Conjugation of a Ru(II) Arene Complex to Neomycin or to Guanidinoneomycin Leads to Compounds with Differential Cytotoxicities and Accumulation between Cancer and Normal Cells

A straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η6-p-cym)RuCl(PPh3)2]+, allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with N,N′-di-Boc-N″-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semipreparative high-performance liquid chromatography (HPLC) and characterized by electrospray ionization (ESI) and matrix-assisted laser desorptionionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy. The cytotoxicity of the compounds was tested in MCF-7 (breast) and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, the neomycinruthenium conjugate (2) displayed moderate antiproliferative activity in both cancer cell lines (IC50 ≈ 80 μM), whereas the neamine conjugate (4) was inactive (IC50 ≈ 200 μM). However, the guanidinylated analogue of the neomycinruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC50 = 7.17 μM in DU-145 and IC50 = 11.33 μM in MCF-7). Although the same ranking in antiproliferative activity was found in the nontumorigenic cell line (3 2 > 4), IC50 values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g., IC50 = 49.4 μM for 2) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC50 = 12.75 μM for 3) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates 2 and 3 revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher antiproliferative activity of 3. Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycinruthenium conjugate (3) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycinruthenium conjugate (2). Such differences in cytotoxic activity and cellular accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium(II) arene derivatives to guanidinoglycosides.

Differential pattern of glycogen accumulation after protein phosphatase 1 glycogen-targeting subunit PPP1R6 overexpression, compared to PPP1R3C and PPP1R3A, in skeletal muscle cells

Background PPP1R6 is a protein phosphatase 1 glycogen-targeting subunit (PP1-GTS) abundant in skeletal muscle with an undefined metabolic control role. Here PPP1R6 effects on myotube glycogen metabolism, particle size and subcellular distribution are examined and compared with PPP1R3C/PTG and PPP1R3A/GM. Results PPP1R6 overexpression activates glycogen synthase (GS), reduces its phosphorylation at Ser-641/0 and increases the extracted and cytochemically-stained glycogen content, less than PTG but more than GM. PPP1R6 does not change glycogen phosphorylase activity. All tested PP1-GTS-cells have more glycogen particles than controls as found by electron microscopy of myotube sections. Glycogen particle size is distributed for all cell-types in a continuous range, but PPP1R6 forms smaller particles (mean diameter 14.4 nm) than PTG (36.9 nm) and GM (28.3 nm) or those in control cells (29.2 nm). Both PPP1R6- and GM-derived glycogen particles are in cytosol associated with cellular structures; PTG-derived glycogen is found in membrane- and organelle-devoid cytosolic glycogen-rich areas; and glycogen particles are dispersed in the cytosol in control cells. A tagged PPP1R6 protein at the C-terminus with EGFP shows a diffuse cytosol pattern in glucose-replete and -depleted cells and a punctuate pattern surrounding the nucleus in glucose-depleted cells, which colocates with RFP tagged with the Golgi targeting domain of β-1,4-galactosyltransferase, according to a computational prediction for PPP1R6 Golgi location. Conclusions PPP1R6 exerts a powerful glycogenic effect in cultured muscle cells, more than GM and less than PTG. PPP1R6 protein translocates from a Golgi to cytosolic location in response to glucose. The molecular size and subcellular location of myotube glycogen particles is determined by the PPP1R6, PTG and GM scaffolding.

Differential effects of immunosuppressive drugs on effector and regulatory T cells.

