι/λ functions downstream of SMO to phosphorylate and activate GLI1, resulting in maximal DNA binding and transcriptional activation. Activated aPKC-ι/λ is upregulated in SMO-inhibitor-resistant tumours and targeting aPKC-ι/λ suppresses signalling and growth of resistant BCC cell lines. These results demonstrate that aPKC-ι/λ is critical for HH-dependent processes and implicates aPKC-ι/λ as a new, tumour-selective therapeutic target for the treatment of SMO-inhibitor-resistant cancers.

Herbal remedy magnolol suppresses IL-6-induced STAT3 activation and gene expression in endothelial cells

PubMed Central

Chen, Shih-Chung; Chang, Ying-Ling; Wang, Danny Ling; Cheng, Jing-Jy

2006-01-01

Magnolol (Mag), an active constituent isolated from the Chinese herb Hou p'u (Magnolia officinalis) has long been used to suppress inflammatory processes. Chronic inflammation is well known to be involved in vascular injuries such as atherosclerosis in which interleukin (IL)-6 may participate. Signal transducer and activator of transcription protein 3 (STAT3), a transcription factor involved in inflammation and the cell cycle, is activated by IL-6. In this study, we evaluated whether Mag can serve as an anti-inflammatory agent during endothelial injuries. The effects of Mag on IL-6-induced STAT3 activation and downstream target gene induction in endothelial cells (ECs) were examined. Pretreatment of ECs with Mag dose dependently inhibited IL-6-induced Tyr705 and Ser727 phosphorylation in STAT3 without affecting the phosphorylation of JAK1, JAK2, and ERK1/2. Mag pretreatment of these ECs dose dependently suppressed IL-6-induced promoter activity of intracellular cell adhesion molecule (ICAM)-1 that contains functional IL-6 response elements (IREs). An electrophoretic mobility shift assay (EMSA) revealed that Mag treatment significantly reduced STAT3 binding to the IRE region. Consistently, Mag treatment markedly inhibited ICAM-1 expression on the endothelial surface. As a result, reduced monocyte adhesion to IL-6-activated ECs was observed. Furthermore, Mag suppressed IL-6-induced promoter activity of cyclin D1 and monocyte chemotactic protein (MCP)-1 for which STAT3 activation plays a role. In conclusion, our results indicate that Mag inhibits IL-6-induced STAT3 activation and subsequently results in the suppression of downstream target gene expression in ECs. These results provide a therapeutic basis for the development of Mag as an anti-inflammatory agent for vascular disorders including atherosclerosis. PMID:16520748

Identification of Rice Genes Associated With Enhanced Cold Tolerance by Comparative Transcriptome Analysis With Two Transgenic Rice Plants Overexpressing DaCBF4 or DaCBF7, Isolated From Antarctic Flowering Plant Deschampsia antarctica

PubMed Central

Byun, Mi Young; Cui, Li Hua; Lee, Jungeun; Park, Hyun; Lee, Andosung; Kim, Woo Taek; Lee, Hyoungseok

2018-01-01

Few plant species can survive in Antarctica, the harshest environment for living organisms. Deschampsia antarctica is the only natural grass species to have adapted to and colonized the maritime Antarctic. To investigate the molecular mechanism of the Antarctic adaptation of this plant, we identified and characterized D. antarctica C-repeat binding factor 4 (DaCBF4), which belongs to monocot CBF group IV. The transcript level of DaCBF4 in D. antarctica was markedly increased by cold and dehydration stress. To assess the roles of DaCBF4 in plants, we generated a DaCBF4-overexpressing transgenic rice plant (Ubi:DaCBF4) and analyzed its abiotic stress response phenotype. Ubi:DaCBF4 displayed enhanced tolerance to cold stress without growth retardation under any condition compared to wild-type plants. Because the cold-specific phenotype of Ubi:DaCBF4 was similar to that of Ubi:DaCBF7 (Byun et al., 2015), we screened for the genes responsible for the improved cold tolerance in rice by selecting differentially regulated genes in both transgenic rice lines. By comparative transcriptome analysis using RNA-seq, we identified 9 and 15 genes under normal and cold-stress conditions, respectively, as putative downstream targets of the two D. antarctica CBFs. Overall, our results suggest that Antarctic hairgrass DaCBF4 mediates the cold-stress response of transgenic rice plants by adjusting the expression levels of a set of stress-responsive genes in transgenic rice plants. Moreover, selected downstream target genes will be useful for genetic engineering to enhance the cold tolerance of cereal plants, including rice. PMID:29774046

Isolation of a thyroid hormone-responsive gene by immunoprecipitation of thyroid hormone receptor-DNA complexes.

PubMed Central

Bigler, J; Eisenman, R N

1994-01-01

Thyroid hormone (T3) receptor (TR) is a ligand-dependent transcription factor that acts through specific binding sites in the promoter region of target genes. In order to identify new genes that are regulated by T3, we used anti-TR antiserum to immunoprecipitate TR-DNA complexes from GH4 cell nuclei that had previously been treated with a restriction enzyme. Screening of the immunopurified, cloned DNA for TR binding sites by electrophoretic mobility shift assay yielded 53 positive clones. A subset of these clones was specifically immunoprecipitated with anti-TR antiserum and may therefore represent biologically significant binding sites. One of these clones, clone 122, was characterized in detail. It includes sequences highly related to the NICER long terminal repeat-like element and contains three TR binding sites as determined by DNase I footprinting. Two of the clone 122 TR binding sites are located upstream of the TATA box, and one is located downstream. The TR binding site downstream from the promoter was necessary and sufficient to confer T3-dependent regulation in transient transfection experiments. Expression of a reporter construct under the control of the clone 122 promoter region was activated by TR in the absence of ligand and returned to basal levels after T3 addition. Clone 122 sequences hybridize to at least two different mRNAs of approximately 6 and 10 kb from GH4 cells. The levels of both of these mRNAs increased upon removal of T3. Our studies suggest that specific immunoprecipitation of chromatin allows identification of binding sites and target genes for transcription factors. Images PMID:7935476

Identification of Rice Genes Associated With Enhanced Cold Tolerance by Comparative Transcriptome Analysis With Two Transgenic Rice Plants Overexpressing DaCBF4 or DaCBF7, Isolated From Antarctic Flowering Plant Deschampsia antarctica.

PubMed

Byun, Mi Young; Cui, Li Hua; Lee, Jungeun; Park, Hyun; Lee, Andosung; Kim, Woo Taek; Lee, Hyoungseok

2018-01-01

Few plant species can survive in Antarctica, the harshest environment for living organisms. Deschampsia antarctica is the only natural grass species to have adapted to and colonized the maritime Antarctic. To investigate the molecular mechanism of the Antarctic adaptation of this plant, we identified and characterized D. antarctica C-repeat binding factor 4 ( DaCBF4 ), which belongs to monocot CBF group IV. The transcript level of DaCBF4 in D. antarctica was markedly increased by cold and dehydration stress. To assess the roles of DaCBF4 in plants, we generated a DaCBF4 -overexpressing transgenic rice plant ( Ubi:DaCBF4 ) and analyzed its abiotic stress response phenotype. Ubi:DaCBF4 displayed enhanced tolerance to cold stress without growth retardation under any condition compared to wild-type plants. Because the cold-specific phenotype of Ubi:DaCBF4 was similar to that of Ubi:DaCBF7 (Byun et al., 2015), we screened for the genes responsible for the improved cold tolerance in rice by selecting differentially regulated genes in both transgenic rice lines. By comparative transcriptome analysis using RNA-seq, we identified 9 and 15 genes under normal and cold-stress conditions, respectively, as putative downstream targets of the two D. antarctica CBFs. Overall, our results suggest that Antarctic hairgrass DaCBF4 mediates the cold-stress response of transgenic rice plants by adjusting the expression levels of a set of stress-responsive genes in transgenic rice plants. Moreover, selected downstream target genes will be useful for genetic engineering to enhance the cold tolerance of cereal plants, including rice.

Phosphorylation of HSP27 by Protein Kinase D is Essential for Mediating Neuroprotection Against Ischemic Neuronal Injury

PubMed Central

Stetler, R. Anne; Gao, Yanqin; Zhang, Lili; Weng, Zhongfang; Zhang, Feng; Hu, Xiaoming; Wang, Suping; Vosler, Peter; Cao, Guodong; Sun, Dandan; Graham, Steven H.; Chen, Jun

2012-01-01

Heat shock protein 27 (HSP27, or HSPB1) exerts cytoprotection against many cellular insults, including cerebral ischemia. We previously identified apoptosis signal-regulating kinase 1 (ASK1) as a critical downstream target of HSP27 conferring the neuroprotective effects of HSP27 against neuronal ischemia. However, the function of HSP27 is highly influenced by post-translational modification, with differential cellular effects based on phosphorylation at specific serine residues. The role of phosphorylation in neuronal ischemic neuroprotection is currently unknown. We have created transgenic mice and viral vectors containing HSP27 mutated at three critical serine residues (Ser15, Ser78 and Ser82) to either alanine (HSP27-A, non-phosphorylatable) or aspartate (HSP27-D, phospho-mimetic) residues. Under both in vitro and in vivo neuronal ischemic settings, overexpression of wild-type HSP27 (HSP27) and HSP27-D, but not HSP27-A, was neuroprotective and inhibited downstream ASK1 signaling pathways. Consistently, overexpressed HSP27 was phosphorylated by endogenous mechanisms when neurons were under ischemic stress, and single point mutations identified Ser15 and Ser82 as critical for neuroprotection. Using a panel of inhibitors and gene knockdown approaches, we identified the upstream kinase protein kinase D (PKD) as the primary kinase targeting HSP27 directly for phosphorylation. PKD and HSP27 co-immunoprecipitated, and inhibition or knockdown of PKD abrogated the neuroprotective effects of HSP27 as well as the interaction with and inhibition of ASK1 signaling. Taken together, these data demonstrate that HSP27 requires PKD-mediated phosphorylation for its suppression of ASK1 cell death signaling and neuroprotection against ischemic injury. PMID:22357851

Pertussis Toxin Exploits Host Cell Signaling Pathways Induced by Meningitis-Causing E. coli K1-RS218 and Enhances Adherence of Monocytic THP-1 Cells to Human Cerebral Endothelial Cells.

PubMed

Starost, Laura Julia; Karassek, Sascha; Sano, Yasuteru; Kanda, Takashi; Kim, Kwang Sik; Dobrindt, Ulrich; Rüter, Christian; Schmidt, Marcus Alexander

2016-10-13

Pertussis toxin (PTx), the major virulence factor of the whooping cough-causing bacterial pathogen Bordetella pertussis , permeabilizes the blood-brain barrier (BBB) in vitro and in vivo. Breaking barriers might promote translocation of meningitis-causing bacteria across the BBB, thereby facilitating infection. PTx activates several host cell signaling pathways exploited by the neonatal meningitis-causing Escherichia coli K1-RS218 for invasion and translocation across the BBB. Here, we investigated whether PTx and E. coli K1-RS218 exert similar effects on MAPK p38, NF-κB activation and transcription of downstream targets in human cerebral endothelial TY10 cells using qRT-PCR, Western blotting, and ELISA in combination with specific inhibitors. PTx and E. coli K1-RS218 activate MAPK p38, but only E. coli K1-RS218 activates the NF-κB pathway. mRNA and protein levels of p38 and NF-κB downstream targets including IL-6, IL-8, CxCL-1, CxCL-2 and ICAM-1 were increased. The p38 specific inhibitor SB203590 blocked PTx-enhanced activity, whereas E. coli K1-RS218's effects were inhibited by the NF-κB inhibitor Bay 11-7082. Further, we found that PTx enhances the adherence of human monocytic THP-1 cells to human cerebral endothelial TY10 cells, thereby contributing to enhanced translocation. These modulations of host cell signaling pathways by PTx and meningitis-causing E. coli support their contributions to pathogen and monocytic THP-1 cells translocation across the BBB.

Pertussis Toxin Exploits Host Cell Signaling Pathways Induced by Meningitis-Causing E. coli K1-RS218 and Enhances Adherence of Monocytic THP-1 Cells to Human Cerebral Endothelial Cells

PubMed Central

Starost, Laura Julia; Karassek, Sascha; Sano, Yasuteru; Kanda, Takashi; Kim, Kwang Sik; Dobrindt, Ulrich; Rüter, Christian; Schmidt, Marcus Alexander

2016-01-01

Pertussis toxin (PTx), the major virulence factor of the whooping cough-causing bacterial pathogen Bordetella pertussis, permeabilizes the blood–brain barrier (BBB) in vitro and in vivo. Breaking barriers might promote translocation of meningitis-causing bacteria across the BBB, thereby facilitating infection. PTx activates several host cell signaling pathways exploited by the neonatal meningitis-causing Escherichia coli K1-RS218 for invasion and translocation across the BBB. Here, we investigated whether PTx and E. coli K1-RS218 exert similar effects on MAPK p38, NF-κB activation and transcription of downstream targets in human cerebral endothelial TY10 cells using qRT-PCR, Western blotting, and ELISA in combination with specific inhibitors. PTx and E. coli K1-RS218 activate MAPK p38, but only E. coli K1-RS218 activates the NF-κB pathway. mRNA and protein levels of p38 and NF-κB downstream targets including IL-6, IL-8, CxCL-1, CxCL-2 and ICAM-1 were increased. The p38 specific inhibitor SB203590 blocked PTx-enhanced activity, whereas E. coli K1-RS218’s effects were inhibited by the NF-κB inhibitor Bay 11-7082. Further, we found that PTx enhances the adherence of human monocytic THP-1 cells to human cerebral endothelial TY10 cells, thereby contributing to enhanced translocation. These modulations of host cell signaling pathways by PTx and meningitis-causing E. coli support their contributions to pathogen and monocytic THP-1 cells translocation across the BBB. PMID:27754355

Infarct-remodeled myocardium is receptive to protection by isoflurane postconditioning: role of protein kinase B/Akt signaling.

PubMed

Feng, Jianhua; Fischer, Gregor; Lucchinetti, Eliana; Zhu, Min; Bestmann, Lukas; Jegger, David; Arras, Margarete; Pasch, Thomas; Perriard, Jean-Claude; Schaub, Marcus C; Zaugg, Michael

2006-05-01

Postinfarct remodeled myocardium exhibits numerous structural and biochemical alterations. So far, it is unknown whether postconditioning elicited by volatile anesthetics can also provide protection in the remodeled myocardium. Myocardial infarct was induced in male Wistar rats by ligation of the left anterior descending coronary artery. Six weeks later, hearts were buffer-perfused and exposed to 40 min of ischemia followed by 90 min of reperfusion. Anesthetic postconditioning was induced by 15 min of 2.1 vol% isoflurane. In some experiments, LY294002 (15 microM), a phosphatidylinositol 3-kinase inhibitor, was coadministered with isoflurane. Masson's trichrome staining, immunohistochemistry, Western blot analysis, and reverse-transcription polymerase chain reaction served to confirm remodeling. In buffer-perfused hearts, functional recovery was recorded, and acute infarct size was measured using 1% triphenyltetrazolium chloride staining and lactate dehydrogenase release during reperfusion. Western blot analysis was used to determine phosphorylation of reperfusion injury salvage kinases including protein kinase B/Akt and its downstream targets after 15 min of reperfusion. Infarct hearts exhibited typical macroscopic and molecular changes of remodeling. Isoflurane postconditioning improved functional recovery and decreased acute infarct size, as determined by triphenyltetrazolium (35 +/- 5% in unprotected hearts vs. 8 +/- 3% in anesthetic postconditioning; P < 0.05) and lactate dehydrogenase release. This protection was abolished by LY294002, which inhibited phosphorylation of protein kinase B/Akt and its downstream targets glycogen synthase kinase 3beta, endothelial nitric oxide synthase, and p70S6 kinase. Infarct-remodeled myocardium is receptive to protection by isoflurane postconditioning via protein kinase B/Akt signaling. This is the first time to demonstrate that anesthetic postconditioning retains its marked protection in diseased myocardium.

Anti-inflammatory Effects of Cardamonin in Ovarian Cancer Cells Are Mediated via mTOR Suppression.

PubMed

Chen, Huajiao; Shi, Daohua; Niu, Peiguang; Zhu, Yanting; Zhou, Jintuo

2018-05-17

Cardamonin exhibits a variety of pharmacological activities including anti-inflammatory and antitumor, which are correlated with the inhibition of nuclear factor-kappaB and the mammalian target of rapamycin, respectively. However, whether the anti-inflammatory effects of cardamonin are mediated by the mammalian target of rapamycin remains unknown. In this study, ovarian cancer SKOV3 cells were cultured with lipopolysaccharide to induce inflammation, and the inhibitory effects and underlying molecular mechanisms of cardamonin were investigated using specific inhibitors of the mammalian target of rapamycin and the nuclear factor-kappaB pathway (rapamycin and pyrrolidine dithiocarbamate, respectively). Our results indicated that cardamonin inhibited the viability of normal and lipopolysaccharide-pretreated SKOV3 cells in a concentration-dependent manner. In accordance with rapamycin, the activation of the mammalian target of rapamycin and its downstream target, ribosomal protein S6 kinase 1, was inhibited by cardamonin, while pyrrolidine dithiocarbamate substantially blocked nuclear factor-kappaB activation and mildly inhibited the phosphorylation of the mammalian target of rapamycin and ribosomal protein S6 kinase 1. Pretreated with pyrrolidine dithiocarbamate, the effect of cardamonin on the mammalian target of rapamycin signalling was not affected, but the expression of inflammatory factors was further reduced. In cells pretreated with rapamycin, the inhibitory effects of cardamonin were completely suppressed with regards to the phosphorylation of the mammalian target of rapamycin, ribosomal protein S6 kinase 1, TNF- α , and interleukin-6, and nuclear factor-kappaB p65 protein expression was decreased. In conclusion, our findings indicate that the anti-inflammatory effects of cardamonin are correlated with mammalian target of rapamycin inhibition. Georg Thieme Verlag KG Stuttgart · New York.

Inhibiting the Epidermal Growth Factor Receptor | Center for Cancer Research

Cancer.gov

The Epidermal Growth Factor Receptor (EGFR) is a widely distributed cell surface receptor that responds to several extracellular signaling molecules through an intracellular tyrosine kinase, which phosphorylates target enzymes to trigger a downstream molecular cascade. Since the discovery that EGFR mutations and amplifications are critical in a number of cancers, efforts have

Novel angiogenesis inhibitory activity in cinnamon extract blocks VEGFR2 kinase and downstream signaling

USDA-ARS?s Scientific Manuscript database

VEGF is one of the most critical factors that induce angiogenesis, and has thus become an attractive target for anti-angiogenesis treatment. However, most of the current anti-VEGF agents that often cause side effects cannot be recommended for long term use. Identification of natural VEGF inhibitors...

A Memory Advantage for Untrustworthy Faces

ERIC Educational Resources Information Center

Rule, Nicholas O.; Slepian, Michael L.; Ambady, Nalini

2012-01-01

Inferences of others' social traits from their faces can influence how we think and behave towards them, but little is known about how perceptions of people's traits may affect downstream cognitions, such as memory. Here we explored the relationship between targets' perceived social traits and how well they were remembered following a single brief…

Targeting the r(CGG) repeats that cause FXTAS with modularly assembled small molecules and oligonucleotides.

PubMed

Tran, Tuan; Childs-Disney, Jessica L; Liu, Biao; Guan, Lirui; Rzuczek, Suzanne; Disney, Matthew D

2014-04-18

We designed small molecules that bind the structure of the RNA that causes fragile X-associated tremor ataxia syndrome (FXTAS), an incurable neuromuscular disease. FXTAS is caused by an expanded r(CGG) repeat (r(CGG)(exp)) that inactivates a protein regulator of alternative pre-mRNA splicing. Our designed compounds modulate r(CGG)(exp) toxicity in cellular models of FXTAS, and pull-down experiments confirm that they bind r(CGG)(exp) in vivo. Importantly, compound binding does not affect translation of the downstream open reading frame (ORF). We compared molecular recognition properties of our optimal compound to oligonucleotides. Studies show that r(CGG)(exp)'s self-structure is a significant energetic barrier for oligonucleotide binding. A fully modified 2'-OMethyl phosphorothioate is incapable of completely reversing an FXTAS-associated splicing defect and inhibits translation of the downstream ORF, which could have deleterious effects. Taken together, these studies suggest that a small molecule that recognizes structure may be more well suited for targeting highly structured RNAs that require strand invasion by a complementary oligonucleotide.

Targeting the r(CGG) Repeats That Cause FXTAS with Modularly Assembled Small Molecules and Oligonucleotides

PubMed Central

2015-01-01

We designed small molecules that bind the structure of the RNA that causes fragile X-associated tremor ataxia syndrome (FXTAS), an incurable neuromuscular disease. FXTAS is caused by an expanded r(CGG) repeat (r(CGG)exp) that inactivates a protein regulator of alternative pre-mRNA splicing. Our designed compounds modulate r(CGG)exp toxicity in cellular models of FXTAS, and pull-down experiments confirm that they bind r(CGG)expin vivo. Importantly, compound binding does not affect translation of the downstream open reading frame (ORF). We compared molecular recognition properties of our optimal compound to oligonucleotides. Studies show that r(CGG)exp’s self-structure is a significant energetic barrier for oligonucleotide binding. A fully modified 2′-OMethyl phosphorothioate is incapable of completely reversing an FXTAS-associated splicing defect and inhibits translation of the downstream ORF, which could have deleterious effects. Taken together, these studies suggest that a small molecule that recognizes structure may be more well suited for targeting highly structured RNAs that require strand invasion by a complementary oligonucleotide. PMID:24506227

Nutrient/TOR-dependent regulation of RNA polymerase III controls tissue and organismal growth in Drosophila

PubMed Central

Marshall, Lynne; Rideout, Elizabeth J; Grewal, Savraj S

2012-01-01

The nutrient/target-of-rapamycin (TOR) pathway has emerged as a key regulator of tissue and organismal growth in metazoans. The signalling components of the nutrient/TOR pathway are well defined; however, the downstream effectors are less understood. Here, we show that the control of RNA polymerase (Pol) III-dependent transcription is an essential target of TOR in Drosophila. We find that TOR activity controls Pol III in growing larvae via inhibition of the repressor Maf1 and, in part, via the transcription factor Drosophila Myc (dMyc). Moreover, we show that loss of the Pol III factor, Brf, leads to reduced tissue and organismal growth and prevents TOR-induced cellular growth. TOR activity in the larval fat body, a tissue equivalent to vertebrate fat or liver, couples nutrition to insulin release from the brain. Accordingly, we find that fat-specific loss of Brf phenocopies nutrient limitation and TOR inhibition, leading to decreased systemic insulin signalling and reduced organismal growth. Thus, stimulation of Pol III is a key downstream effector of TOR in the control of cellular and systemic growth. PMID:22367393

Physical and functional interactions between Drosophila TRAF2 and Pelle kinase contribute to Dorsal activation.

PubMed

Shen, B; Liu, H; Skolnik, E Y; Manley, J L

2001-07-17

Signaling through the Toll receptor is required for dorsal/ventral polarity in Drosophila embryos, and also plays an evolutionarily conserved role in the immune response. Upon ligand binding, Toll appears to multimerize and activate the associated kinase, Pelle. However, the immediate downstream targets of Pelle have not been identified. Here we show that Drosophila tumor necrosis factor receptor-associated factor 2 (dTRAF2), a homologue of human TRAF6, physically and functionally interacts with Pelle, and is phosphorylated by Pelle in vitro. Importantly, dTRAF2 and Pelle cooperate to activate Dorsal synergistically in cotransfected Schneider cells. Deletion of the C-terminal TRAF domain of dTRAF2 enhances Dorsal activation, perhaps reflecting the much stronger interaction of the mutant protein with phosphorylated, active Pelle. Taken together, our results indicate that Pelle and dTRAF2 physically and functionally interact, and that the TRAF domain acts as a regulator of this interaction. dTRAF2 thus appears to be a downstream target of Pelle. We discuss these results in the context of Toll signaling in flies and mammals.

Discovery and preclinical pharmacology of a selective ATP-competitive Akt inhibitor (GDC-0068) for the treatment of human tumors.

PubMed

Blake, James F; Xu, Rui; Bencsik, Josef R; Xiao, Dengming; Kallan, Nicholas C; Schlachter, Stephen; Mitchell, Ian S; Spencer, Keith L; Banka, Anna L; Wallace, Eli M; Gloor, Susan L; Martinson, Matthew; Woessner, Richard D; Vigers, Guy P A; Brandhuber, Barbara J; Liang, Jun; Safina, Brian S; Li, Jun; Zhang, Birong; Chabot, Christine; Do, Steven; Lee, Leslie; Oeh, Jason; Sampath, Deepak; Lee, Brian B; Lin, Kui; Liederer, Bianca M; Skelton, Nicholas J

2012-09-27

The discovery and optimization of a series of 6,7-dihydro-5H-cyclopenta[d]pyrimidine compounds that are ATP-competitive, selective inhibitors of protein kinase B/Akt is reported. The initial design and optimization was guided by the use of X-ray structures of inhibitors in complex with Akt1 and the closely related protein kinase A. The resulting compounds demonstrate potent inhibition of all three Akt isoforms in biochemical assays and poor inhibition of other members of the cAMP-dependent protein kinase/protein kinase G/protein kinase C extended family and block the phosphorylation of multiple downstream targets of Akt in human cancer cell lines. Biological studies with one such compound, 28 (GDC-0068), demonstrate good oral exposure resulting in dose-dependent pharmacodynamic effects on downstream biomarkers and a robust antitumor response in xenograft models in which the phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin pathway is activated. 28 is currently being evaluated in human clinical trials for the treatment of cancer.

Inhibition of mammalian target of rapamycin activation in the rostral anterior cingulate cortex attenuates pain-related aversion in rats.

