Discovery of a structural class of antibiotics with explainable deep learning.

The discovery of novel structural classes of antibiotics is urgently needed to address the ongoing antibiotic resistance crisis1-9. Deep learning approaches have aided in exploring chemical spaces1,10-15; these typically use black box models and do not provide chemical insights. Here we reasoned that the chemical substructures associated with antibiotic activity learned by neural network models can be identified and used to predict structural classes of antibiotics. We tested this hypothesis by developing an explainable, substructure-based approach for the efficient, deep learning-guided exploration of chemical spaces. We determined the antibiotic activities and human cell cytotoxicity profiles of 39,312 compounds and applied ensembles of graph neural networks to predict antibiotic activity and cytotoxicity for 12,076,365 compounds. Using explainable graph algorithms, we identified substructure-based rationales for compounds with high predicted antibiotic activity and low predicted cytotoxicity. We empirically tested 283 compounds and found that compounds exhibiting antibiotic activity against Staphylococcus aureus were enriched in putative structural classes arising from rationales. Of these structural classes of compounds, one is selective against methicillin-resistant S. aureus (MRSA) and vancomycin-resistant enterococci, evades substantial resistance, and reduces bacterial titres in mouse models of MRSA skin and systemic thigh infection. Our approach enables the deep learning-guided discovery of structural classes of antibiotics and demonstrates that machine learning models in drug discovery can be explainable, providing insights into the chemical substructures that underlie selective antibiotic activity.

Entropic repulsion of cholesterol-containing layers counteracts bioadhesion.

Control of adhesion is a striking feature of living matter that is of particular interest regarding technological translation1-3. We discovered that entropic repulsion caused by interfacial orientational fluctuations of cholesterol layers restricts protein adsorption and bacterial adhesion. Moreover, we found that intrinsically adhesive wax ester layers become similarly antibioadhesive when containing small quantities (under 10 wt%) of cholesterol. Wetting, adsorption and adhesion experiments, as well as atomistic simulations, showed that repulsive characteristics depend on the specific molecular structure of cholesterol that encodes a finely balanced fluctuating reorientation at the interface of unconstrained supramolecular assemblies: layers of cholesterol analogues differing only in minute molecular variations showed markedly different interfacial mobility and no antiadhesive effects. Also, orientationally fixed cholesterol layers did not resist bioadhesion. Our insights provide a conceptually new physicochemical perspective on biointerfaces and may guide future material design in regulation of adhesion.

Breast cancer cells compete with hematopoietic stem and progenitor cells for intercellular adhesion molecule 1-mediated binding to the bone marrow microenvironment.

Adhesion-based cellular interactions involved in breast cancer metastasis to the bone marrow remain elusive. We identified that breast cancer cells directly compete with hematopoietic stem and progenitor cells (HSPCs) for retention in the bone marrow microenvironment. To this end, we established two models of competitive cell adhesion-simultaneous and sequential-to study a potential competition for homing to the niche and displacement of the endogenous HSPCs upon invasion by tumor cells. In both models, breast cancer cells but not non-tumorigenic cells competitively reduced adhesion of HSPCs to bone marrow-derived mesenchymal stromal cells (MSCs) in a tumor cell number-dependent manner. Higher adhesive force between breast cancer cells and MSCs, as compared with HSPCs, assessed by quantitative atomic force microscopy-based single-cell force spectroscopy could partially account for tumor cell mediated reduction in HSPC adhesion to MSCs. Genetic inactivation and blockade studies revealed that homophilic interactions between intercellular adhesion molecule 1 (ICAM-1) expressed on tumor cells and MSCs, respectively, regulate the competition between tumor cells and HSPCs for binding to MSCs. Moreover, tumor cell-secreted soluble ICAM-1(sICAM-1) also impaired HSPC adhesion via blocking CD18-ICAM-1 binding between HSPCs and MSCs. Xenotransplantation studies in NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice revealed reduction of human HSPCs in the bone marrow via metastatic breast cancer cells. These findings point to a direct competitive interaction between disseminated breast cancer cells and HSPCs within the bone marrow micro environment. This interaction might also have implications on niche-based tumor support. Therefore, targeting this cross talk may represent a novel therapeutic strategy.

