Biology subjects

Dong, Z.

Publications and source records attributed to Dong, Z..

At least 109 records · Page 6Linked to original sources

bioRxiv2021

Spatiotemporal mapping of gene expression landscapes and developmental trajectories during zebrafish embryogenesis

Vertebrate embryogenesis is a remarkably dynamic process during which numerous cell types of different lineages generate, change, or disappear within a short period of time. A major challenge in understanding this process is the lack of topographical transcriptomic information that can help correlate microenvironmental cues within the hierarchy of cell fate decisions. Here, we employed Stereo-seq, a high-definition spatially resolved transcriptomic technology, to dissect the spatiotemporal dynamics of gene expression and regulatory networks in the developing zebrafish embryos. We profiled 91 embryo sections covering six critical time points during the first 24 hours of development, obtaining a total of 139,391 spots at cellular size ([~]100 m2) with spatial coordinates. Meanwhile, we identified spatial modules and co-varying genes for specific tissue organizations. By performing the integrative analysis of the Stereo-seq and scRNA-seq data from each time point, we reconstructed the spatially resolved developmental trajectories of cell fate transitions and molecular changes during zebrafish embryogenesis. We further investigated the spatial distribution of ligand-receptor pairs for major signaling pathways and identified novel interactions that potentially crosstalk with the Notch signaling pathway during zebrafish development. Our study constitutes a fundamental reference for further studies aiming to understand vertebrate development.

developmental biology↗

bioRxiv2021

Calling the Amino Acid Sequence of a Protein/Peptide from the Nanospectrum Produced by a Sub-nanometer Diameter Pore

The blockade current that develops when a protein translocates across a thin membrane through a sub-nanometer diameter pore (i.e., a nanospectrum) informs with extreme sensitivity on the sequence of amino acids that constitute the protein. Whereas mass spectrometry (MS) is still the dominant technology for protein identification, it suffers limitations. In proteome-wide studies, MS fails to sequence proteins de novo, but merely classifies a protein and it is not very sensitive requiring about a femtomole to do that. Compared with MS, a sub-nanometer diameter pore (i.e. a sub-nanopore) directly reads the amino acids constituting a single protein molecule, but efficient computational tools are still required for processing and interpreting the blockade current. Here, we delineate computational methods for processing sub-nanopore nanospectra and predicting electrical blockade currents from protein sequences, which are essential for protein identification.

bioinformatics↗

bioRxiv2021

Improved Ultrasound Localization Microscopy based on Microbubble Uncoupling via Transmit Excitation (MUTE)

Ultrasound localization microscopy (ULM) demonstrates great potential for visualization of tissue microvasculature at depth with high spatial resolution. The success of ULM heavily depends on the robust localization of isolated microbubbles (MBs), which can be challenging in vivo especially within larger vessels where MBs can overlap and cluster close together. While MB dilution alleviates the issue of MB overlap to a certain extent, it drastically increases the data acquisition time needed for MBs to populate the microvasculature, which is already on the order of several minutes using recommended MB concentrations. Inspired by optical super-resolution imaging based on stimulated emission depletion (STED), here we propose a novel ULM imaging sequence based on microbubble uncoupling via transmit excitation (MUTE). MUTE "silences" MB signals by creating acoustic nulls to facilitate MB separation, which leads to robust localization of MBs especially under high concentrations. The efficiency of localization accomplished via the proposed technique was first evaluated in simulation studies with conventional ULM as a benchmark. Then an in vivo study based on the chorioallantoic membrane (CAM) of chicken embryos showed that MUTE could reduce the data acquisition time by half thanks to the enhanced MB separation and localization. Finally, the performance of MUTE was validated in an in vivo mouse brain study. These results demonstrate the high MB localization efficacy of MUTE-ULM, which contributes to a reduced data acquisition time and improved temporal resolution for ULM.

bioengineering↗

bioRxiv2021

Boundary domain genes were recruited to suppress bract growth and promote branching in maize

