Novel intein-based self-cleaving affinity tag for recombinant protein production in Escherichia coli

We evaluated several intein-based self-cleaving affinity tags for expression and single-step affinity chromatography purification of recombinant proteins produced in Escherichia coli. We used human growth hormone (hGH) as target protein that contains two internal disulfide bridges and an N-terminal phenylalanine. Use of N-terminal thiol-induced Sce VMA1 intein affinity tag resulted in purified hGH deficient in disulfide bonds. Inteins with self-cleavage inducible by pH and/or temperature shift were analyzed. N-terminal Ssp DnaX intein affinity tag resulted in a completely cleaved cytosolic protein, whereas N-terminal Ssp DnaB intein affinity tag resulted in a cytosolic fusion protein incapable of releasing hGH. Periplasmic expression of target protein was analyzed using an N-terminal signal peptide and C-terminal Ssp DnaX pH-inducible self-cleaving affinity tag. The fusion protein was properly expressed in pH 8 buffered culture medium. Fusion of a periplasmic signal peptide to the N-terminus of the POI allowed secretion to the periplasmic region and presence of the natural N-terminal amino acid of the POI following cleavage. Periplasmic expression of hGH fused to this novel C-terminal DnaX intein-based self-cleaving affinity tag made possible expression and purification of hGH protein containing disulfide bonds and free of extra amino acids.Fil: Amaranto, Marilla. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Vaccarello, Paula. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; ArgentinaFil: Correa, Elisa María Eugenia. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Barra, Jose Luis. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Godino, Agustina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentin

Prebiotic iron–sulfur peptide catalysts generate a pH gradient across model membranes of late protocells

Prebiotic chemistry was likely mediated by metals, but how such prebiotic chemistry progressed into the metabolic-like networks needed to sustain life remains unclear. Here we experimentally delineate a potential path from prebiotically plausible iron–sulfur peptide catalysts to the types of pH gradients exploited by all known living organisms. Iron–sulfur peptides cooperatively accept electrons from NADH in a manner that is only partially mediated by ionic interactions. The electrons are then either passed to a terminal electron acceptor, such as hydrogen peroxide, or to an intermediate carrier, such as ubiquinone. The reduction of hydrogen peroxide leads to the production of hydroxide, which then contributes to the formation of a pH gradient across late protocell membranes. The data are consistent with the activity of prebiotic iron–sulfur peptide catalysts providing a selective advantage by equipping protocells with a pathway that connects catabolism to anabolism

Assisted Walking Program on Walking Ability in In-Hospital Geriatric Patients: A Randomized Trial

The main aim of this study was to evaluate if an individualized assisted walking program (IAWP) for hospitalized older patients could improve walking ability compared with usual geriatric care and rehabilitation

[A three-months follow up of a randomized controlled trial of Assisted Walking Program for in-hospital geriatric patients]

: . A three-months follow up of a randomized controlled trial of Assisted Walking Program for in-hospital geriatric patients. Introduction: Functional decline is common among older hospitalized patients. In fact, low mobility and bed rest during hospitalization have been considered as predictable causes of independent ambulation decline in older hospitalized patients. Aim: Primary endpoint: the older patients' walking ability change, compared with usual care, from hospital pre-admission/admission to discharge and 90 days follow-up, assessed with the Braden Activity subscale. The secondary end point was the occurrence of re-hospitalization and mortality. Methods: A 90-days follow-up randomized controlled trial, open labeled was conducted in a geriatric ward. Results: A total of 307 hospitalized patients (>65 years) were included. The intervention group received an Individualized Assisted Walking Program (IAWP), which significantly improved walking ability at discharge (p<.001) and 90-day follow-up (p=0.009), compared to the control group, which received the usual care. There were no significant differences in terms of mortality and re-hospitalizations. Conclusions: An individualized assisted walking program improves walking abilitiy during hospitalization and over time. For this, a nurse staffing and workload reorganization, a multidisciplinary approach, and an early nurses' planning, could be relevant factors in influencing successfully the older patients' healthcare

