Partial Nitrification of Wastewater from an Aminoplastic Resin Producing Factory

Nitrification via nitrite was studied in two aerobic reactors treating wastewater from an aminoplastic resin producing factory at HRT varying between 1.37-1.89 and 2.45-3.63 days. Both reactors were fed with concentrations of 366, 450, 1099 and 1899 mg N-NH4+/L. In general in the reactor operated at a lower HRT, the nitritation percentage decreased from 87.2 to 21.6%, while the nitratation percentage remained always lower than 2.5% (except in the last period) when the ammonium concentration was increased. This behaviour could be due to the inhibition of the ammonium and nitrite oxidation produced by high free ammonia concentrations up to 179.3 mg N-NH3/L. In the reactor operated at a higher HRT, the nitritation percentage decreased and the nitratation percentage increased from 88.6 to 39.6% and from 0.65 to 35.7%, respectively, due to an increase of the dissolved oxygen concentration from 0.76 to 1.02 mg O2/L. However, when ammonium was fed at a concentration of 1898.7 mg N-NH4+/L, the nitritation increased and the nitratation decreased, probably as a result of the accumulation of free ammonia up to 2.04 mg N-NH3/L, meaning that nitrite oxidizers were inhibited. Nitrite build-up was observed after each modification of ammonium concentration in the feed

Innovación en los talleres de entrenamiento de memoria. Evolución y procesos de mejora de la Fundación Pere Tarrés

La pèrdua de la memòria és una de les preocupacions de les persones grans. Des de fa uns anys, els casals i els equipaments per a la gent gran realitzen una activitat de caràcter socioeducatiu anomenada tallers de memòria, que té com a objectiu proporcionar els coneixements i recursos perquè les persones grans puguin entrenar satisfactòriament aquesta funció cognitiva i millorar-ne el rendiment. Els últims anys, les necessitats de les persones grans i les demandes de les organitzacions que imparteixen aquest tipus d’activitats han anat variant. Conscients d’aquest situació, un equip, constituït per docents i tècnics de casals de la Fundació Pere Tarrés, ens plantejàrem iniciar un procés d’innovació per adequar els tallers d’entrenament de la memòria a les persones grans d’avui.Memory loss is one of the concerns of the elderly. For a number of years now, facilities for older people have been running memory workshops as a socio-educational activity that aims to provide the knowledge and resources to enable seniors to train and improve the performance of this cognitive function successfully. In recent years, the needs of older people and the demands of the organizations that provide these types of activities have changed. In response to this situation, a team of teachers and technicians from the Pere Tarrés Foundation is preparing to initiate a process of innovation to adapt training workshops to the needs of older people of today.La pérdida de la memoria es una de las preocupaciones de las personas mayores. Desde hace algunos años, los casales y los equipamientos para gente mayor realizan una actividad de carácter socioeducativo denominada talleres de memoria, que tiene como objetivo proporcionar los conocimientos y recursos para que las personas mayores puedan entrenar satisfactoriamente esta función cognitiva y mejorar su rendimiento. En los últimos años, las necesidades de las personas mayores y las demandas de las organizaciones que imparten este tipo de actividades han ido variando. Conscientes de esta situación, un equipo constituido por docentes y técnicos de casales de la Fundación Pere Tarrés, nos planteamos iniciar un proceso de innovación para adecuar los talleres de entrenamiento a la gente mayor de hoy

Projecting wildfire occurrence at regional scale from Land Use/Cover and climate change scenarios

LUC4FIRE: Project funded by the Spanish Ministry of Science, Innovation and Universities (CSO2015-73407-JIN)

Importance of the vaporization margin during CO2 laser treatment of oral leukoplakia: a survival study

Objectives: the main purpose of this study was to assess the response of oral leukoplakia to CO2 laser vaporization treatment, as well as determining possible factors that may affect recurrence of lesions. Materials and methods: a retrospective study was conducted, in which the medical records of patients who had been clinically and histologically diagnosed with oral leukoplakia and treated with CO2 laser between 1996 and 2019 at the Oral Medicine Teaching Unit of the Faculty of Dentistry of the University of Santiago de Compostela were reviewed. Results: fifty-eight patients were included: 36 female and 22 male subjects, with a mean age of 63.7 years old (SD ±13.1). The average follow-up time was 57.5 months (SD ±57.9). A relapse rate of 52.6% was determined. Of all the studied variables, the margin was the only one for which a statistically significant association with recurrence of lesions was demonstrated (p < 0.05). Conclusion: the vaporization of lesions using CO2 laser with a safety margin of at least 3 mm from the clinical limits of OL is a key factor in preventing recurrenceS

