Biology subjects

Wang, J. W.-T.

Publications and source records attributed to Wang, J. W.-T..

3 recordsLinked to original sources

bioRxiv2023

Mapping the Morphology of DNA on Carbon Nanotube-Based Sensors in Solution using X-ray Scattering Interferometry

Single-walled carbon nanotubes (SWCNTs) with adsorbed single-stranded DNA (ssDNA) are applied as sensors to investigate biological systems, with applications ranging from clinical diagnostics to agricultural biotechnology. Unique ssDNA sequences render SWCNTs selectively responsive to target analytes. However, it remains unclear how the ssDNA conformation on the SWCNT surface contributes to their ultimate functionality, as observations have been constrained to computational models or experiments under dehydrated states that differ substantially from the aqueous biological environments in which the nanosensors are applied. Herein, we demonstrate a direct mode of measuring in-solution ssDNA geometries on SWCNTs via X-ray scattering interferometry (XSI), which leverages the interference pattern produced by AuNP tags conjugated to ssDNA on the SWCNT surface. We employ XSI to quantify distinct surface-adsorbed morphologies for two ssDNA oligomer lengths, conformational changes as a function of ionic strength, and the mechanism of dopamine sensing for a previously established ssDNA-SWCNT nanosensor, with corresponding ab initio modeling for visualization. We show that the shorter oligomer, (GT)6, adopts a highly ordered structure of stacked rings along the SWCNT axis, compared to the longer, less periodic (GT)15 wrapping. The presence of dopamine elicits a simultaneous axial elongation and radial constriction of the ssDNA closer to the SWCNT surface. Application of XSI to probe solution-phase morphologies of nanoparticle-based tools will yield insights into sensing mechanisms and inform future design strategies for polymer-functionalized SWCNT technologies.

biophysics↗

bioRxiv2022

Synaptic scale dopamine disruption in Huntington's Disease model mice imaged with near infrared catecholamine nanosensors

Dopamine neuromodulation is a critical process that facilitates learning, motivation, and motor control. Disruption of these processes has been implicated in several neurological and psychiatric disorders including Huntingtons Disease (HD). While several treatments for physical and psychiatric HD symptoms target dopaminergic neuromodulation, the mechanism by which dopaminergic dysfunction occurs during HD is unknown. This is partly due to limited capability to visualize dopamine dynamics at the spatiotemporal resolution of both neuromodulator release (ms) and dopaminergic boutons ({micro}m). Here we employ near-infrared fluorescent catecholamine nanosensors (nIRCats) to image dopamine release within the brain striatum of R6/2 Huntingtons Disease Model (R6/2) mice. We find that stimulated dorsal striatal dopamine release decreases with progressive motor degeneration and that these decreases are primarily driven by a decrease in the number of dopamine hotspots combined with decreased release intensity and decreased release fidelity. Using nIRCats high spatial resolution, we show that dopamine hotspots in late HD show increased ability to add new dopamine hotspots at high extracellular calcium concentrations and track individual dopamine hotspots over repeated stimulations and pharmacological wash to measure dopamine hotspots release fidelity. Compellingly, we demonstrate that antagonism of D2-autoreceptors using Sulpiride and direct blocking of Kv1.2 channels using 4-Aminopyradine (4-AP) increases the fidelity of dopamine hotspot activity in WT striatum but not in late HD striatum, indicating that D2-autoreceptor regulation of dopamine release through Kv1.2 channels is compromised in late HD. These findings, enabled by nIRCats, provide a more detailed look into how dopamine release is disrupted and dysregulated during Huntingtons Disease to alter the coverage of dopamine modulation across the dorsal striatum. SIGNIFICANCE STATEMENTHuntingtons Disease (HD) is a neurodegenerative disorder with no cure. Dysfunction of dopamine signaling is known to deteriorate in HD but has not been studied at the spatial level of individual release sites. Here, we image dopamine release from individual hotspots in brain slices from R6/2 HD mice at early and late disease timepoints with dopamine nanosensors. We track single dopamine hotspots and find that dopamine hotspot number, release intensity, and release fidelity decrease in HD, and demonstrate that changes in D2-autoreceptor regulation manifest through changes in hotspot release fidelity thus compromising dopamine coverage across the dorsal lateral striatum. These findings highlight dopaminergic neurons in cortico-striatal signaling during HD as a promising new therapeutic target for HD treatment.

neuroscience↗

bioRxiv2022

Quantification of cell penetrating peptide mediated delivery of proteins in plant leaves

Protein delivery to plants offers many opportunities for plant bioengineering via gene editing and through direction of protein-protein interactions. However, the delivery and confirmation of successful protein delivery to plants presents both practical and analytical challenges. We present a GFP bimolecular fluorescence complementation-based tool, delivered complementation in planta (DCIP), which allows for unambiguous and quantitative measurement of protein delivery in leaves. Using DCIP, we demonstrate cell-penetrating peptide mediated cytosolic delivery of peptides and recombinant proteins in Nicotiana benthamiana. We show that DCIP enables measurement of delivery efficiency and enables functional screening of cell penetrating peptide efficacies for in-plant protein delivery. Finally, we demonstrate that DCIP detects cell penetrating peptide mediated delivery of recombinantly expressed proteins such as mCherry and Lifeact into intact leaves. Finally, we also demonstrate, for the first time, delivery of a recombinant plant transcription factor, WUSCHEL (AtWUS), in N. benthamiana. RT-qPCR analysis of AtWUS delivery in Arabidopsis seedlings also suggests delivered WUS can recapitulate AtWUS-overexpression transcriptional changes. All combined, DCIP offers a new and powerful tool for interrogating cytosolic delivery of proteins in plants and highlights future avenues for engineering plant physiology.

plant biology↗