Ran is a potential therapeutic target for cancer cells with molecular changes associated with activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways

Purpose Cancer cells have been shown to be more susceptible to Ran knockdown than normal cells. We now investigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK [mitogen-activated protein/extracellular signal-regulated kinase (ERK; MEK)] and phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 activities. Experimental Design Apoptosis was measured by flow cytometry [propidium iodide (PI) and Annexin V staining] and MTT assay in cancer cells grown under different conditions after knockdown of Ran. The correlations between Ran expression and patient survival were examined in breast and lung cancers. Results Cancer cells with their PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways inhibited are less susceptible to Ran silencing-induced apoptosis. K-Ras-mutated, c-Met-amplified, and Pten-deleted cancer cells are also more susceptible to Ran silencing-induced apoptosis than their wild-type counterparts and this effect is reduced by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Overexpression of Ran in clinical specimens is significantly associated with poor patient outcome in both breast and lung cancers. This association is dramatically enhanced in cancers with increased c-Met or osteopontin expression, or with oncogenic mutations of K-Ras or PIK3CA, all of which are mutations that potentially correlate with activation of the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways. Silencing Ran also results in dysregulation of nucleocytoplasmic transport of transcription factors and downregulation of Mcl-1 expression, at the transcriptional level, which are reversed by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Conclusion Ran is a potential therapeutic target for treatment of cancers with mutations/changes of expression in protooncogenes that lead to activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways. ©2011 AACR.

A satellite RNA of barley yellow dwarf virus contains a novel hammerhead structure in the self-cleavage domain

An RNA molecule with properties of a satellite RNA was found in an isolate of barley yellow dwarf virus (BYDV), RPV serotype. It is 322 nucleotides long, single-stranded, and does not hybridize to the viral genome. Dimers of the RNA, which presumably represent replicative intermediates, were able to self-cleave into monomers. In vitro transcripts from cDNA clones were capable of self-cleavage in both the plus (encapsidated) and minus orientations. The sequence flanking the minus strand cleavage site contained a consensus " hammerhead" structure, similar to those found in other self-cleaving satellite RNAs. Although related to the hammerhead structure, sequences flanking the plus strand termini showed differences from the consensus and may be folded into a different structure containing a pseudoknot. © 1991.

Fault symptoms of rolling element bearings under variable operating conditions - a multi domain analysis

Diagnostics of rolling element bearings have been traditionally developed for constant operating conditions, and sophisticated techniques, like Spectral Kurtosis or Envelope Analysis, have proven their effectiveness by means of experimental tests, mainly conducted in small-scale laboratory test-rigs. Algorithms have been developed for the digital signal processing of data collected at constant speed and bearing load, with a few exceptions, allowing only small fluctuations of these quantities. Owing to the spreading of condition based maintenance in many industrial fields, in the last years a need for more flexible algorithms emerged, asking for compatibility with highly variable operating conditions, such as acceleration/deceleration transients. This paper analyzes the problems related with significant speed and load variability, discussing in detail the effect that they have on bearing damage symptoms, and propose solutions to adapt existing algorithms to cope with this new challenge. In particular, the paper will i) discuss the implication of variable speed on the applicability of diagnostic techniques, ii) address quantitatively the effects of load on the characteristic frequencies of damaged bearings and iii) finally present a new approach for bearing diagnostics in variable conditions, based on envelope analysis. The research is based on experimental data obtained by using artificially damaged bearings installed on a full scale test-rig, equipped with actual train traction system and reproducing the operation on a real track, including all the environmental noise, owing to track irregularity and electrical disturbances of such a harsh application.

The art of mooting : mooting and the cognitive domain

Mooting is modeled principally on appellate advocacy. However, the skill set developed by participating in a moot program – being that necessary to persuade someone to your preferred position – is indispensible to anyone practising law. Developing effective mooting skills in students necessitates the engagement of coaches with an appropriate understanding of the theories underlying mooting and advocacy practice and their interconnection with each other. This article explains the relevance of the cognitive domain to mooting performance and places it in context with the psychomotor and affective domains.