Grass inflorescence development is diverse and complex and involves sophisticated but poorly understood interactions of genes regulating branch determinacy and leaf growth. Here, we use a combination of transcript profiling, genetic and phylogenetic analyses to investigate tasselsheath1 (tsh1) and tsh4, two maize genes that simultaneously suppress inflorescence leaf growth and inhibit branching. We identify a regulatory network of inflorescence leaf suppression that involves the phase change gene tsh4 upstream of tsh1 and the ligule identity gene liguleless2 (lg2). We also find that a series of duplications in the tsh1 gene lineage facilitated its shift from boundary domain in non-grasses to suppressed inflorescence leaves of grasses. Collectively, these results suggest that the boundary domain genes tsh1 and lg2 were recruited to inflorescence leaves where they suppress growth and regulate a non-autonomous signaling center that promotes inflorescence branching, an important component of yield in cereal grasses.

plant biology↗

bioRxiv2021

Localization free super-resolution microbubble velocimetry using a long short-term memory neural network

Ultrasound localization microscopy is a super-resolution imaging technique that exploits the unique characteristics of contrast microbubbles to side-step the fundamental trade-off between imaging resolution and penetration depth. However, the conventional reconstruction technique is confined to low microbubble concentrations to avoid localization and tracking errors. Several research groups have introduced sparsity- and deep learning-based approaches to overcome this constraint to extract useful vascular structural information from overlapping microbubble signals, but these solutions have not been demonstrated to produce blood flow velocity maps of the microcirculation. Here, we introduce Deep-SMV, a localization free super-resolution microbubble velocimetry technique, based on a long short-term memory neural network, that provides high imaging speed and robustness to high microbubble concentrations, and directly outputs blood velocity measurements at a super-resolution. Deep-SMV is trained efficiently using microbubble flow simulation on real in vivo vascular data and demonstrates real-time velocity map reconstruction suitable for functional vascular imaging and pulsatility mapping at super-resolution. The technique is successfully applied to a wide variety of imaging scenarios, include flow channel phantoms, chicken embryo chorioallantoic membranes, and mouse kidney, tumor, and brain imaging.

bioengineering↗

bioRxiv2021

SNR-efficient distortion-free diffusion relaxometry imaging using ACcelerated Echo-train shifted EPTI (ACE-EPTI)

PurposeTo develop an efficient acquisition technique for distortion-free diffusion MRI and diffusion-relaxometry. MethodsA new ACcelerated Echo-train shifted Echo-Planar Time-resolved Imaging (ACE-EPTI) technique is developed to achieve high-SNR, distortion- and blurring-free diffusion and diffusion-relaxometry imaging. ACE-EPTI employs a newly designed variable density spatiotemporal encoding with self-navigation capability, that allows submillimeter in-plane resolution using only 3-shot. Moreover, an echo-train-shifted acquisition is developed to achieve minimal TE, together with an SNR-optimal readout length, leading to ~30% improvement in SNR efficiency over single-shot EPI. To recover the highly accelerated data with high image quality, a tailored subspace image reconstruction framework is developed, that corrects for odd/even-echo phase difference, shot-to-shot phase variation, and the B0 field changes due to field drift and eddy currents across different dynamics. After the phase-corrected subspace reconstruction, artifacts-free high-SNR diffusion images at multiple TEs are obtained with varying T2* weighting. ResultsSimulation, phantom and in-vivo experiments were performed, which validated the 3-shot spatiotemporal encoding provides accurate reconstruction at submillimeter resolution. The use of echo-train shifting and optimized readout length improves the SNR-efficiency by 27-36% over single-shot EPI. The reconstructed multi-TE diffusion images were demonstrated to be free from distortion (susceptibility and eddy currents) and phase/field variation induced artifacts. These improvements of ACE-EPTI enable improved diffusion tensor imaging and rich multi-TE information for diffusion-relaxometry analysis. ConclusionACE-EPTI was demonstrated to be an efficient and powerful technique for high-resolution diffusion imaging and diffusion-relaxometry, which provides high SNR, distortion- and blurring-free, and time-resolved multi-echo images by a fast 3-shot acquisition.

bioengineering↗

bioRxiv2021

Heterogeneity in the transcriptional response of the human pathogen Aspergillus fumigatus to the antifungal agent caspofungin

