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1.  Caveolin-1 is required for TGF-β-induced transactivation of the EGF receptor pathway in hepatocytes through the activation of the metalloprotease TACE/ADAM17 
Cell Death & Disease  2014;5(7):e1326-.
Transforming growth factor-beta (TGF-β) plays a dual role in hepatocytes, inducing both pro- and anti-apoptotic responses, whose balance decides cell fate. Survival signals are mediated by the epidermal growth factor receptor (EGFR) pathway, which is activated by TGF-β in these cells. Caveolin-1 (Cav1) is a structural protein of caveolae linked to TGF-β receptors trafficking and signaling. Previous results have indicated that in hepatocytes, Cav1 is required for TGF-β-induced anti-apoptotic signals, but the molecular mechanism is not fully understood yet. In this work, we show that immortalized Cav1−/− hepatocytes were more sensitive to the pro-apoptotic effects induced by TGF-β, showing a higher activation of caspase-3, higher decrease in cell viability and prolonged increase through time of intracellular reactive oxygen species (ROS). These results were coincident with attenuation of TGF-β-induced survival signals in Cav1−/− hepatocytes, such as AKT and ERK1/2 phosphorylation and NFκ-B activation. Transactivation of the EGFR pathway by TGF-β was impaired in Cav1−/− hepatocytes, which correlated with lack of activation of TACE/ADAM17, the metalloprotease responsible for the shedding of EGFR ligands. Reconstitution of Cav1 in Cav1−/− hepatocytes rescued wild-type phenotype features, both in terms of EGFR transactivation and TACE/ADAM17 activation. TACE/ADAM17 was localized in detergent-resistant membrane (DRM) fractions in Cav1+/+ cells, which was not the case in Cav1−/− cells. Disorganization of lipid rafts after treatment with cholesterol-binding agents caused loss of TACE/ADAM17 activation after TGF-β treatment. In conclusion, in hepatocytes, Cav1 is required for TGF-β-mediated activation of the metalloprotease TACE/ADAM17 that is responsible for shedding of EGFR ligands and activation of the EGFR pathway, which counteracts the TGF-β pro-apoptotic effects. Therefore, Cav1 contributes to the pro-tumorigenic effects of TGF-β in liver cancer cells.
doi:10.1038/cddis.2014.294
PMCID: PMC4123087  PMID: 25032849
2.  Oxidative stress and its downstream signaling in aging eyes 
Background
Oxidative stress (OS) and its biomarkers are the biochemical end point of the imbalance between reactive oxygen species (ROS) production and the ability of the antioxidant (AOX) biological systems to fight against oxidative injury.
Objective
We reviewed the role of OS and its downstream signaling in aging eyes.
Methods
A search of the literature and current knowledge on the physiological and pathological mechanisms of OS were revisited in relation to the eyes and the aging process. Most prevalent ocular diseases have been analyzed herein in relation to OS and nutraceutic supplements, such as dry-eye disorders, glaucoma, age-related macular degeneration, and diabetic retinopathy.
Results
Clinical, biochemical, and molecular data from anterior and posterior eye segment diseases point to OS as the common pathogenic mechanism in the majority of these ocular disorders, many of which are pathologies causing visual impairment, blindness, and subsequent loss of life quality. Studies with nutraceutic supplements in aging eye-related pathologies have also been reviewed.
Conclusion
OS, nutritional status, and nutraceutic supplements have to be considered within the standards of care of older ophthalmologic patients. OS biomarkers and surrogate end points may help in managing the aging population with ocular diseases.
doi:10.2147/CIA.S52662
PMCID: PMC3990383  PMID: 24748782
oxidative stress; eyes; aging; ophthalmic diseases; antioxidants; epidemiology
3.  Label-free magnetic resonance imaging to locate live cells in three-dimensional porous scaffolds 
Porous scaffolds are widely tested materials used for various purposes in tissue engineering. A critical feature of a porous scaffold is its ability to allow cell migration and growth on its inner surface. Up to now, there has not been a method to locate live cells deep inside a material, or in an entire structure, using real-time imaging and a non-destructive technique. Herein, we seek to demonstrate the feasibility of the magnetic resonance imaging (MRI) technique as a method to detect and locate in vitro non-labelled live cells in an entire porous material. Our results show that the use of optimized MRI parameters (4.7 T; repetition time = 3000 ms; echo time = 20 ms; resolution 39 × 39 µm) makes it possible to obtain images of the scaffold structure and to locate live non-labelled cells in the entire material, with a signal intensity higher than that obtained in the culture medium. In the current study, cells are visualized and located in different kinds of porous scaffolds. Moreover, further development of this MRI method might be useful in several three-dimensional biomaterial tests such as cell distribution studies, routine qualitative testing methods and in situ monitoring of cells inside scaffolds.
doi:10.1098/rsif.2012.0068
PMCID: PMC3405747  PMID: 22442095
magnetic resonance imaging; scaffolds; cell culture; in vitro
4.  Production of engineered long-life and male sterile Pelargonium plants 
BMC Plant Biology  2012;12:156.