Fibrogenic stresses activate different mitogen-activated protein kinase pathways in renal epithelial, endothelial or fibroblast cell populations

Fibrogenic stresses promote progression of renal tubulointerstitial fibrosis, disparately affecting survival, proliferation and trans-differentiation of intrinsic renal cell populations through ill-defined biomolecular pathways. We investigated the effect of fibrogenic stresses on the activation of cell-specific mitogen-activated protein kinase (MAPK) in renal fibroblast, epithelial and endothelial cell populations. The relative outcomes (cell death, proliferation, trans-differentiation) associated with activation or inhibition of extracellular-regulated protein kinase (ERK) or stress activated/c-Jun N terminal kinase (JNK) were analysed in each renal cell population after challenge with oxidative stress (1 mmol/L H2O2), transforming growth factor-beta1 (TGF-beta1, 10 ng/mL) or tumour necrosis factor-alpha (TNF-alpha, 50 ng/mL) over 0-20 h. Apoptosis increased significantly in all cell types after oxidative stress (P < 0.05). In fibroblasts, oxidative stress caused the activation of ERK (pERK) but not JNK (pJNK). Inhibition of ERK by PD98059 supported its role in a fibroblast death pathway. In epithelial and endothelial cells, oxidative stress-induced apoptosis was preceded by early induction of pERK, but its inhibition did not support a pro-apoptotic role. Early ERK activity may be conducive to their survival or promote the trans-differentiation of epithelial cells. In epithelial and endothelial cells, oxidative stress induced pJNK acutely. Pretreatment with SP600125 (JNK inhibitor) verified its pro-apoptotic activity only in epithelial cells. Transforming growth factor-beta1 did not significantly alter mitosis or apoptosis in any of the cell types, nor did it alter MAPK activity. Tumor necrosis factor-alpha caused increased apoptosis with no associated change in MAPK activity. Our results demonstrate renal cell-specific differences in the activation of ERK and JNK following fibrotic insult, which may be useful for targeting excessive fibroblast proliferation in chronic fibrosis.

Selective regulation of p38β protein and signaling by integrin-linked kinase mediates bladder cancer cell migration

Integrin-linked kinase (ILK) and p38MAPK are protein kinases that transduce extracellular signals regulating cell migration and actin cytoskeletal organization. ILK-dependent regulation of p38MAPK is critical for mammalian kidney development and in smooth muscle cell migration, however, specific p38 isoforms has not been previously examined in ILK-regulated responses. Signaling by ILK and p38MAPK is often dysregulated in bladder cancer, and here we report a strong positive correlation between protein levels of ILK and p38β, which is the predominant isoform found in bladder cancer cells, as well as in patient-matched normal bladder and tumor samples. Knockdown by RNA interference of either p38β or ILK disrupts serum-induced, Rac1-dependent migration and actin cytoskeletal organization in bladder cancer cells. Surprisingly, ILK knockdown causes the selective reduction in p38β cellular protein level, without inhibiting p38β messenger RNA (mRNA) expression. The loss of p38β protein in ILK-depleted cells is partially rescued by the 26S proteasomal inhibitor MG132. Using co-precipitation and bimolecular fluorescent complementation assays, we find that ILK selectively forms cytoplasmic complexes with p38β. In situ proximity ligation assays further demonstrate that serum-stimulated assembly of endogenous ILK–p38β complexes is sensitive to QLT-0267, a small molecule ILK kinase inhibitor. Finally, inhibition of ILK reduces the amplitude and period of serum-induced activation of heat shock protein 27 (Hsp27), a target of p38β implicated in actin cytoskeletal reorganization. Our work identifies Hsp27 as a novel target of ILK–p38β signaling complexes, playing a key role in bladder cancer cell migration.

Phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations

Purpose The LUX-Lung 3 study investigated the efficacy of chemotherapy compared with afatinib, a selective, orally bioavailable ErbB family blocker that irreversibly blocks signaling from epidermal growth factor receptor (EGFR/ErbB1), human epidermal growth factor receptor 2 (HER2/ErbB2), and ErbB4 and has wide-spectrum preclinical activity against EGFR mutations. A phase II study of afatinib in EGFR mutation-positive lung adenocarcinoma demonstrated high response rates and progression-free survival (PFS). Patients and Methods In this phase III study, eligible patients with stage IIIB/IV lung adenocarcinoma were screened for EGFR mutations. Mutation-positive patients were stratified by mutation type (exon 19 deletion, L858R, or other) and race (Asian or non-Asian) before two-to-one random assignment to 40 mg afatinib per day or up to six cycles of cisplatin plus pemetrexed chemotherapy at standard doses every 21 days. The primary end point was PFS by independent review. Secondary end points included tumor response, overall survival, adverse events, and patient-reported outcomes (PROs). Results A total of 1,269 patients were screened, and 345 were randomly assigned to treatment. Median PFS was 11.1 months for afatinib and 6.9 months for chemotherapy (hazard ratio [HR], 0.58; 95% CI, 0.43 to 0.78; P = .001). Median PFS among those with exon 19 deletions and L858R EGFR mutations (n = 308) was 13.6 months for afatinib and 6.9 months for chemotherapy (HR, 0.47; 95% CI, 0.34 to 0.65; P = .001). The most common treatmentrelated adverse events were diarrhea, rash/acne, and stomatitis for afatinib and nausea, fatigue, and decreased appetite for chemotherapy. PROs favored afatinib, with better control of cough, dyspnea, and pain. Conclusion Afatinib is associated with prolongation of PFS when compared with standard doublet chemotherapy in patients with advanced lung adenocarcinoma and EGFR mutations.

Symptom control and quality of life in lux-lung 3 : a phase III study of afatinib or cisplatin/pemetrexed in patients with advanced lung adenocarcinoma with EGFR mutations

Purpose Patient-reported symptoms and health-related quality of life (QoL) benefits were investigated in a randomized, phase III trial of afatinib or cisplatin/pemetrexed. Patients and Methods Three hundred forty-five patients with advanced epidermal growth factor receptor (EGFR) mutation-positive lung adenocarcinoma were randomly assigned 2:1 to afatinib 40 mg per day or up to six cycles of cisplatin/pemetrexed. Lung cancer symptoms and health-related QoL were assessed every 21 days until progression using the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire C30 and Lung Cancer-13 questionnaires. Analyses of cough, dyspnea, and pain were preplanned, including percentage of patients who improved on therapy, time to deterioration of symptoms, and change in symptoms over time. Results Questionnaire compliance was high. Compared with chemotherapy, afatinib significantly delayed the time to deterioration for cough (hazard ratio [HR], 0.60; 95% CI, 0.41 to 0.87; P = .007) and dyspnea (HR, 0.68; 95% CI, 0.50 to 0.93; P = .015), but not pain (HR, 0.83; 95% CI, 0.62 to 1.10; P = .19). More patients on afatinib (64%) versus chemotherapy (50%) experienced improvements in dyspnea scores (P lt; .010). Differences in mean scores over time significantly favored afatinib over chemotherapy for cough (P lt; .001) and dyspnea (P = .001). Afatinib showed significantly better mean scores over time in global health status/QoL (P = .015) and physical (P = .001), role (P = .004), and cognitive (P lt; .007) functioning compared with chemotherapy. Fatigue and nausea were worse with chemotherapy, whereas diarrhea, dysphagia, and sore mouth were worse with afatinib (all P = .01). Conclusion In patients with lung adenocarcinoma with EGFR mutations, first-line afatinib was associated with better control of cough and dyspnea compared with chemotherapy, although diarrhea, dysphagia, and sore mouth were worse. Global health status/QoL was also improved over time with afatinib compared with chemotherapy.