PubMed

Lu, Bo; Jiang, Jingyan; Sun, Jianliang; Xiao, Chun; Meng, Bo; Zheng, Jinwei; Li, Xiaoyu; Wang, Ruichun; Wu, Guorong; Chen, Junping

2016-09-01

Pain is a complex experience that comprises both sensory and affective dimensions. Mammalian target of rapamycin (mTOR) plays an important role in the modulation of neuronal plasticity associated with the pathogenesis of pain sensation. However, the role of mTOR in pain affect is unclear. Using a formalin-induced conditioned place avoidance (F-CPA) test, the current study investigated the effects of the mTOR specific inhibitor rapamycin on noxious stimulation induced aversion in the rostral anterior cingulate cortex (rACC). Intraplantar injection of 5% formalin was associated with significant activation of mTOR, as well as p70 ribosomal S6 protein (p70S6K), its downstream effector, in the rACC. The inhibition of mTOR activation with rapamycin disrupted pain-related aversion; however, this inhibition did not affect formalin-induced spontaneous nociceptive behaviors in rats. These findings demonstrated for the first time that mTOR and its downstream pathway in the rACC contribute to the induction of pain-related negative emotion. Copyright © 2016 Elsevier B.V. All rights reserved.

PECAM1 regulates flow-mediated Gab1 tyrosine phosphorylation and signaling

PubMed Central

Xu, Suowen; Ha, Chang Hoon; Wang, Weiye; Xu, Xiangbin; Yin, Meimei; Jin, Felix Q.; Mastrangelo, Michael; Koroleva, Marina; Fujiwara, Keigi; Jin, Zheng Gen

2016-01-01

Endothelial dysfunction, characterized by impaired activation of endothelial nitric oxide (NO) synthase (eNOS) and ensued decrease of NO production, is a common mechanism of various cardiovascular pathologies, including hypertension and atherosclerosis. Laminar blood flow-mediated specific signaling cascades modulate vascular endothelial cells (ECs) structure and functions. We have previously shown that flow-stimulated Gab1 (Grb2-associated binder-1) tyrosine phosphorylation mediates eNOS activation in ECs, which in part confers laminar flow atheroprotective action. However, the molecular mechanisms whereby flow regulates Gab1 tyrosine phosphorylation and its downstream signaling events remain unclear. Here we show that platelet endothelial cell adhesion molecule-1 (PECAM1), a key molecule in an endothelial mechanosensing complex, specifically mediates Gab1 tyrosine phosphorylation and its downstream Akt and eNOS activation in ECs upon flow rather than hepatocyte growth factor (HGF) stimulation. Small interfering RNA (siRNA) targeting PECAM1 abolished flow- but not HGF-induced Gab1 tyrosine phosphorylation and Akt, eNOS activation as well as Gab1 membrane translocation. Protein-tyrosine phosphatase SHP2, which has been shown to interact with Gab1, was involved in flow signaling and HGF signaling, as SHP2 siRNA diminished the flow- and HGF-induced Gab1 tyrosine phosphorylation, membrane localization and downstream signaling. Pharmacological inhibition of PI3K decreased flow-, but not HGF-mediated Gab1 phosphorylation and membrane localization as well as eNOS activation. Finally, we observed that flow-mediated Gab1 and eNOS phosphorylation in vivo induced by voluntary wheel running was reduced in PECAM1 knockout mice. These results demonstrate a specific role of PECAM1 in flow-mediated Gab1 tyrosine phosphorylation and eNOS signaling in ECs. PMID:26706435

Downstream effects of ROCK signaling in cultured human corneal stromal cells: microarray analysis of gene expression.

PubMed

Harvey, Stephen A K; Anderson, Susan C; SundarRaj, Nirmala

2004-07-01

Rho-associated coiled-coil-containing protein kinase (ROCK) is a downstream target of Rho GTPase signaling and regulates the assembly of stress fibers. Previous reports indicate that Rho/ROCK signaling is involved in the regulation of several cellular processes, some of which may be cell-type specific and are probably critical to corneal stromal cell activation. The present study identified ROCK-regulated gene expression in corneal stromal cells. Corneal stromal cells derived from eyes of three different donors were cultured to yield the following designated phenotypes: baseline fibroblasts (DMEM with 10% serum), activated fibroblasts (10% serum+bFGF+heparin), and myofibroblasts (1% serum+TGF-beta 1). Cells were exposed to the ROCK inhibitor Y-27632 or vehicle for 12 hours, and transcript levels altered by ROCK inhibition were identified with oligonucleotide microarrays (GeneChips; Affymetrix, Santa Clara, CA). In these phenotypes, Y-27632 caused marked (twofold or more) increases or decreases in 14/4, 12/3, and 15/10 transcripts. In both fibroblast groups Y-27632-treatment increased expression of endothelin receptors and of parathyroid hormone-like hormone. The upregulation of alpha-smooth muscle actin in myofibroblasts was attenuated by Y-27632. Combining data from all groups identified ROCK-supported (Y-27632 inhibitable) expression of 10 transcripts, including ribonucleotide reductase M2, the cyclin B1-CDC2-CKS2 system, and four mitotic spindle-associated proteins. ROCK inhibition causes broad inhibition of DNA synthesis and mitosis and causes changes that are different between (bFGF-activated) fibroblasts and (TGF-beta 1-induced) myofibroblasts. Thus, Rho/ROCK signaling regulates both common and distinct downstream events in corneal stromal cells activated (differentiated) to fibroblast or myofibroblast phenotype.

Growth of chronic myeloid leukemia cells is inhibited by infection with Ad-SH2-HA adenovirus that disrupts Grb2-Bcr-Abl complexes.

PubMed

Peng, Zhi; Luo, Hong-Wei; Yuan, Ying; Shi, Jing; Huang, Shi-Feng; Li, Chun-Li; Cao, Wei-Xi; Huang, Zong-Gan; Feng, Wen-Li

2011-05-01

The persistence of Bcr-Abl-positive cells in patients on imatinib therapy indicates that inhibition of the Bcr-Abl kinase activity alone might not be sufficient to eradicate the leukemia cells. Many downstream effectors of Bcr-Abl have been described, including activation of both the Grb2-SoS-Ras-MAPK and Grb2-Gab2-PI3K-Akt pathways. The Bcr-Abl-Grb2 interaction, which is mediated by the direct interaction of the Grb2 SH2 domain with the phospho-Bcr-Abl Y177, is required for activation of these signaling pathways. Therefore, disrupting their interaction represents a potential therapeutic strategy for inhibiting the oncogenic downstream signals of Bcr-Abl. Adenovirus Ad-SH2-HA expressing the Grb2 SH2 domain was constructed and applied in this study. As expected, Ad-SH2-HA efficiently infected CML cells and functioned by binding to the phospho-Bcr-Abl Y177 site, competitively disrupting the Grb2 SH2-phospho-Bcr-Abl Y177 complex. They induced potent anti-proliferation and apoptosis-inducing effects in CML cell lines. Moreover, the Ras, MAPK and Akt activities were significantly reduced in the Ad-SH2-HA treated cells. These were not observed with the point-mutated control adenovirus Ad-Sm-HA with abolished phospho-Bcr-Abl Y177 binding sites. These data indicate that, in addition to the direct targeting of Bcr-Abl, selective inhibition of its downstream signaling pathways may be a therapeutic option for CML, and the Ad-SH2-HA-mediated killing strategy could be explored as a promising anti-leukemia agent in CML.

Ewing’s Sarcoma: Overcoming the Therapeutic Plateau

PubMed Central

Subbiah, Vivek; Kurzrock, Razelle

2013-01-01

The hallmark of Ewing’s sarcoma (EWS) is a translocation -- t(11;22)(q24;q12) -- that most frequently results in the EWS/FLI1 aberrant chimeric gene. Because EWS afflicts young patients, it stands out among the diverse sarcoma subtypes. The frontline, standard-of-care cytotoxic chemotherapy regimens produce minimal benefit in patients with metastases at presentation or those with relapsed disease. While the outcomes of chemorefractory EWS patients are poor, recent developments have led to the promising use of targeted therapy. Specifically, inhibition of insulin-like growth factor 1 receptor (IGF1R) signaling and the mammalian target of rapamycin (mTOR) pathways has emerged as a targeted therapy in EWS, with select patients experiencing dramatic therapeutic responses. However, targeted therapies in general, and these responders in particular, are faced with the ultimate conundrum of eventual resistance. To optimize response, combining IGF1R and mTOR inhibitor-based regimens with chemotherapy in the upfront setting in newly diagnosed high-risk EWS may clarify the true benefit of IGF1R inhibitors in these patients. Another option is to explore novel targeted multikinase inhibitors and poly(ADP-ribose) polymerase (PARP) inhibitors, which have experienced a surge in supporting preclinical data. Drugs inhibiting the downstream targets of EWS/FLI1 are also in preclinical development. However, ultimately, the underlying biomarker correlates of resistance and response must be delineated along with ways to overcome them. Novel agents, together with integration of advances in multimodal approaches (including surgery and radiation), as well as offering targeted therapies early in the disease course represent new strategies for confronting the challenges of EWS. PMID:22742646

[Perspectives of the stomach cancer treatment: the introduction of molecular targeted therapy and the hope for cure].

PubMed

Cheung, Dae Young; Kim, Jae Kwang

2013-03-25

The overall survival of patients with gastric cancer has increased markedly in Korea, even higher than those of developed nations in Western world. It is due to the virtue of Korean National Cancer Screening Program and nowadays more than half of patients are diagnosed at the early stage of gastric cancer. However, for patients with unresectable gastric cancer, the outcomes of traditional cytotoxic chemotherapy regimens stay at a median survival of 9-11 months. The knowledge of cancer biology and the data from gene expression profiling has explosively expanded. Alternations in the expression of receptor tyrosine kinases pathways including Human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), phosphatydyl inositol 3 kinase/mammalian target of rapamycin (PI3K/mTOR), hepatocyte growth factor receptor (HGFR/MET), and fibroblast growth factor receptor (FGFR) were proved to be critical in cancer cell survival and biological agents targeting those altered receptor tyrosine kinases, their ligands and downstream effector molecules are developed for anti-cancer purpose. Until now, only trastuzumab succeeded to significantly increase overall survival of patients with HER2 overexpressing gastric cancer. Other agents including bevacizumab, gefitinib, erlotinib, and lapatinib failed to achieve the efficacy in survival gain over standard chemotherapy. Insights about the variations between regions, races, and individuals call for the effort to find reliable predictive biomarkers for drug efficacy and to design finely stratified clinical trials. Compared to current treatment paradigms, it is hoped that molecularly targeted treatment along with conventional cytotoxic chemotherapy will lead to significant gains in survival.

Optimized scalable network switch

DOEpatents

Blumrich, Matthias A [Ridgefield, CT; Chen, Dong [Croton On Hudson, NY; Coteus, Paul W [Yorktown Heights, NY; Gara, Alan G [Mount Kisco, NY; Giampapa, Mark E [Irvington, NY; Heidelberger, Philip [Cortlandt Manor, NY; Steinmacher-Burow, Burkhard D [Mount Kisco, NY; Takken, Todd E [Mount Kisco, NY; Vranas, Pavlos M [Bedford Hills, NY

2007-12-04

In a massively parallel computing system having a plurality of nodes configured in m multi-dimensions, each node including a computing device, a method for routing packets towards their destination nodes is provided which includes generating at least one of a 2m plurality of compact bit vectors containing information derived from downstream nodes. A multilevel arbitration process in which downstream information stored in the compact vectors, such as link status information and fullness of downstream buffers, is used to determine a preferred direction and virtual channel for packet transmission. Preferred direction ranges are encoded and virtual channels are selected by examining the plurality of compact bit vectors. This dynamic routing method eliminates the necessity of routing tables, thus enhancing scalability of the switch.

Optimized scalable network switch

DOEpatents

Blumrich, Matthias A.; Chen, Dong; Coteus, Paul W.

2010-02-23

In a massively parallel computing system having a plurality of nodes configured in m multi-dimensions, each node including a computing device, a method for routing packets towards their destination nodes is provided which includes generating at least one of a 2m plurality of compact bit vectors containing information derived from downstream nodes. A multilevel arbitration process in which downstream information stored in the compact vectors, such as link status information and fullness of downstream buffers, is used to determine a preferred direction and virtual channel for packet transmission. Preferred direction ranges are encoded and virtual channels are selected by examining the plurality of compact bit vectors. This dynamic routing method eliminates the necessity of routing tables, thus enhancing scalability of the switch.

European downstream oil industry safety performance : statistical summary of reported incidents, 1996

DOT National Transportation Integrated Search

1997-12-01

This report is the third by CONCAWE reviewing the safety performance of the downstream oil industry in Western Europe. It includes the results of 28 companies which together represent over 90% of the oil refining capacity in Europe. It is therefore a...

Direct binding of arsenic trioxide to AMPK and generation of inhibitory effects on acute myeloid leukemia precursors

PubMed Central

Beauchamp, Elspeth M.; Kosciuczuk, Ewa M.; Serrano, Ruth; Nanavati, Dhaval; Swindell, Elden P.; Viollet, Benoit; O'Halloran, Thomas V.; Altman, Jessica K.; Platanias, Leonidas C.

2014-01-01

Arsenic trioxide (As2O3) exhibits potent antineoplastic effects and is used extensively in clinical oncology for the treatment of a subset of patients with acute myeloid leukemia (AML). Although As2O3 is known to regulate activation of several signaling cascades, the key events, accounting for its anti-leukemic properties, remain to be defined. We provide evidence that arsenic can directly bind to cysteine 299 in AMPKα and inhibit its activity. This inhibition of AMPK by arsenic is required in part for its cytotoxic effects on primitive leukemic progenitors from patients with AML, while concomitant treatment with an AMPK activator antagonizes in vivo the arsenic-induced antileukemic effects in a xenograft AML mouse model. A consequence of AMPK inhibition is activation of the mTOR pathway as a negative regulatory feedback loop. However, when AMPK expression is lost, arsenic-dependent activation of the kinase RSK downstream of MAPK activity compensates the generation of regulatory feedback signals through phosphorylation of downstream mTOR targets. Thus, therapeutic regimens with arsenic trioxide will need to include inhibitors of both the mTOR and RSK pathways in combination to prevent engagement of negative feedback loops and maximize antineoplastic responses. PMID:25344585

The role of Mixer in patterning the early Xenopus embryo.

PubMed

Kofron, Matt; Wylie, Chris; Heasman, Janet

2004-05-01

The transcription factor VegT, is required in early Xenopus embryos for the formation of both the mesoderm and endoderm germ layers. Inherited as a maternal mRNA localized only in vegetal cells, VegT activates the transcription of a large number of transcription factors, as well as signaling ligands that induce cells in the vegetal mass to form endoderm, and the marginal zone to form mesoderm. It is important now to understand the extent to which transcription factors downstream of VegT play individual, or overlapping, roles in the specification and patterning of the endoderm and mesoderm. In addition, it is important to understand the mechanism that specifies the boundary between endoderm and mesoderm. One of the downstream targets of VegT, the homeodomain protein Mixer, is expressed at high levels at the mesoderm/endoderm boundary at the late blastula stage. We therefore examined its functions by blocking its translation using morpholino oligos. In Mixer-depleted embryos, the expression of many signaling ligands and transcription factors was affected. In particular, we found that the expression of several genes, including several normally expressed in mesoderm, was upregulated. Functional assays of Mixer-depleted vegetal cells showed that they have increased mesoderm-inducing activity. This demonstrates that Mixer plays an essential role in controlling the amount of mesoderm induction by the vegetal cells.

Erosional Consequence of Saltcedar Control

NASA Astrophysics Data System (ADS)

Vincent, Kirk R.; Friedman, Jonathan M.; Griffin, Eleanor R.

2009-08-01

Removal of nonnative riparian trees is accelerating to conserve water and improve habitat for native species. Widespread control of dominant species, however, can lead to unintended erosion. Helicopter herbicide application in 2003 along a 12-km reach of the Rio Puerco, New Mexico, eliminated the target invasive species saltcedar ( Tamarix spp.), which dominated the floodplain, as well as the native species sandbar willow ( Salix exigua Nuttall), which occurred as a fringe along the channel. Herbicide application initiated a natural experiment testing the importance of riparian vegetation for bank stability along this data-rich river. A flood three years later eroded about 680,000 m3 of sediment, increasing mean channel width of the sprayed reach by 84%. Erosion upstream and downstream from the sprayed reach during this flood was inconsequential. Sand eroded from channel banks was transported an average of 5 km downstream and deposited on the floodplain and channel bed. Although vegetation was killed across the floodplain in the sprayed reach, erosion was almost entirely confined to the channel banks. The absence of dense, flexible woody stems on the banks reduced drag on the flow, leading to high shear stress at the toe of the banks, fluvial erosion, bank undercutting, and mass failure. The potential for increased erosion must be included in consideration of phreatophyte control projects.

Erosional consequence of saltcedar control

USGS Publications Warehouse

Vincent, K.R.; Friedman, J.M.; Griffin, E.R.

2009-01-01

Removal of nonnative riparian trees is accelerating to conserve water and improve habitat for native species. Widespread control of dominant species, however, can lead to unintended erosion. Helicopter herbicide application in 2003 along a 12-km reach of the Rio Puerco, New Mexico, eliminated the target invasive species saltcedar (Tamarix spp.), which dominated the floodplain, as well as the native species sandbar willow (Salix exigua Nuttall), which occurred as a fringe along the channel. Herbicide application initiated a natural experiment testing the importance of riparian vegetation for bank stability along this data-rich river. A flood three years later eroded about 680,000 m3 of sediment, increasing mean channel width of the sprayed reach by 84%. Erosion upstream and downstream from the sprayed reach during this flood was inconsequential. Sand eroded from channel banks was transported an average of 5 km downstream and deposited on the floodplain and channel bed. Although vegetation was killed across the floodplain in the sprayed reach, erosion was almost entirely confined to the channel banks. The absence of dense, flexible woody stems on the banks reduced drag on the flow, leading to high shear stress at the toe of the banks, fluvial erosion, bank undercutting, and mass failure. The potential for increased erosion must be included in consideration of phreatophyte control projects. ?? 2009 U.S. Government.

Microfluidic immunocapture of circulating pancreatic cells using parallel EpCAM and MUC1 capture: characterization, optimization and downstream analysis.

PubMed

Thege, Fredrik I; Lannin, Timothy B; Saha, Trisha N; Tsai, Shannon; Kochman, Michael L; Hollingsworth, Michael A; Rhim, Andrew D; Kirby, Brian J

2014-05-21

We have developed and optimized a microfluidic device platform for the capture and analysis of circulating pancreatic cells (CPCs) and pancreatic circulating tumor cells (CTCs). Our platform uses parallel anti-EpCAM and cancer-specific mucin 1 (MUC1) immunocapture in a silicon microdevice. Using a combination of anti-EpCAM and anti-MUC1 capture in a single device, we are able to achieve efficient capture while extending immunocapture beyond single marker recognition. We also have detected a known oncogenic KRAS mutation in cells spiked in whole blood using immunocapture, RNA extraction, RT-PCR and Sanger sequencing. To allow for downstream single-cell genetic analysis, intact nuclei were released from captured cells by using targeted membrane lysis. We have developed a staining protocol for clinical samples, including standard CTC markers; DAPI, cytokeratin (CK) and CD45, and a novel marker of carcinogenesis in CPCs, mucin 4 (MUC4). We have also demonstrated a semi-automated approach to image analysis and CPC identification, suitable for clinical hypothesis generation. Initial results from immunocapture of a clinical pancreatic cancer patient sample show that parallel capture may capture more of the heterogeneity of the CPC population. With this platform, we aim to develop a diagnostic biomarker for early pancreatic carcinogenesis and patient risk stratification.

Ecological effects on streams from forest fertilization; literature review and conceptual framework for future study in the western Cascades

USGS Publications Warehouse

Anderson, Chauncey W.

2002-03-19

Studies of the responses of stream biota to fertilization have been rare and have targeted either immediate, toxicity-based responses or used methods insensitive to ongoing ecological processes. This report reviews water-quality studies following forest fertilizations, emphasizing Cascade streams in the Pacific Northwest and documented biological responses in those streams. A conceptual model predicting potential ecological response to fertilization, which includes effects on algal growth and primary production, is presented. In this model, applied fertilizer nitrogen reaching streams is mostly exported during winter. However, some nitrogen retained in soils or stream and riparian areas may become available to aquatic biota during spring and summer. Biological responses may be minimal in small streams nearest to application because of light limitation, but may be elevated downstream where light is sufficient to allow algal growth. Ultimately, algal response could be greatest in downstream reaches, although ambient nutrient concentrations remain low due to uptake and benthic nutrient recycling. Ground-water flow paths and hyporheic processing could be critical in determining the fate of applied nitrogen. A framework is provided for testing this response in the Little River watershed, a tributary to the North Umpqua River, Oregon, at basic and intensive levels of investigation.

Mitochondrial Protein PGAM5 Regulates Mitophagic Protection against Cell Necroptosis.

PubMed

Lu, Wei; Sun, Junhui; Yoon, Jeong Seon; Zhang, Yan; Zheng, Lixin; Murphy, Elizabeth; Mattson, Mark P; Lenardo, Michael J

2016-01-01

Necroptosis as a molecular program, rather than simply incidental cell death, was established by elucidating the roles of receptor interacting protein (RIP) kinases 1 and 3, along with their downstream partner, mixed lineage kinase-like domain protein (MLKL). Previous studies suggested that phosphoglycerate mutase family member 5 (PGAM5), a mitochondrial protein that associates with RIP1/RIP3/MLKL complex, promotes necroptosis. We have generated mice deficient in the pgam5 gene and surprisingly found PGAM5-deficiency exacerbated rather than reduced necroptosis in response to multiple in vitro and in vivo necroptotic stimuli, including ischemic reperfusion injury (I/R) in the heart and brain. Electron microscopy, biochemical, and confocal analysis revealed that PGAM5 is indispensable for the process of PINK1 dependent mitophagy which antagonizes necroptosis. The loss of PGAM5/PINK1 mediated mitophagy causes the accumulation of abnormal mitochondria, leading to the overproduction of reactive oxygen species (ROS) that worsen necroptosis. Our results revise the former proposal that PGAM5 acts downstream of RIP1/RIP3 to mediate necroptosis. Instead, PGAM5 protects cells from necroptosis by independently promoting mitophagy. PGAM5 promotion of mitophagy may represent a therapeutic target for stroke, myocardial infarction and other diseases caused by oxidative damage and necroptosis.

Particle Velocity Measuring System

NASA Technical Reports Server (NTRS)

Arndt, G. Dickey (Inventor); Carl, James R. (Inventor)

1998-01-01

Method and apparatus are provided for determining the velocity of individual food particles within a liquid/solid food mixture that is cooked by an aseptic cooking method whereby the food mixture is heated as it flows through a flowline. At least one upstream and at least one downstream microwave transducer are provided to determine the minimum possible travel time of the fastest food particle through the flowline. In one embodiment, the upstream detector is not required. In another embodiment, a plurality of small dipole antenna markers are secured to a plurality of food particles to provide a plurality of signals as the markers pass the upstream and downstream transducers. The dipole antenna markers may also include a non-linear element to reradiate a harmonic frequency of a transmitter frequency. Upstream and downstream transducers include dipole antennas that are matched to the impedance of the food slurry and a signal transmission cable by various impedance matching means including unbalanced feed to the antennas.

Solanum tuberosum StCDPK1 is regulated by miR390 at the posttranscriptional level and phosphorylates the auxin efflux carrier StPIN4 in vitro, a potential downstream target in potato development.

PubMed

Santin, Franco; Bhogale, Sneha; Fantino, Elisa; Grandellis, Carolina; Banerjee, Anjan K; Ulloa, Rita M

2017-02-01

Among many factors that regulate potato tuberization, calcium and calcium-dependent protein kinases (CDPKs) play an important role. CDPK activity increases at the onset of tuber formation with StCDPK1 expression being strongly induced in swollen stolons. However, not much is known about the transcriptional and posttranscriptional regulation of StCDPK1 or its downstream targets in potato development. To elucidate further, we analyzed its expression in different tissues and stages of the life cycle. Histochemical analysis of StCDPK1::GUS (β-glucuronidase) plants demonstrated that StCDPK1 is strongly associated with the vascular system in stems, roots, during stolon to tuber transition, and in tuber sprouts. In agreement with the observed GUS profile, we found specific cis-acting elements in StCDPK1 promoter. In silico analysis predicted miR390 to be a putative posttranscriptional regulator of StCDPK1. Quantitative real time-polymerase chain reaction (qRT-PCR) analysis showed ubiquitous expression of StCDPK1 in different tissues which correlated well with Western blot data except in leaves. On the contrary, miR390 expression exhibited an inverse pattern in leaves and tuber eyes suggesting a possible regulation of StCDPK1 by miR390. This was further confirmed by Agrobacterium co-infiltration assays. In addition, in vitro assays showed that recombinant StCDPK1-6xHis was able to phosphorylate the hydrophilic loop of the auxin efflux carrier StPIN4. Altogether, these results indicate that StCDPK1 expression is varied in a tissue-specific manner having significant expression in vasculature and in tuber eyes; is regulated by miR390 at posttranscriptional level and suggest that StPIN4 could be one of its downstream targets revealing the overall role of this kinase in potato development. © 2016 Scandinavian Plant Physiology Society.

Biological Role and Therapeutic Targeting of TGF-β3 in Glioblastoma.

