"Be sustainable": EOSC-Life recommendations for implementation of FAIR principles in life science data handling.

The main goals and challenges for the life science communities in the Open Science framework are to increase reuse and sustainability of data resources, software tools, and workflows, especially in large-scale data-driven research and computational analyses. Here, we present key findings, procedures, effective measures and recommendations for generating and establishing sustainable life science resources based on the collaborative, cross-disciplinary work done within the EOSC-Life (European Open Science Cloud for Life Sciences) consortium. Bringing together 13 European life science research infrastructures, it has laid the foundation for an open, digital space to support biological and medical research. Using lessons learned from 27 selected projects, we describe the organisational, technical, financial and legal/ethical challenges that represent the main barriers to sustainability in the life sciences. We show how EOSC-Life provides a model for sustainable data management according to FAIR (findability, accessibility, interoperability, and reusability) principles, including solutions for sensitive- and industry-related resources, by means of cross-disciplinary training and best practices sharing. Finally, we illustrate how data harmonisation and collaborative work facilitate interoperability of tools, data, solutions and lead to a better understanding of concepts, semantics and functionalities in the life sciences.

Phosphatidylethanolamine and Phosphatidylserine Synergize To Enhance GAS6/AXL-Mediated Virus Infection and Efferocytosis.

Phosphatidylserine (PS) receptors mediate clearance of apoptotic cells-efferocytosis-by recognizing the PS exposed on those cells. They also mediate the entry of enveloped viruses by binding PS in the virion membrane. Here, we show that phosphatidylethanolamine (PE) synergizes with PS to enhance PS receptor-mediated efferocytosis and virus entry. The presence of PE on the same surface as PS dramatically enhances recognition of PS by PS-binding proteins such as GAS6, PROS, and TIM1. Liposomes containing both PE and PS bound to GAS6 and were engulfed by AXL-expressing cells much more efficiently than those containing PS alone. Further, infection of AXL-expressing cells by infectious Zika virus or Ebola, Chikungunya, or eastern equine encephalitis pseudoviruses was inhibited with greater efficiency by the liposomes containing both PS and PE compared to a mixture of liposomes separately composed of PS and PE. These data demonstrate that simultaneous recognition of PE and PS maximizes PS receptor-mediated virus entry and efferocytosis and underscore the important contribution of PE in these major biological processes.IMPORTANCE Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are usually sequestered to the inner leaflet of the plasma membrane of the healthy eukaryotic cells. During apoptosis, these phospholipids move to the cell's outer leaflet where they are recognized by so-called PS receptors on surveilling phagocytes. Several pathogenic families of enveloped viruses hijack these PS receptors to gain entry into their target cells. Here, we show that efficiency of these processes is enhanced, namely, PE synergizes with PS to promote PS receptor-mediated virus infection and clearance of apoptotic cells. These findings deepen our understanding of how these fundamental biological processes are executed.

AXL-dependent infection of human fetal endothelial cells distinguishes Zika virus from other pathogenic flaviviruses.

Although a causal relationship between Zika virus (ZIKV) and microcephaly has been established, it remains unclear why ZIKV, but not other pathogenic flaviviruses, causes congenital defects. Here we show that when viruses are produced in mammalian cells, ZIKV, but not the closely related dengue virus (DENV) or West Nile virus (WNV), can efficiently infect key placental barrier cells that directly contact the fetal bloodstream. We show that AXL, a receptor tyrosine kinase, is the primary ZIKV entry cofactor on human umbilical vein endothelial cells (HUVECs), and that ZIKV uses AXL with much greater efficiency than does DENV or WNV. Consistent with this observation, only ZIKV, but not WNV or DENV, bound the AXL ligand Gas6. In comparison, when DENV and WNV were produced in insect cells, they also infected HUVECs in an AXL-dependent manner. Our data suggest that ZIKV, when produced from mammalian cells, infects fetal endothelial cells much more efficiently than other pathogenic flaviviruses because it binds Gas6 more avidly, which in turn facilitates its interaction with AXL.

Antimicrobial resistance profiles of microorganisms isolated from hospitalized patients in Dominican Republic.

