Abstract Fungal surfaces must remain hydrophobic to enable growth, dispersal, and survival under fluctuating environmental conditions, yet the molecular basis of this property remains incompletely understood. Here, we identify fungisporins, fusahexins, and related cyclic non-ribosomal peptides (NRPs) as members of a conserved functional class of fungal metabolites, termed WAter Repellent Peptides (WARPs), that are required for fungal surface hydrophobicity. Across filamentous fungi, WARPs vary substantially in sequence and length but share conserved structural features, including cyclization, hydrophobic amino acid composition, and alternating D- and L-configurations, consistent with a flexible amphiphilic scaffold. Loss of WARP-producing non-ribosomal peptide synthetases results in rapid collapse of aerial hyphae upon water exposure, demonstrating that these peptides are required for maintenance of hydrophobic aerial structures. Using phage-display-derived antibodies, we localize WARPs to the hyphal surface, supporting their role as surface-associated structural components. Together, these findings identify a conserved NRPS-encoded peptide system that contributes to fungal hydrophobicity and establish WARPs as a broadly distributed class of surface-associated metabolites with structural function in filamentous fungi.

Melting and glass formation of metal-organic frameworks (MOFs) allow them to be processed into bulk materials. However, two major challenges remain: only a small fraction of MOF crystals undergo melting and glass-formation, and no well-established strategies exist for tuning MOF glass structures and properties. Here, we address both challenges through co-melting of zeolitic imidazole frameworks (ZIFs), a subset of MOFs, with heterocycle-based halide salts. The salt acts as a chemical "modifier", akin to the role of alkali modifiers in traditional silicate glasses, e.g., allowing the melting of ZIF-8 that otherwise decomposes prior to melting. Through experimental and computational analyses, we show that the salts depolymerize the ZIFs, enabling continuous tuning of the fraction of bridging to non-bridging imidazolate linkers and, thereby, the thermal and mechanical properties. The proposed strategy enables diversification of MOF glass chemistry, tunable structures and properties, and ultimately an increased number of glass-forming MOFs with improved functionalities.

Water is one of the most reactive and abundant molecules on Earth, and it is thus crucial to understand its reactivity with various material families. One of the big unknown questions is how water in liquid and vapor forms impact the fast-emerging class of metal-organic frameworks (MOFs). Here, we discover that high-pressure water vapor drastically modifies the structure and hence the dynamic, thermodynamic, and mechanical properties of MOF glasses. In detail, we find that an archetypical MOF (ZIF-62) is extremely sensitive to heat treatments performed at 460 °C and water vapor pressures up to ∼110 bar. Both the melting and glass transition temperatures decrease remarkably (by >100 °C), and simultaneously, hardness and Young's modulus increase by up to 100% under very mild treatment conditions (<20 bar of hydrothermal pressure). Structural analyses suggest water to partially coordinate to Zn in the form of a hydroxide ion by replacing a bridging imidazolate-based linker. The work provides insight into the role of hot-compressed water in influencing the structure and properties of MOF glasses and opens a new route for systematically changing the thermodynamics and kinetics of MOF liquids and thus altering the thermal and mechanical properties of the resulting MOF glasses.

Bispecific antibodies (bsAbs) have attracted significant attention due to their dual binding activity, which permits simultaneous targeting of antigens and synergistic binding effects beyond what can be obtained even with combinations of conventional monospecific antibodies. Despite the tremendous therapeutic potential, the design and construction of bsAbs are often hampered by practical issues arising from the increased structural complexity as compared to conventional monospecific antibodies. The issues are diverse in nature, spanning from decreased biophysical stability from fusion of exogenous antigen-binding domains to antibody chain mispairing leading to formation of antibody-related impurities that are very difficult to remove. The added complexity requires judicious design considerations as well as extensive molecular engineering to ensure formation of high quality bsAbs with the intended mode of action and favorable drug-like qualities. In this review, we highlight and summarize some of the key considerations in design of bsAbs as well as state-of-the-art engineering principles that can be applied in efficient construction of bsAbs with diverse molecular formats.