The rise in telework : impact on working conditions and regulations

Aquesta publicació s'elabora a partir de les contribucions de cadascú dels membres nacionals que integren la Network of Eurofound Correspondents. Pel cas d'Espanya la contribució ha estat realitzada per l'Alejandro Godino (veure annex 4 Network of Eurofound Correspondents)This report presents Eurofound's research on telework during the COVID-19 pandemic in 2020 and 2021. It explores changes in the incidence of telework, working conditions experienced by employees working from home and changes to regulations addressing issues related to this working arrangement. The findings reveal a rapid escalation of telework triggered by the pandemic: in 2021, 2 out of 10 European employees were teleworking - a figure that most likely would not have been reached before 2027 had the pandemic not occurred. The health crisis unleashed the social and technological potential for flexibility in terms of working time and place. The impacts of telework on working conditions were initially difficult to determine because it was difficult to disentangle them from pandemic-induced factors, such as lockdowns and school closures. However, both the positive impacts, such as the contribution of telework to improving work-life balance, and the negative impacts, such as reduced social interaction and an increase in overtime worked, have become more evident. The rise in telework and an awareness of its implications for working conditions have prompted a renewed focus on regulatory frameworks, with new telework regulations passed in several EU Member State

2-year outcomes of MitraClip as a bridge to heart transplantation: The international MitraBridge registry

Background: In the first report from the MitraBridge registry, MitraClip as a bridge to heart transplantation (HTx) proved to be at 1-year an effective treatment strategy for 119 patients with advanced heart failure (HF) who were potential candidates for HTx. We aimed to determine if benefits of MitraClip procedure as a bridge-to-transplant persist up to 2-years. Methods: By the end of the enrollment period, a total of 153 advanced HF patients (median age 59 years, left ventricular ejection fraction 26.9 ± 7.7%) with significant secondary mitral regurgitation, who were potential candidates for HTx and were treated with MitraClip as a bridge-to-transplant strategy, were included in the MitraBridge registry. The primary endpoint was the 2-year composite adverse events rate of all-cause death, first hospitalization for HF, urgent HTx or LVAD implantation. Results: Procedural success was achieved in 89.5% of cases. Thirty-day mortality was 0%. At 2-year, Kaplan-Meier estimates of freedom from primary endpoint was 47%. Through 24 months, the annualized rate of HF rehospitalization per patient-year was 44%. After an overall median follow-up time of 26 (9-52) months, elective HTx was successfully performed in 30 cases (21%), 19 patients (13.5%) maintained or obtained the eligibility for transplant, and 32 patients (22.5%) no longer had an indication for HTx because of significant clinical improvement. Conclusions: After 2-years of follow-up, the use of MitraClip as a bridge-to-transplant was confirmed as an effective strategy, allowing elective HTx or eligibility for transplant in one third of patients, and no more need for transplantation in 22.5% of cases

Common breast cancer susceptibility alleles are associated with tumor subtypes in BRCA1 and BRCA2 mutation carriers: results from the Consortium of Investigators of Modifiers of BRCA1/2.

Abstract Introduction Previous studies have demonstrated that common breast cancer susceptibility alleles are differentially associated with breast cancer risk for BRCA1 and/or BRCA2 mutation carriers. It is currently unknown how these alleles are associated with different breast cancer subtypes in BRCA1 and BRCA2 mutation carriers defined by estrogen (ER) or progesterone receptor (PR) status of the tumour. Methods We used genotype data on up to 11,421 BRCA1 and 7,080 BRCA2 carriers, of whom 4,310 had been affected with breast cancer and had information on either ER or PR status of the tumour, to assess the associations of 12 loci with breast cancer tumour characteristics. Associations were evaluated using a retrospective cohort approach. Results The results suggested stronger associations with ER-positive breast cancer than ER-negative for 11 loci in both BRCA1 and BRCA2 carriers. Among BRCA1 carriers, single nucleotide polymorphism (SNP) rs2981582 (FGFR2) exhibited the biggest difference based on ER status (per-allele hazard ratio (HR) for ER-positive = 1.35, 95% CI: 1.17 to 1.56 vs HR = 0.91, 95% CI: 0.85 to 0.98 for ER-negative, P-heterogeneity = 6.5 × 10-6). In contrast, SNP rs2046210 at 6q25.1 near ESR1 was primarily associated with ER-negative breast cancer risk for both BRCA1 and BRCA2 carriers. In BRCA2 carriers, SNPs in FGFR2, TOX3, LSP1, SLC4A7/NEK10, 5p12, 2q35, and 1p11.2 were significantly associated with ER-positive but not ER-negative disease. Similar results were observed when differentiating breast cancer cases by PR status. Conclusions The associations of the 12 SNPs with risk for BRCA1 and BRCA2 carriers differ by ER-positive or ER-negative breast cancer status. The apparent differences in SNP associations between BRCA1 and BRCA2 carriers, and non-carriers, may be explicable by differences in the prevalence of tumour subtypes. As more risk modifying variants are identified, incorporating these associations into breast cancer subtype-specific risk models may improve clinical management for mutation carriers

Reproducibility of fluorescent expression from engineered biological constructs in E. coli

We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices.Peer reviewe