Zoledronate inhibits endothelial cell adhesion, migration and survival through the suppression of multiple, prenylation-dependent signaling pathways.

BACKGROUND: Recent evidence indicates that zoledronate, a nitrogen-containing bisphosphonate used to treat conditions of increased bone resorption, may have anti-angiogenic activity. The endothelial cells signaling events modulated by zoledronate remain largely elusive. OBJECTIVES: The aim of this work was to identify signaling events suppressed by zoledronate in endothelial cells and responsible for some of its biological effects. METHODS: Human umbilical vein endothelial cells (HUVEC) were exposed to zoledronate, isoprenoid analogs (i.e. farnesol and geranylgeraniol) and various inhibitors of signaling, and the effect on adhesion, survival, migration, actin cytoskeleton and signaling events characterized. RESULTS: Zoledronate reduced Ras prenylation, Ras and RhoA translocation to the membrane, and sustained ERK1/2 phosphorylation and tumor necrosis factor (TNF) induced JNK phosphorylation. Isoprenoid analogs attenuated zoledronate effects on HUVEC adhesion, actin stress fibers and focal adhesions, migration and survival. Isoprenoid analogs also restored Ras prenylation, RhoA translocation to the membrane, sustained FAK and ERK1/2 phosphorylation and prevented suppression of protein kinase B (PKB) and JNK phosphorylation in HUVEC exposed to TNF in the presence of zoledronate. Pharmacological inhibition of Rock, a RhoA target mediating actin fiber formation, phosphatidylinositol 3-kinase, an activator of PKB, MEK1/2, an activator of ERK1/2, and JNK, recapitulated individual zoledronate effects, consistent with the involvement of these molecules and pathways and their inhibition in the zoledronate effects. CONCLUSIONS: This work has demonstrated that zoledronate inhibits HUVEC adhesion, survival, migration and actin stress fiber formation by interfering with protein prenylation and has identified ERK1/2, JNK, Rock, FAK and PKB as kinases affected by zoledronate in a prenylation-dependent manner.

Automatic classification of MR scans in Alzheimer's disease.

To be diagnostically useful, structural MRI must reliably distinguish Alzheimer's disease (AD) from normal aging in individual scans. Recent advances in statistical learning theory have led to the application of support vector machines to MRI for detection of a variety of disease states. The aims of this study were to assess how successfully support vector machines assigned individual diagnoses and to determine whether data-sets combined from multiple scanners and different centres could be used to obtain effective classification of scans. We used linear support vector machines to classify the grey matter segment of T1-weighted MR scans from pathologically proven AD patients and cognitively normal elderly individuals obtained from two centres with different scanning equipment. Because the clinical diagnosis of mild AD is difficult we also tested the ability of support vector machines to differentiate control scans from patients without post-mortem confirmation. Finally we sought to use these methods to differentiate scans between patients suffering from AD from those with frontotemporal lobar degeneration. Up to 96% of pathologically verified AD patients were correctly classified using whole brain images. Data from different centres were successfully combined achieving comparable results from the separate analyses. Importantly, data from one centre could be used to train a support vector machine to accurately differentiate AD and normal ageing scans obtained from another centre with different subjects and different scanner equipment. Patients with mild, clinically probable AD and age/sex matched controls were correctly separated in 89% of cases which is compatible with published diagnosis rates in the best clinical centres. This method correctly assigned 89% of patients with post-mortem confirmed diagnosis of either AD or frontotemporal lobar degeneration to their respective group. Our study leads to three conclusions: Firstly, support vector machines successfully separate patients with AD from healthy aging subjects. Secondly, they perform well in the differential diagnosis of two different forms of dementia. Thirdly, the method is robust and can be generalized across different centres. This suggests an important role for computer based diagnostic image analysis for clinical practice.

Multiple filtering devices for the estimation of cyclical DSGE models

We propose a method to estimate time invariant cyclical DSGE models using the informationprovided by a variety of filters. We treat data filtered with alternative procedures as contaminated proxies of the relevant model-based quantities and estimate structural and non-structuralparameters jointly using a signal extraction approach. We employ simulated data to illustratethe properties of the procedure and compare our conclusions with those obtained when just onefilter is used. We revisit the role of money in the transmission of monetary business cycles.

Protonation controls ASIC1a activity via coordinated movements in multiple domains.

