A Versatile Chemoenzymatic Nanoreactor that Mimics NAD(P)H Oxidase for the In Situ Regeneration of Cofactors

Herein, we report a multifunctional chemoenzymatic nanoreactor (NanoNOx) for the glucose-controlled regeneration of natural and artificial nicotinamide cofactors. NanoNOx are built of glucose oxidase-polymer hybrids that assemble in the presence of an organometallic catalyst: hemin. The design of the hybrid is optimized to increase the effectiveness and the directional channeling at low substrate concentration. Importantly, NanoNOx can be reutilized without affecting the catalytic properties, can show high stability in the presence of organic solvents, and can effectively oxidize assorted natural and artificial enzyme cofactors. Finally, the hybrid was successfully coupled with NADH-dependent dehydrogenases in one-pot reactions, using a strategy based on the sequential injection of a fuel, namely, glucose. Hence, this study describes the first example of a hybrid chemoenzymatic nanomaterial able to efficiently mimic NOx enzymes in cooperative one-pot cascade reactions.The authors gratefully acknowledge the financial support from the Spanish Research Agency (AEI) for the financial support (PID2019-110239RB-I00 from I+D call and RYC2018-025923-I from RyC program), including FEDER funds; IKERBASQUE-Basque Foundation for Science, BBVA foundation (Leonardo Fellowships, IN[21]_CBB_QUI_0086) and Maria de Maeztu Units of Excellence Programme: Grant Number CEX2020-001038-M. AR-A thanks the Basque Government for his Ph.D. fellowship (PRE_2021_2_0151). Open Access funding is provided by the University of the Basque Country (UPV/EHU)

Enzymatic upgrading of nanochitin using an ancient lytic polysaccharide monooxygenase

Numerous enzymes have the potential to upgrade biomass, converting it into high-tech materials for new applications. However, the features of natural enzymes often limit their use beyond chemical conversion of the substrate. The development of strategies for the enzymatic conversion of biomass into high-value materials may broaden the range of applications of enzymes and enzyme design techniques. A relevant case is lytic polysaccharide monooxygenase (LPMO), a class of enzymes that catalyzes the oxidative cleavage of glycosidic bonds. Here, we show that an ancestral LPMO can generate chitin nanocrystals. Physicochemical characterization of the chitin nanocrystals demonstrates modifications that make it superior compared to chitin obtained by chemical treatments. We show that the nanocrystals are suitable for controlled 2D and 3D cell cultures, as well as for engineering a biomatrix that combines with graphene oxide, forming a hybrid conductive bioink.This work has been supported by grants PID2019-109087RB-I00 to R.P.-J. from Spanish Ministry of Science and Innovation. This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 964764 to R.P.-J. ‘Materials + Technologies’ Research Group also acknowledges UPV/EHU and the Basque Government in the frame of “Research Group” (GIU 18/216) and “Grupos Consolidados” (IT776-13), respectively. We also thank Gipuzkoako Foru Aldundia for financial Support. HEK293T cells were a kind gift from Dr. Maria Muñoz Caffarel (Biodonostia, San Sebastian, Spain)

Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR–Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.This work has been supported by grant nos. PID2019-109087RB-I00 (to R.P.-J.) and RTI2018-101223-B-I00 and PID2021-127644OB-I00 (to L.M.) from the Spanish Ministry of Science and Innovation. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 964764 (to R.P.-J.). The content presented in this document represents the views of the authors, and the European Commission has no liability in respect to the content. We acknowledge financial support from the Spanish Foundation for the Promotion of Research of Amyotrophic Lateral Sclerosis. A.F. acknowledges Spanish Center for Biomedical Network Research on Rare Diseases (CIBERE) intramural funds (no. ER19P5AC756/2021). F.J.M.M. acknowledges research support by Conselleria d’Educació, Investigació, Cultura i Esport from Generalitat Valenciana, research project nos. PROMETEO/2017/129 and PROMETEO/2021/057. M.M. acknowledges funding from CIBERER (grant no. ER19P5AC728/2021). The work has received funding from the Regional Government of Madrid (grant no. B2017/BMD3721 to M.A.M.-P.) and from Instituto de Salud Carlos III, cofounded with the European Regional Development Fund ‘A way to make Europe’ within the National Plans for Scientific and Technical Research and Innovation 2017–2020 and 2021–2024 (nos. PI17/1659, PI20/0429 and IMP/00009; to M.A.M.-P. B.P.K. was supported by an MGH ECOR Howard M. Goodman Award and NIH P01 HL142494

New strategies for writing Degree Dissertations in English: pair-tutoring and teaching mini-videos