Aspergillus fumigatus is the main causative agent of invasive pulmonary aspergillosis (IPA), a severe disease that affects immunosuppressed patients worldwide. The fungistatic drug caspofungin is the second line therapy against IPA but has increasingly been used against clinical strains that are resistant to azoles, the first line antifungal therapy. In high concentrations, caspofungin induces a tolerance phenotype with partial reestablishment of fungal growth called caspofungin paradoxical effect (CPE), resulting from a change in the composition of the cell wall. An increasing number of studies has shown that different isolates of A. fumigatus exhibit phenotypic heterogeneity, including heterogeneity in their CPE response. To gain insights into the underlying molecular mechanisms of CPE response heterogeneity, we analyzed the transcriptomes of two A. fumigatus reference strains, Af293 and CEA17, exposed to low and high caspofungin concentrations. We found that there is a core transcriptional response that involves genes related to cell wall remodeling processes, mitochondrial function, transmembrane transport, and amino acid and ergosterol metabolism, and a variable response related to secondary metabolite (SM) biosynthesis and iron homeostasis. Specifically, we show here that the overexpression of a SM pathway that works as an iron chelator extinguishes the CPE in both backgrounds, whereas iron depletion is detrimental for the CPE in Af293 but not in CEA17. We next investigated the function of the transcription factor CrzA, whose deletion was previously shown to result in heterogeneity in the CPE response of the Af293 and CEA17 strains. We found that CrzA constitutively binds to and modulates the expression of several genes related to processes involved in caspofungin tolerance, and that crzA deletion differentially impacts the SM production and growth of Af293 and CEA17. As opposed to the {Delta}crzACEA17 mutant, the {Delta}crzAAf293 mutant fails to activate cell wall remodeling genes upon caspofungin exposure, which most likely severely affects its macrostructure and extinguishes its CPE. This work describes how heterogeneity in the response to an antifungal agent between A. fumigatus strains stems from heterogeneity in the function of a transcription factor and its downstream target genes.

microbiology↗

bioRxiv2021

Aging-related cerebral microvascular changes visualized using Ultrasound Localization Microscopy in the living mouse

Aging-related cognitive decline is an emerging health crisis; however, no established unifying mechanism has been identified for the cognitive impairments seen in an aging population. A vascular hypothesis of cognitive decline has been proposed but is difficult to test given the contradictory radiologic needs of high-fidelity microvascular imaging resolution and a broad and deep brain imaging field of view. Super-resolution ultrasound localization microscopy (ULM) offers a potential solution by exploiting circulating microbubbles to achieve a vascular resolution approaching the capillary scale without sacrificing imaging depth. In this report, we apply ULM imaging to a mouse model of aging and quantify differences in cerebral vascularity, blood velocity, and vessel tortuosity across several brain regions. We found significant decreases in blood velocity, and significant increases in vascular tortuosity, across all brain regions in the aged cohort, and significant decreases in blood volume in the cortex. These data provide the first-ever measurements of subcortical microvascular dynamics in vivo and reveal that aging has a major impact on these measurements.

neuroscience↗

bioRxiv2021

A comprehensive mass spectral library for human thyroid tissues

Thyroid nodules occur in about 60% of the population. Current diagnostic strategies, however, often fail at distinguishing malignant nodules before surgery, thus leading to unnecessary, invasive treatments. As proteins are involved in all physio/pathological processes, a proteome investigation of biopsied nodules may help correctly classify and identify malignant nodules and discover therapeutic targets. Quantitative mass spectrometry data-independent acquisition (DIA) enables highly reproducible and rapid throughput investigation of proteomes. An exhaustive spectral library of thyroid nodules is essential for DIA yet still unavailable. This study presents a comprehensive thyroid spectral library covering five types of thyroid tissue: multinodular goiter, follicular adenoma, follicular and papillary thyroid carcinoma, and normal thyroid tissue. Our library includes 925,330 transition groups, 157,548 peptide precursors, 121,960 peptides, 9941 protein groups, and 9826 proteins from proteotypic peptides. This library resource was evaluated using three papillary thyroid carcinoma samples and their corresponding adjacent normal thyroid tissue, leading to effective quantification of up to 7863 proteins from biopsy-level thyroid tissues.

systems biology↗

bioRxiv2021

Simultaneous pure T2 and varying T2'-weighted BOLD fMRI using Echo Planar Time-resolved Imaging (EPTI) for mapping laminar fMRI responses

