Beatriz García

Versatile Nanostructured Materials via Direct Reaction of Functionalized Catechols

A facile one-step polymerization strategy is explored to achieve novel catechol-based materials. Depending on the functionality of the catechol, the as-prepared product can be used to modify at will the surface tension of nano and bulk structures, from oleo-/hydrophobic to highly hydrophilic. A hydrophobic catechol prepared thus polymerized shows the ability to self-assemble as solid nanoparticles with sticky properties in polar solvent media. Such a versatile concept is ideal for the development of catechol-based multifunctional materials.

Human TRIB2 is a repressor of FOXO that contributes to the malignant phenotype of melanoma cells

FOXO transcription factors are evolutionarily conserved proteins that orchestrate gene expression programs known to control a variety of cellular processes such as cell cycle, apoptosis, DNA repair and protection from oxidative stress. As the abrogation of FOXO function is a key feature of many tumor cells, regulation of FOXO factors is receiving increasing attention in cancer research. In order to discover genes involved in the regulation of FOXO activity, we performed a large-scale RNA-mediated interference (RNAi) screen using cell-based reporter systems that monitor transcriptional activity and subcellular localization of FOXO. We identified genes previously implicated in phosphoinositide 3-kinase/Akt signaling events, which are known to be important for FOXO function. In addition, we discovered a previously unrecognized FOXO-repressor function of TRIB2, the mammalian homolog of the Drosophila gene tribbles. A cancer-profiling array revealed specific overexpression of TRIB2 in malignant melanoma, but not in other types of skin cancer. We provide experimental evidence that TRIB2 transcript levels correlate with the degree of cytoplasmic localization of FOXO3a. Moreover, we show that TRIB2 is important in the maintenance of the oncogenic properties of melanoma cells, as its silencing reduces cell proliferation, colony formation and wound healing. Tumor growth was also substantially reduced upon RNAi-mediated TRIB2 knockdown in an in vivo melanoma xenograft model. Our studies suggest that TRIB2 provides the melanoma cells with growth and survival advantages through the abrogation of FOXO function. Altogether, our results show the potential of large-scale cell-based RNAi screens to identify promising diagnostic markers and therapeutic targets.

Chemical Genetic Analysis of FOXO Nuclear–Cytoplasmic Shuttling by Using Image-Based Cell Screening

FOXO proteins are direct targets of PI3K/Akt signaling and they integrate the signals of several other transduction pathways at the transcriptional level. FOXO transcription factors are involved in normal cell homeostasis and neoplasia, and they are regulated by multiple post‐transcriptional modifications. In cancer research, the regulation of the FOXO factors is receiving increasing attention as their activation has been linked to cell‐cycle arrest and apoptosis. Hence, FOXO proteins have been proposed to act as tumor suppressors. Here, we applied a chemical biology approach to study the mechanisms that influence the intracellular localization of the FOXO family member FOXO3a. We established a high‐throughput cellular‐imaging assay that monitors the nuclear–cytoplasmic translocation of a GFP–FOXO3a fusion protein in tumor cells. Nuclear accumulation of fluorescent signals upon treatment with the known PI3K inhibitors LY294002, wortmannin, PIK‐75, and PI‐103 was dose dependent and agreed well with the IC50 values reported for PI3Kα inhibition in vitro. Additionally, we identified 17 compounds from a panel of 73 low‐molecular‐weight compounds capable of inducing the nuclear accumulation of GFP–FOXO. These compounds include chemicals known to interfere with components of the PI3K/Akt signaling pathway, as well as with nuclear export and Ca2+/calmodulin (CaM)‐dependent signaling events. Interestingly, the therapeutic agent vinblastine induced efficient nuclear translocation of the FOXO reporter protein. Our data illustrate the potential of chemical genetics when combined with robust and sensitive high‐content‐screening technology.