La redacción de textos académicos en inglés está íntimamente relacionada con la competencia lingüística que el alumnado universitario ha de desarrollar (B1 como requisito lingüístico mínimo previo a su graduación) y que, de manera lógica, está encadenada a la mejora de su empleabilidad (tanto a niveles nacionales como internacionales), de sus competencias comunicativas en segunda lengua y, por supuesto, la posibilidad de que los resultados de su trabajo académico trasciendan fronteras. El proyecto que aquí se presenta surge como evolución de un proyecto de innovación docente anterior, aprobado e implementado en la UCO (“La tutoría piramidal como estrategia para el diseño y desarrollo del trabajo final de grado en educación”, 2015-2-6011), y cuyo resultado ha permitido identificar la necesidad que con esta propuesta pretendemos cubrir. El objetivo final de este proyecto es enseñar al alumnado universitario a redactar sus TFG en inglés, al tiempo que el profesorado también participa en los seminarios, en los que se abordan también técnicas que faciliten la corrección de estos trabajos que se redactan en una lengua no materna o principal. La metodología se basa en seminarios participativos, así como en la elaboración de recursos, en los que alumnado (tutores y tutorizados) y profesorado (tutores y tutorizados) colaborarán para la consecución final del objetivo: la redacción y corrección de TFG, principalmente en lengua inglesa. Entre estos recursos, destacamos el diseño y la elaboración de minivídeos docentes modulares con la participación del profesorado implicado en el proyecto, así como del alumnado mentor, para explicar los aspectos clave de la elaboración de un TFG.Writing academic texts in English is closely related to the linguistic competence that university students need to develop (B1 level as a minimum linguistic requirement prior to their graduation). This competence entails an improvement of their future employability (both at the national and international levels), their communicative competences in a second language (L2), and obviously also the possibility that the results derived from their academic projects spill across national boundaries. This innovation project arises as the evolution of a previous innovation strategy, approved and implemented at the University of Córdoba (“La tutoría piramidal como estrategia para el diseño y desarrollo del trabajo final de grado en educación”, 2015-2-6011), whose results allowed the identification of the weaknesses that we aim to focus on during the development of this project. The final objective is to teach university students how to write their end-of-degree dissertations in English. At the same time teachers of the UCO also participate in a series of seminars to know different techniques about how to review these projects that are written in a language different from their mother tongue or main language. The methodology of this project is based on participative seminars, as well as on the development of resources, where students (mentors and mentees) and teachers (mentors and mentees) collaborate towards a common objective: writing and reviewing end-of-degree dissertations, mainly written in English. Among these resources, it can be highlighted the design and elaboration of teaching mini-videos, with the participation of the teachers involved in the project, as well as the mentor students, in order to explain the key aspects to be considered when writing an end-of-degree dissertation

Protein nanomechanics: from fast-folding proteins to microbial infections

256 p.Esta tesis explora los límites de la técnica de SMFS (Single-molecule force spectroscopy) AFM (atomicforce microscopy), estudiando la estabilidad mecánica de proteínas en todo su espectro, desde las lábiles proteínas de plegamiento rápido hasta las más resistentes, implicadas en procesos de infección microbianos. Utilizando este conocimiento, hemos diseñado un protocolo pionero basado en métodos de molecular docking y hight-throughput screening, utilizados habitualmente en la industria farmacéutica,para identificar moléculas con potencial mecano regulador. Estas moléculas nos permiten modular la estabilidad y el mecanismo de plegamiento de proteínas. En esta tesis también hemos iniciado el desarrollo de una técnica que combina MT (magnetic tweezers) y bioquímica de proteínas para estudiarlas etapas iniciales de la infección bacteriana. En esta tesis, se han examinado cuatro proteínas, desde la lábil EnHD, pasando por CD4 hasta las más mecánicamente resistentes como Caf1 o FnBPA. Estas proteínas están relacionadas con procesos de infección que originan enfermedades como SIDA, peste yendocarditis. El presente trabajo conecta la nanomecánica de proteínas con importantes preguntas biológicas, como los procesos de infección microbianos y alteraciones en la estabilidad de las proteínas

Linear basal cell carcinoma: Report of three cases with dermoscopic findings.

Linear basal cell carcinoma was first described as a new clinical subtype in 1985 by Lewis. We were able to find published reports of only 43 other cases since then. Certain unique clinical and histological characteristics differentiate it from other basal cell carcinomas hence, some authors consider it as a different clinical entity.[1,2] Herein, we report three cases of linear basal cell carcinoma

Compliant mechanical response of the ultrafast folding protein EnHD under force

Exhibiting low-energy (un)folding barriers and fast kinetics, ultrafast folding proteins are enticing models to study protein dynamics. The authors use single molecule force spectroscopy AFM to capture the compliant behaviour hallmarking the dynamics of ultrafast folding proteins under force