PubMed

Seystahl, Katharina; Papachristodoulou, Alexandros; Burghardt, Isabel; Schneider, Hannah; Hasenbach, Kathy; Janicot, Michel; Roth, Patrick; Weller, Michael

2017-06-01

Transforming growth factor (TGF)-β contributes to the malignant phenotype of glioblastoma by promoting invasiveness and angiogenesis and creating an immunosuppressive microenvironment. So far, TGF-β 1 and TGF-β 2 isoforms have been considered to act in a similar fashion without isoform-specific function in glioblastoma. A pathogenic role for TGF-β 3 in glioblastoma has not been defined yet. Here, we studied the expression and functional role of endogenous and exogenous TGF-β 3 in glioblastoma models. TGF-β 3 mRNA is expressed in human and murine long-term glioma cell lines as well as in human glioma-initiating cell cultures with expression levels lower than TGF-β 1 or TGF-β 2 in most cell lines. Inhibition of TGF-β 3 mRNA expression by ISTH2020 or ISTH2023, two different isoform-specific phosphorothioate locked nucleic acid (LNA)-modified antisense oligonucleotide gapmers, blocks downstream SMAD2 and SMAD1/5 phosphorylation in human LN-308 cells, without affecting TGF-β 1 or TGF-β 2 mRNA expression or protein levels. Moreover, inhibition of TGF-β 3 expression reduces invasiveness in vitro Interestingly, depletion of TGF-β 3 also attenuates signaling evoked by TGF-β 1 or TGF-β 2 In orthotopic syngeneic (SMA-560) and xenograft (LN-308) in vivo glioma models, expression of TGF-β 3 as well as of the downstream target, plasminogen-activator-inhibitor (PAI)-1 , was reduced, while TGF-β 1 and TGF-β 2 levels were unaffected following systemic treatment with TGF-β 3 -specific antisense oligonucleotides. We conclude that TGF-β 3 might function as a gatekeeper controlling downstream signaling despite high expression of TGF-β 1 and TGF-β 2 isoforms. Targeting TGF-β 3 in vivo may represent a promising strategy interfering with aberrant TGF-β signaling in glioblastoma. Mol Cancer Ther; 16(6); 1177-86. ©2017 AACR . ©2017 American Association for Cancer Research.

Target of rapamycin complex 2 signals to downstream effector yeast protein kinase 2 (Ypk2) through adheres-voraciously-to-target-of-rapamycin-2 protein 1 (Avo1) in Saccharomyces cerevisiae.

PubMed

Liao, Hsien-Ching; Chen, Mei-Yu

2012-02-24

The conserved Ser/Thr kinase target of rapamycin (TOR) serves as a central regulator in controlling cell growth-related functions. There exist two distinct TOR complexes, TORC1 and TORC2, each coupling to specific downstream effectors and signaling pathways. In Saccharomyces cerevisiae, TORC2 is involved in regulating actin organization and maintaining cell wall integrity. Ypk2 (yeast protein kinase 2), a member of the cAMP-dependent, cGMP-dependent, and PKC (AGC) kinase family, is a TORC2 substrate known to participate in actin and cell wall regulation. Employing avo3(ts) mutants with defects in TORC2 functions that are suppressible by active Ypk2, we investigated the molecular interactions involved in mediating TORC2 signaling to Ypk2. GST pulldown assays in yeast lysates demonstrated physical interactions between Ypk2 and components of TORC2. In vitro binding assays revealed that Avo1 directly binds to Ypk2. In avo3(ts) mutants, the TORC2-Ypk2 interaction was reduced and could be restored by AVO1 overexpression, highlighting the important role of Avo1 in coupling TORC2 to Ypk2. The interaction was mapped to an internal region (amino acids 600-840) of Avo1 and a C-terminal region of Ypk2. Ypk2(334-677), a truncated form of Ypk2 containing the Avo1-interacting region, was able to interfere with Avo1-Ypk2 interaction in vitro. Overexpressing Ypk2(334-677) in yeast cells resulted in a perturbation of TORC2 functions, causing defective cell wall integrity, aberrant actin organization, and diminished TORC2-dependent Ypk2 phosphorylation evidenced by the loss of an electrophoretic mobility shift. Together, our data support the conclusion that the direct Avo1-Ypk2 interaction is crucial for TORC2 signaling to the downstream Ypk2 pathway.

MicroRNA-200b Downregulates Oxidation Resistance 1 (Oxr1) Expression in the Retina of Type 1 Diabetes Model

PubMed Central

Murray, Anne R.; Chen, Qian; Takahashi, Yusuke; Zhou, Kevin K.; Park, Kyoungmin; Ma, Jian-xing

2013-01-01

Purpose. MicroRNAs (miRNAs) are known to participate in post-transcriptional regulation of gene expression and are involved in multiple pathogenic processes. Here, we identified miRNA expression changes in the retinas of Akita mice, a genetic model of type 1 diabetes, and investigated the potential role of miRNA in diabetic retinopathy. Methods. Visual function of Akita and control mice was evaluated by electroretinography. MiRNA expression changes in the retinas of Akita mice were identified by miRNA-specific microarray and confirmed by quantitative RT-PCR (qRT-PCR). The potential downstream targets of identified miRNAs were predicted by bioinformatic analysis using web-based applications and confirmed by dual luciferase assay. The mRNA and protein changes of identified downstream targets were examined by qRT-PCR and Western blot analysis. Results. MiRNA-specific microarray and qRT-PCR showed that miR-200b was upregulated significantly in the Akita mouse retina. Sequence analysis and luciferase assay identified oxidation resistance 1 (Oxr1) as a downstream target gene regulated by miR-200b. In a human Müller cell line, MIO-M1, transfection of a miR-200b mimic downregulated Oxr1 expression. Conversely, transfection of MIO-M1 with a miR-200b inhibitor resulted in upregulated Oxr1. Furthermore, overexpression of recombinant Oxr1 attenuated oxidative stress marker, nitration of cellular proteins, and ameliorated apoptosis induced by 4-hydroxynonenal (4-HNE), an oxidative stressor. Similarly, transfection of a miR-200b inhibitor decreased, whereas transfection of miR-200b mimic increased the number of apoptotic cells following 4-HNE treatment. Conclusions. These results suggested that miR-200b–regulated Oxr1 potentially has a protective role in diabetic retinopathy. PMID:23404117

The miR-106b-25 cluster targets Smad7, activates TGF-β signaling, and induces EMT and tumor initiating cell characteristics downstream of Six1 in human breast cancer

PubMed Central

Smith, Anna L.; Iwanaga, Ritsuko; Drasin, David J.; Micalizzi, Douglas S.; Vartuli, Rebecca L; Tan, Aik-Choon; Ford, Heide L.

2012-01-01

The role of TGF-β signaling in tumorigenesis is paradoxical: it can be tumor suppressive or tumor promotional, depending on context. The metastatic regulator, Six1, was recently shown to mediate this switch, providing a novel means to explain this elusive “TGF-β paradox”. Herein, we identify a mechanism by which Six1 activates the tumor promotional arm of TGF-β signaling, via its ability to upregulate the miR-106b-25 microRNA cluster, and further identify a novel function for this cluster of microRNAs. While expression of the miR-106b-25 cluster is known to overcome TGF-β-mediated growth suppression via targeting p21 and BIM, we demonstrate for the first time that this same cluster can additionally target the inhibitory Smad7 protein, resulting in increased levels of the TGF-β type I receptor (TβRI) and downstream activation of TGF-β signaling. We further show that the miR-106b-25 cluster is sufficient to induce an epithelial to mesenchymal transition and a tumor initiating cell phenotype, and that it is required downstream of Six1 to induce these phenotypes. Finally, we demonstrate a significant correlation between miR-106b, Six1, and activated TGF-β signaling in human breast cancers, and further show that high levels of miR-106b and miR-93 in breast tumors significantly predicts shortened time to relapse. These findings expand the spectrum of oncogenic functions of miR-106b-25, and may provide a novel molecular explanation, through the Six1 regulated miR-106b-25 cluster, by which TGF-β signaling shifts from tumor suppressive to tumor promoting. PMID:22286770

Plasma spray nozzle with low overspray and collimated flow

NASA Technical Reports Server (NTRS)

Beason, Jr., George P. (Inventor); McKechnie, Timothy N. (Inventor); Power, Christopher A. (Inventor)

1996-01-01

An improved nozzle for reducing overspray in high temperature supersonic plasma spray devices comprises a body defining an internal passageway having an upstream end and a downstream end through which a selected plasma gas is directed. The nozzle passageway has a generally converging/diverging Laval shape with its upstream end converging to a throat section and its downstream end diverging from the throat section. The upstream end of the passageway is configured to accommodate a high current cathode for producing an electrical arc in the passageway to heat and ionize the gas flow to plasma form as it moves along the passageway. The downstream end of the nozzle is uniquely configured through the methodology of this invention to have a contoured bell-shape that diverges from the throat to the exit of the nozzle. Coating material in powder form is injected into the plasma flow in the region of the bell-shaped downstream end of the nozzle and the powder particles become entrained in the flow. The unique bell shape of the nozzle downstream end produces a plasma spray that is ideally expanded at the nozzle exit and thus virtually free of shock phenomena, and that is highly collimated so as to exhibit significantly reduced fanning and diffusion between the nozzle and the target. The overall result is a significant reduction in the amount of material escaping from the plasma stream in the form of overspray and a corresponding improvement in the cost of the coating operation and in the quality and integrity of the coating itself.

Nonmarket resource valuation in the postfire environment

Treesearch

David Calkin; Greg Jones; Kevin Hyde

2008-01-01

After the containment of large wildland fires, major onsite and downstream effects including lost soil productivity, watershed response, increased vulnerability to invasive weeds, and downstream sedimentation can cause threats to human life and property. Burned Area Emergency Response (BAER) teams are responsible for developing treatment plans to mitigate negative...

p21-activated kinases in cancer.

PubMed

Kumar, Rakesh; Gururaj, Anupama E; Barnes, Christopher J

2006-06-01

The pivotal role of kinases in signal transduction and cellular regulation has lent them considerable appeal as pharmacological targets across a broad spectrum of cancers. p21-activated kinases (Paks) are serine/threonine kinases that function as downstream nodes for various oncogenic signalling pathways. Paks are well-known regulators of cytoskeletal remodelling and cell motility, but have recently also been shown to promote cell proliferation, regulate apoptosis and accelerate mitotic abnormalities, which results in tumour formation and cell invasiveness. Alterations in Pak expression have been detected in human tumours, which makes them an attractive new therapeutic target.

Deja Vu: EGF receptors drive resistance to BRAF inhibitors.

PubMed

Girotti, Maria Romina; Marais, Richard

2013-05-01

The promise of personalized medicine is upon us, and in some cancers, targeted therapies are rapidly becoming the mainstay of treatment for selected patients based on their molecular profile. The protein kinase BRAF is a driver oncogene in both thyroid cancer and melanoma, but while drugs that target BRAF and its downstream signaling pathway are effective in melanoma, they are ineffective in thyroid cancer. In this issue of Cancer Discovery, Montero-Conde and colleagues investigate why thyroid cancer is resistant to BRAF inhibitors despite the presence of BRAF mutation.

Dana-Farber Cancer Institute: Identification of Therapeutic Targets in KRAS Driven Lung Cancer | Office of Cancer Genomics

Cancer.gov

The CTD2 Center at Dana Farber Cancer Institute focuses on the use of high-throughput genetic and bioinformatic approaches to identify and credential oncogenes and co-dependencies in cancers. This Center aims to provide the cancer research community with information that will facilitate the prioritization of targets based on both genomic and functional evidence, inform the most appropriate genetic context for downstream mechanistic and validation studies, and enable the translation of this information into therapeutics and diagnostics.

siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs.

PubMed

Vickers, Timothy A; Crooke, Stanley T

2012-07-01

While most siRNAs induce sequence-specific target mRNA cleavage and degradation in a process mediated by Ago2/RNA-induced silencing complex (RISC), certain siRNAs have also been demonstrated to direct target RNA reduction through deadenylation and subsequent degradation of target transcripts in a process which involves Ago1/RISC and P-bodies. In the current study, we present data suggesting that a third class of siRNA exist, which are capable of promoting target RNA reduction that is independent of both Ago and RISC. These siRNAs bind the target messenger RNA at the polyA signal and are capable of redirecting a small amount of polyadenylation to downstream polyA sites when present, however, the majority of the activity appears to be due to inhibition of polyadenylation or deadenylation of the transcript, followed by exosomal degradation of the immature mRNA.

Kinetics of Hydrogen Oxidation Downstream of Lean Propane and Hydrogen Flames

NASA Technical Reports Server (NTRS)

Fine, Burton

1961-01-01

The decay of hydrogen was measured downstream of lean, flat, premixed hydrogen and propane-air flames seated on cooled porous burners. Experimental variables included temperature, pressure, initial equivalence ratio and diluent. Sampling of burned gas was done through uncooled quartz orifice probes, and the analysis was based on gas chromatography. An approximate treatment of the data in which diffusion was neglected led to the following rate expression for the zone downstream of hydrogen flames d[H (sub 2)] divided by (d times t) equals 1.7 times 10 (sup 10) [H (sub 2)] (sup 3) divided by (sub 2) [O (sub 2)]e (sup (-8100 divided by RT)) moles per liters per second. On the basis of a rate expression of this form, the specific rate constant for the reaction downstream of hydrogen flames was about three times as great as that determined downstream of propane flames. This result was explained on the basis of the existence of a steady state between hydrogen and carbon monoxide in the burned gas downstream of propane flames.

Assessing downstream flood impacts due to a potential GLOF from Imja Tsho in Nepal

NASA Astrophysics Data System (ADS)

Somos-Valenzuela, M. A.; McKinney, D. C.; Byers, A. C.; Rounce, D. R.; Portocarrero, C.; Lamsal, D.

2015-03-01

Glacial-dominated areas pose unique challenges to downstream communities in adapting to recent and continuing global climate change, including increased threats of glacial lake outburst floods (GLOFs) that can increase risk due to flooding of downstream communities and cause substantial impacts on regional social, environmental and economic systems. The Imja glacial lake (or Imja Tsho) in Nepal, which has the potential to generate a GLOF, was studied using a two-dimensional debris-flow inundation model in order to evaluate the effectiveness of proposed measures to reduce possible flooding impacts to downstream communities by lowering the lake level. The results indicate that only minor flood impact reduction is achieved in the downstream community of Dingboche with modest (~3 m) lake lowering. Lowering the lake by 10 m shows a significant reduction in inundated area. However, lowering the lake by 20 m almost eliminates all flood impact at Dingboche. Further downstream at Phakding, the impact of the GLOF is significant and similar reductions in inundation are likely as a result of lake lowering.

Identifying antimalarial compounds targeting dihydrofolate reductase-thymidylate synthase (DHFR-TS) by chemogenomic profiling.

PubMed

Aroonsri, Aiyada; Akinola, Olugbenga; Posayapisit, Navaporn; Songsungthong, Warangkhana; Uthaipibull, Chairat; Kamchonwongpaisan, Sumalee; Gbotosho, Grace O; Yuthavong, Yongyuth; Shaw, Philip J

2016-07-01

The mode of action of many antimalarial drugs is unknown. Chemogenomic profiling is a powerful method to address this issue. This experimental approach entails disruption of gene function and phenotypic screening for changes in sensitivity to bioactive compounds. Here, we describe the application of reverse genetics for chemogenomic profiling in Plasmodium. Plasmodium falciparum parasites harbouring a transgenic insertion of the glmS ribozyme downstream of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene were used for chemogenomic profiling of antimalarial compounds to identify those which target DHFR-TS. DHFR-TS expression can be attenuated by exposing parasites to glucosamine. Parasites with attenuated DHFR-TS expression were significantly more sensitive to antifolate drugs known to target DHFR-TS. In contrast, no change in sensitivity to other antimalarial drugs with different modes of action was observed. Chemogenomic profiling was performed using the Medicines for Malaria Venture (Switzerland) Malaria Box compound library, and two compounds were identified as novel DHFR-TS inhibitors. We also tested the glmS ribozyme in Plasmodium berghei, a rodent malaria parasite. The expression of reporter genes with downstream glmS ribozyme could be attenuated in transgenic parasites comparable with that obtained in P. falciparum. The chemogenomic profiling method was applied in a P. berghei line expressing a pyrimethamine-resistant Toxoplasma gondii DHFR-TS reporter gene under glmS ribozyme control. Parasites with attenuated expression of this gene were significantly sensitised to antifolates targeting DHFR-TS, but not other drugs with different modes of action. In conclusion, these data show that the glmS ribozyme reverse genetic tool can be applied for identifying primary targets of antimalarial compounds in human and rodent malaria parasites. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Evaluation of targeted and untargeted effects-based monitoring tools to assess impacts of contaminants of emerging concern on fish in the South Platte River, CO.

PubMed

Ekman, Drew R; Keteles, Kristen; Beihoffer, Jon; Cavallin, Jenna E; Dahlin, Kenneth; Davis, John M; Jastrow, Aaron; Lazorchak, James M; Mills, Marc A; Murphy, Mark; Nguyen, David; Vajda, Alan M; Villeneuve, Daniel L; Winkelman, Dana L; Collette, Timothy W

2018-08-01

Rivers in the arid Western United States face increasing influences from anthropogenic contaminants due to population growth, urbanization, and drought. To better understand and more effectively track the impacts of these contaminants, biologically-based monitoring tools are increasingly being used to complement routine chemical monitoring. This study was initiated to assess the ability of both targeted and untargeted biologically-based monitoring tools to discriminate impacts of two adjacent wastewater treatment plants (WWTPs) on Colorado's South Platte River. A cell-based estrogen assay (in vitro, targeted) determined that water samples collected downstream of the larger of the two WWTPs displayed considerable estrogenic activity in its two separate effluent streams. Hepatic vitellogenin mRNA expression (in vivo, targeted) and NMR-based metabolomic analyses (in vivo, untargeted) from caged male fathead minnows also suggested estrogenic activity downstream of the larger WWTP, but detected significant differences in responses from its two effluent streams. The metabolomic results suggested that these differences were associated with oxidative stress levels. Finally, partial least squares regression was used to explore linkages between the metabolomics responses and the chemical contaminants that were detected at the sites. This analysis, along with univariate statistical approaches, identified significant covariance between the biological endpoints and estrone concentrations, suggesting the importance of this contaminant and recommending increased focus on its presence in the environment. These results underscore the benefits of a combined targeted and untargeted biologically-based monitoring strategy when used alongside contaminant monitoring to more effectively assess ecological impacts of exposures to complex mixtures in surface waters. Published by Elsevier Ltd.

Downregulation of the Sonic Hedgehog/Gli pathway transcriptional target Neogenin-1 is associated with basal cell carcinoma aggressiveness.

PubMed

Casas, Bárbara S; Adolphe, Christelle; Lois, Pablo; Navarrete, Nelson; Solís, Natalia; Bustamante, Eva; Gac, Patricio; Cabané, Patricio; Gallegos, Ivan; Wainwright, Brandon J; Palma, Verónica

2017-10-13

Basal Cell Carcinoma (BCC) is one of the most diagnosed cancers worldwide. It develops due to an unrestrained Sonic Hedgehog (SHH) signaling activity in basal cells of the skin. Certain subtypes of BCC are more aggressive than others, although the molecular basis of this phenomenon remains unknown. We have previously reported that Neogenin-1 (NEO1) is a downstream target gene of the SHH/GLI pathway in neural tissue. Given that SHH participates in epidermal homeostasis, here we analyzed the epidermal expression of NEO1 in order to identify whether it plays a role in adult epidermis or BCC. We describe the mRNA and protein expression profile of NEO1 and its ligands (Netrin-1 and RGMA) in human and mouse control epidermis and in a broad range of human BCCs. We identify in human BCC a significant positive correlation in the levels of NEO1 receptor, NTN-1 and RGMA ligands with respect to GLI1 , the main target gene of the canonical SHH pathway. Moreover, we show via cyclopamine inhibition of the SHH/GLI pathway of ex vivo cultures that NEO1 likely functions as a downstream target of SHH/GLI signaling in the skin. We also show how Neo1 expression decreases throughout BCC progression in the K14-Cre:Ptch1 lox/lox mouse model and that aggressive subtypes of human BCC exhibit lower levels of NEO1 than non-aggressive BCC samples. Taken together, these data suggest that NEO1 is a SHH/GLI target in epidermis. We propose that NEO1 may be important in tumor onset and is then down-regulated in advanced BCC or aggressive subtypes.

Downregulation of the Sonic Hedgehog/Gli pathway transcriptional target Neogenin-1 is associated with basal cell carcinoma aggressiveness

PubMed Central

Casas, Bárbara S.; Adolphe, Christelle; Lois, Pablo; Navarrete, Nelson; Solís, Natalia; Bustamante, Eva; Gac, Patricio; Cabané, Patricio; Gallegos, Ivan; Wainwright, Brandon J.; Palma, Verónica

2017-01-01

Basal Cell Carcinoma (BCC) is one of the most diagnosed cancers worldwide. It develops due to an unrestrained Sonic Hedgehog (SHH) signaling activity in basal cells of the skin. Certain subtypes of BCC are more aggressive than others, although the molecular basis of this phenomenon remains unknown. We have previously reported that Neogenin-1 (NEO1) is a downstream target gene of the SHH/GLI pathway in neural tissue. Given that SHH participates in epidermal homeostasis, here we analyzed the epidermal expression of NEO1 in order to identify whether it plays a role in adult epidermis or BCC. We describe the mRNA and protein expression profile of NEO1 and its ligands (Netrin-1 and RGMA) in human and mouse control epidermis and in a broad range of human BCCs. We identify in human BCC a significant positive correlation in the levels of NEO1 receptor, NTN-1 and RGMA ligands with respect to GLI1, the main target gene of the canonical SHH pathway. Moreover, we show via cyclopamine inhibition of the SHH/GLI pathway of ex vivo cultures that NEO1 likely functions as a downstream target of SHH/GLI signaling in the skin. We also show how Neo1 expression decreases throughout BCC progression in the K14-Cre:Ptch1lox/lox mouse model and that aggressive subtypes of human BCC exhibit lower levels of NEO1 than non-aggressive BCC samples. Taken together, these data suggest that NEO1 is a SHH/GLI target in epidermis. We propose that NEO1 may be important in tumor onset and is then down-regulated in advanced BCC or aggressive subtypes. PMID:29137400

Method of and apparatus for testing the integrity of filters

DOEpatents

Herman, R.L.

1985-05-07

A method of and apparatus are disclosed for testing the integrity of individual filters or filter stages of a multistage filtering system including a diffuser permanently mounted upstream and/or downstream of the filter stage to be tested for generating pressure differentials to create sufficient turbulence for uniformly dispersing trace agent particles within the airstream upstream and downstream of such filter stage. Samples of the particle concentration are taken upstream and downstream of the filter stage for comparison to determine the extent of particle leakage past the filter stage. 5 figs.

Method of and apparatus for testing the integrity of filters

DOEpatents

Herman, Raymond L [Richland, WA

1985-01-01

A method of and apparatus for testing the integrity of individual filters or filter stages of a multistage filtering system including a diffuser permanently mounted upstream and/or downstream of the filter stage to be tested for generating pressure differentials to create sufficient turbulence for uniformly dispersing trace agent particles within the airstream upstream and downstream of such filter stage. Samples of the particle concentration are taken upstream and downstream of the filter stage for comparison to determine the extent of particle leakage past the filter stage.

Methods of and apparatus for testing the integrity of filters

DOEpatents

Herman, R.L.

1984-01-01

A method of and apparatus for testing the integrity of individual filters or filter stages of a multistage filtering system including a diffuser permanently mounted upstream and/or downstream of the filter stage to be tested for generating pressure differentials to create sufficient turbulence for uniformly dispersing trace agent particles within the airstram upstream and downstream of such filter stage. Samples of the particel concentration are taken upstream and downstream of the filter stage for comparison to determine the extent of particle leakage past the filter stage.

Fell-Muir lecture: connective tissue growth factor (CCN2) – a pernicious and pleiotropic player in the development of kidney fibrosis

PubMed Central

Mason, Roger M

2013-01-01

Connective tissue growth factor (CTGF, CCN2) is a member of the CCN family of matricellular proteins. It interacts with many other proteins, including plasma membrane proteins, modulating cell function. It is expressed at low levels in normal adult kidney cells but is increased in kidney diseases, playing important roles in inflammation and in the development of glomerular and interstitial fibrosis in chronic disease. This review reports the evidence for its expression in human and animal models of chronic kidney disease and summarizes data showing that anti-CTGF therapy can successfully attenuate fibrotic changes in several such models, suggesting that therapies targeting CTGF and events downstream of it in renal cells may be useful for the treatment of human kidney fibrosis. Connective tissue growth factor stimulates the development of fibrosis in the kidney in many ways including activating cells to increase extracellular matrix synthesis, inducing cell cycle arrest and hypertrophy, and prolonging survival of activated cells. The relationship between CTGF and the pro-fibrotic factor TGFβ is examined and mechanisms by which CTGF promotes signalling by the latter are discussed. No specific cellular receptors for CTGF have been discovered but it interacts with and activates several plasma membrane proteins including low-density lipoprotein receptor-related protein (LRP)-1, LRP-6, tropomyosin-related kinase A, integrins and heparan sulphate proteoglycans. Intracellular signalling and downstream events triggered by such interactions are reviewed. Finally, the relationships between CTGF and several anti-fibrotic factors, such as bone morphogenetic factor-4 (BMP4), BMP7, hepatocyte growth factor, CCN3 and Oncostatin M, are discussed. These may determine whether injured tissue heals or progresses to fibrosis. PMID:23110747

Double-stranded RNA transcribed from vector-based oligodeoxynucleotide acts as transcription factor decoy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiao, Xiao; Gang, Yi; Department of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, Shaanxi Province

2015-02-06

Highlights: • A shRNA vector based transcription factor decoy, VB-ODN, was designed. • VB-ODN for NF-κB inhibited cell viability in HEK293 cells. • VB-ODN inhibited expression of downstream genes of target transcription factors. • VB-ODN may enhance nuclear entry ratio for its feasibility of virus production. - Abstract: In this study, we designed a short hairpin RNA vector-based oligodeoxynucleotide (VB-ODN) carrying transcription factor (TF) consensus sequence which could function as a decoy to block TF activity. Specifically, VB-ODN for Nuclear factor-κB (NF-κB) could inhibit cell viability and decrease downstream gene expression in HEK293 cells without affecting expression of NF-κB itself.more » The specific binding between VB-ODN produced double-stranded RNA and NF-κB was evidenced by electrophoretic mobility shift assay. Moreover, similar VB-ODNs designed for three other TFs also inhibit their downstream gene expression but not that of themselves. Our study provides a new design of decoy for blocking TF activity.« less

A biochemical approach to identifying microRNA targets

PubMed Central

Karginov, Fedor V.; Conaco, Cecilia; Xuan, Zhenyu; Schmidt, Bryan H.; Parker, Joel S.; Mandel, Gail; Hannon, Gregory J.

