Biology subjects

Poudel, C.

Publications and source records attributed to Poudel, C..

12 recordsLinked to original sources

bioRxiv2026

A Human Precision Cut Kidney Slice Platform for Studying Glomerular Biology and Therapeutic Responses

Introduction: Short term human precision cut kidney slice cultures (PCKS) represent a tractable experimental that contains mature kidney cell types within their native anatomic context and that can be manipulated in vitro. In this study, we employ genomic tools to analyze the PCKS response to culture conditions and their suitability for study of glomerular disease biology. Methods: The impact of culture medium and oxygenation on PCKS viability and gene expression was explored using spatial transcriptomics and traditional histologic approaches. The biological response of PCKS to IFN{gamma} stimulation and signaling suppression by the JAK1/2 inhibitor baricitinib were also explored. Results: A genome wide atlas of glomerular and tubulointerstitial gene expression patterns was generated from PCKS cultured in different media and oxygenation conditions and at various timepoints in culture. IFN{gamma} induced APOL1 gene and protein expression in glomeruli, which could be suppressed by baricitinib. Expression changes were reproducible in PCKS derived from multiple donors. Conclusions: PCKS are reproducibly responsive to IFN{gamma} stimulation, which will allow dissection of APOL1 mediated glomerular disease mechanisms in a human tissue model system. Spatial transcriptomic profiling of tissue responses helps to tailor culture medium/oxygenation to optimize the PCKS system to address specific biological questions.

cell biology↗

bioRxiv2026

High-throughput, organ-scale 3D tubule tracking using TubuleMAP

Advances in tissue clearing and lightsheet microscopy enable mesoscale imaging of intact and convoluted tubular networks, yet analytical tools to map tubule continuity and assess injury patterns within and across tubules are limited. Here, we introduce TubuleMAP, a semi-automated pipeline for 3D tubule tracking and reconstruction that adapts to various morphological and staining patterns, leverages parallel processing of terabyte-scale data for large-scale analysis of tubular networks, and uses a napari interface for human oversight. Using TubuleMAP, we reconstruct 1,000 intact mouse nephrons in [~]1-millimeter-thick kidney slab with [~]400-fold higher throughput and <1% human effort compared to prior approaches. These reconstructions enable analysis of mesoscale nephron organization, quantitative profiling of pathologic morphologies, whole-nephron cytometry, and identification of rare morphologies at unprecedented scales. We demonstrate generalizability by reconstructing all seminiferous tubules in a mouse testis within a day. TubuleMAP is released as an open-source Python package.

bioengineering↗

bioRxiv2024

Nanoscale Optical Imaging, Reconstruction, and Spatial Analysis of Whole Mouse Glomeruli

Renal glomeruli have traditionally been studied by micrometer-scale optical microscopy to interrogate overall physiology or molecular distributions and by nanoscale electron microscopy to interrogate the ultrastructure of thin sections. While these approaches are powerful, they have been limited in their ability to obtain detailed views of the glomeruli as holistic 3D functional units. To fill this knowledge gap, we have developed a novel pipeline for imaging, reconstructing, and analyzing whole mouse glomeruli at 100 nm resolution using super-resolution fluorescence microscopy. This pipeline integrates both manual and machine learning approaches to annotate and analyze glomerular structures. Using this method, we created 18 detailed glomerulus models, from a range of healthy, aged, and model diseased mice, that outline all major structures and cell types. These models have been made publicly accessible in an online repository, providing a valuable resource for further studies. Our results also uncovered a diverse set of novel phenotypes including nuclear enlargement in all glomerular cell types in aging and disease, as well as an aging-related pattern of regional thickening of the Bowmans capsule basement membrane near the tubular-glomerular junction.

physiology↗

bioRxiv2023

Highly Multiplexed Fluorescence Microscopy with Spectrally Tunable Semiconducting Polymer Dots

