Single-Aminoacid Discrimination in Proteins with Homogeneous Nanopore Sensors and Neural Networks

A technology capable of sequencing individual protein molecules would revolutionize our understanding of biological processes. Nanopore technology can analyze single heteropolymer molecules such as DNA by measuring the ionic current flowing through a single nanometer hole made in an electrically insulating membrane. This current is sensitive to the monomer sequence. However, proteins are remarkably complex and identifying a single residue change in a protein remains a challenge. In this work, I show that simple neural networks can be trained to recognize protein mutants. Although these networks are quickly and efficiently trained, their ability to generalize in an independent experiment is poor. Using a thermal annealing protocol on the nanopore sample, and examining many mutants with the same nanopore sensor are measures aimed at reducing training data variability which produce an increase in the generalizability of the trained neural network. Using this approach, we obtain a 100% correct assignment among 9 mutants in >50% of the experiments. Interestingly, the neural network performance, compared to a random guess, improves as more mutants are included in the dataset for discrimination. Engineered nanopores prepared with high homogeneity coupled with state-of-the-art analysis of the ionic current signals may enable single-molecule protein sequencing.I thank Andrina Chambers for critical reading of the manuscript and helpful suggestions. I thank G. Celaya for the preparation of hemolysin monomers and erythrocyte membranes. DRL work was supported by grants BIO201788946R, BFU2016-81754-ERC from MINECO (FEDER funds) , IT1201-19 from the Basque Government, and a Ramon y Cajal fellowship (RYC20312799

DNA-binding miniproteins based on zinc fingers. Assessment of the interaction using nanopores

Obtaining artificial proteins that mimic the DNA binding properties of natural transcription factors could open new ways of manipulating gene expression at will. In this context it is particularly interesting to develop simple synthetic systems. Inspired by the modularity of natural transcription factors, we have designed synthetic miniproteins that combine the zinc finger module of the transcription factor GAGA and AT-hook peptide domains. These constructs are capable of binding to composite DNA sequences of up to 14 base pairs with high affinity and good selectivity. In particular, we have synthesized three different chimeras and characterized their DNA binding properties by electrophoresis and fluorescence anisotropy. We have also used, for the first time in the study of peptide-based DNA binders, nanopore force spectroscopy to obtain further data on the DNA interaction

Racial and Ethnic Disparities in Health in Latin America and the Caribbean

There is increasing awareness that race and ethnicity play an important role in the poverty and social marginalization of Latin American and Caribbean populations.Health Care

T cells isolated from G-CSF-treated multiple myeloma patients are suitable for the generation of BCMA-directed CAR-T cells

Autologous cell immunotherapy using B cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR)-T cells is an effective novel treatment for multiple myeloma (MM). This therapy has only been used for relapsed and refractory patients, at which stage the endogenous T cells used to produce the CAR-T cells are affected by the immunosuppressive nature of advanced MM and/or side effects of previous therapies. An alternative pool of fitter T cells is found in leukocytoapheresis products that are routinely collected to obtain hematopoietic progenitor cells for autologous stem cell transplantation (ASCT) early in the treatment of MM. However, to mobilize the progenitor cells, patients are dosed with granulocyte colony-stimulating factor (G-CSF), which is reported to adversely affect T cell proliferation, function, and differentiation. Here, we aimed to first establish whether G-CSF treatment negatively influences T cell phenotype and to ascertain whether previous exposure of T cells to G-CSF is deleterious for anti-BCMA CAR-T cells. We observed that G-CSF had a minimal impact on T cell phenotype when added in vitro or administered to patients. Moreover, we found that CAR-T cell fitness and anti-tumor activity were unaffected when generated from G-CSF-exposed T cells. Overall, we showed that ASCT apheresis products are a suitable source of T cells for anti-BCMA CAR-T cell manufacture

Regulation of the transcriptional program by DNA methylation during human αβ T-cell development

© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. Thymocyte differentiation is a complex process involving well-defined sequential developmental stages that ultimately result in the generation of mature T-cells. In this study, we analyzed DNA methylation and gene expression profiles at successive human thymus developmental stages. Gain and loss of methylation occurred during thymocyte differentiation, but DNA demethylation was much more frequent than de novo methylation and more strongly correlated with gene expression. These changes took place in CpG-poor regions and were closely associated with T-cell differentiation and TCR function. Up to 88 genes that encode transcriptional regulators, some of whose functions in T-cell development are as yet unknown, were differentially methylated during differentiation. Interestingly, no reversion of accumulated DNA methylation changes was observed as differentiation progressed, except in a very small subset of key genes (RAG1, RAG2, CD8A, PTCRA, etc.), indicating that methylation changes are mostly unique and irreversible events. Our study explores the contribution of DNA methylation to T-cell lymphopoiesis and provides a fine-scale map of differentially methylated regions associated with gene expression changes. These can lay the molecular foundations for a better interpretation of the regulatory networks driving human thymopoiesis.Plan Nacional de [I+D+I 2008–2011]; Instituto de Salud Carlos III [grant number PI12/02587]; Red Española de Investigación Renal (REDinREN) [grant number RD12/0021/0018 and RD12/0021/0021]; Spanish Ministry of Science and Innovation [grant number SAF2010- 15106 and PLE2009-0110]; European Union [Fondos FEDER]Peer Reviewe

Long-term Responders after autologous stem cell transplantation in multiple myeloma

Introduction: Multiple myeloma (MM) is considered an incurable hematological neoplasm. For transplant-eligible patients, initial treatment includes an induction phase followed by an autologous stem cell transplantation (ASCT). Despite the introduction of several drugs in the past years, relapses still occur. Nevertheless, some patients achieve sustained responses after successful induction treatment and ASCT. Methods: We retrospectively evaluated all patients diagnosed with MM in our institution who underwent induction treatment and ASCT between 1990 and 2015. The subset of patients who achieved a sustained response (any degree) for 5 or more years after ASCT without further treatment or signs of progression were distinguished as 'long-term responders' (LTRs). In the non-LTR group, a cohort referred to as 'prolonged responders' (PLRs) showed sustained response of at least 5 years after ASCT but eventually relapsed. We collected and analyzed clinical and laboratory data. Results: Two hundred and fifty patients were diagnosed with MM and received induction treatment and ASCT at our institution in the study period. Among them, 54 (21.6%) patients met the criteria for LTR. Some diagnostic features such as a younger age, female gender, ECOG performance status of 0, lower International Staging System (ISS) stage, lower bone marrow plasma cell infiltration, and lower serum levels of calcium, C-reactive protein, and lactate dehydrogenase (LDH) were found to be more prevalent in LTR. Female gender, an ECOG performance status of 0, a localized Durie-Salmon stage, an ISS of I-II, the absence of bone disease, and an LDH within normal range were also predictive of longer progression-free survival (PFS) and overall survival (OS) in the whole cohort. The depth of the response achieved after induction and ASCT as well as the administration of an IMID-based maintenance regimen may play a role in the differences observed on PFS between cohorts. A detectable M-protein with a monoclonal gammopathy of undetermined significance (MGUS)-like behavior was detected in one-third of LTR after ASCT. Although relapses continue to occur in patients who achieve a 5-year treatment-free period after ASCT, a plateau is observed in the survival curves at approximately 21 years of follow-up

Design and implementation of Integrated Didactic Units: CLIL in action

Este trabajo desarrolla el diseño y la implementación de Unidades Didácticas Integradas (UDI) en el marco de la educación superior siguiendo el enfoque AICLE (Aprendizaje Integrado de Contenidos y Lenguas Extranjeras). El diseño y la implementación de una metodología conjunta ha permitido poner en práctica dichas UDI durante el curso 2015/2016 en diferentes ámbitos de contenido. Ello ha ofrecido datos relevantes sobre el nivel de lengua y contenidos que el alumnado ha adquirido. La integración de diferentes áreas en las dos ramas de conocimiento (Ciencias Sociales e Ingeniería) ha constituido un marco interesante y especialmente adecuado para la puesta en práctica de estas UDI en el marco de la Educación Superior Europea.This paper addresses the design and implementation of Integrated Didactic Units (IDUs) within Higher Education following the CLIL (Content and Language Integrated Learning) approach. The design of a joint methodology for different content areas has allowed the teaching staff herein to implement different IDUs along the 2015/2016 academic year. The data collected on initial and final language level and contents from the joint and complementary work of two different content areas (Social Sciences and Engineering) has offered an interesting working framework which we consider suitable for an EHEA teaching innovation after the analysis and discussion of our results

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