FAIR+E pathogen data for surveillance and research: lessons from COVID-19.

The COVID-19 pandemic has exemplified the importance of interoperable and equitable data sharing for global surveillance and to support research. While many challenges could be overcome, at least in some countries, many hurdles within the organizational, scientific, technical and cultural realms still remain to be tackled to be prepared for future threats. We propose to (i) continue supporting global efforts that have proven to be efficient and trustworthy toward addressing challenges in pathogen molecular data sharing; (ii) establish a distributed network of Pathogen Data Platforms to (a) ensure high quality data, metadata standardization and data analysis, (b) perform data brokering on behalf of data providers both for research and surveillance, (c) foster capacity building and continuous improvements, also for pandemic preparedness; (iii) establish an International One Health Pathogens Portal, connecting pathogen data isolated from various sources (human, animal, food, environment), in a truly One Health approach and following FAIR principles. To address these challenging endeavors, we have started an ELIXIR Focus Group where we invite all interested experts to join in a concerted, expert-driven effort toward sustaining and ensuring high-quality data for global surveillance and research.

Protein domains provide a new layer of information for classifying human variations in rare diseases.

Introduction: Using the ACMG-AMP guidelines for the interpretation of sequence variants, it remains difficult to meet the criterion associated with the protein domain, PM1, which is assigned in only about 10% of cases, whereas the criteria related to variant frequency, PM2/BA1/BS1, is reported in 50% of cases. To improve the classification of human missense variants using protein domains information, we developed the DOLPHIN system (https://dolphin.mmg-gbit.eu). Methods: We used Pfam alignments of eukaryotes to define DOLPHIN scores to identify protein domain residues and variants that have a significant impact. In parallel, we enriched gnomAD variants frequencies for each domains' residue. These were validated using ClinVar data. Results: We applied this method to all potential human transcripts' variants, resulting in 30.0% being assigned a PM1 label, whereas 33.2% were eligible for a new benign support criterion, BP8. We also showed that DOLPHIN provides an extrapolated frequency for 31.8% of the variants, compared to the original frequency available in gnomAD for 7.6% of them. Discussion: Overall, DOLPHIN allows a simplified use of the PM1 criterion, an expanded application of the PM2/BS1 criteria and the creation of a new BP8 criterion. DOLPHIN could facilitate the classification of amino acid substitutions in protein domains that cover nearly 40% of proteins and represent the sites of most pathogenic variants.

Transportation as a Disease Vector in COVID-19: Border Mobility and Disease Spread.

More than a year after COVID-19 was declared a pandemic by the World Health Organization, the U.S.A. and Mexico rank first and fourth, respectively, with regard to the number of deaths. From March 2020, nonessential travelers were not allowed to cross the border into the U.S.A. from Mexico via international land ports of entry, which resulted in a more than 50% decrease in the number of people crossing the border. However, border communities still face a higher number of cases and faster community spread compared with those without international land ports of entry. This paper established an econometric model to understand the effects of cross-border mobility and other socioeconomic parameters on the speed of spread. The model was developed at the U.S. county level using data from all 3,141 counties in the U.S.A. Additionally, a follow-up U.S. county comparative analysis was developed to examine the significance of having a border crossing between the U.S.A. and Mexico for U.S. counties. The findings of the analysis revealed that the variables having a significant effect are as follows: population density; number of people per household; population in the 15-65 age group; median household income; mask use; number of visits to transit stations; number of visits to workplace; overall mobility; and having a border crossing to Mexico within county limits. The comparative analysis found that U.S. counties with border crossings have an average of 123 cases per 1,000 population whereas their counterparts without border crossings only have 90 cases per 1,000 population.

Electrical Conductive Properties of 3D-PrintedConcrete Composite with Carbon Nanofibers.

Electrical conductive properties in cement-based materials have received attention in recent years due to their key role in many innovative application (i.e., energy harvesting, deicing systems, electromagnetic shielding, and self-health monitoring). In this work, we explore the use 3D printing as an alternative method for the preparation of electrical conductive concretes. With this aim, the conductive performance of cement composites with carbon nanofibers (0, 1, 2.5, and 4 wt%) was explored by means of a combination of thermogravimetric analysis (TGA) and dielectric spectroscopy (DS) and compared with that of specimens prepared with the traditional mold method. The combination of TGA and DS gave us a unique insight into the electrical conductive properties, measuring the specimens' performance while monitoring the amount in water confined in the porous network. Experimental evidence of an additional contribution to the electrical conductivity due to sample preparation is provided. In particular, in this work, a strong correlation between water molecules in interconnected pores and the σ(ω) values is shown, originating, mainly, from the use of the 3D printing technique.

