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Ultrasound (US) neuromodulation, especially sonogenetics, has been demonstrated with potential applications in noninvasive and targeted treatment of various neurological disorders. Despite the growing interest, the mechanism for US neuromodulation remains elusive, and the optimal condition for eliciting a neural response with minimal adverse effect has not been identified. Here, we investigate the Piezo1 activation and intracellular calcium response elicited by acoustical streaming induced shear stress under various US exposure conditions. We find that Piezo1 activation and resultant intracellular calcium response depend critically on shear stress amplitude and pulse length of the stimulation. Under the same insonification acoustic energy, we further identify an optical pulse length that leads to maximum cell deformation, Piezo1 activation, and calcium response with minimal injury, confirmed by numerical modeling of Piezo1 channel gating dynamics. Our results provide insight into the mechanism of ultrasonic activation of Piezo1 and highlight the importance of optimizing US exposure conditions in sonogenetics applications.

One of the earliest events in cellular mechanotransduction is often an increase in intracellular calcium concentration associated with intracellular calcium waves (ICWs) in various physiologic or pathophysiologic processes. Although cavitation-induced calcium responses are believed to be important for modulating downstream bioeffects such as cell injury and mechanotransduction in ultrasound therapy, the fundamental mechanisms of these responses have not been elucidated. In this study, we investigated mechanistically the ICWs elicited in single HeLa cells by the tandem bubble-induced jetting flow in a microfluidic system. We identified two distinct (fast and slow) types of ICWs at varying degrees of flow shear stress-induced membrane deformation, as determined by different bubble standoff distances. We showed that ICWs were initiated by an extracellular calcium influx across the cell membrane nearest to the jetting flow, either primarily through poration sites for fast ICWs or opening of mechanosensitive ion channels for slow ICWs, which then propagated in the cytosol via a reaction–diffusion process from the endoplasmic reticulum. The speed of ICW (CICW ) was found to correlate strongly with the severity of cell injury, with CICW in the range of 33 μm/s to 93 μm/s for fast ICWs and 1.4 μm/s to 12 μm/s for slow ICWs. Finally, we demonstrated that micrometer-sized beads attached to the cell membrane integrin could trigger ICWs under mild cavitation conditions without collateral injury. The relation between the characteristics of ICW and cell injury, and potential strategies to mitigate cavitation-induced injury while evoking an intracellular calcium response, may be particularly useful for exploiting ultrasound-stimulated mechanotransduction applications in the future.

Therapeutic ultrasound or shockwave has shown its great potential to stimulate neural and muscle tissue, where cavitation microbubble induced Ca signaling is believed to play an important role. However, the pertinent mechanisms are unknown, especially at the single-cell level. Particularly, it is still a major challenge to get a comprehensive understanding of the effect of potential mechanosensitive molecular players on the cellular responses, including mechanosensitive ion channels, purinergic signaling and integrin ligation by extracellular matrix. Here, laser-induced cavitation microbubble was used to stimulate individual HEK293T cells either genetically knocked out or expressing Piezo1 ion channels with different normalized bubble-cell distance. Ca signaling and potential membrane poration were evaluated with a real-time fluorescence imaging system. Integrin-binding microbeads were attached to the apical surface of the cells at mild cavitation conditions, where the effect of Piezo1, P2X receptors and integrin ligation on single cell intracellular Ca signaling was assessed. Ca responses were rare at normalized cell-bubble distances that avoided membrane poration, even with overexpression of Piezo1, but could be increased in frequency to 42% of cells by attaching integrin-binding beads. We identified key molecular players in the bead-enhanced Ca response: increased integrin ligation by substrate ECM triggered ATP release and activation of P2X-but not Piezo1-ion channels. The resultant Ca influx caused dynamic changes in cell spread area. This approach to safely eliciting a Ca response with cavitation microbubbles and the uncovered mechanism by which increased integrin-ligation mediates ATP release and Ca signaling will inform new strategies to stimulate tissues with ultrasound and shockwaves.

