Directed Evolution of Acoustic Reporter Genes Using High-Throughput Acoustic Screening.

A major challenge in the fields of biological imaging and synthetic biology is noninvasively visualizing the functions of natural and engineered cells inside opaque samples such as living animals. One promising technology that addresses this limitation is ultrasound (US), with its penetration depth of several cm and spatial resolution on the order of 100 µm. Within the past decade, reporter genes for US have been introduced and engineered to link cellular functions to US signals via heterologous expression in commensal bacteria and mammalian cells. These acoustic reporter genes (ARGs) represent a novel class of genetically encoded US contrast agent, and are based on air-filled protein nanostructures called gas vesicles (GVs). Just as the discovery of fluorescent proteins was followed by the improvement and diversification of their optical properties through directed evolution, here we describe the evolution of GVs as acoustic reporters. To accomplish this task, we establish high-throughput, semiautomated acoustic screening of ARGs in bacterial cultures and use it to screen mutant libraries for variants with increased nonlinear US scattering. Starting with scanning site saturation libraries for two homologues of the primary GV structural protein, GvpA/B, two rounds of evolution resulted in GV variants with 5- and 14-fold stronger acoustic signals than the parent proteins. We anticipate that this and similar approaches will help high-throughput protein engineering play as large a role in the development of acoustic biomolecules as it has for their fluorescent counterparts.

Directed Evolution of Acoustic Reporter Genes Using High-Throughput Acoustic Screening.

A major challenge in the fields of biological imaging and synthetic biology is noninvasively visualizing the functions of natural and engineered cells inside opaque samples such as living animals. One promising technology that addresses this limitation is ultrasound (US), with its penetration depth of several cm and spatial resolution on the order of 100 µm. 1 Within the past decade, reporter genes for US have been introduced 2,3 and engineered 4,5 to link cellular functions to US signals via heterologous expression in commensal bacteria and mammalian cells. These acoustic reporter genes (ARGs) represent a novel class of genetically encoded US contrast agent, and are based on air-filled protein nanostructures called gas vesicles (GVs). 6 Just as the discovery of fluorescent proteins was followed by the improvement and diversification of their optical properties through directed evolution, here we describe the evolution of GVs as acoustic reporters. To accomplish this task, we establish high-throughput, semi-automated acoustic screening of ARGs in bacterial cultures and use it to screen mutant libraries for variants with increased nonlinear US scattering. Starting with scanning site saturation libraries for two homologs of the primary GV structural protein, GvpA/B, two rounds of evolution resulted in GV variants with 5- and 14-fold stronger acoustic signals than the parent proteins. We anticipate that this and similar approaches will help high-throughput protein engineering play as large a role in the development of acoustic biomolecules as it has for their fluorescent counterparts.

Bioorthogonal Labeling Enables In Situ Fluorescence Imaging of Expressed Gas Vesicle Nanostructures.

Gas vesicles (GVs) are proteinaceous nanostructures that, along with virus-like particles, encapsulins, nanocages, and other macromolecular assemblies, are being developed for potential biomedical applications. To facilitate such development, it would be valuable to characterize these nanostructures' subcellular assembly and localization. However, traditional fluorescent protein fusions are not tolerated by GVs' primary constituent protein, making optical microscopy a challenge. Here, we introduce a method for fluorescently visualizing intracellular GVs using the bioorthogonal label FlAsH, which becomes fluorescent upon reaction with the six-amino acid tetracysteine (TC) tag. We engineered the GV subunit protein, GvpA, to display the TC tag and showed that GVs bearing TC-tagged GvpA can be successfully assembled and fluorescently visualized in HEK 293T cells. Importantly, this was achieved by replacing only a fraction of GvpA with the tagged version. We used fluorescence images of the tagged GVs to study the GV size and distance distributions within these cells. This bioorthogonal and fractional labeling approach will enable research to provide a greater understanding of GVs and could be adapted to similar proteinaceous nanostructures.

Bioorthogonal labeling enables in situ fluorescence imaging of expressed gas vesicle nanostructures.

Gas vesicles (GVs) are proteinaceous nanostructures that, along with virus-like particles, encapsulins, nano-cages, and other macromolecular assemblies are being developed for potential biomedical applications. To facilitate such development, it would be valuable to characterize these nanostructures' sub-cellular assembly and localization. However, traditional fluorescent protein fusions are not tolerated by GVs' primary constituent protein, making optical microscopy a challenge. Here, we introduce a method for fluorescently visualizing intracellular GVs using the bioorthogonal label FlAsH, which becomes fluorescent upon binding the six-amino acid tetracysteine (TC) tag. We engineered the GV subunit protein, GvpA, to display the TC tag, and showed that GVs bearing TC-tagged GvpA can be successfully assembled and fluorescently visualized in HEK 293T cells. We used fluorescence images of the tagged GVs to study GV size and distance distributions within these cells. This bioorthogonal labeling approach will enable research to provide a greater understanding of GVs and could be adapted to similar proteinaceous nanostructures.

