Trends in the design and use of elastin-like recombinamers as biomaterials

Producción CientíficaElastin-like recombinamers (ELRs), which derive from one of the repetitive domains found in natural elastin, have been intensively studied in the last few years from several points of view. In this mini review, we discuss all the recent works related to the investigation of ELRs, starting with those that define these polypeptides as model intrinsically disordered proteins or regions (IDPs or IDRs) and its relevance for some biomedical applications. Furthermore, we summarize the current knowledge on the development of drug, vaccine and gene delivery systems based on ELRs, while also emphasizing the use of ELR-based hydrogels in tissue engineering and regenerative medicine (TERM). Finally, we show different studies that explore applications in other fields, and several examples that describe biomaterial blends in which ELRs have a key role. This review aims to give an overview of the recent advances regarding ELRs and to encourage further investigation of their properties and applications.Comisión Europea (project NMP-2014-646075)Ministerio de Economía, Industria y Competitividad (projects PCIN-2015-010 / MAT2016-78903-R / BES-2014-069763)Junta de Castilla y León (project VA317P18

Bioengineered dynamic systems based on elastin-like recombinamers

La ingeniería tisular y la medicina regenerativa son campos interdisciplinares que tienen como objetivo desarrollar sustitutos biológicos que restauren, mantengan o mejoren la función de un tejido o de un órgano perdida por lesión o enfermedad. Los biomateriales utilizados en aplicaciones biomédicas persiguen alcanzar características esenciales que promuevan la nueva morfogénesis tisular creando un entorno favorable para el crecimiento celular, así como las propiedades físicas y químicas adecuadas requeridas para un tejido determinado. En décadas pasadas, se han encontrado varios obstáculos para seleccionar biomateriales funcionales que imiten la matriz extracelular natural (MEC). A la inversa, los polímeros naturales derivados de proteínas de la matriz extracelular ofrecen la ventaja de la señalización biológica, pero pueden ser antigénicas y sus propiedades físicas son más difíciles de modular que en los polímeros sintéticos. La biodegradación es una característica crucial en los implantes biomédicos y su modulación ha sido ampliamente estudiada. Presentar un tiempo de degradación sincronizado con la formación de los nuevos tejidos es un objetivo altamente perseguido. La tasa de degradación está influenciada por muchas características de los polímeros, incluida la morfología y la orientación de la cadena, la cristalinidad inicial, el peso molecular, la presencia de monómeros y oligómeros residuales y la pureza. Existen varios métodos para cambiar y controlar la velocidad de degradación de los polímeros sintéticos y naturales, como la copolimerización, la mezcla, los aditivos y la irradiación, pero estos métodos presentan diferentes inconvenientes, como el cambio en las propiedades mecánicas, la pobre biocompatibilidad y el retraso en la bioabsorción. Debido a estos inconvenientes, puede ser útil producir biomateriales que sean sensibles a ciertos enzimas que se valgan de la respuesta inflamatoria que se produce por la propia lesión o introducción del implante. Los avances en ingeniería genética han facilitado la producción de proteínas modificadas que abarcan una amplia gama de propiedades y funcionalidades, como la adhesión celular, la señalización celular, la elasticidad y la biodegradabilidad. Actualmente, los recombinamers de tipo elastina (ELR) muestran propiedades sobresalientes para su aplicación en regeneración de tejidos. Los ELR son polipéptidos artificiales con un alto contenido en elastina que los convirtió en materiales capaces de imitar la ECM. Tienen interesantes propiedades mecánicas que van desde los elastómeros ideales hasta el plástico que permiten, por lo tanto, un esfuerzo mecánico repetitivo y confieren resistencia mecánica a través de un mecanismo separado que soporta varias funciones del tejido. Por último, son extremadamente biocompatibles debido a su