Comprehensive Characterization of the Viscoelastic Properties of Bovine Submaxillary Mucin (BSM) Hydrogels and the Effect of Additives.

This study presents a comprehensive characterization of the viscoelastic and structural properties of bovine submaxillary mucin (BSM), which is widely used as a commercial source to conduct mucus-related research. We conducted concentration studies of BSM and examined the effects of various additives, NaCl, CaCl2, MgCl2, lysozyme, and DNA, on its rheological behavior. A notable connection between BSM concentration and viscoelastic properties was observed, particularly under varying ionic conditions. The rheological spectra could be well described by a fractional Kelvin-Voigt model with a minimum of model parameters. A detailed proteomics analysis provided insight into the protein, especially mucin composition within BSM, showing MUC19 as the main component. Cryo-scanning electron microscopy enabled the visualization of the porous BSM network structure. These investigations give us a more profound comprehension of the BSM properties, especially those pertaining to viscoelasticity, and how they are influenced by concentration and environmental conditions, aspects relevant to the field of mucus research.

Mucus-Inspired Self-Healing Hydrogels: A Protective Barrier for Cells against Viral Infection.

Mucus is a dynamic biological hydrogel, composed primarily of the glycoprotein mucin, exhibits unique biophysical properties and forms a barrier protecting cells against a broad-spectrum of viruses. Here, this work develops a polyglycerol sulfate-based dendronized mucin-inspired copolymer (MICP-1) with ≈10% repeating units of activated disulfide as cross-linking sites. Cryo-electron microscopy (Cryo-EM) analysis of MICP-1 reveals an elongated single-chain fiber morphology. MICP-1 shows potential inhibitory activity against many viruses such as herpes simplex virus 1 (HSV-1) and SARS-CoV-2 (including variants such as Delta and Omicron). MICP-1 produces hydrogels with viscoelastic properties similar to healthy human sputum and with tuneable microstructures using linear and branched polyethylene glycol-thiol (PEG-thiol) as cross-linkers. Single particle tracking microrheology, electron paramagnetic resonance (EPR) and cryo-scanning electron microscopy (Cryo-SEM) are used to characterize the network structures. The synthesized hydrogels exhibit self-healing properties, along with viscoelastic properties that are tuneable through reduction. A transwell assay is used to investigate the hydrogel's protective properties against viral infection against HSV-1. Live-cell microscopy confirms that these hydrogels can protect underlying cells from infection by trapping the virus, due to both network morphology and anionic multivalent effects. Overall, this novel mucin-inspired copolymer generates mucus-mimetic hydrogels on a multi-gram scale. These hydrogels can be used as models for disulfide-rich airway mucus research, and as biomaterials.

Mucin-Inspired Polymeric Fibers for Herpes Simplex Virus Type 1 Inhibition.

Mucus lines the epithelial cells at the biological interface and is the first line of defense against multiple viral infections. Mucins, the gel-forming components of mucus, are high molecular weight glycoproteins and crucial for preventing infections by binding pathogens. Consequently, mimicking mucins is a promising strategy for new synthetic virus inhibitors. In this work, synthetic mucin-inspired polymers (MIPs) as potential inhibitors of herpes simplex virus 1 (HSV-1) are investigated. By using a telechelic reversible addition-fragmentation chain-transfer (RAFT) polymerization technique, a new dendronized polysulfate p(G1AAm-OSO3)PDS with an amide-backbone similar to the native mucin glycoproteins is synthesized. p(G1AAm-OSO3)PDS shows mucin-like elongated fiber structure, as revealed in cryo-electron microscopy (cryo-EM) imaging, and its HSV-1 inhibition activity together with its previously reported methacrylate analogue p(G1MA-OSO3)PDS is tested. Both of the sulfated MIPs show strong HSV-1 inhibition in plaque reduction assays with IC50 values in lower nanomolar range (<3 × 10-9 m) and demonstrate a high cell compatibility (CC50 > 1.0 mg mL-1) with lower anticoagulant activity than heparin. In addition, the prophylactic and therapeutic activity of both MIPs is assessed in pre- and post-infection inhibition assays and clearly visualize their high potential for application using fluorescent microscopy imaging of infected cells.

Microfluidics-Based Force Spectroscopy Enables High-Throughput Force Experiments with Sub-Nanometer Resolution and Sub-Piconewton Sensitivity.

