Explore the vast range of titles available.

Protein nanoparticles are effective platforms for antigen presentation and targeting effector immune cells in vaccine development. Encapsulins are a class of protein-based microbial nanocompartments that self-assemble into icosahedral structures with external diameters ranging from 24 to 42 nm. Encapsulins from were designed to package bacterial RNA when produced in and were shown to have immunogenic and self-adjuvanting properties enhanced by this RNA. We genetically incorporated a 20-mer peptide derived from a mutant strain of the SARS-CoV-2 receptor binding domain (RBD) into the encapsulin protomeric coat protein for presentation on the exterior surface of the particle. This immunogen elicited conformationally-relevant humoral responses to the SARS-CoV-2 RBD. Immunological recognition was enhanced when the same peptide was presented in a heterologous prime/boost vaccination strategy using the engineered encapsulin and a previously reported variant of the PP7 virus-like particle, leading to the development of a selective antibody response against a SARS-CoV-2 RBD point mutant. While generating epitope-focused antibody responses is an interplay between inherent vaccine properties and B/T cells, here we demonstrate the use of orthogonal nanoparticles to fine-tune the control of epitope focusing.

Cryo-electron microscopy data analysis can yield multiple structures from a single heterogeneous dataset. Here, we show a workflow we used for the identification of a contaminant from a cryoEM grid without prior knowledge of protein sequence. We determined the tail structure of Escherichia phage YDC107 from only several thousand particles. The workflow combines high-resolution single-particle data processing with de novo model determination using ML-based methods. Structural analysis revealed that the central part of the phage tail has a C6 symmetry, however the overall symmetry of each segment is C3 due to dimerization of a flexible domain.Competing Interest StatementThe authors have declared no competing interest.

Nature has been shown to repeatedly employ proteinaceous containers spanning a broad range of length scales (i.e. approximately 10 to >2000 nm) to suit myriad biological functions including the propagation and infectious behavior of viruses, creating distinct microenvironments designed to facilitate specific metabolic processes, and the generation of metabolite storehouses for maintaining intracellular homeostasis. Such macromolecular cage assemblies are evolutionary marvels, forming highly symmetrical and monodisperse architectures in a hierarchical fashion from either singular or small subsets of structurally-related protein building blocks. In recognition of their vast diversity in terms of sizes, morphologies, physiochemical attributes, and dynamic functional behaviors, synthetic biologists have increasingly sought to repurpose naturally occurring protein containers for applications in a breadth of biotechnologically-relevant fields. Along these lines, this dissertation specifically focuses on the rational engineering of a recently discovered class of proteinaceous nanocontainers, referred to as encapsulins, in order to generate catalytically functional multienzyme nanoreactors.The first chapter provides general context for this dissertation by presenting a broad overview of select protein-based container structures found in nature, followed by several brief reviews of therapeutic, catalytic, and biomaterials applications for which these protein-based containers have been employed in recent decades. The second chapter describes efforts to rationally engineer the exterior surface of encapsulin nanocontainers derived from the hyperthermophilic bacterium Thermotoga maritima to present a series of solvent-exposed peptide interaction domains. T. maritima encapsulins (TmE) presenting external SpyCatcher covalent interaction domains were shown to capture up to 60 copies of an Escherichia coli dihydrofolate reductase (DHFR) variant enzyme both in vitro and in vivo. Surface-tethered DHFR enzymes maintained catalytic functionality with minimal deviations from their untethered Michaelis-Menten profiles. The third chapter expands upon the DHFR-decorated nanocontainers generated in chapter 2 to construct a bi-enzymatic nanoreactor metabolon in which the reduction of dihydrofolate by DHFR is used to fuel the demethylation of an aryl substrate by LigM, a tetrahydrofolate-dependent aryl-O-demethylase enzyme isolated from Sphingomonas paucimobilis SYK-6, which was encapsulated within the TmE lumen. The resulting bi-enzymatic nanoreactors were shown to be functional, though mutations previously used to enlarge the 5-fold symmetry pores natively distributed throughout the TmE shell were needed to facilitate efficient exchange of pathway metabolites between the interior and exterior spaces.The fourth chapter shifts focus to describe attempts to generate a novel in vitro cargo loading mechanism for TmE under benign solvent conditions by abrogating the native in vivo container assembly process using engineered steric obstructions. Recombinant fusion of a bulky protein domain to the lumen-oriented N-terminus of TmE was shown to prevent full container assembly, and subsequent proteolytic liberation of the TmE coat proteins resulted in rapid initiation of container assembly. However, cargo loading attempts in tandem with protease treatment proved unsuccessful, highlighting the need for refinement of the steric-based assembly design.The final chapter provides a general summary of the works presented in the preceding chapter. Additionally, several commentaries concerning the successes and failures of the nanocontainer engineering strategies employed in this dissertation are presented, along with general opinions pertaining to possible future directions within the field of nanocontainer engineering.