Current studies of biological tissues require visualizing diverse cell types and molecular interactions, creating a growing need for versatile techniques to simultaneously probe numerous targets. Traditional multiplexed imaging is limited to around five targets at once. Emerging methods utilizing sequential rounds of staining, imaging, and signal removal can probe tens of targets but require specialized hardware, time-consuming workflows, and face some challenges with sample distortion and artifacts. Here we present a new method for highly-multiplexed fluorescence microscopy using semiconducting polymer dots (Pdots) in a single round of staining and imaging. Pdots are small, bright, and photostable fluorescent probes with a wide range of tunable Stokes shifts (20-450 nm). Multiple series of Pdots with varying excitation wavelengths allow for fast (<1 minute) and single-round imaging of up to 21 targets in brain and kidney. This method is based on a simple immunofluorescence workflow, efficient use of spectral space, standard hardware, and straightforward analysis, making it widely applicable for bioimaging laboratories.

biochemistry↗

bioRxiv2023

An end-to-end workflow for non-destructive 3D pathology

Recent advances in 3D pathology offer the ability to image orders-of-magnitude more tissue than conventional pathology while providing a volumetric context that is lacking with 2D tissue sections, all without requiring destructive tissue sectioning. Generating high-quality 3D pathology datasets on a consistent basis is non-trivial, requiring careful attention to many details regarding tissue preparation, imaging, and data/image processing in an iterative process. Here we provide an end-to-end protocol covering all aspects of a 3D pathology workflow (using light-sheet microscopy as an illustrative imaging platform) with sufficient detail to perform well-controlled preclinical and clinical studies. While 3D pathology is compatible with diverse staining protocols and computationally generated color palettes for visual analysis, this protocol will focus on a fluorescent analog of hematoxylin and eosin (H&E), which remains the most common stain for gold-standard diagnostic determinations. We present our guidelines for a broad range of end-users (e.g., biologists, clinical researchers, and engineers) in a simple tutorial format.

pathology↗

bioRxiv2022

A unified in vitro to in vivo fluorescence lifetime screening platform yields amyloid β aggregation inhibitors

Inhibiting the aggregation of amyloid {beta} (1-42) is a promising strategy for the development of disease-modifying Alzheimers disease therapeutics. To date, however, no sufficiently efficacious inhibitors have been identified, despite the best efforts of >200 advanced drug development campaigns. This failure can be attributed to limitations in current compound screening and in vivo validation assays. Here, we report an in vitro to in vivo screening platform based on the use of a fluorescence lifetime aggregation sensor. The microfluidic "nanoFLIM" assay developed circumvents issues that plague conventional assays, such as lack of reproducibility, high cost and artefactual false read-outs. The fluorescence lifetime sensor can also dynamically monitor peptide aggregation in cellular and Caenorhabditis elegans disease models, providing directly comparable aggregation kinetics, which is not achievable by any other method. The power of this unified system for accelerating hit-to-lead strategies, lowering attrition rates and expediting in vivo screening, was demonstrated with a pilot screening campaign of 445 compounds, revealing a new inhibitor that can inhibit amyloid {beta} self-assembly in vitro as well as in cellular and whole organism disease models.

molecular biology↗

bioRxiv2020

Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies

Misfolded -synuclein is a major component of Lewy bodies, which are a hallmark of Parkinsons disease. A large body of evidence shows that -synuclein can self-assemble into amyloid fibrils, but the relationship between amyloid formation and Lewy body formation still remains unclear. Here we show, both in vitro and in a C. elegans model of Parkinsons disease, that -synuclein undergoes liquid-liquid phase separation by forming a liquid droplet state, which converts into an amyloid-rich hydrogel. This maturation process towards the amyloid state is delayed in the presence of model synaptic vesicles in vitro. Taken together, these results suggest that the formation of Lewy bodies is linked to the arrested maturation of -synuclein condensates in the presence of lipids and other cellular components.

molecular biology↗

bioRxiv2020

Satellite repeat transcripts modulate heterochromatin condensates and safeguard chromosome stability in mouse embryonic stem cells

Heterochromatin maintains genome integrity and function, and is organised into distinct nuclear domains. Some of these domains are proposed to form by phase separation through the accumulation of HP1. Mammalian heterochromatin contains noncoding major satellite repeats (MSR), which are highly transcribed in mouse embryonic stem cells (ESCs). Here, we report that MSR transcripts can drive the formation of HP1 droplets in vitro, and scaffold heterochromatin into dynamic condensates in ESCs, leading to the formation of large nuclear domains that are characteristic of pluripotent cells. Depleting MSR transcripts causes heterochromatin to transition into a more compact and static state. Unexpectedly, changing heterochromatins biophysical properties has severe consequences for ESCs, including chromosome instability and mitotic defects. These findings uncover an essential role for MSR transcripts in modulating the organisation and properties of heterochromatin to preserve genome stability. They also provide new insights into the processes that could regulate phase separation and the functional consequences of disrupting the properties of heterochromatin condensates.

