NEVUS DE SPITZ: REVISIÓN DE CASOS Y EXPRESIÓN DE LA PROTEÍNA ALK

Los nevus de Spitz son un grupo de lesiones melanocíticas que afectan normalmente a gente joven. Son lesiones benignas, sin embargo su principal diagnóstico diferencial es el melanoma, por lo que es muy importante diferenciarlos con seguridad. Recientemente se han documentado fusiones de cinasas en nevus de Spitz, pero hay escasa información sobre las características clínicas y microscópicas asociadas a las mismas. El objetivo de este trabajo es describir la morfología de los nevus de Spitz y relacionarla con la expresión del gen ALK. Informamos una serie de 40 casos diagnosticados de nevus de Spitz (20 varones y 20 mujeres) en los cuales revisamos las características morfológicas y las fusiones de cinasa. El rango de edad de los pacientes era de uno a 56 años (media=15,9 moda=9 y mediana=13 años). La mayoría de las lesiones se localizaban en extremidades inferiores y se presentaban clínicamente como nódulos polipoides. En ningún caso se observaron recidivas de la lesión. El espesor de los tumores era de 0,16mm a 4,06mm, (media= 1,12mm mediana = 1,02mm). Las características histopatológicas en general presentaban celularidad tanto epitelioide como fusiforme, crecimiento expansivo y escaso pleomorfismo. El 35% de los casos fueron positivos para la recombinación del gen ALK, pero no mostraban más características en común entre sí. Ante estos resultados, concluimos que dado que no han mostrado diferentes características histopatológicas que puedan ayudarnos a mejorar la clasificación de estas lesiones, las fusiones del gen ALK no son útiles en la práctica clínica habitual

Tuning the helical sense and elongation of polymers through the combined action of the two components of tetraalkylammonium-anion salts

The helical sense and elongation of a helical polymer that bears the 4-ethynylanilide of (R)-α‑methoxy-α-phenylacetic acid [m-(R)-1] can be tuned by the action of the two components of tetraalkylammonium-anion salts. Thus, while the supramolecular PPA-anilide/anion interaction creates a chiroptical switch, inducing either P or M helical senses in the poly(phenylacetylene) (PPA) depending on the nature of the anion —P helix: fluoride (F−) or cyanide (CN−); M helix: acetate (OAc−), benzoate (OBz−) or azide (N3−)—, deprotonation of the anilide group by these anions in dry conditions makes it possible to play with the elongation of the PPA, depending on the size of the tetraalkylammonium group, which results in a colorimetric switch. Thus, a color change from yellow to deep red is obtained by deprotonation of the anilide group when a tetraabutylammonium salt is used, while yellow/light-red and yellow/orange color changes are obtained when the size of the tetraalkylammonium group is reduced. Structural and mechanistic studies confirm that the color change occurs when the counterion accommodates itself close to the pendant group once the anilide hydrogen is removed. This PPA/tetraalkylammonium association is responsible of elongation changes in the PPA and therefore on the conjugation of the polyene backbone that is accompanied with color changes. Moreover, this colorimetric switch also operates in the solid state and can be switched in a vacuum/air moisture cycleFinancial support from MINECO (PID2019-109733GB-I00), Xunta de Galicia (ED431C 2018/30; Centro singular de investigación de Galicia accreditation 2016–2019, ED431G/09 and the European Regional Development Fund (ERDF) is gratefully acknowledged. We also thank Servicio de Nanotecnología y Análisis de Superficies (CACTI, UVIGO)S

Near Infrared Spectroscopy for Bacterial Detection in the Dairy Industry

[EN] This article discusses the use of near-infrared (NIR) spectroscopy combined with multivariate classification methods for detecting bacterial contamination in milk in the dairy industry. In the first experiment, the study found that NIR was accurate and reliable in detecting the presence of biofilms in milk. Our results showed that the technology was effective in distinguishing between contaminated and uncontaminated samples with an area under the receiver operating characteristic (ROC) curve (AUC) greater than 99%. It was also effective in classifying the samples belonging to different strains. In a second experiment, we used the same methodology to assess their effectiveness in detecting bacterial contamination proportions in milk. Our results showed that the technology was effective in classifying milk samples contaminated with four different bacteria and uncontaminated controls with an AUC greater than 97%. Moreover, results were still good when data from all bacteria were analyzed together, even at low bacterial concentrations, obtaining an average precision of 70%. These results demonstrate the potential of this technology to be used as a rapid and accurate method for identifying bacterial contamination in the dairy sector.SIPrincipado de Asturia

Regulation of BDNF Release by ARMS/Kidins220 through Modulation of Synaptotagmin-IV Levels

BDNF is a growth factor with important roles in the nervous system in both physiological and pathological conditions, but the mechanisms controlling its secretion are not completely understood. Here, we show that ARMS/Kidins220 negatively regulates BDNF secretion in neurons from the CNS and PNS. Downregulation of the ARMS/Kidins220 protein in the adult mouse brain increases regulated BDNF secretion, leading to its accumulation in the striatum. Interestingly, two mouse models of Huntington's disease (HD) showed increased levels of ARMS/Kidins220 in the hippocampus and regulated BDNF secretion deficits. Importantly, reduction of ARMS/Kidins220 in hippocampal slices from HD mice reversed the impaired regulated BDNF release. Moreover, there are increased levels of ARMS/Kidins220 in the hippocampus and PFC of patients with HD. ARMS/Kidins220 regulates Synaptotagmin-IV levels, which has been previously observed to modulate BDNF secretion. These data indicate that ARMS/Kidins220 controls the regulated secretion of BDNF and might play a crucial role in the pathogenesis of HD.SIGNIFICANCE STATEMENT BDNF is an important growth factor that plays a fundamental role in the correct functioning of the CNS. The secretion of BDNF must be properly controlled to exert its functions, but the proteins regulating its release are not completely known. Using neuronal cultures and a new conditional mouse to modulate ARMS/Kidins220 protein, we report that ARMS/Kidins220 negatively regulates BDNF secretion. Moreover, ARMS/Kidins220 is overexpressed in two mouse models of Huntington's disease (HD), causing an impaired regulation of BDNF secretion. Furthermore, ARMS/Kidins220 levels are increased in brain samples from HD patients. Future studies should address whether ARMS/Kidins220 has any function on the pathophysiology of HD