Background
Pelargonium is one of the most popular garden plants in the world. Moreover, it has a considerable economic importance in the ornamental plant market. Conventional cross-breeding strategies have generated a range of cultivars with excellent traits. However, gene transfer via Agrobacterium tumefaciens could be a helpful tool to further improve Pelargonium by enabling the introduction of new genes/traits. We report a simple and reliable protocol for the genetic transformation of Pelargonium spp. and the production of engineered long-life and male sterile Pelargonium zonale plants, using the pSAG12::ipt and PsEND1::barnase chimaeric genes respectively.
Results
The pSAG12::ipt transgenic plants showed delayed leaf senescence, increased branching and reduced internodal length, as compared to control plants. Leaves and flowers of the pSAG12::ipt plants were reduced in size and displayed a more intense coloration. In the transgenic lines carrying the PsEND1::barnase construct no pollen grains were observed in the modified anther structures, which developed instead of normal anthers. The locules of sterile anthers collapsed 3–4 days prior to floral anthesis and, in most cases, the undeveloped anther tissues underwent necrosis.
Conclusion
The chimaeric construct pSAG12::ipt can be useful in Pelargonium spp. to delay the senescence process and to modify plant architecture. In addition, the use of engineered male sterile plants would be especially useful to produce environmentally friendly transgenic plants carrying new traits by preventing gene flow between the genetically modified ornamentals and related plant species. These characteristics could be of interest, from a commercial point of view, both for pelargonium producers and consumers.
doi:10.1186/1471-2229-12-156
PMCID: PMC3492168  PMID: 22935247
Pelargonium zonale; Pelargonium peltatum; pSAG12 promoter; ipt gene; Engineered long-lived plants; Delayed senescence; Engineered male sterility; PsEND1 promoter; Barnase; Anther ablation; Biosafe ornamentals
5.  Biological Properties of Solid Free Form Designed Ceramic Scaffolds with BMP-2: In Vitro and In Vivo Evaluation 
PLoS ONE  2012;7(3):e34117.
Porous ceramic scaffolds are widely studied in the tissue engineering field due to their potential in medical applications as bone substitutes or as bone-filling materials. Solid free form (SFF) fabrication methods allow fabrication of ceramic scaffolds with fully controlled pore architecture, which opens new perspectives in bone tissue regeneration materials. However, little experimentation has been performed about real biological properties and possible applications of SFF designed 3D ceramic scaffolds. Thus, here the biological properties of a specific SFF scaffold are evaluated first, both in vitro and in vivo, and later scaffolds are also implanted in pig maxillary defect, which is a model for a possible application in maxillofacial surgery. In vitro results show good biocompatibility of the scaffolds, promoting cell ingrowth. In vivo results indicate that material on its own conducts surrounding tissue and allow cell ingrowth, thanks to the designed pore size. Additional osteoinductive properties were obtained with BMP-2, which was loaded on scaffolds, and optimal bone formation was observed in pig implantation model. Collectively, data show that SFF scaffolds have real application possibilities for bone tissue engineering purposes, with the main advantage of being fully customizable 3D structures.
doi:10.1371/journal.pone.0034117
PMCID: PMC3314706  PMID: 22470527
6.  An insertional mutagenesis programme with an enhancer trap for the identification and tagging of genes involved in abiotic stress tolerance in the tomato wild-related species Solanum pennellii 
Plant Cell Reports  2011;30(10):1865-1879.