Estimation of vibration amplitude in Fourier domain optical coherence tomography interferometric signals from Doppler spectrum

In presented method combination of Fourier and Time domain detection enables to broaden the effective bandwidth for time dependent Doppler Signal that allows for using higher-order Bessel functions to calculate unambiguously the vibration amplitudes.

Myosin light chain kinase regulates synaptic plasticity and fear learning in the lateral amygdala

Learning and memory depend on signaling mole- cules that affect synaptic efficacy. The cytoskeleton has been implicated in regulating synaptic transmission but its role in learning and memory is poorly understood. Fear learning depends on plasticity in the lateral nucleus of the amygdala. We therefore examined whether the cytoskeletal-regulatory protein, myosin light chain kinase, might contribute to fear learning in the rat lateral amygdala. Microinjection of ML-7, a specific inhibitor of myosin light chain kinase, into the lateral nucleus of the amygdala before fear conditioning, but not immediately afterward, enhanced both short-term memory and long-term memory, suggesting that myosin light chain kinase is involved specifically in memory acquisition rather than in posttraining consolidation of memory. Myosin light chain kinase inhibitor had no effect on memory retrieval. Furthermore, ML-7 had no effect on behavior when the train- ing stimuli were presented in a non-associative manner. An- atomical studies showed that myosin light chain kinase is present in cells throughout lateral nucleus of the amygdala and is localized to dendritic shafts and spines that are postsynaptic to the projections from the auditory thalamus to lateral nucleus of the amygdala, a pathway specifically impli- cated in fear learning. Inhibition of myosin light chain kinase enhanced long-term potentiation, a physiological model of learning, in the auditory thalamic pathway to the lateral nu- cleus of the amygdala. When ML-7 was applied without as- sociative tetanic stimulation it had no effect on synaptic responses in lateral nucleus of the amygdala. Thus, myosin light chain kinase activity in lateral nucleus of the amygdala appears to normally suppress synaptic plasticity in the cir- cuits underlying fear learning, suggesting that myosin light chain kinase may help prevent the acquisition of irrelevant fears. Impairment of this mechanism could contribute to pathological fear learning.

Integrin αvβ3 upregulates integrin-linked kinase expression in human ovarian cancer cells via enhancement of ILK gene transcription

We previously showed that integrin alphavbeta3 overexpression and engagement by its ligand vitronectin increased adhesion, motility, and proliferation of human ovarian cancer cells. In search of differentially regulated genes involved in these tumor biological events, we previously identified the integrin-linked kinase (ILK) to be under control of alphavbeta3. In the present investigation we demonstrated significantly upregulated ILK protein as a function of alphavbeta3 in two ovarian cancer cell lines, OV-MZ-6 and OVCAR-3, and proved co-localization at the surface of alphavbeta3-overexpressing cells adherent to vitronectin. Increase of ILK protein was reflected by enhanced ILK promoter activity, an effect, which we further characterized with regard to transcriptional response elements involved. Abrogation of NF-kappaB/c-rel or p53 binding augmented ILK promoter activity and preserved induction by alphavbeta3. The AP1-mutant exhibited decreased promoter activity but was also still inducible by alphavbeta3. Disruption of the two DNA consensus motifs for Ets proteins led to divergent observations: mutation of the Ets motif at promoter position -462 bp did not significantly alter promoter activity but still allowed response to alphavbeta3. In contrast, disruption of the second Ets motif at position -85 bp did not only lead to slightly diminished promoter activity but also, in that case, abrogated ILK promoter induction by alphavbeta3. Subsequent co-transfection studies with ets-1 in the presence of the second Ets motif led to additional induction of ILK promoter activity. Taken together, these data suggest that ets-1 binding to the second Ets DNA motif strongly contributes to alphavbeta3-mediated ILK upregulation. By increasing ILK as an important integrin-proximal kinase, alphavbeta3 may promote its intracellular signaling and tumor biological processes arising thereof in favor of ovarian cancer metastasis.

