SARS-CoV-2 Mpro responds to oxidation by forming disulfide and NOS/SONOS bonds.

The main protease (Mpro) of SARS-CoV-2 is critical for viral function and a key drug target. Mpro is only active when reduced; turnover ceases upon oxidation but is restored by re-reduction. This suggests the system has evolved to survive periods in an oxidative environment, but the mechanism of this protection has not been confirmed. Here, we report a crystal structure of oxidized Mpro showing a disulfide bond between the active site cysteine, C145, and a distal cysteine, C117. Previous work proposed this disulfide provides the mechanism of protection from irreversible oxidation. Mpro forms an obligate homodimer, and the C117-C145 structure shows disruption of interactions bridging the dimer interface, implying a correlation between oxidation and dimerization. We confirm dimer stability is weakened in solution upon oxidation. Finally, we observe the protein's crystallization behavior is linked to its redox state. Oxidized Mpro spontaneously forms a distinct, more loosely packed lattice. Seeding with crystals of this lattice yields a structure with an oxidation pattern incorporating one cysteine-lysine-cysteine (SONOS) and two lysine-cysteine (NOS) bridges. These structures further our understanding of the oxidative regulation of Mpro and the crystallization conditions necessary to study this structurally.

Assembly Requirements for the Construction of Large-Scale Binary Protein Structures.

The precise assembly of multiple biomacromolecules into well-defined structures and materials is of great importance for various biomedical and nanobiotechnological applications. In this study, we investigate the assembly requirements for two-component materials using charged protein nanocages as building blocks. To achieve this, we designed several variants of ferritin nanocages to determine the surface characteristics necessary for the formation of large-scale binary three-dimensional (3D) assemblies. These nanocage variants were employed in protein crystallization experiments and macromolecular crystallography analyses, complemented by computational methods. Through the screening of nanocage variant combinations at various ionic strengths, we identified three essential features for successful assembly: (1) the presence of a favored crystal contact region, (2) the presence of a charged patch not involved in crystal contacts, and (3) sufficient distinctiveness between the nanocages. Surprisingly, the absence of noncrystal contact mediating patches had a detrimental effect on the assemblies, highlighting their unexpected importance. Intriguingly, we observed the formation of not only binary structures but also both negatively and positively charged unitary structures under previously exclusively binary conditions. Overall, our findings will inform future design strategies by providing some design rules, showcasing the utility of supercharging symmetric building blocks in facilitating the assembly of biomacromolecules into large-scale binary 3D assemblies.

Neural network enhanced aging time measurements of diary product remaining with infrared spectroscopy.

The determination of the drying degree of food residues on surfaces is an important step before efficient cleaning can be achieved. To accomplish this goal, a rapid evaluation based on a neural network and non-invasive measurement technique is introduced. Two common starch-based products and various yogurts from different manufacturers are used as example contaminants to determine the aging time of dried food residue. Near-infrared spectroscopy serves as a modern and fast measurement technique for investigating food compositions. Two analysis methods were compared for processing the measured near-infrared spectral data. The raw data were analyzed using partial least squares regression in conjunction with necessary preprocessing steps. As an alternative method, three different types of neural networks are employed. The aim of this approach is to compensate for the filtering steps before regression, which are typically necessary for multivariate regression. The challenge is to measure three different types of food and obtain a reliable prediction of moisture content in order to draw conclusions about the drying time. The experiments have shown that simple flat neural networks have similar accuracy compared to conventional regression. The use of a convolutional layer in advance demonstrates a significant improvement in prediction compared to other neural networks and even manages to surpass the accuracy of PLS regression. A network with a convolutional layer can also compensate for the sometimes strong variations between food types.

Calpeptin is a potent cathepsin inhibitor and drug candidate for SARS-CoV-2 infections.

Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (Mpro), its moderate activity in Mpro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptin's efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight reduces the viral load in the trachea. Despite a higher risk of side effects, an intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets. Here we show that the inhibition of cathepsins, a protein family of the host organism, by calpeptin is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections.

Crystalline Biohybrid Materials Based on Protein Cages.

Highly ordered superstructures of nanomaterials can be synthesized using protein cages as templates for the assembly of inorganic nanoparticles. Here, we describe in detail the creation of these biohybrid materials. The approach involves computational redesign of ferritin cages, followed by recombinant protein production and purification of the new variants. Metal oxide nanoparticles are synthesized inside the surface-charged variants. The composites are assembled using protein crystallization to yield highly ordered superlattices, which are characterized, for example, with small angle X-ray scattering. This protocol provides a detailed and comprehensive account on our newly established strategy for the synthesis of crystalline biohybrid materials.

Overcoming Non-Specific Interactions for Efficient Encapsulation of Doxorubicin in Ferritin Nanocages for Targeted Drug Delivery.