Spin-echo (SE) BOLD fMRI has high microvascular specificity, and thus provides a more reliable means to localize neural activity compared to conventional gradient-echo BOLD fMRI. However, the most common SE BOLD acquisition method, SE-EPI, is known to suffer from T2' contrast contamination with undesirable draining vein bias. To address this, in this study, we extended a recently developed distortion/blurring-free multi-shot EPI technique, Echo-Planar Time-resolved Imaging (EPTI), to cortical-depth dependent SE-fMRI at 7T to test whether it could provide purer SE BOLD contrast with minimal T2' contamination for improved neuronal specificity. From the same acquisition, the time-resolved feature of EPTI also provides a series of asymmetric SE (ASE) images with varying T2' weightings, and enables extraction of data equivalent to conventional SE EPI with different echo train lengths (ETLs). This allows us to systematically examine how T2'-contribution affects different SE acquisition strategies using a single dataset. A low-rank spatiotemporal subspace reconstruction was implemented for the SE-EPTI acquisition, which incorporates corrections for both shot-to-shot phase variations and dynamic B0 drifts. SE-EPTI was used in a visual task fMRI experiment to demonstrate that i) the pure SE image provided by EPTI results in the highest microvascular specificity; ii) the ASE EPTI series, with a graded introduction of T2' weightings at time points farther away from the pure SE, show a gradual sensitivity increase along with increasing draining vein bias; iii) the longer ETL seen in conventional SE EPI acquisitions will induce more draining vein bias. Consistent results were observed across multiple subjects, demonstrating the robustness of the proposed technique for SE-BOLD fMRI with high specificity.

neuroscience↗

bioRxiv2021

Flexibility-aware graph-based algorithm improves antigen epitopes identification

Epitopes of an antigen are the surface residues in the spatial proximity that can be recognized by antibodies. Identifying such residues has shown promising potentiality in vaccine design, drug development and chemotherapy, thus attracting extensive endeavors. Although great efforts have been made, the epitope prediction performance is still unsatisfactory. One possible issue accounting to this poor performance could be the ignorance of structural flexibility of antigens. Flexibility is a natural characteristic of antigens, which has been widely reported. However, this property has never been used by existing models. To this end, we propose a novel flexibility-aware graph-based computational model to identify epitopes. Unlike existing graph-based approaches that take the static structures of antigens as input, we consider all possible variations of the side chains in graph construction. These flexibility-aware graphs, of which the edges are highly enriched, are further partitioned into subgraphs by using a graph clustering algorithm. These clusters are subsequently expanded into larger graphs for detecting overlapping residues between epitopes if exist. Finally, the expanded graphs are classified as epitopes or non-epitopes via a newly designed graph convolutional network. Experimental results show that our flexibility-aware model markedly outperforms existing approaches and promotes the F1-score to 0.656. Comparing to the state-of-the-art, our approach makes an increment of F1-score by 16.3%. Further in-depth analysis demonstrates that the flexibility-aware strategy contributes the most to the improvement. The source codes of the proposed model is freely available at https://github.com/lzhlab/epitope. Author summaryEpitope prediction is helpful to many biomedical applications so that dozens of models have been proposed aiming at improving prediction efficiency and accuracy. However, the performances are still unsatisfactory due to its complicated nature, particularly the noteworthy flexible structures, which makes the precise prediction even more challenging. The existing approaches have overlooked the flexibility during model construction. To this end, we propose a graph model with flexibility heavily involved. Our model is mainly composed of three parts: i) flexibility-aware graph construction; ii) overlapping subgraph clustering; iii) graph convolutional network-based subgraph classification. Experimental results show that our newly proposed model markedly outperforms the existing best ones, making an increment of F1-score by 16.3%.

bioinformatics↗

bioRxiv2021

Fluorescence-Activated Droplet Sorting of Polyethylene terephthalate Degrading Enzymes

Enzymes that can decompose synthetic plastics such as polyethylene terephthalate (PET) are urgently needed. However, a bottleneck remains due to a lack of techniques for detecting and sorting environmental microorganisms with vast diversity and abundance. Here, we developed a fluorescence-activated droplet sorting (FADS) pipeline for high-throughput screening of PET-degrading microorganisms or enzymes (PETases). The pipeline comprises three steps: generation and incubation of droplets encapsulating single cells, picoinjection of fluorescein dibenzoate (FDBz) as the fluorogenic probe, and screening of droplets to obtain PET-degrading cells. We characterized critical factors associated with this method, including specificity and sensitivity for discriminating PETase from other enzymes. We then optimized its performance and compatibility with environmental samples. The system was used to screen a wastewater sample from a PET textile mill. We successfully obtained PET-degrading species from nine different genera. Moreover, two putative PETases from isolates Kineococcus endophyticus Un-5 and Staphylococcus epidermidis Un-C2-8 were genetically derived, heterologously expressed, and preliminarily validated for PET-degrading activities. We speculate that the FADS pipeline can be widely adopted to discover new PET-degrading microorganisms and enzymes in various environments and may be utilized in the directed evolution of PETases using synthetic biology.