Tightly anchored tissue-mimetic matrices as instructive stem cell microenvironments.

A major obstacle in defining the exact role of extracellular matrix (ECM) in stem cell niches is the lack of suitable in vitro methods that recapitulate complex ECM microenvironments. Here we describe a methodology that permits reliable anchorage of native cell-secreted ECM to culture carriers. We validated our approach by fabricating two types of human bone marrow-specific ECM substrates that were robust enough to support human mesenchymal stem cells (MSCs) and hematopoietic stem and progenitor cells in vitro. We characterized the molecular composition, structural features and nanomechanical properties of the MSC-derived ECM preparations and demonstrated their ability to support expansion and differentiation of bone marrow stem cells. Our methodology enables the deciphering and modulation of native-like multicomponent ECMs of tissue-resident stem cells and will therefore prepare the ground for a more rational design of engineered stem cell niches.

TNF-α-mediated adhesion of monocytes to endothelial cells-The role of ephrinA1.

The ligand ephrin A1 is more often discussed to play a role in the development of the atherosclerotic plaque and in this context especially in the monocyte adhesion to endothelial cells. As tumor necrosis factor-α (TNF-α) is known to induce monocyte adhesion to endothelium and ephrin A1 expression, the present study focuses on the involvement of ephrin A1 in TNF-α-mediated monocyte adhesion. The analysis of different members of the Eph/ephrin system in TNF-α-treated human umbilical vein endothelial cells (HUVEC) revealed that especially ephrinA1 was found to be highly regulated by TNF-α compared to other members of the Eph family. This effect is also present in arterial endothelial cells from the umbilical artery and from the coronary artery. This regulation is dependent on NFκB-activation as shown by the expression of a constitutive-active IκB-mutant. By using siRNA-mediated silencing and adenoviral overexpression of ephrinA1 in HUVEC, the involvement of ephrinA1 in the TNF-α triggered monocyte adhesion to endothelial cells could be demonstrated. In addition, these results could be verified by quantitative adhesion measurement using atomic force microscopy-based single-cell force spectroscopy and under flow conditions. Furthermore, this effect is mediated via the EphA4 receptor. EphrinA1 does not influence the mRNA or protein expression of the adhesion receptors VCAM-1 and ICAM-1 in endothelial cells. However, the surface presentation of these adhesion receptors is modulated in an ephrinA1-dependent manner. In conclusion, these data demonstrate that ephrinA1 plays an important role in the TNF-α-mediated adhesion of monocytes to endothelial cells, which might be of great importance in the context of atherosclerosis.

MiR-134-mediated ß1 integrin expression and function in mesenchymal stem cells.

The composition of the hematopoietic stem cell (HSC) niche within the bone marrow is highly dynamic, tightly regulated, and of importance for various HSC properties. Integrins are important molecules within this niche that influence those properties through the interactions of HSCs and mesenchymal stem cells (MSCs). Here we investigated the function of miR-134 in integrin regulation in MSCs. In MSCs, miR-134 post-transcriptionally regulated ß1 integrin expression. This negative regulation of ß1 integrin was mediated by the binding of miR-134 to its 3' untranslated region, which contains two conserved binding sites for miR-134. The miR-134-mediated silencing of ß1 integrin in MSCs was shown by atomic force microscopy to decrease the adhesion of 32D cells to MSCs transfected with miR-134. Furthermore, the adhesion of MSCs to fibronectin was reduced after transfection with miR-134. MSCs from patients with myelodysplastic syndrome (MDS) revealed highly significant miR-134 overexpression compared with MSCs from healthy bone marrow donors. MSCs from MDS patients showed lower ß1 integrin protein, but not lower mRNA, expression, suggesting post-transcriptional regulation. The present study demonstrates miR-134-mediated negative regulation of ß1 integrin that influences cell adhesion to and of MSCs. These results further contribute to our understanding of the complexity of MDS.

A practical guide to quantify cell adhesion using single-cell force spectroscopy.