Due to its beneficial pharmacological properties, ferritin (Ftn) is considered as an interesting drug delivery vehicle to alleviate the cardiotoxicity of doxorubicin (DOX) in chemotherapy. However, the encapsulation of DOX in Ftn suffers from heavy precipitation and low protein recovery yield which limits its full potential. Here, a new DOX encapsulation strategy by cysteine-maleimide conjugation is proposed. In order to demonstrate that this strategy is more efficient compared to the other approaches, DOX is encapsulated in Ftn variants carrying different surface charges. Furthermore, in contrast to the common belief, this data show that DOX molecules are also found to bind non-specifically to the surface of Ftn. This can be circumvented by the use of Tris(2-carboxyethyl)phosphine (TCEP) during encapsulation or by washing with acidic buffer. The biocompatibility studies of the resulting DOX Ftn variants in MCF-7 and MHS cancer cells shows a complex relationship between the cytotoxicity, the DOX loading and the different surface charges of Ftn. Further investigation on the cell uptake mechanism provides reasonable explanations for the cytotoxicity results and reveals that surface charging of Ftn hinders its transferrin receptor 1 (TfR-1) mediated cellular uptake in MCF-7 cells.

Assembly of chemically modified protein nanocages into 3D materials for the adsorption of uremic toxins.

Hemodialysis fails to remove protein-bound uremic toxins that are attributed with high cardiovascular risk. Application of adsorption materials is a viable strategy, but suitable biocompatible adsorbents are still not available. Here, we demonstrate that adsorbents based on the bottom-up assembly of the intrinsically biocompatible protein cage ferritin are applicable for toxin adsorption. Due to the size-exclusion effect of its pores, only small molecules such as uremic toxins can enter the protein cage. Protein redesign techniques that target selectively the inner surface were used to introduce anchor sites for chemical modification. Porous crystalline adsorbents were fabricated by bottom-up assembly of the protein cage. Linkage of up to 96 phenylic or aliphatic molecules per container was verified by ESI-MS. Materials based on unmodified ferritin cages can already adsorb the uremic toxins. The adsorption capacity could be increased by about 50% through functionalization with hydrophobic molecules reaching 458 µg g-1 for indoxyl sulfate. The biohybrid materials show no contamination with endotoxins and do not activate blood platelets. These findings demonstrate the great potential of protein-based adsorbents for the clearance of uremic toxins: modifications enhance toxin adsorption without diminishing the biocompatibility of the final protein-based material.

Encapsulation of Gold Nanoparticles into Redesigned Ferritin Nanocages for the Assembly of Binary Superlattices Composed of Fluorophores and Gold Nanoparticles.

Nanomaterials with a defined composition and structure can be synthesized by exploiting natural templates or biomolecular matrices. In the present work, we use protein nanocages derived from human ferritin as a nanoscale building block for the assembly of gold nanoparticles and fluorescent molecules in the solid state. As a generalizable strategy, we show that prior to material synthesis, the cargo can be encapsulated into the protein nanocages using a dis- and reassembly approach. Toward this end, a new ligand system for gold nanoparticles enables efficient encapsulation of these particles into the nanocages. The gold nanoparticle-loaded protein nanocages are co-assembled with fluorophore-loaded protein nanocages. Binary superlattices are formed because two oppositely charged ferritin nanocages are used as templates for the assembly. The binary crystals show strong exciton-plasmon coupling between the encapsulated fluorophores and gold nanoparticles, which was spatially resolved with fluorescence lifetime imaging. The strategy outlined here offers a modular approach toward binary nanomaterials with highly ordered building blocks.

Mosquito-Textile Physics: A Mathematical Roadmap to Insecticide-Free, Bite-Proof Clothing for Everyday Life.

Garments treated with chemical insecticides are commonly used to prevent mosquito bites. Resistance to insecticides, however, is threatening the efficacy of this technology, and people are increasingly concerned about the potential health impacts of wearing insecticide-treated clothing. Here, we report a mathematical model for fabric barriers that resist bites from Aedes aegypti mosquitoes based on textile physical structure and no insecticides. The model was derived from mosquito morphometrics and analysis of mosquito biting behavior. Woven filter fabrics, precision polypropylene plates, and knitted fabrics were used for model validation. Then, based on the model predictions, prototype knitted textiles and garments were developed that prevented mosquito biting, and comfort testing showed the garments to possess superior thermophysiological properties. Our fabrics provided a three-times greater bite resistance than the insecticide-treated cloth. Our predictive model can be used to develop additional textiles in the future for garments that are highly bite resistant to mosquitoes.

X-ray-Based Techniques to Study the Nano-Bio Interface.

