Rational stabilization of the C-LytA affinity tag by protein engineering

The C-LytA protein constitutes the choline-binding module of the LytA amidase from Streptococcus pneumoniae. Owing to its affinity for choline and analogs, it is regularly used as an affinity tag for the purification of proteins in a single chromatographic step. In an attempt to build a robust variant against thermal denaturation, we have engineered several salt bridges on the protein surface. All the stabilizing mutations were pooled in a single variant, C-LytAm7, which contained seven changes: Y25K, F27K, M33E, N51K, S52K, T85K and T108K. The mutant displays a 7 degrees C thermal stabilization compared with the wild-type form, together with a complete reversibility upon heating and a higher kinetic stability. Moreover, the accumulation of intermediates in the unfolding of C-LytA is virtually abolished for C-LytAm7. The differences in stability become more evident when the proteins are bound to a DEAE-cellulose affinity column, as most of wild-type C-LytA is denatured at approximately 65 degrees C, whereas C-LytAm7 may stand temperatures up to 90 degrees C. Finally, the change in the isoelectric point of C-LytAm7 enhances its solubility at acidic pHs. Therefore, C-LytAm7 behaves as an improved affinity tag and supports the engineering of surface salt bridges as an effective approach for protein stabilization

El impacto de las respuestas inconsistentes en las medidas de bienestar estimadas con el método del experimento de elección

[EN] Choice experiments have become an important tool to provide guidance about the value of environmental goods and services. Several evidences, however, are pointing toward an important mis-match between the rationality principles assumed by this methodology and real respondents’ behaviour, what may be giving rise to inconsistent choices. This paper studies the effects of such inconsistencies by using data from an online survey aimed at valuing the environmental impacts of organic farming in mountainous olive groves. The results are analysed by means of three random parameter models and provide evidence on the necessity to appropriately consider and treat choice inconsistencies as a result of their influence on welfare estimates.[ES] Los experimentos de elección se han convertido en una importante herramienta para la valoración de bienes y servicios ambientales. Sin embargo, existen evidencias que demuestran que los principios de racionalidad asumidos en esta metodología a menudo no son respetados en las estrategias de elección empleadas por los entrevistados, pudiendo dar lugar a elecciones inconsistentes. Este trabajo examina las consecuencias de este tipo de elecciones mediante una encuesta online destinada a valorar los impactos de la agricultura ecológica en el olivar de montaña. Los resultados son analizados mediante tres modelos de parámetros aleatorios, y ponen de manifiesto la necesidad de considerar y tratar las inconsistencias debido a su influencia en la valoración de los impactos evaluados.Esta investigación forma parte del proyecto INIA RTA2009-00024-00-00 financiado por el INIA en el marco del Subprograma de Proyectos de Investigación Fundamental orientada a los Recursos y Tecnologías Agrarias y cofinanciado por el Fondo FEDER en el marco del programa operativo de economía basada en el conocimiento. Los autores quieren agradecer, sin perjuicio por la responsabilidad de los errores que puedan persistir, los comentarios y sugerencias realizados por los revisores anónimos y por el Comité Editorial, por su importante contribución a la mejora del artículoRocamora Montiel, B.; Colombo, S.; Glenk, K. (2014). The impact of choice inconsistencies in discrete choice experiments’ welfare estimates. Economía Agraria y Recursos Naturales - Agricultural and Resource Economics. 14(2):27-48. https://doi.org/10.7201/earn.2014.02.02SWORD274814

Structural characterization of PaaX, the main repressor of the phenylacetate degradation pathway in Escherichia coli W: A novel fold of transcription regulator proteins

15 p.-8 fig.-2 tab.PaaX is a transcriptional repressor of the phenylacetic acid (PAA) catabolic pathway, a central route for bacterial aerobic degradation of aromatic compounds. Induction of the route is achieved through the release of PaaX from its promoter sequences by the first compound of the pathway, phenylacetyl-coenzyme A (PA-CoA). We report the crystal structure of PaaX from Escherichia coli W. PaaX displays a novel type of fold for transcription regulators, showing a dimeric conformation where the monomers present a three-domain structure: an N-terminal winged helix-turn-helix domain, a dimerization domain similar to the Cas2 protein and a C-terminal domain without structural homologs. The domains are separated by a crevice amenable to harbour a PA-CoA molecule. The biophysical characterization of the protein in solution confirmed several hints predicted from the structure, i.e. its dimeric conformation, a modest importance of cysteines and a high dependence of solubility and thermostability on ionic strength. At a moderately acidic pH, the protein formed a stable folding intermediate with remaining α-helical structure, a disrupted tertiary structure and exposed hydrophobic patches. Our results provide valuable information to understand the stability and mechanism of PaaX and pave the way for further analysis of other regulators with similar structural configurations.This research was funded by the following sources: Grants PID2019-105126RB-I00, PID2022-139209OB-C21 (MCIN/AEI/10.13039/501100011033/and ERDF A way of making Europe), TED2021-129747B-C22 (AEI/10.13039/501100011033/NextGenerationEU/PRTR) and CIBER-Consorcio Centro de Investigación Biomédica en Red (CIBERES, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Spain) to JMS; grants PID2020-115331GB-100 funded by MCIN/AEI/10.13039/501100011033 and CRSII5_198737/1 (Swiss National Science Foundation) to JAH; grant PID2021-128751NB-I00 (MICINN/AEI/FEDER/UE) to IU, and grant RYC2021-030916-I by the Spanish Agencia Estatal de Investigación to RM. VMH-R was supported by a FPU PhD fellowship from Spanish Ministerio de Educación y Ciencia.Peer reviewe

