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The spread of bacterial resistance to antibiotics poses the need for antimicrobial discovery. With traditional search paradigms being exhausted, approaches that are altogether different from antibiotics may offer promising and creative solutions. Here, we introduce a de novo peptide topology that—by emulating the virus architecture—assembles into discrete antimicrobial capsids. Using the combination of high-resolution and real-time imaging, we demonstrate that these artificial capsids assemble as 20-nm hollow shells that attack bacterial membranes and upon landing on phospholipid bilayers instantaneously (seconds) convert into rapidly expanding pores causing membrane lysis (minutes). The designed capsids show broad antimicrobial activities, thus executing one primary function—they destroy bacteria on contact. With the growing threat of antibiotic resistance, unconventional approaches to antimicrobial discovery are needed. Here, the authors present a peptide topology that mimics virus architecture and assembles into antimicrobial capsids that disrupt bacterial membranes upon contact.

Antimicrobial resistance challenges the ability of modern medicine to contain infections. Given the dire need for new antimicrobials, polypeptide antibiotics hold particular promise. These agents hit multiple targets in bacteria starting with their most exposed regions—their membranes. However, suitable approaches to quantify the efficacy of polypeptide antibiotics at the membrane and cellular level have been lacking. Here, we employ two complementary microfluidic platforms to probe the structure–activity relationships of two experimental series of polypeptide antibiotics. We reveal strong correlations between each peptide’s physicochemical activity at the membrane level and biological activity at the cellular level. We achieve this knowledge by assaying the membranolytic activities of the compounds on hundreds of individual giant lipid vesicles, and by quantifying phenotypic responses within clonal bacterial populations with single-cell resolution. Our strategy proved capable of detecting differential responses for peptides with single amino acid substitutions between them, and can accelerate the rational design and development of peptide antimicrobials.

Three-dimensional cell culture systems underpin cell-based technologies ranging from tissue scaffolds for regenerative medicine to tumor models and organoids for drug screening. However, to realise the full potential of these technologies requires analytical methods able to capture the diverse information needed to characterize constituent cells, scaffold components and the extracellular milieu. Here we describe a multimodal imaging workflow which combines fluorescence, vibrational and second harmonic generation microscopy with secondary ion mass spectrometry imaging and transmission electron microscopy to analyse the morphological, chemical and ultrastructural properties of cell-seeded scaffolds. Using cell nuclei as landmarks we register fluorescence with label-free optical microscopy images and high mass resolution with high spatial resolution secondary ion mass spectrometry images, with an accuracy comparable to the intrinsic spatial resolution of the techniques. We apply these methods to investigate relationships between cell distribution, cytoskeletal morphology, scaffold fiber organisation and biomolecular composition in type I collagen scaffolds seeded with human dermal fibroblasts.

Nature constructs matter by employing protein folding motifs, many of which have been synthetically reconstituted to exploit function. A less understood motif whose structure-function relationships remain unexploited is formed by parallel β-strands arranged in a helical repetitive pattern, termed a β-helix. Herein we reconstitute a protein β-helix by design and endow it with biological function. Unlike β-helical proteins, which are contiguous covalent structures, this β-helix self-assembles from an elementary sequence of 18 amino acids. Using a combination of experimental and computational methods, we demonstrate that the resulting assemblies are discrete cylindrical structures exhibiting conserved dimensions at the nanoscale. We provide evidence for the structures to form a carpet-like three-dimensional scaffold promoting and inhibiting the growth of human and bacterial cells, respectively, while being able to mediate intracellular gene delivery. The study introduces a self-assembled β-helix as a self-contained bio- and multi-functional motif for exploring and exploiting mechanistic biology. The structure-function relationships of a β-helix, a folding motif formed by parallel β-strands arranged in a helical repetitive pattern, remain poorly understood and underexploited. Here, the authors reconstitute a protein β-helix by design from an elementary sequence of 18 amino acids, which self-assembles into a self-contained multifunctional motif exhibiting a range of biological functions.

Antimicrobial peptides are postulated to disrupt microbial phospholipid membranes. The prevailing molecular model is based on the formation of stable or transient pores although the direct observation of the fundamental processes is lacking. By combining rational peptide design with topographical (atomic force microscopy) and chemical (nanoscale secondary ion mass spectrometry) imaging on the same samples, we show that pores formed by antimicrobial peptides in supported lipid bilayers are not necessarily limited to a particular diameter, nor they are transient, but can expand laterally at the nano-to-micrometer scale to the point of complete membrane disintegration. The results offer a mechanistic basis for membrane poration as a generic physicochemical process of cooperative and continuous peptide recruitment in the available phospholipid matrix.

Synthetic DNA is of increasing demand across many sectors of research and commercial activities. Engineering biology, therapy, data storage and nanotechnology are set for rapid developments if DNA can be provided at scale and low cost. Stimulated by successes in next generation sequencing and gene editing technologies, DNA synthesis is already a burgeoning industry. However, the synthesis of >200 bp sequences remains unaffordable. To overcome these limitations and start writing DNA as effectively as it is read, alternative technologies have been developed including molecular assembly and cloning methods, template-independent enzymatic synthesis, microarray and rolling circle amplification techniques. Here, we review the progress in developing and commercializing these technologies, which are exemplified by innovations from leading companies. We discuss pros and cons of each technology, the need for oversight and regulatory policies for DNA synthesis as a whole and give an overview of DNA synthesis business models. There is increasing demand for synthetic DNA. However, our ability to make, or write, DNA lags behind our ability to sequence, or read, it. This Review discusses commercialized DNA synthesis technologies in the pursuit of closing the DNA writing gap.

