Biology subjects

Chan, R.

Publications and source records attributed to Chan, R..

11 recordsLinked to original sources

bioRxiv2026

Q-DOAS: A Proximity Quenching Assay for Real-Time Detection of Early Protein Aggregation Events

Accurate measurement of protein aggregation is essential for studying neurodegenerative diseases. The standard ThT assay reports on amyloid formation but is blind to early oligomers and is prone to interference. We describe Q-DOAS, a plate-reader assay that quantifies protein self-assembly in real time via proximity-quenching of a single, site-specifically conjugated dye (BODIPY-TMR). Using mutant Huntingtin-exon 1 (mHTT-Ex1) and -Synuclein A53T, we show Q-DOAS detects pre-amyloid oligomers, yielding quantitative kinetic data compatible with mechanistic analysis. We demonstrate its utility to dissect mutational effects, screen for protein and small-molecule inhibitors, and quantify amyloid seeding activity in cellular and mouse models of Huntingtons disease. Q-DOAS also detects seeds in cerebrospinal fluid from Parkinsons disease patients without amplification. Q-DOAS provides a sensitive, robust, and scalable tool for studying the earliest events in amyloid pathologies and for advancing therapeutic development.

biochemistry↗

bioRxiv2025

SOX9 is part of a combinatorial marker that reveals early development and embryological origins of the mouse brown adipose tissue depots

Brown adipose tissue (BAT) is a mammalian thermogenic tissue that helps to maintain body temperature and provides important metabolic functions. Most brown adipocytes are located in structures called depots, and the development of depots is incompletely understood. Here, we define the embryonic positions in which the three major mouse BAT depots arise using a combination of morphological landmarks and expression of SOX9, a transcription factor that we find, by immunofluorescence and genetic lineage tracing, marks brown adipocyte progenitors from around the time of their exit from dermomyotome. Lineage tracing is also used to confirm the origin of the largest depot, interscapular BAT, in Pax7-positive dermomyotome of somites, and to show that the scapular BAT has a similar embryological origin, while cervical BAT arises from a somitic source outside of the Pax7 lineage. Finally, using Sox9 as part of a novel combinatorial marker, we profile the transcriptome of early brown adipocyte progenitors and pre-adipocytes and model their trajectory, revealing pathways including the paracrine signaling environment, in which brown adipocytes of the major depots likely arise. Our results apply to understanding the earliest stages of development of this novel mammalian tissue.

developmental biology↗

bioRxiv2024

TMEM165 replenishes lysosomal calcium stores, protects cells against calcium overload, and mediates calcium-induced lysosomal H+ leakage

The proper function of lysosomes depends on their ability to store and release calcium. While several lysosomal calcium release channels have been described, how mammalian lysosomes replenish their calcium stores has not been determined. Using genetic depletion and overexpression techniques combined with electrophysiology and visualization of subcellular ion concentrations and their fluxes across the lysosomal membrane, we show here that TMEM165 imports calcium to the lysosomal lumen and mediates calcium-induced lysosomal proton leakage. Accordingly, TMEM165 accelerates the recovery of cells from cytosolic calcium overload thereby enhancing cell survival while causing a significant acidification of the perilysosomal area and the entire cytosol. These data indicate that in addition to its essential role in the glycosylation in the Golgi, a small but significant fraction of TMEM165 localizes on the lysosomal limiting membrane, where it protects cells against cytosolic calcium overload, preserves lysosomal function by refilling lysosomal calcium stores and regulates perilysosomal and lysosomal pH.

cell biology↗

bioRxiv2024

TL1A/DR3 signaling regulates the generation of pathogenic Th9 cells in experimental inflammatory bowel disease