Transporters of the solute carrier superfamily (SLCs) are responsible for the transmembrane traffic of the majority of chemical substances in cells and tissues and are therefore of fundamental biological importance. As is often the case with membrane proteins that can be heavily glycosylated, a lack of reliable high-affinity binders hinders their functional analysis. Purifying and reconstituting transmembrane proteins in their lipidic environments remains challenging and standard approaches to generate binders for multi-transmembrane proteins, such as SLCs, channels or G protein-coupled receptors (GPCRs) are lacking. While generating protein binders to 27 SLCs, we produced full length protein or cell lines as input material for binder generation by selected binder generation platforms. As a result, we obtained 525 binders for 22 SLCs. We validated the binders with a cell-based validation workflow using immunofluorescent and immunoprecipitation methods to process all obtained binders. Finally, we demonstrated the potential applications of the binders that passed our validation pipeline in structural, biochemical, and biological applications using the exemplary protein SLC12A6, an ion transporter relevant in human disease. With this work, we were able to generate easily renewable and highly specific binders against SLCs, which will greatly facilitate the study of this neglected protein family. We hope that the process will serve as blueprint for the generation of binders against the entire superfamily of SLC transporters.

Antibody phage-display technology identifies antibody-antigen interactions through multiple panning rounds, but traditional screening gives no information on enrichment or diversity throughout the process. This results in the loss of valuable binders. Next Generation Sequencing can overcome this problem. We introduce a high accuracy long-read sequencing method based on the recent Oxford Nanopore Technologies (ONT) Q20 + chemistry in combination with dual unique molecular identifiers (UMIs) and an optimized bioinformatic analysis pipeline to monitor the selections. We identified binders from two single-domain antibody libraries selected against a model protein. Traditional colony-picking was compared with our ONT-UMI method. ONT-UMI enabled monitoring of diversity and enrichment before and after each selection round. By combining phage antibody selections with ONT-UMIs, deep mining of output selections is possible. The approach provides an alternative to traditional screening, enabling diversity quantification after each selection round and rare binder recovery, even when the dominating binder was > 99% abundant. Moreover, it can give insights on binding motifs for further affinity maturation and specificity optimizations. Our results demonstrate a platform for future data guided selection strategies.

Microfiltration membranes are increasingly used to retain the cells inside bioreactors while continuous harvest of the desired biopharmaceutical occurs in perfusion processes. One method of microfiltration is Alternating Tangential Flow (ATF) filtration, which involves moving the cultivation broth tangentially back and forth across the membrane, which results in a backwash effect that reduces fouling. In this study, fouling was investigated with asymmetric polysulfone hollow fibers operated in ATF mode attached to a bioreactor producing a recombinant protein in Chinese Hamster Ovary (CHO) cells. Fouling was assessed through different approaches, including determination of critical flux using an improved flux-step method. Fouling was studied through measurements of Transmembrane Pressure (TMP), protein transmission, membrane pore size and staining of the membrane after operation to visualize the distribution of biological fouling inside the membrane. For critical flux determination, fluxes of up to 69 LMH were used without exceeding the critical flux. No sign of fouling was observed for the short term (<3.5 h) critical flux experiment. However, during prolonged operation at 8.3 LMH the TMP jumped to 0.9-0.95 bar, indicating fouling. At this state, the protein transmission remains at the same high level (>88%).

Aluminum nitride (AlN) is a material of growing interest for power electronics, fabrication of sensors, micro-electromechanical systems, and piezoelectric generators. For the latter, the formation of nanowire arrays or nanostructured films is one of the emerging research directions. In the current work, nanostructured AlN films manufactured with normal and glancing angle magnetron sputter depositions have been investigated with scanning and transmission electron microscopy, X-ray diffraction, atomic force microscopy, and optical spectroscopy. Growth of the nanostructures was realized utilizing metal seed particles (Ag, Au, and Al), allowing the control of the nucleation and following growth of AlN. It was demonstrated how variations of seed particle material and size can be used to tune the parameters of nanostructures and morphology of the AlN films. Using normal angle deposition allowed the growth of bud-shaped structures, which consisted of pillars/lamellae with wurtzite-like crystalline structures. Deposition at a glancing angle of 85° led to a film of individual nanostructures located near each other and tilted at an angle of 33° relative to the surface normal. Such films maintained a high degree of wurtzite-like crystallinity but had a more open structure and higher roughness than the nanostructured films grown at normal incidence deposition. The developed production strategies and recipes for controlling parameters of nanostructured films pave the way for the formation of matrices to be used in piezoelectric applications.

