Acquired resistance to anti-EGFR mAb ICR62 in cancer cells is accompanied by an increased EGFR expression, HER-2/HER-3 signalling and sensitivity to pan HER blockers

Our results provide a novel mechanistic insight into the development of acquired resistance to EGFR antibody-based therapy in colorectal cancer cells and justify further investigations on the therapeutic benefits of pan-HER family inhibitors in the treatment of colorectal cancer patients once acquired resistance to EGFR antibody-based therapy is developed

Androgens Contribute to Sex Differences in Myocardial Remodeling under Pressure Overload by a Mechanism Involving TGF-β

Background: In clinical studies, myocardial remodeling in aortic valve stenosis appears to be more favorable in women than in men, even after menopause. In the present study, we assessed whether circulating androgens contribute to a less favorable myocardial remodeling under pressure overload in males. We examined sex-related differences in one-year-old male and female mice. Whereas male mice at this age exhibited circulating androgen levels within the normal range for young adults, the circulating estrogens in females were reduced. The contribution of gonadal androgens to cardiac remodeling was analyzed in a group of same-age castrated mice. Methodology/Principal Findings: Animals were subjected to transverse aortic constriction (TAC). Echocardiography was performed 2 weeks after TAC and myocardial mRNA levels of TGF-bs, Smads 2 and 3, collagens, fibronectin, b-myosin heavy chain and a-myosin heavy chain were determined by q-PCR. Protein detection of p-SMAD2/3 was performed by Western Blot. Histological staining of fibrosis was performed with picrosirius red and Masson’s trichrome. Compared with females, males developed more severe tissue fibrosis, LV dilation and hemodynamic dysfunction. TAC-males showed higher myocardial expression levels of TGF-bs and the treatment with a neutralizing antibody to TGF-b prevented myocardial fibrosis development. Orchiectomy diminished TAC-induced up-regulation of TGF-bs and TGF-b target genes, and it also reduced fibrosis and hemodynamic dysfunction. The capability of androgens to induce TGF-b expression was confirmed i

The oxysterol 27-hydroxycholesterol increases β-amyloid and oxidative stress in retinal pigment epithelial cells

<p>Abstract</p> <p>Background</p> <p>Alzheimer's disease (AD) and age-related macular degeneration (AMD) share several pathological features including β-amyloid (Aβ) peptide accumulation, oxidative damage, and cell death. The causes of AD and AMD are not known but several studies suggest disturbances in cholesterol metabolism as a culprit of these diseases. We have recently shown that the cholesterol oxidation metabolite 27-hydroxycholesterol (27-OHC) causes AD-like pathology in human neuroblastoma SH-SY5Y cells and in organotypic hippocampal slices. However, the extent to which and the mechanisms by which 27-OHC may also cause pathological hallmarks related to AMD are ill-defined. In this study, the effects of 27-OHC on AMD-related pathology were determined in ARPE-19 cells. These cells have structural and functional properties relevant to retinal pigmented epithelial cells, a target in the course of AMD.</p> <p>Methods</p> <p>ARPE-19 cells were treated with 0, 10 or 25 μM 27-OHC for 24 hours. Levels of Aβ peptide, mitochondrial and endoplasmic reticulum (ER) stress markers, Ca²⁺homeostasis, glutathione depletion, reactive oxygen species (ROS) generation, inflammation and cell death were assessed using ELISA, Western blot, immunocytochemistry, and specific assays.</p> <p>Results</p> <p>27-OHC dose-dependently increased Aβ peptide production, increased levels of ER stress specific markers caspase 12 and gadd153 (also called CHOP), reduced mitochondrial membrane potential, triggered Ca²⁺dyshomeostasis, increased levels of the nuclear factor κB (NFκB) and heme-oxygenase 1 (HO-1), two proteins activated by oxidative stress. Additionally, 27-OHC caused glutathione depletion, ROS generation, inflammation and apoptotic-mediated cell death.</p> <p>Conclusions</p> <p>The cholesterol metabolite 27-OHC is toxic to RPE cells. The deleterious effects of this oxysterol ranged from Aβ accumulation to oxidative cell damage. Our results suggest that high levels of 27-OHC may represent a common pathogenic factor for both AMD and AD.</p