Ultrasonic reporters of calcium for deep tissue imaging of cellular signals.

Calcium imaging has enabled major biological discoveries. However, the scattering of light by tissue limits the use of standard fluorescent calcium indicators in living animals. To address this limitation, we introduce the first genetically encoded ultrasonic reporter of calcium (URoC). Based on a unique class of air-filled protein nanostructures called gas vesicles, we engineered URoC to produce elevated nonlinear ultrasound signal upon binding to calcium ions. With URoC expressed in mammalian cells, we demonstrate noninvasive ultrasound imaging of calcium signaling in vivo during drug-induced receptor activation. URoC brings the depth and resolution advantages of ultrasound to the in vivo imaging of dynamic cellular function and paves the way for acoustic biosensing of a broader variety of biological signals.

Structure of Anabaena flos-aquae gas vesicles revealed by cryo-ET.

Gas vesicles (GVs) are gas-filled protein nanostructures employed by several species of bacteria and archaea as flotation devices to enable access to optimal light and nutrients. The unique physical properties of GVs have led to their use as genetically encodable contrast agents for ultrasound and MRI. Currently, however, the structure and assembly mechanism of GVs remain unknown. Here we employ cryoelectron tomography to reveal how the GV shell is formed by a helical filament of highly conserved GvpA subunits. This filament changes polarity at the center of the GV cylinder, a site that may act as an elongation center. Subtomogram averaging reveals a corrugated pattern of the shell arising from polymerization of GvpA into a ß sheet. The accessory protein GvpC forms a helical cage around the GvpA shell, providing structural reinforcement. Together, our results help explain the remarkable mechanical properties of GVs and their ability to adopt different diameters and shapes.

Genomically mined acoustic reporter genes for real-time in vivo monitoring of tumors and tumor-homing bacteria.

Ultrasound allows imaging at a much greater depth than optical methods, but existing genetically encoded acoustic reporters for in vivo cellular imaging have been limited by poor sensitivity, specificity and in vivo expression. Here we describe two acoustic reporter genes (ARGs)-one for use in bacteria and one for use in mammalian cells-identified through a phylogenetic screen of candidate gas vesicle gene clusters from diverse bacteria and archaea that provide stronger ultrasound contrast, produce non-linear signals distinguishable from background tissue and have stable long-term expression. Compared to their first-generation counterparts, these improved bacterial and mammalian ARGs produce 9-fold and 38-fold stronger non-linear contrast, respectively. Using these new ARGs, we non-invasively imaged in situ tumor colonization and gene expression in tumor-homing therapeutic bacteria, tracked the progression of tumor gene expression and growth in a mouse model of breast cancer, and performed gene-expression-guided needle biopsies of a genetically mosaic tumor, demonstrating non-invasive access to dynamic biological processes at centimeter depth.

Focused ultrasound excites cortical neurons via mechanosensitive calcium accumulation and ion channel amplification.

Ultrasonic neuromodulation has the unique potential to provide non-invasive control of neural activity in deep brain regions with high spatial precision and without chemical or genetic modification. However, the biomolecular and cellular mechanisms by which focused ultrasound excites mammalian neurons have remained unclear, posing significant challenges for the use of this technology in research and potential clinical applications. Here, we show that focused ultrasound excites primary murine cortical neurons in culture through a primarily mechanical mechanism mediated by specific calcium-selective mechanosensitive ion channels. The activation of these channels results in a gradual build-up of calcium, which is amplified by calcium- and voltage-gated channels, generating a burst firing response. Cavitation, temperature changes, large-scale deformation, and synaptic transmission are not required for this excitation to occur. Pharmacological and genetic inhibition of specific ion channels leads to reduced responses to ultrasound, while over-expressing these channels results in stronger ultrasonic stimulation. These findings provide a mechanistic explanation for the effect of ultrasound on neurons to facilitate the further development of ultrasonic neuromodulation and sonogenetics as tools for neuroscience research.

Controlled Release of Molecular Intercalants from Two-Dimensional Nanosheet Films.