Quenchbodies (Q-bodies), a type of biosensor, are antibodies labeled with a fluorescent dye near the antigen recognition site. In the absence of an antigen, the dye is quenched by tryptophans in the antibody sequence; however, in its presence, the dye is displaced and therefore de-quenched. Although scFv and Fab are mainly used to create Q-bodies, this is the first report where a single-domain heavy chain VH from a semi-synthetic human antibody library formed the basis. To create a proof of concept “mini Q-body”, a human anti-lysozyme single-domain VH antibody C3 was used. Mini Q-bodies were successfully developed using seven dyes. Different responses were observed depending on the dye and linker length; it was concluded that the optimal linker length for the TAMRA dye was C5, and rhodamine 6G was identified as the dye with the largest de-quenching response. Three single-domain antibodies with sequences similar to that of the C3 antibody were chosen, and the results confirmed the applicability of this method in developing mini Q-bodies. In summary, mini Q-bodies are an easy-to-use and time-saving method for detecting proteins.

A variety of nanostructures capable of generating strong local electromagnetic fields (hot spots) in interaction with radiation have been under intensive investigation towards plasmonic applications in surface-enhanced Raman scattering (SERS), biosensing, broadband absorbers, thermophotovoltaics, photocatalysis, etc. In many cases, these nanostructures are formed on a surface or embedded into a near-surface layer of the dielectric substrate, making some part of the field dissipate into the bulk and not contribute to the desired plasmonic functionality. To reduce such losses, the interface between the metallic nanostructures and the dielectric environment should be engineered. In the current work, Au nanodiscs are fabricated on Si posts of very small diameter (pin-shaped structures), enabling them to decouple the strong optical near fields localized at the nanodiscs from the bulk Si substrate. The Si post diameter is optimized by adjusting the gas flow rates in reactive-ion etching, resulting in a minimum post diameter of 20 nm at the nanodisc interface. The effect of this diameter on the localized surface plasmon resonance of the nanodisc is investigated with linear optical spectroscopic measurements, where a significant spectral blue shift of the resonance band is noticed compared to similar discs formed on bulk Si surface. The experimental results are compared with modelling where a 3-fold increase in the electric field enhancement is demonstrated. The fabricated pin-shaped nanostructures are tested in SERS measurements showing a significant increase in the enhancement factor in the order of 10(6). Thus, this work suggests a way of engineering 3D morphology to tune the substrate influence on the plasmonic properties of nanostructures and to develop efficient nanofabrication technologies.

Surface-enhanced Raman spectroscopy (SERS) detection of analyte molecules at ultra-low concentrations re-quires highly-efficient plasmonic nanostructures enabling a high hot-spot density. However, a facile and cost-effective strategy toward large-area fabrication of efficient nanostructures with significant electromagnetic field enhancement remains a great challenge. Further, SERS faces reliability issues with the molecular fingerprint at ultra-low concentrations. This work shows a one-step rapid fabrication technique utilizing glancing angle deposition for growing 3D nanopillars of Ag or Au, which is facile, scalable and cost-effective. The 3D nanopillar substrates can reliably detect analyte molecules with concentrations as low as 10-18 M with a high signal-to-noise ratio molecular fingerprint proven for Cresyl violet, p-aminothiophenol and Rhodamine 6G. The ultra-high enhancement is realized in conjunction with the formation of a high hot-spot density due to localized surface plasmons and surface plasmons at metal/air interface. A portable handheld Raman spectrometer is used to evaluate the potential application of the nanopillars for on-site diagnostics. It avoids the need for sophisticated tabletop instruments yet provides high-precision molecular specificity outside specialized laboratories. The 3D nanopillar substrates show excellent molecular detection limits at 10-15 M concentrations when tested with a handheld Raman spectrometer. The uniqueness of the 3D nanopillar features with the formation of a high density of hot-spots and one-step nanofabrication methods provide a platform to unravel on-site diagnostics with cost-effective approaches.

