Computational epitope mapping of class I fusion proteins using low complexity supervised learning methods.

Antibody epitope mapping of viral proteins plays a vital role in understanding immune system mechanisms of protection. In the case of class I viral fusion proteins, recent advances in cryo-electron microscopy and protein stabilization techniques have highlighted the importance of cryptic or 'alternative' conformations that expose epitopes targeted by potent neutralizing antibodies. Thorough epitope mapping of such metastable conformations is difficult but is critical for understanding sites of vulnerability in class I fusion proteins that occur as transient conformational states during viral attachment and fusion. We introduce a novel method Accelerated class I fusion protein Epitope Mapping (AxIEM) that accounts for fusion protein flexibility to improve out-of-sample prediction of discontinuous antibody epitopes. Harnessing data from previous experimental epitope mapping efforts of several class I fusion proteins, we demonstrate that accuracy of epitope prediction depends on residue environment and allows for the prediction of conformation-dependent antibody target residues. We also show that AxIEM can identify common epitopes and provide structural insights for the development and rational design of vaccines.

[Preimplantation genetic testing: legal and ethical aspects in clinical practice]. / Tests génétiques préimplantatoires†: enjeux légaux et éthiques dans la pratique clinique.

In Switzerland, since modifications of the law regulating reproductive medicine introduced the 1rst of September 2017, preimplantation genetic testing (PGT) has been legalised. Infertile couples undergoing in vitro fertilization (IVF) can benefit from this technology by detecting which embryos are aneuploid (ie abnormal number of chromosomes, PGT-A). This is performed in order to transfer euploid embryos (normal number of chromosomes) and to optimise success, though data are limited. Couples at risk of transmitting a severe monogenic disease or unbalanced translocation can undergo PGT for monogenic disease or chromosomal structural rearrangements (PGT-M/SR). These tests are subject to strict legal criteria. Their clinical application needs to be approved through a multidisciplinary approach taking into account legal and ethical issues while respecting the autonomy of the couples.

Depuis le 1er septembre 2017, les tests génétiques préimplantatoires (PGT) sont autorisés en Suisse suite aux modifications de la loi sur la procréation médicalement assistée (LPMA). Les couples infertiles qui effectuent une fécondation in vitro (FIV) peuvent bénéficier d'un PGT des aneuploïdies (PGT-A) dans le but de transférer des embryons euploïdes (nombre normal de chromosomes) et ainsi optimiser leurs chances de succès, sous réserve de données encore limitées. Les couples à risque de transmettre une maladie monogénique grave ou une translocation déséquilibrée peuvent avoir recours au PGT d'une anomalie d'un gène unique ou d'une anomalie de structure de chromosome (PGT-M/SR), dans les limites d'un cadre légal strict. Leur mise en pratique doit être décidée de manière pluridisciplinaire en tenant compte des enjeux légaux et éthiques dans le respect de l'autonomie des couples.

Neutrophil extracellular trap formation upon exposure of hydrophobic materials to human whole blood causes thrombogenic reactions.

Neutrophil extracellular trap (NET) formation, a reaction of the innate immune system to fight pathogens, was shown to be involved in thrombus formation. In the present study blood-contacting biomaterials with graded surface characteristics were investigated as a potential cause of NET formation on medical devices. Surface properties are known to govern protein adsorption, cell adhesion and ultimately the activation of several other host defense pathways - potentially also the formation of NETs. Model materials of defined hydrophilic or hydrophobic properties (glass, and thin films of poly(ethylene-alt-maleic anhydride), self-assembled monolayers of methyl terminated alkanethiols, and Teflon AF™) were incubated either with isolated human granulocytes after pre-adsorption with plasma proteins or with human whole blood. NET formation - detected as extracellular DNA, citrullinated histones, elastase and reactive oxygen species (ROS) - was observed on hydrophobic surfaces. Furthermore, NET formation on the hydrophobic surface Teflon AF™ resulted in elevated thrombin generation in hirudin-anticoagulated whole blood, but not in heparinized whole blood. Disintegration of surface-bound NETs by DNase treatment resulted in significantly lower pro-coagulant effects. Thus, NET formation can contribute to the thrombogenicity of clinically applied hydrophobic materials, suggesting NETosis as well as NET surface anchorage as new targets of anticoagulation strategies.

Multilayer hydrogel coatings to combine hemocompatibility and antimicrobial activity.