Salinity and drought have a huge impact on agriculture since there are few areas free of these abiotic stresses and the problem continues to increase. In tomato, the most important horticultural crop worldwide, there are accessions of wild-related species with a high degree of tolerance to salinity and drought. Thus, the finding of insertional mutants with other tolerance levels could lead to the identification and tagging of key genes responsible for abiotic stress tolerance. To this end, we are performing an insertional mutagenesis programme with an enhancer trap in the tomato wild-related species Solanum pennellii. First, we developed an efficient transformation method which has allowed us to generate more than 2,000 T-DNA lines. Next, the collection of S. pennelli T0 lines has been screened in saline or drought conditions and several presumptive mutants have been selected for their salt and drought sensitivity. Moreover, T-DNA lines with expression of the reporter uidA gene in specific organs, such as vascular bundles, trichomes and stomata, which may play key roles in processes related to abiotic stress tolerance, have been identified. Finally, the growth of T-DNA lines in control conditions allowed us the identification of different development mutants. Taking into account that progenies from the lines are being obtained and that the collection of T-DNA lines is going to enlarge progressively due to the high transformation efficiency achieved, there are great possibilities for identifying key genes involved in different tolerance mechanisms to salinity and drought.
doi:10.1007/s00299-011-1094-y
PMCID: PMC3172414  PMID: 21647638
Insertional mutagenesis; Solanum pennellii; Enhancer trap; Salinity; Drought; Gene tagging
7.  Association between a SLC23A2 gene variation, plasma vitamin C levels, and risk of glaucoma in a Mediterranean population 
Molecular Vision  2011;17:2997-3004.
Purpose
Several dietary factors have been associated with glaucoma. Among them, dietary antioxidant intake (i.e., vitamin C and vitamin A) in association with glaucoma has been analyzed, but with mixed results. Genetic factors may play a role in modulating the effect of dietary antioxidant intake on glaucoma; however, nutrigenetic studies in this field are scarce. Our aim was to study the association between selected polymorphisms in key proteins related to vitamin C and vitamin A concentrations and primary open-angle glaucoma (POAG).
Methods
We performed a case-control study matched for age, sex, and bodyweight. We recruited 300 subjects (150 POAG cases and 150 controls) from a Mediterranean population and determined the plasma concentrations of vitamin C and vitamin A for each subject. We selected the following single-nucleotide polymorphisms (SNPs) in genes related to vitamin A and vitamin C concentrations: rs176990 and rs190910 in the retinol-binding protein 1 (RBP1) gene; and rs10063949 and rs1279683 in the Na+-dependent L-ascorbic acid transporters 1 and 2, respectively (encoded by the SLC23A1 and SLC23A2 genes).
Results
We found a statistically significant association between the rs1279386 (A>G) SNP in SLC23A2 and POAG risk. In the crude analysis, homozygous subjects for the G allele (GG subjects) had higher risk of POAG than other genotypes (OR: 1.67; 95% CI: 1.03–2.71). This association remained statistically significant (p=0.010) after multivariate adjustment for potential confounders. We also found that POAG patients had lower plasma vitamin C concentrations than control subjects (9.9±1.7 µg/ml versus 11.7±1.8 µg/ml, p<0.001). Moreover, we consistently detected a significant association between the rs1279386 SNP in SLC23A2 and plasma vitamin C concentrations: GG subjects had significantly lower plasma vitamin C concentrations than the other genotypes (9.0±1.4 µg/ml versus 10.5±1.6 µg/ml, p<0.001 in POAG cases and 10.9±1.6 µg/ml versus 12.1±1.8 µg/ml, p<0.001 in controls). The rs10063949 SNP in SLC23A1 was not associated with either plasma vitamin C concentrations or POAG risk. Similarly, SNPs in RBP1 were not associated with vitamin A concentrations or POAG risk.
Conclusions
The rs1279683 SNP in SLC23A2 was significantly associated with lower plasma concentrations of vitamin C and with higher risk of POAG in GG subjects.
PMCID: PMC3236071  PMID: 22171153
8.  Functional Analysis of the Arlequin Mutant Corroborates the Essential Role of the ARLEQUIN/TAGL1 Gene during Reproductive Development of Tomato 
PLoS ONE  2010;5(12):e14427.
