Search

Using a fluorescence complementation assay, Delivered Complementation in Planta (DCIP), we demonstrate cell-penetrating peptide-mediated cytosolic delivery of peptides and recombinant proteins in Nicotiana benthamiana. We show that DCIP enables quantitative measurement of protein delivery efficiency and enables functional screening of cell-penetrating peptides for in-planta 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. We also demonstrate delivery of a recombinant plant transcription factor, WUSCHEL (AtWUS), into N. benthamiana. RT-qPCR analysis of AtWUS delivery in Arabidopsis seedlings also suggests delivered WUS can recapitulate transcriptional changes induced by overexpression of AtWUS. Taken together, our findings demonstrate that DCIP offers a new and powerful tool for interrogating cytosolic delivery of proteins in plants and highlights future avenues for engineering plant physiology.

Traditional deep fluorescence imaging has primarily focused on red‐shifting imaging wavelengths into the near‐infrared (NIR) windows or implementation of multi‐photon excitation approaches. Here, the advantages of NIR and multiphoton imaging are combined by developing a dual‐infrared two‐photon microscope that enables high‐resolution deep imaging in biological tissues. This study first computationally identifies that photon absorption, as opposed to scattering, is the primary contributor to signal attenuation. A NIR two‐photon microscope is constructed next with a 1640 nm femtosecond pulsed laser and a NIR PMT detector to image biological tissues labeled with fluorescent single‐walled carbon nanotubes (SWNTs). Spatial imaging resolutions are achieved close to the Abbe resolution limit and eliminate blur and background autofluorescence of biomolecules, 300 µm deep into brain slices and through the full 120 µm thickness of a Nicotiana benthamiana leaf. NIR‐II two‐photon microscopy can also measure tissue heterogeneity by quantifying how much the fluorescence power law function varies across tissues, a feature this study exploits to distinguish Huntington's Disease afflicted mouse brain tissues from wildtype. These results suggest dual‐infrared two‐photon microscopy can accomplish in‐tissue structural imaging and biochemical sensing with a minimal background, and with high spatial resolution, in optically opaque or highly autofluorescent biological tissues. [ABSTRACT FROM AUTHOR]

Plants are capable of mounting an immune defense in response to infection, wounding, or environmental stress. The immune response of a plant is often mediated through the recognition of conserved molecular motifs associated with stress. Treating plants with these molecular motifs prior to exposure to stress, termed immune priming, can improve plant resilience to subsequent stressors. Here, we demonstrate the use of cellulose nanocrystals (CNCs) as an immune priming tool for Arabidopsis thaliana combining phenotypic and transcriptomic data to validate the efficacy of CNCs as an immune primer. Pretreatment of Arabidopsis with CNCs reduces the level of infection by the pathogen Pseudomonas syringae up to 65%. RNA sequencing shows that treatment of Arabidopsis with CNCs results in deep transcriptional reprogramming, perturbing over 1300 genes, most of which are associated with immune regulation. We hypothesize plants recognize CNCs as cell-wall damage, potentially explaining the observed immune response. Finally, physiological characterization of the plant response to CNCs demonstrates minimal effects on plant growth without inducing callose deposition or generation of reactive oxygen species. This work provides a foundation for future investigation of cellulose-based nanomaterials as immune primers, particularly in crop species. Given their ease of synthesis, low cost, degradability, and bio/environmental compatibility, cellulose-based materials could serve as a complement to conventional biocides for pathogen control. [ABSTRACT FROM AUTHOR]

It is well known that electric fields occur in wind-blown dust, due to the triboelectric charging of particles as they collide. Triboelectric charging, or contact electrification, is a poorly understood and complex phenomenon. It is especially important in granular systems, as the high surface-to-volume ratio can lead to the build-up of large amounts of charge. A particularly surprising effect, which is important in dust systems, is that charge transfer occurs in systems of a single composition, such that there is a particle-size dependent polarity of the particles. Here, we use a combined experimental and theoretical approach to elucidate the electrostatic charging that occurs during dust storms, and the effects of this electrostatic charging on dust transport. We create laboratory-scale wind-blown dust systems, and study the electrostatic charging. We find that larger particles tend to charge positive and to stay at or near the sand bed, while smaller particles tend to charge negative and get lofted to higher elevations. This self-segregating of charged particles would lead to electric fields within a dust storm. Our results show that electric fields then increase the dust transport by more easily lofting charged particles.

Large amounts of dust are lofted into the atmosphere from arid regions of the world before being transported up to thousands of kilometers. This atmospheric dust interacts with solar radiation causing changes in the climate, with larger-sized particles having a heating effect, and smaller-sized particles having a cooling effect. Previous studies on the long-range transport of dust have found larger particles than expected, without a model to explain their transport. Here, we investigate the effect of electric fields on lofted airborne dust by blowing sand through a vertically-oriented electric field, and characterizing the size distribution as a function of height. We also model this system, considering the gravitational, drag, and electrostatic forces on particles, to understand the effects of the electric field. Our results indicate that electric fields keep particles suspended at higher elevations and enrich the concentration of larger particles at higher elevations. We extend our model from the small-scale system to long-range atmospheric dust transport to develop insights on the effects of electric fields on size distributions of lofted dust in the atmosphere. We show that the presence of electric fields and the resulting electrostatic force on particles can help explain the transport of unexpectedly larger particles and cause the size distribution to become more uniform as a function of elevation. Thus, our experimental and modelling results indicate that electrostatic forces should be considered when determining the effect of atmospheric dust on the climate. [ABSTRACT FROM AUTHOR]

Lipid nanoparticles (LNPs) are the most clinically advanced nonviral RNA-delivery vehicles, though challenges remain in fully understanding how LNPs interact with biological systems. In vivo , proteins form an associated corona on LNPs that redefines their physicochemical properties and influences delivery outcomes. Despite its importance, the LNP protein corona is challenging to study owing to the technical difficulty of selectively recovering soft nanoparticles from biological samples. Herein, we developed a quantitative, label-free mass spectrometry-based proteomics approach to characterize the protein corona on LNPs. Critically, this protein corona isolation workflow avoids artifacts introduced by the presence of endogenous nanoparticles in human biofluids. We applied continuous density gradient ultracentrifugation for protein-LNP complex isolation, with mass spectrometry for protein identification normalized to protein composition in the biofluid alone. With this approach, we quantify proteins consistently enriched in the LNP corona including vitronectin, C-reactive protein, and alpha-2-macroglobulin. We explore the impact of these corona proteins on cell uptake and mRNA expression in HepG2 human liver cells, and find that, surprisingly, increased levels of cell uptake do not correlate with increased mRNA expression in part likely due to protein corona-induced lysosomal trafficking of LNPs. Our results underscore the need to consider the protein corona in the design of LNP-based therapeutics.

A range of techniques including physical property measurements, neutron scattering experiments, ab initio molecular dynamics, and classical molecular dynamics simulations are used to probe the structural, thermodynamic, and transport properties of a deep eutectic solvent comprised of a 1:2 molar ratio of choline chloride and ethylene glycol. This mixture, known as Ethaline, has many desirable properties for use in a range of applications, and therefore, understanding its liquid structure and transport properties is of interest. Simulation results are able to capture experimental densities, diffusivities, viscosities, and structure factors extremely well. The solvation environment is dynamic and dominated by different hydrogen bonding interactions. Dynamic heterogeneities resulting from hydrogen bonding interactions are quantified. Rotational dynamics of molecular dipole moments of choline and ethylene glycol are computed and found to exhibit a fast and slow mode.