Evaluating the Potential of Polygenic Risk Score to Improve Colorectal Cancer Screening

Background: Colorectal cancer has high incidence and associ-ated mortality worldwide. Screening programs are recommended for men and women over 50. Intermediate screens such as fecal immunochemical testing (FIT) select patients for colonoscopy with suboptimal sensitivity. Additional biomarkers could improve the current scenario. Methods: We included 2,893 individuals with a positive FIT test. They were classified as cases when a high-risk lesion for colorectal cancer was detected after colonoscopy, whereas the control group comprised individuals with low-risk or no lesions. 65 colorectal cancer risk genetic variants were geno-typed. Polygenic risk score (PRS) and additive models for risk prediction incorporating sex, age, FIT value, and PRS were generated. Results: Risk score was higher in cases compared with controls [per allele OR = 1.04; 95% confidence interval (CI), 1.02-1.06; P = 65), compared with those in the first decile (<= 54; OR = 2.22; 95% CI, 1.59-3.12; P < 0.0001). The model combining sex, age, FIT value, and PRS reached the highest accuracy for identifying patients with a high-risk lesion [cross-validated area under the ROC curve (AUROC): 0.64; 95% CI, 0.62-0.66]. Conclusions: This is the first investigation analyzing PRS in a two-step colorectal cancer screening program. PRS could improve current colorectal cancer screening, most likely for higher at-risk subgroups. However, its capacity is limited to predict colorectal cancer risk status and should be complemented by additional biomarkers.Impact: PRS has capacity for risk stratification of colorectal cancer suggesting its potential for optimizing screening strategies alongside with other biomarkers

Implementation of a mindfulness-based crisis intervention for frontline healthcare workers during the COVID-19 outbreak in a public general hospital in Madrid, Spain

Introduction: The COVID-19 outbreak is having an impact on the well-being of healthcare workers. Mindfulness-based interventions have shown effectiveness in reducing stress and fostering resilience and recovery in healthcare workers. There are no studies examining the feasibility of brief mindfulness-based interventions during the COVID-19 outbreak. Materials and Methods: This is an exploratory study with a post intervention assessment. We describe an on-site brief mindfulness intervention and evaluate its helpfulness, safety, and feasibility. Results: One thousand out of 7,000 (14%) healthcare workers from La Paz University Hospital in Madrid (Spain) participated in at least one session. One hundred and fifty out of 1,000 (15%) participants filled out a self-report questionnaire evaluating the helpfulness of the intervention for on-site stress reduction. Ninety two subjects (61%) participated in more than one session. Most of the participants were women (80%) with a mean age of 38.6 years. Almost half of the sample were nurses (46%). Sessions were perceived as being helpful with a mean rating of 8.4 on a scale from 0 to 10. Only 3 people (2%) reported a minor adverse effect (increased anxiety or dizziness). Discussion: Our data supports the utility, safety and feasibility of an on-site, brief mindfulness-based intervention designed to reduce stress for frontline health workers during a crisis. There is a need to continue testing this type of interventions, and to integrate emotion regulation strategies as an essential part of health workers' general training. Clinical Trial Registration number: NCT04555005

Development of a novel anti-CD19 chimeric antigen receptor: A paradigm for an affordable CAR T cell production at academic institutions

Genetically modifying autologous T cells to express an anti-CD19 chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19+ B cell malignancies in several clinical trials (CTs). Making this treatment available to our patients prompted us to develop a novel CART19 based on our own anti-CD19 antibody (A3B1), followed by CD8 hinge and transmembrane region, 4-1BB- and CD3z-signaling domains. We show that A3B1 CAR T cells are highly cytotoxic and specific against CD19+ cells in vitro, inducing secretion of pro-inflammatory cytokines and CAR T cell proliferation. In vivo, A3B1 CAR T cells are able to fully control disease progression in an NOD.Cg-Prkdcscid Il2rdtm1Wjl/SzJ (NSG) xenograph B-ALL mouse model. Based on the pre-clinical data, we conclude that our CART19 is clearly functional against CD19+ cells, to a level similar to other CAR19s currently being used in the clinic. Concurrently, we describe the implementation of our CAR T cell production system, using lentiviral vector and CliniMACS Prodigy, within a medium-sized academic institution. The results of the validation phase show our system is robust and reproducible, while maintaining a low cost that is affordable for academic institutions. Our model can serve as a paradigm for similar institutions, and it may help to make CAR T cell treatment available to all patients