Synergism between Wnt3a and heparin enhances osteogenesis via a phosphoinositide 3-kinase/Akt/RUNX2 pathway

A new strategy has emerged to improve healing of bone defects using exogenous glycosaminoglycans by increasing the effectiveness of bone-anabolic growth factors. Wnt ligands play an important role in bone formation. However, their functional interactions with heparan sulfate/heparin have only been investigated in non-osseous tissues. Our study now shows that the osteogenic activity of Wnt3a is cooperatively stimulated through physical interactions with exogenous heparin. N-Sulfation and to a lesser extent O-sulfation of heparin contribute to the physical binding and optimal co-stimulation of Wnt3a. Wnt3a-heparin signaling synergistically increases osteoblast differentiation with minimal effects on cell proliferation. Thus, heparin selectively reduces the effective dose of Wnt3a needed to elicit osteogenic, but not mitogenic responses. Mechanistically, Wnt3a-heparin signaling strongly activates the phosphoinositide 3-kinase/Akt pathway and requires the bone-related transcription factor RUNX2 to stimulate alkaline phosphatase activity, which parallels canonical beta-catenin signaling. Collectively, our findings establish the osteo-inductive potential of a heparin-mediated Wnt3a-phosphoinositide 3-kinase/Akt-RUNX2 signaling network and suggest that heparan sulfate supplementation may selectively reduce the therapeutic doses of peptide factors required to promote bone formation.

A sub‒domain smoothed Galerkin method for solid mechanics problems

A sub‒domain smoothed Galerkin method is proposed to integrate the advantages of mesh‒free Galerkin method and FEM. Arbitrarily shaped sub‒domains are predefined in problems domain with mesh‒free nodes. In each sub‒domain, based on mesh‒free Galerkin weak formulation, the local discrete equation can be obtained by using the moving Kriging interpolation, which is similar to the discretization of the high‒order finite elements. Strain smoothing technique is subsequently applied to the nodal integration of sub‒domain by dividing the sub‒domain into several smoothing cells. Moreover, condensation of DOF can also be introduced into the local discrete equations to improve the computational efficiency. The global governing equations of present method are obtained on the basis of the scheme of FEM by assembling all local discrete equations of the sub‒domains. The mesh‒free properties of Galerkin method are retained in each sub‒domain. Several 2D elastic problems have been solved on the basis of this newly proposed method to validate its computational performance. These numerical examples proved that the newly proposed sub‒domain smoothed Galerkin method is a robust technique to solve solid mechanics problems based on its characteristics of high computational efficiency, good accuracy, and convergence.

Practical aspects of frequency-domain identification of dynamic models of marine structures from hydrodynamic data

The motion response of marine structures in waves can be studied using finite-dimensional linear-time-invariant approximating models. These models, obtained using system identification with data computed by hydrodynamic codes, find application in offshore training simulators, hardware-in-the-loop simulators for positioning control testing, and also in initial designs of wave-energy conversion devices. Different proposals have appeared in the literature to address the identification problem in both time and frequency domains, and recent work has highlighted the superiority of the frequency-domain methods. This paper summarises practical frequency-domain estimation algorithms that use constraints on model structure and parameters to refine the search of approximating parametric models. Practical issues associated with the identification are discussed, including the influence of radiation model accuracy in force-to-motion models, which are usually the ultimate modelling objective. The illustration examples in the paper are obtained using a freely available MATLAB toolbox developed by the authors, which implements the estimation algorithms described.

Time-domain hydroelastic analysis of a flexible marine structure using state-space models

This article deals with time-domain hydroelastic analysis of a marine structure. The convolution terms associated with fluid memory effects are replaced by an alternative state-space representation, the parameters of which are obtained by using realization theory. The mathematical model established is validated by comparison to experimental results of a very flexible barge. Two types of time-domain simulations are performed: dynamic response of the initially inert structure to incident regular waves and transient response of the structure after it is released from a displaced condition in still water. The accuracy and the efficiency of the simulations based on the state-space model representations are compared to those that integrate the convolutions.