Quantitative analysis of cellular interactions with the extracellular environment is necessary to gain an understanding of how cells regulate adhesion in the development and maintenance of multicellular organisms, and how changes in cell adhesion contribute to diseases. We provide a practical guide to quantify the adhesive strength of living animal cells to various substrates using atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS). We describe how to control cell state and attachment to the AFM cantilever, how to functionalize supports for SCFS measurements, how to conduct cell adhesion measurements, and how to analyze and interpret the recorded SCFS data. This guide is intended to assist newcomers in the field to perform AFM-based SCFS measurements.

Precision Culture Scaling to Establish High-Throughput Vasculogenesis Models.

Hydrogel-based 3D cell cultures can recapitulate (patho)physiological phenomena ex vivo. However, due to their complex multifactorial regulation, adapting these tissue and disease models for high-throughput screening workflows remains challenging. In this study, a new precision culture scaling (PCS-X) methodology combines statistical techniques (design of experiment and multiple linear regression) with automated, parallelized experiments and analyses to customize hydrogel-based vasculogenesis cultures using human umbilical vein endothelial cells and retinal microvascular endothelial cells. Variations of cell density, growth factor supplementation, and media composition are systematically explored to induce vasculogenesis in endothelial mono- and cocultures with mesenchymal stromal cells or retinal microvascular pericytes in 384-well plate formats. The developed cultures are shown to respond to vasculogenesis inhibitors in a compound- and dose-dependent manner, demonstrating the scope and power of PCS-X in creating parallelized tissue and disease models for drug discovery and individualized therapies.

Experimental study and simulations of hydrogen cooling effectiveness for aviation PEM fuel cells.

Proton exchange membrane fuel cells (PEMFCs) are seen as one possible future means of driving the change towards a zero-emission society. In a civil aircraft, fuel cell systems can have multiple potential benefits, such as reduced noise, lowered emissions and higher fuel economy compared to jet aircraft. For controlling the fuel cell temperature, thermal management systems are required which can be optimized for aircraft applications regarding their weight and reliability. In this work, a simplified and light-weight thermal management system relying on hydrogen cooling is presented and analysed. To investigate the feasibility, a test rig and a three-dimensional, singular channel model in ANSYS Fluent were designed. Fuel cell temperature could be maintained within the set threshold in the model and the test rig, thus showing that controlling the fuel cell temperature via the hydrogen reactant flow is a viable alternative thermal management system. Results from the model indicate that both the hydrogen mass flow and hydrogen inlet temperature should be used to control the fuel cell temperature. Furthermore, operating the fuel cell at medium to low current densities is favourable for hydrogen cooling. Future studies will explore alternate flow field designs to facilitate thermal management system relying on hydrogen.

Quantifying the effect of covalently immobilized enzymes on biofilm formation by atomic force microscopy-based single-cell force spectroscopy.

A novel atomic force microscopy-based single-cell force spectroscopy assay to quantify the adhesion of bacterial cells to surfaces was developed. The assay was applied to quantify the effect of two biofilm-degrading enzymes, the protease Subtilisin A and glycoside hydrolase cellulase, on the attachment of the biofilm-forming bacterial strain Cobetia marina. Insights on the mechanism of the initial adhesion and on the nature of the adhesion-mediating molecules were gained. The assay can be easily adapted to various other substrates, different bacterial strains and other fouling species (e.g., algae and diatoms).

Microfluidics-assisted synthesis and functionalization of monodisperse colloidal hydrogel particles for optomechanical biosensors.

The soft colloidal probe (SCP) assay is a highly versatile sensing principle employing micrometer-sized hydrogel particles as optomechanical transducer elements. We report the synthesis, optimization, and conjugation of SCPs with defined narrow size distribution and specifically tailored mechanical properties and functionalities for integration into a microinterferometric optomechanical biosensor platform. Droplet-based microfluidics was used to crosslink polyethylene glycol (PEG) macromonomers by photocrosslinking and thiol-Michael addition. The effect of several synthesis parameters, i.e. PEG and radical initiator solid content, molecular weight and architecture of macromonomers, as well as UV exposure time and energy, were examined. SCPs were characterized with regard to the conversion of contained functional groups, morphology and mechanical properties by bright-field, confocal laser scanning and reflection interference contrast microscopy, as well as force spectroscopy. Functional groups were introduced during SCP synthesis and by several post-synthesis procedures, based on photoradical grafting and thiol-Michael addition. Preparation of SCPs by thiol-Michael addition and subsequent coupling of maleimide derivatives to unreacted thiols proved to be the superior strategy, while other approaches were associated with changes in the properties of the SCP. The newly developed SCPs were tested for their sensing capabilities employing the biotin-streptavidin-system. Biotin detection in the range of 10-7 to 10-10 M verified the concept of the synthesis strategy and the advantage of using monodisperse SCPs for easier and faster sensing applications of the SCP assay.