Expression of the Intracellular COPT3-Mediated Cu Transport Is Temporally Regulated by the TCP16 Transcription Factor

[EN] Copper is an essential element in plants. When scarce, copper is acquired from extracellular environment or remobilized from intracellular sites, through members of the high affinity copper transporters family COPT located at the plasma membrane and internal membrane, respectively. Here, we show that COPT3 is an intracellular copper transporter, located at a compartment of the secretory pathway, that is mainly expressed in pollen grains and vascular bundles. Contrary to the COPT1 plasma membrane member, the expression of the internal COPT3 membrane transporter was higher at 12 h than at 0 h of a neutral photoperiod day under copper deficiency. The screening of a library of conditionally overexpressed transcription factors implicated members of the TCP family in the COPT3 differential temporal expression pattern. Particularly, in vitro, TCP16 was found to bind to the COPT3 promoter and down-regulated its expression. Accordingly, TCP16 was mainly expressed at 0 h under copper deficiency and induced at 12 h by copper excess. Moreover, TCP16 overexpression resulted in increased sensitivity to copper deficiency, whereas the tcp16 mutant was sensitive to copper excess. Both copper content and the expression of particular copper status markers were altered in plants with modified levels of TCP16. Consistent with TCP16 affecting pollen development, the lack of COPT3 function led to altered pollen morphology. Furthermore, analysis of copt3 and COPT3 overexpressing plants revealed that COPT3 function exerted a negative effect on TCP16 expression. Taken together, these results suggest a differential daily regulation of copper uptake depending on the external and internal copper pools, in which TCP16 inhibits copper remobilization at dawn through repression of intracellular transporters.This work has been supported by grants BIO2017-87828-C2-1-P (LP) and the TRANSPLANTA Consortium (CSD2007-00057) from the Spanish Ministry of Economy and Competitiveness, and by FEDER funds from the European Union. NA-C and AC-S were recipients of a predoctoral FPI fellowship from the Spanish Ministry of Economy and Competitiveness.Andrés-Colás, N.; Carrió-Seguí, Á.; Abdel-Ghany, SE.; Pilon, M.; Peñarrubia, L. (2018). Expression of the Intracellular COPT3-Mediated Cu Transport Is Temporally Regulated by the TCP16 Transcription Factor. Frontiers in Plant Science. 9. https://doi.org/10.3389/fpls.2018.00910S9Abdel-Ghany, S. E., Müller-Moulé, P., Niyogi, K. K., Pilon, M., & Shikanai, T. (2005). Two P-Type ATPases Are Required for Copper Delivery in Arabidopsis thaliana Chloroplasts. The Plant Cell, 17(4), 1233-1251. doi:10.1105/tpc.104.030452Almeida, D. M., Gregorio, G. B., Oliveira, M. M., & Saibo, N. J. M. (2016). Five novel transcription factors as potential regulators of OsNHX1 gene expression in a salt tolerant rice genotype. Plant Molecular Biology, 93(1-2), 61-77. doi:10.1007/s11103-016-0547-7à lvarez-Fernández, A., Díaz-Benito, P., Abadía, A., López-Millán, A.-F., & Abadía, J. (2014). Metal species involved in long distance metal transport in plants. Frontiers in Plant Science, 5. doi:10.3389/fpls.2014.00105Andrés-Colás, N., Perea-García, A., Mayo de Andrés, S., Garcia-Molina, A., Dorcey, E., Rodríguez-Navarro, S., … Peñarrubia, L. (2013). Comparison of global responses to mild deficiency and excess copper levels in Arabidopsis seedlings. Metallomics, 5(9), 1234. doi:10.1039/c3mt00025gAndrés-Colás, N., Perea-García, A., Puig, S., & Peñarrubia, L. (2010). Deregulated Copper Transport Affects Arabidopsis Development Especially in the Absence of Environmental Cycles. Plant Physiology, 153(1), 170-184. doi:10.1104/pp.110.153676Andrés-Colás, N., Sancenón, V., Rodríguez-Navarro, S., Mayo, S., Thiele, D. J., Ecker, J. R., … Peñarrubia, L. (2006). The Arabidopsis heavy metal P-type ATPase HMA5 interacts with metallochaperones and functions in copper detoxification of roots. The Plant Journal, 45(2), 225-236. doi:10.1111/j.1365-313x.2005.02601.xAndriankaja, M. E., Danisman, S., Mignolet-Spruyt, L. F., Claeys, H., Kochanke, I., Vermeersch, M., … Inzé, D. (2014). Transcriptional coordination between leaf cell differentiation and chloroplast development established by TCP20 and the subgroup Ib bHLH transcription factors. Plant Molecular Biology, 85(3), 233-245. doi:10.1007/s11103-014-0180-2Atamian, H. S., & Harmer, S. L. (2016). Circadian regulation of hormone signaling and plant physiology. Plant Molecular Biology, 91(6), 691-702. doi:10.1007/s11103-016-0477-4Balsemão-Pires, E., Andrade, L. R., & Sachetto-Martins, G. (2013). Functional study of TCP23 in Arabidopsis thaliana during plant development. Plant Physiology and Biochemistry, 67, 120-125. doi:10.1016/j.plaphy.2013.03.009Bar-Peled, M., & Raikhel, N. V. (1997). Characterization of AtSEC12 and AtSAR1 (Proteins Likely Involved in Endoplasmic Reticulum and Golgi Transport). Plant Physiology, 114(1), 315-324. doi:10.1104/pp.114.1.315Bate, N., & Twell, D. (1998). Plant Molecular Biology, 37(5), 859-869. doi:10.1023/a:1006095023050Bemer, M., van Dijk, A. D. J., Immink, R. G. H., & Angenent, G. C. (2017). Cross-Family Transcription Factor Interactions: An Additional Layer of Gene Regulation. Trends in Plant Science, 22(1), 