microbiology↗

bioRxiv2021

Ultrafast high-resolution multi-parametric brain MRI using 3D Echo Planar Time-resolved Imaging

Multi-parametric quantitative MRI has shown great potential to improve the sensitivity and specificity of clinical diagnosis and to enhance our understanding of complex brain processes, but suffers from long scan time especially at high spatial resolution. To address this long-standing challenge, we introduce a novel approach, termed 3D Echo Planar Time-resolved Imaging (3D-EPTI), which significantly increases the acceleration capacity of MRI sampling, and provides high acquisition efficiency for multi-parametric MRI. This is achieved by exploiting the spatiotemporal correlation of MRI data at multiple timescales through new encoding strategies within and between efficient continuous readouts. Specifically, an optimized spatiotemporal CAIPI encoding within the readouts combined with a radial-block sampling strategy across the readouts enables an acceleration rate of 800 folds in the k-t space. A subspace reconstruction was employed to resolve thousands of artifact-free high-quality multi-contrast images spaced at a time interval of ~1 ms. We have demonstrated the ability of 3D-EPTI to provide robust and repeatable whole-brain simultaneous T1, T2, T2*, PD and B1+ mapping at high isotropic resolution within minutes (e.g., 1-mm isotropic resolution in 3 minutes), and to enable submillimeter multi-parametric imaging to study detailed brain structures. HighlightsO_LIUltra-fast acquisition for 3D multi-parametric quantitative MRI. C_LIO_LISimultaneous T1 T2 T2* PD and B1+ mapping. C_LIO_LI3-minute scan at 1-mm isotropic resolution with whole-brain coverage. C_LIO_LIMulti-parametric mapping at 700-m isotropic resolution in 10 minutes. C_LIO_LIRepeatable quantification and cortical-depth analysis. C_LI

bioengineering↗

bioRxiv2021

Minian: An open-source miniscope analysis pipeline

Miniature microscopes have gained considerable traction for in vivo calcium imaging in freely behaving animals. However, extracting calcium signals from raw videos is a computationally complex problem and remains a bottleneck for many researchers utilizing single-photon in vivo calcium imaging. Despite the existence of many powerful analysis packages designed to detect and extract calcium dynamics, most have either key parameters that are hard-coded or insufficient step-by-step guidance and validations to help the users choose the best parameters. This makes it difficult to know whether the output is reliable and meets the assumptions necessary for proper analysis. Moreover, large memory demand is often a constraint for setting up these pipelines since it limits the choice of hardware to specialized computers. Given these difficulties, there is a need for a low memory demand, user-friendly tool offering interactive visualizations of how altering parameters at each step of the analysis affects data output. Our open-source analysis pipeline, Minian (Miniscope Analysis), facilitates the transparency and accessibility of single-photon calcium imaging analysis, permitting users with little computational experience to extract the location of cells and their corresponding calcium traces and deconvolved neural activities. Minian contains interactive visualization tools for every step of the analysis, as well as detailed documentation and tips on parameter exploration. Furthermore, Minian has relatively small memory demands and can be run on a laptop, making it available to labs that do not have access to specialized computational hardware. Minian has been validated to reliably and robustly extract calcium events across different brain regions and from different cell types. In practice, Minian provides an open-source calcium imaging analysis pipeline with user-friendly interactive visualizations to explore parameters and validate results.

neuroscience↗

bioRxiv2021

Dissecting lipid contents in the distinct regions of native retinal rod disk membranes

Photoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating domain-specific samples. We have overcome this by using SMA to co-immunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each samples copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which were enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein-lipid interactions. High-resolution profiling of the ROS disk lipid composition provides a model for future studies of other complex cellular structures, and gives new insights into how intricate membrane structure and protein activity are balanced within the ROS. SUMMARYSander et al. have parsed the lipid composition of native-source photoreceptor disks and find large differences in fatty acid unsaturation and chain length between the center and rim regions. They selectively copurify membrane proteins and lipids from each region in SMALPs using nanobodies and antibodies.