While silver-loaded catheters are widely used to prevent early-onset catheter-related infections [1], long term antimicrobial protection of indwelling catheters remains to be achieved [2] and antiseptic functionalization of coatings often impairs their hemocompatibility characteristics. Therefore, this work aimed to capitalize on the antimicrobial properties of silver nanoparticles, incorporated in anticoagulant poly(ethylene glycol) (PEG)-heparin hydrogel coatings [3] on thermoplastic polyurethane materials. For prolonged antimicrobial activity, the silver-containing starPEG-heparin hydrogel layers were shielded with silver-free hydrogel layers of otherwise similar composition. The resulting multi-layered gel coatings showed long term antiseptic efficacy against Escherichia coli and Staphylococcus epidermidis strains in vitro, and similarly performed well when incubated with freshly drawn human whole blood with respect to hemolysis, platelet activation and plasmatic coagulation. The introduced hydrogel multilayer system thus offers a promising combination of hemocompatibility and long-term antiseptic capacity to meet an important clinical need.

Bio-responsive polymer hydrogels homeostatically regulate blood coagulation.

Bio-responsive polymer architectures can empower medical therapies by engaging molecular feedback-response mechanisms resembling the homeostatic adaptation of living tissues to varying environmental constraints. Here we show that a blood coagulation-responsive hydrogel system can deliver heparin in amounts triggered by the environmental levels of thrombin, the key enzyme of the coagulation cascade, which--in turn--becomes inactivated due to released heparin. The bio-responsive hydrogel quantitatively quenches blood coagulation over several hours in the presence of pro-coagulant stimuli and during repeated incubation with fresh, non-anticoagulated blood. These features enable the introduced material to provide sustainable, autoregulated anticoagulation, addressing a key challenge of many medical therapies. Beyond that, the explored concept may facilitate the development of materials that allow the effective and controlled application of drugs and biomolecules.

The effect of octadecyl chain immobilization on the hemocompatibility of poly (2-hydroxyethyl methacrylate).

Albumin-scavenging surfaces decorated with n-alkyl chains represent an established strategy for blood-contacting applications. To evaluate this concept, a set of poly (2-hydroxyethyl methacrylate) (pHEMA) films modified with different amounts of octadecyl isocyanate (C18) was investigated in an in vitro hemocompatibility assay using freshly drawn human whole blood. In addition, the hydrogel materials were thoroughly characterized with respect to changes in wettability and elasticity, which accompanied the gradual chemical modification of pHEMA. An increase of the surface C18 content induced enhanced hydrophobicity and stiffness. Immobilization of C18 chains was found to substantially reduce the coagulation activation and the complement activation by the pHEMA films. Platelet adhesion and degranulation (PF4 release) were similar on the modified and the unmodified pHEMA. Platelet adhesion to pHEMA hydrogels was lower than the polytetrafluoroethylene reference. We conclude that the immobilization of octadecyl chains improved the hemocompatibility of pHEMA materials under conditions that might be encountered in low shear blood flow.

Substratum compliance regulates human trabecular meshwork cell behaviors and response to latrunculin B.

PURPOSE: To determine the impact of substratum compliance and latrunculin-B (Lat-B), both alone and together, on fundamental human trabecular meshwork (HTM) cell behavior. Lat-B is a reversible actin cytoskeleton disruptor that decreases resistance to aqueous humor outflow and decreases intraocular pressure. METHODS: HTM cells were cultured on polyacrylamide hydrogels possessing values for compliance that mimic those reported for normal and glaucomatous HTM, or tissue culture plastic (TCP). Cells were treated with 0.2 µM or 2.0 µM Lat-B in dimethyl sulfoxide (DMSO) or DMSO alone. The impact of substratum compliance and/or Lat-B treatment on cell attachment, proliferation, surface area, aspect ratio, and migration were investigated. RESULTS: HTM cells had profoundly decreased attachment and proliferation rates when cultured on hydrogels possessing compliance values that mimic those found for healthy HTM. The effect of Lat-B treatment on HTM cell surface area was less for cells cultured on more compliant hydrogels compared with TCP. HTM cell migration was increased on stiffer hydrogels that mimic the compliance of glaucomatous HTM and on TCP in comparison with more compliant hydrogels. Lat-B treatment decreased cellular migration on all surfaces for at least 7 hours after treatment. CONCLUSIONS: Substratum compliance profoundly influenced HTM cell behaviors and modulated the response of HTM cells to Lat-B. The inclusion of substratum compliance that reflects healthy or glaucomatous HTM results in cell behaviors and responses to therapeutic agents in vitro that may more accurately reflect in vivo conditions.