composición aminoácidica que implica la repetición de secuencias basadas en las secuencias encontradas en la elastina de los mamíferos. En el primer estudio de esta tesis se emplearon dos ELRs con una bioactividad complementaria (CC-RGD y CCC-REDV), las cisteínas están incluidas en la región amino terminal, para desarrollar un sistema 2D en el que la adhesión celular selectiva está controlada espacialmente para su posterior aplicación en la tecnología de biosensores como dispositivos de diagnóstico o en la aplicación de ingeniería de tejidos y medicina regenerativa. Específicamente, en este estudio, las superficies modelo de oro se funcionalizaron con diferentes porcentajes de dos ELRs (CC-RGD y CCC-REDV), lo que demuestra que una composición óptima de 75% CCC-REDV y 25% CC-RGD conduce a una adhesión selectiva de células endoteliales en un sistema de co-cultivo. Luego, una vez que se funcionalizó toda la superficie con 100% de CC-RGD, se limpiaron áreas específicas mediante ablación con láser sin interferir en las propiedades topográficas, hidrófobas y mecánicas de las superficies. Estas áreas se funcionalizaron con un 75% de CCC-REDV y un 25% de CC-RGD y después de los estudios in vitro, se observó una distribución bien definida de células HUVEC y HFF1, es decir, un control espacial selectivo sobre la adhesión de las células HUVEC. Estos hallazgos sugieren un enfoque innovador para la generación de biosensores y / o andamios que requieren la organización espacial y temporal de las células. A continuación, el segundo estudio de esta tesis consiste en la mejora de la actividad angiogénica de los hidrogeles basados en ELR cuando se pega químicamente un pequeño péptido llamado QK que mima el factor de acrecimiento VEGF. Además de las secuencias de adhesión celular (RGD y REDV), un ELR utilizado en este trabajo presenta un sitio proteolítico (VGVAPG) que pertenece a la enzima elastasa. La actividad angiogénica de estos hidrogeles se estudió in vitro, demostrando que no altera las propiedades mecánicas y morfológicas de los ELR, sino la actividad biológica, como la adhesión y proliferación celular. Cuando se inyectó in vivo, se observó una mejor formación de microvasculatura que facilita la perfusión y la conexión con el tejido circundante. Estos resultados mostraron que los hidrogeles basados en ELR-QK son construcciones diseñadas óptimas con una potencial actividad angiogénica que podría emplearse en ingeniería de tejidos y medicina regenerativa para el tratamiento de enfermedades cardiovasculares. El tercer estudio de esta tesis tiene como objetivo lo de controlar en tiempo y espacio la infiltración célular y la biodegradación en un sistema estructurado tridimensional (3D). Los ELR empleados en este trabajo, además de una secuencia de adhesión celular universal (RGD), contienen sitios proteolíticos que se degradan en respuesta al activador de plasminógeno uroquinasa (uPA) con diferente tasa de degradación (rápida y lenta). Aprovechando estas características, en este estudio se desarrolló un sistema 3D que consiste en discos de hidrogeles de tres capas, con una capa interna que contiene el componente de degradación rápida y las capas externas que contienen el ELR de degradación lenta. Esos discos se han implantado por vía subcutánea en ratones y su evolución se siguió a lo largo del tiempo. Como resultado del diseño y de la secuencia de infiltración celular programada, la capa interna se colonizó primero siguiendo un patrón de adentro hacia afuera. Estos resultados demostraron que el sistema basado en 3D-ELR podría aplicarse como un sustituto tisular para la aplicación biomédica que requiere una regeneración controlada y vascularización a lo largo del tiempo. En resumen, los estudios mostrados en esta Tesis brindan nuevos conocimientos sobre los hidrogeles basados en ELR que se utilizarán en ingeniería de tejidos y medicina regenerativa.Departamento de Bioquímica y Biología Molecular y FisiologíaDoctorado en Investigación Biomédic

Use of proteolytic sequences with different cleavage kinetics as a way to generate hydrogels with preprogrammed cell-infiltration patterns imparted over their given 3D spatial structure

