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Abstract Detection and remediation of stress in crops is vital to ensure agricultural productivity. Conventional forms of assessing stress in plants are limited by feasibility, delayed phenotypic responses, inadequate specificity, and lack of sensitivity during initial phases of stress. While mass spectrometry is remarkably precise and achieves high-resolution, complex samples, such as plant tissues, require time-consuming and biased depletion strategies to effectively identify low-abundant stress biomarkers. Here, we bypassed these reduction methods via a nano-omics approach, where gold nanoparticles were used to enrich time- and temperature-dependent stress-related proteins through biomolecular corona formation that were subsequently analyzed by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). This nano-omic approach was more effective than a conventional proteomic analysis using UHPLC- MS/MS for resolving biotic-stress induced responses at early stages of pathogen infection inArabidopsis thaliana, well before the development of visible phenotypic symptoms, as well as in distal tissues of pathogen infected plants at early timepoints. The enhanced sensitivity of this nano-omic approach enables the identification of stress-related proteins at early critical timepoints, providing a more nuanced understanding of plant-pathogen interactions that can be leveraged for the development of early intervention strategies for sustainable agriculture. 

Abstract 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. Abstract Figure 

Abstract 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 aNicotiana benthamianaleaf. 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 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.