Using multiplexed regulation of luciferase activity and GFP translocation to screen for FOXO modulators

BackgroundIndependent luciferase reporter assays and fluorescent translocation assays have been successfully used in drug discovery for several molecular targets. We developed U2transLUC, an assay system in which luciferase and fluorescent read-outs can be multiplexed to provide a powerful cell-based high content screening method.ResultsThe U2transLUC system is based on a stable cell line expressing a GFP-tagged FOXO transcription factor and a luciferase reporter gene under the control of human FOXO-responsive enhancers. The U2transLUC assay measures nuclear-cytoplasmic FOXO shuttling and FOXO-driven transcription, providing a means to analyze these two key features of FOXO regulation in the same experiment. We challenged the U2transLUC system with chemical probes with known biological activities and we were able to identify compounds with translocation and/or transactivation capacity.ConclusionCombining different biological read-outs in a single cell line offers significant advantages over conventional cell-based assays. The U2transLUC assay facilitates the maintenance and monitoring of homogeneous FOXO transcription factor expression and allows the reporter gene activity measured to be normalized with respect to cell viability. U2transLUC is suitable for high throughput screening and can identify small molecules that interfere with FOXO signaling at different levels.

Mussel-Inspired Hydrophobic Coatings for Water-Repellent Textiles and Oil Removal

A series of catechol derivatives with a different number of linear alkyl chain substituents, and different length, have been shown to polymerize in the presence of aqueous ammonia and air, yielding hydrophobic coatings that present the ability to provide robust and efficient water repellency on weaved textiles, including hydrophilic cotton. The polymerization strategy presented exemplifies an alternative route to established melanin- and polydopamine-like functional coatings, affording designs in which all catechol (adhesive) moieties support specific functional side chains for maximization of the desired (hydrophobic) functionality. The coatings obtained proved effective in the transformation of polyester and cotton weaves, as well as filter paper, into reusable water-repellent, oil-absorbent materials capable of retaining roughly double their weight in model compounds (n-tetradecane and olive oil), as well as of separating water/oil mixtures by simple filtration.

Use of virus vectors for the expression in plants of active full-length and single chain anti-coronavirus antibodies

Abstract To extend the potential of antibodies and their derivatives to provide passive protection against enteric infections when supplied orally in crude plant extracts, we have expressed both a small immune protein (SIP) and a full‐length antibody in plants using two different plant virus vectors based on potato virus X (PVX) and cowpea mosaic virus (CPMV). The agr;SIP molecule consisted of a single chain antibody (scFv) specific for the porcine coronavirus, transmissible gastroenteritis virus (TGEV) linked to the α‐CH3 domain from human IgA. To express the full‐length IgA, the individual light and heavy chains from the TGEV‐specific mAb 6A.C3 were inserted into separate PVX constructs and plants were co‐infected with both constructs. Western blot analysis revealed the efficient expression of both the SIP and IgA molecules. Analysis of crude plant extracts revealed that both the plant‐expressed αSIP and IgA molecules could bind to and neutralize TGEV in tissue culture, indicating that active molecules were produced. Oral administration of crude extracts from antibody‐expressing plant tissue to 2‐day‐old piglets showed that both the αSIP and full‐length IgA molecules can provide in vivo protection against TGEV.

A Dual-Color Fluorescence-Based Platform to Identify Selective Inhibitors of Akt Signaling

Background Inhibition of Akt signaling is considered one of the most promising therapeutic strategies for many cancers. However, rational target-orientated approaches to cell based drug screens for anti-cancer agents have historically been compromised by the notorious absence of suitable control cells. Methodology/Principal Findings In order to address this fundamental problem, we have developed BaFiso, a live-cell screening platform to identify specific inhibitors of this pathway. BaFiso relies on the co-culture of isogenic cell lines that have been engineered to sustain interleukin-3 independent survival of the parental Ba/F3 cells, and that are individually tagged with different fluorescent proteins. Whilst in the first of these two lines cell survival in the absence of IL-3 is dependent on the expression of activated Akt, the cells expressing constitutively-activated Stat5 signaling display IL-3 independent growth and survival in an Akt-independent manner. Small molecules can then be screened in these lines to identify inhibitors that rescue IL-3 dependence. Conclusions/Significance BaFiso measures differential cell survival using multiparametric live cell imaging and permits selective inhibitors of Akt signaling to be identified. BaFiso is a platform technology suitable for the identification of small molecule inhibitors of IL-3 mediated survival signaling.