Acid-sensing ion channels (ASICs) are neuronal Na(+)-conducting channels activated by extracellular acidification. ASICs are involved in pain sensation, expression of fear, and neurodegeneration after ischemic stroke. Functional ASICs are composed of three identical or homologous subunits, whose extracellular part has a handlike structure. Currently, it is unclear how protonation of residues in extracellular domains controls ASIC activity. Knowledge of these mechanisms would allow a rational development of drugs acting on ASICs. Protonation may induce conformational changes that control the position of the channel gate. We used voltage-clamp fluorometry with fluorophores attached to residues in different domains of ASIC1a to detect conformational changes. Comparison of the timing of fluorescence and current signals identified residues involved in movements that preceded desensitization and may therefore be associated with channel opening or early steps leading to desensitization. Other residues participated in movements intimately linked to desensitization and recovery from desensitization. Fluorescence signals of all mutants were detected at more alkaline pH than ionic currents. Their midpoint of pH dependence was close to that of steady-state desensitization, whereas the steepness of the pH fluorescence relationship was closer to that of current activation. A sequence of movements was observed upon acidification, and its backward movements during recovery from desensitization occurred in the reverse order, indicating that the individual steps are interdependent. Furthermore, the fluorescence signal of some labeled residues in the finger domain was strongly quenched by a Trp residue in the neighboring β-ball domain. Upon channel activation, their fluorescence intensity increased, indicating that the finger moved away from the β ball. This extensive analysis of activity-dependent conformational changes in ASICs sheds new light on the mechanisms by which protonation controls ASIC activity.

International Union of Pharmacology. XXXV. The glucagon receptor family.

Peptide hormones within the secretin-glucagon family are expressed in endocrine cells of the pancreas and gastrointestinal epithelium and in specialized neurons in the brain, and subserve multiple biological functions, including regulation of growth, nutrient intake, and transit within the gut, and digestion, energy absorption, and energy assimilation. Glucagon, glucagon-like peptide-1, glucagon-like peptide-2, glucose-dependent insulinotropic peptide, growth hormone-releasing hormone and secretin are structurally related peptides that exert their actions through unique members of a structurally related G protein-coupled receptor class 2 family. This review discusses advances in our understanding of how these peptides exert their biological activities, with a focus on the biological actions and structural features of the cognate receptors. The receptors have been named after their parent and only physiologically relevant ligand, in line with the recommendations of the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR).

Simultaneous Optical Recording in Multiple Cells by Digital Holographic Microscopy of Chloride Current Associated to Activation of the Ligand-Gated Chloride Channel GABA(A) Receptor.

Chloride channels represent a group of targets for major clinical indications. However, molecular screening for chloride channel modulators has proven to be difficult and time-consuming as approaches essentially rely on the use of fluorescent dyes or invasive patch-clamp techniques which do not lend themselves to the screening of large sets of compounds. To address this problem, we have developed a non-invasive optical method, based on digital holographic microcopy (DHM), allowing monitoring of ion channel activity without using any electrode or fluorescent dye. To illustrate this approach, GABA(A) mediated chloride currents have been monitored with DHM. Practically, we show that DHM can non-invasively provide the quantitative determination of transmembrane chloride fluxes mediated by the activation of chloride channels associated with GABA(A) receptors. Indeed through an original algorithm, chloride currents elicited by application of appropriate agonists of the GABA(A) receptor can be derived from the quantitative phase signal recorded with DHM. Finally, chloride currents can be determined and pharmacologically characterized non-invasively simultaneously on a large cellular sampling by DHM.

Semi-supervised remote sensing image classification with cluster kernels

A semisupervised support vector machine is presented for the classification of remote sensing images. The method exploits the wealth of unlabeled samples for regularizing the training kernel representation locally by means of cluster kernels. The method learns a suitable kernel directly from the image and thus avoids assuming a priori signal relations by using a predefined kernel structure. Good results are obtained in image classification examples when few labeled samples are available. The method scales almost linearly with the number of unlabeled samples and provides out-of-sample predictions.