66-80. doi:10.1016/j.tplants.2016.10.007Bernal, M., Casero, D., Singh, V., Wilson, G. T., Grande, A., Yang, H., … Krämer, U. (2012). Transcriptome Sequencing Identifies SPL7-Regulated Copper Acquisition Genes FRO4/FRO5 and the Copper Dependence of Iron Homeostasis in Arabidopsis. The Plant Cell, 24(2), 738-761. doi:10.1105/tpc.111.090431Blaby-Haas, C. E., & Merchant, S. S. (2014). Lysosome-related Organelles as Mediators of Metal Homeostasis. Journal of Biological Chemistry, 289(41), 28129-28136. doi:10.1074/jbc.r114.592618Bock, K. W., Honys, D., Ward, J. M., Padmanaban, S., Nawrocki, E. P., Hirschi, K. D., … Sze, H. (2006). Integrating Membrane Transport with Male Gametophyte Development and Function through Transcriptomics. Plant Physiology, 140(4), 1151-1168. doi:10.1104/pp.105.074708Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1-2), 248-254. doi:10.1016/0003-2697(76)90527-3Brancaccio, D., Gallo, A., Piccioli, M., Novellino, E., Ciofi-Baffoni, S., & Banci, L. (2017). [4Fe-4S] Cluster Assembly in Mitochondria and Its Impairment by Copper. Journal of the American Chemical Society, 139(2), 719-730. doi:10.1021/jacs.6b09567Bruinsma, J. (1961). A comment on the spectrophotometric determination of chlorophyll. Biochimica et Biophysica Acta, 52(3), 576-578. doi:10.1016/0006-3002(61)90418-8Carrió-Seguí, A., Garcia-Molina, A., Sanz, A., & Peñarrubia, L. (2014). Defective Copper Transport in the copt5 Mutant Affects Cadmium Tolerance. Plant and Cell Physiology, 56(3), 442-454. doi:10.1093/pcp/pcu180Chen, Y.-Y., Wang, Y., Shin, L.-J., Wu, J.-F., Shanmugam, V., Tsednee, M., … Yeh, K.-C. (2013). Iron Is Involved in the Maintenance of Circadian Period Length in Arabidopsis. Plant Physiology, 161(3), 1409-1420. doi:10.1104/pp.112.212068Coego, A., Brizuela, E., Castillejo, P., Ruíz, S., Koncz, C., … del Pozo, J. C. (2014). The TRANSPLANTA collection of Arabidopsis lines: a resource for functional analysis of transcription factors based on their conditional overexpression. The Plant Journal, 77(6), 944-953. doi:10.1111/tpj.12443Cubas, P., Lauter, N., Doebley, J., & Coen, E. (1999). The TCP domain: a motif found in proteins regulating plant growth and development. The Plant Journal, 18(2), 215-222. doi:10.1046/j.1365-313x.1999.00444.xDanisman, S. (2016). TCP Transcription Factors at the Interface between Environmental Challenges and the Plant’s Growth Responses. Frontiers in Plant Science, 7. doi:10.3389/fpls.2016.01930Dhadi, S. R., Krom, N., & Ramakrishna, W. (2009). Genome-wide comparative analysis of putative bidirectional promoters from rice, Arabidopsis and Populus. Gene, 429(1-2), 65-73. doi:10.1016/j.gene.2008.09.034Dhaka, N., Bhardwaj, V., Sharma, M. K., & Sharma, R. (2017). Evolving Tale of TCPs: New Paradigms and Old Lacunae. Frontiers in Plant Science, 8. doi:10.3389/fpls.2017.00479Franco-Zorrilla, J. M., López-Vidriero, I., Carrasco, J. L., Godoy, M., Vera, P., & Solano, R. (2014). DNA-binding specificities of plant transcription factors and their potential to define target genes. Proceedings of the National Academy of Sciences, 111(6), 2367-2372. doi:10.1073/pnas.1316278111Garcia-Molina, A., Andrés-Colás, N., Perea-García, A., del Valle-Tascón, S., Peñarrubia, L., & Puig, S. (2011). The intracellular Arabidopsis COPT5 transport protein is required for photosynthetic electron transport under severe copper deficiency. The Plant Journal, 65(6), 848-860. doi:10.1111/j.1365-313x.2010.04472.xGarcia-Molina, A., Andrés-Colás, N., Perea-García, A., Neumann, U., Dodani, S. C., Huijser, P., … Puig, S. (2013). The Arabidopsis COPT6 Transport Protein Functions in Copper Distribution Under Copper-Deficient Conditions. Plant and Cell Physiology, 54(8), 1378-1390. doi:10.1093/pcp/pct088Garcia-Molina, A., Xing, S., & Huijser, P. (2014). Functional characterisation of Arabidopsis SPL7 conserved protein domains suggests novel regulatory mechanisms in the Cu deficiency response. BMC Plant Biology, 14(1). doi:10.1186/s12870-014-0231-5Giraud, E., Ng, S., Carrie, C., Duncan, O., Low, J., Lee, C. P., … Whelan, J. (2010). TCP Transcription Factors Link the Regulation of Genes Encoding Mitochondrial Proteins with the Circadian Clock in Arabidopsis thaliana. The Plant Cell, 22(12), 3921-3934. doi:10.1105/tpc.110.074518Guan, P., Ripoll, J.-J., Wang, R., Vuong, L., Bailey-Steinitz, L. J., Ye, D., & Crawford, N. M. (2017). Interacting TCP and NLP transcription factors control plant responses to nitrate availability. Proceedings of the National Academy of Sciences, 114(9), 2419-2424. doi:10.1073/pnas.1615676114Guan, P., Wang, R., Nacry, P., Breton, G., Kay, S. A., Pruneda-Paz, J. L., … Crawford, N. M. (2014). Nitrate foraging byArabidopsisroots is mediated by the transcription factor TCP20 through the systemic signaling pathway. Proceedings of the National Academy of Sciences, 111(42), 15267-15272. doi:10.1073/pnas.1411375111Harmer, S. L., Hogenesch, J. B., Straume, M., Chang, H.