Ultrasound (US) neuromodulation has the unique advantage to noninvasively manipulate neural activity in deep brain with high spatial resolution and flexibility in beam steering. In recent years, there is a growing interest in producing accurate and cell-type specific US neuromodulation using sonogenetics, in which the targeted cells/neurons are genetically modified via overexpression of mechanosensitive (MS) ion channels that can be activated by US. This emerging technique has spurred extensive research activities with the hope for potential clinical treatment of neurological disorders, such as Parkinson’s disease, epilepsy and depression. It has been proposed by mainstream journals that the neuromodulating effects of US are associated with changes in membrane potential due to US-induced cell/neuron membrane deformation and the activation of MS ion channels, in which the latter mechanism is given greater prominence in sonogenetics. In this process, US exerts its mechanical effects in different forms including acoustic radiation force (ARF, associated with momentum transfer from the US wave field to the medium), acoustic streaming (displacement of fluid), and cavitation (generation of bubbles within the tissue). Despite much efforts in the field, the physical mechanism by which US is converted into an effective energy form to elicit neuromodulation remains poorly understood, and there is little consensus about optimal US parameters required to evoke a sonogenetic response with minimal adverse effect. Understanding how US interacts with cell membrane with specific US parameters/configurations will be important to optimize this technology. A significant barrier to advancing the sonogenetics is the lack of technologies and experimental systems to capture and dissect the dynamic interaction of ultrasound with target cells and the resultant cell membrane deformation (or strain) and its correlation to MS channel dynamics at the single cell level. To resolve these technical challenges, we have developed a novel vertically deployed surface acoustic wave (VD-SAW) transducer platform that can be readily integrated with a fluorescence microscope for simultaneous observation and monitoring of the interaction of US waves with target cells, the mechanical strain and stress in cell membrane, and the resultant bioeffects at the single cell level. In Chapter 1, we introduced the background of US neuromodulation and sonogenetics, followed by a description of the major challenges in this field and the key questions we’re going to address in this dissertation. In Chapter 2, we investigated the activation of Piezo1, one of the few eukaryotic channels known to be responsible to US, by monitoring the intracellular calcium response. We observed that Piezo1 activation is highly determined by shear stress amplitude and pulse length (PL) of the stimulation. Under the same acoustic energy, we identified an optimal PL that leads to maximum cell deformation, and Piezo1 activation rate with minimal injury. Our results suggested the optimal PL is related to the viscoelastic response of cell membrane and the gating dynamics of Piezo1 which has not been considered in previous sonogenetics studies. In Chapter 3, we further constructed a 3D cell culture model in collagen hydrogel to better mimic the realistic cell culture condition. The hydrogel ruled out the involvement of acoustic streaming and thus facilitated the investigation of the role of ARF in sonogenetics which is a more practical form of US energy in vivo. The VD-SAW array integrated with 3D cell culture model was translated to a confocal fluorescence microscope for acquiring the initiation of intracellular calcium response and the cell membrane deformation in 3D. With the system upgrade, we found ARF is more efficient than acoustic streaming for activating Piezo1 channel. We also observed that the Piezo2, a homolog to Piezo1, requires higher power of US than Piezo1 for activation. Interestingly, we found Piezo2 is sensitive to membrane compression, while Piezo1 is sensitive to membrane tension. The observed differences of mechanical sensitivities and activating schemes between Piezo1 and Piezo2 were consistent with previous evidence in cell mechanotransduction studies using patch clamp. We further combined numerical modeling and 3D confocal imaging with digital volume correlation to analyze cell membrane stress under insonification. We established the relationship between US-induced mechanical effect and cellular bioeffects (Ca2+ signaling via MS channel activation) by a key parameter: the total strain energy, which take accounts of magnitude of stress, volume of cell under stress, and sonification time. Our results suggested that the stress distribution and total strain energy induced by US were strongly correlated to the directions of ARF. We’ve shown that the total strain energy could highly recapitulate the effectiveness of ARF on Piezo1 activation. Overall, we have developed a miniatured, highly compatible and controllable VD-SAW transducer for studying sonogenetics at the single cell level. Our preliminary results provide new insights into the mechanisms of ultrasonic activation of Piezo. By virtue of its dimensions, compatibility, and targetability, the VD-SAW transducer can be readily applied for studying the mechanisms and key parameters underlying the activation of other MS channels by US in various types of cell/neuron. We also expect that VD-SAW can be translated to ex vivo (e.g. brain slice) or in vivo application with the advancement of fabrication and proper compensation of the skull-induced US attenuation.