OBJECTIVE: To define the antimicrobial resistance profiles of the microorganisms most commonly isolated from hospitalized adult patients in Dominican Republic (DR). METHODS: A retrospective, cross-sectional study of phenotypic antimicrobial susceptibility patterns was conducted using data from 3 802 clinical microbiology reports specifying positive bacterial cultures in samples collected from patients admitted to the clinical, surgery, and intensive care units (ICU) at three tertiary-level care hospitals in the city of Santiago de los Caballeros from 1 January 2016 - 31 December 2017. Descriptive statistics and chi-square test (P ≤ 0.05) were used to analyze the qualitative variables. RESULTS: At the three hospitals, there were 932, 1 090, and 1 780 microbiology reports analyzed. Of the total, 1274 were from the ICU, 1 042 from the surgery unit, and 1 486 from the clinical unit. Methicillin resistance was found in 57.3% of the Staphylococcus aureus isolates and 75.3% of the coagulase-negative staphylococci. Third-generation cephalosporin resistance was detected in 54.4% of isolates identified as members of the Enterobacteriaceae family, 67.3% of the Acinetobacter spp., and 91.7% of the Pseudomonas, while carbapenem resistance was shown by 8.0%, 23.8%, and 51.0% of these, respectively. Most of the resistant Acinetobacter spp. isolates were found in just one hospital and the prevalence of Enterobacteriaceae resistant to carbapenems was highest in the ICU. CONCLUSION: Antimicrobial resistance levels are high among hospitalized patients in Dominican Republic and may cause enhanced risk factors that impact clinical outcomes. Urgent measures are needed to address antimicrobial resistance in DR.

Virion-associated phosphatidylethanolamine promotes TIM1-mediated infection by Ebola, dengue, and West Nile viruses.

Phosphatidylserine (PS) receptors contribute to two crucial biological processes: apoptotic clearance and entry of many enveloped viruses. In both cases, they recognize PS exposed on the plasma membrane. Here we demonstrate that phosphatidylethanolamine (PE) is also a ligand for PS receptors and that this phospholipid mediates phagocytosis and viral entry. We show that a subset of PS receptors, including T-cell immunoglobulin (Ig) mucin domain protein 1 (TIM1), efficiently bind PE. We further show that PE is present in the virions of flaviviruses and filoviruses, and that the PE-specific cyclic peptide lantibiotic agent Duramycin efficiently inhibits the entry of West Nile, dengue, and Ebola viruses. The inhibitory effect of Duramycin is specific: it inhibits TIM1-mediated, but not L-SIGN-mediated, virus infection, and it does so by blocking virus attachment to TIM1. We further demonstrate that PE is exposed on the surface of apoptotic cells, and promotes their phagocytic uptake by TIM1-expressing cells. Together, our data show that PE plays a key role in TIM1-mediated virus entry, suggest that disrupting PE association with PS receptors is a promising broad-spectrum antiviral strategy, and deepen our understanding of the process by which apoptotic cells are cleared.

Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication.

The human immunodeficiency virus type 1 (HIV-1) capsid plays crucial roles in HIV-1 replication and thus represents an excellent drug target. We developed a high-throughput screening method based on a time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) assay, using the C-terminal domain (CTD) of HIV-1 capsid to identify inhibitors of capsid dimerization. This assay was used to screen a library of pharmacologically active compounds, composed of 1,280in vivo-active drugs, and identified ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], an organoselenium compound, as an inhibitor of HIV-1 capsid CTD dimerization. Nuclear magnetic resonance (NMR) spectroscopic analysis confirmed the direct interaction of ebselen with the HIV-1 capsid CTD and dimer dissociation when ebselen is in 2-fold molar excess. Electrospray ionization mass spectrometry revealed that ebselen covalently binds the HIV-1 capsid CTD, likely via a selenylsulfide linkage with Cys198 and Cys218. This compound presents anti-HIV activity in single and multiple rounds of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells. Ebselen inhibits early viral postentry events of the HIV-1 life cycle by impairing the incoming capsid uncoating process. This compound also blocks infection of other retroviruses, such as Moloney murine leukemia virus and simian immunodeficiency virus, but displays no inhibitory activity against hepatitis C and influenza viruses. This study reports the use of TR-FRET screening to successfully identify a novel capsid inhibitor, ebselen, validating HIV-1 capsid as a promising target for drug development.

An iterative and interdisciplinary categorisation process towards FAIRer digital resources for sensitive life-sciences data.