Spatially Explicit Analysis of Metal Transfer to Biota: Influence of Soil Contamination and Landscape

Concepts and developments for a new field in ecotoxicology, referred to as “landscape ecotoxicology,” were proposed in the 1990s; however, to date, few studies have been developed in this emergent field. In fact, there is a strong interest in developing this area, both for renewing the concepts and tools used in ecotoxicology as well as for responding to practical issues, such as risk assessment. The aim of this study was to investigate the spatial heterogeneity of metal bioaccumulation in animals in order to identify the role of spatially explicit factors, such as landscape as well as total and extractable metal concentrations in soils. Over a smelter-impacted area, we studied the accumulation of trace metals (TMs: Cd, Pb and Zn) in invertebrates (the grove snail Cepaea sp and the glass snail Oxychilus draparnaudi) and vertebrates (the bank vole Myodes glareolus and the greater white-toothed shrew Crocidura russula). Total and CaCl2-extractable concentrations of TMs were measured in soils from woody patches where the animals were captured. TM concentrations in animals exhibited a high spatial heterogeneity. They increased with soil pollution and were better explained by total rather than CaCl2-extractable TM concentrations, except in Cepaea sp. TM levels in animals and their variations along the pollution gradient were modulated by the landscape, and this influence was species and metal specific. Median soil metal concentrations (predicted by universal kriging) were calculated in buffers of increasing size and were related to bioaccumulation. The spatial scale at which TM concentrations in animals and soils showed the strongest correlations varied between metals, species and landscapes. The potential underlying mechanisms of landscape influence (community functioning, behaviour, etc.) are discussed. Present results highlight the need for the further development of landscape ecotoxicology and multi-scale approaches, which would enhance our understanding of pollutant transfer and effects in ecosystems

Chemical genetics strategies for identification of molecular targets

Chemical genetics is an emerging field that can be used to study the interactions of chemical compounds, including natural products, with proteins. Usually, the identification of molecular targets is the starting point for studying a drug’s mechanism of action and this has been a crucial step in understanding many biological processes. While a great variety of target identification methods have been developed over the last several years, there are still many bioactive compounds whose target proteins have not yet been revealed because no routine protocols can be adopted. This review contains information concerning the most relevant principles of chemical genetics with special emphasis on the different genomic and proteomic approaches used in forward chemical genetics to identify the molecular targets of the bioactive compounds, the advantages and disadvantages of each and a detailed list of successful examples of molecular targets identified with these approaches

The transcriptional regulator BBX24 impairs DELLA activity to promote shade avoidance in Arabidopsis thaliana