We have established a designed system comprising two peptides that coassemble to form long, thickened protein fibers in water. This system can be rationally engineered to alter fiber assembly, stability, and morphology. Here, we show that rational mutations to our original peptide designs lead to structures with a remarkable level of order on the nanoscale that mimics certain natural fibrous assemblies. In the engineered system, the peptides assemble into two-stranded α-helical coiled-coil rods, which pack in axial register in a 3D hexagonal lattice of size 1.824 nm, and with a periodicity of 4.2 nm along the fiber axis. This model is supported by both electron microscopy and x-ray diffraction. Specifically, the fibers display surface striations separated by nanoscale distances that precisely match the 4.2-nm length expected for peptides configured as α-helices as designed. These patterns extend unbroken across the widths (>=50 nm) and lengths (>10 μm) of the fibers. Furthermore, the spacing of the striations can be altered predictably by changing the length of the peptides. These features reflect a high level of internal order within the fibers introduced by the peptide-design process. To our knowledge, this exceptional order, and its persistence along and across the fibers, is unique in a biomimetic system. This work represents a step toward rational bottom-up assembly of nanostructured fibrous biomaterials for potential applications in synthetic biology and nanobiotechnology.

Biocompatible surfaces hold key to a variety of biomedical problems that are directly related to the competition between host-tissue cell integration and bacterial colonisation. A saving solution to this is seen in the ability of cells to uniquely respond to physical cues on such surfaces thus prompting the search for cell-instructive nanoscale patterns. Here we introduce a generic rationale engineered into biocompatible, titanium, substrates to differentiate cell responses. The rationale is inspired by cicada wing surfaces that display bactericidal nanopillar patterns. The surfaces engineered in this study are titania (TiO 2 ) nanowire arrays that are selectively bactericidal against motile bacteria, while capable of guiding mammalian cell proliferation according to the type of the array. The concept holds promise for clinically relevant materials capable of differential physico-mechanical responses to cellular adhesion.

Regenerative medicine has become an extremely valuable tool offering an alternative to conventional therapies for the repair and regeneration of tissues. The re-establishment of tissue and organ functions can be carried out by tissue engineering strategies or by using medical devices such as implants. However, with any material being implanted inside the human body, one of the conundrums that remains is the ease with which these materials can get contaminated by bacteria. Bacterial adhesion leads to the formation of mature, alive and complex three-dimensional biofilm structures, further infection of surrounding tissues and consequent development of complicated chronic infections. Hence, novel tissue engineering strategies delivering biofilm-targeted therapies, while at the same time allowing tissue formation are highly relevant. In this study our aim was to develop surface modified polyhydroxyalkanoate-based fiber meshes with enhanced bacterial anti-adhesive and juvenile biofilm disrupting properties for tissue regeneration purposes. Using reactive and amphiphilic star-shaped macromolecules as an additive to a polyhydroxyalkanoate spinning solution, a synthetic antimicrobial peptide, Amhelin, with strong bactericidal and anti-biofilm properties, and Dispersin B, an enzyme promoting the disruption of exopolysaccharides found in the biofilm matrix, were covalently conjugated to the fibers by addition to the solution before the spinning process. is one of the most problematic pathogens responsible for tissue-related infections. The initial antibacterial screening showed that Amhelin proved to be strongly bactericidal at 12 μg/ml and caused >50% reductions of biofilm formation at 6 μg/ml, while Dispersin B was found to disperse >70% of pre-formed biofilms at 3 μg/ml. Regarding the cytotoxicity of the agents toward L929 murine fibroblasts, a CC of 140 and 115 μg/ml was measured for Amhelin and Dispersin B, respectively. Optimization of the electrospinning process resulted in aligned fibers. Surface activated fibers with Amhelin and Dispersin B resulted in 83% reduction of adhered bacteria on the surface of the fibers. Additionally, the materials developed were found to be cytocompatible toward L929 murine fibroblasts. The strategy reported in this preliminary study suggests an alternative approach to prevent bacterial adhesion and, in turn biofilm formation, in materials used in regenerative medicine applications such as tissue engineering.

Electron microscopy plays an important role in the analysis of functional nano-to-microstructures. Substrates and staining procedures present common sources of variation for the analysis. However, systematic investigations on the impact of these sources on data interpretation are lacking. Here we pinpoint key determinants associated with reproducibility issues in the imaging of archetypal protein assemblies, protein shells, and filaments. The effect of staining on the morphological characteristics of the assemblies was assessed to reveal differential features for anisotropic (filaments) and isotropic (shells) forms. Commercial substrates and coatings under the same staining conditions gave comparable results for the same model assembly, while highlighting intrinsic sample variations including the density and heterogenous distribution of assemblies on the substrate surface. With no aberrant or disrupted structures observed, and putative artefacts limited to substrate-associated markings, the study emphasizes that reproducible imaging must correlate with an optimal combination of substrate stability, stain homogeneity, accelerating voltage, and magnification.