Miguel Angel Garcia

Allelic polymorphisms in the transcriptional regulatory region of apolipoprotein E gene

In this work, we explored the existence of genetic variants within the apolipoprotein E gene transcriptional regulatory region, using a denaturing gradient gel electrophoresis screening of a region comprising nucleotides −1017 to +406. Upon a population study, three new polymorphic sites (−491, −427 and −219) and two mutations were found. Functional effects of the polymorphisms, assayed by transient transfection and electrophoretic mobility shift assays in a human hepatoma cell line, showed that polymorphisms at sites −491 and −219 of the APOE promoter produce variations in the transcriptional activity of the gene, most probably through differential binding of nuclear proteins.

Subnanometer Local Temperature Probing and Remotely Controlled Drug Release Based on Azo-Functionalized Iron Oxide Nanoparticles

Local heating can be produced by iron oxide nanoparticles (IONPs) when exposed to an alternating magnetic field (AMF). To measure the temperature profile at the nanoparticle surface with a subnanometer resolution, here we present a molecular temperature probe based on the thermal decomposition of a thermo-sensitive molecule, namely, azobis[N-(2-carboxyethyl)-2-methylpropionamidine]. Fluoresceineamine (FA) was bound to the azo molecule at the IONP surface functionalized with poly(ethylene glycol) (PEG) spacers of different molecular weights. Significant local heating, with a temperature increase up to 45 °C, was found at distances below 0.5 nm from the surface of the nanoparticle, which decays exponentially with increasing distance. Furthermore, the temperature increase was found to scale linearly with the applied field at all distances. We implemented these findings in an AMF-triggered drug release system in which doxorubicin was covalently linked at different distances from the IONP surface bearing the same thermo-labile azo molecule. We demonstrated the AMF triggered distance-dependent release of the drug in a cytotoxicity assay on KB cancer cells.

Analysis of focus measure operators for shape-from-focus

Shape-from-focus (SFF) has widely been studied in computer vision as a passive depth recovery and 3D reconstruction method. One of the main stages in SFF is the computation of the focus level for every pixel of an image by means of a focus measure operator. In this work, a methodology to compare the performance of different focus measure operators for shape-from-focus is presented and applied. The selected operators have been chosen from an extensive review of the state-of-the-art. The performance of the different operators has been assessed through experiments carried out under different conditions, such as image noise level, contrast, saturation and window size. Such performance is discussed in terms of the working principles of the analyzed operators.

Population structure of mycobionts and photobionts of the widespread lichen Cetraria aculeata.

Lichens are symbioses between fungi (mycobionts) and photoautotrophic green algae or cyanobacteria (photobionts). Many lichens occupy large distributional ranges covering several climatic zones. So far, little is known about the large‐scale phylogeography of lichen photobionts and their role in shaping the distributional ranges of lichens. We studied south polar, temperate and north polar populations of the widely distributed fruticose lichen Cetraria aculeata. Based on the DNA sequences from three loci for each symbiont, we compared the genetic structure of mycobionts and photobionts. Phylogenetic reconstructions and Bayesian clustering methods divided the mycobiont and photobiont data sets into three groups. An amova shows that the genetic variance of the photobiont is best explained by differentiation between temperate and polar regions and that of the mycobiont by an interaction of climatic and geographical factors. By partialling out the relative contribution of climate, geography and codispersal, we found that the most relevant factors shaping the genetic structure of the photobiont are climate and a history of codispersal. Mycobionts in the temperate region are consistently associated with a specific photobiont lineage. We therefore conclude that a photobiont switch in the past enabled C. aculeata to colonize temperate as well as polar habitats. Rare photobiont switches may increase the geographical range and ecological niche of lichen mycobionts by associating them with locally adapted photobionts in climatically different regions and, together with isolation by distance, may lead to genetic isolation between populations and thus drive the evolution of lichens.

Fission yeast ch‐TOG/XMAP215 homologue Alp14 connects mitotic spindles with the kinetochore and is a component of the Mad2‐dependent spindle checkpoint

The TOG/XMAP215‐related proteins play a role in microtubule dynamics at its plus end. Fission yeast Alp14, a newly identified TOG/XMAP215 family protein, is essential for proper chromosome segregation in concert with a second homologue Dis1. We show that the alp14 mutant fails to progress towards normal bipolar spindle formation. Intriguingly, Alp14 itself is a component of the Mad2‐dependent spindle checkpoint cascade, as upon addition of microtubule‐destabilizing drugs the alp14 mutant is incapable of maintaining high H1 kinase activity, which results in securin destruction and premature chromosome separation. Live imaging of Alp14–green fluorescent protein shows that during mitosis, Alp14 is associated with the peripheral region of the kinetochores as well as with the spindle poles. This is supported by ChIP (chromatin immunoprecipitation) and overlapping localization with the kinetochore marker Mis6. An intact spindle is required for Alp14 localization to the kinetochore periphery, but not to the poles. These results indicate that the TOG/XMAP215 family may play a central role as a bridge between the kinetochores and the plus end of pole to chromosome microtubules.