Purpose: Current experimental data suggest that CD4+CD25+Foxp3+regulatory T cells (Tregs) based immunotherapy would be of greatinterest to promote donor-specific immune tolerance in transplantation(Tx). Whether and how adoptive transfer of Tregs could be bestcombined with current immunosuppressive regimens in clinicalsettings remains to be defined. Using an experimental Tx model,we had previously shown that the transfer of antigen-specific Tregspromoted long-term skin allograft acceptance in lymphopenic mice,in the absence of any immunosuppressive drug. However, allograftsurvival was only slightly prolonged when Tregs were transferredalone into non-lymphopenic mice, suggesting that in more stringentconditions such as in clinical settings adjuvant therapies may beneeded to effectively control alloreactive T cells (Teff).Methods and Materials: Here we have investigated the effects ofvarious immunosuppressive drugs on the survival, proliferation andeffector function of Teff and Tregs in response to alloantigens in in vitroassays and in our in vivo Tx model.Results: Teff proliferation was inhibited in a dose-dependant mannerby rapamycin and cyclosporine A, while anti-CD154 only marginallyaffected Teff proliferation and survival in vitro. Rapamycin promotedapoptosis of Teff as compared to Tregs that were more resistant underthe same culture conditions. In vivo, the transfer of donor-specificTregs could be advantageously combined with rapamycin andanti-CD154 to significantly prolong MHC-mismatched skin allograftsurvival in non-lymphopenic recipients.Conclusion: Taken together, our data indicate thatimmunosuppressive drugs differentially target T-cell subsets and couldpromote Tregs expansion and/or function while controlling the Teff pool.

Interaction of Spiral Waves in the Complex Ginzburg-Landau Equation

Solutions of the general cubic complex Ginzburg-Landau equation comprising multiple spiral waves are considered, and laws of motion for the centers are derived. The direction of the motion changes from along the line of centers to perpendicular to the line of centers as the separation increases, with the strength of the interaction algebraic at small separations and exponentially small at large separations. The corresponding asymptotic wave number and frequency are also determined, which evolve slowly as the spirals move

Differential pharmacologic properties of the two C75 enantiomers: (+)-C75 is a strong anorectic drug. (-)-C75 has antitumour activity

C75 is a synthetic compound described as having antitumoral properties. It produces hypophagia and weight loss in rodents, limiting its use in cancer therapy but identify- ing it as a potential anti-obesity drug. C75 is a fatty acid synthase (FAS) inhibitor and, through its coenzyme A (CoA) derivative, it acts as a carnitine palmitoyltransferase (CPT) 1 inhibitor. Racemic mixtures of C75 have been used in all the previous studies; however, the potential dif- ferent biological activities of C75 enantiomers have not been examined yet. To address this question we synthesized the two C75 enantiomers separately. Our results showed that ( )- C75 inhibits FAS activity in vitro and has a cytotoxic effect on tumor cell lines, without affecting food consumption. (+)-C75 inhibits CPT1 and its administration produces anorexia, suggesting that central inhibition of CPT1 is essential for the anorectic effect of C75. The differential activity of C75 enantiomers may lead to the development of potential new specific drugs for cancer and obesity.

Differential pharmacologic properties of the two C75 enantiomers: (+)-C75 is a strong anorectic drug. (-)-C75 has antitumour activity

C75 is a synthetic compound described as having antitumoral properties. It produces hypophagia and weight loss in rodents, limiting its use in cancer therapy but identify- ing it as a potential anti-obesity drug. C75 is a fatty acid synthase (FAS) inhibitor and, through its coenzyme A (CoA) derivative, it acts as a carnitine palmitoyltransferase (CPT) 1 inhibitor. Racemic mixtures of C75 have been used in all the previous studies; however, the potential dif- ferent biological activities of C75 enantiomers have not been examined yet. To address this question we synthesized the two C75 enantiomers separately. Our results showed that ( )- C75 inhibits FAS activity in vitro and has a cytotoxic effect on tumor cell lines, without affecting food consumption. (+)-C75 inhibits CPT1 and its administration produces anorexia, suggesting that central inhibition of CPT1 is essential for the anorectic effect of C75. The differential activity of C75 enantiomers may lead to the development of potential new specific drugs for cancer and obesity.