2007-01-01

Identifying the downstream targets of microRNAs (miRNAs) is essential to understanding cellular regulatory networks. We devised a direct biochemical method for miRNA target discovery that combined RNA-induced silencing complex (RISC) purification with microarray analysis of bound mRNAs. Because targets of miR-124a have been analyzed, we chose it as our model. We honed our approach both by examining the determinants of stable binding between RISC and synthetic target RNAs in vitro and by determining the dependency of both repression and RISC coimmunoprecipitation on miR-124a seed sites in two of its well characterized targets in vivo. Examining the complete spectrum of miR-124 targets in 293 cells yielded both a set that were down-regulated at the mRNA level, as previously observed, and a set whose mRNA levels were unaffected by miR-124a. Reporter assays validated both classes, extending the spectrum of mRNA targets that can be experimentally linked to the miRNA pathway. PMID:18042700

Purification of Proteins Fused to Maltose-Binding Protein.

PubMed

Lebendiker, Mario; Danieli, Tsafi

2017-01-01

Maltose-Binding Protein (MBP) is one of the most popular fusion partners being used for producing recombinant proteins in bacterial cells. MBP allows the use of a simple capture affinity step on Amylose-Agarose or Dextrin-Sepharose columns, resulting in a protein that is often 70-90 % pure in a single step. In addition to protein isolation applications, MBP provides a high degree of translation, and facilitates the proper folding and solubility of the target protein. This paper describes efficient procedures for isolating highly purified MBP target proteins. Special attention is given to considerations for downstream applications such as structural determination studies, protein activity assays, and assessing the chemical characteristics of the target protein.

Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma.

PubMed

Calero, R; Morchon, E; Martinez-Argudo, I; Serrano, R

2017-10-10

Drug resistance by MAPK signaling recovery or activation of alternative signaling pathways, such as PI3K/AKT/mTOR, is an important factor that limits the long-term efficacy of targeted therapies in melanoma patients. In the present study, we investigated the phospho-proteomic profile of RTKs and its correlation with downstream signaling pathways in human melanoma. We found that tyrosine kinase receptors expression correlated with the expression of pivotal downstream components of the RAS/RAF/MAPK and PI3K/AKT/mTOR pathways in melanoma cell lines and tumors. We also found high expression of HSP90 and the PI3K/AKT/mTOR pathway proteins, 4EBP1 and AKT compared with healthy tissue and this correlated with poor overall survival of melanoma patients. The combination of the HSP90 inhibitor 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 showed a synergistic activity decreasing melanoma cell growth, inducing apoptosis and targeting simultaneously the MAPK and PI3K/AKT/mTOR pathways. These results demonstrate that the combination of HSP90 and PI3K/mTOR inhibitors could be an effective therapeutic strategy that target the main survival pathways in melanoma and must be considered to overcome resistance to BRAF inhibitors in melanoma patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Overexpression of nucleostemin contributes to an advanced malignant phenotype and a poor prognosis in oral squamous cell carcinoma

PubMed Central

Yoshida, R; Nakayama, H; Nagata, M; Hirosue, A; Tanaka, T; Kawahara, K; Nakagawa, Y; Matsuoka, Y; Sakata, J; Arita, H; Hiraki, A; Shinohara, M; Ito, T

2014-01-01

Background: Nucleostemin (NS) is essential for the maintenance of stem cell properties, the functions of which remain poorly understood in cancer cells. The purpose of this study was to explore the impact of NS on malignancy and its clinical significance in oral squamous cell carcinoma (OSCC) patients. Methods: We investigated the effects of NS on the proliferation and invasion of OSCC using NS-overexpressing or -knockdown OSCC cells. We assessed the activation of the STAT3 (signal transducer and activator of transcription 3) signalling pathway and the downstream targets in the cells with different expression levels of NS. An immunohistochemical analysis of NS was also performed in 54 OSCC patients who were treated with preoperative chemoradiotherapy and surgery. Results: The overexpression of NS significantly enhanced the proliferation and invasive potential of OSCC cells. On the other hand, downregulation of NS suppressed the invasiveness of the cells. The alterations of these malignant phenotypes were associated with the activation of STAT3 signalling and its downstream targets. An immunohistochemical analysis demonstrated that a high NS tumour expression level significantly correlated with an advanced T-stage and N-stage. Furthermore, a Cox regression analysis revealed that the NS status (hazard ratio, 9.09; P=0.002) was a significant progression factor for OSCC patients. Conclusions: Our results suggest that targeting NS may provide a promising treatment for highly malignant OSCC. PMID:25314067

Evaluation of target efficiencies for solid-liquid separation steps in biofuels production.

PubMed

Kochergin, Vadim; Miller, Keith

2011-01-01

Development of liquid biofuels has entered a new phase of large scale pilot demonstration. A number of plants that are in operation or under construction face the task of addressing the engineering challenges of creating a viable plant design, scaling up and optimizing various unit operations. It is well-known that separation technologies account for 50-70% of both capital and operating cost. Additionally, reduction of environmental impact creates technological challenges that increase project cost without adding to the bottom line. Different technologies vary in terms of selection of unit operations; however, solid-liquid separations are likely to be a major contributor to the overall project cost. Despite the differences in pretreatment approaches, similar challenges arise for solid-liquid separation unit operations. A typical process for ethanol production from biomass includes several solid-liquid separation steps, depending on which particular stream is targeted for downstream processing. The nature of biomass-derived materials makes it either difficult or uneconomical to accomplish complete separation in a single step. Therefore, setting realistic efficiency targets for solid-liquid separations is an important task that influences overall process recovery and economics. Experimental data will be presented showing typical characteristics for pretreated cane bagasse at various stages of processing into cellulosic ethanol. Results of generic material balance calculations will be presented to illustrate the influence of separation target efficiencies on overall process recoveries and characteristics of waste streams.

Next-generation transcriptome analysis in transgenic birch overexpressing and suppressing APETALA1 sheds lights in reproduction development and diterpenoid biosynthesis.

PubMed

Huang, Haijiao; Chen, Su; Li, Huiyu; Jiang, Jing

2015-09-01

Overexpression of BpAP1 could cause early flowering in birch. BpAP1 affected the expression of many flowering-related unigenes and diterpenoid biosynthesis in transgenic birch, and BpPI was a putative target gene of BpAP1. APETALA1 (AP1) is an MADS-box transcription factor that is involved in the flowering process in plants and has been a focus of genetic studies examining flower development. Here, we carried out transcriptome analysis of birch (Betula platyphylla Suk.), including BpAP1 overexpression lines, BpAP1 suppression lines, and non-transgenic line (NT). Compared with NT, we detected 8302 and 7813 differentially expressed unigenes in 35S::BpAP1 and 35S::BpAP1RNAi transgenic lines, respectively. Overexpression and suppression of BpAP1 in birch affected diterpenoid biosynthesis and altered expression of many flowering-related unigenes. Moreover, combining information from the RNA-seq database and the birch genome, we predicted downstream target genes of BpAP1. Among the 166 putative target genes of BpAP1, there was a positive correlation between BpAP1 and BpPI. These results provide references for further examining the relationship between BpAP1 and its target genes, and reveal that BpAP1 functions as a transcription regulator in birch.

O-GlcNAc in cancer: An Oncometabolism-fueled vicious cycle.

PubMed

Hanover, John A; Chen, Weiping; Bond, Michelle R

2018-06-01

Cancer cells exhibit unregulated growth, altered metabolism, enhanced metastatic potential and altered cell surface glycans. Fueled by oncometabolism and elevated uptake of glucose and glutamine, the hexosamine biosynthetic pathway (HBP) sustains glycosylation in the endomembrane system. In addition, the elevated pools of UDP-GlcNAc drives the O-GlcNAc modification of key targets in the cytoplasm, nucleus and mitochondrion. These targets include transcription factors, kinases, key cytoplasmic enzymes of intermediary metabolism, and electron transport chain complexes. O-GlcNAcylation can thereby alter epigenetics, transcription, signaling, proteostasis, and bioenergetics, key 'hallmarks of cancer'. In this review, we summarize accumulating evidence that many cancer hallmarks are linked to dysregulation of O-GlcNAc cycling on cancer-relevant targets. We argue that onconutrient and oncometabolite-fueled elevation increases HBP flux and triggers O-GlcNAcylation of key regulatory enzymes in glycolysis, Kreb's cycle, pentose-phosphate pathway, and the HBP itself. The resulting rerouting of glucose metabolites leads to elevated O-GlcNAcylation of oncogenes and tumor suppressors further escalating elevation in HBP flux creating a 'vicious cycle'. Downstream, elevated O-GlcNAcylation alters DNA repair and cellular stress pathways which influence oncogenesis. The elevated steady-state levels of O-GlcNAcylated targets found in many cancers may also provide these cells with a selective advantage for sustained growth, enhanced metastatic potential, and immune evasion in the tumor microenvironment.

Occurrence and removal of antibiotics and the corresponding resistance genes in wastewater treatment plants: effluents' influence to downstream water environment.

PubMed

Li, Jianan; Cheng, Weixiao; Xu, Like; Jiao, Yanan; Baig, Shams Ali; Chen, Hong

2016-04-01

In this study, the occurrence of 8 antibiotics [3 tetracyclines (TCs), 4 sulfonamides, and 1 trimethoprim (TMP)], 12 antibiotic resistance genes (ARGs) (10 tet, 2 sul), 4 types of bacteria [no antibiotics, anti-TC, anti-sulfamethoxazole (SMX), and anti-double], and intI1 in two wastewater treatment plants (WWTPs) were assessed and their influences in downstream lake were investigated. Both WWTPs' effluent demonstrated some similarities, but the abundance and removal rate varied significantly. Results revealed that biological treatment mainly removed antibiotics and ARGs, whereas physical techniques were found to eliminate antibiotic resistance bacteria (ARBs) abundance (about 1 log for each one). UV disinfection did not significantly enhance the removal efficiency, and the release of the abundantly available target contaminants from the excess sludge may pose threats to human and the environment. Different antibiotics showed diverse influences on the downstream lake, and the concentrations of sulfamethazine (SM2) and SMX were observed to increase enormously. The total ARG abundance ascended about 0.1 log and some ARGs (e.g., tetC, intI1, tetA) increased due to the high input of the effluent. In addition, the abundance of ARB variation in the lake also changed, but the abundance of four types of bacteria remained stable in the downstream sampling sites.

Modeling the combined influence of host dispersal and waterborne fate and transport on pathogen spread in complex landscapes

PubMed Central

Lu, Ding; McDowell, Julia Z.; Davis, George M.; Spear, Robert C.; Remais, Justin V.

2012-01-01

Environmental models, often applied to questions on the fate and transport of chemical hazards, have recently become important in tracing certain environmental pathogens to their upstream sources of contamination. These tools, such as first order decay models applied to contaminants in surface waters, offer promise for quantifying the fate and transport of pathogens with multiple environmental stages and/or multiple hosts, in addition to those pathogens whose environmental stages are entirely waterborne. Here we consider the fate and transport capabilities of the human schistosome Schistosoma japonicum, which exhibits two waterborne stages and is carried by an amphibious intermediate snail host. We present experimentally-derived dispersal estimates for the intermediate snail host and fate and transport estimates for the passive downstream diffusion of cercariae, the waterborne, human-infective parasite stage. Using a one dimensional advective transport model exhibiting first-order decay, we simulate the added spatial reach and relative increase in cercarial concentrations that dispersing snail hosts contribute to downstream sites. Simulation results suggest that snail dispersal can substantially increase the concentrations of cercariae reaching downstream locations, relative to no snail dispersal, effectively putting otherwise isolated downstream sites at increased risk of exposure to cercariae from upstream sources. The models developed here can be applied to other infectious diseases with multiple life-stages and hosts, and have important implications for targeted ecological control of disease spread. PMID:23162675

Aberrations and Emittance Growth in the DARHT 2nd Axis Downstream Transport

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schulze, Martin E.

The emittance of the DARHT 2 nd Axis has been inferred from solenoid scans performed in the downstream transport (DST) region using a short kicked pulse. The beam spot size is measured by viewing optical transition radiation (OTR) in the near field as a function of the field (current) of a solenoid magnet (S4). The imaging station containing the OTR target is located about 100 cm downstream of the solenoid magnet. The emittance is then inferred using a beam optics code such as LAMDA or XTR by fitting the data to initial conditions upstream of the S4 solenoid magnet. Themore » initial conditions are the beam size, beam convergence and emittance. The beam energy and current are measured. In preparation for a solenoid scan, the magnets upstream of the solenoid are adjusted to produce a round beam with no beam losses due to scraping in the beam tube. This is different from the standard tune in which the beam tune is adjusted to suppress the effects of ions and rf in the septum dump. In this standard tune, approximately 10% of the beam is lost due to scraping as the beam enters the small 3.75” ID beam tube after the septum. The normalized emittance inferred from recent solenoid scans typically ranges from 600 to 800 π(mm-mrad). This larger beam size increases the sensitivity to any non-linear fields in the Collins quadrupoles that are mounted along the small diameter beam tube. The primary magnet used to adjust the beam size in this region is the S3 solenoid magnet. Measurements made of the beam shape as the beam size was decreased showed significant structure consistent with non-linear fields. Using the measured magnetic fields in the Collins quadrupoles including higher order multipoles, the beam transport through the Collins quadrupoles is simulated and compared to the observed OTR images. The simulations are performed using the beam optics codes TRANSPORT [1] and TURTLE [2]. Estimates of the emittance growth and beam losses are made as a function of the S3 magnet setting. The increase in the spot size on the x-ray production target resulting from this emittance growth is examined for different DST tunes.« less

PIK3CA mutations, phosphatase and tensin homolog, human epidermal growth factor receptor 2, and insulin-like growth factor 1 receptor and adjuvant tamoxifen resistance in postmenopausal breast cancer patients.

PubMed

Beelen, Karin; Opdam, Mark; Severson, Tesa M; Koornstra, Rutger H T; Vincent, Andrew D; Wesseling, Jelle; Muris, Jettie J; Berns, Els M J J; Vermorken, Jan B; van Diest, Paul J; Linn, Sabine C

2014-01-27

Inhibitors of the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway can overcome endocrine resistance in estrogen receptor (ER) α-positive breast cancer, but companion diagnostics indicating PI3K/AKT/mTOR activation and consequently endocrine resistance are lacking. PIK3CA mutations frequently occur in ERα-positive breast cancer and result in PI3K/AKT/mTOR activation in vitro. Nevertheless, the prognostic and treatment-predictive value of these mutations in ERα-positive breast cancer is contradictive. We tested the clinical validity of PIK3CA mutations and other canonic pathway drivers to predict intrinsic resistance to adjuvant tamoxifen. In addition, we tested the association between these drivers and downstream activated proteins. Primary tumors from 563 ERα-positive postmenopausal patients, randomized between adjuvant tamoxifen (1 to 3 years) versus observation were recollected. PIK3CA hotspot mutations in exon 9 and exon 20 were assessed with Sequenom Mass Spectometry. Immunohistochemistry was performed for human epidermal growth factor receptor 2 (HER2), phosphatase and tensin homolog (PTEN), and insulin-like growth factor 1 receptor (IGF-1R). We tested the association between these molecular alterations and downstream activated proteins (like phospho-protein kinase B (p-AKT), phospho-mammalian target of rapamycin (p-mTOR), p-ERK1/2, and p-p70S6K). Recurrence-free interval improvement with tamoxifen versus control was assessed according to the presence or absence of canonic pathway drivers, by using Cox proportional hazard models, including a test for interaction. PIK3CA mutations (both exon 9 and exon 20) were associated with low tumor grade. An enrichment of PIK3CA exon 20 mutations was observed in progesterone receptor- positive tumors. PIK3CA exon 20 mutations were not associated with downstream-activated proteins. No significant interaction between PIK3CA mutations or any of the other canonic pathway drivers and tamoxifen-treatment benefit was found. PIK3CA mutations do not have clinical validity to predict intrinsic resistance to adjuvant tamoxifen and may therefore be unsuitable as companion diagnostic for PI3K/AKT/mTOR inhibitors in ERα- positive, postmenopausal, early breast cancer patients.

Initiated chemical vapor deposition polymers for high peak-power laser targets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baxamusa, Salmaan H.; Lepro, Xavier; Lee, Tom

2016-12-05

Here, we report two examples of initiated chemical vapor deposition (iCVD) polymers being developed for use in laser targets for high peak-power laser systems. First, we show that iCVD poly(divinylbenzene) is more photo-oxidatively stable than the plasma polymers currently used in laser targets. Thick layers (10–12 μm) of this highly crosslinked polymer can be deposited with near-zero intrinsic film stress. Second, we show that iCVD epoxy polymers can be crosslinked after deposition to form thin adhesive layers for assembling precision laser targets. The bondlines can be made as thin as ~ 1 μm, approximately a factor of 2 thinner thanmore » achievable using viscous resin-based adhesives. These bonds can withstand downstream coining and stamping processes.« less

Control of Maillard Reactions in Foods: Strategies and Chemical Mechanisms.

PubMed

Lund, Marianne N; Ray, Colin A

2017-06-14

Maillard reactions lead to changes in food color, organoleptic properties, protein functionality, and protein digestibility. Numerous different strategies for controlling Maillard reactions in foods have been attempted during the past decades. In this paper, recent advances in strategies for controlling the Maillard reaction and subsequent downstream reaction products in food systems are critically reviewed. The underlying mechanisms at play are presented, strengths and weaknesses of each strategy are discussed, and reasonable reaction mechanisms are proposed to reinforce the evaluations. The review includes strategies involving addition of functional ingredients, such as plant polyphenols and vitamins, as well as enzymes. The resulting trapping or modification of Maillard targets, reactive intermediates, and advanced glycation endproducts (AGEs) are presented with their potential unwanted side effects. Finally, recent advances in processing for control of Maillard reactions are discussed.

European downstream oil industry safety performance : statistical summary of reported incidents, 1997 and overview 1993 to 1997

DOT National Transportation Integrated Search

1998-10-01

This report is the fourth by CONCAWE reviewing the safety performance of the downstream ol industry in Western Europe. It includes the results of 27 companies which together represent over 90% of the oil refining capacity in the region. Of the 27 com...

77 FR 53905 - Notice of Filing of Plats of Survey; Montana

Federal Register 2010, 2011, 2012, 2013, 2014

2012-09-04

... original meanders of the former left bank of the Missouri River, downstream, through section [[Page 53906... present left bank of the Missouri River, downstream, through sections 29 and 30 and certain division of... final, including decisions or appeals. Authority: 43 U.S.C. Chap. 3. Steve L. Toth, Acting Chief...

Expression of Hormonal Carcinogenesis Genes and Related Regulatory microRNAs in Uterus and Ovaries of DDT-Treated Female Rats.

PubMed

Kalinina, T S; Kononchuk, V V; Gulyaeva, L F

2017-10-01

The insecticide dichlorodiphenyltrichloroethane (DDT) is a nonmutagenic xenobiotic compound able to exert estrogen-like effects resulting in activation of estrogen receptor-α (ERα) followed by changed expression of its downstream target genes. In addition, studies performed over recent years suggest that DDT may also influence expression of microRNAs. However, an impact of DDT on expression of ER, microRNAs, and related target genes has not been fully elucidated. Here, using real-time PCR, we assessed changes in expression of key genes involved in hormonal carcinogenesis as well as potentially related regulatory oncogenic/tumor suppressor microRNAs and their target genes in the uterus and ovaries of female Wistar rats during single and chronic multiple-dose DDT exposure. We found that applying DDT results in altered expression of microRNAs-221, -222, -205, -126a, and -429, their target genes (Pten, Dicer1), as well as genes involved in hormonal carcinogenesis (Esr1, Pgr, Ccnd1, Cyp19a1). Notably, Cyp19a1 expression seems to be also regulated by microRNAs-221, -222, and -205. The data suggest that epigenetic effects induced by DDT as a potential carcinogen may be based on at least two mechanisms: (i) activation of ERα followed by altered expression of the target genes encoding receptor Pgr and Ccnd1 as well as impaired expression of Cyp19a1, affecting, thereby, cell hormone balance; and (ii) changed expression of microRNAs resulting in impaired expression of related target genes including reduced level of Cyp19a1 mRNA.

GIV/Girdin Links Vascular Endothelial Growth Factor Signaling to Akt Survival Signaling in Podocytes Independent of Nephrin

PubMed Central

Wang, Honghui; Misaki, Taro; Taupin, Vanessa; Eguchi, Akiko; Ghosh, Pradipta

2015-01-01

Podocytes are critically involved in the maintenance of the glomerular filtration barrier and are key targets of injury in many glomerular diseases. Chronic injury leads to progressive loss of podocytes, glomerulosclerosis, and renal failure. Thus, it is essential to maintain podocyte survival and avoid apoptosis after acute glomerular injury. In normal glomeruli, podocyte survival is mediated via nephrin-dependent Akt signaling. In several glomerular diseases, nephrin expression decreases and podocyte survival correlates with increased vascular endothelial growth factor (VEGF) signaling. How VEGF signaling contributes to podocyte survival and prevents apoptosis remains unknown. We show here that Gα–interacting, vesicle-associated protein (GIV)/girdin mediates VEGF receptor 2 (VEGFR2) signaling and compensates for nephrin loss. In puromycin aminonucleoside nephrosis (PAN), GIV expression increased, GIV was phosphorylated by VEGFR2, and p-GIV bound and activated Gαi3 and enhanced downstream Akt2, mammalian target of rapamycin complex 1 (mTORC1), and mammalian target of rapamycin complex-2 (mTORC2) signaling. In GIV-depleted podocytes, VEGF-induced Akt activation was abolished, apoptosis was triggered, and cell migration was impaired. These effects were reversed by introducing GIV but not a GIV mutant that cannot activate Gαi3. Our data indicate that after PAN injury, VEGF promotes podocyte survival by triggering assembly of an activated VEGFR2/GIV/Gαi3 signaling complex and enhancing downstream PI3K/Akt survival signaling. Because of its important role in promoting podocyte survival, GIV may represent a novel target for therapeutic intervention in the nephrotic syndrome and other proteinuric diseases. PMID:25012178

Targeted repression of AXIN2 and MYC gene expression using designer TALEs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rennoll, Sherri A.; Scott, Samantha A.; Yochum, Gregory S., E-mail: gsy3@psu.edu

Highlights: • We designed TALE–SID fusion proteins to target AXIN2 and MYC. • TALE–SIDs bound the chromosomal AXIN2 and MYC genes and repressed their expression. • TALE–SIDs repress β-catenin{sup S45F}-dependent AXIN2 and MYC transcription. - Abstract: Designer TALEs (dTALEs) are chimeric transcription factors that can be engineered to regulate gene expression in mammalian cells. Whether dTALEs can block gene transcription downstream of signal transduction cascades, however, has yet to be fully explored. Here we tested whether dTALEs can be used to target genes whose expression is controlled by Wnt/β-catenin signaling. TALE DNA binding domains were engineered to recognize sequences adjacentmore » to Wnt responsive enhancer elements (WREs) that control expression of axis inhibition protein 2 (AXIN2) and c-MYC (MYC). These custom DNA binding domains were linked to the mSin3A interaction domain (SID) to generate TALE–SID chimeric repressors. The TALE–SIDs repressed luciferase reporter activity, bound their genomic target sites, and repressed AXIN2 and MYC expression in HEK293 cells. We generated a novel HEK293 cell line to determine whether the TALE–SIDs could function downstream of oncogenic Wnt/β-catenin signaling. Treating these cells with doxycycline and tamoxifen stimulates nuclear accumulation of a stabilized form of β-catenin found in a subset of colorectal cancers. The TALE–SIDs repressed AXIN2 and MYC expression in these cells, which suggests that dTALEs could offer an effective therapeutic strategy for the treatment of colorectal cancer.« less

Effects of Packstock Use and Backpackers on Water Quality in Yosemite National Park, California

NASA Astrophysics Data System (ADS)

Forrester, H.; Clow, D. W.; Roche, J. W.; Heyvaert, A.

2016-12-01

Visitor use, primarily backpacker camping, packstock (horse and mule) trail use, and packstock grazing, in designated Wilderness, increases the potential for negative effects on water quality. To determine the effects of visitor use on water quality in Wilderness in Yosemite National Park, we collected and analyzed surface-water samples for water quality indicators, consisting of fecal indicator bacteria (Escherichia coli), nutrients (nitrogen, phosphorus), suspended sediment concentration (SSC), and hormones (e.g. estrogen compounds) during the summers of 2012-2014. We collected samples upstream and downstream from different types of visitor use at routine intervals (weekly or biweekly) and during storms. Additionally, we sampled upstream and downstream from meadows, and targeted different types of visitor use during a park-wide synoptic sampling campaign (n=63). At packstock stream crossings, statistically significant (P≤0.05) increases in Escherichia coli (E. coli) and SSC occurred downstream from crossings compared to upstream conditions during routine sampling (median difference: 3 CFU 100ml-1, and >0.3 mg l-1, respectively) and during storms (median difference: 32 CFU 100ml-1, and 2.9 mg l-1). At backpacker campsites, during routine sampling, significant increases occurred downstream from backpacker camping for E. coli (median difference: 1 CFU 100ml-1), and estrogen hormones were detected. At packstock grazing areas, which are located in meadows, no significant increases were detected for any of the measured water quality indicators downstream from grazing. Most synoptic sample concentrations were near or below detection limits. Our results indicate that under current use levels: 1) packstock trail use and backpacker camping are associated with detectable effects on water quality, which are most pronounced during storms; 2) increases in water quality indicators were not detected downstream from meadows where packstock were grazed; and 3) environmental processes in meadows provide a valuable ecosystem service by reducing human related sources of microbial contamination.