Producción CientíficaControl over biodegradation processes is crucial to generate advanced functional structures with a more interactive and efficient role for biomedical applications. Herein, a simple, high-throughput approach is developed based on a 3D-structured system that allows a preprogramed spatial-temporal control over cell infiltration and biodegradation. The 3D-structured system is based on elastin-like recombinamers (ELRs) characterized by differences in the kinetics of their peptide cleavage and consists of a three-layer hydrogel disk comprising an internal layer containing a rapidly degrading component, with the external layers containing a slow-degrading ELR. This structure is intended to invert the conventional pattern of cell infiltration, which goes from the outside to the inside of the implant, to allow an anti-natural process in which infiltration takes place first in the internal layer and later progresses to the outer layers. Time-course in vivo studies proved this hypothesis, i.e. that it is possible to drive the infiltration of cells over time in a given 3D-structured implant in a controlled and predesigned way that is able to overcome the natural tendency of conventional cell infiltration. The results obtained herein open up the possibility of applying this concept to more complex systems with multiple biological functions.European Commission (NMP-2014-646075, PITNGA-2012-317306)Ministerio de Economía, Industria y Competitividad ( grants PCIN-2015-010, MAT2015-68901-R, MAT2016-78903-R, MAT2016-79435-R)Junta de Castilla y León (VA015U16)Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y Leó

Tethering QK peptide to enhance angiogenesis in elastin-like recombinamer (ELR) hydrogels

Producción CientíficaThe development of new capillary networks in engineered constructs is essential for their survival and their integration with the host tissue. It has recently been demonstrated that ELR-based hydrogels encoding different bioactivities are able to modulate their interaction with the host after injection or implantation, as indicated by an increase in cell adhesion and the ability to trigger vascularization processes. Accordingly, the aim of this study was to increase their angiogenic ability both in vitro and in vivo using a small VEGF mimetic peptide named QK, which was tethered chemically to ELR-based hydrogels containing cell-adhesion sequences in their backbone, such as REDV and RGD, as well as a proteolytic site (VGVAPG). In vitro studies were performed using a co-culture of endothelial and fibroblast cells encapsulated into the ELR-based hydrogels in order to determine cell proliferation after 21 days of culture, as well as the number of cell-cell interactions. It was found that although the presence of this peptide does not influence the morphological and rheological properties of these hydrogels, it has an effect on cell behaviour, inducing an increase in cell proliferation and the formation of endothelial cell clusters. In vivo studies demonstrate that the QK peptide enhances the formation of prominent functional capillaries at three weeks post-injection, as confirmed by H&E staining and CD31 immunohistochemistry. The newly formed functional microvasculature ensures perfusion and connection with surrounding tissues. These results show that ELR-QK hydrogels increase capillary network formation and are therefore attractive candidates for application in tissue regeneration, for example for the treatment of cardiovascular diseases such as myocardial infarction or ischemia.European Commission (NMP-2014-646075, PITN-GA-2012-317306)Ministerio de Economía, Industria y Competitividad (Projects PCIN-2015-010, MAT2015- 68901-R, MAT2016-78903-R)Junta de Castilla y León (programa de apoyo a proyectos de investigación - Ref. VA015U16)Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y Leó

Spatial control and cell adhesion selectivity on model gold surfaces grafted with elastin-like recombinamers

Producción CientíficaA simple system for cell selectivity and spatially controlled cell adhesion has been developed using model gold surfaces grafted with a combination of two ELRs containing into their backbone cell-adhesion domains such as RGD and REDV. Grafting onto gold was achieved via redox reaction through thiol groups present in amino terminal cysteine tails of the ELRs. The correlation among contact angle, SEM micrographs, AFM, XPS and QCM-D have been carried out. After in-depth adhesion studies, a mixture of 75% ELR-REDV and 25% ELR-RGD was found to exhibit high selectivity for endothelial cells, promoting strong adhesion thereof. Consequently, certain areas of gold surfaces (strips) were cleaned by laser ablation and functionalized with the mixture 75% ELR-REDV - 25% ELR-RGD leading to a spatial segregation of the co-culture made of HUVEC and HFF1 cells. This platform therefore exhibits selective spatial control over cell adhesion associated with the bioactive epitopes (RGD and REDV) contained in the ELR sequence, since each functionalized surface (including strips) have similar topographic, hydrophobic and mechanical properties.2020-092020-09Comisión Europea (NMP-2014-646075, MSCA-ITN-2014-642687)Ministerio de Economía, Industria y Competitividad (PCIN-2015-010, MAT2015-68901-R, MAT2016-78903-R and MAT2016-79435-R)Junta de Castilla y León (programa de apoyo a proyectos de investigación - Ref. Project VA015U16)Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y Leó