Drift compensation of gas sensor array data by Orthogonal Signal Correction

Drift is an important issue that impairs the reliability of gas sensing systems. Sensor aging, memory effects and environmental disturbances produce shifts in sensor responses that make initial statistical models for gas or odor recognition useless after a relatively short period (typically few weeks). Frequent recalibrations are needed to preserve system accuracy. However, when recalibrations involve numerous samples they become expensive and laborious. An interesting and lower cost alternative is drift counteraction by signal processing techniques. Orthogonal Signal Correction (OSC) is proposed for drift compensation in chemical sensor arrays. The performance of OSC is also compared with Component Correction (CC). A simple classification algorithm has been employed for assessing the performance of the algorithms on a dataset composed by measurements of three analytes using an array of seventeen conductive polymer gas sensors over a ten month period.

Multiple roles of mouse Numb in tuning developmental cell fates.

BACKGROUND: Notch signaling regulates multiple differentiation processes and cell fate decisions during both invertebrate and vertebrate development. Numb encodes an intracellular protein that was shown in Drosophila to antagonize Notch signaling at binary cell fate decisions of certain cell lineages. Although overexpression experiments suggested that Numb might also antagonize some Notch activity in vertebrates, the developmental processes in which Numb is involved remained elusive. RESULTS: We generated mice with a homozygous inactivation of Numb. These mice died before embryonic day E11.5, probably because of defects in angiogenic remodeling and placental dysfunction. Mutant embryos had an open anterior neural tube and impaired neuronal differentiation within the developing cranial central nervous system (CNS). In the developing spinal cord, the number of differentiated motoneurons was reduced. Within the peripheral nervous system (PNS), ganglia of cranial sensory neurons were formed. Trunk neural crest cells migrated and differentiated into sympathetic neurons. In contrast, a selective differentiation anomaly was observed in dorsal root ganglia, where neural crest--derived progenitor cells had migrated normally to form ganglionic structures, but failed to differentiate into sensory neurons. CONCLUSIONS: Mouse Numb is involved in multiple developmental processes and required for cell fate tuning in a variety of lineages. In the nervous system, Numb is required for the generation of a large subset of neuronal lineages. The restricted requirement of Numb during neural development in the mouse suggests that in some neuronal lineages, Notch signaling may be regulated independently of Numb.

Role of peroxynitrite in the redox regulation of cell signal transduction pathways.

Peroxynitrite is a potent oxidant and nitrating species formed from the reaction between the free radicals nitric oxide and superoxide. An excessive formation of peroxynitrite represents an important mechanism contributing to cell death and dysfunction in multiple cardiovascular pathologies, such as myocardial infarction, heart failure and atherosclerosis. Whereas initial works focused on direct oxidative biomolecular damage as the main route of peroxynitrite toxicity, more recent evidence, mainly obtained in vitro, indicates that peroxynitrite also behaves as a potent modulator of various cell signal transduction pathways. Due to its ability to nitrate tyrosine residues, peroxynitrite affects cellular processes dependent on tyrosine phosphorylation. Peroxynitrite also exerts complex effects on the activity of various kinases and phosphatases, resulting in the up- or downregulation of signalling cascades, in a concentration- and cell-dependent manner. Such roles of peroxynitrite in the redox regulation of key signalling pathways for cardiovascular homeostasis, including protein kinase B and C, the MAP kinases, Nuclear Factor Kappa B, as well as signalling dependent on insulin and the sympatho-adrenergic system are presented in detail in this review.

Manipulating the sensitivity of signal-induced repression: quantification and consequences of altered brinker gradients.

Traditionally, the analysis of gene regulatory regions suffered from the caveat that it was restricted to artificial contexts (e.g. reporter constructs of limited size). With the advent of the BAC recombineering technique, genomic constructs can now be generated to test regulatory elements in their endogenous environment. The expression of the transcriptional repressor brinker (brk) is negatively regulated by Dpp signaling. Repression is mediated by small sequence motifs, the silencer elements (SEs), that are present in multiple copies in the regulatory region of brk. In this work, we manipulated the SEs in the brk locus. We precisely quantified the effects of the individual SEs on the Brk gradient in the wing disc by employing a 1D data extraction method, followed by the quantification of the data with reference to an internal control. We found that mutating the SEs results in an expansion of the brk expression domain. However, even after mutating all predicted SEs, repression could still be observed in regions of maximal Dpp levels. Thus, our data point to the presence of additional, low affinity binding sites in the brk locus.

