344 research outputs found
Membrane-tethered Drosophila Armadillo cannot transduce Wingless signal on its own
Drosophila Armadillo and its vertebrate homolog beta-catenin are key effectors of Wingless/Wnt signaling. In the current model, Wingless/Wnt signal stabilizes Armadillo/beta-catenin, which then accumulates in nuclei and binds TCF/LEF family proteins, forming bipartite transcription factors which activate transcription of Wingless/Wnt responsive genes. This model was recently challenged. Overexpression in Xenopus of membrane-tethered beta-catenin or its paralog plakoglobin activates Wnt signaling, suggesting that nuclear localization of Armadillo/beta-catenin is not essential for signaling. Tethered plakoglobin or beta-catenin might signal on their own or might act indirectly by elevating levels of endogenous beta-catenin. We tested these hypotheses in Drosophila by removing endogenous Armadillo. We generated a series of mutant Armadillo proteins with altered intracellular localizations, and expressed these in wild-type and armadillo mutant backgrounds. We found that membrane-tethered Armadillo cannot signal on its own; however it can function in adherens junctions. We also created mutant forms of Armadillo carrying heterologous nuclear localization or nuclear export signals. Although these signals alter the subcellular localization of Arm when overexpressed in Xenopus, in Drosophila they have little effect on localization and only subtle effects on signaling. This supports a model in which Armadillo's nuclear localization is key for signaling, but in which Armadillo intracellular localization is controlled by the availability and affinity of its binding partners
Hybrid optimization method with general switching strategy for parameter estimation
This article is available from: http://www.biomedcentral.com/1752-0509/2/26[Background] Modeling and simulation of cellular signaling and metabolic pathways as networks of
biochemical reactions yields sets of non-linear ordinary differential equations. These models usually
depend on several parameters and initial conditions. If these parameters are unknown, results from
simulation studies can be misleading. Such a scenario can be avoided by fitting the model to
experimental data before analyzing the system. This involves parameter estimation which is usually
performed by minimizing a cost function which quantifies the difference between model predictions
and measurements. Mathematically, this is formulated as a non-linear optimization problem which
often results to be multi-modal (non-convex), rendering local optimization methods detrimental.[Results] In this work we propose a new hybrid global method, based on the combination of an
evolutionary search strategy with a local multiple-shooting approach, which offers a reliable and
efficient alternative for the solution of large scale parameter estimation problems.[Conclusion] The presented new hybrid strategy offers two main advantages over previous
approaches: First, it is equipped with a switching strategy which allows the systematic
determination of the transition from the local to global search. This avoids computationally
expensive tests in advance. Second, using multiple-shooting as the local search procedure reduces
the multi-modality of the non-linear optimization problem significantly. Because multiple-shooting
avoids possible spurious solutions in the vicinity of the global optimum it often outperforms the
frequently used initial value approach (single-shooting). Thereby, the use of multiple-shooting yields
an enhanced robustness of the hybrid approach.This work was supported by the European Community as part of the FP6
COSBICS Project (STREP FP6-512060), the German Federal Ministry of
Education and Research, BMBF-project FRISYS (grant 0313921) and Xunta
de Galicia (PGIDIT05PXIC40201PM).Peer reviewe
A mathematical model for breath gas analysis of volatile organic compounds with special emphasis on acetone
Recommended standardized procedures for determining exhaled lower respiratory
nitric oxide and nasal nitric oxide have been developed by task forces of the
European Respiratory Society and the American Thoracic Society. These
recommendations have paved the way for the measurement of nitric oxide to
become a diagnostic tool for specific clinical applications. It would be
desirable to develop similar guidelines for the sampling of other trace gases
in exhaled breath, especially volatile organic compounds (VOCs) which reflect
ongoing metabolism. The concentrations of water-soluble, blood-borne substances
in exhaled breath are influenced by: (i) breathing patterns affecting gas
exchange in the conducting airways; (ii) the concentrations in the
tracheo-bronchial lining fluid; (iii) the alveolar and systemic concentrations
of the compound. The classical Farhi equation takes only the alveolar
concentrations into account. Real-time measurements of acetone in end-tidal
breath under an ergometer challenge show characteristics which cannot be
explained within the Farhi setting. Here we develop a compartment model that
reliably captures these profiles and is capable of relating breath to the
systemic concentrations of acetone. By comparison with experimental data it is
inferred that the major part of variability in breath acetone concentrations
(e.g., in response to moderate exercise or altered breathing patterns) can be
attributed to airway gas exchange, with minimal changes of the underlying blood
and tissue concentrations. Moreover, it is deduced that measured end-tidal
breath concentrations of acetone determined during resting conditions and free
breathing will be rather poor indicators for endogenous levels. Particularly,
the current formulation includes the classical Farhi and the Scheid series
inhomogeneity model as special limiting cases.Comment: 38 page
Photoactivatable prodrugs of antimelanoma agent Vemurafenib
In this study, we report on novel
photoactivatable caged prodrugs
of vemurafenib. This kinase inhibitor was the first approved drug
for the personalized treatment of BRAF-mutated melanoma and showed
impressive results in clinical studies. However, the occurrence of
severe side effects and drug resistance illustrates the urgent need
for innovative therapeutic approaches. To conquer these limitations,
we implemented photoremovable protecting groups into vemurafenib.