cell biology↗

bioRxiv2020

Mammalian-unique eIF4E2 maintains GSK3β proline kinase activity to resist senescence against hypoxia

Cellular senescence is a stable state of cell cycle arrest elicited by various stresses. Hypoxia modulates senescence, but its consequences and implications in living organisms remains unknown. Here we identified the eIF4E2-GSK3{beta} pathway regulated by hypoxia to maintain p53 proline-directed phosphorylation (S/T-P) to prevent senescence. We previously knew that GSK3{beta} activates p53 translation through phosphorylation of RBM38 Ser195 (-Pro196). Unexpectedly, eIF4E2 directly binds to GSK3{beta} via a conserved motif, mediating Ser195 phosphorylation. Phosphoproteomics revealed that eIF4E2-GSK3{beta} specifically regulates proline-directed phosphorylation. Peptide e2-I or G3-I that disrupts this pathway dephosphorylates p53 at multiple S/T-P, which accelerate senescence by transcriptional suppressing TOPBP1 and TRX1. Consistently, peptides induce liver senescence that is rescued by TOPBP1 expression, and mediate senescence-dependent tumor regression. Furthermore, hypoxia inhibits eIF4E2-GSK3{beta}. Inspiringly, eIF4E2-GSK3{beta} is unique to mammals, which maintains mice viability and prevents liver senescence against physiological hypoxia. Interestingly, this mammalian eIF4E2 protects heart of zebrafish against hypoxia. Together, we identified a mammalian -unique eIF4E2-GSK3{beta} pathway preventing senescence and guarding against hypoxia in vivo.

cell biology↗

bioRxiv2020

In vivo human whole-brain Connectom diffusion MRI dataset at 760 μm isotropic resolution

We present a whole-brain in vivo diffusion MRI (dMRI) dataset acquired at 760 m isotropic resolution and sampled at 1260 q-space points across 9 two-hour sessions on a single healthy subject. The creation of this benchmark dataset is possible through the synergistic use of advanced acquisition hardware and software including the high-gradient-strength Connectom scanner, a custom-built 64-channel phased-array coil, a personalized motion-robust head stabilizer, a recently developed SNR-efficient dMRI acquisition method, and parallel imaging reconstruction with advanced ghost reduction algorithm. With its unprecedented resolution, SNR and image quality, we envision that this dataset will have a broad range of investigational, educational, and clinical applications that will advance the understanding of human brain structures and connectivity. This comprehensive dataset can also be used as a test bed for new modeling, sub-sampling strategies, denoising and processing algorithms, potentially providing a common testing platform for further development of in vivo high resolution dMRI techniques. Whole brain anatomical T1-weighted and T2-weighted images at submillimeter scale along with field maps are also made available.

neuroscience↗

bioRxiv2020

Enabling in vivo Analysis Via Nanoparticle-mediated Intracellular Assay Probe Delivery: Using RAS as the Prototype

Many experimental protocols must be executed in vitro due to a lack of cell-permeable analysis probes. For instance, the cellular signaling moderator RAS proteins alternate between the active GTP-binding and the inactive GDP-binding states. Though many GTP analogs can serve as probes for RAS activity analysis, their cell impermeability renders in vivo analysis impossible. On the other hand, the lipid/calcium/phosphate (LCP) nanoparticle has enabled efficient intracellular delivery of a nucleotide analog as a chemotherapy agent. Thus, using RAS analysis and LCP nanoparticle as the prototype, we tackled the cell-impermeability issue via nanoparticle-mediated intracellular delivery of the analysis probe. Briefly, BODIPY-FT-GTP-{gamma}-S, a GTP analog that becomes fluorescent only upon protein binding, was chosen as the analysis probe, so that GTP binding can be quantified by fluorescent activity. BODIPY-FT-GTP-{gamma}-S-loaded LCP-nanoparticle was synthesized for efficient intracellular BODIPY-FT-GTP-{gamma}-S delivery. Binding of the delivered BODIPY-FT-GTP-{gamma}-S to the RAS proteins were consistent with previously reported observations; the RAS GTP binding activity was reduced in serum-starved cells; and a transient activation peak of the binding activity was observed upon subsequent serum reactivation of the cells. In a word, nanoparticle-mediated probe delivery enabled an in vivo RAS analysis method. The approach should be applicable to a wide variety of analysis protocols.

biochemistry↗