Martin Humenik

Silk-like secretions are well-studied in many insects but remain poorly characterized in Diptera. Larvae of the dipteran Keroplatus testaceus produce two distinct silk-like materials—a mucus web and a cocoon—from their labial salivary glands, differing in composition and function. Here we combined histology, transcriptomics, electron microscopy, proteomics, mechanical testing, and FTIR spectroscopy to characterize these secretions. Neither contains fibroin; instead, both comprise hydrophilic proteins including collagen-like, mucin, and novel proline-rich proteins. Structural predictions and FTIR analyses reveal β-sheets, α-helices, and polyproline II helices, contributing to mechanical robustness despite high hydration. The mucus web facilitates larval mobility and spore capture, while the cocoon provides mechanical protection during pupation. These findings reveal a fibroin-independent silk system adapted to humid, microbe-rich environments, expanding understanding of insect silk diversity and offering insights into novel biomaterials.

Incorporation of silk fibroin into polycaprolactone (PCL) matrices introduces significant alterations to the crystalline structure and surface morphology of blended polymeric materials, presenting opportunities for advanced biomaterial design. This study investigates fibroin‐PCL blends prepared either as films via solution casting or solvent‐based extrusion printing. Fibroin dispersed within the PCL matrix was found to modulate crystalline organization, inducing distinct morphological transformations. The structural and morphological changes were comprehensively characterized using wide‐angle X‐ray scattering (WAXS), atomic force microscopy (AFM), optical microscopy, and scanning electron microscopy (SEM). Low fibroin concentrations in the blends (50% w/w) resulted in the emergence of pits and porous surface topography. Despite the significant morphological changes on the blends’ surface, the type of PCL‐crystallinity remained unchanged, as shown by WAXS. Furthermore, the evaporation rate of the solvent during processing critically influences the texture patterns of the films and the printed grids. By leveraging the interplay between fibroin and PCL in the blended constructs, this work provides insights into the control of surface features in fibroin–PCL systems, with implications for the rational design of biomaterials.

Many diseases that threaten vision originate from imbalances in the production or signaling of vascular endothelial growth factor (VEGF) and nerve growth factor (NGF). While VEGF inhibitors can suppress pathological blood vessel growth and NGF therapies can promote tissue regeneration, treatments employing the growth factors are often invasive, inefficient, or not suitable for long‐term use. For example, the use of NGF‐containing eye drops requires high drug loads and frequent administration, thereby increasing the potential for side effects and high costs. Based on a critical overview of currently used experimental and clinical approaches for the delivery or inhibition of growth factors, we highlight aptamers as promising yet underexplored therapeutic tools. Aptamers combine high target specificity, tunable structure and affinity, stability, low immunogenicity, and scalable chemical synthesis. These properties allow aptamers to be readily incorporated into biomaterial carriers, such as hydrogels, implants, or contact lenses, to improve the controlled binding of growth factors for precise modulation of their levels in ocular tissues via release or sequestration strategies. Leveraging these advantages in future developments could facilitate the aptamer‐functionalized platforms to overcome the limitations of current therapies and advance more sustainable, targeted, and patient‐oriented treatment strategies in ophthalmology.

The process of molecular self‐assembly is an omnipresent mechanism in nature to generate a variety of efficient and functional hierarchical architectures, and inspires tailored material design and development. Thereby, self‐assembly is based on a controlled interplay and association of monomers into highly ordered structures triggered by different non‐covalent interactions. However, in the context of structural protein self‐assembly, this association process could be influenced by the underlying amino acid sequence as well as external triggers including pH value, protein concentration, or ionic composition. Thus, understanding their impact on protein conformation and assembly is indispensable for controlled protein processing and functional materials' engineering. Here, we analyzed the self‐assembly behavior of the intrinsically unstructured, recombinant spider silk proteins eADF4(Ω16) and eADF4(C16), which only differ in one amino acid residue in their repetitive module (glutamine and glutamic acid, respectively), depending on the concentration of kosmotropic potassium phosphate (KPi) and the pH value. The low protein charge in eADF4(Ω16) at neutral pH led to a compacted protein conformation and a significantly increased sensitivity to phosphate resulting in faster assembly kinetics of nanofibrils and precipitation of particles at lower KPi concentrations. In contrast, the presence of glutamic acid residues in eADF4(C16) enhanced the solubility and stability of protein monomers above physiological pH but led to an enhanced assembly/aggregation along with decreasing pH‐values. Interestingly, deprotonation of tyrosine residues at pH 10 introduced negative charges resulting in decreased hydrophobic interactions and thus decelerated restructuring and assembly of eADF4(Ω16). Our results enabled the identification of Pi‐ and pH‐dependent conformation and assembly models of eADF4‐based spider silk proteins allowing controllable processing into fibrils, particles, or hydrogels for specific applications.