Architectural Control of Seeded-Grown Magnetic−Semicondutor Iron Oxide−TiO2 Nanorod Heterostructures: The Role of Seeds in Topology Selection

A colloidal nonaqueous approach to semiconductor-magnetic hybrid nanocrystals (HNCs) with selectable heterodimer topologies and tunable geometric parameters is demonstrated. Brookite TiO(2) nanorods, distinguished by a curved shape-tapered profile with richly faceted terminations, are exploited as substrate seeds onto which a single spherical domain of inverse spinel iron oxide can be epitaxially grown at either one apex or any location along their longitudinal sidewalls in a hot surfactant environment. The topologically controlled arrangement of the component material lattices, the crystallographic relationships holding between them, and strain distribution across individual heterostructures have been studied by combining X-ray diffraction and absorption techniques with high-resolution transmission electron microscopy investigations. Supported by such structural knowledge, the synthetic achievements are interpreted within the frame of various mechanistic models offering complementary views of HNC formation. The different HNC architectures are concluded to be almost equivalent in terms of surface-interface energy balance associated with their formation. HNC topology selection is rationalized on the basis of a diffusion-limited mechanism allowing iron oxide heterogeneous nucleation and growth on the TiO(2) nanorods to switch from a thermodynamically controlled to a kinetically overdriven deposition regime, in which the anisotropic reactivity offered by the uniquely structured seeds is accentuated under high spatially inhomogeneous monomer fluxes. Finally, the multifunctional capabilities of the heterostructures are highlighted through illustration of their magnetic and photocatalytic properties, which have been found to diverge from those otherwise exhibited by their individual material components and physical mixture counterparts.

Spindle–kinetochore attachment requires the combined action of Kin I-like Klp5/6 and Alp14/Dis1-MAPs in fission yeast

Fission yeast Klp5 and Klp6 belong to the microtubule‐destabilizing Kin I family. In klp5 mutants, spindle checkpoint proteins Mad2 and Bub1 are recruited to mitotic kinetochores for a prolonged duration, indicating that these kinetochores are unattached. Further analysis shows that there are kinetochores to which only Bub1, but not Mad2, localizes. These kinetochores are likely to have been captured, yet lack tension. Thus Klp5 and Klp6 play a role in a spindle–kinetochore interaction at dual steps, capture and generation of tension. The TOG/XMAP215 family, Alp14 and Dis1 are known to stabilize microtubules and be required for the bivalent attachment of the kinetochore to the spindle. Despite apparent opposing activities towards microtubule stability, Klp5/Klp6 and Alp14/Dis1 share an essential function, as either dis1klp or alp14klp mutants are synthetically lethal, like alp14dis1. Defective phenotypes are similar to each other, characteristic of attachment defects and chromosome mis‐segregation. Furthermore Alp14 is of significance for kinetochore localization of Klp5. We propose that Klp5/Klp6 and Alp14/Dis1 play a collaborative role in bipolar spindle formation during prometaphase through producing spindle dynamism.

Phylogenetic relationships among plant and animal parasites, and saprotrophs in Aphanomyces (Oomycetes)

Molecular phylogenetic relationships among 12 species of Aphanomyces de Bary (Oomycetes) were analyzed based on 108 ITS sequences of nuclear rDNA. Sequences used in the analyses belonged to the major species currently available in pure culture and GenBank. Bayesian, maximum likelihood, and maximum parsimony analyses support that Aphanomyces constitutes a monophyletic group. Three independent lineages were found: (i) plant parasitic, (ii) animal parasitic, and (iii) saprotrophic or opportunistic parasitic. Sexual reproduction appeared to be critical in plant parasites for survival in soil environments while asexual reproduction seemed to be advantageous for exploiting specialization in animal parasitism. Repeated zoospore emergence seems to be an advantageous property for both plant and animal parasitic modes of life. Growth in unspecific media was generally faster in saprotrophs compared with parasitic species. A number of strains and GenBank sequences were found to be misidentified. It was confirmed molecularly that Aphanomyces piscicida and Aphanomyces invadans appear to be conspecific, and found that Aphanomyces iridis and Aphanomyces euteiches are closely related, if not the same, species. This study has shown a clear evolutionary separation between Aphanomyces species that are plant parasites and those that parasitize animals. Saprotrophic or opportunistic species formed a separate evolutionary lineage except Aphanomyces stellatus whose evolutionary position has not yet been resolved.

Generation of All-in-Focus Images by Noise-Robust Selective Fusion of Limited Depth-of-Field Images

The limited depth-of-field of some cameras prevents them from capturing perfectly focused images when the imaged scene covers a large distance range. In order to compensate for this problem, image fusion has been exploited for combining images captured with different camera settings, thus yielding a higher quality all-in-focus image. Since most current approaches for image fusion rely on maximizing the spatial frequency of the composed image, the fusion process is sensitive to noise. In this paper, a new algorithm for computing the all-in-focus image from a sequence of images captured with a low depth-of-field camera is presented. The proposed approach adaptively fuses the different frames of the focus sequence in order to reduce noise while preserving image features. The algorithm consists of three stages: 1) focus measure; 2) selectivity measure; 3) and image fusion. An extensive set of experimental tests has been carried out in order to compare the proposed algorithm with state-of-the-art all-in-focus methods using both synthetic and real sequences. The obtained results show the advantages of the proposed scheme even for high levels of noise.

Transcription factor AP‐2 activity is modulated by protein kinase A‐mediated phosphorylation

We recently reported that APOE promoter activity is stimulated by cAMP, this effect being mediated by factor AP‐2 [Garcı́a et al. (1996) J. Neurosci. 16, 7550–7556]. Here, we study whether cAMP‐induced phosphorylation modulates the activity of AP‐2. Recombinant AP‐2 was phosphorylated in vitro by protein kinase A (PKA) at Ser239. Mutation of Ser239 to Ala abolished in vitro phosphorylation of AP‐2 by PKA, but not the DNA binding activity of AP‐2. Cotransfection studies showed that PKA stimulated the effect of AP‐2 on the APOE promoter, but not that of the S239A mutant. Therefore, cAMP may modulate AP‐2 activity by PKA‐induced phosphorylation of this factor.