X-ray-based analytics are routinely applied in many fields, including physics, chemistry, materials science, and engineering. The full potential of such techniques in the life sciences and medicine, however, has not yet been fully exploited. We highlight current and upcoming advances in this direction. We describe different X-ray-based methodologies (including those performed at synchrotron light sources and X-ray free-electron lasers) and their potentials for application to investigate the nano-bio interface. The discussion is predominantly guided by asking how such methods could better help to understand and to improve nanoparticle-based drug delivery, though the concepts also apply to nano-bio interactions in general. We discuss current limitations and how they might be overcome, particularly for future use in vivo.

Protecting redesigned supercharged ferritin containers against protease by integration into acid-cleavable polyelectrolyte microgels.

HYPOTHESIS: The application of ferritin containers as a promising drug delivery vehicle is limited by their low bioavailability in blood circulation due to unfavorable environments, such as degradation by protease. The integration of ferritin containers into the polymeric network of microgels through electrostatic interactions is expected to be able to protect ferritin against degradation by protease. Furthermore, a stimuli-responsive microgel system can be designed by employing an acid-degradable crosslinker during the microgel synthesis. This should enable ferritin release in an acidic environment, which will be useful for future drug delivery applications. EXPERIMENTS: Nanoparticle/fluorophores-loaded ferritin was integrated into microgels during precipitation polymerization. The integration was monitored by transmission electron microscopy (TEM)2 and fluorescence microscopy, respectively. After studying ferritin release in acidic solutions, we investigated the stability of ferritin inside microgels against degradation by chymotrypsin. FINDINGS: About 80% of the applied ferritin containers were integrated into microgels and around 85% and 50% of them could be released in buffer pH 2.5 and 4.0, respectively. Total degradation of the microgels was not achieved due to the self-crosslinking of N-isopropylacrylamide (NIPAM). Finally, we prove that microgels could protect ferritin against degradation by chymotrypsin at 37 °C.

Molecular basis for protein-protein interactions.

This minireview provides an overview on the current knowledge of protein-protein interactions, common characterisation methods to characterise them, and their role in protein complex formation with some examples. A deep understanding of protein-protein interactions and their molecular interactions is important for a number of applications, including drug design. Protein-protein interactions and their discovery are thus an interesting avenue for understanding how protein complexes, which make up the majority of proteins, work.

An Acute Spinal Intradural Hematoma after an Extraforaminal Wiltse Approach: A Case Report and Review of the Literature.

A nontraumatic spontaneous spinal acute subdural hematoma (sSDH) is a rare complication after spinal surgery. Although an sSDH is often associated with anticoagulation therapy, vascular malformations, or lumbar puncture, the pathogenesis of nontraumatic spontaneous sSDH remains unclear. We present the case of an intradural hematoma after an extraforaminal surgery through the Wiltse approach for an extraforaminal disk herniation at L5/S1. This 58-year-old woman experienced hypoesthesia and progressive motor dysfunction in the left leg several hours postoperation. Urgent magnetic resonance imaging revealed an intradural hematoma at the L1/L2 to L2/L3 level in the ventral dural sac proximal to the surgical level. Surgical decompression was performed. There was no evidence of trauma, coagulopathy, or anticoagulation therapy. To our knowledge, this case is the first to report an acute sSDH proximal to the surgery level after an extraforaminal spinal surgery through the Wiltse approach for an extraforaminal disk herniation. It illustrates that attentive postoperative neurologic monitoring, even in the absence of intraoperative irregularities, remains important to diagnose and treat this complication at the early stage.

Influence of storage on the quality of conventional CT and µCT-imaging for the middle and inner cat ear.

The aim of this study was to analyze whether different fixation methods such as freezing or formaldehyde storage for different periods of time have an influence on the recognition of anatomical relevant structures in the middle and inner ear of the cat with conventional computed tomography (cCT) and micro-computed tomography (µCT). Besides, effects of freeze-thaw cycles on determined structures of the ear were investigated by means of histological slices. Three veterinarians with different radiologic expertise evaluated the scans of 30 dissected cat ears anonymously and scored predefined structures in a five-point scale with reference to visually sharp reproducibility and perfect image quality. The total scores of the different fixation groups as well as the ears within a group were compared with each other. Furthermore, an intra-reader examination including an evaluation of the identifiability of specified structures was performed for both imaging methods. cCT as well as µCT-scans have a very low variation coefficient of 1.6% and 2.3%, respectively. The results for the alterations between the different fixation methods show that the changes for cCT-scans are negligible, as the percentage alteration compared to fresh samples ranges in a very small interval with values from 1.0% better to 1.2% worse. µCT-scans are more influenced by the fixation method with a range from 1.3% better to 6.9% worse values. The scans mostly deteriorated after two freeze-thaw cycles (1.8% worse) and after storing the samples for 1 (2.4% worse), respectively, and 3 weeks in formaldehyde (6.9% worse).

Occurrence of tympanic bone spicules and bone crests in domestic cats.