ObjectiveDeath receptor 3 (DR3) and its ligand tumor necrosis factor like ligand 1A (TL1A), are involved in the regulation of the balance between effector and regulatory T cells in IBD. New evidence suggests a role of IL-9-secreting Th9 cells in the pathogenesis of ulcerative colitis (UC), although the molecular pathways through which IL-9 and Th9 cells may mediate intestinal inflammation in Crohns disease (CD) are still unclear. DesignWe investigated the role of DR3 signaling in the differentiation of Th9 cells in mouse models of CD-like ileitis and colitis, including SAMP1/YitFc (SAMP) mice. ResultsPolarized-Th9 cells with functional DR3 from SAMP WT (Th9WT) harbor a pro-inflammatory signature compared to DR3-deficient Th9 cells that were obtained from DR3-/-xSAMP mice (Th9KO). Conversely, ablation of DR3 signaling generated anti-inflammatory responses, as reflected by higher numbers of IL-10 producing cells in DR3-/-xSAMP mice. Additionally, RNA-seq and phosphoproteomic analyses showed that inflammatory pathways are significantly more activated in Th9WT than in Th9KO cells. Finally, in the T-cell adoptive transfer model, Th9KO cells were less colitogenic than Th9WT, while IL-9 blockade diminished the severity of intestinal inflammation, indicating a crucial role of functional DR3 receptor in Th9 cells pathogenicity. ConclusionWe describe herein that a functional DR3 receptor is required for the pathogenicity of Th9 cells, thus, constituting a novel mechanism by which TL1A/DR3 signaling mediates experimental CD-like ileitis. The TL1A/DR3/Th9 pro-inflammatory pathway may offer a novel therapeutic target for patients with CD. What is already known on this topic - TL1A/DR3 system plays a pivotal role in the pathogenesis of Crohns disease like ileitis. - Th9 cells are a novel subset of T lymphocytes mainly producing the pro-inflammatory cytokine IL-9 which contributes to intestinal inflammation. - Those finding provided us with a strong rationale to investigate IL-9-producing cells in our SAMP1/YitFc mouse model of CD-like ileitis. What this study adds - DR3 receptor is involved in the regulation and progression of intestinal inflammation by promoting Th9 cell differentiation and pathogenicity. - Using RNA-seq and phosphoproteomic comparative analyses we were able to characterize Th9 cells with and without functional DR3 receptor showing that presence of DR3 confers a pro-inflammatory signature to Th9 cells. - We describe a novel role of DR3 in Th9 cells development that appears to regulate their pro-inflammatory phenotype in models of CD-like ileitis and colitis. How this study might affect research, practice or policy - TLA1/DR3 axis and Th9 cells may be useful as therapeutic targets in CD.

immunology↗

bioRxiv2022

Strain dropouts reveal interactions that govern the metabolic output of the gut microbiome

The gut microbiome is complex, raising questions about the role of individual strains in the community. Here, we address this question by focusing on a functional unit within the community, the metabolic niche that controls bile acid 7-dehydroxylation. By constructing variants of a complex defined community in which we drop out strains that occupy this niche, we explore how interactions within and between niches shape community-level metabolism. Omitting both members of the niche, Clostridium scindens (Cs) and Clostridium hylemonae (Ch), eliminates secondary bile acid production and reshapes the community in a highly specific manner: eight strains go up or down in relative abundance by >100-fold, while the remaining strains are largely unaffected. In single-strain dropout communities (i.e., a strain swap within the niche), Cs and Ch reach the same relative abundance and dehydroxylate bile acids to a similar extent. However, the effect on strains in other niches differs markedly: Clostridium sporogenes increases >1000-fold in the {Delta}Cs but not {Delta}Ch dropout, reshaping the pool of microbiome-derived phenylalanine metabolites. Thus, strains that are functionally redundant within a niche can have widely varying impacts outside the niche, and a strain swap can ripple through the community in an unpredictable manner, resulting in a large impact on an unrelated community-level phenotype. Mice colonized by the {Delta}Cs{Delta}Ch community show decreased liver steatosis relative to those colonized by the {Delta}Ch community, demonstrating that a single strain from the microbiome can have a substantive impact on host physiology. Our work opens the door to the mechanistic studies of the role of an individual strain on community ecology and host physiology.

microbiology↗

bioRxiv2022

Spatial transcriptomics of FFPE pancreatic intraepithelial neoplasias reveals cellular and molecular alterations of progression to pancreatic ductal carcinoma

Spatial transcriptomics (ST) is a powerful new approach to characterize the cellular and molecular architecture of the tumor microenvironment. Previous single-cell RNA-sequencing (scRNA-seq) studies of pancreatic ductal adenocarcinoma (PDAC) have revealed a complex immunosuppressive environment characterized by numerous cancer associated fibroblasts (CAFs) subtypes that contributes to poor outcomes. Nonetheless, the evolutionary processes yielding that microenvironment remain unknown. Pancreatic intraepithelial neoplasia (PanIN) is a premalignant lesion with potential to develop into PDAC, but the formalin-fixed and paraffin-embedded (FFPE) specimens required for PanIN diagnosis preclude scRNA-seq profiling. We developed a new experimental pipeline for FFPE ST analysis of PanINs that preserves clinical specimens for diagnosis. We further developed novel multi-omics analysis methods for threefold integration of imaging, ST, and scRNA-seq data to analyze the premalignant microenvironment. The integration of ST and imaging enables automated cell type annotation of ST spots at a single-cell resolution, enabling spot selection and deconvolution for unique cellular components of the tumor microenvironment (TME). Overall, this approach demonstrates that PanINs are surrounded by the same subtypes of CAFs present in invasive PDACs, and that the PanIN lesions are predominantly of the classical PDAC subtype. Moreover, this new experimental and computational protocol for ST analysis suggests a biological model in which CAF-PanIN interactions promote inflammatory signaling in neoplastic cells which transitions to proliferative signaling as PanINs progress to PDAC. SummaryPancreatic intraepithelial neoplasia (PanINs) are pre-malignant lesions that progress into pancreatic ductal adenocarcinoma (PDAC). Recent advances in single-cell technologies have allowed for detailed insights into the molecular and cellular processes of PDAC. However, human PanINs are stored as formalin-fixed and paraffin-embedded (FFPE) specimens limiting similar profiling of human carcinogenesis. Here, we describe a new analysis protocol that enables spatial transcriptomics (ST) analysis of PanINs while preserving the FFPE blocks required for clinical assessment. The matched H&E imaging for the ST data enables novel machine learning approaches to automate cell type annotations at a single-cell resolution and isolate neoplastic regions on the tissue. Transcriptional profiles of these annotated cells enable further refinement of imaging-based cellular annotations, showing that PanINs are predominatly of the classical subtype and surrounded by PDAC cancer associated fibroblast (CAF) subtypes. Applying transfer learning to integrate ST PanIN data with PDAC scRNA-seq data enables the analysis of cellular and molecular progression from PanINs to PDAC. This analysis identified a transition between inflammatory signaling induced by CAFs and proliferative signaling in PanIN cells as they become invasive cancers. Altogether, this integration of imaging, ST, and scRNA-seq data provides an experimental and computational approach for the analysis of cancer development and progression.

cancer biology↗

bioRxiv2021

γδ-Thymocyte Maturation and Emigration

The thymus is the site of both {beta} and {gamma}{delta}-T cell development. After several unique waves of {gamma}{delta}-T cells are generated in, and exported from, the fetal/neonatal thymus, the adult thymus continues to produce a stream of {gamma}{delta}-T cells throughout life. One intriguing feature of {gamma}{delta} T cell development is the coordination of differentiation and acquisition of effector function within the fetal thymus, however, it is less clear whether this paradigm holds true in adult animals. To investigate the relationship between maturation and time since V(D)J recombination in adult-derived {gamma}{delta}-thymocytes, we used the Rag2pGFP model. Immature (CD24+) {gamma}{delta}-thymocytes expressed high levels of GFP while only a small minority of mature (CD24-) {gamma}{delta}-thymocytes were GFP+. Similarly, most GFP+ {gamma}{delta}-splenocytes were immature, while some were mature. Analysis of {gamma}{delta}-recent thymic emigrants (RTEs) indicated that most {gamma}{delta}-T cell RTEs were CD24+ and GFP+ and adoptive transfer experiments showed that immature {gamma}{delta}-thymocytes could be maintained in the periphery for at least 3 days over which time they matured. With respect to the mature {gamma}{delta}-thymocytes that were GFP-, parabiosis experiments demonstrated that mature {gamma}{delta}-T cells did not recirculate from the periphery. Instead, a population of mature {gamma}{delta}-thymocytes remained resident in the thymus for at least 60 days while mature {gamma}{delta}-thymocytes derived solely from adult hematopoiesis were mostly lost from the thymus within 60 days. Collectively, these data demonstrate two streams of actively developing {gamma}{delta}-T cells in adult mice: an immature subset that quickly leaves the thymus and matures in the periphery, and one that completes maturation within the thymus over a longer period of time. Furthermore, there is a fetal-derived and heterogeneous population of resident {gamma}{delta}-thymocytes of unknown functional importance.

immunology↗

bioRxiv2020

Quantitative study of unsaturated transport of glycerol through aquaglyceroporin that has high affinity for glycerol

Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=37 SRC="FIGDIR/small/152512v1_ufig1.gif" ALT="Figure 1"> View larger version (9K): org.highwire.dtl.DTLVardef@19dcc7forg.highwire.dtl.DTLVardef@dce74dorg.highwire.dtl.DTLVardef@d599b4org.highwire.dtl.DTLVardef@15e24f5_HPS_FORMAT_FIGEXP M_FIG C_FIG ABSTRACTThe structures of several aquaglyceroporins have been resolved to atomic resolution showing two or more glycerols bound inside a channel and confirming a glycerol-facilitators affinity for its substrate glycerol. However, the kinetics data of glycerol transport experiments all point to unsaturated transport that is characteristic of low substrate affinity in terms of the Michaelis-Menten kinetics. In this article, we present an in silico-in vitro research focused on AQP3, one of the human aquaglyceroporins that is natively expressed in the abundantly available erythrocytes. We conducted 2.1 s in silico simulations of AQP3 embedded in a model erythrocyte membrane with intracellular-extracellular asymmetries in leaflet lipid compositions and compartment salt ions. From the equilibrium molecular dynamics (MD), we elucidated the mechanism of glycerol transport at high substrate concentrations. From the steered MD simulations, we computed the Gibbs free-energy profile throughout the AQP3 channel. From the free-energy profile, we quantified the kinetics of glycerol transport that is unsaturated due to glycerol-glycerol interaction mediated by AQP3 resulting in the concerted movement of two glycerol molecules for the transport of one glycerol molecule across the cell membrane. We conducted in vitro experiments on glycerol uptake into human erythrocytes for a wide range of substrate concentrations and various temperatures. The experimental data quantitatively validated our theoretical-computational conclusions on the unsaturated glycerol transport through AQP3 that has high affinity for glycerol.

biophysics↗

bioRxiv2020

In silico simulations of erythrocyte aquaporins with quantitative in vitro validation

Modelling water and membrane lipids is an essential element in the computational research of biophysical/biochemical processes such as water transport across the cell membrane. In this study, we examined the accuracies of two popular water models, TIP3P and TIP4P, in the molecular dynamics simulations of erythrocyte aquaporins (AQP1 and AQP3). We modelled the erythrocyte membrane as an asymmetric lipid bilayer with appropriate lipid compositions of its inner and outer leaflet, in comparison with a symmetric lipid bilayer of a single lipid type. We computed the AQP1/3 permeabilities with the transition state theory with full correction for recrossing events. We also conducted cell swelling assays for water transport across the erythrocyte membrane. The experimental results agree with the TIP3P water-erythrocyte membrane model, in confirmation of the expected accuracy of the erythrocyte membrane model, the TIP3P water model, and the CHARMM parameters for water-protein interactions.

biophysics↗

bioRxiv2018

Weighted Network Density Predicts Range of Latent Variable Model Accuracy

Current experimental techniques impose spatial limits on the number of neuronal units that can be recorded in-vivo. To model the neural dynamics utilizing these sampled data, Latent Variable Models (LVMs) have been proposed to study the common unobserved processes within the system that drives neural activities, through an implicit network with hidden states. Yet, relationships between these latent variable models and widely-studied network connectivity measures remained unclear. In this paper, a biologically plausible latent variable model was first fit to neural activity recorded via 2-photon microscopic calcium imaging in the murine primary visual cortex. Graph theoretic measures were then applied to quantify network properties in the recorded sub-regions. Comparison of weighted network measures with LVM prediction accuracy shows some network measures having a strong relationship with LVM prediction accuracy, while other measures do not have a robust relationship with LVM prediction accuracy. Results show LVM will achieve high accuracy in dense networks.

neuroscience↗

bioRxiv2017

Long-term dysbiosis promotes insulin resistance during obesity despite rapid diet-induced changes in the gut microbiome of mice

The intestinal microbiota and insulin sensitivity are rapidly altered in response to a high fat diet (HFD). It is unclear if gut dysbiosis precedes insulin resistance or vice versa. The initial triggers of diet-induced insulin resistance can differ from mechanisms underlying chronic dysglycemia during prolonged obesity. It is not clear if intestinal dysbiosis contributes to insulin resistance during short-term or long-term HFD-feeding. We found that diet-induced changes in the composition of the fecal microbiome preceded changes in glucose and insulin tolerance at both the onset and removal of a HFD in mice. Dysbiosis occurred after 1-3 days of HFD-feeding, whereas insulin and glucose intolerance manifested by 3-4 days. Antibiotic treatment did not alter glucose tolerance during this short-term HFD period. Conversely, antibiotics improved glucose tolerance in mice with protracted obesity caused by long-term HFD feeding for over 2 months. We also found that microbiota transmissible glucose intolerance only occurred after prolonged diet-induced dysbiosis. Germ-free mice had impaired glucose tolerance when reconstituted with the microbiota from long-term, but not short-term HFD-fed animals. Our results are consistent with intestinal microbiota contributing to chronic insulin resistance and dysglycemia during prolonged obesity, despite rapid diet-induced changes in the taxonomic composition of the fecal microbiota.

biochemistry↗