Sonogenetic Modulation of Cellular Activities Using an Engineered Auditory-Sensing Protein.

Biomolecules that respond to different external stimuli enable the remote control of genetically modified cells. We report herein a sonogenetic approach that can manipulate target cell activities by focused ultrasound stimulation. This system requires an ultrasound-responsive protein derived from an engineered auditory-sensing protein prestin. Heterologous expression of mouse prestin containing two parallel amino acid substitutions, N7T and N308S, that frequently exist in prestins from echolocating species endowed transfected mammalian cells with the ability to sense ultrasound. An ultrasound pulse of low frequency and low pressure efficiently evoked cellular calcium responses after transfecting with prestin(N7T, N308S). Moreover, pulsed ultrasound can also noninvasively stimulate target neurons expressing prestin(N7T, N308S) in deep regions of mouse brains. Our study delineates how an engineered auditory-sensing protein can cause mammalian cells to sense ultrasound stimulation. Moreover, our sonogenetic tools will serve as new strategies for noninvasive therapy in deep tissues.

Detection of Pathogenic and Beneficial Microbes for Roselle Wilt Disease.

Wilt disease of roselle (Hibiscus sabdariffa L.) is common in Taiwan; however, the causative agent remains unknown. The stems of wilted roselle are browned, slightly constricted, and covered by white aerial hyphae, suggesting that potential pathogens may originate from soil. To identify the potential pathogens, we conducted a rhizosphere microbiota survey in phenotypically healthy and diseased plants through fungal internal transcribed spacer (ITS) and bacterial 16S rRNA amplicon sequencing for uncovering the microbial compositions in the roselle rhizosphere. The fungal family Nectriaceae exhibited significantly higher abundance in diseased rhizospheres than in healthy rhizospheres, and this bacterial community was more specific to geography (i.e., plot-dependent) than to rhizosphere disease status. However, a few bacterial groups such as Bacilli were associated with the healthy rhizosphere. Fusarium species were the most dominant species of Nectriaceae in the survey and became the main target for potential pathogen isolation. We successfully isolated 119 strains from diseased plants in roselle fields. Koch's postulates were used to evaluate the pathogenicity of these strains; our results indicated that Fusarium solani K1 (FsK1) can cause wilting and a rotted pith in roselles, which was consistent with observations in the fields. This is the first demonstration that F. solani can cause roselle wilt in Taiwan. Furthermore, these newly isolated strains are the most dominant operational taxonomic units detected in ITS amplicon sequencing in diseased rhizospheres, which serves as further evidence that F. solani is the main pathogen causing the roselle wilt disease. Administration of Bacillus velezensis SOI-3374, a strain isolated from a healthy roselle rhizosphere, caused considerable anti-FsK1 activity, and it can serve as a potential biocontrol agent against roselle wilt disease.

Targeted delivery of engineered auditory sensing protein for ultrasound neuromodulation in the brain.

Sonogenetics is a promising approach for in vivo neuromodulation using ultrasound (US) to non-invasively stimulate cells in deep tissue. However, sonogenetics requires accurate transduction of US-responsive proteins into target cells. Here, we introduce a non-invasive and non-viral approach for intracerebral gene delivery. This approach utilizes temporary ultrasonic disruption of the blood-brain barrier (BBB) to transfect neurons at specific sites in the brain via DNA that encodes engineered US-responsive protein (murine Prestin (N7T, N308S))-loaded microbubbles (pPrestin-MBs). Prestin is a transmembrane protein that exists in the mammalian auditory system and functions as an electromechanical transducer. We further improved the US sensitivity of Prestin by introducing specific amino acid substitutions that frequently occur in sonar species into the mouse Prestin protein. We demonstrated this concept in mice using US with pPrestin-MBs to non-invasively modify and activate neurons within the brain for spatiotemporal neuromodulation. Method: MBs composed of cationic phospholipid and C3F8 loaded with mouse Prestin plasmid (pPrestin) via electrostatic interactions. The mean concentration and size of the pPrestin-MBs were (16.0 ± 0.2) × 109 MBs/mL and 1.1 ± 0.2 µm, respectively. SH-SY5Y neuron-like cells and C57BL mice were used in this study. We evaluated the gene transfection efficiency and BBB-opening region resulting from pPrestin-MBs with 1-MHz US (pressure = 0.1-0.5 MPa, cycle = 50-10000, pulse repetition frequency (PRF): 0.5-5 Hz, sonication time = 60 s) using green fluorescence protein (Venus) and Evans blue staining. Results: The maximum pPrestin expression with the highest cell viability occurred at a pressure of 0.5 MPa, cycle number of 5000, and PRF of 1 Hz. The cellular transfection rate with pPrestin-MBs and US was 20.2 ± 2.5%, which was 1.5-fold higher than that of commercial transfection agents (LT-1). In vivo data suggested that the most profound expression of pPrestin occurred at 2 days after performing pPrestin-MBs with US (0.5 MPa, 240 s sonication time). In addition, no server erythrocyte extravasations and apoptosis cells were observed at US-sonicated region. We further found that with 0.5-MHz US stimulation, cells with Prestin expression were 6-fold more likely to exhibit c-Fos staining than cells without Prestin expression. Conclusion: Successful activation of Prestin-expressing neurons suggests that this technology provides non-invasive and spatially precise selective modulation of one or multiple specific brain regions.

A preliminary study of Parkinson's gene therapy via sono-magnetic sensing gene vector for conquering extra/intracellular barriers in mice.

BACKGROUND: Non-virus genetic treatment for Parkinson's disease (PD) via plasmid glial cell-line derived neurotrophic factor (pGDNF) has shown potential for repairing damaged dopaminergic neurons. However, development of this gene therapy is largely hampered by the insufficient transfection efficiency as a result of the cell membrane, lysosome, and cytoskeleton meshwork. METHODS: In this study, we propose the use of polyethylenimine (PEI)-superparamagnetic iron oxide-plasmid DNA (pDNA)-loaded microbubbles (PSp-MBs) in conjunction with focused ultrasound (FUS) and two-step magnetic navigation to provide cavitation, proton sponge effect and magnetic effects to increase the efficiency of gene delivery. RESULTS: The gene transfection rate in the proposed system was 2.2-fold higher than that of the commercial agent (TransIT®-LT1). The transfection rate could be boosted ∼11%, ∼10%, and 6% by cavitation-magnetic hybrid enhanced cell membrane permeabilization, proton sponge effect, and magnetic-assisted cytoskeleton-reorganization, respectively. In vivo data suggested that effective gene delivery with this system results in a 3.2-fold increase in recovery of dopaminergic neurons and a 3.9-fold improvement in the motor behavior when compared to untreated genetic PD mice. CONCLUSIONS: We proposed that this novel FUS-magnetic hybrid gene delivery platform could be integrated with a variety of therapeutic genes for treating neurodegenerative diseases in the future.