Blueberry and malvidin inhibit cell cycle progression and induce mitochondrial-mediated apoptosis by abrogating the JAK/STAT-3 signalling pathway.

PubMed

Baba, Abdul Basit; Nivetha, Ramesh; Chattopadhyay, Indranil; Nagini, Siddavaram

2017-11-01

Blueberries, a rich source of anthocyanins have attracted considerable attention as functional foods that confer immense health benefits including anticancer properties. Herein, we assessed the potential of blueberry and its major constituent malvidin to target STAT-3, a potentially druggable oncogenic transcription factor with high therapeutic index. We demonstrate that blueberry abrogates the JAK/STAT-3 pathway and modulates downstream targets that influence cell proliferation and apoptosis in a hamster model of oral oncogenesis. Further, we provide mechanistic evidence that blueberry and malvidin function as STAT-3 inhibitors in the oral cancer cell line SCC131. Blueberry and malvidin suppressed STAT-3 phosphorylation and nuclear translocation thereby inducing cell cycle arrest and mitochondrial-mediated apoptosis. However, the combination of blueberry and malvidin with the STAT-3 inhibitor S3I-201 was more efficacious in STAT-3 inhibition relative to single agents. The present study has provided leads for the development of novel combinations of compounds that can serve as inhibitors of STAT-mediated oncogenic signalling. Copyright © 2017 Elsevier Ltd. All rights reserved.

Rapamycin and the transcription factor C/EBPbeta as a switch in osteoclast differentiation: implications for lytic bone diseases.

PubMed

Smink, Jeske J; Leutz, Achim

2010-03-01

Lytic bone diseases and in particular osteoporosis are common age-related diseases characterized by enhanced bone fragility due to loss of bone density. Increasingly, osteoporosis poses a major global health-care problem due to the growth of the elderly population. Recently, it was found that the gene regulatory transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) is involved in bone metabolism. C/EBPbeta occurs as different protein isoforms of variable amino terminal length, and regulation of the C/EBPbeta isoform ratio balance was found to represent an important factor in osteoclast differentiation and bone homeostasis. Interestingly, adjustment of the C/EBPbeta isoform ratio by the process of translational control is downstream of the mammalian target of rapamycin kinase (mTOR), a sensor of the nutritional status and a target of immunosuppressive and anticancer drugs. The findings imply that modulating the process of translational control of C/EBPbeta isoform expression could represent a novel therapeutic approach in osteolytic bone diseases, including cancer and infection-induced bone loss.

Proteomic snapshot of the EGF-induced ubiquitin network

PubMed Central

Argenzio, Elisabetta; Bange, Tanja; Oldrini, Barbara; Bianchi, Fabrizio; Peesari, Raghunath; Mari, Sara; Di Fiore, Pier Paolo; Mann, Matthias; Polo, Simona

2011-01-01

The activity, localization and fate of many cellular proteins are regulated through ubiquitination, a process whereby one or more ubiquitin (Ub) monomers or chains are covalently attached to target proteins. While Ub-conjugated and Ub-associated proteomes have been described, we lack a high-resolution picture of the dynamics of ubiquitination in response to signaling. In this study, we describe the epidermal growth factor (EGF)-regulated Ubiproteome, as obtained by two complementary purification strategies coupled to quantitative proteomics. Our results unveil the complex impact of growth factor signaling on Ub-based intracellular networks to levels that extend well beyond what might have been expected. In addition to endocytic proteins, the EGF-regulated Ubiproteome includes a large number of signaling proteins, ubiquitinating and deubiquitinating enzymes, transporters and proteins involved in translation and transcription. The Ub-based signaling network appears to intersect both housekeeping and regulatory circuitries of cellular physiology. Finally, as proof of principle of the biological relevance of the EGF-Ubiproteome, we demonstrated that EphA2 is a novel, downstream ubiquitinated target of epidermal growth factor receptor (EGFR), critically involved in EGFR biological responses. PMID:21245847

Temsirolimus in the treatment of renal cell carcinoma associated with Xp11.2 translocation/TFE gene fusion proteins: a case report and review of literature.

PubMed

Parikh, Jigarkumar; Coleman, Teresa; Messias, Nidia; Brown, James

2009-12-28

Xp11.2 translocation renal cell carcinomas (TRCCs) are a rare family of tumors newly recognized by the World Health Organization (WHO) in 2004. These tumors result in the fusion of partner genes to the TFE3 gene located on Xp11.2. They are most common in the pediatric population, but have been recently implicated in adult renal cell carcinoma (RCC) presenting at an early age. TFE3-mediated direct transcriptional upregulation of the Met tyrosine kinase receptor triggers dramatic activation of downstream signaling pathways including the protein kinase B (Akt)/phosphatidylinositol-3 kinase (PI3K) and mammalian target of rapamycin (mTOR) pathways. Temsirolimus is an inhibitor of mammalian target of rapamycin (mTOR) kinase, a component of intracellular signaling pathways involved in the growth and proliferation of malignant cells. Here we present a case of a 22-year old female who has been treated with temsirolimus for her Xp11.2/TFE3 gene fusion RCC.

Temsirolimus in the treatment of renal cell carcinoma associated with Xp11.2 translocation/TFE gene fusion proteins: a case report and review of literature

PubMed Central

Parikh, Jigarkumar; Coleman, Teresa; Messias, Nidia; Brown, James

2009-01-01

Xp11.2 translocation renal cell carcinomas (TRCCs) are a rare family of tumors newly recognized by the World Health Organization (WHO) in 2004. These tumors result in the fusion of partner genes to the TFE3 gene located on Xp11.2. They are most common in the pediatric population, but have been recently implicated in adult renal cell carcinoma (RCC) presenting at an early age. TFE3-mediated direct transcriptional upregulation of the Met tyrosine kinase receptor triggers dramatic activation of downstream signaling pathways including the protein kinase B (Akt)/phosphatidylinositol-3 kinase (PI3K) and mammalian target of rapamycin (mTOR) pathways. Temsirolimus is an inhibitor of mammalian target of rapamycin (mTOR) kinase, a component of intracellular signaling pathways involved in the growth and proliferation of malignant cells. Here we present a case of a 22-year old female who has been treated with temsirolimus for her Xp11.2/TFE3 gene fusion RCC. PMID:21139932

Regulators and effectors of bone morphogenetic protein signalling in the cardiovascular system.

PubMed

Luo, Jiang-Yun; Zhang, Yang; Wang, Li; Huang, Yu

2015-07-15

Bone morphogenetic proteins (BMPs) play key roles in the regulation of cell proliferation, differentiation and apoptosis in various tissues and organs, including the cardiovascular system. BMPs signal through both Smad-dependent and -independent cascades to exert a wide spectrum of biological activities. Cardiovascular disorders such as abnormal angiogenesis, atherosclerosis, pulmonary hypertension and cardiac hypertrophy have been linked to aberrant BMP signalling. To correct the dysregulated BMP signalling in cardiovascular pathogenesis, it is essential to get a better understanding of how the regulators and effectors of BMP signalling control cardiovascular function and how the dysregulated BMP signalling contributes to cardiovascular dysfunction. We hence highlight several key regulators of BMP signalling such as extracellular regulators of ligands, mechanical forces, microRNAs and small molecule drugs as well as typical BMP effectors like direct downstream target genes, mitogen-activated protein kinases, reactive oxygen species and microRNAs. The insights into these molecular processes will help target both the regulators and important effectors to reverse BMP-associated cardiovascular pathogenesis. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

A multiple-alignment based primer design algorithm for genetically highly variable DNA targets

PubMed Central

2013-01-01

Background Primer design for highly variable DNA sequences is difficult, and experimental success requires attention to many interacting constraints. The advent of next-generation sequencing methods allows the investigation of rare variants otherwise hidden deep in large populations, but requires attention to population diversity and primer localization in relatively conserved regions, in addition to recognized constraints typically considered in primer design. Results Design constraints include degenerate sites to maximize population coverage, matching of melting temperatures, optimizing de novo sequence length, finding optimal bio-barcodes to allow efficient downstream analyses, and minimizing risk of dimerization. To facilitate primer design addressing these and other constraints, we created a novel computer program (PrimerDesign) that automates this complex procedure. We show its powers and limitations and give examples of successful designs for the analysis of HIV-1 populations. Conclusions PrimerDesign is useful for researchers who want to design DNA primers and probes for analyzing highly variable DNA populations. It can be used to design primers for PCR, RT-PCR, Sanger sequencing, next-generation sequencing, and other experimental protocols targeting highly variable DNA samples. PMID:23965160

Rett syndrome treatment in mouse models: searching for effective targets and strategies.

PubMed

Ricceri, Laura; De Filippis, Bianca; Laviola, Giovanni

2013-05-01

Rett syndrome (RTT) is a pervasive developmental disorder, primarily affecting girls with a prevalence of 1 in every 10,000 births; it represents the second most common cause of intellectual disability in females. Mutations in the gene encoding methyl-CpG-binding protein 2 (MECP2) have been identified as clear etiological factors in more than 90% of classical RTT cases. Whereas the mechanisms leading to the severe, progressive and specific neurological dysfunctions when this gene is mutated still remain to be elucidated, a series of different mouse models have been generated, bearing different Mecp2 mutation. Neurobehavioural analysis in these mouse lines have been carried out and phenotyping analysis can be now utilised to preclinically evaluate the effects of potential RTT treatments. This review summarizes the different results achieved in this research field taking into account different key targets identified to ameliorate RTT phenotype in mouse models, including those not directly downstream of MeCP2 and those limited to the early phases of postnatal development. This article is part of the Special Issue entitled 'Neurodevelopmental Disorders'. Copyright © 2012 Elsevier Ltd. All rights reserved.

A coupled modelling effort to study the fate of contaminated sediments downstream of the Coles Hill deposit, Virginia, USA

NASA Astrophysics Data System (ADS)

Castro-Bolinaga, C. F.; Zavaleta, E. R.; Diplas, P.

2015-03-01

This paper presents the preliminary results of a coupled modelling effort to study the fate of tailings (radioactive waste-by product) downstream of the Coles Hill uranium deposit located in Virginia, USA. The implementation of the overall modelling process includes a one-dimensional hydraulic model to qualitatively characterize the sediment transport process under severe flooding conditions downstream of the potential mining site, a two-dimensional ANSYS Fluent model to simulate the release of tailings from a containment cell located partially above the local ground surface into the nearby streams, and a one-dimensional finite-volume sediment transport model to examine the propagation of a tailings sediment pulse in the river network located downstream. The findings of this investigation aim to assist in estimating the potential impacts that tailings would have if they were transported into rivers and reservoirs located downstream of the Coles Hill deposit that serve as municipal drinking water supplies.

Feeding by whiteflies suppresses downstream jasmonic acid signaling by eliciting salicylic acid signaling.

PubMed

Zhang, Peng-Jun; Li, Wei-Di; Huang, Fang; Zhang, Jin-Ming; Xu, Fang-Cheng; Lu, Yao-Bin

2013-05-01

Phloem-feeding whiteflies in the species complex Bemisia tabaci cause extensive crop damage worldwide. One of the reasons for their "success" is their ability to suppress the effectual jasmonic acid (JA) defenses of the host plant. However, little is understood about the mechanisms underlying whitefly suppression of JA-regulated defenses. Here, we showed that the expression of salicylic acid (SA)-responsive genes (EDS1 and PR1) in Arabidopsis thaliana was significantly enhanced during feeding by whitefly nymphs. Whereas upstream JA-responsive genes (LOX2 and OPR3) also were induced, the downstream JA-responsive gene (VSP1) was repressed, i.e., whiteflies only suppressed downstream JA signaling. Gene-expression analyses with various Arabidopsis mutants, including NahG, npr-1, ein2-1, and dde2-2, revealed that SA signaling plays a key role in the suppression of downstream JA defenses by whitefly feeding. Assays confirmed that SA activation enhanced whitefly performance by suppressing downstream JA defenses.

The Role of the Melanocortin System in Metabolic Disease: New Developments and Advances.

PubMed

Hill, Jennifer W; Faulkner, Latrice D

2017-01-01

Obesity is increasing in prevalence across all sectors of society, and with it a constellation of associated ailments including hypertension, type 2 diabetes, and eating disorders. The melanocortin system is a critical neural system underlying the control of body weight and other functions. Deficits in the melanocortin system may promote or exacerbate the comorbidities of obesity. This system has therefore generated great interest as a potential target for treatment of obesity. However, drugs targeting melanocortin receptors are plagued by problematic side effects, including undesirable increases in sympathetic nervous system activity, heart rate, and blood pressure. Circumnavigating this roadblock will require a clearer picture of the precise neural circuits that mediate the functions of melanocortins. Recent, novel experimental approaches have significantly advanced our understanding of these pathways. We here review the latest advances in our understanding of the role of melanocortins in food intake, reward pathways, blood pressure, glucose control, and energy expenditure. The evidence suggests that downstream melanocortin-responsive circuits responsible for different physiological actions do diverge. Ultimately, a more complete understanding of melanocortin pathways and their myriad roles should allow treatments tailored to the mix of metabolic disorders in the individual patient. © 2016 The Author(s) Published by S. Karger AG, Basel.

Dermal Blimp1 Acts Downstream of Epidermal TGFβ and Wnt/β-Catenin to Regulate Hair Follicle Formation and Growth.

PubMed

Telerman, Stephanie B; Rognoni, Emanuel; Sequeira, Inês; Pisco, Angela Oliveira; Lichtenberger, Beate M; Culley, Oliver J; Viswanathan, Priyalakshmi; Driskell, Ryan R; Watt, Fiona M

2017-11-01

B-lymphocyte-induced maturation protein 1 (Blimp1) is a transcriptional repressor that regulates cell growth and differentiation in multiple tissues, including skin. Although in the epidermis Blimp1 is important for keratinocyte and sebocyte differentiation, its role in dermal fibroblasts is unclear. Here we show that Blimp1 is dynamically regulated in dermal papilla cells during hair follicle (HF) morphogenesis and the postnatal hair cycle, preceding dermal Wnt/β-catenin activation. Blimp1 ablation in E12.5 mouse dermal fibroblasts delayed HF morphogenesis and growth and prevented new HF formation after wounding. By combining targeted quantitative PCR screens with bioinformatic analysis and experimental validation we demonstrated that Blimp1 is both a target and a mediator of key dermal papilla inductive signaling pathways including transforming growth factor-β and Wnt/β-catenin. Epidermal overexpression of stabilized β-catenin was able to override the HF defects in Blimp1 mutant mice, underlining the close reciprocal relationship between the dermal papilla and adjacent HF epithelial cells. Overall, our study reveals the functional role of Blimp1 in promoting the dermal papilla inductive signaling cascade that initiates HF growth. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Yorkie Facilitates Organ Growth and Metamorphosis in Bombyx

PubMed Central

Liu, Shumin; Zhang, Panli; Song, Hong-Sheng; Qi, Hai-Sheng; Wei, Zhao-Jun; Zhang, Guozheng; Zhan, Shuai; Liu, Zhihong; Li, Sheng

2016-01-01

The Hippo pathway, which was identified from genetic screens in the fruit fly, Drosophila melanogaster, has a major size-control function in animals. All key components of the Hippo pathway, including the transcriptional coactivator Yorkie that is the most critical substrate and downstream effector of the Hippo kinase cassette, are found in the silkworm, Bombyx mori. As revealed by microarray and quantitative real-time PCR, expression of Hippo pathway genes is particularly enriched in several mitotic tissues, including the ovary, testis, and wing disc. Developmental profiles of Hippo pathway genes are generally similar (with the exception of Yorkie) within each organ, but vary greatly in different tissues showing nearly opposing expression patterns in the wing disc and the posterior silk gland (PSG) on day 2 of the prepupal stage. Importantly, the reduction of Yorkie expression by RNAi downregulated Yorkie target genes in the ovary, decreased egg number, and delayed larval-pupal-adult metamorphosis. In contrast, baculovirus-mediated YorkieCA overexpression upregulated Yorkie target genes in the PSG, increased PSG size, and accelerated larval-pupal metamorphosis. Together the results show that Yorkie potentially facilitates organ growth and metamorphosis, and suggest that the evolutionarily conserved Hippo pathway is critical for size control, particularly for PSG growth, in the silkworm. PMID:27489496

Potential Therapeutic Targets of Epithelial-Mesenchymal Transition in Melanoma

PubMed Central

Pearlman, Ross L.; de Oca, Mary Katherine Montes; Pal, Harish Chandra; Afaq, Farrukh

2017-01-01

Melanoma is a cutaneous neoplastic growth of melanocytes with great potential to invade and metastasize, especially when not treated early and effectively. Epithelial-mesenchymal transition (EMT) is the process by which melanocytes lose their epithelial characteristics and acquire mesenchymal phenotypes. Mesenchymal protein expression increases the motility, invasiveness, and metastatic potential of melanoma. Many pathways play a role in promotion of mesenchymal protein expression including RAS/RAF/MEK/ERK, PI3K/AKT/mTOR, Wnt/β-catenin, and several others. Downstream effectors of these pathways induce expression of EMT transcription factors including Snail, Slug, Twist, and Zeb that promote repression of epithelial and induction of mesenchymal character. Emerging research has demonstrated that a variety of small molecule inhibitors as well as phytochemicals can influence the progression of EMT and may even reverse the process, inducing re-expression of epithelial markers. Phytochemicals are of particular interest as supplementary treatment options because of their relatively low toxicities and anti-EMT properties. Modulation of EMT signaling pathways using synthetic small molecules and phytochemicals is a potential therapeutic strategy for reducing the aggressive progression of metastatic melanoma. In this review, we discuss the emerging pathways and transcription factor targets that regulate EMT and evaluate potential synthetic small molecules and naturally occurring compounds that may reduce metastatic melanoma progression. PMID:28131904

Coupling growth-factor engineering with nanotechnology for therapeutic angiogenesis.

PubMed

Sinha Roy, Rituparna; Soni, Shivani; Harfouche, Rania; Vasudevan, Pooja R; Holmes, Oliver; de Jonge, Hugo; Rowe, Arthur; Paraskar, Abhimanyu; Hentschel, Dirk M; Chirgadze, Dimitri; Blundell, Tom L; Gherardi, Ermanno; Mashelkar, Raghunath A; Sengupta, Shiladitya

2010-08-03

Therapeutic angiogenesis is an emerging paradigm for the management of ischemic pathologies. Proangiogenic Therapy is limited, however, by the current inability to deliver angiogenic factors in a sustained manner at the site of pathology. In this study, we investigated a unique nonglycosylated active fragment of hepatocyte growth factor/scatter factor, 1K1, which acts as a potent angiogenic agent in vitro and in a zebrafish embryo and a murine matrigel implant model. Furthermore, we demonstrate that nanoformulating 1K1 for sustained release temporally alters downstream signaling through the mitogen activated protein kinase pathway, and amplifies the angiogenic outcome. Merging protein engineering and nanotechnology offers exciting possibilities for the treatment of ischemic disease, and furthermore allows the selective targeting of downstream signaling pathways, which translates into discrete phenotypes.

Subcellular Redox Signaling.

PubMed

Zhu, Liping; Lu, Yankai; Zhang, Jiwei; Hu, Qinghua

2017-01-01

Oxidative and antioxidative system of cells and tissues maintains a balanced state under physiological conditions. A disruption in this balance of redox status has been associated with numerous pathological processes. Reactive oxygen species (ROS) as a major redox signaling generates in a spatiotemporally dependent manner. Subcellular organelles such as mitochondria, endoplasmic reticulum, plasma membrane and nuclei contribute to the production of ROS. In addition to downstream effects of ROS signaling regulated by average ROS changes in cytoplasm, whether subcelluar ROS mediate biological effect(s) has drawn greater attentions. With the advance in redox-sensitive probes targeted to different subcellular compartments, the investigation of subcellular ROS signaling and its associated cellular function has become feasible. In this review, we discuss the subcellular ROS signaling, with particular focus on mechanisms of subcellular ROS production and its downstream effects.

Taste Receptor Signaling-- From Tongues to Lungs

PubMed Central

Kinnamon, Sue C.

2013-01-01

Taste buds are the transducing endorgans of gustation. Each taste bud comprises 50–100 elongated cells, which extend from the basal lamina to the surface of the tongue, where their apical microvilli encounter taste stimuli in the oral cavity. Salts and acids utilize apically located ion channels for transduction, while bitter, sweet and umami (glutamate) stimuli utilize G protein coupled receptors (GPCRs) and second messenger signaling mechanisms. This review will focus on GPCR signaling mechanisms. Two classes of taste GPCRs have been identified, the T1Rs for sweet and umami (glutamate) stimuli, and the T2Rs for bitter stimuli. These low affinity GPCRs all couple to the same downstream signaling effectors that include Gβγ activation of PLCβ2, IP3-mediated release of Ca2+ from intracellular stores, and Ca2+-dependent activation of the monovalent selective cation channel, TrpM5. These events lead to membrane depolarization, action potentials, and release of ATP as a transmitter to activate gustatory afferents. The Gα subunit, α-gustducin, activates a phosphodiesterase to decrease intracellular cAMP levels, although the precise targets of cAMP have not been identified. With the molecular identification of the taste GPCRs, it has become clear that taste signaling is not limited to taste buds, but occurs in many cell types of the airways. These include solitary chemosensory cells, ciliated epithelial cells, and smooth muscle cells. Bitter receptors are most abundantly expressed in the airways, where they respond to irritating chemicals and promote protective airway reflexes, utilizing the same downstream signaling effectors as taste cells. PMID:21481196

A 5′ Splice Site-Proximal Enhancer Binds SF1 and Activates Exon Bridging of a Microexon

PubMed Central

Carlo, Troy; Sierra, Rebecca; Berget, Susan M.

2000-01-01

Internal exon size in vertebrates occurs over a narrow size range. Experimentally, exons shorter than 50 nucleotides are poorly included in mRNA unless accompanied by strengthened splice sites or accessory sequences that act as splicing enhancers, suggesting steric interference between snRNPs and other splicing factors binding simultaneously to the 3′ and 5′ splice sites of microexons. Despite these problems, very small naturally occurring exons exist. Here we studied the factors and mechanism involved in recognizing a constitutively included six-nucleotide exon from the cardiac troponin T gene. Inclusion of this exon is dependent on an enhancer located downstream of the 5′ splice site. This enhancer contains six copies of the simple sequence GGGGCUG. The enhancer activates heterologous microexons and will work when located either upstream or downstream of the target exon, suggesting an ability to bind factors that bridge splicing units. A single copy of this sequence is sufficient for in vivo exon inclusion and is the binding site for the known bridging mammalian splicing factor 1 (SF1). The enhancer and its bound SF1 act to increase recognition of the upstream exon during exon definition, such that competition of in vitro reactions with RNAs containing the GGGGCUG repeated sequence depress splicing of the upstream intron, assembly of the spliceosome on the 3′ splice site of the exon, and cross-linking of SF1. These results suggest a model in which SF1 bridges the small exon during initial assembly, thereby effectively extending the domain of the exon. PMID:10805741

Turbine and method for reducing shock losses in a turbine

DOEpatents

Ristau, Neil

2015-09-01

A turbine includes a rotor and a casing that circumferentially surrounds at least a portion of the rotor. The rotor and the casing at least partially define a gas path through the turbine. A last stage of rotating blades is circumferentially arranged around the rotor and includes a downstream swept portion radially outward from the rotor. A method for reducing shock losses in a turbine includes removing a last stage of rotating blades circumferentially arranged around a rotor and replacing the last stage of rotating blades with rotating blades having a downstream swept portion radially outward from the rotor.

A New Strategy to Control and Eradicate “Undruggable” Oncogenic K-RAS-Driven Pancreatic Cancer: Molecular Insights and Core Principles Learned from Developmental and Evolutionary Biology

PubMed Central

Lee, Michael P.; Lee, Caroline Dasom; Lafever, Alex C.; Svyatova, Elizaveta; Kanda, Kevin; Collier, Amber L.; Siewertsz van Reesema, Lauren L.; Tang-Tan, Angela M.; Zheleva, Vasilena; Bwayi, Monicah N.; Bian, Minglei; Schmidt, Rebecca L.; Petersen, Gloria M.

2018-01-01

Oncogenic K-RAS mutations are found in virtually all pancreatic cancers, making K-RAS one of the most targeted oncoproteins for drug development in cancer therapies. Despite intense research efforts over the past three decades, oncogenic K-RAS has remained largely “undruggable”. Rather than targeting an upstream component of the RAS signaling pathway (i.e., EGFR/HER2) and/or the midstream effector kinases (i.e., RAF/MEK/ERK/PI3K/mTOR), we propose an alternative strategy to control oncogenic K-RAS signal by targeting its most downstream signaling module, Seven-In-Absentia Homolog (SIAH). SIAH E3 ligase controls the signal output of oncogenic K-RAS hyperactivation that drives unchecked cell proliferation, uncontrolled tumor growth, and rapid cancer cell dissemination in human pancreatic cancer. Therefore, SIAH is an ideal therapeutic target as it is an extraordinarily conserved downstream signaling gatekeeper indispensable for proper RAS signaling. Guided by molecular insights and core principles obtained from developmental and evolutionary biology, we propose an anti-SIAH-centered anti-K-RAS strategy as a logical and alternative anticancer strategy to dampen uncontrolled K-RAS hyperactivation and halt tumor growth and metastasis in pancreatic cancer. The clinical utility of developing SIAH as both a tumor-specific and therapy-responsive biomarker, as well as a viable anti-K-RAS drug target, is logically simple and conceptually innovative. SIAH clearly constitutes a major tumor vulnerability and K-RAS signaling bottleneck in pancreatic ductal adenocarcinoma (PDAC). Given the high degree of evolutionary conservation in the K-RAS/SIAH signaling pathway, an anti-SIAH-based anti-PDAC therapy will synergize with covalent K-RAS inhibitors and direct K-RAS targeted initiatives to control and eradicate pancreatic cancer in the future. PMID:29757973

Targeting the Epigenome in Lung Cancer: Expanding Approaches to Epigenetic Therapy

PubMed Central

Jakopovic, Marko; Thomas, Anish; Balasubramaniam, Sanjeeve; Schrump, David; Giaccone, Giuseppe; Bates, Susan E.