-S., Han, B., Zhu, T., … Kay, S. A. (2000). Orchestrated Transcription of Key Pathways in Arabidopsis by the Circadian Clock. Science, 290(5499), 2110-2113. doi:10.1126/science.290.5499.2110Hermans, C., Vuylsteke, M., Coppens, F., Craciun, A., Inzé, D., & Verbruggen, N. (2010). Early transcriptomic changes induced by magnesium deficiency in Arabidopsis thaliana reveal the alteration of circadian clock gene expression in roots and the triggering of abscisic acid-responsive genes. New Phytologist, 187(1), 119-131. doi:10.1111/j.1469-8137.2010.03258.xHirayama, T., Kieber, J. J., Hirayama, N., Kogan, M., Guzman, P., Nourizadeh, S., … Ecker, J. R. (1999). RESPONSIVE-TO-ANTAGONIST1, a Menkes/Wilson Disease–Related Copper Transporter, Is Required for Ethylene Signaling in Arabidopsis. Cell, 97(3), 383-393. doi:10.1016/s0092-8674(00)80747-3Hong, S., Kim, S. A., Guerinot, M. L., & McClung, C. R. (2012). Reciprocal Interaction of the Circadian Clock with the Iron Homeostasis Network in Arabidopsis. Plant Physiology, 161(2), 893-903. doi:10.1104/pp.112.208603Hong-Hermesdorf, A., Miethke, M., Gallaher, S. D., Kropat, J., Dodani, S. C., Chan, J., … Merchant, S. S. (2014). Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas. Nature Chemical Biology, 10(12), 1034-1042. doi:10.1038/nchembio.1662Jefferson, R. A., Kavanagh, T. A., & Bevan, M. W. (1987). GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. The EMBO Journal, 6(13), 3901-3907. doi:10.1002/j.1460-2075.1987.tb02730.xKushnir, S. (1995). Molecular Characterization of a Putative Arabidopsis thaliana Copper Transporter and Its Yeast Homologue. Journal of Biological Chemistry, 270(47), 28479-28486. doi:10.1074/jbc.270.47.28479Kieffer, M., Master, V., Waites, R., & Davies, B. (2011). TCP14 and TCP15 affect internode length and leaf shape in Arabidopsis. The Plant Journal, 68(1), 147-158. doi:10.1111/j.1365-313x.2011.04674.xKim, H., Wu, X., & Lee, J. (2013). SLC31 (CTR) family of copper transporters in health and disease. Molecular Aspects of Medicine, 34(2-3), 561-570. doi:10.1016/j.mam.2012.07.011Klaumann, S., Nickolaus, S. D., Fürst, S. H., Starck, S., Schneider, S., Ekkehard Neuhaus, H., & Trentmann, O. (2011). The tonoplast copper transporter COPT5 acts as an exporter and is required for interorgan allocation of copper in Arabidopsis thaliana. New Phytologist, 192(2), 393-404. doi:10.1111/j.1469-8137.2011.03798.xKosugi, S., & Ohashi, Y. (2002). DNA binding and dimerization specificity and potential targets for the TCP protein family. The Plant Journal, 30(3), 337-348. doi:10.1046/j.1365-313x.2002.01294.xKrom, N., & Ramakrishna, W. (2008). Comparative Analysis of Divergent and Convergent Gene Pairs and Their Expression Patterns in Rice, Arabidopsis, and Populus. Plant Physiology, 147(4), 1763-1773. doi:10.1104/pp.108.122416Li, S. (2015). The Arabidopsis thaliana TCP transcription factors: A broadening horizon beyond development. Plant Signaling & Behavior, 10(7), e1044192. doi:10.1080/15592324.2015.1044192Martín-Trillo, M., & Cubas, P. (2010). TCP genes: a family snapshot ten years later. Trends in Plant Science, 15(1), 31-39. doi:10.1016/j.tplants.2009.11.003Mitra, A., Han, J., Zhang, Z. J., & Mitra, A. (2009). The intergenic region of Arabidopsis thaliana cab1 and cab2 divergent genes functions as a bidirectional promoter. Planta, 229(5), 1015-1022. doi:10.1007/s00425-008-0859-1Mockler, T. C., Michael, T. P., Priest, H. D., Shen, R., Sullivan, C. M., Givan, S. A., … Chory, J. (2007). The Diurnal Project: Diurnal and Circadian Expression Profiling, Model-based Pattern Matching, and Promoter Analysis. Cold Spring Harbor Symposia on Quantitative Biology, 72(1), 353-363. doi:10.1101/sqb.2007.72.006Mukhopadhyay, P., & Tyagi, A. K. (2015). OsTCP19 influences developmental and abiotic stress signaling by modulatingABI4-mediated pathways. Scientific Reports, 5(1). doi:10.1038/srep09998Nicolas, M., & Cubas, P. (2016). TCP factors: new kids on the signaling block. Current Opinion in Plant Biology, 33, 33-41. doi:10.1016/j.pbi.2016.05.006Nohales, M. A., & Kay, S. A. (2016). Molecular mechanisms at the core of the plant circadian oscillator. Nature Structural & Molecular Biology, 23(12), 1061-1069. doi:10.1038/nsmb.3327Palatnik, J. F., Allen, E., Wu, X., Schommer, C., Schwab, R., Carrington, J. C., & Weigel, D. (2003). Control of leaf morphogenesis by microRNAs. Nature, 425(6955), 257-263. doi:10.1038/nature01958Peñarrubia, L., Andrés-Colás, N., Moreno, J., & Puig, S. (2009). Regulation of copper transport in Arabidopsis thaliana: a biochemical oscillator? JBIC Journal of Biological Inorganic Chemistry, 15(1), 29-36. doi:10.1007/s00775-009-0591-8Peñarrubia, L., Romero, P., Carrió-Seguí, A., Andrés-Bordería, A., Moreno, J., & Sanz, A. (2015). Temporal aspects of copper homeostasis and its crosstalk with hormones. Frontiers in Plant Science, 6. doi:10.3389/fpls.2015.00255Perea-García, A., Andrés-Bordería, A., Mayo de Andrés, S., Sanz, A., Davis, A. M., Davis, S. J., … Peñarrubia, L. (2015). Modulation of copper deficiency responses by diurnal and circadian rhythms inArabidopsis thaliana. Journal of Experimental Botany, 67(1), 391-403. doi:10.1093/jxb/erv474Perea-García, A., Garcia-Molina, A., Andrés-Colás, N., Vera-Sirera, F., Pérez-Amador, M. A., Puig, S., & Peñarrubia, L. (2013). Arabidopsis Copper Transport Protein COPT2 Participates in the Cross Talk between Iron Deficiency Responses and Low-Phosphate Signaling. Plant Physiology, 162(1), 180-194. doi:10.1104/pp.112.212407Perea-García, A., Sanz, A., Moreno, J., Andrés-Bordería, A., de Andrés, S. M., Davis, A. M., … Peñarrubia, L. (2016). Daily rhythmicity of high affinity copper transport. Plant Signaling & Behavior, 11(3), e1140291. doi:10.1080/15592324.2016.1140291Pilon-Smits, E. A. H. (2002). Characterization of a NifS-Like Chloroplast Protein from Arabidopsis. Implications for Its Role in Sulfur and Selenium Metabolism. PLANT PHYSIOLOGY, 130(3), 1309-1318. doi:10.1104/pp.102.010280Pruneda-Paz, J. L., Breton, G., Para, A., & Kay, S. A. (2009). A Functional Genomics Approach Reveals CHE as a Component of the Arabidopsis Circadian Clock. Science, 323(5920), 1481-1485. doi:10.1126/science.1167206Puig, S. (2014). Function and Regulation of the Plant COPT Family of High-Affinity Copper Transport Proteins. Advances in Botany, 2014, 1-9. doi:10.1155/2014/476917Rae, T. D. (1999). Undetectable Intracellular Free Copper: The Requirement of a Copper Chaperone for Superoxide Dismutase. Science, 284(5415), 805-808. doi:10.1126/science.284.5415.805Ravet, K., & Pilon, M. (2013). Copper and Iron Homeostasis in Plants: The Challenges of Oxidative Stress. Antioxidants & Redox Signaling, 19(9), 919-932. doi:10.1089/ars.2012.5084Rawat, R., Xu, Z.