Reproductive development of higher plants comprises successive events of organ differentiation and growth which finally lead to the formation of a mature fruit. However, most of the genetic and molecular mechanisms which coordinate such developmental events are yet to be identified and characterized. Arlequin (Alq), a semi-dominant T-DNA tomato mutant showed developmental changes affecting flower and fruit ripening. Sepals were converted into fleshy organs which ripened as normal fruit organs and fruits displayed altered ripening features. Molecular characterization of the tagged gene demonstrated that it corresponded to the previously reported TOMATO AGAMOUS-LIKE 1 (TAGL1) gene, the tomato ortholog of SHATTERPROOF MADS-box genes of Arabidopsis thaliana, and that the Alq mutation promoted a gain-of-function phenotype caused by the ectopic expression of TAGL1. Ectopic overexpression of TAGL1 resulted in homeotic alterations affecting floral organ identity that were similar to but stronger than those observed in Alq mutant plants. Interestingly, TAGL1 RNAi plants yielded tomato fruits which were unable to ripen. They displayed a yellow-orange color and stiffness appearance which are in accordance with reduced lycopene and ethylene levels, respectively. Moreover, pericarp cells of TAGL1 RNAi fruits showed altered cellular and structural properties which correlated to both decreased expression of genes regulating cell division and lignin biosynthesis. Over-expression of TAGL1 is able to rescue the non-ripening phenotype of rin and nor mutants, which is mediated by the transcriptional activation of several ripening genes. Our results demonstrated that TAGL1 participates in the genetic control of flower and fruit development of tomato plants. Furthermore, gene silencing and over-expression experiments demonstrated that the fruit ripening process requires the regulatory activity of TAGL1. Therefore, TAGL1 could act as a linking factor connecting successive stages of reproductive development, from flower development to fruit maturation, allowing this complex process to be carried out successfully.
doi:10.1371/journal.pone.0014427
PMCID: PMC3009712  PMID: 21203447
9.  Smoking, an additional risk factor in elder women with primary open-angle glaucoma 
Molecular Vision  2009;15:2953-2959.
Purpose
Smoking is a serious public health problem worldwide. Some authors refer to it as the “silent epidemic of the 20th century.” It constitutes an important risk factor for ocular pathologies such as age-related macular degeneration (ARMD), diabetic retinopathy, and neuropathy because the toxic effects of tobacco play a key role in the deterioration of eye tissue. Damage to trabecular meshwork cells (TMC) and retinal ganglion cells (RGC), involving inflammation and apoptosis mechanisms, has been proved in glaucoma. The aim of this study was to determine whether smoking influences the progression of primary open angle glaucoma (POAG) in women.
Methods
This experimental study involved a sampling of consecutive cases of smokers, ex-smokers and non-smokers women with POAG. One hundred and twenty women with POAG, aged 40–90 years, were enrolled (40 smokers, 40 ex-smokers and 40 non-smokers). Samples of aqueous humor (AH) and plasma from each subject were obtained at the beginning of the surgical procedures. Both inflammation and apoptosis processes in the subjects were studied by means of enzyme immunoassay and western blot procedures respectively. We analyzed the interleukin-6 (IL-6) levels as an inflammation marker and the expression of caspase-3 and poly (ADP-ribose) polymerase 1 (PARP-1) as apoptosis markers.
Results
IL-6, caspase-3, and PARP-1 levels were significantly higher in the smoker women who smoked than in the ex-smoker and non-smoker glaucomatous groups of the same gender (p<0,05).
Conclusions
Inflammation and apoptosis marker levels increase with smoking in the aqueous humor and plasma samples of POAG women. Smoking could be an important additional risk factor for glaucoma progression in elderly women.
PMCID: PMC2802290  PMID: 20057902
10.  Intra‐ and Interspecific Variation in DNA Content in Cistus (Cistaceae) 
Annals of Botany  2002;90(3):345-351.
Flow cytometry, using propidium iodide and 4′,6‐diamidano‐2-phenylindole staining, was used to estimate the nuclear DNA content (2C) and the proportion of A–T base pairs in 16 species of the Mediterranean genus Cistus. Genome sizes were shown to be constant within species, since no significant intraspecific variation in 2C DNA content was detected. At the genus level, up to about 1·5‐fold differences in absolute DNA amounts were observed, ranging from 3·92 pg in C. crispus to 5·88 pg in C. monspeliensis. The (AT) : (GC) ratio was close to 1, and was similar for all species examined, ranging from 47·87 % A–T content in C. clusii, to 50·67 % in C. populifolius. Pink‐flowered species (subgenus Cistus) had lower DNA amounts than white‐flowered species (subgenera Leucocistus and Halimioides). However, the distribution of DNA amounts in Cistus appeared to be continuous and did not permit a clear separation of infra‐generic ranks in the genus.
doi:10.1093/aob/mcf194
PMCID: PMC4240394  PMID: 12234146
Flow cytometry; nuclear DNA content; genome size; A–T content; Cistus; Cistaceae

Results 1-10 (10)