For life science infrastructures, sensitive data generate an additional layer of complexity. Cross-domain categorisation and discovery of digital resources related to sensitive data presents major interoperability challenges. To support this FAIRification process, a toolbox demonstrator aiming at support for discovery of digital objects related to sensitive data (e.g., regulations, guidelines, best practice, tools) has been developed. The toolbox is based upon a categorisation system developed and harmonised across a cluster of 6 life science research infrastructures. Three different versions were built, tested by subsequent pilot studies, finally leading to a system with 7 main categories (sensitive data type, resource type, research field, data type, stage in data sharing life cycle, geographical scope, specific topics). 109 resources attached with the tags in pilot study 3 were used as the initial content for the toolbox demonstrator, a software tool allowing searching of digital objects linked to sensitive data with filtering based upon the categorisation system. Important next steps are a broad evaluation of the usability and user-friendliness of the toolbox, extension to more resources, broader adoption by different life-science communities, and a long-term vision for maintenance and sustainability.

Uncovering a conserved vulnerability site in SARS-CoV-2 by a human antibody.

An essential step for SARS-CoV-2 infection is the attachment to the host cell receptor by its Spike receptor-binding domain (RBD). Most of the existing RBD-targeting neutralizing antibodies block the receptor-binding motif (RBM), a mutable region with the potential to generate neutralization escape mutants. Here, we isolated and structurally characterized a non-RBM-targeting monoclonal antibody (FD20) from convalescent patients. FD20 engages the RBD at an epitope distal to the RBM with a KD of 5.6 nM, neutralizes SARS-CoV-2 including the current Variants of Concern such as B.1.1.7, B.1.351, P.1, and B.1.617.2 (Delta), displays modest cross-reactivity against SARS-CoV, and reduces viral replication in hamsters. The epitope coincides with a predicted "ideal" vulnerability site with high functional and structural constraints. Mutation of the residues of the conserved epitope variably affects FD20-binding but confers little or no resistance to neutralization. Finally, in vitro mode-of-action characterization and negative-stain electron microscopy suggest a neutralization mechanism by which FD20 destructs the Spike. Our results reveal a conserved vulnerability site in the SARS-CoV-2 Spike for the development of potential antiviral drugs.

A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection.

SARS-CoV-2, the causative agent of COVID-191, features a receptor-binding domain (RBD) for binding to the host cell ACE2 protein1-6. Neutralizing antibodies that block RBD-ACE2 interaction are candidates for the development of targeted therapeutics7-17. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages in bioavailability, amenability, and production and storage owing to their small sizes and high stability. Here, we report the rapid selection of 99 synthetic nanobodies (sybodies) against RBD by in vitro selection using three libraries. The best sybody, MR3 binds to RBD with high affinity (KD = 1.0 nM) and displays high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.42 µg mL-1). Structural, biochemical, and biological characterization suggests a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency have been generated by structure-based design, biparatopic construction, and divalent engineering. Two divalent forms of MR3 protect hamsters from clinical signs after live virus challenge and a single dose of the Fc-fusion construct of MR3 reduces viral RNA load by 6 Log10. Our results pave the way for the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid development of targeted medical interventions during an outbreak.

Parvovirus H-1 induces cytopathic effects in breast carcinoma-derived cultures.

Parvovirus H-1 (H-1 PV) preferentially replicates in malignant cells resulting in their death by cytolysis. It has often been considered a potential candidate for use in novel anticancer therapy. To evaluate its potential in a model of natural tumors, we assayed in vitro the effect exerted by H-1 PV on short-term cultures derived from breast tumor samples freshly excised from patients. Our results show that H-1 PV effectively kills tumor-derived cells, whereas normal tissue-derived cells showed no H-1 PV-induced cytopathic effects (CPE). We also determined that the H-1 PV sensitivity (up to 67% sensitive cultures) is related with the quantities of virus assayed. We further examined the expression and phosphorylation state of the parvoviral nonstructural protein 1 (NS1), known to be associated with parvoviruses-induced CPE. Both appear to be impaired in normal tissue-derived cells and resistant cultures. Finally, we show that H-1 PV sensitivity in cultures correlates significantly with higher tumor grades (Nottingham combined histologic grade 2 or 3). This report confirms that H-1 PV can efficiently induce CPE in primary breast tumor cells in vitro. It identifies tumor characteristics representing potential criteria for recruiting patients for clinical evaluation of H-1 PV antitumor effects.