-F., Yao, K.-M., & Chye, M.-L. (2005). Identification of cis-elements for ethylene and circadian regulation of the Solanum melongena gene encoding cysteine proteinase. Plant Molecular Biology, 57(5), 629-643. doi:10.1007/s11103-005-0954-7RODRIGO-MORENO, A., ANDRÉS-COLÁS, N., POSCHENRIEDER, C., GUNSÉ, B., PEÑARRUBIA, L., & SHABALA, S. (2012). Calcium- and potassium-permeable plasma membrane transporters are activated by copper inArabidopsisroot tips: linking copper transport with cytosolic hydroxyl radical production. Plant, Cell & Environment, 36(4), 844-855. doi:10.1111/pce.12020Rogers, H. J., Bate, N., Combe, J., Sullivan, J., Sweetman, J., Swan, C., … Twell, D. (2001). Plant Molecular Biology, 45(5), 577-585. doi:10.1023/a:1010695226241Rubio-Somoza, I., Zhou, C.-M., Confraria, A., Martinho, C., von Born, P., Baena-Gonzalez, E., … Weigel, D. (2014). Temporal Control of Leaf Complexity by miRNA-Regulated Licensing of Protein Complexes. Current Biology, 24(22), 2714-2719. doi:10.1016/j.cub.2014.09.058Salomé, P. A., Oliva, M., Weigel, D., & Krämer, U. (2012). Circadian clock adjustment to plant iron status depends on chloroplast and phytochrome function. The EMBO Journal, 32(4), 511-523. doi:10.1038/emboj.2012.330Sancenón, V., Puig, S., Mateu-Andrés, I., Dorcey, E., Thiele, D. J., & Peñarrubia, L. (2004). TheArabidopsisCopper Transporter COPT1 Functions in Root Elongation and Pollen Development. Journal of Biological Chemistry, 279(15), 15348-15355. doi:10.1074/jbc.m313321200Sancenón, V., Puig, S., Mira, H., Thiele, D. J., & Peñarrubia, L. (2003). Plant Molecular Biology, 51(4), 577-587. doi:10.1023/a:1022345507112Seila, A. C., Calabrese, J. M., Levine, S. S., Yeo, G. W., Rahl, P. B., Flynn, R. A., … Sharp, P. A. (2008). Divergent Transcription from Active Promoters. Science, 322(5909), 1849-1851. doi:10.1126/science.1162253Takeda, T., Amano, K., Ohto, M., Nakamura, K., Sato, S., Kato, T., … Ueguchi, C. (2006). RNA Interference of the Arabidopsis Putative Transcription Factor TCP16 Gene Results in Abortion of Early Pollen Development. Plant Molecular Biology, 61(1-2), 165-177. doi:10.1007/s11103-006-6265-9Terzaghi, W. B., & Cashmore, A. R. (1995). Photomorphenesis: Seeing the light in plant development. Current Biology, 5(5), 466-468. doi:10.1016/s0960-9822(95)00092-3Uberti-Manassero, N. G., Coscueta, E. R., & Gonzalez, D. H. (2016). Expression of a repressor form of the Arabidopsis thaliana transcription factor TCP16 induces the formation of ectopic meristems. Plant Physiology and Biochemistry, 108, 57-62. doi:10.1016/j.plaphy.2016.06.031Viola, I. L., Camoirano, A., & Gonzalez, D. H. (2015). Redox-Dependent Modulation of Anthocyanin Biosynthesis by the TCP Transcription Factor TCP15 during Exposure to High Light Intensity Conditions in Arabidopsis. Plant Physiology, 170(1), 74-85. doi:10.1104/pp.15.01016Viola, I. L., Guttlein, L. N., & Gonzalez, D. H. (2013). Redox Modulation of Plant Developmental Regulators from the Class I TCP Transcription Factor Family. PLANT PHYSIOLOGY, 162(3), 1434-1447. doi:10.1104/pp.113.216416Viola, I. L., Reinheimer, R., Ripoll, R., Manassero, N. G. U., & Gonzalez, D. H. (2011). Determinants of the DNA Binding Specificity of Class I and Class II TCP Transcription Factors. Journal of Biological Chemistry, 287(1), 347-356. doi:10.1074/jbc.m111.256271Wakano, C., Byun, J. S., Di, L.-J., & Gardner, K. (2012). The dual lives of bidirectional promoters. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1819(7), 688-693. doi:10.1016/j.bbagrm.2012.02.006Wang, H., Mao, Y., Yang, J., & He, Y. (2015). TCP24 modulates secondary cell wall thickening and anther endothecium development. Frontiers in Plant Science, 6. doi:10.3389/fpls.2015.00436Wang, H.-Y., Klatte, M., Jakoby, M., Bäumlein, H., Weisshaar, B., & Bauer, P. (2007). Iron deficiency-mediated stress regulation of four subgroup Ib BHLH genes in Arabidopsis thaliana. Planta, 226(4), 897-908. doi:10.1007/s00425-007-0535-xWang, S., Sun, X., Hoshino, Y., Yu, Y., Jia, B., Sun, Z., … Zhu, Y. (2014). MicroRNA319 Positively Regulates Cold Tolerance by Targeting OsPCF6 and OsTCP21 in Rice (Oryza sativa L.). PLoS ONE, 9(3), e91357. doi:10.1371/journal.pone.0091357Weiss, D., & Ori, N. (2007). Mechanisms of Cross Talk between Gibberellin and Other Hormones. Plant Physiology, 144(3), 1240-1246. doi:10.1104/pp.107.100370Welchen, E., & Gonzalez, D. H. (2006). Overrepresentation of Elements Recognized by TCP-Domain Transcription Factors in the Upstream Regions of Nuclear Genes Encoding Components of the Mitochondrial Oxidative Phosphorylation Machinery. Plant Physiology, 141(2), 540-545. doi:10.1104/pp.105.075366Wu,

