Biology subjects

Chang, H.-J.

Publications and source records attributed to Chang, H.-J..

8 recordsLinked to original sources

bioRxiv2026

Single Cell-Type Spatial Proteomics Uncovers Regional Heterogeneity of Astrocytes

Astrocytes are a subset of glial cells in the central nervous system (CNS) that support numerous processes essential for brain function. Their functional diversity is thought to arise from specialized subpopulations with distinct molecular profiles. Although single-cell and single-nucleus RNA sequencing (scRNA-seq and snRNA-seq) have greatly advanced our understanding of astrocyte transcriptomic heterogeneity, mRNA abundance does not always correlate with protein levels because of post-transcriptional and translational regulation. Therefore, studying protein profiles remains essential to accurately capture astrocyte functional states and heterogeneity. Here, we used Microscoop Mint, a microscopy-guided spatial proteomics platform that integrates subcellular, region-specific sample preparation with LC-MS/MS-based mass spectrometry, enabling direct protein profiling of astrocytes in paraformaldehyde-fixed, optimal cutting temperature (OCT)-embedded mouse brain tissue. By applying this approach, we uncovered distinct regional-associated astrocyte proteomic signatures in the cerebral cortex and hippocampus and selected novel candidate protein markers for subsequent validation by immunofluorescence. Notably, MINK1 and PLEKHB1 showed preferential expression in hippocampal and cortical astrocytes, respectively, highlighting their potential as region-specific astrocyte markers. Overall, this strategy enables high-precision, unbiased spatial proteomic discovery at subcellular resolution, providing a powerful framework for linking molecular diversity to functional specialization in astrocyte biology.

neuroscience↗

bioRxiv2025

Microscopy-Guided Spatial Proteomics Reveals Novel Proteins at the Mitochondria-Lipid Droplet Interface and Their Role in Lipid Metabolism

Mitochondria-lipid droplet (LD) interactions play a critical role in lipid metabolism and the progression of metabolic diseases such as non-alcoholic fatty liver disease (NAFLD). However, the dynamic nature of these interactions has hindered the identification of novel protein constituents and their functional roles. Here, we employed Microscoop Mint, an advanced microscopy-guided spatial proteomics platform, to isolate proteins localized at mitochondria-LD contact sites in oleic acid (OA)-treated HepG2 cells, an in vitro model for studying fatty liver disease. Microscoop Mint integrates high-resolution image analysis and two-photon illumination to achieve precise biotinylation of proteins at subcellular regions of interest. Coupled with mass spectrometry, this approach identified a proteome enriched at the mitochondria-LD interface, including both well-characterized lipid-associated proteins and previously unrecognized candidates. Among the 373 common proteins identified across replicates, five novel candidates with no prior association to lipid metabolism were selected for validation. Immunofluorescence staining confirmed their localization at mitochondria-LD contact sites, with more pronounced association in OA-treated cells. Notably, suppression of one candidate, FHL3, led to reduced LD size, elongated mitochondrial morphology, and diminished mitochondria-LD interactions, suggesting its role in regulating fatty acid {beta}-oxidation. Our findings demonstrate the utility of Microscoop Mint in unveiling novel molecular players at organelle contact sites. This study not only provides insights into the functional dynamics of mitochondria-LD interactions but also highlights potential therapeutic targets for lipid metabolic disorders and NAFLD.

cell biology↗

bioRxiv2024

p53 protein abundance is a therapeutic window across TP53 mutant cancers and is targetable with proximity inducing small molecules

TP53 mutant cancers are associated with approximately half of cancer deaths. The most common mechanism of p53 inactivation involves missense mutations. Such mutations in TP53 result in a robust upregulation of the p53 protein. Here, we demonstrate an induced proximity approach to selectively kill TP53 mutant cells. This approach uses the increased abundance of p53 protein in TP53 mutant cancer cells to concentrate toxic molecules in these cells. We demonstrate the first generalizable strategy using a small molecule to selectively kill TP53 mutant cells. This molecule binds the Y220C mutant of p53 and concentrates a PLK1 inhibitor in cells harboring TP53 Y220C mutations. Together, these data demonstrate that the abundance of p53 protein provides a therapeutic window for TP53 missense mutant cancers that can be translated into a cell death signal using proximity-inducing small molecules.

biochemistry↗

bioRxiv2024

Pulsed sounds caused by internal oxygen transport during photosynthesis in the seagrass Halophila ovalis

Oxygen bubbles that leak from seagrass blades during photosynthesis have been hypothesized to cause cavitation sounds in aquatic plants. Here we investigate low-amplitude sounds with regular pulse rates produced during photosynthesis in seagrass beds of Halophila ovalis (Qitou Bay, Penghu islands and Cigu Lagoon, Taiwan). Sound pulses appear in the morning when illumination exceeds 10,000 Lux, peak at midday and decrease in midafternoon on a sunny day. Frequencies peak between 1 to 4 kHz, durations range between ca. 1.8 to 4.8 ms, and sound pressure level 1 cm from the bed is 80.3{+/-}2.0 dB re 1Pa (1-3 kHz band, 11:00 on a cloudy day). Sounds attenuate rapidly, disappearing beyond 15 cm. Blocking sunlight or administering herbicide stops ongoing sounds. Gas bubbles are not typically seen during sound production ruling out cavitation, and external force (finger pressing or scissor cutting) applied to the substrate of seagrass patch or leaves, petioles, or rhizomes generally increases pulse rate. We suggest sound emission is caused by internal oxygen transport through pores in diaphragms (a whistle mechanism) at the leaf base and nodes of the rhizome.