Bone marrow mesenchymal stromal cell-derived extracellular matrix displays altered glycosaminoglycan structure and impaired functionality in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of hematologic malignancies characterized by clonal hematopoiesis, one or more cytopenias such as anemia, neutropenia, or thrombocytopenia, abnormal cellular maturation, and a high risk of progression to acute myeloid leukemia. The bone marrow microenvironment (BMME) in general and mesenchymal stromal cells (MSCs) in particular contribute to both the initiation and progression of MDS. However, little is known about the role of MSC-derived extracellular matrix (ECM) in this context. Therefore, we performed a comparative analysis of in vitro deposited MSC-derived ECM of different MDS subtypes and healthy controls. Atomic force microscopy analyses demonstrated that MDS ECM was significantly thicker and more compliant than those from healthy MSCs. Scanning electron microscopy showed a dense meshwork of fibrillar bundles connected by numerous smaller structures that span the distance between fibers in MDS ECM. Glycosaminoglycan (GAG) structures were detectable at high abundance in MDS ECM as white, sponge-like arrays on top of the fibrillar network. Quantification by Blyscan assay confirmed these observations, with higher concentrations of sulfated GAGs in MDS ECM. Fluorescent lectin staining with wheat germ agglutinin and peanut agglutinin demonstrated increased deposition of N-acetyl-glucosamine GAGs (hyaluronan (HA) and heparan sulfate) in low risk (LR) MDS ECM. Differential expression of N-acetyl-galactosamine GAGs (chondroitin sulfate, dermatan sulfate) was observed between LR- and high risk (HR)-MDS. Moreover, increased amounts of HA in the matrix of MSCs from LR-MDS patients were found to correlate with enhanced HA synthase 1 mRNA expression in these cells. Stimulation of mononuclear cells from healthy donors with low molecular weight HA resulted in an increased expression of various pro-inflammatory cytokines suggesting a contribution of the ECM to the inflammatory BMME typical of LR-MDS. CD34+ hematopoietic stem and progenitor cells (HSPCs) displayed an impaired differentiation potential after cultivation on MDS ECM and modified morphology accompanied by decreased integrin expression which mediate cell-matrix interaction. In summary, we provide evidence for structural alterations of the MSC-derived ECM in both LR- and HR-MDS. GAGs may play an important role in this remodeling processes during the malignant transformation which leads to the observed disturbance in the support of normal hematopoiesis.

Retinal pigment epithelium cell alignment on nanostructured collagen matrices.

We investigated attachment and migration of human retinal pigment epithelial cells (primary, SV40-transfected and ARPE-19) on nanoscopically defined, two-dimensional matrices composed of parallel-aligned collagen type I fibrils. These matrices were used non-cross-linked (native) or after riboflavin/UV-A cross-linking to study cell attachment and migration by time-lapse video microscopy. Expression of collagen type I and IV, MMP-2 and of the collagen-binding integrin subunit α(2) were examined by immunofluorescence and Western blotting. SV40-RPE cells quickly attached to the nanostructured collagen matrices and aligned along the collagen fibrils. However, they disrupted both native and cross-linked collagen matrices within 5 h. Primary RPE cells aligned more slowly without destroying either native or cross-linked substrates. Compared to primary RPE cells, ARPE-19 cells showed reduced alignment but partially disrupted the matrices within 20 h after seeding. Expression of the collagen type I-binding integrin subunit α(2) was highest in SV40-RPE cells, lower in primary RPE cells and almost undetectable in ARPE-19 cells. Thus, integrin α(2) expression levels directly correlated with the degree of cell alignment in all examined RPE cell types. Specific integrin subunit α(2)-mediated matrix binding was verified by preincubation with an α(2)-function-blocking antibody, which impaired cell adhesion and alignment to varying degrees in primary and SV40-RPE cells. Since native matrices supported extended and directed primary RPE cell growth, optimizing the matrix production procedure may in the future yield nanostructured collagen matrices serving as transferable cell sheet carriers.