Solution co-deposition of two-dimensional (2D) nanosheets with chemical solutes yields nanosheet-molecular heterostructures. A feature of these macroscopic layered hybrids is their ability to release the intercalated molecular agent to express chemical functionality on their surfaces or in their near surroundings. Systematic design methods are needed to control this molecular release to match the demand for rate and lifetime in specific applications. We hypothesize that release kinetics are controlled by transport processes within the layered solids, which primarily involve confined molecular diffusion through nanochannels formed by intersheet van der Waals gaps. Here a variety of graphene oxide (GO)/molecular hybrids are fabricated and subject to transient experiments to characterize release kinetics, locations, and mechanisms. The measured release rate profiles can be successfully described by a numerical model of internal transport processes, and the results used to extract effective Z-directional diffusion coefficients for various film types. The diffusion coefficients are found to be 8 orders of magnitude lower than those in free solution due to nanochannel confinement and serpentine path effects, and this retardation underlies the ability of 2D materials to control and extend release over useful time scales. In-plane texturing of the heterostructured films by compressive wrinkling or crumpling is shown to be a useful design tool to control the release rate for a given film type and molecular intercalant. The potential of this approach is demonstrated through case studies on the controlled release of chemical virucidal agents.

Acoustically triggered mechanotherapy using genetically encoded gas vesicles.

Recent advances in molecular engineering and synthetic biology provide biomolecular and cell-based therapies with a high degree of molecular specificity, but limited spatiotemporal control. Here we show that biomolecules and cells can be engineered to deliver potent mechanical effects at specific locations inside the body through ultrasound-induced inertial cavitation. This capability is enabled by gas vesicles, a unique class of genetically encodable air-filled protein nanostructures. We show that low-frequency ultrasound can convert these biomolecules into micrometre-scale cavitating bubbles, unleashing strong local mechanical effects. This enables engineered gas vesicles to serve as remotely actuated cell-killing and tissue-disrupting agents, and allows genetically engineered cells to lyse, release molecular payloads and produce local mechanical damage on command. We demonstrate the capabilities of biomolecular inertial cavitation in vitro, in cellulo and in vivo, including in a mouse model of tumour-homing probiotic therapy.

Measuring gas vesicle dimensions by electron microscopy.

Gas vesicles (GVs) are cylindrical or spindle-shaped protein nanostructures filled with air and used for flotation by various cyanobacteria, heterotrophic bacteria, and Archaea. Recently, GVs have gained interest in biotechnology applications due to their ability to serve as imaging agents and actuators for ultrasound, magnetic resonance and several optical techniques. The diameter of GVs is a crucial parameter contributing to their mechanical stability, buoyancy function and evolution in host cells, as well as their properties in imaging applications. Despite its importance, reported diameters for the same types of GV differ depending on the method used for its assessment. Here, we provide an explanation for these discrepancies and utilize electron microscopy (EM) techniques to accurately estimate the diameter of the most commonly studied types of GVs. We show that during air drying on the EM grid, GVs flatten, leading to a ~1.5-fold increase in their apparent diameter. We demonstrate that GVs' diameter can be accurately determined by direct measurements from cryo-EM samples or alternatively indirectly derived from widths of flat collapsed and negatively stained GVs. Our findings help explain the inconsistency in previously reported data and provide accurate methods to measure GVs dimensions.

Controlling pore structure and conductivity in graphene nanosheet films through partial thermal exfoliation.

Thermal exfoliation is an efficient and scalable method for the production of graphene nanosheets or nanoplatelets, which are typically re-assembled or blended to form new macroscopic "graphene-based materials". Thermal exfoliation can be applied to these macroscopic graphene-based materials after casting to create internal porosity, but this process variant has not been widely studied, and can easily lead to destruction of the physical form of the original cast body. Here we explore how the partial thermal exfoliation of graphene oxide (GO) multilayer nanosheet films can be used to control pore structure and electrical conductivity of planar, textured, and confined GO films. The GO films are shown to exfoliate explosively when the instrument-set heating rates are 100 K/min and above leading to complete destruction of the film geometry. Textured films with engineered micro-wrinkling and crumpling show similar thermal behavior to planar films. Here, we also demonstrate a novel method to produce fairly large size intact rGO films of high electrical conductivity and microporosity based on confinement. Sandwiching GO precursor films between inert plates during partial exfoliation at 250°C produces high conductivity and porosity material in the form of a flexible film that preserves the macroscopic structure of the original cast body.

Emergency preparation and mitigation for COVID-19 response in an integrated pharmacy practice model.