Bispecific antibodies (bsAbs) enable dual binding of different antigens with potential synergistic targeting effects and innovative therapeutic possibilities. The formation of bsAbs is, however, often dependent on complex engineering strategies with a high risk of antibody chain mispairing leading to contamination of the final product with incorrectly assembled antibody species. This study demonstrates formation of bsAbs in a generic and conceptually easy manner through fusion of single-domain antibodies (sdAbs) onto IgG scaffolds through flexible 10 amino acid linkers to form high-quality bsAbs with both binding functionalities intact and minimal product-related impurities. SdAbs are attractive fusion partners due to their small and monomeric nature combined with antigen-binding capabilities comparable to conventional human antibodies. By systematically comparing a comprehensive panel of symmetric αPD-L1×αHER2 antibodies, including reversely mirrored antigen specificities, we investigate how the molecular geometry affects production, stability, antigen binding and CD16a binding. SdAb fusion of the heavy chain was generally preferred over light chain fusion for promoting good expression and high biophysical stability as well as maintaining efficient binding to both antigens. We find that N-terminal sdAb fusion might sterically hinder antigen-binding to the Fv region of the IgG scaffold, whereas C-terminal fusion might disturb antigen-binding to the fused sdAb. Our work demonstrates a toolbox of complementary methods for in-depth analysis of key features, such as in-solution dual antigen binding, thermal stability, and aggregation propensity, to ensure high bsAb quality. These techniques can be executed at high-throughput and/or with very low material consumption and thus represent valuable tools for bsAb screening and development.

The human immune system uses antibodies to neutralize foreign antigens. They are composed of heavy and light chains, both with constant and variable regions. The variable region has six hypervariable loops, also known as complementary-determining regions (CDRs) that determine antibody diversity and antigen specificity. Knowledge of their significance, and certain residues present in these areas, is vital for antibody therapeutics development. This study includes an analysis of more than 11,000 human antibody sequences from the International Immunogenetics information system (IMGT). The analysis included parameters such as length distribution, overall amino acid diversity, amino acid frequency per CDR and residue position within antibody chains. Overall, our findings confirm existing knowledge, such as CDRH3's high length diversity and amino acid variability, increased aromatic residue usage, particularly tyrosine, charged and polar residues like aspartic acid, serine, and the flexible residue glycine. Specific residue positions within each CDR influence these occurrences, implying a unique amino acid type distribution pattern. We compared amino acid type usage in CDRs and non-CDR regions, both in globular and transmembrane proteins, which revealed distinguishing features, such as increased frequency of tyrosine, serine, aspartic acid, and arginine. These findings should prove useful for future optimization, improvement of affinity, synthetic antibody library design, or the creation of antibodies de-novo in silico.

Large, randomized libraries are a key technology for many biotechnological applications. While genetic diversity is the main parameter most libraries direct their resources on, less focus is devoted to ensuring functional IN-frame expression. This study describes a faster and more efficient system based on a split β-lactamase complementation for removal of OFF-frame clones and increase of functional diversity, suitable for construction of randomized libraries. The gene of interest is inserted between two fragments of the β-lactamase gene, conferring resistance to β-lactam drugs only upon expression of an inserted IN-frame gene without stop codons or frameshifts. The preinduction-free system was capable of eliminating OFF-frame clones in starting mixtures of as little as 1% IN-frame clones and enriching to about 70% IN-frame clones, even when their starting rate was as low as 0.001%. The curation system was verified by constructing a single-domain antibody phage display library using trinucleotide phosphoramidites for randomizing a complementary determining region, while eliminating OFF-frame clones and maximizing functional diversity.

Antibodies are attractive therapeutic candidates due to their ability to bind cognate antigens with high affinity and specificity. Still, the underlying molecular rules governing the antibody-antigen interface remain poorly understood, making in silico antibody design inherently difficult and keeping the discovery and design of novel antibodies a costly and laborious process. This study investigates the characteristics of antibody-antigen binding interfaces through a computational analysis of more than 850,000 atom-atom contacts from the largest reported set of antibody-antigen complexes with 1833 nonredundant, experimentally determined structures. The analysis compares binding characteristics of conventional antibodies and single-domain antibodies (sdAbs) targeting both protein- and peptide antigens. We find clear patterns in the number antibody-antigen contacts and amino acid frequencies in the paratope. The direct comparison of sdAbs and conventional antibodies helps elucidate the mechanisms employed by sdAbs to compensate for their smaller size and the fact that they harbor only half the number of complementarity-determining regions compared to conventional antibodies. Furthermore, we pinpoint antibody interface hotspot residues that are often found at the binding interface and the amino acid frequencies at these positions. These findings have direct potential applications in antibody engineering and the design of improved antibody libraries.