Small and hyperostotic tympanic bone spicules (STBS and HTBS) extending from the tympanic wall or from the septum bullae into the tympanic cavity have been described in large feline species such as Siberian tigers or African lions and in canids such as dogs, red foxes and wolves. Detailed descriptions of prevalence, location and orientation were performed for dogs and African lions by means of necropsy and conventional computed tomography (cCT). Aims of the current study were to describe same characteristics for domestic cats by means of microcomputed tomography (µCT) and cCT. A total of 15 cats or rather 30 ears were examined. Furthermore, new bone formations extending into the tympanic cavity shaped like small lamellae were found and named "tympanic bone crests" (TBCs). The registered phenomenon of a thickened tympanic wall in some cats was named "tympanic wall thickening" (TWT). STBS appeared in 43% of the ears being bilateral in 44% of the cases, whereas HTBS appeared in one ear. TBC was present in 33% of the ears with a bilateral prevalence of 67%, and TWT could be detected in 13% of the ears. The mentioned structures are just barely or not at all detectable with cCT; therefore, µCT is necessary for an identification and detailed description. The origin and the factors inducing the development of examined phenomena are unknown, and it is hypothesized that the occurrence can be assessed as anatomical norm variations.

A Bifunctional Adsorber Particle for the Removal of Hydrophobic Uremic Toxins from Whole Blood of Renal Failure Patients.

Hydrophobic uremic toxins accumulate in patients with chronic kidney disease, contributing to a highly increased cardiovascular risk. The clearance of these uremic toxins using current hemodialysis techniques is limited due to their hydrophobicity and their high binding affinity to plasma proteins. Adsorber techniques may be an appropriate alternative to increase hydrophobic uremic toxin removal. We developed an extracorporeal, whole-blood bifunctional adsorber particle consisting of a porous, activated charcoal core with a hydrophilic polyvinylpyrrolidone surface coating. The adsorption capacity was quantified using analytical chromatography after perfusion of the particles with an albumin solution or blood, each containing mixtures of hydrophobic uremic toxins. A time-dependent increase in hydrophobic uremic toxin adsorption was depicted and all toxins showed a high binding affinity to the adsorber particles. Further, the particle showed a sufficient hemocompatibility without significant effects on complement component 5a, thrombin-antithrombin III complex, or thrombocyte concentration in blood in vitro, although leukocyte counts were slightly reduced. In conclusion, the bifunctional adsorber particle with cross-linked polyvinylpyrrolidone coating showed a high adsorption capacity without adverse effects on hemocompatibility in vitro. Thus, it may be an interesting candidate for further in vivo studies with the aim to increase the efficiency of conventional dialysis techniques.

Differences between µCT-imaging and conventional CT for the diagnosis of possible diseases of the middle and inner cat ear.

The aim of this study was to check the relevance of using in-vivo micro computed tomography (µCT) for the diagnosis of possible diseases of the middle and inner ear of the cat. Therefore, on the one hand, differences of the detail detectability between the two imaging methods conventional computed tomography (cCT) and in-vivo µCT were analyzed. Six healthy cat ears were dissected and scanned several times and the obtained images were compared with each other. On the other hand, histological slices of all ears were prepared and pictures of defined anatomical structures were taken and compared with the identical sectional plane of the µCT-images. This way it was possible to evaluate the quality and clinical limitations of the in-vivo µCT. The results show that an in-vivo µCT is suitable to analyze even the smallest osseous structures, such as the semicircular ducts, the spiral osseous lamina or the ossicles whereas with the help of cCT it is not possible to identify such small osseous structures because of their blurred and less detailed representation. Delicate soft tissue structures as the membranous labyrinth including hearing and vestibular organ cannot be differentiated with as well in-vivo µCT- as with cCT-images. In-vivo µCT represent a good possibility for more detailed diagnosis of extremely fine structures which cannot be detected with cCT. Histological slices can nonetheless not be replaced by in-vivo µCT due to a too low spatial resolution and the limitations of the in-vivo µCT with regard to the evaluation of soft tissue dense structures.

Correction: Peptide-directed encapsulation of inorganic nanoparticles into protein containers.

Correction for 'Peptide-directed encapsulation of inorganic nanoparticles into protein containers' by Tobias Beck et al., Nanoscale, 2018, 10, 22917-22926.

Higher-Order Structures Based on Molecular Interactions for the Formation of Natural and Artificial Biomaterials.

The assembly of molecular building blocks into highly ordered structures is crucial, both in nature and for the development of novel functional materials. In nature, noncovalent interactions, such as hydrogen bonds or hydrophobic interactions, enable the reversible assembly of biopolymers, such as DNA or proteins. Inspired by these design principles, scientists have created biohybrid materials that employ natural building blocks and their assembly properties. Thus, structures and materials are attainable that cannot be made through other synthetic procedures. Herein, we review current concepts and highlight recent advances.