Ectopic expression of H-1 parvovirus NS1 protein induces alterations in actin filaments and cell death in human normal MRC-5 and transformed MRC-5 SV2 cells.

When grown in human cell lines, oncolytic H-1 parvovirus (H-1PV) replication preferentially occurs in transformed cells, which ultimately die upon infection. H-1PV-induced cytotoxicity is mainly due to P4 promoter-driven NS1 protein expression. Infection of untransformed cells generally does not induce deleterious effects because the P4 promoter is not activated. Here, we show that ectopic CMV-driven NS1 protein expression in normal human MRC-5 cells results in alterations of actin filaments and cell death, and both effects are prevented by a serine 473 mutation. The same substitution preserves actin filaments of transfected MRC-5 SV2 cells, that are MRC-5 transformed counterparts, but does not impair NS1-induced cytotoxicity.

Effect of the Organic Semiconductor Side Groups on the Structural and Electronic Properties of Their Interface with Dopants.

Two derivatives of [1]benzothieno[3,2-b][1]benzothiophene (BTBT), namely, 2,7-dioctyl-BTBT (C8-BTBT) and 2,7-diphenyl-BTBT (DPh-BTBT), belonging to one of the best performing organic semiconductor (OSC) families, have been employed to investigate the influence of the substitutional side groups on the properties of the interface created when they are in contact with dopant molecules. As a molecular p-dopant, the fluorinated fullerene C60F48 is used because of its adequate electronic levels and its bulky molecular structure. Despite the dissimilarity introduced by the OSC film termination, dopant thin films grown on top adopt the same (111)-oriented FCC crystalline structure in the two cases. However, the early stage distribution of the dopant on each OSC film surface is dramatically influenced by the group side, leading to distinct host-dopant interfacial morphologies that strongly affect the nanoscale local work function. In this context, Kelvin probe force microscopy and photoelectron emission spectroscopy provide a comprehensive picture of the interfacial electronic properties. The extent of charge transfer and energy level alignment between OSCs and dopant are debated in light of the differences in the ionization potential of the OSC in the films, the interface nanomorphology, and the electronic coupling with the substrate.

AAV vectors engineered to target insulin receptor greatly enhance intramuscular gene delivery.

Adeno-associated virus (AAV) is one of the most commonly used vectors for gene therapy, and the applications for AAV-delivered therapies are numerous. However, the current state of technology is limited by the low efficiency with which most AAV vectors transduce skeletal muscle tissue. We demonstrate that vector efficiency can be enhanced by modifying the AAV capsid with a peptide that binds a receptor highly expressed in muscle tissue. When an insulin-mimetic peptide, S519, previously characterized for its high affinity to insulin receptor (IR), was inserted into the capsid, the AAV9 transduction efficiency of IR-expressing cell lines as well as differentiated primary human muscle cells was dramatically enhanced. This vector also exhibited efficient transduction of mouse muscle in vivo, resulting in up to 18-fold enhancement over AAV9. Owing to its superior transduction efficiency in skeletal muscle, we named this vector "enhanced AAV9" (eAAV9). We also found that the modification enhanced the transduction efficiency of several other AAV serotypes. Together, these data show that AAV transduction of skeletal muscle can be improved by targeting IR. They also show the broad utility of this modular strategy and suggest that it could also be applied to next-generation vectors that have yet to be engineered.

Chasing the "Killer" Phonon Mode for the Rational Design of Low-Disorder, High-Mobility Molecular Semiconductors.

Molecular vibrations play a critical role in the charge transport properties of weakly van der Waals bonded organic semiconductors. To understand which specific phonon modes contribute most strongly to the electron-phonon coupling and ensuing thermal energetic disorder in some of the most widely studied high-mobility molecular semiconductors, state-of-the-art quantum mechanical simulations of the vibrational modes and the ensuing electron-phonon coupling constants are combined with experimental measurements of the low-frequency vibrations using inelastic neutron scattering and terahertz time-domain spectroscopy. In this way, the long-axis sliding motion is identified as a "killer" phonon mode, which in some molecules contributes more than 80% to the total thermal disorder. Based on this insight, a way to rationalize mobility trends between different materials and derive important molecular design guidelines for new high-mobility molecular semiconductors is suggested.

Zika Virus-Immune Plasmas from Symptomatic and Asymptomatic Individuals Enhance Zika Pathogenesis in Adult and Pregnant Mice.