Identification of additional risk loci for stroke and small vessel disease: a meta-analysis of genome-wide association studies

BACKGROUND: Genetic determinants of stroke, the leading neurological cause of death and disability, are poorly understood and have seldom been explored in the general population. Our aim was to identify additional loci for stroke by doing a meta-analysis of genome-wide association studies. METHODS: For the discovery sample, we did a genome-wide analysis of common genetic variants associated with incident stroke risk in 18 population-based cohorts comprising 84 961 participants, of whom 4348 had stroke. Stroke diagnosis was ascertained and validated by the study investigators. Mean age at stroke ranged from 45·8 years to 76·4 years, and data collection in the studies took place between 1948 and 2013. We did validation analyses for variants yielding a significant association (at p<5 × 10(-6)) with all-stroke, ischaemic stroke, cardioembolic ischaemic stroke, or non-cardioembolic ischaemic stroke in the largest available cross-sectional studies (70 804 participants, of whom 19 816 had stroke). Summary-level results of discovery and follow-up stages were combined using inverse-variance weighted fixed-effects meta-analysis, and in-silico lookups were done in stroke subtypes. For genome-wide significant findings (at p<5 × 10(-8)), we explored associations with additional cerebrovascular phenotypes and did functional experiments using conditional (inducible) deletion of the probable causal gene in mice. We also studied the expression of orthologs of this probable causal gene and its effects on cerebral vasculature in zebrafish mutants. FINDINGS: We replicated seven of eight known loci associated with risk for ischaemic stroke, and identified a novel locus at chromosome 6p25 (rs12204590, near FOXF2) associated with risk of all-stroke (odds ratio [OR] 1·08, 95% CI 1·05-1·12, p=1·48 × 10(-8); minor allele frequency 21%). The rs12204590 stroke risk allele was also associated with increased MRI-defined burden of white matter hyperintensity-a marker of cerebral small vessel disease-in stroke-free adults (n=21 079; p=0·0025). Consistently, young patients (aged 2-32 years) with segmental deletions of FOXF2 showed an extensive burden of white matter hyperintensity. Deletion of Foxf2 in adult mice resulted in cerebral infarction, reactive gliosis, and microhaemorrhage. The orthologs of FOXF2 in zebrafish (foxf2b and foxf2a) are expressed in brain pericytes and mutant foxf2b(-/-) cerebral vessels show decreased smooth muscle cell and pericyte coverage. INTERPRETATION: We identified common variants near FOXF2 that are associated with increased stroke susceptibility. Epidemiological and experimental data suggest that FOXF2 mediates this association, potentially via differentiation defects of cerebral vascular mural cells. Further expression studies in appropriate human tissues, and further functional experiments with long follow-up periods are needed to fully understand the underlying mechanisms

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2

Height and body-mass index trajectories of school-aged children and adolescents from 1985 to 2019 in 200 countries and territories: a pooled analysis of 2181 population-based studies with 65 million participants