[EN] In response to canopy shade, plant vegetative structures elongate to gain access to light. However, the mechanism that allows a plastic transcriptional response to canopy shade light is not fully elucidated. Here we propose that the activity of PIF4, a key transcription factor in the shade signalling network, is modulated by the interplay between the BBX24 transcriptional regulator and DELLA proteins, which are negative regulators of the gibberellin (GA) signalling pathway. We show that GA-related targets are enriched among genes responsive to BBX24 under shade and that the shade-response defect in bbx24 mutants is rescued by a GA treatment that promotes DELLA degradation. BBX24 physically interacts with DELLA proteins and alleviates DELLA-mediated repression of PIF4 activity. The proposed molecular mechanism provides reversible regulation of the activity of a key transcription factor that may prove especially relevant under fluctuating light conditions.We thank Santiago Mora Garcia for valuable initial discussions and Peter Quail for the PIL1::LUC construct. This work was supported by grants from Agencia Nacional de Promocion Cientifica y Tecnologica, and Universidad de Buenos Aires (to J.F.B), and the Spanish Ministry of Science, BIO2010-15071 (to M.A.B.).Crocco, C.; Locascio ., AAM.; Escudero, CM.; Alabadí Diego, D.; Blazquez Rodriguez, MA.; Botto, J. (2015). The transcriptional regulator BBX24 impairs DELLA activity to promote shade avoidance in Arabidopsis thaliana. Nature Communications. 6:1-10. https://doi.org/10.1038/ncomms7202S1106Valladares, F. & Niinemets, U. Shade tolerance, a key plant feature of complex nature and consequences. Annu. Rev. Ecol. Evol. Syst. 39, 237–257 (2008).Casal, J. J. Photoreceptor signaling networks in plant responses to shade. Annu. Rev. Plant Biol. 64, 403–427 (2013).Botto, J. F. & Coluccio, M. P. Seasonal and plant-density dependency for quantitative trait loci affecting flowering time in multiple populations of Arabidopsis thaliana. Plant Cell Environ. 30, 1465–1479 (2007).Coluccio, M. P., Sánchez, S., Kasulin, L., Yanovsky, M. J. & Botto, J. F. Genetic mapping of natural variation in a shade avoidance response: ELF3 is the candidate gene for a QTL in hypocotyl growth regulation. J. Exp. Bot. 62, 167–176 (2011).Filiault, D. L. & Maloof, J. N. A genome-wide association study identifies variants underlying the Arabidopsis thaliana shade avoidance response. PLoS. Genet. 8, e1002589 (2012).Kasulin, L., Agrofoglio, Y. & Botto, J. F. The receptor-like kinase ERECTA contributes to the shade-avoidance syndrome in a background-dependent manner. Ann. Bot. 111, 811–819 (2013).Leivar, P. & Monte, E. PIFs: systems integrators in plant development. Plant Cell 26, 56–78 (2014).Lorrain, S., Allen, T., Duek, P. D., Whitelam, G. C. & Fankhauser, C. Phytochrome-mediated inhibition of shade avoidance involves degradation of growth-promoting bHLH transcription factors. Plant J. 53, 312–323 (2008).Hornitschek, P., Lorrain, S., Zoete, V., Michielin, O. & Fankhauser, C. Inhibition of the shade avoidance response by formation of non-DNA binding bHLH heterodimers. EMBO J. 28, 3893–3902 (2009).Gangappa, S. N. & Botto, J. F. The BBX family of plant transcription factors. Trends Plant Sci. 19, 460–470 (2014).Crocco, C. D., Holm, M., Yanovsky, M. J. & Botto, J. F. AtBBX21 and COP1 genetically interact in the regulation of shade avoidance. Plant J. 64, 551–562 (2010).Gangappa, S. N. et al. The Arabidopsis B-BOX protein BBX25 interacts with HY5, negatively regulating BBX22 expression to suppress seedling photomorphogenesis. Plant Cell 25, 1243–1257 (2013).Devlin, F. P., Yanovsky, M. J. & Kay, S. A. A genomic analysis of the shade avoidance response in Arabidopsis. Plant Physiol. 133, 1–13 (2003).Hisamatsu, T., King, R. W., Helliwell, C. A. & Koshioka, M. The involvement of gibberellin 20-oxidase genes in phytochrome-regulated petiole elongation of Arabidopsis. Plant Physiol. 138, 1106–1116 (2005).Locascio, A., Blázquez, M. A. & Alabadí, D. Genomic analysis of DELLA protein activity. Plant Cell Physiol. 54, 1229–1237 (2013).de Lucas, M. et al. A molecular framework for light and gibberellin control of cell elongation. Nature 451, 480–486 (2008).Feng, S. et al. Coordinated regulation of Arabidopsis thaliana development by light and gibberellins. Nature 451, 475–480 (2008).Djakovic-Petrovic, T., de Wit, M., Voesenek, L. A. C. J. & Pierik, R. DELLA protein function in growth responses to canopy signals. Plant J. 51, 117–126 (2007).Pierik, R., de Wit, M. & Voesenek, L. A. C. J. Growth-mediated stress escape: convergence of signal transduction pathways activated upon exposure to two different environmental stresses. New Phytol. 189, 122–134 (2011).Colebrook, E. H., Thomas, S. G., Phillips, A. L. & Hedden, P. The role of gibberellin signalling in plant responses to abiotic stress. J. Exp. Biol. 217, 67–75 (2014).Holtan, H. E. et al. BBX32, an Arabidopsis B-Box protein, functions in light signaling by suppressing HY5-regulated gene expression and interacting with STH2/BBX21. Plant Physiol. 156, 2109–2123 (2011).Xu, D. et al. Convergence of light and ABA signaling on the ABI5 promoter. PLoS. Genet. 