Ultrasound or shockwave-induced cavitation is used therapeutically to stimulate neural and muscle tissue, but the mechanisms underlying this mechanotransduction are unclear. Intracellular Ca2+ signaling is one of the earliest events in mechanotransduction. In this study, we investigate the mechanism of Ca2+ signaling in individual HEK293T cells stimulated by single cavitation bubbles. Ca2+ responses are rare at cell-bubble distance that avoids membrane poration, even with overexpression of the mechanosensitive ion channel Piezo1, but could be increased in frequency to 42% of cells by attaching RGD beads to the apical surface of the cells. By using Piezo1 knockout and Piezo1-expressing cells, integrin-blocking antibodies, and inhibitors of P2X ion channels, key molecular players are identified in the RGD bead-enhanced Ca2+ response: increased integrin ligation by substrate ECM triggers ATP release and activation of P2X—but not Piezo1—ion channels. These molecular players have not been examined previously in cavitation-induced Ca2+ signaling. The resultant Ca2+ influx causes dynamic changes in cell spread area. This approach to eliciting a Ca2+ response with cavitation microbubbles without cell injury, and the uncovered mechanotransduction mechanism by which increased integrin-ligation mediates ATP release and Ca2+ signaling will inform new strategies to stimulate tissues with ultrasound and shockwaves. Competing Interest Statement The authors have declared no competing interest. Footnotes * ↵* E-mail: fenfang.liatduke.edu, pzhongatduke.edu, brenton.hoffmanatduke.edu

Effective capture and safe disposal of radioactive iodine (129I or 131I) during nuclear power generation processes have always been a worldwide environmental concern. Low-cost and high-efficiency iodine removal materials are urgently needed. In this study, we synthesized two aniline-based hypercrosslinked polymers (AHCPs), AHCP-1 and AHCP-2, for iodine capture in both aqueous and gaseous phases. They are obtained by aniline polymerization through Friedel–Crafts alkylation and Scholl coupling reaction, respectively, with high chemical and thermal stability. Notably, AHCP-1 exhibits record-high static iodine adsorption (250 wt%) in aqueous solution. In the iodine vapor adsorption, AHCP-2 presents an excellent total iodine capture (596 wt%), surpassing the most reported amorphous polymer adsorbents. The rich primary amine groups of AHCPs promote the rapid physical capture of iodine from iodine water and iodine vapor. Intrinsic features such as low-cost preparation, good recyclability, as well as excellent performance in iodine capture indicate that the AHCPs can be used as potential candidates for the removal of iodine from radioactive wastewater and gas mixtures.

Nowadays, biological databases are playing an increasingly critical role in biological research. Myceliophthora thermophila is an excellent thermophilic fungal chassis for industrial enzyme production and plant biomass-based chemical synthesis. The lack of a dedicated public database has made access to and reanalysis of M. thermophila data difficult. To bridge this gap, we developed MTD (https://mtd.biodesign.ac.cn/), a cloud-based omics database and interactive platform for M. thermophila. MTD integrates comprehensive genome annotations, sequence-based predictions, transcriptome data, curated experimental descriptions, and bioinformatics analysis tools, offering a comprehensive, one-stop solution with a ‘top-down’ search strategy to streamline M. thermophila research. The platform supports data reproduction, rapid querying, and in-depth mining of existing transcriptome datasets. Based on analyses using data and tools in MTD, we identified shifts in metabolic allocation in a glucoamylase hyperproduction strain of M. thermophila, highlighting changes in fatty acid biosynthesis and amino acids biosynthesis pathways, which provide new insights into the underlying phenotypic alterations. As a pioneering resource, MTD marks a key advancement in M. thermophila research and sets the model for developing similar databases for other species. •MTD is a cloud-based and serverless knowledge database for the non-model fungus M. thermophila, integrating omics data and bioinformatics tools.•Its one-stop platform allows users to rapidly reanalyze and mine data through modular features, driving scientific discoveries.•MTD serves as a reference pipeline for developing databases for other interested non-model species.