Several techniques have been established to quantify the mechanicals of single molecules. However, most of them show only limited capabilities of parallelizing the measurement by performing many individual measurements simultaneously. Herein, a microfluidics-based single-molecule force spectroscopy method, which achieves sub-nanometer spatial resolution and sub-piconewton sensitivity and is capable of simultaneously quantifying hundreds of single-molecule targets in parallel, is presented. It relies on a combination of total internal reflection microscopy and microfluidics, in which monodisperse fluorescent beads are immobilized on the bottom of a microfluidic channel by macromolecular linkers. Application of a flow generates a well-defined shear force acting on the beads, whereas the nanomechanical linker response is quantified based on the force-induced displacement of individual beads. To handle the high amount of data generated, a cluster analysis which is capable of a semi-automatic identification of measurement artifacts and molecular populations is implemented. The method is validated by probing the mechanical response polyethylene glycol linkers and binding strength of biotin-NeutrAvidin complexes. Two energy barriers (at 3 and 5.7 Å, respectively) in the biotin-NeutrAvidin interaction are resolved and the unfolding behavior of talin's rod domain R3 in the force range between 1 to ≈10 pN is probed.

A fast open-source Fiji-macro to quantify virus infection and transfection on single-cell level by fluorescence microscopy.

The ability to automatically analyze large quantities of image data is a valuable tool for many biochemical assays, as it rapidly provides reliable data. Here, we describe a fast and robust Fiji macro for the analysis of cellular fluorescence microscopy images with single-cell resolution. The macro presented here was validated by successful reconstruction of fluorescent and non-fluorescent cell mixing ratios (for fluorescence fractions ranging between 0 and 100%) and applied to quantify the efficiency of transfection and virus infection inhibition. It performed well compared with manually obtained image quantification data. Its use is not limited to the cases shown here but is applicable for most monolayered cellular assays with nuclei staining. We provide a detailed description of how the macro works and how it is applied to image data. It can be downloaded free of charge and may be used by and modified according to the needs of the user. • Rapid, simple, and reproducible segmentation of eukaryotic cells in confluent cellular assays • Open-source software for use without technical or computational expertise • Single-cell analysis allows identification and quantification of virus infected cell populations and infection inhibition.

One-pot gram-scale synthesis of virucidal heparin-mimicking polymers as HSV-1 inhibitors.

A straightforward and gram-scale synthesis method was developed to engineer highly sulfated hyperbranched polyglycerol bearing sulfated alkyl chains. The compounds with shorter alkyl chains showed multivalent virustatic inhibition against herpes simplex virus type 1 (HSV-1), similar to heparin. In contrast, the compound with the longest alkyl chains irreversibly inhibited the virus.

Image analysis-based quantification of fungal sporulation by automatic conidia counting and gray value correlation.

The present work describes a new computer-assisted image analysis method for the rapid, simple, objective and reproducible quantification of actively discharged fungal spores which can serve as a manual for laboratories working in this context. The method can be used with conventional laboratory equipment by using bright field microscopes, standard scanners and the open-source software ImageJ. Compared to other conidia quantification methods by computer-assisted image analysis, the presented method bears a higher potential to be applied for large-scale sample quantities. The key to make quantification faster is the calculation of the linear relationship between the gray value and the automatically counted number of conidia that has only to be performed once in the beginning of analysis. Afterwards, the gray value is used as single parameter for quantification. The fast, easy and objective determination of sporulation capacity enables facilitated quality control of fungal formulations designed for biological pest control.•Rapid, simple, objective and reproducible quantification of fungal sporulation suitable for large-scale sample quantities.•Requires conventional laboratory equipment and open-source software without technical or computational expertise.•The number of automatically counted conidia can be correlated with the gray value and after initial calculation of a linear fit, the gray value can be applied as single quantification parameter.

Polysulfates Block SARS-CoV-2 Uptake through Electrostatic Interactions*.

Here we report that negatively charged polysulfates can bind to the spike protein of SARS-CoV-2 via electrostatic interactions. Using a plaque reduction assay, we compare inhibition of SARS-CoV-2 by heparin, pentosan sulfate, linear polyglycerol sulfate (LPGS) and hyperbranched polyglycerol sulfate (HPGS). Highly sulfated LPGS is the optimal inhibitor, with an IC50 of 67 µg mL-1 (approx. 1.6 µm). This synthetic polysulfate exhibits more than 60-fold higher virus inhibitory activity than heparin (IC50 : 4084 µg mL-1 ), along with much lower anticoagulant activity. Furthermore, in molecular dynamics simulations, we verified that LPGS can bind more strongly to the spike protein than heparin, and that LPGS can interact even more with the spike protein of the new N501Y and E484K variants. Our study demonstrates that the entry of SARS-CoV-2 into host cells can be blocked via electrostatic interactions, therefore LPGS can serve as a blueprint for the design of novel viral inhibitors of SARS-CoV-2.

Heteromultivalent topology-matched nanostructures as potent and broad-spectrum influenza A virus inhibitors.