2013-01-01

Epigenetic aberrations offer dynamic and reversible targets for cancer therapy; increasingly, alteration via overexpression, mutation, or rearrangement is found in genes that control the epigenome. Such alterations suggest a fundamental role in carcinogenesis. Here, we consider three epigenetic mechanisms: DNA methylation, histone tail modification and non-coding, microRNA regulation. Evidence for each of these in lung cancer origin or progression has been gathered, along with evidence that epigenetic alterations might be useful in early detection. DNA hypermethylation of tumor suppressor promoters has been observed, along with global hypomethylation and hypoacetylation, suggesting an important role for tumor suppressor gene silencing. These features have been linked as prognostic markers with poor outcome in lung cancer. Several lines of evidence have also suggested a role for miRNA in carcinogenesis and in outcome. Cigarette smoke downregulates miR-487b, which targets both RAS and MYC; RAS is also a target of miR-let-7, again downregulated in lung cancer. Together the evidence implicates epigenetic aberration in lung cancer and suggests that targeting these aberrations should be carefully explored. To date, DNA methyltransferase and histone deacetylase inhibitors have had minimal clinical activity. Explanations include the possibility that the agents are not sufficiently potent to invoke epigenetic reversion to a more normal state; that insufficient time elapses in most clinical trials to observe true epigenetic reversion; and that doses often used may provoke off-target effects such as DNA damage that prevent epigenetic reversion. Combinations of epigenetic therapies may address those problems. When epigenetic agents are used in combination with chemotherapy or targeted therapy it is hoped that downstream biological effects will provoke synergistic cytotoxicity. This review evaluates the challenges of exploiting the epigenome in the treatment of lung cancer. PMID:24130964

Integrated development of up- and downstream processes supported by the Cherry-Tag™ for real-time tracking of stability and solubility of proteins.

PubMed

Baumann, Pascal; Bluthardt, Nicolai; Renner, Sarah; Burghardt, Hannah; Osberghaus, Anna; Hubbuch, Jürgen

2015-04-20

Product analytics is the bottleneck of most processes in bioprocess engineering, as it is rather time-consuming. Real-time and in-line product tracing without sample pre-treatment is only possible for few products. The Cherry-Tag™ (Delphi Genetics, Belgium) which can be fused to any target protein allows for straightforward product analytics by VIS absorption measurements. When the fused protein becomes unstable or insoluble, the chromophore function of the group is lost, which makes this technology an ideal screening tool for solubility and stability in up- and downstream process development. The Cherry-Tag™ technology will be presented for the tagged enzyme glutathione-S-transferase (GST) from Escherichia coli in a combined up- and downstream process development study. High-throughput cultivations were carried out in a 48-well format in a BioLector system (m2p-Labs, Germany). The best cultivation setup of highest product titer was scaled up to a 2.5L shake flask culture, followed by a selective affinity chromatography product capturing step. In upstream applications the tag was capable of identifying conditions where insoluble and non-native inclusion bodies were formed. In downstream applications the red-colored product was found to be bound effectively to a GST affinity column. Thus, it was identified to be a native and active protein, as the binding mechanism relies on catalytic activity of the enzyme. The Cherry-Tag™ was found to be a reliable and quantitative tool for real-time tracking of stable and soluble proteins in up- and downstream processing applications. Denaturation and aggregation of the product can be detected in-line at any stage of the process. Critical stages can be identified and subsequently changed or replaced. Copyright © 2015 Elsevier B.V. All rights reserved.

Optogenetic analysis of a nociceptor neuron and network reveals ion channels acting downstream of primary sensors.

PubMed

Husson, Steven J; Costa, Wagner Steuer; Wabnig, Sebastian; Stirman, Jeffrey N; Watson, Joseph D; Spencer, W Clay; Akerboom, Jasper; Looger, Loren L; Treinin, Millet; Miller, David M; Lu, Hang; Gottschalk, Alexander

2012-05-08

Nociception generally evokes rapid withdrawal behavior in order to protect the tissue from harmful insults. Most nociceptive neurons responding to mechanical insults display highly branched dendrites, an anatomy shared by Caenorhabditis elegans FLP and PVD neurons, which mediate harsh touch responses. Although several primary molecular nociceptive sensors have been characterized, less is known about modulation and amplification of noxious signals within nociceptor neurons. First, we analyzed the FLP/PVD network by optogenetics and studied integration of signals from these cells in downstream interneurons. Second, we investigated which genes modulate PVD function, based on prior single-neuron mRNA profiling of PVD. Selectively photoactivating PVD, FLP, and downstream interneurons via Channelrhodopsin-2 (ChR2) enabled the functional dissection of this nociceptive network, without interfering signals by other mechanoreceptors. Forward or reverse escape behaviors were determined by PVD and FLP, via integration by command interneurons. To identify mediators of PVD function, acting downstream of primary nocisensor molecules, we knocked down PVD-specific transcripts by RNAi and quantified light-evoked PVD-dependent behavior. Cell-specific disruption of synaptobrevin or voltage-gated Ca(2+) channels (VGCCs) showed that PVD signals chemically to command interneurons. Knocking down the DEG/ENaC channel ASIC-1 and the TRPM channel GTL-1 indicated that ASIC-1 may extend PVD's dynamic range and that GTL-1 may amplify its signals. These channels act cell autonomously in PVD, downstream of primary mechanosensory molecules. Our work implicates TRPM channels in modifying excitability of and DEG/ENaCs in potentiating signal output from a mechano-nociceptor neuron. ASIC-1 and GTL-1 homologs, if functionally conserved, may denote valid targets for novel analgesics. Copyright © 2012 Elsevier Ltd. All rights reserved.

COX-derived prostanoid pathways in gastrointestinal cancer development and progression: novel targets for prevention and intervention.

PubMed

Cathcart, Mary-Clare; O'Byrne, Kenneth J; Reynolds, John V; O'Sullivan, Jacintha; Pidgeon, Graham P

2012-01-01

Arachidonic acid metabolism through cyclooxygenase (COX) pathways leads to the generation of biologically active eicosanoids. Eicosanoid expression levels vary during development and progression of gastrointestinal (GI) malignancies. COX-2 is the major COX-isoform responsible for G.I. cancer development/progression. COX-2 expression increases during progression from a normal to cancerous state. Evidence from observational studies has demonstrated that chronic NSAID use reduces the risk of cancer development, while both incidence and risk of death due to G.I. cancers were significantly reduced by daily aspirin intake. A number of randomized controlled trials (APC trial, Prevention of Sporadic Adenomatous Polyps trial, APPROVe trial) have also shown a significant protective effect in patients receiving selective COX-2 inhibitors. However, chronic use of selective COX-2 inhibitors at high doses was associated with increased cardiovascular risk, while NSAIDs have also been associated with increased risk. More recently, downstream effectors of COX-signaling have been investigated in cancer development/progression. PGE(2), which binds to both EP and PPAR receptors, is the major prostanoid implicated in the carcinogenesis of G.I. cancers. The role of TXA(2) in G.I. cancers has also been examined, although further studies are required to uncover its role in carcinogenesis. Other prostanoids investigated include PGD(2) and its metabolite 15d-PGJ2, PGF(1α) and PGI(2). Targeting these prostanoids in G.I. cancers has the promise of avoiding cardiovascular toxicity associated with chronic selective COX-2 inhibition, while maintaining anti-tumor reactivity. A progressive sequence from normal to pre-malignant to a malignant state has been identified in G.I. cancers. In this review, we will discuss the role of the COX-derived prostanoids in G.I. cancer development and progression. Targeting these downstream prostanoids for chemoprevention and/or treatment of G.I. cancers will also be discussed. Finally, we will highlight the latest pre-clinical technologies as well as avenues for future investigation in this highly topical research field. Copyright © 2011 Elsevier B.V. All rights reserved.

System and method for reducing combustion dynamics in a combustor

DOEpatents

Uhm, Jong Ho; Johnson, Thomas Edward; Zuo, Baifang; York, William David

2015-09-01

A system for reducing combustion dynamics in a combustor includes an end cap having an upstream surface axially separated from a downstream surface, and tube bundles extend from the upstream surface through the downstream surface. A divider inside a tube bundle defines a diluent passage that extends axially through the downstream surface, and a diluent supply in fluid communication with the divider provides diluent flow to the diluent passage. A method for reducing combustion dynamics in a combustor includes flowing a fuel through tube bundles, flowing a diluent through a diluent passage inside a tube bundle, wherein the diluent passage extends axially through at least a portion of the end cap into a combustion chamber, and forming a diluent barrier in the combustion chamber between the tube bundle and at least one other adjacent tube bundle.

Tumor Inhibitory Effect of IRCR201, a Novel Cross-Reactive c-Met Antibody Targeting the PSI Domain.

PubMed

Park, Hyunkyu; Kim, Donggeon; Kim, Eunmi; Sa, Jason K; Lee, Hee Won; Yu, Suji; Oh, Jiwon; Kim, Seok-Hyung; Yoon, Yeup; Nam, Do-Hyun

2017-09-13

Hepatocyte growth factor receptor (HGFR, c-Met) is an essential member of the receptor tyrosine kinase (RTK) family that is often dysregulated during tumor progression, driving a malignant phenotypic state and modulating important cellular functions including tumor growth, invasion, metastasis, and angiogenesis, providing a strong rationale for targeting HGF/c-Met signaling axis in cancer therapy. Based on its protumorigenic potentials, we developed IRCR201, a potent antagonistic antibody targeting the plexin-semaphorin-integrin (PSI) domain of c-Met, using synthetic human antibody phage libraries. We characterized and evaluated the biochemical properties and tumor inhibitory effect of IRCR201 in vitro and in vivo. IRCR201 is a novel fully-human bivalent therapeutic antibody that exhibits cross-reactivity against both human and mouse c-Met proteins with high affinity and specificity. IRCR201 displayed low agonist activity and rapidly depleted total c-Met protein via the lysosomal degradation pathway, inhibiting c-Met-dependent downstream activation and attenuating cellular proliferation in various c-Met-expressing cancer cells. In vivo tumor xenograft models also demonstrated the superior tumor inhibitory responsiveness of IRCR201. Taken together, IRCR201 provides a promising therapeutic agent for c-Met-positive cancer patients through suppressing the c-Met signaling pathway and tumor growth.

A Discrete Ubiquitin-Mediated Network Regulates the Strength of NOD2 Signaling

PubMed Central

Tigno-Aranjuez, Justine T.; Bai, Xiaodong

2013-01-01

Dysregulation of NOD2 signaling is implicated in the pathology of various inflammatory diseases, including Crohn's disease, asthma, and sarcoidosis, making signaling proteins downstream of NOD2 potential therapeutic targets. Inhibitor-of-apoptosis (IAP) proteins, particularly cIAP1, are essential mediators of NOD2 signaling, and in this work, we describe a molecular mechanism for cIAP1's regulation in the NOD2 signaling pathway. While cIAP1 promotes RIP2's tyrosine phosphorylation and subsequent NOD2 signaling, this positive regulation is countered by another E3 ubiquitin ligase, ITCH, through direct ubiquitination of cIAP1. This ITCH-mediated ubiquitination leads to cIAP1's lysosomal degradation. Pharmacologic inhibition of cIAP1 expression in ITCH−/− macrophages attenuates heightened ITCH−/− macrophage muramyl dipeptide-induced responses. Transcriptome analysis, combined with pharmacologic inhibition of cIAP1, further defines specific pathways within the NOD2 signaling pathway that are targeted by cIAP1. This information provides genetic signatures that may be useful in repurposing cIAP1-targeted therapies to correct NOD2-hyperactive states and identifies a ubiquitin-regulated signaling network centered on ITCH and cIAP1 that controls the strength of NOD2 signaling. PMID:23109427

Lifespan extension induced by AMPK and calcineurin is mediated by CRTC-1 and CREB.

PubMed

Mair, William; Morantte, Ianessa; Rodrigues, Ana P C; Manning, Gerard; Montminy, Marc; Shaw, Reuben J; Dillin, Andrew

2011-02-17

Activating AMPK or inactivating calcineurin slows ageing in Caenorhabditis elegans and both have been implicated as therapeutic targets for age-related pathology in mammals. However, the direct targets that mediate their effects on longevity remain unclear. In mammals, CREB-regulated transcriptional coactivators (CRTCs) are a family of cofactors involved in diverse physiological processes including energy homeostasis, cancer and endoplasmic reticulum stress. Here we show that both AMPK and calcineurin modulate longevity exclusively through post-translational modification of CRTC-1, the sole C. elegans CRTC. We demonstrate that CRTC-1 is a direct AMPK target, and interacts with the CREB homologue-1 (CRH-1) transcription factor in vivo. The pro-longevity effects of activating AMPK or deactivating calcineurin decrease CRTC-1 and CRH-1 activity and induce transcriptional responses similar to those of CRH-1 null worms. Downregulation of crtc-1 increases lifespan in a crh-1-dependent manner and directly reducing crh-1 expression increases longevity, substantiating a role for CRTCs and CREB in ageing. Together, these findings indicate a novel role for CRTCs and CREB in determining lifespan downstream of AMPK and calcineurin, and illustrate the molecular mechanisms by which an evolutionarily conserved pathway responds to low energy to increase longevity.

Lifespan extension induced by AMPK and calcineurin is mediated by CRTC-1 and CREB

PubMed Central

Mair, William; Morantte, Ianessa; Rodrigues, Ana P. C.; Manning, Gerard; Montminy, Marc; Shaw, Reuben J.; Dillin, Andrew

2011-01-01

Activating AMPK or inactivating calcineurin slows ageing in Caenorhabditis elegans1,2 and both have been implicated as therapeutic targets for age-related pathology in mammals3–5. However, the direct targets that mediate their effects on longevity remain unclear. In mammals, CREB-regulated transcriptional coactivators (CRTCs)6 are a family of cofactors involved in diverse physiological processes including energy homeostasis7–9, cancer10 and endoplasmic reticulum stress11. Here we show that both AMPK and calcineurin modulate longevity exclusively through post-translational modification of CRTC-1, the sole C. elegans CRTC. We demonstrate that CRTC-1 is a direct AMPK target, and interacts with the CREB homologue-1 (CRH-1) transcription factor in vivo. The pro-longevity effects of activating AMPK or deactivating calcineurin decrease CRTC-1 and CRH-1 activity and induce transcriptional responses similar to those of CRH-1 null worms. Downregulation of crtc-1 increases lifespan in a crh-1-dependent manner and directly reducing crh-1 expression increases longevity, substantiating a role for CRTCs and CREB in ageing. Together, these findings indicate a novel role for CRTCs and CREB in determining lifespan downstream of AMPK and calcineurin, and illustrate the molecular mechanisms by which an evolutionarily conserved pathway responds to low energy to increase longevity. PMID:21331044

DYRK1B as therapeutic target in Hedgehog/GLI-dependent cancer cells with Smoothened inhibitor resistance

PubMed Central

Gruber, Wolfgang; Hutzinger, Martin; Elmer, Dominik Patrick; Parigger, Thomas; Sternberg, Christina; Cegielkowski, Lukasz; Zaja, Mirko; Leban, Johann; Michel, Susanne; Hamm, Svetlana; Vitt, Daniel; Aberger, Fritz

2016-01-01

A wide range of human malignancies displays aberrant activation of Hedgehog (HH)/GLI signaling, including cancers of the skin, brain, gastrointestinal tract and hematopoietic system. Targeting oncogenic HH/GLI signaling with small molecule inhibitors of the essential pathway effector Smoothened (SMO) has shown remarkable therapeutic effects in patients with advanced and metastatic basal cell carcinoma. However, acquired and de novo resistance to SMO inhibitors poses severe limitations to the use of SMO antagonists and urgently calls for the identification of novel targets and compounds. Here we report on the identification of the Dual-Specificity-Tyrosine-Phosphorylation-Regulated Kinase 1B (DYRK1B) as critical positive regulator of HH/GLI signaling downstream of SMO. Genetic and chemical inhibition of DYRK1B in human and mouse cancer cells resulted in marked repression of HH signaling and GLI1 expression, respectively. Importantly, DYRK1B inhibition profoundly impaired GLI1 expression in both SMO-inhibitor sensitive and resistant settings. We further introduce a novel small molecule DYRK1B inhibitor, DYRKi, with suitable pharmacologic properties to impair SMO-dependent and SMO-independent oncogenic GLI activity. The results support the use of DYRK1B antagonists for the treatment of HH/GLI-associated cancers where SMO inhibitors fail to demonstrate therapeutic efficacy. PMID:26784250

Calpain-mediated cleavage of collapsin response mediator protein-2 drives acute axonal degeneration

PubMed Central

Zhang, Jian-Nan; Michel, Uwe; Lenz, Christof; Friedel, Caroline C.; Köster, Sarah; d’Hedouville, Zara; Tönges, Lars; Urlaub, Henning; Bähr, Mathias; Lingor, Paul; Koch, Jan C.

2016-01-01

Axonal degeneration is a key initiating event in many neurological diseases. Focal lesions to axons result in a rapid disintegration of the perilesional axon by acute axonal degeneration (AAD) within several hours. However, the underlying molecular mechanisms of AAD are only incompletely understood. Here, we studied AAD in vivo through live-imaging of the rat optic nerve and in vitro in primary rat cortical neurons in microfluidic chambers. We found that calpain is activated early during AAD of the optic nerve and that calpain inhibition completely inhibits axonal fragmentation on the proximal side of the crush while it attenuates AAD on the distal side. A screening of calpain targets revealed that collapsin response mediator protein-2 (CRMP2) is a main downstream target of calpain activation in AAD. CRMP2-overexpression delayed bulb formation and rescued impairment of axonal mitochondrial transport after axotomy in vitro. In vivo, CRMP2-overexpression effectively protected the proximal axon from fragmentation within 6 hours after crush. Finally, a proteomic analysis of the optic nerve was performed at 6 hours after crush, which identified further proteins regulated during AAD, including several interactors of CRMP2. These findings reveal CRMP2 as an important mediator of AAD and define it as a putative therapeutic target. PMID:27845394

Occurrence and preliminarily environmental risk assessment of selected pharmaceuticals in the urban rivers, China

PubMed Central

Zhou, Haidong; Ying, Tianqi; Wang, Xuelian; Liu, Jianbo

2016-01-01

Twelve selected pharmaceuticals including antibiotics, analgesics, antiepileptics and lipid regulators were analysed and detected in water samples collected from 18 sampling sections along the three main urban rivers in Yangpu District of Shanghai, China during four sampling campaigns. Besides, algal growth inhibition test was conducted to preliminarily assess the eco-toxicology induced by the target pharmaceuticals in the rivers. Mean levels for most of target compounds were generally below 100 ng/L at sampling sections, with the exception of caffeine and paracetamol presenting considerably high concentration. The detected pharmaceuticals in the urban rivers ranged from

Ras oncogenes in oral cancer: the past 20 years.

PubMed

Murugan, Avaniyapuram Kannan; Munirajan, Arasambattu Kannan; Tsuchida, Nobuo

2012-05-01

Oral squamous cell carcinoma (OSCC) of head and neck is associated with high morbidity and mortality in both Western and Asian countries. Several risk factors for the development of oral cancer are very well established, including tobacco chewing, betel quid, smoking, alcohol drinking and human papilloma virus (HPV) infection. Apart from these risk factors, many genetic factors such as oncogenes, tumor suppressor genes and regulatory genes are identified to involve in oral carcinogenesis with these risk factors dependent and independent manner. Ras is one of the most frequently genetically deregulated oncogene in oral cancer. In this review, we analyze the past 22years of literature on genetic alterations such as mutations and amplifications of the isoforms of the ras oncogene in oral cancer. Further, we addressed the isoform-specific role of the ras in oral carcinogenesis. We also discussed how targeting the Akt and MEK, downstream effectors of the PI3K/Akt and MAPK pathways, respectively, would probably pave the possible molecular therapeutic target for the ras driven tumorigenesis in oral cancer. Analysis of these ras isoforms may critically enlighten specific role of a particular ras isoform in oral carcinogenesis, enhance prognosis and pave the way for isoform-specific molecular targeted therapy in OSCC. Copyright © 2011 Elsevier Ltd. All rights reserved.

Occurrence and preliminarily environmental risk assessment of selected pharmaceuticals in the urban rivers, China

NASA Astrophysics Data System (ADS)

Zhou, Haidong; Ying, Tianqi; Wang, Xuelian; Liu, Jianbo

2016-10-01

Twelve selected pharmaceuticals including antibiotics, analgesics, antiepileptics and lipid regulators were analysed and detected in water samples collected from 18 sampling sections along the three main urban rivers in Yangpu District of Shanghai, China during four sampling campaigns. Besides, algal growth inhibition test was conducted to preliminarily assess the eco-toxicology induced by the target pharmaceuticals in the rivers. Mean levels for most of target compounds were generally below 100 ng/L at sampling sections, with the exception of caffeine and paracetamol presenting considerably high concentration. The detected pharmaceuticals in the urban rivers ranged from

Targeting cancer stem-like cells in glioblastoma and colorectal cancer through metabolic pathways.

PubMed

Kahlert, U D; Mooney, S M; Natsumeda, M; Steiger, H-J; Maciaczyk, J

2017-01-01

Cancer stem-like cells (CSCs) are thought to be the main cause of tumor occurrence, progression and therapeutic resistance. Strong research efforts in the last decade have led to the development of several tailored approaches to target CSCs with some very promising clinical trials underway; however, until now no anti-CSC therapy has been approved for clinical use. Given the recent improvement in our understanding of how onco-proteins can manipulate cellular metabolic networks to promote tumorigenesis, cancer metabolism research may well lead to innovative strategies to identify novel regulators and downstream mediators of CSC maintenance. Interfering with distinct stages of CSC-associated metabolics may elucidate novel, more efficient strategies to target this highly malignant cell population. Here recent discoveries regarding the metabolic properties attributed to CSCs in glioblastoma (GBM) and malignant colorectal cancer (CRC) were summarized. The association between stem cell markers, the response to hypoxia and other environmental stresses including therapeutic insults as well as developmentally conserved signaling pathways with alterations in cellular bioenergetic networks were also discussed. The recent developments in metabolic imaging to identify CSCs were also summarized. This summary should comprehensively update basic and clinical scientists on the metabolic traits of CSCs in GBM and malignant CRC. © 2016 UICC.

Using The Corngrass1 Gene To Enhance The Biofuel Properties Of Crop Plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hake, Sarah; Chuck, George

2015-10-29

The development of novel plant germplasm is vital to addressing our increasing bioenergy demands. The major hurdle to digesting plant biomass is the complex structure of the cell walls, the substrate of fermentation. Plant cell walls are inaccessible matrices of macromolecules that are polymerized with lignin, making fermentation difficult. Overcoming this hurdle is a major goal toward developing usable bioenergy crop plants. Our project seeks to enhance the biofuel properties of perennial grass species using the Corngrass1 (Cg1) gene and its targets. Dominant maize Cg1 mutants produce increased biomass by continuously initiating extra axillary meristems and leaves. We cloned Cg1more » and showed that its phenotype is caused by over expression of a unique miR156 microRNA gene that negatively regulates SPL transcription factors. We transferred the Cg1 phenotype to other plants by expressing the gene behind constitutive promoters in four different species, including the monocots, Brachypodium and switchgrass, and dicots, Arabidopsis and poplar. All transformants displayed a similar range of phenotypes, including increased biomass from extended leaf production, and increased vegetative branching. Field grown switchgrass transformants showed that overall lignin content was reduced, the ratio of glucans to xylans was increased, and surprisingly, that starch levels were greatly increased. The goals of this project are to control the tissue and temporal expression of Cg1 by using different promoters to drive its expression, elucidate the function of the SPL targets of Cg1 by generating gain and loss of function alleles, and isolate downstream targets of select SPL genes using deep sequencing and chromatin immunoprecipitation. We believe it is possible to control biomass accumulation, cell wall properties, and sugar levels through manipulation of either the Cg1 gene and/or its SPL targets.« less

Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma.

PubMed

Venkatesh, Humsa S; Tam, Lydia T; Woo, Pamelyn J; Lennon, James; Nagaraja, Surya; Gillespie, Shawn M; Ni, Jing; Duveau, Damien Y; Morris, Patrick J; Zhao, Jean J; Thomas, Craig J; Monje, Michelle

2017-09-28

High-grade gliomas (HGG) are a devastating group of cancers, and represent the leading cause of brain tumour-related death in both children and adults. Therapies aimed at mechanisms intrinsic to glioma cells have translated to only limited success; effective therapeutic strategies will need also to target elements of the tumour microenvironment that promote glioma progression. Neuronal activity promotes the growth of a range of molecularly and clinically distinct HGG types, including adult and paediatric glioblastoma (GBM), anaplastic oligodendroglioma, and diffuse intrinsic pontine glioma (DIPG). An important mechanism that mediates this neural regulation of brain cancer is activity-dependent cleavage and secretion of the synaptic adhesion molecule neuroligin-3 (NLGN3), which promotes glioma proliferation through the PI3K-mTOR pathway. However, the necessity of NLGN3 for glioma growth, the proteolytic mechanism of NLGN3 secretion, and the further molecular consequences of NLGN3 secretion in glioma cells remain unknown. Here we show that HGG growth depends on microenvironmental NLGN3, identify signalling cascades downstream of NLGN3 binding in glioma, and determine a therapeutically targetable mechanism of secretion. Patient-derived orthotopic xenografts of paediatric GBM, DIPG and adult GBM fail to grow in Nlgn3 knockout mice. NLGN3 stimulates several oncogenic pathways, such as early focal adhesion kinase activation upstream of PI3K-mTOR, and induces transcriptional changes that include upregulation of several synapse-related genes in glioma cells. NLGN3 is cleaved from both neurons and oligodendrocyte precursor cells via the ADAM10 sheddase. ADAM10 inhibitors prevent the release of NLGN3 into the tumour microenvironment and robustly block HGG xenograft growth. This work defines a promising strategy for targeting NLGN3 secretion, which could prove transformative for HGG therapy.