In general, this caging concept provides spatial and temporal control
over the activation of molecules triggered by ultraviolet light. Thus,
higher inhibitor concentrations in tumor tissues might be reached
with less systemic effects. Our study describes the first development
of caged vemurafenib prodrugs useful as pharmacological tools. We
investigated their photochemical characteristics and photoactivation. <i>In vitro</i> evaluation proved the intended loss-of-function
and the light-dependent recovery of efficacy in kinase and cellular
assays. The reported vemurafenib photo prodrugs represent a powerful
biological tool for novel pharmacological approaches in cancer research
When TADs go bad: chromatin structure and nuclear organisation in human disease
Chromatin in the interphase nucleus is organised as a hierarchical series of structural domains, including self-interacting domains called topologically associating domains (TADs). This arrangement is thought to bring enhancers into closer physical proximity with their target genes, which often are located hundreds of kilobases away in linear genomic distance. TADs are demarcated by boundary regions bound by architectural proteins, such as CTCF and cohesin, although much remains to be discovered about the structure and function of these domains. Recent studies of TAD boundaries disrupted in engineered mouse models show that boundary mutations can recapitulate human developmental disorders as a result of aberrant promoter-enhancer interactions in the affected TADs. Similar boundary disruptions in certain cancers can result in oncogene overexpression, and CTCF binding sites at boundaries appear to be hyper-mutated across cancers. Further insights into chromatin organisation, in parallel with accumulating whole genome sequence data for disease cohorts, are likely to yield additional valuable insights into the roles of noncoding sequence variation in human disease
Wingless Signalling Alters the Levels, Subcellular Distribution and Dynamics of Armadillo and E-Cadherin in Third Instar Larval Wing Imaginal Discs
Background: Armadillo, the Drosophila orthologue of vertebrate beta-catenin, plays a dual role as the key effector of Wingless/Wnt1 signalling, and as a bridge between E-Cadherin and the actin cytoskeleton. In the absence of ligand, Armadillo is phosphorylated and targeted to the proteasome. Upon binding of Wg to its receptors, the "degradation complex'' is inhibited; Armadillo is stabilised and enters the nucleus to transcribe targets. Methodology/Principal Findings: Although the relationship between signalling and adhesion has been extensively studied, few in vivo data exist concerning how the "transcriptional'' and "adhesive'' pools of Armadillo are regulated to orchestrate development. We have therefore addressed how the subcellular distribution of Armadillo and its association with E-Cadherin change in larval wing imaginal discs, under wild type conditions and upon signalling. Using confocal microscopy, we show that Armadillo and E-Cadherin are spatio-temporally regulated during development, and that a punctate species becomes concentrated in a subapical compartment in response to Wingless. In order to further dissect this phenomenon, we overexpressed Armadillo mutants exhibiting different levels of activity and stability, but retaining E-Cadherin binding. Arm(S10) displaces endogenous Armadillo from the AJ and the basolateral membrane, while leaving E-Cadherin relatively undisturbed. Surprisingly, Delta NArm(1-155) caused displacement of both Armadillo and E-Cadherin, results supported by our novel method of quantification. However, only membrane-targeted Myr-Delta NArm(1-155) produced comparable nuclear accumulation of Armadillo and signalling to Arm(S10). These experiments also highlighted a row of cells at the A/P boundary depleted of E-Cadherin at the AJ, but containing actin. Conclusions/Significance: Taken together, our results provide in vivo evidence for a complex non-linear relationship between Armadillo levels, subcellular distribution and Wingless signalling. Moreover, this study highlights the importance of Armadillo in regulating the subcellular distribution of E-CadherinPublisher PDFPeer reviewe
Benchmarking of Mutation Diagnostics in Clinical Lung Cancer Specimens