Summary Background Comparable global data on health and nutrition of school-aged children and adolescents are scarce. We aimed to estimate age trajectories and time trends in mean height and mean body-mass index (BMI), which measures weight gain beyond what is expected from height gain, for school-aged children and adolescents. Methods For this pooled analysis, we used a database of cardiometabolic risk factors collated by the Non-Communicable Disease Risk Factor Collaboration. We applied a Bayesian hierarchical model to estimate trends from 1985 to 2019 in mean height and mean BMI in 1-year age groups for ages 5–19 years. The model allowed for non-linear changes over time in mean height and mean BMI and for non-linear changes with age of children and adolescents, including periods of rapid growth during adolescence. Findings We pooled data from 2181 population-based studies, with measurements of height and weight in 65 million participants in 200 countries and territories. In 2019, we estimated a difference of 20 cm or higher in mean height of 19-year-old adolescents between countries with the tallest populations (the Netherlands, Montenegro, Estonia, and Bosnia and Herzegovina for boys; and the Netherlands, Montenegro, Denmark, and Iceland for girls) and those with the shortest populations (Timor-Leste, Laos, Solomon Islands, and Papua New Guinea for boys; and Guatemala, Bangladesh, Nepal, and Timor-Leste for girls). In the same year, the difference between the highest mean BMI (in Pacific island countries, Kuwait, Bahrain, The Bahamas, Chile, the USA, and New Zealand for both boys and girls and in South Africa for girls) and lowest mean BMI (in India, Bangladesh, Timor-Leste, Ethiopia, and Chad for boys and girls; and in Japan and Romania for girls) was approximately 9–10 kg/m2. In some countries, children aged 5 years started with healthier height or BMI than the global median and, in some cases, as healthy as the best performing countries, but they became progressively less healthy compared with their comparators as they grew older by not growing as tall (eg, boys in Austria and Barbados, and girls in Belgium and Puerto Rico) or gaining too much weight for their height (eg, girls and boys in Kuwait, Bahrain, Fiji, Jamaica, and Mexico; and girls in South Africa and New Zealand). In other countries, growing children overtook the height of their comparators (eg, Latvia, Czech Republic, Morocco, and Iran) or curbed their weight gain (eg, Italy, France, and Croatia) in late childhood and adolescence. When changes in both height and BMI were considered, girls in South Korea, Vietnam, Saudi Arabia, Turkey, and some central Asian countries (eg, Armenia and Azerbaijan), and boys in central and western Europe (eg, Portugal, Denmark, Poland, and Montenegro) had the healthiest changes in anthropometric status over the past 3·5 decades because, compared with children and adolescents in other countries, they had a much larger gain in height than they did in BMI. The unhealthiest changes—gaining too little height, too much weight for their height compared with children in other countries, or both—occurred in many countries in sub-Saharan Africa, New Zealand, and the USA for boys and girls; in Malaysia and some Pacific island nations for boys; and in Mexico for girls. Interpretation The height and BMI trajectories over age and time of school-aged children and adolescents are highly variable across countries, which indicates heterogeneous nutritional quality and lifelong health advantages and risks

Rising rural body-mass index is the main driver of the global obesity epidemic in adults

Body-mass index (BMI) has increased steadily in most countries in parallel with a rise in the proportion of the population who live in cities(.)(1,2) This has led to a widely reported view that urbanization is one of the most important drivers of the global rise in obesity(3-6). Here we use 2,009 population-based studies, with measurements of height and weight in more than 112 million adults, to report national, regional and global trends in mean BMI segregated by place of residence (a rural or urban area) from 1985 to 2017. We show that, contrary to the dominant paradigm, more than 55% of the global rise in mean BMI from 1985 to 2017-and more than 80% in some low- and middle-income regions-was due to increases in BMI in rural areas. This large contribution stems from the fact that, with the exception of women in sub-Saharan Africa, BMI is increasing at the same rate or faster in rural areas than in cities in low- and middle-income regions. These trends have in turn resulted in a closing-and in some countries reversal-of the gap in BMI between urban and rural areas in low- and middle-income countries, especially for women. In high-income and industrialized countries, we noted a persistently higher rural BMI, especially for women. There is an urgent need for an integrated approach to rural nutrition that enhances financial and physical access to healthy foods, to avoid replacing the rural undernutrition disadvantage in poor countries with a more general malnutrition disadvantage that entails excessive consumption of low-quality calories.Peer reviewe

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries