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Versatile DNA and polypeptide-based structures have been designed based on complementary modules. However, polypeptides can also form higher oligomeric states. We investigated the introduction of tetrameric modules as a substitute for coiled-coil dimerization units used in previous modular nanostructures. Tetramerizing helical bundles can run in parallel or antiparallel orientation, expanding the number of topological solutions for modular nanostructures. Furthermore, this strategy facilitates the construction of nanostructures from two identical polypeptide chains. Importantly, tetrameric modules substantially stabilized protein nanostructures against air-water interface denaturation, enabling the determination of the first cryo-electron microscopy three-dimensional structure of a coiled-coil-based nanostructure, confirming the designed agreement of the modules forming a tetrahedral cage., (© 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Gas vesicles (GVs) are large cylindrical gas-filled protein assemblies found in diverse aquatic bacteria that enable their adaptation of buoyancy. GVs have already been used as ultrasound contrasting agents. Here, we investigate GVs derived from Bacillus megaterium , aiming to minimize the number of accessory Gvps within the GV gene cluster and demonstrate the use of GVs as enhancers of acoustic radiation force administered by ultrasound. Three ( GvpR , GvpT , and GvpU ) out of 11 genes in the cluster were found to be dispensable for functional GV formation, and their omission resulted in narrower GVs. Two essential proteins GvpJ and GvpN were absent from recently determined GV structures, but GvpJ was nevertheless found to be tightly bound to the cylindrical part of GVs in this study. Additionally, the N-terminus of GvpN was observed to play an important role in the formation of mature GVs. The binding of engineered GvpC from Anabaena flos-aquae to HEK293 cells via integrins enhanced the acoustic force delivered by ultrasound and resulted in an increased Ca 2+ influx into cells. Coupling with a synthetic Ca 2+ -dependent signaling pathway GVs efficiently enhanced cell stimulation by ultrasound, which expands the potentials of noninvasive sonogenetics cell stimulation.

The delivery of vaccines plays a pivotal role in influencing the strength and longevity of the immune response and controlling reactogenicity. Mucosal immunization, as compared to parenteral vaccination, could offer greater protection against respiratory infections while being less invasive. While oral vaccination has been presumed less effective and believed to target mainly the gastrointestinal tract, trans-buccal delivery using mucoadhesive films (MAF) may allow targeted delivery to the mucosa. Here we present an effective strategy for mucosal delivery of several vaccine platforms incorporated in MAF, including DNA plasmids, viral vectors, and lipid nanoparticles incorporating mRNA (mRNA/LNP). The mRNA/LNP vaccine formulation targeting SARS-CoV-2 as a proof of concept remained stable within MAF consisting of slowly releasing water-soluble polymers and an impermeable backing layer, facilitating enhanced penetration into the oral mucosa. This formulation elicited antibody and cellular responses comparable to the intramuscular injection, but also induced the production of mucosal IgAs, highlighting its efficacy, particularly for use as a booster vaccine and the potential advantage for protection against respiratory infections. The MAF vaccine preparation demonstrates significant advantages, such as efficient delivery, stability, and simple noninvasive administration with the potential to alleviate vaccine hesitancy., Competing Interests: Declaration of competing interest RJ, HE, DL, KM and ŠM are contributors to the patent application describing the invention in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Membraneless liquid compartments based on phase-separating biopolymers have been observed in diverse cell types and attributed to weak multivalent interactions predominantly based on intrinsically disordered domains. The design of liquid-liquid phase separated (LLPS) condensates based on de novo designed tunable modules that interact in a well-understood, controllable manner could improve our understanding of this phenomenon and enable the introduction of new features. Here we report the construction of CC-LLPS in mammalian cells, based on designed coiled-coil (CC) dimer-forming modules, where the stability of CC pairs, their number, linkers, and sequential arrangement govern the transition between diffuse, liquid and immobile condensates and are corroborated by coarse-grained molecular simulations. Through modular design, we achieve multiple coexisting condensates, chemical regulation of LLPS, condensate fusion, formation from either one or two polypeptide components or LLPS regulation by a third polypeptide chain. These findings provide further insights into the principles underlying LLPS formation and a design platform for controlling biological processes., (© 2023. The Author(s).)

As the causal agent of the grapevine yellows disease Bois noir, ' Candidatus Phytoplasma solani' has a major economic impact on grapevines. To improve the control of Bois noir, it is critical to understand the very complex epidemiological cycles that involve the multiple " Ca . P. solani" host plants and insect vectors, of which Hyalesthes obsoletus is the most important. In the present study, multiple genotyping of the tuf , secY, stamp , and vmp1 genes was performed. This involved archived grapevine samples that were collected during an official survey of grapevine yellows throughout the wine-growing regions of Slovenia (from 2003 to 2016), plus samples from Austrian grapevines, stinging nettle, field bindweed, and insect samples (collected from 2012 to 2019). The data show that the tuf-b2 type of the tuf gene has been present in eastern Slovenia since at least 2003. The hypotheses that the occurrence of the haplotypes varies due to the geographical position of Slovenia on the Italian-Slovenian Karst divide and that the haplotypes are similar between Slovenian and Austrian Styria were confirmed. The data also show haplotype changes for host plants and H . obsoletus associated with ' Ca . P. solani,' which might be linked to new epidemiological cycles of this phytoplasma that involve not just new plant sources and new insect vectors, but also climate and land-use changes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mehle, Kavčič, Mermal, Vidmar, Pompe Novak, Riedle-Bauer, Brader, Kladnik and Dermastia.)

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and β-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small β-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The β-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the β-annulus peptide for vaccine design.

Objective: Congenital heart valve disease is one of the most common abnormalities in children. There are limited technological solutions available for treating children with congenital heart valve diseases. The aim of this study is to provide the details of the consensus reached in terms of pediatric definitions, design approach, in vitro testing, and clinical trials, which may be used as guidance for developing prosthetic heart valves for the pediatric indication., Methods: In stark contrast to the various designs of adult-sized replacement valves available in the market, there are no Food and Drug Administration (FDA)-approved prosthetic heart valves available for use in the pediatric population. There is a pressing need for FDA-approved pediatric valve devices in the United States. The pediatric patient population has been typically excluded from replacement heart valve trials for several reasons. In January 2010, heart valve manufacturers and pediatric clinicians collaborated with academicians and FDA staff in a workshop to suggest ways to successfully evaluate pediatric prosthetic valves and conduct pediatric clinical trials to provide acceptable heart valve replacement options for this patient population., Results: Recommendations, derived from ISO 5840:2005 and the 2010 FDA Draft Replacement Heart Valve Guidance, are provided for hydrodynamic, durability, and fatigue testing., Conclusions: The article specifically addresses in vitro and premarket and postmarket approval clinical studies that should be considered by a heart valve manufacturer for obtaining regulatory approval of pediatric sizes of prosthetic heart valve designs that are already approved for adult clinical use., (Copyright © 2013 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)