Efficient Cas9 multiplex editing using unspaced sgRNA arrays engineering in a Potato virus X vector

[EN] Systems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR-associated proteins (Cas) have revolutionized genome editing in many organisms, including plants. Most CRISPR-Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as Cas nucleases or the synthetic guide RNA (sgRNA). While Cas nucleases are constant elements in editing approaches, sgRNAs are target-specific and a screening process is usually required to identify those most effective. Plant virus-derived vectors are an alternative for the fast and efficient delivery of sgRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus-induced genome editing. We engineered Potato virus X (PVX) to build a vector that easily expresses multiple sgRNAs in adult solanaceous plants. Using the PVX-based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expresses Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced sgRNAs, achieving highly efficient multiplex editing in a few days in adult plant tissues. Moreover, virus-free edited progeny can be obtained from plants regenerated from infected tissues or infected plant seeds, which exhibit a high rate of heritable biallelic mutations. In conclusion, this new PVX vector allows easy, fast and efficient expression of sgRNA arrays for multiplex CRISPR-Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in Solanaceae crops.This work was supported by grants BIO2017-83184-R and PID2019-108203RB-I00 from Ministerio de Ciencia e Innovacion (Spain) through the Agencia Estatal de Investigacion (co-financed European Regional Development Fund), and H2020-760331 Newcotiana from the European Commission. M.U. and S.S. are the recipients of fellowships FPU17/05503 and BES-2017-0890098, respectively, from Ministerio de Ciencia e Innovacion (Spain)Uranga-Ruiz De Eguino, M.; Aragones, V.; Selma García, S.; Vázquez-Vilar, M.; Orzáez Calatayud, DV.; Daròs, J. (2021). Efficient Cas9 multiplex editing using unspaced sgRNA arrays engineering in a Potato virus X vector. The Plant Journal. 106(2):555-565. https://doi.org/10.1111/tpj.15164555565106

A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana

[EN] Background CRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS). Results In this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programmable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold and 245-fold for the endogenous N. benthamiana DFR and PAL2 genes, respectively, with negligible expression in the absence of the trigger. Conclusions The tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.This work has been funded by EU Horizon 2020 Project Newcotiana Grant 760331, PID2019-108203RB-100 Plan Nacional I + D, Spanish Ministry of Economy and Competitiveness and 'ERACoBioTech' Project SUSPHIRE Grant No. 722361. Vazquez-Vilar, M. is recipient of APOSTD/2020/096 (Generalitat Valenciana and Fondo Social Europeo post-doctoral grant). Garcia-Perez, E. is recipient of ACIF-2020 fellowship (Generalitat Valenciana). Diego-Martin, B. and Moreno-Gimenez, E. are recipients of FPU fellowships. Selma, S. is recipient of FPI fellowship.García-Pérez, E.; Diego-Martín, B.; Quijano-Rubio, A.; Moreno-Giménez, E.; Selma, S.; Orzáez Calatayud, DV.; Vázquez-Vilar, M. (2022). A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana. BMC Biotechnology. 22(1):1-13. https://doi.org/10.1186/s12896-022-00741-x11322

Noninvasive detection of microsatellite instability in patients with endometrial cancer