Biocompatible polydopamine-like particles for the removal of heavy metals at extremely low concentrations

A family of catechol-based submicron particles, with sizes between 200 and 300 nm, was tested for the removal of Cd(II), Pb(II) and Cr(VI) in water. The highest adsorption capacity was obtained with catecholbased particles in the case of Pb(II), followed by Cd(II). However, the catechol particles failed to adsorb Cr(VI). Our results indicate an up to four-fold increase of the adsorption capacity of these particles compared to that of activated carbon under the same experimental conditions. To check the biocompatible character of the submicron particles, their stability was evaluated in a phosphate buffer solution (PBS) and in a cell culture medium. The results confirmed that the presence of proteins in the medium favors their stability. A bioluminescent Vibrio fischeri test and a cytotoxicity assay on the HepG2 cell line were used to determine that the catechol particles did not exhibit any substantial toxicity. The results show that these catechol-based particles can be used as an efficient biocompatible adsorbent to remove heavy metals at extremely low concentrations.

The Effect of Cellular Differentiation on HSV-1 Infection of Oligodendrocytic Cells

Herpes simplex type 1 (HSV-1) is a neurotropic virus that infects many types of cells. Previous studies have demonstrated that oligodendrocytic cells are highly susceptible to HSV-1 infection. Here we analysed HSV-1 infection of a human oligodendrocytic cell line, HOG, and oligodendrocyte precursor cells (OPCs) cultured under growth or differentiation conditions. In addition to cell susceptibility, the role of the major cell receptors for viral entry was assessed. Our results revealed that OPCs and HOG cells cultured under differentiation conditions became more susceptible to HSV-1. On the other hand, viral infection induced morphological changes corresponding to differentiated cells, suggesting that HSV-1 might be inducing cell differentiation. We also observed colocalization of HVEM and nectin-1 with viral particles, suggesting that these two major HSV-1 receptors are functional in HOG cells. Finally, electron microscopy assays indicated that HSV-1 may be also entering OLs by macropinocytosis depending on their differentiation stage. In addition, vesicles containing intracellular enveloped virions observed in differentiated cells point to an endocytic mechanism of virus entry. All these data are indicative of diverse entry pathways dependent on the maturation stage of OLs.

Characterizing postural oscillation in children and adolescents with hereditary sensorimotor neuropathy

Charcot Marie Tooth disease (CMT) has negative functional impact on postural control of children; however, it has not been widely studied. Stabilometry can provide insights about postural control and guide preventive interventions in immature perceptual and musculoskeletal systems as those seen in children with CMT. This cross-sectional study aimed to identify and interpret stabilometric variables that reflect the postural control of children with CMT. 53 subjects (age 6–17) were assigned to one of the two groups: CMT (15 males and 14 females with CMT) or Control (13 males and 11 females healthy). Quiet standing was tested in different conditions: with open and closed eyes on regular surface (open-regular, closed-regular) and foam surface (open-foam, closed-foam) using a force platform. The minimum of 2 and maximum of 3 trials of 30 seconds for each test condition provided the classical stabilometric variables and Romberg Quotient (RQv). CMT group showed increase of confidence ellipse area, mean velocity, mediolateral and anteroposterior velocities associated with decreased mean body oscillation frequency, as the complexity of tasks increased. CMT postural deficit was identified by greater and faster sway associated with these lower frequencies, when compared to Control.