The RD-Connect Genome-Phenome Analysis Platform: Accelerating diagnosis, research, and gene discovery for rare diseases.

Rare disease patients are more likely to receive a rapid molecular diagnosis nowadays thanks to the wide adoption of next-generation sequencing. However, many cases remain undiagnosed even after exome or genome analysis, because the methods used missed the molecular cause in a known gene, or a novel causative gene could not be identified and/or confirmed. To address these challenges, the RD-Connect Genome-Phenome Analysis Platform (GPAP) facilitates the collation, discovery, sharing, and analysis of standardized genome-phenome data within a collaborative environment. Authorized clinicians and researchers submit pseudonymised phenotypic profiles encoded using the Human Phenotype Ontology, and raw genomic data which is processed through a standardized pipeline. After an optional embargo period, the data are shared with other platform users, with the objective that similar cases in the system and queries from peers may help diagnose the case. Additionally, the platform enables bidirectional discovery of similar cases in other databases from the Matchmaker Exchange network. To facilitate genome-phenome analysis and interpretation by clinical researchers, the RD-Connect GPAP provides a powerful user-friendly interface and leverages tens of information sources. As a result, the resource has already helped diagnose hundreds of rare disease patients and discover new disease causing genes.

Standardisation of pathogenicity classification for somatic alterations in solid tumours and haematologic malignancies.

BACKGROUND: The difficulty in interpreting somatic alterations is correlated with the increase in sequencing panel size. To correctly guide the clinical management of patients with cancer, there needs to be accurate classification of pathogenicity followed by actionability assessment. Here, we describe a specific detailed workflow for the classification of the pathogenicity of somatic variants in cancer into five categories: benign, likely benign, unknown significance, likely pathogenic and pathogenic. METHODS: Classification is obtained by combining a set of eight relevant criteria in favour of either a pathogenic or a benign effect (pathogenic stand-alone, pathogenic very strong, pathogenic strong, pathogenic moderate, pathogenic supporting, benign supporting, benign strong and benign stand-alone). RESULTS: Our guide is concordant with the ACMG/AMP 2015 guidelines for germline variants. Interpretation of somatic variants requires considering specific criteria, such as the disease and therapeutic context, co-occurring genomic events in the tumour when available and the use of cancer-specific variant databases. In addition, the gene role in tumorigenesis (oncogene or tumour suppressor gene) also needs to be taken into consideration. CONCLUSION: Our classification could contribute to homogenize best practices on somatic variant pathogenicity interpretation and improve interpretation consistency both within and between laboratories.

Maintenance Strategies for Industrial Multi-Stage Machines: The Study of a Thermoforming Machine.

The study of reliability, availability and control of industrial manufacturing machines is a constant challenge in the industrial environment. This paper compares the results offered by several maintenance strategies for multi-stage industrial manufacturing machines by analysing a real case of a multi-stage thermoforming machine. Specifically, two strategies based on preventive maintenance, Preventive Programming Maintenance (PPM) and Improve Preventive Programming Maintenance (IPPM) are compared with two new strategies based on predictive maintenance, namely Algorithm Life Optimisation Programming (ALOP) and Digital Behaviour Twin (DBT). The condition of machine components can be assessed with the latter two proposals (ALOP and DBT) using sensors and algorithms, thus providing a warning value for early decision-making before unexpected faults occur. The study shows that the ALOP and DBT models detect unexpected failures early enough, while the PPM and IPPM strategies warn of scheduled component replacement at the end of their life cycle. The ALOP and DBT strategies algorithms can also be valid for managing the maintenance of other multi-stage industrial manufacturing machines. The authors consider that the combination of preventive and predictive maintenance strategies may be an ideal approach because operating conditions affect the mechanical, electrical, electronic and pneumatic components of multi-stage industrial manufacturing machines differently.