10, e1004197 (2014).Pierik, R., Djakovic-Petrovic, T., Keuskamp, D. H., de Wit, M. & Voesenek, L. A. C. J. Auxin and ethylene regulate elongation responses to neighbor proximity signals independent of gibberellin and DELLA proteins in Arabidopsis. Plant Physiol. 149, 1701–1712 (2009).Keuskamp, D. H. et al. Blue-light-mediated shade avoidance requires combined auxin and brassinosteroid action in Arabidopsis seedlings. Plant J. 67, 208–217 (2011).Li, L. et al. Linking photoreceptor excitation to changes in plant architecture. Genes Dev. 26, 785–790 (2012).Hornitschek, P. et al. Phytochrome interacting factors 4 and 5 control seedling growth in changing light conditions by directly controlling auxin signaling. Plant J. 71, 699–711 (2012).Leivar, P. et al. Dynamic antagonism between phytochromes and PIF family basic helix-loop-helix factors induces selective reciprocal responses to light and shade in a rapidly responsive transcriptional network in Arabidopsis. Plant Cell 24, 1398–1419 (2012).Oh, E., Zhu, J.-Y. & Wang, Z.-Y. Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses. Nat. Cell Biol. 14, 802–809 (2012).Dill, A. & Sun, T. P. Synergistic derepression of gibberellin signaling by removing RGA and GAI function in Arabidopsis thaliana. Genetics 159, 777–785 (2001).Cole, B., Kay, S. A. & Chory, J. Automated analysis of hypocotyl growth dynamics during shade avoidance in Arabidopsis. Plant J. 65, 991–1000 (2011).Zhang, Y. et al. A quartet of PIF bHLH factors provides a transcriptionally centered signaling hub that regulates seedling morphogenesis through differential expression-patterning of shared target genes in Arabidopsis. PLoS. Genet. 9, e1003244 (2013).Leivar, P. et al. Definition of early transcriptional circuitry involved in light-induced reversal of PIF-imposed repression of photomorphogenesis in young Arabidopsis seedlings. Plant Cell 21, 3535–3553 (2009).Willige, B. C. et al. The DELLA domain of GA INSENSITIVE mediates the interaction with the GA INSENSITIVE DWARF1A gibberellin receptor of Arabidopsis. Plant Cell 19, 1209–1220 (2007).Davière, J.-M. & Achard, P. Gibberellin signaling in plants. Develop 140, 1147–1151 (2013).Lim, S. et al. ABA-INSENSITIVE3, ABA-INSENSITIVE5, and DELLAs interact to activate the expression of SOMNUS and other high-temperature-inducible genes in imbibed seeds in Arabidopsis. Plant Cell 25, 4863–4878 (2013).Yoshida, H. et al. DELLA protein functions as a transcriptional activator through the DNA binding of the indeterminate domain family proteins. Proc. Natl Acad. Sci. USA 111, 7861–7866 (2014).Yamaguchi, N. et al. Gibberellin acts positively then negatively to control onset of flower formation in Arabidopsis. Science 344, 638–641 (2014).Stavang, J. et al. Hormonal regulation of temperature-induced growth in Arabidopsis. Plant J. 60, 589–601 (2009).Achard, P. et al. DELLAs contribute to plant photomorphogenesis. Plant Physiol. 143, 1163–1172 (2007).Arana, M. V., Marín-de la Rosa, N., Maloof, J. N., Blázquez, M. A. & Alabadí, D. Circadian oscillation of gibberellin signaling in Arabidopsis. Proc. Natl Acad. Sci. USA 108, 9292–9297 (2011).Bai, M.-Y., Fan, M., Oh, E. & Wang, Z.-Y. A triple helix-loop-helix/basic helix-loop-helix cascade controls cell elongation downstream of multiple hormonal and environmental signaling pathways in Arabidopsis. Plant Cell 24, 4917–4929 (2012).Ikeda, M., Fujiwara, S., Mitsuda, N. & Ohme-Takagi, M. A triantagonistic basic helix-loop-helix system regulates cell elongation in Arabidopsis. Plant Cell 24, 4483–4497 (2012).Yang, D.-L. et al. Plant hormone jasmonate prioritizes defense over growth by interfering with gibberellin signaling cascade. Proc. Natl Acad. Sci. USA 109, E1192–E1200 (2012).Ciolfi, A. et al. Dynamics of the shade-avoidance response in Arabidopsis. Plant Physiol. 163, 331–353 (2013).Indorf, M., Cordero, J., Neuhaus, G. & Rodríguez-Franco, M. Salt tolerance (STO), a stress-related protein, has a major role in light signalling. Plant J. 51, 563–574 (2007).Gallego-Bartolomé, J., Kami, C., Fankhauser, C., Alabadí, D. & Blázquez, M. A. A hormonal regulatory module that provides flexibility to tropic responses. Plant Physiol. 156, 1819–1825 (2011).Earley, K. W. et al. Gateway-compatible vectors for plant functional genomics and proteomics. Plant J. 45, 616–629 (2006).Tusher, V. G., Tibshirani, R. & Chu, G. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl Acad. Sci. USA 98, 5116–5121 (2001).Gallego-Bartolomé, J. et al. Molecular mechanism for the interaction between gibberellin and brassinosteroid signaling pathways in Arabidopsis. Proc. Natl Acad. Sci. USA 109, 13446–13451 (2012).Belda-Palazón, B. et al. Aminopropyltransferases involved in polyamine biosynthesis localize preferentially in the nucleus of plant cells. PLoS ONE 7, e46907 (2012).Gallego-Bartolomé, J., Alabadí, D. & Blázquez, M. A. DELLA-induced early transcriptional changes during etiolated development in Arabidopsis thaliana. PLoS ONE 6, e23918 (2011).Piskurewicz, U. et al. The gibberellic acid signaling repressor RGL2 inhibits Arabidopsis seed germination by stimulating abscisic acid synthesis and ABI5 activity. Plant Cell 20, 2729–2745 (2008).Paz-Ares, J. REGIA, an EU project on functional genomics of transcription factors from Arabidopsis thaliana. Comp. Funct. Genomics 3, 102–108 (2002)