Robust Nonlinear Mapping of Soil Contamination Using Support Regression

Spatial data analysis mapping and visualization is of great importance in various fields: environment, pollution, natural hazards and risks, epidemiology, spatial econometrics, etc. A basic task of spatial mapping is to make predictions based on some empirical data (measurements). A number of state-of-the-art methods can be used for the task: deterministic interpolations, methods of geostatistics: the family of kriging estimators (Deutsch and Journel, 1997), machine learning algorithms such as artificial neural networks (ANN) of different architectures, hybrid ANN-geostatistics models (Kanevski and Maignan, 2004; Kanevski et al., 1996), etc. All the methods mentioned above can be used for solving the problem of spatial data mapping. Environmental empirical data are always contaminated/corrupted by noise, and often with noise of unknown nature. That's one of the reasons why deterministic models can be inconsistent, since they treat the measurements as values of some unknown function that should be interpolated. Kriging estimators treat the measurements as the realization of some spatial randomn process. To obtain the estimation with kriging one has to model the spatial structure of the data: spatial correlation function or (semi-)variogram. This task can be complicated if there is not sufficient number of measurements and variogram is sensitive to outliers and extremes. ANN is a powerful tool, but it also suffers from the number of reasons. of a special type ? multiplayer perceptrons ? are often used as a detrending tool in hybrid (ANN+geostatistics) models (Kanevski and Maignank, 2004). Therefore, development and adaptation of the method that would be nonlinear and robust to noise in measurements, would deal with the small empirical datasets and which has solid mathematical background is of great importance. The present paper deals with such model, based on Statistical Learning Theory (SLT) - Support Vector Regression. SLT is a general mathematical framework devoted to the problem of estimation of the dependencies from empirical data (Hastie et al, 2004; Vapnik, 1998). SLT models for classification - Support Vector Machines - have shown good results on different machine learning tasks. The results of SVM classification of spatial data are also promising (Kanevski et al, 2002). The properties of SVM for regression - Support Vector Regression (SVR) are less studied. First results of the application of SVR for spatial mapping of physical quantities were obtained by the authorsin for mapping of medium porosity (Kanevski et al, 1999), and for mapping of radioactively contaminated territories (Kanevski and Canu, 2000). The present paper is devoted to further understanding of the properties of SVR model for spatial data analysis and mapping. Detailed description of the SVR theory can be found in (Cristianini and Shawe-Taylor, 2000; Smola, 1996) and basic equations for the nonlinear modeling are given in section 2. Section 3 discusses the application of SVR for spatial data mapping on the real case study - soil pollution by Cs137 radionuclide. Section 4 discusses the properties of the modelapplied to noised data or data with outliers.

Differential diagnosis of leukocoria and strabismus, first presenting signs of retinoblastoma.

Leukocoria in infants is always a danger signal as retinoblastoma, a malignant retinal tumor, is responsible for half of the cases in this age group. More common signs should also be considered suspicious until proved otherwise, such as strabismus, the second most frequent sign of retinoblastoma. Less frequent manifestations are inflammatory conditions resistant to treatment, hypopyon, orbital cellulitis, hyphema or heterochromia. Other causal pathologies, including persistent hyperplastic primary vitreous (PHPV), Coats' disease, ocular toxocariasis or retinopathy of prematurity, may also manifest the same warning signs and require specialized differential diagnosis. Members of the immediate family circle are most likely to notice the first signs, the general practitioner, pediatrician or general ophthalmologist the first to be consulted. On their attitude will depend the final outcome of this vision and life-threatening disease. Early diagnosis is vital.

Differential gene expression in the pathogenic dermatophyte Arthroderma benhamiae in vitro versus during infection.

Although dermatophytes are the most common agents of superficial mycoses in humans and animals, the molecular basis of the pathogenicity of these fungi is largely unknown. In vitro digestion of keratin by dermatophytes is associated with the secretion of multiple proteases, which are assumed to be responsible for their particular specialization to colonize and degrade keratinized host structures during infection. To investigate the role of individual secreted proteases in dermatophytosis, a guinea pig infection model was established for the zoophilic dermatophyte Arthroderma benhamiae, which causes highly inflammatory cutaneous infections in humans and rodents. By use of a cDNA microarray covering approximately 20-25 % of the A. benhamiae genome and containing sequences of at least 23 protease genes, we revealed a distinct in vivo protease gene expression profile in the fungal cells, which was surprisingly different from the pattern elicited during in vitro growth on keratin. Instead of the major in vitro -expressed proteases, others were activated specifically during infection. These enzymes are therefore suggested to fulfil important functions that are not exclusively associated with the degradation of keratin. Most notably, the gene encoding the serine protease subtilisin 6, which is a known major allergen in the related dermatophyte Trichophyton rubrum and putatively linked to host inflammation, was found to be the most strongly upregulated gene during infection. In addition, our approach identified other candidate pathogenicity-related factors in A. benhamiae, such as genes encoding key enzymes of the glyoxylate cycle and an opsin-related protein. Our work provides what we believe to be the first broad-scale gene expression profile in human pathogenic dermatophytes during infection, and points to putative virulence-associated mechanisms that make these micro-organisms the most successful aetiological agents of superficial mycoses.