Endometrial cancer; Liquid biopsy; Uterine aspirateCàncer d'endometri; Biòpsia líquida; Aspirat uteríCáncer de endometrio; Biopsia líquida; Aspirado uterinoThe analysis of mismatch repair proteins in solid tissue is the standard of care (SoC) for the microsatellite instability (MSI) characterization in endometrial cancer (EC). Uterine aspirates (UAs) or circulating-DNA (cfDNA) samples capture the intratumor heterogeneity and provide a more comprehensive and dynamic molecular diagnosis. Thus, MSI analysis by droplet-digital PCR (ddPCR) in UAs and cfDNA can provide a reliable tool to characterize and follow-up the disease. The UAs, paraffin-embedded tumor tissue (FFPE) and longitudinal plasma samples from a cohort of 90 EC patients were analyzed using ddPCR panel and compared to the SoC. A high concordance (96.67%) was obtained between the analysis of MSI markers in UAs and the SoC. Three discordant cases were validated as unstable by ddPCR on FFPE samples. Besides, a good overall concordance (70.27%) was obtained when comparing the performance of the ddPCR assay on UAs and cfDNA in high-risk tumors. Importantly, our results also evidenced the value of MSI analysis to monitor the disease evolution. MSI evaluation in minimally invasive samples shows great accuracy and sensitivity and provides a valuable tool for the molecular characterization and follow-up of endometrial tumors, opening new opportunities for personalized management of EC.Centro de Investigación Biomédica en Red de Cáncer, Grant/Award Numbers: CB16/12/00295, CB16/12/00328; Fundación Científica Asociación Española Contra el Cáncer, Grant/Award Numbers: FC_AECC PROYE19036MOR, 2018-AECC, INVES20051COLA; Fundación Instituto de Investigación Sanitaria de Santiago de Compostela; Instituto de Salud Carlos III and FEDER, Grant/Award Numbers: CM19/00087, CP20/00119, PI20/00969, PI20/01566, PI21/00990; Spanish Ministry of Economy and Innovation, Grant/Award Number: PID2019-104644RB-I0

Multigene Engineering by GoldenBraid Cloning: From Plants to Filamentous Fungi and Beyond