The lncRNA 44s2 Study Applicability to the Design of 45-55 Exon Skipping Therapeutic Strategy for DMD.

In skeletal muscle, long noncoding RNAs (lncRNAs) are involved in dystrophin protein stabilization but also in the regulation of myocytes proliferation and differentiation. Hence, they could represent promising therapeutic targets and/or biomarkers for Duchenne and Becker muscular dystrophy (DMD/BMD). DMD and BMD are X-linked myopathies characterized by a progressive muscular dystrophy with or without dilatative cardiomyopathy. Two-thirds of DMD gene mutations are represented by deletions, and 63% of patients carrying DMD deletions are eligible for 45 to 55 multi-exons skipping (MES), becoming BMD patients (BMDΔ45-55). We analyzed the genomic lncRNA presence in 38 BMDΔ45-55 patients and characterized the lncRNA localized in introns 44 and 55 of the DMD gene. We highlighted that all four lncRNA are differentially expressed during myogenesis in immortalized and primary human myoblasts. In addition, the lncRNA44s2 was pointed out as a possible accelerator of differentiation. Interestingly, lncRNA44s expression was associated with a favorable clinical phenotype. These findings suggest that lncRNA44s2 could be involved in muscle differentiation process and become a potential disease progression biomarker. Based on these results, we support MES45-55 therapy and propose that the design of the CRISPR/Cas9 MES45-55 assay consider the lncRNA sequences bordering the exonic 45 to 55 deletion.

TGFß signalling acts as a molecular brake of myoblast fusion.

Fusion of nascent myoblasts to pre-existing myofibres is critical for skeletal muscle growth and repair. The vast majority of molecules known to regulate myoblast fusion are necessary in this process. Here, we uncover, through high-throughput in vitro assays and in vivo studies in the chicken embryo, that TGFß (SMAD2/3-dependent) signalling acts specifically and uniquely as a molecular brake on muscle fusion. While constitutive activation of the pathway arrests fusion, its inhibition leads to a striking over-fusion phenotype. This dynamic control of TGFß signalling in the embryonic muscle relies on a receptor complementation mechanism, prompted by the merging of myoblasts with myofibres, each carrying one component of the heterodimer receptor complex. The competence of myofibres to fuse is likely restored through endocytic degradation of activated receptors. Altogether, this study shows that muscle fusion relies on TGFß signalling to regulate its pace.

Identification of a peripheral blood gene signature predicting aortic valve calcification.

Calcific aortic valve disease (CAVD) is a significant cause of illness and death worldwide. Identification of early predictive markers could help optimize patient management. RNA-sequencing was carried out on human fetal aortic valves at gestational weeks 9, 13, and 22 and on a case-control study with adult noncalcified and calcified bicuspid and tricuspid aortic valves. In dimension reduction and clustering analyses, diseased valves tended to cluster with fetal valves at week 9 rather than normal adult valves, suggesting that part of the disease program might be due to reiterated developmental processes. The analysis of groups of coregulated genes revealed predominant immune-metabolic signatures, including innate and adaptive immune responses involving lymphocyte T-cell metabolic adaptation. Cytokine and chemokine signaling, cell migration, and proliferation were all increased in CAVD, whereas oxidative phosphorylation and protein translation were decreased. Discrete immune-metabolic gene signatures were present at fetal stages and increased in adult controls, suggesting that these processes intensify throughout life and heighten in disease. Cellular stress response and neurodegeneration gene signatures were aberrantly expressed in CAVD, pointing to a mechanistic link between chronic inflammation and biological aging. Comparison of the valve RNA-sequencing data set with a case-control study of whole blood transcriptomes from asymptomatic individuals with early aortic valve calcification identified a highly predictive gene signature of CAVD and of moderate aortic valve calcification in overtly healthy individuals. These data deepen and broaden our understanding of the molecular basis of CAVD and identify a peripheral blood gene signature for the early detection of aortic valve calcification.

Hox-dependent coordination of mouse cardiac progenitor cell patterning and differentiation.

