Zhiyang Jin

Protein kinases are an essential class of enzymes that regulate cellular signaling pathways, with their dysregulation implicated in pathologies such as cancer and neurodegenerative diseases. Despite the existence of high-performance fluorescent biosensors of kinase activity, it remains challenging to study the function and regulatory pathways of kinases in opaque tissues due to the limited tissue penetration of light. To address this limitation, we report an ultrasonic reporter of kinase activity (UReKA) designed to detect protein kinase A (PKA) activity by altering the ultrasound scattering of gas vesicles (GVs), a unique class of air-filled protein nanostructures. We engineer a GV shell protein to respond to PKA, demonstrate the functionality of UReKA both in purified protein format and expressed in mammalian cells, and showcase its capacity to monitor PKA signaling in response to pharmacological stimulation or genetic mutations. This approach provides a means to visualize cellular functional activity in opaque media, with broad potential for future applications in cancer biology, cellular development, and drug discovery.

Protein kinases are an essential class of enzymes that regulate cellular signaling pathways, with their dysregulation implicated in pathologies such as cancer and neurodegenerative diseases. Despite the existence of high-performance fluorescent biosensors of kinase activity, it remains challenging to study the function and regulation pathways of kinases in opaque tissues due to the limited tissue penetration of light. To address this limitation, we introduce the first ultrasonic reporter of kinase activity (UReKA), designed to detect protein kinase A (PKA) activity by altering the ultrasound scattering of gas vesicles, a unique class of air-filled protein nanostructures. We engineer a gas vesicle shell protein to respond to PKA, demonstrate the functionality of UReKA both in purified protein format and expressed in mammalian cells, and showcase its capacity to monitor PKA signaling in response to pharmacological stimulation or genetic mutation. This work makes it possible to visualize cellular functional activity in opaque media, with broad potential for future applications in cancer biology, cellular development, and drug discovery.

Three-dimensional printing offers promise for patient-specific implants and therapies but is often limited by the need for invasive surgical procedures. To address this, we developed an imaging-guided deep tissue in vivo sound printing (DISP) platform. By incorporating cross-linking agent–loaded low-temperature–sensitive liposomes into bioinks, DISP enables precise, rapid, on-demand cross-linking of diverse functional biomaterials using focused ultrasound. Gas vesicle–based ultrasound imaging provides real-time monitoring and allows for customized pattern creation in live animals. We validated DISP by successfully printing near diseased areas in the mouse bladder and deep within rabbit leg muscles in vivo, demonstrating its potential for localized drug delivery and tissue replacement. DISP’s ability to print conductive, drug-loaded, cell-laden, and bioadhesive biomaterials demonstrates its versatility for diverse biomedical applications.

Gas vesicles (GVs) based on acoustic reporter genes have emerged as potent contrast agents for cellular and molecular ultrasound imaging. These air-filled, genetically encoded protein nanostructures can be expressed in a variety of cell types in vivo to visualize cell location and activity or injected systemically to label and monitor tissue function. Distinguishing GV signal from tissue deep inside intact organisms requires imaging approaches such as amplitude modulation (AM) or collapse-based pulse sequences. However, these approaches have limitations either in sensitivity or require the destruction of GVs, restricting the imaging of dynamic cellular processes. To address these limitations, we developed harmonic imaging to enhance the sensitivity of nondestructive GV imaging. We hypothesized that harmonic imaging, integrated with AM, could significantly elevate GV detection sensitivity by leveraging the nonlinear acoustic response of GVs. We tested this hypothesis by imaging tissue-mimicking phantoms embedded with purified GVs, mammalian cells genetically modified to express GVs, and mice liver in vivo post-systemic infusion of GVs. Our findings reveal that harmonic cross-propagating wave AM (HxAM) imaging markedly surpasses traditional xAM in isolating GVs' nonlinear acoustic signature, demonstrating significant (p < 0.05) enhancements in imaging performance. HxAM imaging improves detection of GV producing cells up to three folds in vitro, enhances in vivo imaging performance by over 10 dB, while extending imaging depth by up to 20%. Investigation into the backscattered spectra further elucidates the advantages of harmonic imaging. These advancements bolster ultrasound's capability in molecular and cellular imaging, underscoring the potential of harmonic signals to improve GV detection.