Simultaneous targeting of different antigens by bispecific antibodies (bsAbs) is permitting synergistic binding functionalities with high therapeutic potential, but is also rendering their analysis challenging. We introduce flow-induced dispersion analysis (FIDA) for the in-depth characterization of bsAbs with diverse molecular architectures and valencies under near-native conditions without potentially obstructive surface immobilization. Individual equilibrium dissociation constants are determined in solution, even in higher-order complexes with both antigens involved, hereby allowing the analysis of binding cooperativity and elucidation of a potential interference between the interactions. We further illustrate bispecific binding functionality as incremental increases in complex sizes when the bsAbs are exposed to one or two antigens. The possibility for comprehensive binding analysis with low material consumption and high matrix tolerability irrespective of molecular format and with little optimization renders FIDA a versatile tool for format selection and characterization of complex bi/multispecific protein therapeutics throughout the drug development and biomanufacturing pipeline.

Wide bandgap semiconductors such as group III-nitrides and SiC are considered as key materials for the fabrication of smaller, more reliable and efficient power electronics. Fabrication of robust and durable power devices requires an optimized design based on the understanding of the interfacial adhesion properties of the constituent thin-film heterostructures. In this study, the adhesion properties of GaN/AlN layers grown on Si substrates were investigated. Particularly, the influence of the AlN buffer layers, necessary for GaN growth on Si, on the delamination response was determined. The interfacial adhesion strength was obtained using cross-sectional nanoindentation (CSN) and four-point bending (4PB) tests. Analytical models based on beam- and elastic plate theory which were applied respectively to calculate the interfacial fracture energy (Gic) for both methods are found to be in good agreement provided the loading conditions are similar. Detailed transmission and scanning electron microscopy investigations prior and subsequent to delamination reveal the microstructural details of the relevant interfaces and provide insights into the encountered mechanisms of interfacial failure. Finally, the probability of delamination along the weakest interface is discussed based on a fracture mechanics model.

We have investigated the function of human topoisomerase 1 (TOP1) in regulation of G-quadruplex (G4) formation in the Pu27 region of the MYC P1 promoter. Pu27 is among the best characterized G4 forming sequences in the human genome and it is well known that promoter activity is inhibited upon G4 formation in this region. We found that TOP1 downregulation stimulated transcription from a promoter with wildtype Pu27 but not if the G4 motif in Pu27 was interrupted by mutation(s). The effect was not specific to the MYC promoter and similar results were obtained for the G4 forming promoter element WT21. The other major DNA topoisomerases with relaxation activity, topoisomerases 2α and β, on the other hand, did not affect G4 dependent promoter activity. The cellular studies were supported by in vitro investigations demonstrating a high affinity of TOP1 for wildtype Pu27 but not for mutant sequences unable to form G4. Moreover, TOP1 was able to induce G4 formation in Pu27 inserted in double stranded plasmid DNA in vitro. This is the first time TOP1 has been demonstrated capable of inducing G4 formation in double stranded DNA and of influencing G4 formation in cells.

Simultaneously improving the strength and toughness of materials is a major challenge. Inorganic-polymer hybrids offer the potential to combine mechanical properties of a stiff inorganic glass with a flexible organic polymer. However, the toughening mechanism at the atomic scale remains largely unknown. Based on combined experimental and molecular dynamics simulation results, we find that the deformation and fracture behavior of hybrids are governed by noncovalent intermolecular interactions between polymer and silica networks rather than the breakage of covalent bonds. We then attempt three methods to improve the balance between strength and toughness of hybrids, namely the total inorganic/organic (I/O) weight ratio, the size of silica nanoparticles, and the ratio of -C-O vs -C-C bonds in the polymer chains. Specifically, for a hybrid with matched silica size and I/O ratio, we demonstrate optimized mechanical properties in terms of strength (1.75 MPa at breakage), degree of elongation at the fracture point (31%), toughness (219 kPa), hardness (1.08 MPa), as well as Young's modulus (3.0 MPa). We also demonstrate that this hybrid material shows excellent biocompatibility and ability to support cell attachment as well as proliferation. This supports the possible application of this material as a strong yet tough bone scaffold material.