Push-Pull and Feedback Mechanisms Can Align Signaling System Outputs with Inputs.

PubMed

Andrews, Steven S; Peria, William J; Yu, Richard C; Colman-Lerner, Alejandro; Brent, Roger

2016-11-23

Many cell signaling systems, including the yeast pheromone response system, exhibit "dose-response alignment" (DoRA), in which output of one or more downstream steps closely matches the fraction of occupied receptors. DoRA can improve the fidelity of transmitted dose information. Here, we searched systematically for biochemical network topologies that produced DoRA. Most networks, including many containing feedback and feedforward loops, could not produce DoRA. However, networks including "push-pull" mechanisms, in which the active form of a signaling species stimulates downstream activity and the nominally inactive form reduces downstream activity, enabled perfect DoRA. Networks containing feedbacks enabled DoRA, but only if they also compared feedback to input and adjusted output to match. Our results establish push-pull as a non-feedback mechanism to align output with variable input and maximize information transfer in signaling systems. They also suggest genetic approaches to determine whether particular signaling systems use feedback or push-pull control. Copyright © 2016 Elsevier Inc. All rights reserved.

Analysis of dissolved gas and fluid chemistry in mountainous region of Goaping river watershed in southern Taiwan

NASA Astrophysics Data System (ADS)

Tang, Kai-Wen; Chen, Cheng-Hong; Liu, Tsung-Kwei

2016-04-01

Annual rainfall in Taiwan is up to 2500 mm, about 2.5 times the average value of the world. However due to high topographic relief of the Central Mountain Range in Taiwan, groundwater storage is critical for water supply. Mountain region of the Goaping river watershed in southern Taiwan is one of the potential areas to develop groundwater recharge model. Therefore the target of this study is to understand sources of groundwater and surface water using dissolved gas and fluid chemistry. Four groundwater and 6 surface water samples were collected from watershed, 5 groundwater and 13 surface water samples were collected from downstream. All samples were analyzed for stable isotopes (hydrogen and oxygen), dissolved gases (including nitrogen, oxygen, argon, methane and carbon dioxide), noble gases (helium and radon) and major ions. Hydrogen and oxygen isotopic ratios of surface water and groundwater samples aligned along meteoric water line. For surface water, dissolved gases are abundant in N2 (>80%) and O2 (>10%); helium isotopic ratio is approximately equal to 1 RA (RA is 3He/4He ratio of air); radon-222 concentration is below the detection limit (<200 Bq/m3); and concentrations of major anions and cations are low (Na+ <20 ppm, Ca2+ < 60 ppm, Cl- <2 ppm). All these features indicate that surface waters are predominately recharged by precipitation. For groundwater, helium isotopic ratios (0.9˜0.23 RA) are lower and radon-222 concentrations (300˜6000 Bq/m3) are much higher than the surface water. Some samples have high amounts of dissolved gases, such as CH4 (>20%) or CO2 (>10%), most likely contributed by biogenic or geogenic sources. On the other hand, few samples that have temperature 5° higher than the average of other samples, show significantly high Na+ (>1000 ppm), Ca2+ (>150 ppm) and Cl- (>80 ppm) concentrations. An interaction between such groundwater and local hot springs is inferred. Watershed and downstream samples differ in dissolved gas species and fluid chemistry for groundwater and surface water. The higher hydrogen and oxygen isotopic ratios for surface water from downstream are most probably caused by evaporation. Low radon-222 concentrations of some groundwater from downstream may represent sources from different aquifers. Therefore, we conclude that surface water from downstream are recharged directly from its watershed, but groundwater are influenced by the local geological environment. Keywords: groundwater, dissolved gas, noble gas, radon in water, 3He/4He

Null EPAC Mutants Reveal a Sequential Order of Versatile cAMP Effects during "Drosophila" Aversive Odor Learning

ERIC Educational Resources Information Center

Richlitzki, Antje; Latour, Philipp; Schwärzel, Martin

2017-01-01

Here, we define a role of the cAMP intermediate EPAC in "Drosophila" aversive odor learning by means of null epac mutants. Complementation analysis revealed that EPAC acts downstream from the "rutabaga" adenylyl cyclase and in parallel to protein kinase A. By means of targeted knockdown and genetic rescue we identified mushroom…

A Novel Differentiation Therapy Approach to Reduce the Metastatic Potential of Basal, Highly Metastatic, Triple-Negative Breast Cancers

DTIC Science & Technology

2011-05-01

task 1 b) GATA3 was shown to directly modulate expression of genes regulating the cell cycle (Pei et al., 2009; Molenaar et al., 2010) and GATA3...downstream target of GATA3 and restrains mammary luminal progenitor cell proliferation and tumorigenesis. Cancer Ce/l15:389-401. Molenaar JJ, Ebus

Signaling Pathways in Pathogenesis of Diamond Blackfan Anemia

DTIC Science & Technology

2015-12-01

abnormalities, and predisposition to cancer. The current treatment of steroids and chronic transfusions leads to significant morbidity. Approximately 25...more rigorously test this hypothesis and identify new downstream targets and microRNAs, we propose three specific aims. In Aim 1, we will...aplasia, congenital abnormalities, and predisposition to cancer. The current treatment of steroids and chronic transfusions leads to significant

40 CFR 92.114 - Exhaust gas and particulate sampling and analytical system.

Code of Federal Regulations, 2011 CFR

2011-07-01

... downstream of the analyzer. The gauge tap must be within 2 inches of the analyzer exit port. Gauge... must be used. The gauge tap must be within 2 inches of the analyzer entrance port. (vi) Calibration or.... Equivalent loadings (0.5 mg/1075 mm2 stain area) shall be used as target loadings when other filter sizes are...

40 CFR 92.114 - Exhaust gas and particulate sampling and analytical system.

Code of Federal Regulations, 2010 CFR

2010-07-01

... downstream of the analyzer. The gauge tap must be within 2 inches of the analyzer exit port. Gauge... must be used. The gauge tap must be within 2 inches of the analyzer entrance port. (vi) Calibration or.... Equivalent loadings (0.5 mg/1075 mm2 stain area) shall be used as target loadings when other filter sizes are...

Epidermal growth factor receptor and integrins control force-dependent vinculin recruitment to E-cadherin junctions.

PubMed

Sehgal, Poonam; Kong, Xinyu; Wu, Jun; Sunyer, Raimon; Trepat, Xavier; Leckband, Deborah

2018-03-20

This study reports novel findings that link E-cadherin (also known as CDH1)-mediated force-transduction signaling to vinculin targeting to intercellular junctions via epidermal growth factor receptor (EGFR) and integrins. These results build on previous findings that demonstrated that mechanically perturbed E-cadherin receptors activate phosphoinositide 3-kinase and downstream integrins in an EGFR-dependent manner. Results of this study show that this EGFR-mediated kinase cascade controls the force-dependent recruitment of vinculin to stressed E-cadherin complexes - a key early signature of cadherin-based mechanotransduction. Vinculin targeting requires its phosphorylation at tyrosine 822 by Abl family kinases (hereafter Abl), but the origin of force-dependent Abl activation had not been identified. We now present evidence that integrin activation, which is downstream of EGFR signaling, controls Abl activation, thus linking E-cadherin to Abl through a mechanosensitive signaling network. These findings place EGFR and integrins at the center of a positive-feedback loop, through which force-activated E-cadherin signals regulate vinculin recruitment to cadherin complexes in response to increased intercellular tension.This article has an associated First Person interview with the first author of the paper. © 2018. Published by The Company of Biologists Ltd.

6-Gingerol Suppresses Adipocyte-Derived Mediators of Inflammation In Vitro and in High-Fat Diet-Induced Obese Zebra Fish.

PubMed

Choi, Jia; Kim, Kui-Jin; Kim, Byung-Hak; Koh, Eun-Jeong; Seo, Min-Jung; Lee, Boo-Yong

2017-02-01

The present study was performed to investigate the molecular mechanism of 6-gingerol on adipocyte-mediated systemic inflammation in vitro and in high-fat diet-induced obese zebra fish. 6-Gingerol decreased adipogenesis due to the suppression of adipocyte differentiation markers, including peroxisome proliferator-activated receptor gamma, CCAATT enhancer binding protein α , and adipocyte protein 2, and triglyceride synthesis enzymes, including sterol regulatory element-binding protein-1, fatty acid synthase, lysophosphatidic acid acyltransferase, and acyl-coA : diacylglycerol acyltransferase 1, in 3T3-L1. A coculture insert system using 3T3-L1 with RAW 264.7 (coculture insert system using fully differentiated 3T3-L1 cells with RAW 264.7 macrophages) revealed that 6-gingerol increased anti-inflammatory cytokine interleukin-10. The expression of TNF α , monocyte chemotactic protein-1, interleukin-1 β , and interleukin-6 were decreased in the coculture insert system using fully differentiated 3T3-L1 cells with RAW 264.7 macrophages treated with 6-gingerol. Moreover, the coculture insert system using fully differentiated 3T3-L1 cells with RAW 264.7 macrophages treated with 6-gingerol inhibited the protein expression of TNF α and monocyte chemotactic protein-1 in RAW 264.7. 6-Gingerol decreased c-JUN N-terminal kinase and I kappa B kinase beta and its downstream target AP-1 expression in the coculture insert system using fully differentiated 3T3-L1 cells with RAW 264.7 macrophages. Furthermore, 6-gingerol decreased the expression of inducible nitric oxide synthase stimulated by the coculture insert system using fully differentiated 3T3-L1 cells with RAW 264.7 macrophages in RAW 264.7 and attenuated nitric oxide production in diet-induced obese zebra fish. Our results suggest that 6-gingerol suppresses inflammation through the regulation of the c-JUN N-terminal kinase-I kappa B kinase beta and its downstream targets. Georg Thieme Verlag KG Stuttgart · New York.

Downscaling catchment scale flood risk to contributing sub-catchments to determine the optimum location for flood management.

NASA Astrophysics Data System (ADS)

Pattison, Ian; Lane, Stuart; Hardy, Richard; Reaney, Sim

2010-05-01

The recent increase in flood frequency and magnitude has been hypothesised to have been caused by either climate change or land management. Field scale studies have found that changing land management practices does affect local runoff and streamflow, but upscaling these effects to the catchment scale continues to be problematic, both conceptually and more importantly methodologically. The impact on downstream flood risk is highly dependent upon where the changes are in the catchment, indicating that some areas of the catchment are more important in determining downstream flood risk than others. This is a major flaw in the traditional approach to studying the effect of land use on downstream flood risk: catchment scale hydrological models, which treat every cell in the model equally. We are proposing an alternative ideological approach for doing flood management research, which is underpinned by downscaling the downstream effect (problem i.e. flooding) to the upstream causes (contributing sub-catchments). It is hoped that this approach could have several benefits over the traditional upscaling approach. Firstly, it provides an efficient method to prioritise areas for land use management changes to be implemented to reduce downstream flood risk. Secondly, targets for sub-catchment hydrograph change can be determined which will deliver the required downstream effect. Thirdly, it may be possible to detect the effect of land use changes in upstream areas on downstream flood risk, by weighting the areas of most importance in hydrological models. Two methods for doing this downscaling are proposed; 1) data-based statistical analysis; and 2) hydraulic modelling-based downscaling. These will be outlined using the case study of the River Eden, Cumbria, NW England. The data-based methodology uses the timing and magnitude of floods for each sub-catchment. Principal components analysis (PCA) is used to simplify sub-catchment interactions and optimising stepwise regression is used to predict downstream flood magnitude from the significant principal components. Two particular sub-catchments, the Eamont and the Upper Eden were highlighted as explaining the highest proportion of downstream flood risk, with 21.0% and 19.6% respectively. This approach uses the concept of data mining, whereby commonly available discharge data is used in an innovative way to learn about catchment behaviour. An alternative downscaling approach is hydraulic modelling whereby the input hydrographs from each tributary are changed in turn, both in terms of the magnitudes and the timing of the flows. This basic scenario testing approach can be used to assess the sensitivity of downstream flood risk to upstream contributing tributaries. This approach also highlighted the Upper Eden and Eamont as the most sensitive sub-catchments. A 25% reduction in the flows from these sub-catchments resulted in a 33.1cm and 21.9cm stage reduction downstream respectively, while an 8 hour delay of the peak flow caused a 32.3cm and 27.4cm decrease in downstream stage respectively. This alternative flood management approach is not a replacement to traditional hydrological modelling (upscaling), but a pre-step which allows for more focussed and informed investigation of land management scenarios, in the area where they are most likely to have beneficial impacts on downstream flooding.

A Functional Assay for GPR55: Envision Protocol.

PubMed

Anavi-Goffer, Sharon; Ross, Ruth A

2016-01-01

AlphaScreen(®) SureFire(®) assay is a novel technology that combines luminescent oxygen channeling technology, nano-beads, and monocloncal antibodies to detect the level of a selected protein in a volume lower than 5 μl. This method is more sensitive compared with the traditional enzyme-linked immunosorbent assays (ELISA), and can detect an increasing number of new targets. Here, we described a method for AlphaScreen(®) SureFire(®) assay that targets ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys activation of GPR55 by L-α-lysophosphatidylinositol (LPI) and certain cannabinoids.

Rolling Band Artifact Flagging in the Kepler Data Pipeline

NASA Astrophysics Data System (ADS)

Clarke, Bruce; Kolodziejczak, Jeffery J; Caldwell, Douglas A.

2014-06-01

Instrument-induced artifacts in the raw Kepler pixel data include time-varying crosstalk from the fine guidance sensor (FGS) clock signals, manifestations of drifting moiré pattern as locally correlated nonstationary noise and rolling bands in the images. These systematics find their way into the calibrated pixel time series and ultimately into the target flux time series. The Kepler pipeline module Dynablack models the FGS crosstalk artifacts using a combination of raw science pixel data, full frame images, reverse-clocked pixel data and ancillary temperature data. The calibration module (CAL) uses the fitted Dynablack models to remove FGS crosstalk artifacts in the calibrated pixels by adjusting the black level correction per cadence. Dynablack also detects and flags spatial regions and time intervals of strong time-varying black-level. These rolling band artifact (RBA) flags are produced on a per row per cadence basis by searching for transit signatures in the Dynablack fit residuals. The Photometric Analysis module (PA) generates per target per cadence data quality flags based on the Dynablack RBA flags. Proposed future work includes using the target data quality flags as a basis for de-weighting in the Presearch Data Conditioning (PDC), Transiting Planet Search (TPS) and Data Validation (DV) pipeline modules. We discuss the effectiveness of RBA flagging for downstream users and illustrate with some affected light curves. We also discuss the implementation of Dynablack in the Kepler data pipeline and present results regarding the improvement in calibrated pixels and the expected improvement in cotrending performance as a result of including FGS corrections in the calibration. Funding for the Kepler Mission has been provided by the NASA Science Mission Directorate.

Control of vascular smooth muscle function by Src-family kinases and reactive oxygen species in health and disease

PubMed Central

MacKay, Charles E; Knock, Greg A

2015-01-01

Abstract Reactive oxygen species (ROS) are now recognised as second messenger molecules that regulate cellular function by reversibly oxidising specific amino acid residues of key target proteins. Amongst these are the Src-family kinases (SrcFKs), a multi-functional group of non-receptor tyrosine kinases highly expressed in vascular smooth muscle (VSM). In this review we examine the evidence supporting a role for ROS-induced SrcFK activity in normal VSM contractile function and in vascular remodelling in cardiovascular disease. VSM contractile responses to G-protein-coupled receptor stimulation, as well as hypoxia in pulmonary artery, are shown to be dependent on both ROS and SrcFK activity. Specific phosphorylation targets are identified amongst those that alter intracellular Ca2+ concentration, including transient receptor potential channels, voltage-gated Ca2+ channels and various types of K+ channels, as well as amongst those that regulate actin cytoskeleton dynamics and myosin phosphatase activity, including focal adhesion kinase, protein tyrosine kinase-2, Janus kinase, other focal adhesion-associated proteins, and Rho guanine nucleotide exchange factors. We also examine a growing weight of evidence in favour of a key role for SrcFKs in multiple pro-proliferative and anti-apoptotic signalling pathways relating to oxidative stress and vascular remodelling, with a particular focus on pulmonary hypertension, including growth-factor receptor transactivation and downstream signalling, hypoxia-inducible factors, positive feedback between SrcFK and STAT3 signalling and positive feedback between SrcFK and NADPH oxidase dependent ROS production. We also discuss evidence for and against the potential therapeutic targeting of SrcFKs in the treatment of pulmonary hypertension. PMID:25384773

Dual sphingosine kinase inhibitor SKI-II enhances sensitivity to 5-fluorouracil in hepatocellular carcinoma cells via suppression of osteopontin and FAK/IGF-1R signalling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grbčić, Petra; Tomljanović, Ivana; Klobučar, Marko

Hepatocellular carcinoma (HCC) represents the third leading cause of cancer-related deaths globally. Although 5-Fluorouracil (5-FU) is used as the first choice treatment for advanced HCC, it exerts poor efficacy and is associated with acquired and intrinsic resistance. Sphingosine kinases (Sphk) 1 and 2 play tumour-promoting roles in different cancer types including HCC and thus represent promising pharmacological targets. In the present study, we have investigated for the first time the anticancer efficacy and underlying molecular mechanisms of combined administration of 5-FU and dual Sphk1/Sphk2 inhibitor SKI-II (4-[[4-(4-chlorophenyl)-1,3-thiazol-2-yl]amino]phenol) in HepG2 hepatocellular carcinoma cells. Here, we report that co-administration of 5-FU andmore » SKI-II at low sub-toxic concentrations of 20 μM and 5 μM, respectively, synergistically inhibit cell proliferation, markedly reduce cell migration and the clonogenic survival, and increase apoptosis induction in HepG2 cells. Additional Western blot analyses have shown that possible mechanisms underlying enhanced sensitivity to 5-FU induced by dual Sphk 1/2 inhibition could include abrogation of FAK-regulated IGF-1R activity and down-regulation of osteopontin expression culminating in the inhibition of NF-κB activity and its downstream signalling mediated by sirtuin 1 and p38 MAPK. Our results clearly show that pharmacological blockade of both Sphk isoforms represents a promising strategy to boost the anti-tumour efficacy of 5-FU and provide a rationale for further in vivo studies into the possible use of SKI-II inhibitor as an adjunct to 5-FU treatment in HCC. - Highlights: • Pharmacological inhibition of sphingosine kinase 1/2 augments cytotoxic effects of 5-FU in HepG2 cells. • Co-administration of 5-FU and SKI-II synergistically inhibits cell proliferation, reduces cell migration and clonogenic survival and increases apoptosis induction in HepG2 cells. • The mechanism underlying chemosensitization effects includes attenuation of FAK/IGF-1R and osteopontin signaling leading to the inhibition of NF-κB activity and its downstream targets SIRT1 and p38 MAPK.« less

The Double Flybys of the Cassini Mission: Navigation Challenges and Lessons Learned

NASA Technical Reports Server (NTRS)

Wagner, Sean; Buffington, Brent

2014-01-01

Since 2004, the Cassini spacecraft has flown by Titan and other Saturn moons numerous times, successfully accomplishing its 100th targeted encounter of Titan in March 2014. The navigation of Cassini is challenging, even more so with "double flybys," two encounters separated by at most a few days. Because of this tight spacing, there is not enough time for a maneuver in between. Additionally, maneuvers prior to a double flyby only target one of the two encounters. This paper discusses the challenges faced by the Cassini Navigation Team with each double flyby, as well as lessons learned during operational support of each dual encounter. The strengths and weaknesses of the targeting strategies considered for each double flyby are also detailed, by comparing downstream ?V costs and changes to the non-targeted flyby conditions.

Immunoglobulin class switch DNA recombination: induction, targeting and beyond

PubMed Central

Xu, Zhenming; Zan, Hong; Pone, Egest J.; Mai, Thach; Casali, Paolo

2012-01-01

Class switch DNA recombination (CSR) of the immunoglobulin heavy chain (IgH) locus is central to the maturation of the antibody response and critically requires the AID cytidine deaminase. CSR entails changes of the chromatin state and transcriptional activation of the IgH locus upstream and downstream switch (S) regions that are to undergo S-S DNA recombination, induction of AID, and targeting of CSR factors to S regions by 14-3-3 adaptors and as enabled by the transcription machinery and histone modifications. In this Review, we focus on recent advances in CSR induction and targeting. We also outline an integrated model of the assembly of macromolecular complexes that transduce critical epigenetic information to enzymatic effectors of the CSR machinery. PMID:22728528

Improved Algorithms for Blending Dam Releases to Meet Downstream Water-Temperature Targets in the CE-QUAL-W2 Water-Quality Model

NASA Astrophysics Data System (ADS)

Rounds, S. A.; Buccola, N. L.

2014-12-01

The two-dimensional (longitudinal, vertical) water-quality model CE-QUAL-W2, version 3.7, was enhanced with new features to help dam operators and managers efficiently explore and optimize potential solutions for temperature management downstream of thermally stratified reservoirs. Such temperature management often is accomplished by blending releases from multiple dam outlets that access water of different temperatures at different depths in the reservoir. The original blending algorithm in this version of the model was limited to mixing releases from two outlets at a time, and few constraints could be imposed. The new enhanced blending algorithm allows the user to (1) specify a time-series of target release temperatures, (2) designate from 2 to 10 floating or fixed-elevation outlets for blending, (3) impose maximum head constraints as well as minimum and maximum flow constraints for any blended outlet, and (4) set a priority designation for each outlet that allows the model to choose which outlets to use and how to balance releases among them. The modified model was tested against a previously calibrated model of Detroit Lake on the North Santiam River in northwestern Oregon, and the results compared well. The enhanced model code is being used to evaluate operational and structural scenarios at multiple dam/reservoir systems in the Willamette River basin in Oregon, where downstream temperature management for endangered fish is a high priority for resource managers and dam operators. These updates to the CE-QUAL-W2 blending algorithm allow scenarios involving complicated dam operations and/or hypothetical outlet structures to be evaluated more efficiently with the model, with decreased need for multiple/iterative model runs or preprocessing of model inputs to fully characterize the operational constraints.

IL-15 Activates the Jak3/STAT3 Signaling Pathway to Mediate Glucose Uptake in Skeletal Muscle Cells

PubMed Central

Krolopp, James E.; Thornton, Shantaé M.; Abbott, Marcia J.

2016-01-01

Myokines are specialized cytokines that are secreted from skeletal muscle (SKM) in response to metabolic stimuli, such as exercise. Interleukin-15 (IL-15) is a myokine with potential to reduce obesity and increase lean mass through induction of metabolic processes. It has been previously shown that IL-15 acts to increase glucose uptake in SKM cells. However, the downstream signals orchestrating the link between IL-15 signaling and glucose uptake have not been fully explored. Here we employed the mouse SKM C2C12 cell line to examine potential downstream targets of IL-15-induced alterations in glucose uptake. Following differentiation, C2C12 cells were treated overnight with 100 ng/ml of IL-15. Activation of factors associated with glucose metabolism (Akt and AMPK) and known downstream targets of IL-15 (Jak1, Jak3, STAT3, and STAT5) were assessed with IL-15 stimulation. IL-15 stimulated glucose uptake and GLUT4 translocation to the plasma membrane. IL-15 treatment had no effect on phospho-Akt, phospho-Akt substrates, phospho-AMPK, phospho-Jak1, or phospho-STAT5. However, with IL-15, phospho-Jak3 and phospho-STAT3 levels were increased along with increased interaction of Jak3 and STAT3. Additionally, IL-15 induced a translocation of phospho-STAT3 from the cytoplasm to the nucleus. We have evidence that a mediator of glucose uptake, HIF1α, expression was dependent on IL-15 induced STAT3 activation. Finally, upon inhibition of STAT3 the positive effects of IL-15 on glucose uptake and GLUT4 translocation were abolished. Taken together, we provide evidence for a novel signaling pathway for IL-15 acting through Jak3/STAT3 to regulate glucose metabolism. PMID:28066259

Differential receptor dependencies: expression and significance of muscarinic M1 receptors in the biology of prostate cancer.

PubMed

Mannan Baig, Abdul; Khan, Naveed A; Effendi, Vardah; Rana, Zohaib; Ahmad, H R; Abbas, Farhat

2017-01-01

Recent reports on acetylcholine muscarinic receptor subtype 3 (CHRM3) have shown its growth-promoting role in prostate cancer. Additional studies report the proliferative effect of the cholinergic agonist carbachol on prostate cancer by its agonistic action on CHRM3. This study shows that the type 1 acetylcholine muscarinic receptor (CHRM1) contributes toward the proliferation and growth of prostate cancer. We used growth and cytotoxic assays, the prostate cancer microarray database and CHRM downstream pathways' homology of CHRM subtypes to uncover multiple signals leading to the growth of prostate cancer. Growth assays showed that pilocarpine stimulates the proliferation of prostate cancer. Moreover, it shows that carbachol exerts an additional agonistic action on nicotinic cholinergic receptor of prostate cancer cells that can be blocked by tubocurarine. With the use of selective CHRM1 antagonists such as pirenzepine and dicyclomine, a considerable inhibition of proliferation of prostate cancer cell lines was observed in dose ranging from 15-60 µg/ml of dicyclomine. The microarray database of prostate cancer shows a dominant expression of CHRM1 in prostate cancer compared with other cholinergic subtypes. The bioinformatics of prostate cancer and CHRM pathways show that the downstream signalling include PIP3-AKT-CaM-mediated growth in LNCaP and PC3 cells. Our study suggests that antagonism of CHRM1 may be a potential therapeutic target against prostate cancer.

Recent Progress in Understanding Subtype Specific Regulation of NMDA Receptors by G Protein Coupled Receptors (GPCRs)

PubMed Central

Yang, Kai; Jackson, Michael F.; MacDonald, John F.