Acoustic reporter genes (ARGs) have enabled the imaging of gene expression with ultrasound, which provides high-resolution access to deep, optically opaque living tissues. However, unlike their fluorescent counterparts, ARGs have so far been limited to a single “color,” preventing multiplexed imaging of cellular states or populations. Here, we use rational protein design and directed evolution to develop two novel ARGs that can be distinguished from each other based on their acoustic pressure response profiles, enabling “two-color” ultrasound imaging of gene expression. We demonstrate the utility of multiplexed ARGs for delineating bacterial cell species and cell states in vitro , and then apply them towards imaging distinct subpopulations of probiotics in the mouse gastrointestinal tract and in tumor-colonizing bacterial agents in vivo . Just as the first wavelength-shifted derivatives of fluorescent proteins opened a vivid world for optical microscopy, our next-generation acoustic proteins set the stage for a richer symphony of ultrasound signals from living subjects.

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.

Gas vesicles (GVs) based on acoustic reporter genes have emerged as potent contrast agents for cellular and molecular ultrasound imaging. These air-filled, genetically encoded protein nanostructures can be expressed in a variety of cell types in vivo to visualize cell location and activity or injected systemically to label and monitor tissue function. Distinguishing GVs from tissue signal deep inside intact organisms requires imaging approaches such as amplitude modulation (AM) or collapse-based pulse sequences, however they have limitations in sensitivity or require irreversible collapse of the GVs that restricts its scope for imaging dynamic cellular processes. To address these limitations, this study explores the utility of harmonic imaging to enhance the sensitivity of non-destructive imaging of GVs and cellular processes. Traditional fundamental-frequency imaging utilizing cross-wave AM (xAM) sequences has been deemed optimal for GV imaging. Contrary to this, we hypothesize that harmonic imaging, integrated with xAM could significantly elevate GV detection sensitivity. To verify our hypothesis, we conducted imaging on tissue-mimicking phantoms embedded with purified GVs, mammalian cells genetically modified to express GVs, and live mice after systemic GV infusion. Our findings reveal that harmonic xAM (HxAM) imaging markedly surpasses traditional xAM in isolating GVs’ nonlinear acoustic signature, showcasing significant enhancements in signal-to-background and contrast-to-background ratios across all tested samples. Further investigation into the backscattered spectra elucidates the efficacy of harmonic imaging in conjunction with xAM. HxAM imaging enables the detection of lower concentrations of GVs and cells with ultrasound and extends the imaging depth in vivo by up to 20% and imaging performance metrics by up to 10dB. These advancements bolster the capabilities of ultrasound for molecular and cellular imaging, underscoring the potential of using harmonic signals to amplify GV detection.

Devices that can be remote-controlled under image guidance to precisely deliver biomedicines to sites of disease are a major goal of biomedical research. However, most existing externally triggered delivery systems are based on complex micromachines that are controlled with electromagnetic waves and require custom external instrumentation. Here we present a drug delivery platform comprising a simple protein-containing hydrogel that can be both imaged and triggered to release drugs at specific locations using widely available ultrasound imaging devices. This technology is based on the addition of air-filled protein nanostructures called gas vesicles (GVs) to hydrogel delivery vehicles. While intact, GVs sterically block the release of drug payloads and allow the vehicle to be imaged with ultrasound. An increase in ultrasound pressure causes the collapse of GVs within hydrogels present at the desired anatomical location, instantly creating percolation channels and triggering rapid drug release. Both the imaging and release are performed using a common diagnostic ultrasound probe. We implement this concept by establishing ultrasound-controlled drug diffusion and release from hydrogels in vitro and demonstrating targeted image-guided protein delivery in vivo following oral administration. We use this approach to deliver anti-inflammatory antibodies to treat gastrointestinal inflammation in a rat model of colitis. Targeted acoustic percolation switches (TAPS) open a conduit for local, image-guided drug delivery with a simple formulation and commonplace ultrasound equipment.

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.

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.