PURPOSE: The purpose of this descriptive report is to share experiences in crisis response planning and risk mitigation at a university health system department of pharmacy with an integrated clinical practice model in the early months of the coronarvirus disease 2019 (COVID-19) pandemic. SUMMARY: The department of pharmacy's COVID-19 pandemic response included successful planning and implementation of measures to maintain pharmacy operations and minimize COVID-19 exposure of patients and staff. These measures included ensuring adequate personnel staffing using flexible staffing solutions, ongoing assessment of supply chain integrity, and continuation of integrated clinical pharmacy services 24/7 throughout the initial phase of the COVID-19 pandemic. Information technology (IT) and educational program modifications are also discussed. CONCLUSION: This report describes successful crisis planning and risk mitigation in the setting of COVID-19, which was facilitated by the department of pharmacy's integrated clinical practice model. This model enabled uninterrupted personnel scheduling, supply chain integrity, continued provision of 24/7 integrated clinical services, adaptive use of IT tools, and continuation of educational programs. The experiences described may be instructive to other pharmacy departments in evaluating their response to the COVID-19 pandemic and in planning for similar pandemic or other emergency scenarios.

Manganese dioxide nanosheets induce mitochondrial toxicity in fish gill epithelial cells.

The development and production of engineered 2D nanomaterials are expanding exponentially, increasing the risk of their release into the aquatic environment. A recent study showed 2D MnO2 nanosheets, under development for energy and biomedical applications, dissolve upon interaction with biological reducing agents, resulting in depletion of intracellular glutathione levels within fish gill cells. However, little is known concerning their toxicity and interactions with subcellular organelles. To address this gap, we examined cellular uptake, cytotoxicity and mitochondrial effects of 2D MnO2 nanosheets using an in vitro fish gill cell line to represent a target tissue of rainbow trout, a freshwater indicator species. The data demonstrate cellular uptake of MnO2 nanosheets into lysosomes and potential mechanisms of dissolution within the lysosomal compartment. MnO2 nanosheets induced severe mitochondrial dysfunction at sub-cytotoxic doses. Quantitative, single cell fluorescent imaging revealed mitochondrial fission and impaired mitochondrial membrane potential following MnO2 nanosheet exposure. Seahorse analyses for cellular respiration revealed that MnO2 nanosheets inhibited basal respiration, maximal respiration and the spare respiratory capacity of gill cells, indicating mitochondrial dysfunction and reduced cellular respiratory activity. MnO2 nanosheet exposure also inhibited ATP production, further supporting the suppression of mitochondrial function and cellular respiration. Together, these observations indicate that 2D MnO2 nanosheets impair the ability of gill cells to respond to energy demands or prolonged stress. Finally, our data demonstrate significant differences in the toxicity of the 2D MnO2 nanosheets and their microparticle counterparts. This exemplifies the importance of considering the unique physical characteristics of 2D nanomaterials when conducting safety assessments.

Controlling nanochannel orientation and dimensions in graphene-based nanofluidic membranes.

There is great interest in exploiting van der Waals gaps in layered materials as nanofluidic channels. Graphene oxide (GO) nanosheets are known to spontaneously assemble into stacked planar membranes with transport properties that are highly selective to molecular structure. Use of conventional GO membranes in liquid-phase applications is often limited by low flux values, due to intersheet nanochannel alignment perpendicular to the desired Z-directional transport, which leads to circuitous fluid pathways that are orders of magnitude longer than the membrane thickness. Here we demonstrate an approach that uses compressive instability in Zr-doped GO thin films to create wrinkle patterns that rotate nanosheets to high angles. Capturing this structure in polymer matrices and thin sectioning produce fully dense membranes with arrays of near-vertically aligned nanochannels. These robust nanofluidic devices offer pronounced reduction in fluid path-length, while retaining the high selectivity for water over non-polar molecules characteristic of GO interlayer nanochannels.

An all-inorganic, fully dense, stretchable ceramic magnetic film.

There is widespread interest in new materials-based approaches for introducing flexibility to electromagnetic devices, such as displays, human-machine interfaces, smart textiles, and biomedical implants. From fabrication to application, incorporating ceramic components is particularly challenging due to their extreme stiffness. Here, we introduce a new approach for designing flexible ceramic films and demonstrate it by fabricating fully dense, pre-wrinkled magnetic cobalt ferrite films composed of tiled nanoplatelets. The method relies on the colloidal engineering of metalized graphene nanosheets, which are cast and compressed into wrinkled composite films with accurate control of composition and morphology. Removal of the graphene template by thermal oxidation yields free-standing cobalt ferrite films that can be stretched up to 200% and bent to radii of 2.5 mm while maintaining their magnetic properties. Magnetization retention of 73% is documented after 150% linear mechanical stretching over 100 cycles. The significant stretchability and flexibility in this hard magnetic material is achieved at near full metal oxide crystal density without addition of significant void space or a polymeric elastomer matrix.