Preexisting immunity against dengue virus or West Nile virus was previously reported to mediate antibody-dependent enhancement (ADE) of Zika virus (ZIKV) infection in a mouse model. We show here that ZIKV-immune plasma samples from both symptomatic and asymptomatic individuals mediated ZIKV ADE of infection in vitro and in mice. In a lethal infection model with a viral inoculum 10 times higher, both ADE and protection were observed, depending on the amount of infused immune plasma. In a vertical-transmission model, ZIKV-immune plasma infused to timed pregnant mice increased fetal demise and decreased the body weight of surviving fetuses. Depletion of IgG from an immune plasma abolished ADE of infection, and the presence of purified IgG alone mediated ADE of infection. Higher viral loads and proinflammatory cytokines were detected in mice treated with ZIKV-immune plasma samples compared to those receiving control plasma. Together, these data show that passive immunization with homotypic ZIKV antibodies, depending on the concentration, could either worsen or limit a subsequent ZIKV infection.IMPORTANCE Antibody-dependent enhancement (ADE) of virus infection is common to many viruses and is problematic when plasma antibody levels decline to subneutralizing concentrations. ADE of infection is especially important among flaviviruses, many of which are the cause of global health problems. Recently, human plasma samples immune to heterologous flaviviruses were shown to promote Zika virus (ZIKV) infection. Here we showed in immunocompromised mouse models that homologous immune plasma samples protect mice from subsequent infection at high antibody concentrations but that they mediate ADE of infection and increase ZIKV pathogenesis in adult mice and fetal demise during pregnancy at low concentrations.

Rotator side chains trigger cooperative transition for shape and function memory effect in organic semiconductors.

Martensitic transition is a solid-state phase transition involving cooperative movement of atoms, mostly studied in metallurgy. The main characteristics are low transition barrier, ultrafast kinetics, and structural reversibility. They are rarely observed in molecular crystals, and hence the origin and mechanism are largely unexplored. Here we report the discovery of martensitic transition in single crystals of two different organic semiconductors. In situ microscopy, single-crystal X-ray diffraction, Raman and nuclear magnetic resonance spectroscopy, and molecular simulations combined indicate that the rotating bulky side chains trigger cooperative transition. Cooperativity enables shape memory effect in single crystals and function memory effect in thin film transistors. We establish a molecular design rule to trigger martensitic transition in organic semiconductors, showing promise for designing next-generation smart multifunctional materials.

Activation of a helper and not regulatory human CD4+ T cell response by oncolytic H-1 parvovirus.

BACKGROUND: H-1 parvovirus (H-1 PV), a rodent autonomous oncolytic parvovirus, has emerged as a novel class of promising anticancer agents, because of its ability to selectively find and destroy malignant cells. However, to probe H-1 PV multimodal antitumor potential one of the major prerequisites is to decipher H-1 PV direct interplay with human immune system, and so prevent any risk of impairment. METHODOLOGY/PRINCIPAL FINDINGS: Non activated peripheral blood mononuclear cells (PBMCs) are not sensitive to H-1 PV cytotoxic effect. However, the virus impairs both activated PBMC proliferation ability and viability. This effect is related to H-1 PV infection as evidenced by Western blotting detection of H-1 PV main protein NS1. However, TCID50 experiments did not allow newly generated virions to be detected. Moreover, flow cytometry has shown that H-1 PV preferentially targets B lymphocytes. Despite seeming harmful at first sight, H-1 PV seems to affect very few NK cells and CD8+ T lymphocytes and, above all, clearly does not affect human neutrophils and one of the major CD4+ T lymphocyte subpopulation. Very interestingly, flow cytometry analysis and ELISA assays proved that it even activates human CD4+ T cells by increasing activation marker expression (CD69 and CD30) and both effective Th1 and Th2 cytokine secretion (IL-2, IFN-γ and IL-4). In addition, H-1 PV action does not come with any sign of immunosuppressive side effect. Finally, we have shown the efficiency of H-1 PV on xenotransplanted human nasopharyngeal carcinoma, in a SCID mouse model reconstituted with human PBMC. CONCLUSIONS/SIGNIFICANCE: Our results show for the first time that a wild-type oncolytic virus impairs some immune cell subpopulations while directly activating a Helper CD4+ T cell response. Thus, our data open numerous gripping perspectives of investigation and strongly argue for the use of H-1 PV as an anticancer treatment.