Perturbation of addition of second heart field (SHF) cardiac progenitor cells to the poles of the heart tube results in congenital heart defects (CHD). The transcriptional programs and upstream regulatory events operating in different subpopulations of the SHF remain unclear. Here, we profile the transcriptome and chromatin accessibility of anterior and posterior SHF sub-populations at genome-wide levels and demonstrate that Hoxb1 negatively regulates differentiation in the posterior SHF. Spatial mis-expression of Hoxb1 in the anterior SHF results in hypoplastic right ventricle. Activation of Hoxb1 in embryonic stem cells arrests cardiac differentiation, whereas Hoxb1-deficient mouse embryos display premature cardiac differentiation. Moreover, ectopic differentiation in the posterior SHF of embryos lacking both Hoxb1 and its paralog Hoxa1 results in atrioventricular septal defects. Our results show that Hoxb1 plays a key role in patterning cardiac progenitor cells that contribute to both cardiac poles and provide new insights into the pathogenesis of CHD.

Transgenesis and web resources in quail.

Due to its amenability to manipulations, to live observation and its striking similarities to mammals, the chicken embryo has been one of the major animal models in biomedical research. Although it is technically possible to genome-edit the chicken, its long generation time (6 months to sexual maturity) makes it an impractical lab model and has prevented it widespread use in research. The Japanese quail (Coturnix coturnix japonica) is an attractive alternative, very similar to the chicken, but with the decisive asset of a much shorter generation time (1.5 months). In recent years, transgenic quail lines have been described. Most of them were generated using replication-deficient lentiviruses, a technique that presents diverse limitations. Here, we introduce a novel technology to perform transgenesis in quail, based on the in vivo transfection of plasmids in circulating Primordial Germ Cells (PGCs). This technique is simple, efficient and allows using the infinite variety of genome engineering approaches developed in other models. Furthermore, we present a website centralizing quail genomic and technological information to facilitate the design of genome-editing strategies, showcase the past and future transgenic quail lines and foster collaborative work within the avian community.

Piezo1 is required for outflow tract and aortic valve development.

AIMS: During embryogenesis, the onset of circulatory blood flow generates a variety of hemodynamic forces which reciprocally induce changes in cardiovascular development and performance. It has been known for some time that these forces can be detected by as yet unknown mechanosensory systems which in turn promote cardiogenic events such as outflow tract and aortic valve development. PIEZO1 is a mechanosensitive ion channel present in endothelial cells where it serves to detect hemodynamic forces making it an ideal candidate to play a role during cardiac development. We sought to determine whether PIEZO1 is required for outflow tract and aortic valve development. METHODS AND RESULTS: By analysing heart development in zebrafish we have determined that piezo1 is expressed in the developing outflow tract where it serves to detect hemodynamic forces. Consequently, disrupting Piezo1 signalling leads to defective outflow tract and aortic valve development and indicates this gene may be involved in the etiology of congenital heart diseases. Based on these findings, we analysed genomic data generated from patients who suffer from left ventricular outflow tract obstructions (LVOTO) and identified 3 probands who each harboured potentially pathogenic variants in PIEZO1. Subsequent in vitro and in vivo assays indicates that these variants behave as dominant negatives leading to an inhibition of normal PIEZO1 mechanosensory activity. Expressing these dominant negative PIEZO1 variants in zebrafish endothelium leads to defective aortic valve development. CONCLUSION: These data indicate that the mechanosensitive ion channel piezo1 is required for outflow tract and aortic valve development.

Advances in Orthotic and Prosthetic Manufacturing: A Technology Review.

In this work, the recent advances for rapid prototyping in the orthoprosthetic industry are presented. Specifically, the manufacturing process of orthoprosthetic aids are analysed, as thier use is widely extended in orthopedic surgery. These devices are devoted to either correct posture or movement (orthosis) or to substitute a body segment (prosthesis) while maintaining functionality. The manufacturing process is traditionally mainly hand-crafted: The subject's morphology is taken by means of plaster molds, and the manufacture is performed individually, by adjusting the prototype over the subject. This industry has incorporated computer aided design (CAD), computed aided engineering (CAE) and computed aided manufacturing (CAM) tools; however, the true revolution is the result of the application of rapid prototyping technologies (RPT). Techniques such as fused deposition modelling (FDM), selective laser sintering (SLS), laminated object manufacturing (LOM), and 3D printing (3DP) are some examples of the available methodologies in the manufacturing industry that, step by step, are being included in the rehabilitation engineering market-an engineering field with growth and prospects in the coming years. In this work we analyse different methodologies for additive manufacturing along with the principal methods for collecting 3D body shapes and their application in the manufacturing of functional devices for rehabilitation purposes such as splints, ankle-foot orthoses, or arm prostheses.