This is the peer reviewed version of the following article: Vazquez-Vilar, M., Gandía, M., García-Carpintero, V., Marqués, E., Sarrion-Perdigones, A., Yenush, L., Polaina, J., Manzanares, P., Marcos, J. F., & Orzaez, D. (2020). Multigene engineering by goldenbraid cloning: from plants to filamentous fungi and beyond. Current Protocols in Molecular Biology, 130, e116, doi: 10.1002/cpmb.116, which has been published in final form at https://doi.org/10.1002/cpmb.116. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] Many synthetic biologists have adopted methods based on Type IIS restriction enzymes and Golden Gate technology in their cloning procedures, as these enable the combinatorial assembly of modular elements in a very efficient way following standard rules. GoldenBraid (GB) is a Golden Gate¿based modular cloning system that, in addition, facilitates the engineering of large multigene constructs and the exchange of DNA parts as result of its iterative cloning scheme. GB was initially developed specifically for plant synthetic biology, and it has been subsequently extended and adapted to other organisms such as Saccharomyces cerevisiae, filamentous fungi, and human cells by incorporating a number of host¿specific features into its basic scheme. Here we describe the general GB cloning procedure and provide detailed protocols for its adaptation to filamentous fungi¿a GB variant known as FungalBraid. The assembly of a cassette for gene disruption by homologous recombination, a fungal¿specific extension of the GB utility, is also shown. Development of FungalBraid was relatively straightforward, as both plants and fungi can be engineered using the same binary plasmids via Agrobacterium¿mediated transformation. We also describe the use of a set of web¿based tools available at the GB website that assist users in all cloning procedures. The availability of plant and fungal versions of GB will facilitate genetic engineering in these industrially relevant organisms.This article is dedicated to the memory of our friend and colleague Dr. Alejandro Sarrion-Perdigones, an early developer of GoldenBraid. We acknowledge the excellent technical assistance provided by Marisol Gascón (IBMCP, Valencia, Spain) with the fluorescent images. This work was funded by Grant BIO2013- 42193 and Grant BIO2016-78601-R, Plan Nacional I+D, Spanish Ministry of Economy and Competitiveness, RTI2018-101115-B-C21 from the Ministerio de Ciencia, Innovación y Universidades (Spain) (MICINN/FEDER Funds), and PROMETEO/ 2018/066 from Conselleria d'Educació (Generalitat Valenciana, Comunitat Valenciana, Spain) and SUSPHIRE PCI2018- 092893-ERA CoBioTech (109) (MCIU/FEDER).Vázquez-Vilar, M.; Gandía, M.; García-Carpintero, V.; Marqués, E.; Sarrion-Perdigones, A.; Yenush, L.; Polaina, J.... (2020). Multigene Engineering by GoldenBraid Cloning: From Plants to Filamentous Fungi and Beyond. Current Protocols in Molecular Biology. 130(1):1-31. https://doi.org/10.1002/cpmb.116S1311301Bernabé‐Orts, J. M., Casas‐Rodrigo, I., Minguet, E. G., Landolfi, V., Garcia‐Carpintero, V., Gianoglio, S., … Orzaez, D. (2019). Assessment of Cas12a‐mediated gene editing efficiency in plants. Plant Biotechnology Journal, 17(10), 1971-1984. doi:10.1111/pbi.13113Ballester, A.-R., Marcet-Houben, M., Levin, E., Sela, N., Selma-Lázaro, C., Carmona, L., … Gabaldón, T. (2015). Genome, Transcriptome, and Functional Analyses of Penicillium expansum Provide New Insights Into Secondary Metabolism and Pathogenicity. Molecular Plant-Microbe Interactions®, 28(3), 232-248. doi:10.1094/mpmi-09-14-0261-fiKhang, C. H., Park, S.-Y., Lee, Y.-H., & Kang, S. (2005). A dual selection based, targeted gene replacement tool for Magnaporthe grisea and Fusarium oxysporum. Fungal Genetics and Biology, 42(6), 483-492. doi:10.1016/j.fgb.2005.03.004Chen, C., Liu, J., Duan, C., Pan, Y., & Liu, G. (2020). Improvement of the CRISPR-Cas9 mediated gene disruption and large DNA fragment deletion based on a chimeric promoter in Acremonium chrysogenum. Fungal Genetics and Biology, 134, 103279. doi:10.1016/j.fgb.2019.103279Bai Flagfeldt, D., Siewers, V., Huang, L., & Nielsen, J. (2009). Characterization of chromosomal integration sites for heterologous gene expression inSaccharomyces cerevisiae. Yeast, 26(10), 545-551. doi:10.1002/yea.1705Fräbel, S., Wagner, B., Krischke, M., Schmidts, V., Thiele, C. M., Staniek, A., & Warzecha, H. (2018). Engineering of new-to-nature halogenated indigo precursors in plants. Metabolic Engineering, 46, 20-27. doi:10.1016/j.ymben.2018.02.003Fresquet-Corrales, S., Roque, E., Sarrión-Perdigones, A., Rochina, M., López-Gresa, M. P., Díaz-Mula, H. M., … Cañas, L. A. (2017). Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp. PLOS ONE, 12(9), e0184839. doi:10.1371/journal.pone.0184839Garrigues, S., Gandía, M., & Marcos, J. F. (2015). Occurrence and function of fungal antifungal proteins: a case study of the citrus postharvest pathogen Penicillium digitatum. Applied Microbiology and Biotechnology, 100(5), 2243-2256. doi:10.1007/s00253-015-7110-3Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343-345. doi:10.1038/nmeth.1318Gurgel, I. L. da S., Jorge, K. T. de O. S., Malacco, N. L. S. de O., Souza, J. A. M., Rocha, M. C., Fernandes, M. F., … Soriani, F. M. (2019). The Aspergillus fumigatus Mucin MsbA Regulates the Cell Wall Integrity Pathway and Controls Recognition of the Fungus by the Immune System. mSphere, 4(3). doi:10.1128/msphere.00350-19Hernanz-Koers, M., Gandía, M., Garrigues, S., Manzanares, P., Yenush, L., Orzaez, D., & Marcos, J. F. (2018). FungalBraid: A GoldenBraid-based modular cloning platform for the assembly and exchange of DNA elements tailored to fungal synthetic biology. Fungal Genetics and Biology, 116, 51-61. doi:10.1016/j.fgb.2018.04.010Juarez, P., Huet-Trujillo, E., Sarrion-Perdigones, A., Falconi, E., Granell, A., & Orzaez, D. (2013). Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants. International Journal of Molecular Sciences, 14(3), 6205-6222. doi:10.3390/ijms14036205Kramer, M. F., & Coen, D. M. (2001). Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization. Current Protocols in Molecular Biology. doi:10.1002/0471142727.mb1501s56Marcet-Houben, M., Ballester, A.-R., de la Fuente, B., Harries, E., Marcos, J. F., González-Candelas, L., & Gabaldón, T. (2012). Genome sequence of the necrotrophic fungus Penicillium digitatum, the main postharvest pathogen of citrus. BMC Genomics, 13(1). doi:10.1186/1471-2164-13-646Michielse, C. B., J Hooykaas, P. J., J J van den Hondel, C. A. M., & J Ram, A. F. (2008). Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori. Nature Protocols, 3(10), 1671-1678. doi:10.1038/nprot.2008.154Müller, K. M., & Arndt, K. M. (2011). Standardization in Synthetic Biology. Synthetic Gene Networks, 23-43. doi:10.1007/978-1-61779-412-4_2Patron, N. J., Orzaez, D., Marillonnet, S., Warzecha, H., Matthewman, C., Youles, M., … Rogers, C. (2015). Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist, 208(1), 13-19. doi:10.1111/nph.13532Pérez-González, A., Kniewel, R., Veldhuizen, M., Verma, H. K., Navarro-Rodríguez, M., Rubio, L. M., & Caro, E. (2017). Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of heterologous proteins in yeast mitochondria. BMC Biotechnology, 17(1). doi:10.1186/s12896-017-0393-yPérez-Nadales, E., & Di Pietro, A. (2011). The Membrane Mucin Msb2 Regulates Invasive Growth and Plant Infection in Fusarium oxysporum  . The Plant Cell, 23(3), 1171-1185. doi:10.1105/tpc.110.075093Salazar-Cerezo, S., Kun, R. S., de Vries, R. P., & Garrigues, S. (2020). CRISPR/Cas9 technology enables the development of the filamentous ascomycete fungus Penicillium subrubescens as a new industrial enzyme producer. Enzyme and Microbial Technology, 133, 109463. doi:10.1016/j.enzmictec.2019.109463Sarrion-Perdigones, A., Chang, L., Gonzalez, Y., Gallego-Flores, T., Young, D. W., & Venken, K. J. T. (2019). Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying. Nature Communications, 10(1). doi:10.1038/s41467-019-13651-ySarrion-Perdigones, A., Falconi, E. E., Zandalinas, S. I., Juárez, P., Fernández-del-Carmen, A., Granell, A., & Orzaez, D. (2011). GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules. PLoS ONE, 6(7), e21622. doi:10.1371/journal.pone.0021622Sarrion-Perdigones, A., Vazquez-Vilar, M., Palaci, J., Castelijns, B., Forment, J., Ziarsolo, P., … Orzaez, D. (2013). GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology. 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A Modular Cloning Toolbox for the Generation of Chloroplast Transformation Vectors. PLoS ONE, 9(10), e110222. doi:10.1371/journal.pone.0110222Vazquez-Vilar, M., Bernabé-Orts, J. M., Fernandez-del-Carmen, A., Ziarsolo, P., Blanca, J., Granell, A., & Orzaez, D. (2016). A modular toolbox for gRNA–Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods, 12(1). doi:10.1186/s13007-016-0101-2Villiers, B. R. M., Stein, V., & Hollfelder, F. (2009). USER friendly DNA recombination (USERec): a simple and flexible near homology-independent method for gene library construction. Protein Engineering, Design and Selection, 23(1), 1-8. doi:10.1093/protein/gzp063Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE, 6(2), e16765. doi:10.1371/journal.pone.0016765Sarrion‐Perdigones et al. (2013). See above.Hernanz‐Koersetal. (2018). See above.https://gbcloning.upv.es/https://benchling.co

Effect of sex in systemic psoriasis therapy: Differences in prescription, effectiveness and safety in the BIOBADADERM prospective cohort

The effect of sex on systemic therapy for psoriasis has not been well studied. The aim of this study was to analyse a large multicentre Spanish cohort of 2,881 patients with psoriasis (58.3% males), followed from January 2008 to November 2018, to determine whether sex influences prescription, effectiveness of therapy, and the risk of adverse events. The results show that women are more likely than men to be pre-scribed biologics. There were no differences between men and women in effectiveness of therapy, measur-ed in terms of drug survival. Women were more likely to develop adverse events, but the difference in risk was small and does not justify different management. Study limitations include residual confounding and the use of drug survival as a proxy for effectiveness.The BIOBADADERM project is promoted by the Fundación Piel Sana Academia Española de Dermatología y Venereología, which receives financial support from the Spanish Medicines and Health Products Agency (Agencia Española de Medicamentos y Productos Sanitarios) and from pharmaceutical companies (Abbott/Abbvie, Pfizer, MSD, Novartis, Lilly, Janssen and Almirall)