Multiple Geotechnological Tools Applied to Digital Mapping of Tropical Soils

ABSTRACT In recent years, geotechnologies as remote and proximal sensing and attributes derived from digital terrain elevation models indicated to be very useful for the description of soil variability. However, these information sources are rarely used together. Therefore, a methodology for assessing and specialize soil classes using the information obtained from remote/proximal sensing, GIS and technical knowledge has been applied and evaluated. Two areas of study, in the State of São Paulo, Brazil, totaling approximately 28.000 ha were used for this work. First, in an area (area 1), conventional pedological mapping was done and from the soil classes found patterns were obtained with the following information: a) spectral information (forms of features and absorption intensity of spectral curves with 350 wavelengths -2,500 nm) of soil samples collected at specific points in the area (according to each soil type); b) obtaining equations for determining chemical and physical properties of the soil from the relationship between the results obtained in the laboratory by the conventional method, the levels of chemical and physical attributes with the spectral data; c) supervised classification of Landsat TM 5 images, in order to detect changes in the size of the soil particles (soil texture); d) relationship between classes relief soils and attributes. Subsequently, the obtained patterns were applied in area 2 obtain pedological classification of soils, but in GIS (ArcGIS). Finally, we developed a conventional pedological mapping in area 2 to which was compared with a digital map, ie the one obtained only with pre certain standards. The proposed methodology had a 79 % accuracy in the first categorical level of Soil Classification System, 60 % accuracy in the second category level and became less useful in the categorical level 3 (37 % accuracy).

Triiodothyronine has diverse and multiple stimulating effects on expression of the major myelin protein genes.

If the importance of triiodothyronine (T3) on brain development including myelinogenesis has long been recognized, its mechanism of action at the gene level is still not fully elucidated. We studied the effect of T3 on the expression of myelin protein genes in aggregating brain cell cultures. T3 increases the concentrations of mRNA transcribed from the following four myelin protein genes: myelin basic protein (Mbp), myelin-associated glycoprotein (Mag), proteolipid protein (Plp), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (Cnp). T3 is not only a triggering signal for oligodendrocyte differentiation, but it has continuous stimulatory effects on myelin gene expression. Transcription in isolated nuclei experiments shows that T3 increases Mag and Cnp transcription rates. After inhibiting transcription with actinomycin D, we measured the half-lives of specific mRNAs. Our results show that T3 increases the stability of mRNA for myelin basic protein, and probably proteolipid protein. In vitro translation followed by myelin basic protein-specific immunoprecipitation showed a direct stimulatory effect of T3 on myelin basic protein mRNA translation. Moreover, this stimulation was higher when the mRNA was already stabilized in culture, indicating that stabilization is achieved through mRNA structural modifications. These results demonstrate the diverse and multiple mechanisms of T3 stimulation of myelin protein genes.

First consumption ever of multiple substances: applying an expert-based taxonomy to a Swiss national sample of adolescents.

The use of multiple legal and illegal substances by adolescents is a growing concern in all countries, but since no consensus about a taxonomy did emerge yet, it is difficult to understand the different patterns of consumption and to implement tailored prevention and treatment programs directed towards specific subgroups of the adolescent population. Using data from a Swiss survey on adolescent health, we analyzed the age at which ten legal and illegal substances were consumed for the first time ever by applying a method combining the strength of both automatic clustering and use of substance experts. Results were then compared to 30 socio-economic factors to establish the usefulness of and to validate our taxonomy. We also analyzed the succession of substance first use for each group. The final taxonomy consists of eight groups ranging from non-consumers to heavy drug addicts. All but four socio-economic factors were significantly associated with the taxonomy, the strongest associations being observed with health, behavior, and sexuality factors. Numerous factors influence adolescents in their decision to first try substances or to use them on a regular basis, and no factor alone can be considered as an absolute marker of problematic behavior regarding substance use. Different processes of experimentation with substances are associated with different behaviors, therefore focusing on only one substance or only one factor is not efficient. Prevention and treatment programs can then be tailored to address specific issues related to different youth subgroups.

Characterization and classification of matrix effects in biological samples analyses.

An exhaustive classification of matrix effects occurring when a sample preparation is performed prior to liquid-chromatography coupled to mass spectrometry (LC-MS) analyses was proposed. A total of eight different situations were identified allowing the recognition of the matrix effect typology via the calculation of four recovery values. A set of 198 compounds was used to evaluate matrix effects after solid phase extraction (SPE) from plasma or urine samples prior to LC-ESI-MS analysis. Matrix effect identification was achieved for all compounds and classified through an organization chart. Only 17% of the tested compounds did not present significant matrix effects.