Stability and adaptability of runner peanut genotypes based on nonlinear regression and AMMI analysis

The objective of this work was to estimate the stability and adaptability of pod and seed yield in runner peanut genotypes based on the nonlinear regression and AMMI analysis. Yield data from 11 trials, distributed in six environments and three harvests, carried out in the Northeast region of Brazil during the rainy season were used. Significant effects of genotypes (G), environments (E), and GE interactions were detected in the analysis, indicating different behaviors among genotypes in favorable and unfavorable environmental conditions. The genotypes BRS Pérola Branca and LViPE‑06 are more stable and adapted to the semiarid environment, whereas LGoPE‑06 is a promising material for pod production, despite being highly dependent on favorable environments.

Differential miRNA and IncRNA expression in embryonic stem cells lacking the Notch1 receptor

Embryonic stem (ES) cells-derived cardiomyocytes represent an attractive source of cells in cell replacement therapies for heart disease. However, controlled cardiogenic differentiation of ES cells requires a complete understanding of the complex molecular mechanisms regulating the differentiation process. We have previously shown that differentiation of ES cells into cardiomyocytes is favored by inactivation of the Notch 1 receptor pathway. In the present study, we therefore compared two ES cell lines, one with normal Notchl expression and one carrying deleted Notchl receptor alleles (Notchl-deleted ES cells) in order to identify genes responsible for the increased propensity of Notchl-deleted ES cells to produce cardiomyocytes. Using RNA-sequencing, we found approximately 300 coding and noncoding transcripts, which are differently expressed in undifferentiated Notchl-deleted ES cells. Since accumulating evidences indicate that long noncoding RNAs (IncRNAs) play important roles in ES cell pluripotency and differentiation, we focused our analysis on modulated IncRNAs. In particular, two IncRNAs, named here lnc 1230 and lnc 1335, are highly induced in the absence of Notchl receptor expression. These represent therefore prime candidates that could favor cardiogenic commitment in undifferentiated ES cells. Indeed, we demonstrate that forced expression of these two IncRNAs in wild-type ES cells result in a significant increase of the number of cardiac progenitor cells and cardiomyocytes in the differentiated progeny of these ES cells. Furthermore, we also identify several microRNAs that are differentially modulated in absence of Notchl expression. Among these are miR-142-5p and miR- 381-3p. Interestingly, both lncl230 and lncl335 are targets of these two microRNAs. Altogether, these data suggest that Notchl-dependent noncoding gene networks, implicating microRNAs and IncRNAs, control embryonic stem cell commitment into the mesodermal and cardiac lineages already at the undifferentiated state. - Les cardiomyocytes issus cellules souches embryonnaires sont une source très prometteuse pour les thérapies cellulaire de remplacement dans le cadre des maladies cardiaques. Cependant, l'utilisation de telles cellules requiert une compréhension poussée des mécanismes moléculaire régulant la différenciation. Nous avons par le passé démontré que la différenciation des cellules souches embryonnaires en cardiomyocytes est favorisée par l'inactivation de la voie d'activation intracellulaire dépendante du récepteur Notch 1. Nous avons donc comparé deux lignées de cellules souches embryonnaires, une présentant une voie d'activation Notchl normale et une chez laquelle les allèles codant pour le récepteur Notchl avaient été invalidés, de façon à identifier les gènes impliqués dans la capacité augmentée des cellules déficientes à produire des cardiomyocytes. En utilisant du séquençage d'ARN à haut débit, nous avons trouvé environ 300 gènes différemment exprimés dans les cellules déficientes pour Notchl. Par ailleurs, des évidences de plus en plus nombreuses suggèrent qu'une nouvelle classe de molécules appelée « long noncoding RNAs » joue un rôle prépondérant dans la maintenance de l'état non différencié et de la capacité de différenciation des cellules souches embryonnaires. Nous avons trouvé que plusieurs « long noncoding RNAs » étaient modulés en l'absence de Notchl, et en particulier deux molécules que nous avons appelées lncl230 et lncl335. Ces derniers représentent des candidats potentiels devant permettre de favoriser la production de cardiomyocytes. Nous avons en effet démontré que la surexpression de ces deux candidats dans des cellules souches embryonnaires résultait en une surproduction de cardiomyocytes. De plus, nous avons également identifié plusieurs microRNAs dont l'expression était modulée dans les cellules souches embryonnaires déficientes dans la voie Notchl. De façon intéressante, parmi ces microRNAs, le miR-142-5p et le miR-381-3p sont capables de cibler lncl230 and lncl335. Dans l'ensemble, ces résultats indiquent donc que des réseaux d'interaction dépendant de la voie d'activation Notch 1 et impliquant des ARNs non codant existent dans les cellules souches embryonnaires pour réguler leur différenciation en différent types cellulaires spécifiques.