Contact with the air-water interface can bias the orientation of macromolecules during cryo-EM sample preparation, leading to uneven sample distribution, preferred orientation, and damage to the molecules of interest. To prevent this, we describe a method to encapsulate target proteins within highly hydrophilic, structurally homogeneous, and stable protein shells, which we refer to as \"nanocrates\" for this purpose. Here, we describe packaging, data acquisition, and reconstruction of three proof-of-principle examples, each illuminating a different aspect of the method: apoferritin (ApoF, demonstrating high-resolution), thyroglobulin (Tg, solving a known preferred orientation problem), and 7,8-dihydroneopterin aldolase (DHNA, a structure previously uncharacterized by cryo-EM).

Periodically-modulated potentials in the form of light fields have previously been applied to induce reversible phase transitions in dilute colloidal systems with long-range interactions. Here we investigate whether similar transitions can be induced in very dense systems, where inter-particle contacts are important. Using microscopy we show that particles in such systems are indeed strongly affected by modulated potentials. We discuss technical aspects relevant to generating the light-induced potentials and to imaging simultaneously the particles. We also consider what happens when the particle size is comparable with the modulation wavelength. The effects of selected modulation wavelengths as well as pure radiation pressure are illustrated.

Over the last decade, the light microscope has become increasingly useful as a quantitative tool for studying colloidal systems. The ability to obtain particle coordinates in bulk samples from micrographs is particularly appealing. In this paper we review and extend methods for optimal image formation of colloidal samples, which is vital for particle coordinates of the highest accuracy, and for extracting the most reliable coordinates from these images. We discuss in depth the accuracy of the coordinates, which is sensitive to the details of the colloidal system and the imaging system. Moreover, this accuracy can vary between particles, particularly in dense systems. We introduce a previously unreported error estimate and use it to develop an iterative method for finding particle coordinates. This individual-particle accuracy assessment also allows comparison between particle locations obtained from different experiments. Though aimed primarily at confocal microscopy studies of colloidal systems, the methods outlined here should transfer readily to many other feature extraction problems, especially where features may overlap one another.

A spatial light modulator (SLM) and a pair of galvanometer-mounted mirrors (GMM) were combined into an optical tweezers set-up. This provides great flexibility as the SLM creates an array of traps which can be moved smoothly and quickly with the GMM. To optimise performance, the effect of the incidence angle on the SLM with respect to phase and intensity response was investigated. Although it is common to use the SLM at an incidence angle of 45 degrees, smaller angles give a full 2pi phase shift and an output intensity which is less dependent on the magnitude of the phase shift. The traps were calibrated using an active oscillatory technique and a passive probability distribution method.

We identify putative load-bearing structures (bridges) in experimental colloidal systems studied by confocal microscopy. Bridges are co-operative structures that have been used to explain stability and inhomogeneous force transmission in simulated granular packings with a range of densities. We show that bridges similar to those found in granular simulations are present in real experimental colloidal packings. We describe critically the bridge-finding procedure for real experimental data and propose a new criterion-Lowest Mean Squared Separation (LSQS)-for selecting optimum stabilisations.

Agriculture is expanding into regions that are affected by salinity. This review considers the energetic costs of salinity tolerance in crop plants and provides a framework for a quantitative assessment of costs. Different sources of energy, and modifications of root system architecture that would maximize water vs ion up take are addressed. Energy requirements for transport of salt (NaCl) to leaf vacuoles for osmotic adjustment could be small if there are no substantial leaks back across plasma membrane and tonoplast in root and leaf. The coupling ratio of the H⁺ -ATPase also is a critical component. One proposed leak, that of Na⁺ influx across the plasma membrane through certain aquaporin channels, might be coupled to water flow, thus conserving energy. For the tonoplast, control of two types of cation channels is required for energy efficiency. Transporters controlling the Na⁺ and Cl⁻ concentrations in mitochondria and chloroplasts are largely unknown and could be a major energy cost. The complexity of the system will require a sophisticated modelling approach to identify critical transporters, apoplastic barriers and root structures. This modelling approach will inform experimentation and allow a quantitative assess ment of the energy costs of Na Cl tolerance to guide breeding and engineering of molecular components.

Evidence-based guidelines for implementation and measurement of antibiotic stewardship interventions in inpatient populations including long-term care were prepared by a multidisciplinary expert panel of the Infectious Diseases Society of America and the Society for Healthcare Epidemiology of America. The panel included clinicians and investigators representing internal medicine, emergency medicine, microbiology, critical care, surgery, epidemiology, pharmacy, and adult and pediatric infectious diseases specialties. These recommendations address the best approaches for antibiotic stewardship programs to influence the optimal use of antibiotics.