Desarrollo de un modelo experimental de estrés oxidativo in vivo

[ES]Los efectos nocivos de las especies reactivas de oxígeno (ROS) se producen durante la vida adulta , y se ha sugerido que el exceso de ROS- estrés oxidativo puede ser un factor que contribuye a los procesos neurodegenerativos . El glutatión ( GSH ) es uno de los más abundantes antioxidantes y , en enfermedades neurológicas tales como la enfermedad de Parkinson o trastornos mentales , la deficiencia de GSH es el conocido indicador bioquímico más temprana de degeneration.This neuronales observación ha llevado a la sugerencia de que el estrés oxidativo puede estar detrás de la causas de la disfunción neuronal asociada con estos trastornos neurológicos . Desafortunadamente , debido a la falta de una herramienta suficientemente robusto , el efecto específico de la deficiencia de GSH en la patogénesis de enfermedades neurológicas nunca se ha demostrado in vivo , por lo tanto el papel real de GSH estrés oxidativo pérdida mediada en estos trastornos sigue siendo difícil de alcanzar . El glutatión es un tripéptido ( g - glutamylcysteinilglycine ) sintetizado por dos reacciones dependientes de ATP consecutivos . El glutamato - cisteína ligasa ( GCL o sintetasa g - glutamilcisteína ; CE 6.3.2.2 ) cataliza la primera y limitante de la velocidad a paso , formando g - glutamilcisteína a partir del glutamato y cisteína . Esto es seguido por la glutatión sintetasa (CE 6.3.2.3 ) - reacción catalizada , que se une a G glicina - glutamilcisteína , la formación de glutatión . GCL es una enzima heterodimérica compuesta de un catalizador (pesado , 73 kDa ) y un modulador ( luz, 27,7 kDa ) subunidad . Estudios realizados con purificada GCL han demostrado que el sitio activo reside en la subunidad catalítica , mientras que la subunidad modulador aumenta la afinidad de la subunidad catalítica para el glutamato y disminuye la sensibilidad a la inhibición por retroalimentación por GSH . En el cerebro , donde - a lo mejor de nuestro , actividad GCL conocimiento - la enzima no ha sido purificada es muy débil , aunque es más alta en astrocitos en comparación con neurons.This contribuye a la mayor resistencia de los astrocitos , en comparación con las neuronas , contra el estrés oxidativo . Los astrocitos cooperan con las neuronas de la biosíntesis de GSH neuronal antioxidante mediante el suministro de los precursores de GSH . Por lo tanto , ya sea limitando el suministro de los precursores , o la capacidad de las neuronas para usarlos , provoca el estrés oxidativo en las neuronas que conducen a la neurodegeneración , por lo menos en la cultura . Esto nos ha llevado a la hipótesis de que desmontables neuronal específica y controlada temporalmente de GCL in vivo puede provocar una disfunción neurológica espontánea , lo que posiblemente imitando los problemas neurológicos asociados con el Parkinson o las enfermedades mentales y potencialmente útil para la identificación de proteínas redox-sensibles novedosos involucrado en trastornos neurológicos . Los modelos existentes in vivo para el estudio de GCL , subunidad catalítica , la deficiencia en el cerebro son escasos y fracasaron. Ratones knockout homocigotos contra GCL , subunidad catalítica , no son viables más allá del día 8 de embriones , y los heterocigotos presentan mecanismos de compensación como el aumento de la biosíntesis de ascorbato. Además , este sistema genético disponible no knockout tejido GCL específicamente o temporalmente controlada , siendo por tanto inadecuados para investigar el papel del estrés oxidativo en el sistema nervioso central durante la edad adulta . Nos habíamos identificado previamente una pequeña horquilla de ARN ( shRNA ) dirigido contra GCL , subunidad catalítica de que, en las neuronas cultivadas, desencadena el estrés oxidativo espontáneo. Hemos implementado estrategia de ARNi in vivo para producir un ratón de doble condicional que expresa la GCL shRNA en las células del sistema nervioso central , en particular las neuronas del hipocampo , para volver a crear el estrés oxidativo in vivo t tejido de manera específica y en inducible . Utilizamos la tecnología Cre - LoxP para crear ratones que expresan shGCL en las neuronas in vivo , y se caracterizaron bioquímicamente , inmuno - histológica y comportamiento. Se encontró una disminución en la GCL , y un aumento de los marcadores oxidativos . También encontramos efectos dependientes del sexo en la caracterización del comportamiento para las tareas de ansiedad , la capacidad motora y memoria. En conclusión , aquí se describe una estrategia novedosa para el estudio de estrés oxidativo in vivo de una manera específica y controlada en el tiempo el tejido . Este modelo puede abrir nuevas posibilidades para el estudio de la implicación de los elevados de ROS en las enfermedades mentales , como la enfermedad o la ansiedad de Alzheimer, que son intrínsecos a una serie de trastornos psiquiátricos como la depresión , ataques de pánico , fobias, trastorno obsesivo -compulsivo y el estrés postraumático , sin embargo , estas enfermedades carecen actualmente de apropiado en modelos in vivo para la investigación de nuevas estrategias terapéuticas. Además, puesto que hemos diseñado la herramienta genética con dos sitios de restricción únicos que flanquean la secuencia de ARNhc , nuevos modelos de ratones transgénicos podrían ser sencilla generada a desmontables cualquier otra proteína específica de tejido in vivo . Creemos que el modelo de ratón transgénico aquí descrito puede ser útil para una mejor comprensión de las consecuencias del estrés oxidativo en células específicas in vivo , así como para la evaluación de nuevos enfoques farmacológicos.