The effect of biophysical attributes of the ocular trabecular meshwork associated with glaucoma on the cell response to therapeutic agents.

Glaucoma is a devastating neurodegenerative disease, which can lead to vision loss and is associated with irreversible damage to retinal ganglion cells. Although the mechanism of disease onset remains unknown, we have recently demonstrated that the stiffness of the ocular trabecular meshwork (HTM) increases dramatically in human donor eyes with a history of glaucoma. Here we report that polyacrylamide hydrogels, which mimic the compliant conditions of normal and glaucomatous HTM, profoundly modulate cytoskeletal dynamics and the elastic modulus of the overlying HTM cells. Substratum compliance also modulates HTM cell response to Latrunculin-B, a cytoskeletal disrupting agent currently in human clinical trials for the treatment of glaucoma. Additionally, we observed a compliance-dependent rebound effect of Latrunculin-B with an unexpected increase in HTM cell elastic modulus being observed upon withdrawal of the drug. The results predict that cytoskeletal disrupting drugs may be more potent in advanced stages of glaucoma.

The ability of surface characteristics of materials to trigger leukocyte tissue factor expression.

Biomaterial-induced thrombosis is usually attributed to blood coagulation initiated by contact phase and platelet-related reactions. Considering the major role of extrinsic initiation in blood coagulation in vivo, we studied the material related-induction of this pathway by investigating the relevance of surface properties for the expression of Tissue Factor (TF), a critical initiator of the extrinsic pathway of coagulation. We incubated materials with self-assembled monolayers of alkylthiols (SAMs) displaying various ratios of -CH(3), -OH, and -COOH terminations with fresh heparinized whole human blood in vitro. The transcription of TF-mRNA in leukocytes showed clear differences in relation to surface properties and increased over time. In addition, a positive correlation between TF transcription and its presence on leukocytes, granulocyte activation, and complement activation was found. Cells displaying the highest TF expression after material contact had significantly lower intracellular TF, pointing to previous TF release. Yet under the conditions of our whole blood incubation set-up within the limited time frame the observed initiation of the extrinsic pathway did not trigger blood coagulation.

Synergistic effect of hydrophobic and anionic surface groups triggers blood coagulation in vitro.

Biomaterial induced coagulation encompasses plasmatic and cellular processes. The functional loss of biomedical devices possibly resulting from these thrombotic reactions motivates the need for a better understanding of processes occurring at blood-biomaterial interfaces. Well defined model surfaces providing specific chemical-physical properties (self assembled monolayers (SAMs)) displaying hydrophobic or/and acidic terminal groups were used to uncover initial mechanisms of biomaterial induced coagulation. We investigated the influence of electrical charge and wettability on platelet- and contact activation, the two main actors of blood coagulation, which are often considered as separate mechanisms in biomaterials research. Our results show a dependence of contact activation on acidic surface groups and a correlation of platelet adhesion to surface hydrophobicity. Clot formation resulting from the interplay of blood platelets and contact activation was only found on surfaces combining both acidic and hydrophobic surface groups but not on monolayers displaying extreme hydrophobic/acidic properties.

Blood coagulation on biomaterials requires the combination of distinct activation processes.

The rational design of hemocompatible materials requires a mechanistic understanding of activation processes induced at the blood-material interface. Binary self-assembled monolayers of alkyl thiols (SAMs) with various ratios of -CH3 and -COOH terminations were used to study the relevance of hydrophobic and negatively charged surfaces for the initiation of blood coagulation. Platelet adhesion and activation of the intrinsic coagulation pathway scaled with the surface composition: the numbers of adherent platelets were highest on the 100%-CH3 surface whereas the greatest contact activation was seen on 100%-COOH surfaces. In vitro whole blood incubation assays showed, however, that the surfaces exposing either -CH3 or -COOH groups induced comparably low levels of thrombin formation while the surfaces with intermediate contents of both terminating groups had significantly higher values. These results reveal that contact activation and platelet adhesion have a strong synergistic effect on coagulation on blood-contacting materials even though these events in isolation are not sufficient to induce substantial thrombin formation. Successful surface design strategies for hemocompatible materials therefore need to carefully consider the interplay of both processes.