Here, we report the topology-matched design of heteromultivalent nanostructures as potent and broad-spectrum virus entry inhibitors based on the host cell membrane. Initially, we investigate the virus binding dynamics to validate the better binding performance of the heteromultivalent moieties as compared to homomultivalent ones. The heteromultivalent binding moieties are transferred to nanostructures with a bowl-like shape matching the viral spherical surface. Unlike the conventional homomultivalent inhibitors, the heteromultivalent ones exhibit a half maximal inhibitory concentration of 32.4 ± 13.7 µg/ml due to the synergistic multivalent effects and the topology-matched shape. At a dose without causing cellular toxicity, >99.99% reduction of virus propagation has been achieved. Since multiple binding sites have also been identified on the S protein of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), we envision that the use of heteromultivalent nanostructures may also be applied to develop a potent inhibitor to prevent coronavirus infection.

Topology-Matching Design of an Influenza-Neutralizing Spiky Nanoparticle-Based Inhibitor with a Dual Mode of Action.

In this study, we demonstrate the concept of "topology-matching design" for virus inhibitors. With the current knowledge of influenza A virus (IAV), we designed a nanoparticle-based inhibitor (nano-inhibitor) that has a matched nanotopology to IAV virions and shows heteromultivalent inhibitory effects on hemagglutinin and neuraminidase. The synthesized nano-inhibitor can neutralize the viral particle extracellularly and block its attachment and entry to the host cells. The virus replication was significantly reduced by 6 orders of magnitude in the presence of the reverse designed nano-inhibitors. Even when used 24 hours after the infection, more than 99.999 % inhibition is still achieved, which indicates such a nano-inhibitor might be a potent antiviral for the treatment of influenza infection.

Topology-Matching Design of an Influenza-Neutralizing Spiky Nanoparticle-Based Inhibitor with a Dual Mode of Action.

In this study, we demonstrate the concept of "topology-matching design" for virus inhibitors. With the current knowledge of influenza A virus (IAV), we designed a nanoparticle-based inhibitor (nano-inhibitor) that has a matched nanotopology to IAV virions and shows heteromultivalent inhibitory effects on hemagglutinin and neuraminidase. The synthesized nano-inhibitor can neutralize the viral particle extracellularly and block its attachment and entry to the host cells. The virus replication was significantly reduced by 6 orders of magnitude in the presence of the reverse designed nano-inhibitors. Even when used 24 hours after the infection, more than 99.999 % inhibition is still achieved, which indicates such a nano-inhibitor might be a potent antiviral for the treatment of influenza infection.

Analysis and refinement of 2D single-particle tracking experiments.

In recent decades, single particle tracking (SPT) has been developed into a sophisticated analytical approach involving complex instruments and data analysis schemes to extract information from time-resolved particle trajectories. Very often, mobility-related properties are extracted from these particle trajectories, as they often contain information about local interactions experienced by the particles while moving through the sample. This tutorial aims to provide a comprehensive overview about the accuracies that can be achieved when extracting mobility-related properties from 2D particle trajectories and how these accuracies depend on experimental parameters. Proper interpretation of SPT data requires an assessment of whether the obtained accuracies are sufficient to resolve the effect under investigation. This is demonstrated by calculating mean square displacement curves that show an apparent super- or subdiffusive behavior due to poor measurement statistics instead of the presence of true anomalous diffusion. Furthermore, the refinement of parameters involved in the design or analysis of SPT experiments is discussed and an approach is proposed in which square displacement distributions are inspected to evaluate the quality of SPT data and to extract information about the maximum distance over which particles should be tracked during the linking process.

A microfluidic co-cultivation platform to investigate microbial interactions at defined microenvironments.

Interspecies interactions inside microbial communities bear a tremendous diversity of complex chemical processes that are by far not understood. Even for simplified, often synthetic systems, the interactions between two microbes are barely revealed in detail. Here, we present a microfluidic co-cultivation platform for the analysis of growth and interactions inside microbial consortia with single-cell resolution. Our device allows the spatial separation of two different microbial organisms inside adjacent microchambers facilitating sufficient exchange of metabolites via connecting nanochannels. Inside the cultivation chambers cell growth can be observed with high spatio-temporal resolution by live-cell imaging. In contrast to conventional approaches, in which single-cell activity is typically fully masked by the average bulk behavior, the small dimensions of the microfluidic cultivation chambers enable accurate environmental control and observation of cellular interactions with full spatio-temporal resolution. Our method enables one to study phenomena in microbial interactions, such as gene transfer or metabolic cross-feeding. We chose two different microbial model systems to demonstrate the wide applicability of the technology. First, we investigated commensalistic interactions between an industrially relevant l-lysine-producing Corynebacterium glutamicum strain and an l-lysine auxotrophic variant of the same species. Spatially separated co-cultivation of both strains resulted in growth of the auxotrophic strain due to secreted l-lysine supplied by the producer strain. As a second example we investigated bacterial conjugation between Escherichia coli S17-1 and Pseudomonas putida KT2440 cells. We could show that direct cell contact is essential for the successful gene transfer via conjugation and was hindered when cells were spatially separated. The presented device lays the foundation for further studies on contactless and contact-based interactions of natural and synthetic microbial communities.