Customizing biohybrid cryogels to serve as ready-to-use delivery systems of signaling proteins.

Macroporous cryogels have recently gained increasing interest for the controlled administration of signaling proteins in tissue engineering due to an advantageous combination of material properties. However, most of the previously reported cryogel systems did not allow for tunable, sustained protein release. We therefore designed a set of ready-to-use multi-armed polyethylene glycol (starPEG)-heparin cryogel systems containing different amounts of the protein-affine glycosaminoglycan component heparin to enable systematically tunable long-term delivery of different signaling proteins without affecting other cell-instructive properties. Experimental data and mathematical modeling indicate that the macroporous structure causes local differences in the concentration of proteins released into the pores and in the surrounding of the cryogels. As a proof-of-concept for their ready-to-use potential, cryogels pre-functionalized with signaling proteins and cell adhesion-peptides were demonstrated to induce the neuronal differentiation of colonizing pheochromocytoma cells. The elaborated approach opens up new perspectives for cryogels as easily storable and applicable systems for the precision delivery of signaling proteins.

Cytoplasmic condensation induced by membrane damage is associated with antibiotic lethality.

Bactericidal antibiotics kill bacteria by perturbing various cellular targets and processes. Disruption of the primary antibiotic-binding partner induces a cascade of molecular events, leading to overproduction of reactive metabolic by-products. It remains unclear, however, how these molecular events contribute to bacterial cell death. Here, we take a single-cell physical biology approach to probe antibiotic function. We show that aminoglycosides and fluoroquinolones induce cytoplasmic condensation through membrane damage and subsequent outflow of cytoplasmic contents as part of their lethality. A quantitative model of membrane damage and cytoplasmic leakage indicates that a small number of nanometer-scale membrane defects in a single bacterium can give rise to the cellular-scale phenotype of cytoplasmic condensation. Furthermore, cytoplasmic condensation is associated with the accumulation of reactive metabolic by-products and lipid peroxidation, and pretreatment of cells with the antioxidant glutathione attenuates cytoplasmic condensation and cell death. Our work expands our understanding of the downstream molecular events that are associated with antibiotic lethality, revealing cytoplasmic condensation as a phenotypic feature of antibiotic-induced bacterial cell death.

Amphiphilic Copolymers for Versatile, Facile, and In Situ Tunable Surface Biofunctionalization.

Precision surface engineering is key to advanced biomaterials. A new platform of PEGylated styrene-maleic acid copolymers for adsorptive surface biofunctionalization is reported. Balanced amphiphilicity renders the copolymers water-soluble but strongly affine for surfaces. Fine-tuning of their molecular architecture provides control over adsorptive anchorage onto specific materials-which is why they are referred to as "anchor polymers" (APs)-and over structural characteristics of the adsorbed layers. Conjugatable with an array of bioactives-including cytokine-complexing glycosaminoglycans, cell-adhesion-mediating peptides and antimicrobials-APs can be applied to customize materials for demanding biotechnologies in uniquely versatile, simple, and robust ways. Moreover, homo- and heterodisplacement of adsorbed APs provide unprecedented means of in situ alteration and renewal of the functionalized surfaces. The related options are exemplified with proof-of-concept experiments of controlled bacterial adhesion, human umbilical vein endothelial cell, and induced pluripotent cell growth on AP-functionalized surfaces.

Stimulated single-cell force spectroscopy to quantify cell adhesion receptor crosstalk.

To control their attachment to substrates and other cells, cells regulate their adhesion receptors. One regulatory process is receptor crosstalk, where the binding of one type of cell adhesion molecule influences the activity of another type. To identify such crosstalk and gain insight into their mechanisms, we developed the stimulated single-cell force spectroscopy assay. In this assay, the influence of a cells adhesion to one substrate on the strength of its adhesion to a second substrate is examined. The assay quantifies the adhesion of the cell and the contributions of specific adhesion receptors. This allows mechanisms by which the adhesion is regulated to be determined. Using the assay we identified crosstalk between collagen-binding integrin alpha(1)beta(1) and fibronectin-binding integrin alpha(5)beta(1) in HeLa cells. This crosstalk was unidirectional, from integrin alpha(1)beta(1) to integrin alpha(5)beta(1), and functioned by regulating the endocytosis of integrin alpha(5)beta(1). The single-cell assay should be expandable for the screening and quantification of crosstalk between various cell adhesion molecules and other cell surface receptors.