[EN]The deleterious effects of reactive oxygen species (ROS) occur during adulthood, and it has been suggested that excess ROS -oxidative stress- may be a contributing factor in neurodegenerative processes. Glutathione (GSH) is one of the most abundant antioxidants and, in neurological diseases such as Parkinson's disease or mental disorders, GSH deficiency is the earliest known biochemical indicator of neuronal degeneration.This observation has led to the suggestion that oxidative stress may be behind the causes of neuronal dysfunction associated with these neurological disorders. Unfortunately, due to the lack of a sufficiently robust tool, the specific effect of GSH deficiency in the pathogenesis of neurological diseases has never been shown in vivo, thus the actual role of GSH loss-mediated oxidative stress in these disorders still remains elusive. Glutathione is a tripeptide (g-glutamylcysteinilglycine) synthesized by two consecutive ATP-dependent reactions. Glutamate-cysteine ligase (GCL or g-glutamylcysteine synthetase; EC 6.3.2.2) catalyzes the first -and rate-limiting- step, forming g-glutamylcysteine from glutamate and cysteine. This is followed by the glutathione synthetase (EC 6.3.2.3)-catalyzed reaction, which binds glycine to g-glutamylcysteine, forming glutathione. GCL is a heterodimeric enzyme composed of a catalytic (heavy; 73 kDa) and a modulatory (light; 27.7 kDa) subunit. Studies performed with purified GCL have shown that the active site resides at the catalytic subunit, whereas the modulatory subunit increases the affinity of the catalytic subunit for glutamate and decreases the sensitivity to feedback inhibition by GSH . In the brain, where -to the best of our knowledge- the enzyme has never been purified, GCL activity is very weak, although it is higher in astrocytes when compared with neurons.This contributes to the higher resistance of astrocytes, when compared with neurons, against oxidative stress. Astrocytes co-operate with neurons for neuronal antioxidant GSH biosynthesis by supplying GSH precursors. Thus, limiting either the supply of precursors, or the ability of neurons to use them, triggers oxidative stress in neurons leading to neurodegeneration, at least in culture. This has led us to hypothesize that neuronal-specific and temporally-controlled knockdown of GCL in vivo may lead to spontaneous neurological dysfunction, thus possibly mimicking the neurological problems associated with Parkinson's or mental diseases and potentially useful for the identification of novel redox-sensitive proteins involved in neurological disorders. The existing in vivo models for studying GCL, catalytic subunit, deficiency in the brain are scarce and failed. Homozygous knockout mice against GCL, catalytic subunit, are not viable beyond embryonic day 8th, and the heterozygous ones display compensation mechanisms such as increased ascorbate biosynthesis. In addition, this available genetic system does not knockout GCL tissue specifically or temporally controlled, thus being unsuitable to investigate the role of oxidative stress in central nervous system during adulthood. We had previously identified a small hairpin RNA (shRNA) targeted against GCL, catalytic subunit that, in cultured neurons, triggered spontaneous oxidative stress. We have implemented RNAi strategy in vivo to produce a double-conditional mouse expressing the GCL shRNA in the cells of the central nervous system, particularly hippocampal neurons, to recreate oxidative stress in vivo t tissue specific and inducible way. We used Cre-LoxP technology to create mice expressing shGCL in neurons in vivo, and they were characterized biochemically, immuno-histologically and behaviorally. We found a decrease in GCL, and an increase in oxidative markers. We also found sex-dependent effects in behavioural characterization for anxiety, motor ability and memory tasks. In conclusion, here we describe a novel strategy for studying oxidative stress in vivo in a tissue specific and time-controlled manner. This model may open new possibilities to study the involvement of elevated ROS in mental illnesses, such as Alzheimer's disease or anxiety, which are intrinsic to a number of psychiatric disorders including depression, panic attacks, phobias, obsessive-compulsive disorder and post-traumatic stress; however, these diseases currently lack of appropriate in vivo models for research on new therapeutic strategies. Furthermore, since we designed the genetic tool with two unique restriction sites flanking the shRNA sequence, new transgenic mice models could be straightforward generated to knockdown any other protein tissue-specifically in vivo. We believe that the transgenic mouse model herein described may be useful for a better understanding of the consequences of oxidative stress in specific cells in vivo, as well as for assessing novel pharmacological approaches