2014-01-01

G Protein Coupled Receptors (GPCRs) are the largest family of receptors whose ligands constitute nearly a third of prescription drugs in the market. They are widely involved in diverse physiological functions including learning and memory. NMDA receptors (NMDARs), which belong to the ionotropic glutamate receptor family, are likewise ubiquitously expressed in the central nervous system (CNS) and play a pivotal role in learning and memory. Despite its critical contribution to physiological and pathophysiological processes, few pharmacological interventions aimed directly at regulating NMDAR function have been developed to date. However, it is well established that NMDAR function is precisely regulated by cellular signalling cascades recruited downstream of G protein coupled receptor (GPCR) stimulation. Accordingly, the downstream regulation of NMDARs likely represents an important determinant of outcome following treatment with neuropsychiatric agents that target selected GPCRs. Importantly, the functional consequence of such regulation on NMDAR function varies, based not only on the identity of the GPCR, but also on the cell type in which relevant receptors are expressed. Indeed, the mechanisms responsible for regulating NMDARs by GPCRs involve numerous intracellular signalling molecules and regulatory proteins that vary from one cell type to another. In the present article, we highlight recent findings from studies that have uncovered novel mechanisms by which selected GPCRs regulate NMDAR function and consequently NMDAR-dependent plasticity. PMID:24562329

Hydrogen sulfide as an oxygen sensor.

PubMed

Olson, Kenneth R

2015-02-10

Although oxygen (O2)-sensing cells and tissues have been known for decades, the identity of the O2-sensing mechanism has remained elusive. Evidence is accumulating that O2-dependent metabolism of hydrogen sulfide (H2S) is this enigmatic O2 sensor. The elucidation of biochemical pathways involved in H2S synthesis and metabolism have shown that reciprocal H2S/O2 interactions have been inexorably linked throughout eukaryotic evolution; there are multiple foci by which O2 controls H2S inactivation, and the effects of H2S on downstream signaling events are consistent with those activated by hypoxia. H2S-mediated O2 sensing has been demonstrated in a variety of O2-sensing tissues in vertebrate cardiovascular and respiratory systems, including smooth muscle in systemic and respiratory blood vessels and airways, carotid body, adrenal medulla, and other peripheral as well as central chemoreceptors. Information is now needed on the intracellular location and stoichometry of these signaling processes and how and which downstream effectors are activated by H2S and its metabolites. Development of specific inhibitors of H2S metabolism and effector activation as well as cellular organelle-targeted compounds that release H2S in a time- or environmentally controlled way will not only enhance our understanding of this signaling process but also provide direction for future therapeutic applications.

Global Phosphoproteomic Analysis of Insulin/Akt/mTORC1/S6K Signaling in Rat Hepatocytes.

PubMed

Zhang, Yuanyuan; Zhang, Yajie; Yu, Yonghao

2017-08-04

Insulin resistance is a hallmark of type 2 diabetes. Although multiple genetic and physiological factors interact to cause insulin resistance, deregulated signaling by phosphorylation is a common underlying mechanism. In particular, the specific phosphorylation-dependent regulatory mechanisms and signaling outputs of insulin are poorly understood in hepatocytes, which represents one of the most important insulin-responsive cell types. Using primary rat hepatocytes as a model system, we performed reductive dimethylation (ReDi)-based quantitative mass spectrometric analysis and characterized the phosphoproteome that is regulated by insulin as well as its key downstream kinases including Akt, mTORC1, and S6K. We identified a total of 12 294 unique, confidently localized phosphorylation sites and 3805 phosphorylated proteins in this single cell type. Detailed bioinformatic analysis on each individual data set identified both known and previously unrecognized targets of this key insulin downstream effector pathway. Furthermore, integrated analysis of the hepatic Akt/mTORC1/S6K signaling axis allowed the delineation of the substrate specificity of several close-related kinases within the insulin signaling pathway. We expect that the data sets will serve as an invaluable resource, providing the foundation for future hypothesis-driven research that helps delineate the molecular mechanisms that underlie the pathogenesis of type 2 diabetes and related metabolic syndrome.

Radiant zone heated particulate filter

DOEpatents

Gonze, Eugene V [Pinckney, MI

2011-12-27

A system includes a particulate matter (PM) filter including an upstream end for receiving exhaust gas and a downstream end. A radiant zoned heater includes N zones, where N is an integer greater than one, wherein each of the N zones includes M sub-zones, where M is an integer greater than or equal to one. A control module selectively activates at least a selected one of the N zones to initiate regeneration in downstream portions of the PM filter from the one of the N zones, restricts exhaust gas flow in a portion of the PM filter that corresponds to the selected one of the N zones, and deactivates non-selected ones of the N zones.

An integrative approach unveils FOSL1 as an oncogene vulnerability in KRAS-driven lung and pancreatic cancer.

PubMed

Vallejo, Adrian; Perurena, Naiara; Guruceaga, Elisabet; Mazur, Pawel K; Martinez-Canarias, Susana; Zandueta, Carolina; Valencia, Karmele; Arricibita, Andrea; Gwinn, Dana; Sayles, Leanne C; Chuang, Chen-Hua; Guembe, Laura; Bailey, Peter; Chang, David K; Biankin, Andrew; Ponz-Sarvise, Mariano; Andersen, Jesper B; Khatri, Purvesh; Bozec, Aline; Sweet-Cordero, E Alejandro; Sage, Julien; Lecanda, Fernando; Vicent, Silve

2017-02-21

KRAS mutated tumours represent a large fraction of human cancers, but the vast majority remains refractory to current clinical therapies. Thus, a deeper understanding of the molecular mechanisms triggered by KRAS oncogene may yield alternative therapeutic strategies. Here we report the identification of a common transcriptional signature across mutant KRAS cancers of distinct tissue origin that includes the transcription factor FOSL1. High FOSL1 expression identifies mutant KRAS lung and pancreatic cancer patients with the worst survival outcome. Furthermore, FOSL1 genetic inhibition is detrimental to both KRAS-driven tumour types. Mechanistically, FOSL1 links the KRAS oncogene to components of the mitotic machinery, a pathway previously postulated to function orthogonally to oncogenic KRAS. FOSL1 targets include AURKA, whose inhibition impairs viability of mutant KRAS cells. Lastly, combination of AURKA and MEK inhibitors induces a deleterious effect on mutant KRAS cells. Our findings unveil KRAS downstream effectors that provide opportunities to treat KRAS-driven cancers.

Broad Detection of Alterations Predicted to Confer Lack of Benefit From EGFR Antibodies or Sensitivity to Targeted Therapy in Advanced Colorectal Cancer.

PubMed

Rankin, Andrew; Klempner, Samuel J; Erlich, Rachel; Sun, James X; Grothey, Axel; Fakih, Marwan; George, Thomas J; Lee, Jeeyun; Ross, Jeffrey S; Stephens, Philip J; Miller, Vincent A; Ali, Siraj M; Schrock, Alexa B

2016-09-28

A KRAS mutation represented the first genomic biomarker to predict lack of benefit from anti-epidermal growth factor receptor (EGFR) antibody therapy in advanced colorectal cancer (CRC). Expanded RAS testing has further refined the treatment approach, but understanding of genomic alterations underlying primary and acquired resistance is limited and further study is needed. We prospectively analyzed 4,422 clinical samples from patients with advanced CRC, using hybrid-capture based comprehensive genomic profiling (CGP) at the request of the individual treating physicians. Comparison with prior molecular testing results, when available, was performed to assess concordance. We identified a RAS/RAF pathway mutation or amplification in 62% of cases, including samples harboring KRAS mutations outside of the codon 12/13 hotspot region in 6.4% of cases. Among cases with KRAS non-codon 12/13 alterations for which prior test results were available, 79 of 90 (88%) were not identified by focused testing. Of 1,644 RAS/RAF wild-type cases analyzed by CGP, 31% harbored a genomic alteration (GA) associated with resistance to anti-EGFR therapy in advanced CRC including mutations in PIK3CA, PTEN, EGFR, and ERBB2. We also identified other targetable GA, including novel kinase fusions, receptor tyrosine kinase amplification, activating point mutations, as well as microsatellite instability. Extended genomic profiling reliably detects alterations associated with lack of benefit to anti-EGFR therapy in advanced CRC, while simultaneously identifying alterations potentially important in guiding treatment. The use of CGP during the course of clinical care allows for the refined selection of appropriate targeted therapies and clinical trials, increasing the chance of clinical benefit and avoiding therapeutic futility. Comprehensive genomic profiling (CGP) detects diverse genomic alterations associated with lack of benefit to anti-epidermal growth factor receptor therapy in advanced colorectal cancer (CRC), as well as targetable alterations in many other genes. This includes detection of a broad spectrum of activating KRAS alterations frequently missed by focused molecular hotspot testing, as well as other RAS/RAF pathway alterations, mutations shown to disrupt antibody binding, RTK activating point mutations, amplifications, and rearrangements, and activating alterations in downstream effectors including PI3K and MEK1. The use of CGP in clinical practice is critical to guide appropriate selection of targeted therapies for patients with advanced CRC. ©AlphaMed Press.

Power Plant Bromide Discharges and Downstream Drinking Water Systems in Pennsylvania.

PubMed

Good, Kelly D; VanBriesen, Jeanne M

2017-10-17

Coal-fired power plants equipped with wet flue gas desulfurization (FGD) systems have been implicated in increasing bromide levels and subsequent increases in disinfection byproducts at downstream drinking water plants. Bromide was not included as a regulated constituent in the recent steam electric effluent limitations guidelines and standards (ELGs) since the U.S. EPA analysis suggested few drinking water facilities would be affected by bromide discharges from power plants. The present analysis uses a watershed approach to identify Pennsylvania drinking water intakes downstream of wet FGD discharges and to assess the potential for bromide discharge effects. Twenty-two (22) public drinking water systems serving 2.5 million people were identified as being downstream of at least one wet FGD discharge. During mean August conditions (generally low-flow, minimal dilution) in receiving rivers, the median predicted bromide concentrations contributed by wet FGD at Pennsylvania intake locations ranged from 5.2 to 62 μg/L for the Base scenario (including only natural bromide in coal) and from 16 to 190 μg/L for the Bromide Addition scenario (natural plus added bromide for mercury control); ranges depend on bromide loads and receiving stream dilution capacity.

Flow conditioner for fuel injector for combustor and method for low-NO.sub.x combustor

DOEpatents

Dutta, Partha; Smith, Kenneth O.; Ritz, Frank J.

2013-09-10

An injector for a gas turbine combustor including a catalyst coated surface forming a passage for feed gas flow and a channel for oxidant gas flow establishing an axial gas flow through a flow conditioner disposed at least partially within an inner wall of the injector. The flow conditioner includes a length with an interior passage opening into upstream and downstream ends for passage of the axial gas flow. An interior diameter of the interior passage smoothly reduces and then increases from upstream to downstream ends.

High-throughput micro-scale cultivations and chromatography modeling: Powerful tools for integrated process development.

PubMed

Baumann, Pascal; Hahn, Tobias; Hubbuch, Jürgen

2015-10-01

Upstream processes are rather complex to design and the productivity of cells under suitable cultivation conditions is hard to predict. The method of choice for examining the design space is to execute high-throughput cultivation screenings in micro-scale format. Various predictive in silico models have been developed for many downstream processes, leading to a reduction of time and material costs. This paper presents a combined optimization approach based on high-throughput micro-scale cultivation experiments and chromatography modeling. The overall optimized system must not necessarily be the one with highest product titers, but the one resulting in an overall superior process performance in up- and downstream. The methodology is presented in a case study for the Cherry-tagged enzyme Glutathione-S-Transferase from Escherichia coli SE1. The Cherry-Tag™ (Delphi Genetics, Belgium) which can be fused to any target protein allows for direct product analytics by simple VIS absorption measurements. High-throughput cultivations were carried out in a 48-well format in a BioLector micro-scale cultivation system (m2p-Labs, Germany). The downstream process optimization for a set of randomly picked upstream conditions producing high yields was performed in silico using a chromatography modeling software developed in-house (ChromX). The suggested in silico-optimized operational modes for product capturing were validated subsequently. The overall best system was chosen based on a combination of excellent up- and downstream performance. © 2015 Wiley Periodicals, Inc.

The Akt signaling pathway

PubMed Central

Madhunapantula, SubbaRao V; Mosca, Paul J

2011-01-01

Studies using cultured melanoma cells and patient tumor biopsies have demonstrated deregulated PI3 kinase-Akt3 pathway activity in ∼70% of melanomas. Furthermore, targeting Akt3 and downstream PRAS40 has been shown to inhibit melanoma tumor development in mice. Although these preclinical studies and several other reports using small interfering RNAs and pharmacological agents targeting key members of this pathway have been shown to retard melanoma development, analysis of early Phase I and Phase II clinical trials using pharmacological agents to target this pathway demonstrate the need for (1) selection of patients whose tumors have PI3 kinase-Akt pathway deregulation, (2) further optimization of therapeutic agents for increased potency and reduced toxicity, (3) the identification of additional targets in the same pathway or in other signaling cascades that synergistically inhibit the growth and progression of melanoma, and (4) better methods for targeted delivery of pharmaceutical agents inhibiting this pathway. In this review we discuss key potential targets in PI3K-Akt3 signaling, the status of pharmacological agents targeting these proteins, drugs under clinical development, and strategies to improve the efficacy of therapeutic agents targeting this pathway. PMID:22157148

Cooling system having dual suction port compressor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Guolian

2017-08-29

A cooling system for appliances, air conditioners, and other spaces includes a compressor, and a condenser that receives refrigerant from the compressor. The system also includes an evaporator that receives refrigerant from the condenser. Refrigerant received from the condenser flows through an upstream portion of the evaporator. A first portion of the refrigerant flows to the compressor without passing through a downstream portion of the evaporator, and a second portion of the refrigerant from the upstream portion of the condenser flows through the downstream portion of the evaporator after passing through the upstream portion of the evaporator. The second portionmore » of the refrigerant flows to the compressor after passing through the downstream portion of the evaporator. The refrigeration system may be configured to cool an appliance such as a refrigerator and/or freezer, or it may be utilized in air conditioners for buildings, motor vehicles, or other such spaces.« less

Turbofan gas turbine engine with variable fan outlet guide vanes

NASA Technical Reports Server (NTRS)

Wood, Peter John (Inventor); LaChapelle, Donald George (Inventor); Grant, Carl (Inventor); Zenon, Ruby Lasandra (Inventor); Mielke, Mark Joseph (Inventor)

2010-01-01

A turbofan gas turbine engine includes a forward fan section with a row of fan rotor blades, a core engine, and a fan bypass duct downstream of the forward fan section and radially outwardly of the core engine. The forward fan section has only a single stage of variable fan guide vanes which are variable fan outlet guide vanes downstream of the forward fan rotor blades. An exemplary embodiment of the engine includes an afterburner downstream of the fan bypass duct between the core engine and an exhaust nozzle. The variable fan outlet guide vanes are operable to pivot from a nominal OGV position at take-off to an open OGV position at a high flight Mach Number which may be in a range of between about 2.5-4+. Struts extend radially across a radially inwardly curved portion of a flowpath of the engine between the forward fan section and the core engine.

Refrigeration system having dual suction port compressor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Guolian

A cooling system for appliances, air conditioners, and other spaces includes a compressor, and a condenser that receives refrigerant from the compressor. The system also includes an evaporator that receives refrigerant from the condenser. Refrigerant received from the condenser flows through an upstream portion of the evaporator. A first portion of the refrigerant flows to the compressor without passing through a downstream portion of the evaporator, and a second portion of the refrigerant from the upstream portion of the condenser flows through the downstream portion of the evaporator after passing through the upstream portion of the evaporator. The second portionmore » of the refrigerant flows to the compressor after passing through the downstream portion of the evaporator. The refrigeration system may be configured to cool an appliance such as a refrigerator and/or freezer, or it may be utilized in air conditioners for buildings, motor vehicles, or other such spaces.« less

Absorber for terahertz radiation management

DOEpatents

Biallas, George Herman; Apeldoorn, Cornelis; Williams, Gwyn P.; Benson, Stephen V.; Shinn, Michelle D.; Heckman, John D.

2015-12-08

A method and apparatus for minimizing the degradation of power in a free electron laser (FEL) generating terahertz (THz) radiation. The method includes inserting an absorber ring in the FEL beam path for absorbing any irregular THz radiation and thus minimizes the degradation of downstream optics and the resulting degradation of the FEL output power. The absorber ring includes an upstream side, a downstream side, and a plurality of wedges spaced radially around the absorber ring. The wedges form a scallop-like feature on the innermost edges of the absorber ring that acts as an apodizer, stopping diffractive focusing of the THz radiation that is not intercepted by the absorber. Spacing between the scallop-like features and the shape of the features approximates the Bartlett apodization function. The absorber ring provides a smooth intensity distribution, rather than one that is peaked on-center, thereby eliminating minor distortion downstream of the absorber.

Continuous Manufacturing of Recombinant Therapeutic Proteins: Upstream and Downstream Technologies.

PubMed

Patil, Rohan; Walther, Jason

2017-03-07

Continuous biomanufacturing of recombinant therapeutic proteins offers several potential advantages over conventional batch processing, including reduced cost of goods, more flexible and responsive manufacturing facilities, and improved and consistent product quality. Although continuous approaches to various upstream and downstream unit operations have been considered and studied for decades, in recent years interest and application have accelerated. Researchers have achieved increasingly higher levels of process intensification, and have also begun to integrate different continuous unit operations into larger, holistically continuous processes. This review first discusses approaches for continuous cell culture, with a focus on perfusion-enabling cell separation technologies including gravitational, centrifugal, and acoustic settling, as well as filtration-based techniques. We follow with a review of various continuous downstream unit operations, covering categories such as clarification, chromatography, formulation, and viral inactivation and filtration. The review ends by summarizing case studies of integrated and continuous processing as reported in the literature.

A Screen for Modifiers of Hedgehog Signaling in Drosophila melanogaster Identifies swm and mts

PubMed Central

Casso, David J.; Liu, Songmei; Iwaki, D. David; Ogden, Stacey K.; Kornberg, Thomas B.

2008-01-01

Signaling by Hedgehog (Hh) proteins shapes most tissues and organs in both vertebrates and invertebrates, and its misregulation has been implicated in many human diseases. Although components of the signaling pathway have been identified, key aspects of the signaling mechanism and downstream targets remain to be elucidated. We performed an enhancer/suppressor screen in Drosophila to identify novel components of the pathway and identified 26 autosomal regions that modify a phenotypic readout of Hh signaling. Three of the regions include genes that contribute constituents to the pathway—patched, engrailed, and hh. One of the other regions includes the gene microtubule star (mts) that encodes a subunit of protein phosphatase 2A. We show that mts is necessary for full activation of Hh signaling. A second region includes the gene second mitotic wave missing (swm). swm is recessive lethal and is predicted to encode an evolutionarily conserved protein with RNA binding and Zn+ finger domains. Characterization of newly isolated alleles indicates that swm is a negative regulator of Hh signaling and is essential for cell polarity. PMID:18245841

Soil Investigation of Lower East Fork Poplar Creek

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dickson, Johnbull O.; Mayes, Melanie; Earles, Jennifer E.

2017-03-01

Mercury is regarded by the US Department of Energy (DOE) Oak Ridge Office of Environmental Management as a priority contaminant on the Oak Ridge Reservation because of the environmental risks associated with substantial losses from buildings, soils, and surface waters at the Y-12 National Security Complex (Y-12). As a result of historical releases of mercury from Y-12 primarily in the 1950s and early 1960s, the lower East Fork Poplar Creek (LEFPC) stream channel and bank soil margins are contaminated with mercury (Brooks and Southworth 2011; Tennessee Valley Authority 1985b, a). A Mercury Remediation Technology Development project is underway to evaluatemore » the nature of downstream mercury contamination and to develop targeted site-specific remedial technologies that can mitigate mercury release and biological uptake. It is known that mercury concentration varies longitudinally and with depth in LEFPC bank soils; however, soil types and soil physical properties are not well known, especially relative to the zones of mercury contamination. Moreover, there are no soil maps for the downstream reaches of LEFPC in Roane County (i.e. from the Chestnut Hill Road downstream) and this work represents the first ever soil mapping along this section of LEFPC.« less

Prolyl isomerase Pin1 acts downstream of miR-200 to promote cancer stem-like cell traits in breast cancer

PubMed Central

Luo, Man-Li; Gong, Chang; Chen, Chun-Hau; Lee, Daniel Y.; Hu, Hai; Huang, Pengyu; Yao, Yandan; Guo, Wenjun; Reinhardt, Ferenc; Wulf, Gerburg; Lieberman, Judy; Zhou, Xiao Zhen; Song, Erwei; Lu, Kun Ping

2014-01-01

Breast cancer stem-like cells (BCSC) have been implicated in tumor growth, metastasis, drug resistance and relapse but druggable targets in appropriate subsets of this cell population have yet to be identified. Here we identify a fundamental role for the prolyl isomerase Pin1 in driving BCSC expansion, invasiveness and tumorigenicity, defining it as a key target of miR-200c which is known to be a critical regluator in BSCS. Pin1 overexpression expanded the growth and tumorigenicity of BCSC and triggered epithelial-mesenchymal transition (EMT). Conversely, genetic or pharmaacological inhibition of Pin1 reduced the abundance and self-renewal activity of BCSC. Moreover, moderate overexpression of miR-200c-resistant Pin1 rescued the BCSC defect in miR-200c-expressing cells. Genetic deletion of Pin1 also decreased the abundance and repopulating capability of normal mouse mammary stem cells. In human cells freshly isolated from reduction mammoplasty tissues, Pin1 overexpression endowed BCSC traits to normal breast epithelial cells, expanding both luminal and basal/myoepithelial lineages in these cells. In contrast, Pin1 silencing in primary breast cancer cells isolated from clinical samples inhibited the expansion, self-renewal activity and tumorigenesis of BCSC in vitro and in vivo. Overall, our work demonstrated that Pin1 is a pivotal regulator acting downstream of miR-200c to drive BCSC and breast tumorigenicity, highlighting a new therapeutic target to eradicate BCSC. PMID:24786790

Convergent, not serial, striatal and pallidal circuits regulate opioid-induced food intake

PubMed Central

Taha, Sharif A.; Katsuura, Yoshihiro; Noorvash, David; Seroussi, Ariel; Fields, Howard L.

2009-01-01

Mu opioid receptor (MOR) signaling in the nucleus accumbens (NAcc) elicits marked increases in the consumption of palatable tastants. However, the mechanism and circuitry underlying this effect are not fully understood. Multiple downstream target regions have been implicated in mediating this effect but the role of the ventral pallidum (VP), a primary target of NAcc efferents, has not been well defined. To probe the mechanisms underlying increased consumption, we identified behavioral changes in licking patterns following NAcc MOR stimulation. Because the temporal structure of licking reflects the physiological substrates modulating consumption, these measures provide a useful tool in dissecting the cause of increased consumption following NAcc MOR stimulation. Next, we used a combination of pharmacological inactivation and lesions to define the role of the VP in hyperphagia following infusion of the MOR-specific agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) into the NAcc. In agreement with previous studies, results from lick microstructure analysis suggest that NAcc MOR stimulation augments intake through a palatability-driven mechanism. Our results also demonstrate an important role for the VP in normal feeding behavior: pharmacological inactivation of the VP suppresses baseline and NAcc DAMGO-induced consumption. However, this interaction does not occur through a serial circuit requiring direct projections from the NAcc to the VP. Rather, our results indicate that NAcc and VP circuits converge on a common downstream target that regulates food intake. PMID:19336249

Role of NF-E2 related factor 2 (Nrf2) on chemotherapy resistance in acute myeloid leukemia (AML) and the effect of pharmacological inhibition of Nrf2.

PubMed

Karathedath, Sreeja; Rajamani, Bharathi M; Musheer Aalam, Syed Mohammed; Abraham, Ajay; Varatharajan, Savitha; Krishnamurthy, Partha; Mathews, Vikram; Velayudhan, Shaji Ramachandran; Balasubramanian, Poonkuzhali

2017-01-01

Cytarabine (Ara-C) and Daunorubicin (Dnr) forms the backbone of acute myeloid leukemia (AML) therapy. Drug resistance and toxic side effects pose a major threat to treatment success and hence alternate less toxic therapies are warranted. NF-E2 related factor-2 (Nrf2), a master regulator of antioxidant response is implicated in chemoresistance in solid tumors. However, little is known about the role of Nrf2 in AML chemoresistance and the effect of pharmacological inhibitor brusatol in modulating this resistance. Primary AML samples with high ex-vivo IC50 to Ara-C, ATO, Dnr had significantly high NRF2 RNA expression. Gene-specific knockdown of NRF2 improved sensitivity to these drugs in resistant AML cell lines by decreasing the expression of downstream antioxidant targets of Nrf2 by compromising the cell's ability to scavenge the ROS. Treatment with brusatol, a pharmacological inhibitor of Nrf2, improved sensitivity to Ara-C, ATO, and Dnr and reduced colony formation capacity. AML cell lines stably overexpressing NRF2 showed increased resistance to ATO, Dnr and Ara-C and increased expression of downstream targets. This study demonstrates that Nrf2 could be an ideal druggable target in AML, more so to the drugs that function through ROS, suggesting the possibility of using Nrf2 inhibitors in combination with chemotherapeutic agents to modulate drug resistance in AML.

Vav family exchange factors: an integrated regulatory and functional view

PubMed Central

Bustelo, Xosé R

2014-01-01

The Vav family is a group of tyrosine phosphorylation-regulated signal transduction molecules hierarchically located downstream of protein tyrosine kinases. The main function of these proteins is to work as guanosine nucleotide exchange factors (GEFs) for members of the Rho GTPase family. In addition, they can exhibit a variety of catalysis-independent roles in specific signaling contexts. Vav proteins play essential signaling roles for both the development and/or effector functions of a large variety of cell lineages, including those belonging to the immune, nervous, and cardiovascular systems. They also contribute to pathological states such as cancer, immune-related dysfunctions, and atherosclerosis. Here, I will provide an integrated view about the evolution, regulation, and effector properties of these signaling molecules. In addition, I will discuss the pros and cons for their potential consideration as therapeutic targets. PMID:25483299