Chaotic systems with a null conditional Lyapunov exponent under nonlinear driving.

Control of a chaotic system by homogeneous nonlinear driving, when a conditional Lyapunov exponent is zero, may give rise to special and interesting synchronizationlike behaviors in which the response evolves in perfect correlation with the drive. Among them, there are the amplification of the drive attractor and the shift of it to a different region of phase space. In this paper, these synchronizationlike behaviors are discussed, and demonstrated by computer simulation of the Lorentz model [E. N. Lorenz, J. Atmos. Sci. 20 130 (1963)] and the double scroll [T. Matsumoto, L. O. Chua, and M. Komuro, IEEE Trans. CAS CAS-32, 798 (1985)].

Linear and nonlinear terrain deformation maps from a reduced set of interferometric SAR images

In this paper, an advanced technique for the generation of deformation maps using synthetic aperture radar (SAR) data is presented. The algorithm estimates the linear and nonlinear components of the displacement, the error of the digital elevation model (DEM) used to cancel the topographic terms, and the atmospheric artifacts from a reduced set of low spatial resolution interferograms. The pixel candidates are selected from those presenting a good coherence level in the whole set of interferograms and the resulting nonuniform mesh tessellated with the Delauney triangulation to establish connections among them. The linear component of movement and DEM error are estimated adjusting a linear model to the data only on the connections. Later on, this information, once unwrapped to retrieve the absolute values, is used to calculate the nonlinear component of movement and atmospheric artifacts with alternate filtering techniques in both the temporal and spatial domains. The method presents high flexibility with respect to the required number of images and the baselines length. However, better results are obtained with large datasets of short baseline interferograms. The technique has been tested with European Remote Sensing SAR data from an area of Catalonia (Spain) and validated with on-field precise leveling measurements.

General electromagnetic simulation tool to predict the microwave nonlinear response of planar, arbitrarily-shaped HTS structures

This work describes a simulation tool being developed at UPC to predict the microwave nonlinear behavior of planar superconducting structures with very few restrictions on the geometry of the planar layout. The software is intended to be applicable to most structures used in planar HTS circuits, including line, patch, and quasi-lumped microstrip resonators. The tool combines Method of Moments (MoM) algorithms for general electromagnetic simulation with Harmonic Balance algorithms to take into account the nonlinearities in the HTS material. The Method of Moments code is based on discretization of the Electric Field Integral Equation in Rao, Wilton and Glisson Basis Functions. The multilayer dyadic Green's function is used with Sommerfeld integral formulation. The Harmonic Balance algorithm has been adapted to this application where the nonlinearity is distributed and where compatibility with the MoM algorithm is required. Tests of the algorithm in TM010 disk resonators agree with closed-form equations for both the fundamental and third-order intermodulation currents. Simulations of hairpin resonators show good qualitative agreement with previously published results, but it is found that a finer meshing would be necessary to get correct quantitative results. Possible improvements are suggested.