Revealing early steps of alpha2beta1 integrin-mediated adhesion to collagen type I by using single-cell force spectroscopy.

We have characterized early steps of alpha(2)beta(1) integrin-mediated cell adhesion to a collagen type I matrix by using single-cell force spectroscopy. In agreement with the role of alpha(2)beta(1) as a collagen type I receptor, alpha(2)beta(1)-expressing Chinese hamster ovary (CHO)-A2 cells spread rapidly on the matrix, whereas alpha(2)beta(1)-negative CHO wild-type cells adhered poorly. Probing CHO-A2 cell detachment forces over a contact time range of 600 s revealed a nonlinear adhesion response. During the first 60 s, cell adhesion increased slowly, and forces associated with the smallest rupture events were consistent with the breakage of individual integrin-collagen bonds. Above 60 s, a fraction of cells rapidly switched into an activated adhesion state marked by up to 10-fold increased detachment forces. Elevated overall cell adhesion coincided with a rise of the smallest rupture forces above the value required to break a single-integrin-collagen bond, suggesting a change from single to cooperative receptor binding. Transition into the activated adhesion mode and the increase of the smallest rupture forces were both blocked by inhibitors of actomyosin contractility. We therefore propose a two-step mechanism for the establishment of alpha(2)beta(1)-mediated adhesion as weak initial, single-integrin-mediated binding events are superseded by strong adhesive interactions involving receptor cooperativity and actomyosin contractility.

Surface morphology and mechanical properties of fibroblasts from scleroderma patients.

Overproduction of extracellular matrix components by fibroblasts plays a key role in the pathogenesis of scleroderma. To investigate whether these functional alterations are accompanied by changes in the mechanical properties and morphology of fibroblast, atomic force microscopy was applied to dermal fibroblasts derived either from scleroderma patients or from healthy donors. No significant morphological differences could be observed among the different cell strains showing long cytoskeleton fibres similar in length and irregularly distributed protrusions on the cell surface. In contrast, significant differences in cellular stiffness of dermal fibroblasts derived from scleroderma lesions were detected. Compared to fibroblasts from healthy donors, diseased cells were characterized by a reduced elastic constant both when the global and local mechanical properties were probed. The altered stiffness of scleroderma fibroblasts may be important in the pathogenesis of the disease as it could lead to the abnormal response of fibroblasts to mechanical stimuli.

Temperature-Induced Mechanomodulation of Interpenetrating Networks of Star Poly(ethylene glycol)-Heparin and Poly(N-isopropylacrylamide).

Thermoresponsive interpenetrating networks (IPNs) were prepared by sequential synthesis of a biohybrid network of star-shaped poly(ethylene glycol) [starPEG] and heparin and a poly(N-isopropylacrylamide)-polymer network. Amide bond formation was used for cross-linking of the starPEG-heparin network and photo-cross-linking with N,N'-methylenebis(acrylamide) was applied for the formation of the second polymer network. Both networks were linked by chain entanglements and hydrogen bonds only. The obtained sequential IPNs (seq-IPNs) showed temperature-dependent network properties as reflected by swelling and elasticity data as well as by the release of glycosaminoglycan-binding growth factors. The elastic modulus of the seq-IPNs was found to be amplified up to 50-fold upon temperature change from 22 to 37 °C compared to the intrinsic elastic moduli of the two combined networks. The heparin concentration (as well as the complexation of growth factors with the hydrogel-contained heparin) was demonstrated to be variably independent from the mechanical properties (elastic moduli) of the hydrogels. Illustrating the usability of the developed seq-IPN platform for cell fate control, the thermo-modulation of the release of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2) is shown as well as the osteogenic differentiation of human mesenchymal stem cells exposed to stiff and BMP-2 releasing seq-IPNs.