Treatment of EGFR-mutant non-small cell lung cancer patients with the tyrosine kinase inhibitors erlotinib or gefitinib results in high response rates and prolonged progression-free survival. Despite the development of sensitive mutation detection approaches, a thorough validation of these in a clinical setting has so far been lacking. We performed, in a clinical setting, a systematic validation of dideoxy βSangerβ sequencing and pyrosequencing against massively parallel sequencing as one of the most sensitive mutation detection technologies available. Mutational annotation of clinical lung tumor samples revealed that of all patients with a confirmed response to EGFR inhibition, only massively parallel sequencing detected all relevant mutations. By contrast, dideoxy sequencing missed four responders and pyrosequencing missed two responders, indicating a dramatic lack of sensitivity of dideoxy sequencing, which is widely applied for this purpose. Furthermore, precise quantification of mutant alleles revealed a low correlation (r2β=β0.27) of histopathological estimates of tumor content and frequency of mutant alleles, thereby questioning the use of histopathology for stratification of specimens for individual analytical procedures. Our results suggest that enhanced analytical sensitivity is critically required to correctly identify patients responding to EGFR inhibition. More broadly, our results emphasize the need for thorough evaluation of all mutation detection approaches against massively parallel sequencing as a prerequisite for any clinical implementation
Nonsense and missense mutation of mitochondrial ND6 gene promotes cell migration and invasion in human lung adenocarcinoma
Impaired CK1 Delta Activity Attenuates SV40-Induced Cellular Transformation In Vitro and Mouse Mammary Carcinogenesis In Vivo
Simian virus 40 (SV40) is a powerful tool to study cellular transformation in vitro, as well as tumor development and progression in vivo. Various cellular kinases, among them members of the CK1 family, play an important role in modulating the transforming activity of SV40, including the transforming activity of T-Ag, the major transforming protein of SV40, itself. Here we characterized the effects of mutant CK1Ξ΄ variants with impaired kinase activity on SV40-induced cell transformation in vitro, and on SV40-induced mammary carcinogenesis in vivo in a transgenic/bi-transgenic mouse model. CK1Ξ΄ mutants exhibited a reduced kinase activity compared to wtCK1Ξ΄ in in vitro kinase assays. Molecular modeling studies suggested that mutation N172D, located within the substrate binding region, is mainly responsible for impaired mutCK1Ξ΄ activity. When stably over-expressed in maximal transformed SV-52 cells, CK1Ξ΄ mutants induced reversion to a minimal transformed phenotype by dominant-negative interference with endogenous wtCK1Ξ΄. To characterize the effects of CK1Ξ΄ on SV40-induced mammary carcinogenesis, we generated transgenic mice expressing mutant CK1Ξ΄ under the control of the whey acidic protein (WAP) gene promoter, and crossed them with SV40 transgenic WAP-T-antigen (WAP-T) mice. Both WAP-T mice as well as WAP-mutCK1Ξ΄/WAP-T bi-transgenic mice developed breast cancer. However, tumor incidence was lower and life span was significantly longer in WAP-mutCK1Ξ΄/WAP-T bi-transgenic animals. The reduced CK1Ξ΄ activity did not affect early lesion formation during tumorigenesis, suggesting that impaired CK1Ξ΄ activity reduces the probability for outgrowth of in situ carcinomas to invasive carcinomas. The different tumorigenic potential of SV40 in WAP-T and WAP-mutCK1Ξ΄/WAP-T tumors was also reflected by a significantly different expression of various genes known to be involved in tumor progression, specifically of those involved in wnt-signaling and DNA repair. Our data show that inactivating mutations in CK1Ξ΄ impair SV40-induced cellular transformation in vitro and mouse mammary carcinogenesis in vivo
Segment-Specific Neuronal Subtype Specification by the Integration of Anteroposterior and Temporal Cues
To address the question of how neuronal diversity is achieved throughout the CNS, this study provides evidence of modulation of neural progenitor cell βoutputβ along the body axis by integration of local anteroposterior and temporal cues
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