A graphene-based hydrogel monolith with tailored surface chemistry for PFAS passive sampling.

Aquatic contamination by per- and polyfluorinated alkyl substances (PFAS) has attracted global attention due to their environmental and health concerns. Current health advisories and surface water regulatory limits require PFAS detection in the parts per trillion (ppt) range. One way to achieve those low detection limits is to use a reliable passive sampling-based monitoring tool for PFAS, as exists for numerous nonpolar persistent organic pollutants. Here we introduce a new graphene-based hydrogel monolith and describe its synthesis, chemical functionalization, property characterization, and testing as a PFAS equilibrium passive sampler. The graphene monoliths were self-assembled by hydrothermal treatment from graphene oxide (GO) aqueous dispersions to produce free standing cylinders of ~563 mm3 volume consisting of ~4 wt-% thin-walled porous graphene and ~96 wt-% water. The uptake of 23 PFAS was measured on the as-produced monoliths, and equilibrium partition coefficients (KSW), were derived for longer chain (C≥8) perfluoroalkyl acids (PFAA) and neutral precursors such as sulfonamides (log KSW range 1.9 - 3.6). To increase the KSW for shorter chain PFAA, the monoliths were chemically modified by a new diazonium-based grafting reaction that introduces positive surface charge without damage to the graphenic backbone. Introduction of benzylamine moieties through the diazonium intermediate switches zeta potential at pH 7 from -45mV (as-produced graphene) to + 5mV. This modification increased the sorption of short and middle chain PFAA by ten-fold (e.g. log KSW for PFBA increased from 1.3 to 2.2), thereby improving the functionality of the passive sampler device for a wider range of PFAS. Field deployments demonstrated that the graphene monoliths were capable of detecting key PFAS in the Delaware River.

Shear Failure in Supported Two-Dimensional Nanosheet Van der Waals Thin Films.

Liquid-phase deposition of exfoliated 2D nanosheets is the basis for emerging technologies that include writable electronic inks, molecular barriers, selective membranes, and protective coatings against fouling or corrosion. These nanosheet thin films have complex internal structures that are discontinuous assemblies of irregularly tiled micron-scale sheets held together by van der Waals (vdW) forces. On stiff substrates, nanosheet vdW films are stable to many common stresses, but can fail by internal delamination under shear stress associated with handling or abrasion. This "re-exfoliation" pathway is an intrinsic feature of stacked vdW films and can limit nanosheet-based technologies. Here we investigate the shear stability of graphene oxide and MoSe2 nanosheet vdW films through lap shear experiments on polymer-nanosheet-polymer laminates. These sandwich laminate structures fail in mixed cohesive and interfacial mode with critical shear forces from 40 - 140 kPa and fracture energies ranging from 0.2 - 6 J/m2. Surprisingly these energies are higher than delamination energies reported for smooth peeling of ordered stacks of continuous 2D sheets, which we propose is due to energy dissipation and chaotic crack motion during nanosheet film disassembly at the crack tip. Experiment results also show that film thickness plays a key role in determining critical shear force (maximum load before failure) and dissipated energy for different nanosheet vdW films. Using a mechanical model with an edge crack in the thin nanosheet film, we propose a shear-to-tensile failure mode transition to explain a maximum in critical shear force for graphene oxide films but not MoSe2 films. This transition reflects a weakening of the substrate confinement effect and increasing rotational deformation near the film edge as the film thickness increases. For graphene oxide, the critical shear force can be increased by electrostatic cross-linking achieved through interlayer incorporation of metal cations. These results have important implications for the stability of functional devices that employ 2D nanosheet coatings.

Ultrasound Technologies for Imaging and Modulating Neural Activity.

Visualizing and perturbing neural activity on a brain-wide scale in model animals and humans is a major goal of neuroscience technology development. Established electrical and optical techniques typically break down at this scale due to inherent physical limitations. In contrast, ultrasound readily permeates the brain, and in some cases the skull, and interacts with tissue with a fundamental resolution on the order of 100 µm and 1 ms. This basic ability has motivated major efforts to harness ultrasound as a modality for large-scale brain imaging and modulation. These efforts have resulted in already-useful neuroscience tools, including high-resolution hemodynamic functional imaging, focused ultrasound neuromodulation, and local drug delivery. Furthermore, recent breakthroughs promise to connect ultrasound to neurons at the genetic level for biomolecular imaging and sonogenetic control. In this article, we review the state of the art and ongoing developments in ultrasonic neurotechnology, building from fundamental principles to current utility, open questions, and future potential.