plant biology↗

bioRxiv2023

Microscopy-guided subcellular proteomic discovery by high-speed ultra-content photo-biotinylation

Microscopy-guided proteomics at an organelle-dimension resolution is desired for revealing unknown protein constituents at specific disease- or functional-associated regions at the molecular-molecular interactions level. Here, we achieve protein spatial purification by introducing a firmware-integrated microscopy platform that triggers in situ subcellular photo-biotinylation of proteins at user-defined regions of interest (ROIs) one field of view (FOV) at a time for thousands of FOVs fully automatically. An illumination pattern at the analogous ROIs of each FOV is calculated on the fly by either machine learning or traditional image processing. Photoactivatable amino acid crosslinkers are activated by a two-photon focal light one spot at a time at a sub-millisecond illumination duration per spot. Imaging, pattern generation, targeted illumination, and FOV movement are coordinated and cycled with high-speed mechatronic control to complete illumination on millions of ROI spots within hours. Once enough proteins are biotinylated in a cell or tissue sample, the sample is scraped and lysed, and avidin pulldown is used to enrich proteins to achieve spatial protein scooping at a 240-nm precision. Subsequent LC-MS/MS is implemented to reveal the subcellular proteome in high sensitivity, specificity, and resolution. Using this technology termed optoproteomics, we have revealed novel stress granule-localized and amyloid {beta}-localized proteins validated by immunostaining. Together, spatial purification by ultra-content, high-speed microscopy-targeted photo-biotinylation enables unprecedented subcellular spatial proteomics discovery in any microscopically recognizable regions.

cell biology↗

bioRxiv2022

An Optimized Electrotransformation Protocol For Lactobacillus Jensenii.

Engineered bacteria are promising candidates for in situ detection and treatment of diseases. The female uro-genital tract presents several pathologies, such as sexually transmitted diseases or genital cancer, that could benefit from such technology. While bacteria from the gut microbiome are increasingly engineered, the use of chassis isolated from the female uro-genital resident flora has been limited. A major hurdle to implement the experimental throughput required for efficient engineering in these non-model bacteria is their low transformability. Here we report an optimized electrotransformation protocol for Lactobacillus jensenii, one the most widespread species across vaginal microflora. Starting from classical conditions, we optimized buffers, electric field parameters, cuvette type and DNA quantity to achieve an 80-fold improvement in transformation efficiency, with up to 3.5{middle dot}103 CFUs/{micro}g of DNA in L. jensenii ATCC 25258. We also identify several plasmids that are maintained and support reporter gene expression in L. jensenii. Finally, we demonstrate that our protocol provides increased transformability in three independent clinical isolates of L. jensenii. This work will facilitate the genetic engineering of L. jensenii and enable its use for addressing challenges in gynecological healthcare.

microbiology↗

bioRxiv2021

A synthetic receptor platform enables rapid and portable monitoring of liver dysfunction via engineered bacteria.

Bacterial biosensors, or bactosensors, are promising field-deployable agents for medical and environmental diagnostics. However, the lack of scalable frameworks to systematically program ligand detection limits their applications. Here we present a synthetic receptor platform, termed EMeRALD (Engineered Modularized Receptors Activated via Ligand-induced Dimerization) which supports the modular assembly of sensing modules onto a high-performance, generic signaling scaffold controlling gene expression in E. coli. We applied EMeRALD to detect bile salts, a biomarker of liver dysfunction, by repurposing sensing modules from enteropathogenic Vibrio species. We improved the sensitivity and lowered the limit-of-detection of the sensing module by directed evolution. We then engineered a colorimetric bactosensor detecting pathological bile salt levels in serum from patients having undergone liver transplant, providing an output detectable by the naked-eye. The EMeRALD technology enables functional exploration of natural sensing modules and rapid engineering of synthetic receptors for diagnostics, environmental monitoring, and control of therapeutic microbes.

synthetic biology↗

bioRxiv2021

An engineered lactate responding promoter system operating in glucose-rich and anaerobic environments.

Bacteria equipped with genetically-encoded lactate biosensors would support several applications in biopharmaceutical production, diagnosis, or therapeutics. However, many applications involve glucose-rich and anaerobic environments, in which current whole-cell lactate biosensors have low performance. Here we engineered a synthetic lactate biosensor system by repurposing the natural LldPRD promoter regulated by the LldR transcriptional regulator. We removed glucose catabolite repression by designing a hybrid promoter, containing LldR operators and tuned both regulator and reporter gene expression to optimize biosensor signal-to-noise ratio. The resulting lactate biosensor, termed ALPaGA (A Lactate Promoter Operating in Glucose and Anaerobia) can operate in glucose rich, aerobic and anaerobic conditions. Our work provides a versatile lactate biosensing platform suitable for many environmental conditions.

synthetic biology↗