Actividad estafilocócica de las endolisinas LysRODI y LysRODI¿Ami en leche y queso fresco

Trabajo presentado en la VII Reunión Red Española de Bacteriófagos y Elementos Transductores - RED FAGOMA, celebrada en Trujillo, Cáceres (España), del 26 al 28 de octubre de 202

Deletion of the amidase domain of endolysin LysRODI enhances antistaphylococcal activity in milk and during fresh cheese production

Milk contamination with Staphylococcus aureus can lead to food poisoning in consumers. One strategy to minimize this risk is the use of phage-derived lysins, which are innocuous for humans and do not readily select for resistant variants. However, it remains necessary to find new candidate lysins and define the conditions for their utilization. This study compares the potential of LysRODI and its derivative LysRODIΔAmi (lacking the amidase domain), which displays high activity and storage stability, to successfully decrease staphylococcal contamination in milk under different conditions. Our results show that the engineered protein is more efficacious than the parent endolysin in practically all cases. For instance, while LysRODI only decreased the number of cells by 1–2 log units in different types of commercial milk and contamination levels, the chimeric lysin eliminated them below detection. Also, LysRODIΔAmi was more active against four strains with varying degrees of susceptibility. Regarding incubation temperature, both proteins were faster at 32 °C and 37 °C. Significantly, the engineered lysin eliminated detectable contamination in just 15 min. Finally, LysRODIΔAmi proved very successful at reducing staphylococcal contamination below detection during lab-scale fresh cheese production by enzymatic coagulation. Our data show that LysRODIΔAmi is a promising candidate for biocontrol in milk.This work was funded by grants PID2019-105311 RB-I00 (MICIU/AEI/FEDER, EU, Spain501100011033) to P.G. and A.R, and AYUD/2021/52120 (Program of Science, Technology and Innovation, 2021–2023 and FEDER EU, Principado de Asturias, Spain). S.A. has an FPI fellowship (Ministry of Science and Innovation, Spain)

Staphylococcus aureus biofilms formed in milk display resistance to the phage lytic protein LysRODI¿Ami

Trabajo presentado en la ASM Conference on Biofilms, celebrada en Charlotte, NC (Estados Unidos), del 13 al 17 de noviembre de 202

Reversible assembly of enantiomeric helical polymers: from fibers to gels

A novel class of stereocomplexes is described by the interaction of helically complementary poly(phenylacetylene)s (PPAs) carrying an a-methoxy-a-trifluoromethylphenylacetamide pendant group. The formation of the stereocomplex requires the presence of cis amide bonds on the external crest of the polymer to provide efficient cooperative supramolecular hydrogen bonding between matching enantiomeric helical structures. The interlocking of the chains gives rise to supramolecular fiber-like aggregates that, at higher concentrations, result in gels. The modification of the cis–trans amide conformation at the pendant groups allows the controlled formation and cleavage of the stereocomplex due to a dramatic change between the intermolecular and intramolecular hydrogen bond interactionsThis work was supported by grants from Ministerio de Ciencia e Innovación [CTQ2009-08632/BQU, CTQ2012-33436, CTQ2012-31381], and Xunta de Galicia (PGIDIT09CSA029209PR, CN2011/037, EM2013/0032S