The ELIXIR Human Copy Number Variations Community: building bioinformatics infrastructure for research.

Copy number variations (CNVs) are major causative contributors both in the genesis of genetic diseases and human neoplasias. While "High-Throughput" sequencing technologies are increasingly becoming the primary choice for genomic screening analysis, their ability to efficiently detect CNVs is still heterogeneous and remains to be developed. The aim of this white paper is to provide a guiding framework for the future contributions of ELIXIR's recently established human CNV Community, with implications beyond human disease diagnostics and population genomics. This white paper is the direct result of a strategy meeting that took place in September 2018 in Hinxton (UK) and involved representatives of 11 ELIXIR Nodes. The meeting led to the definition of priority objectives and tasks, to address a wide range of CNV-related challenges ranging from detection and interpretation to sharing and training. Here, we provide suggestions on how to align these tasks within the ELIXIR Platforms strategy, and on how to frame the activities of this new ELIXIR Community in the international context.

Author Correction: Leveraging European infrastructures to access 1 million human genomes by 2022.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In vivo measurement of surface skin strain during human gait to improve the design of rehabilitation devices.

When designing any rehabilitation, sportswear or exoskeleton device the mechanical behaviour of the body segment must be known, specifically the skin, because an excessive tissue strain may lead to ulceration and bedsores. To date, it is not known if the kinematic variability between subjects have an effect on the skin strain field, and therefore, in the design and manufacturing of rehabilitation products, such as orthoses. Several studies have analysed the skin deformation during human motion, nevertheless, the comparison between the skin strain field in different subjects during normal or pathological gait has not been reported yet. This work presents a comparison of skin strain analysis for different gait patterns to study the differences between people and, specifically, if it is possible to standardize the orthotic design between subjects with the same gait disorder. Moreover, the areas with relatively minimum strain during the ankle-foot motion are compared to improve the design of structural parts of rehabilitation devices. In this case, a validated 3D digital image correlation system has been used for this purpose combined with strain ellipse theory. The results demonstrate variations in the skin strain field between subjects with the same pathology and similarities between subjects with normal gait. However, more studies and experiments are necessaries to validate this hypothesis and also to test it between different gait pathologies.

Leveraging European infrastructures to access 1 million human genomes by 2022.

Human genomics is undergoing a step change from being a predominantly research-driven activity to one driven through health care as many countries in Europe now have nascent precision medicine programmes. To maximize the value of the genomic data generated, these data will need to be shared between institutions and across countries. In recognition of this challenge, 21 European countries recently signed a declaration to transnationally share data on at least 1 million human genomes by 2022. In this Roadmap, we identify the challenges of data sharing across borders and demonstrate that European research infrastructures are well-positioned to support the rapid implementation of widespread genomic data access.

Human pre-valvular endocardial cells derived from pluripotent stem cells recapitulate cardiac pathophysiological valvulogenesis.

Genetically modified mice have advanced our understanding of valve development and disease. Yet, human pathophysiological valvulogenesis remains poorly understood. Here we report that, by combining single cell sequencing and in vivo approaches, a population of human pre-valvular endocardial cells (HPVCs) can be derived from pluripotent stem cells. HPVCs express gene patterns conforming to the E9.0 mouse atrio-ventricular canal (AVC) endocardium signature. HPVCs treated with BMP2, cultured on mouse AVC cushions, or transplanted into the AVC of embryonic mouse hearts, undergo endothelial-to-mesenchymal transition and express markers of valve interstitial cells of different valvular layers, demonstrating cell specificity. Extending this model to patient-specific induced pluripotent stem cells recapitulates features of mitral valve prolapse and identified dysregulation of the SHH pathway. Concurrently increased ECM secretion can be rescued by SHH inhibition, thus providing a putative therapeutic target. In summary, we report a human cell model of valvulogenesis that faithfully recapitulates valve disease in a dish.