Natural spider silk fibers are recognized for their outstanding mechanical properties. The production of underlying recombinant spider silk proteins in scalable quantities unlocks their potential for technical and biomedical applications. The recombinant spider silk technology further enables the introduction of new functions via genetic encoding. In the present study, the non‐canonical amino acid L‐azidohomoalanine is incorporated into the engineered Araneus diadematus fibroin 4, eADF4(C16), at positions explicitly defined by methionine codons in an N‐terminal peptide tag. The azido‐functionalized eADF4(C16) is processed into particles, films, or electrospun into nanofibers. As functional entities, high molecular weight molecules, such as antibodies, are modified with bio‐orthogonal groups suitable for strain‐promoted cycloaddition to the exposed azido‐groups on the spider silk supports. The antibody‐activated spider silk nonwoven meshes exemplarily show their applicability as bio‐capturing membranes.

Spider silks are proteinaceous fiber materials inspiring material design in various technical and biomedical fields due to their exceptional toughness, which exceeds that of most natural and artificial fibers. Solid‐state nuclear magnetic resonance (NMR) spectroscopy has been used herein to obtain insights into the structure and dynamics of ¹³ C/ ¹⁵ N isotope‐labeled nanofibrils and microparticles made of the recombinantly produced, engineered spider silk protein eADF4(C16), for which structural information was still lacking. Although these two β‐sheet‐rich morphologies differ substantially in their microscopic appearance (nanofibrils vs. microparticles), the solid‐state NMR spectra reveal high structural and dynamic similarities at the atomic level. For both morphologies, it was found that the rigid alanine stretch in the eADF4 sequence forms a mixture of rectangular and staggered β‐sheets extending to the flanking serine residues. In addition, our data reveal that the tyrosine sidechains are rigidified, which suggests their engagement in π–π‐stacking interactions. All of the glutamic acid residues were found to be deprotonated, which implies their localization on the outside of the fibril, where their negative charge can be compensated. Trans ‐ as well as cis ‐conformations were observed for proline residues, which suggests that they might further control the formation and extension of the poly‐alanine β‐sheet region during the self‐assembly process. The gained understanding of structure, dynamics, and assembly of the engineered spider silk protein eADF4(C16) will enable the tailored design of functional spider silk‐based biomaterials in the future. It will be especially useful in context of chemical modifications and genetic fusions supporting the development of fibril‐based hydrogel systems in the field of biosensing and tissue engineering.

Silk materials have been extensively studied across several insect groups; however, dipterans remain largely unexplored. In this study, we examined the silk-like secretions of Keroplatus testaceus larvae using histochemistry, transcriptomics, proteomics, mechanical assays, and Fourier-transform infrared (FTIR) spectroscopy. Our results show that a single pair of labial salivary glands produces two distinct secretions, the mucus web and the cocoon, which differ in both composition and function. Neither secretion contains fibroin. Instead, both are composed of a complex mixture of hydrophilic proteins, including collagen-like, mucin-like, novel proline-rich proteins, peroxidases, protease inhibitors, and other proteins with no clear homologs in other species. Structural predictions and FTIR analysis of the cocoon reveal the presence of secondary structures, including β-sheets, α-helices, and polyproline II helices. Although the secretion lacks the characteristic fibrillar core of fibroin-based silks, it is highly hydrated, rich in proline-containing proteins, and exhibits relatively high mechanical strength. These findings confirm that K. testaceus produces a highly specialized silk-like material that is structurally and compositionally adapted to larval life within wood-decomposing fungal fruiting bodies.

Due to excellent mechanical properties and biocompatibility, materials based on silk fibroin are increasingly included in advanced biomedical research and applications. However, their poor supporting properties for cell adhesion and proliferation represent limiting factors of the utilization. To eliminate this deficiency, we developed a series of phase-separation approaches allowing for tunable texturing of planar and 3D printed fibroin surfaces from nano to macro levels. The formation of surface structures presented is based on a combination of good and poor solvents, whereas no potentially problematic templates or additives, diminishing biocompatibility of the resulting material, are required. A critical factor in obtaining and scaling of the textures is control over the degree of transformation of fibroin secondary structures between prevalently amorphous Silk I and semicrystalline Silk II forms before and during surface treatment. Employing a set of optimized procedures, selectively or hierarchically structured fibroin surfaces can be prepared at the nano, micro, and macro level, which are characterized by long-term stability in physiological environments, allowing enhanced adhesion and proliferation of human keratinocytes as well as skin fibroblast cultivations.

This article was originally published online on 7 September 2023 with an error in the affiliation. The affiliation appears correct above. AIP Publishing apologi