Surviving in isolation: genetic variation, bottlenecks and reproductive strategies in the Canarian endemic Limonium macrophyllum (Plumbaginaceae)

Oceanic archipelagos are typically rich in endemic taxa, because they offer ideal conditions for diversification and speciation in isolation. One of the most remarkable evolutionary radiations on the Canary Islands comprises the 16 species included in Limonium subsection Nobiles, all of which are subject to diverse threats, and legally protected. Since many of them are single-island endemics limited to one or a few populations, there exists a risk that a loss of genetic variation might limit their longterm survival. In this study, we used eight newly developed microsatellite markers to characterize the levels of genetic variation and inbreeding in L. macrophyllum, a species endemic to the North-east of Tenerife that belongs to Limonium subsection Nobiles. We detected generally low levels of genetic variation over all populations (HT = 0.363), and substantial differentiation among populations (FST = 0.188;RST = 0.186) coupled with a negligible degree of inbreeding (F = 0.042). Obligate outcrossing may have maintained L. macrophyllum relatively unaffected by inbreeding despite the species’ limited dispersal ability and the genetic bottlenecks likely caused by a prolonged history of grazing. Although several factors still constitute a risk for the conservation of L. macrophyllum, the lack of inbreeding and the recent positive demographic trends observed in the populations of this species are factors that favour its future persistence