molecular biology↗

bioRxiv2020

Illuminating chromatin compaction in live cells and fixed tissues using SiR-DNA fluorescence lifetime

The global compaction state of chromatin in a nucleus is an important component of cell identity that has been difficult to measure. We have developed a quantitative method to measure the chromatin compaction state in both live and fixed cells, without the need for genetic modification, using the fluorescence lifetime of SiR-DNA dye. After optimising this method using live cancer cell lines treated to induce chromatin compaction or decompaction, we observed chromatin compaction in differentiating epithelial cells in fixed tissue sections, as well as local decompaction foci that may represent transcription factories. In addition, we shed new light on chromatin decompaction during embryonic stem cell transition out of their naive pluripotent state. This method will be useful to studies of nuclear architecture, and may be easy, cheap, and accessible enough to serve as a general assay of stem-ness.

cell biology↗

bioRxiv2020

Pyruvate kinase controls signal strength in the insulin secretory pathway

Pancreatic {beta}-cells couple nutrient metabolism with appropriate insulin secretion. Here, we show that pyruvate kinase (PK), which converts ADP and phosphoenolpyruvate (PEP) into ATP and pyruvate, underlies {beta}-cell sensing of both glycolytic and mitochondrial fuels. PK present at the plasma membrane is sufficient to close KATP channels and initiate calcium influx. Small-molecule PK activators increase {beta}-cell oscillation frequency and potently amplify insulin secretion. By cyclically depriving mitochondria of ADP, PK restricts oxidative phosphorylation in favor of the mitochondrial PEP cycle with no net impact on glucose oxidation. Our findings support a compartmentalized model of {beta}-cell metabolism in which PK locally generates the ATP/ADP threshold required for insulin secretion, and identify a potential therapeutic route for diabetes based on PK activation that would not be predicted by the {beta}-cell consensus model. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=164 HEIGHT=200 SRC="FIGDIR/small/907790v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1f6503forg.highwire.dtl.DTLVardef@e9393org.highwire.dtl.DTLVardef@fab08borg.highwire.dtl.DTLVardef@5c7c55_HPS_FORMAT_FIGEXP M_FIG C_FIG The consensus model for {beta}-cell glucose sensing supports a dominant role for OxPhos. This model doesnt fully explain the observed metabolic and electrophysiologic oscillations associated with glucose-stimulated insulin secretion. Lewandowski et al. challenge this model by mechanistically connecting the anaplerotic PEP cycle to the electrically silent triggering phase, and OxPhos to the electrically active secretory phase. Here, the allosteric recruitment of pyruvate kinase directs metabolic traffic between the two cycles and identifies potential therapeutic strategies for diabetes based on pharmacologic pyruvate kinase activation. HIGHLIGHTSO_LICompartmentalized pyruvate kinase (PK) activity underlies {beta}-cell fuel sensing C_LIO_LIMembrane-associated PK closes KATP channels and controls calcium influx C_LIO_LIBy lowering ADP, PK toggles mitochondria between OxPhos and PEP biosynthesis C_LIO_LIPharmacologic PK activation increases oscillatory frequency and amplifies secretion C_LI

cell biology↗

bioRxiv2018

Intrinsically aggregation-prone proteins form amyloid-like aggregates and contribute to tissue aging in C. elegans

Reduced protein homeostasis and increased protein instability is a common feature of aging. Yet it remains unclear whether protein instability is a cause of aging. In neurodegenerative diseases and amyloidoses, specific proteins self-assemble into amyloid fibrils and accumulate as pathological solid aggregates in a variety of tissues. More recently, widespread protein aggregation has been described during normal aging, in the absence of disease processes. Until now, an extensive characterization of the nature of age-dependent protein aggregation and its consequences for aging has been lacking. Here, we show that age-dependent aggregates are rapidly formed by newly synthesized proteins and contain amyloid-like structures similar to disease-associated protein aggregates. Moreover, we demonstrate that age-dependent protein aggregation accelerates the functional decline of different tissues in C. elegans. Together, these finding reveal that the formation of amyloid aggregates is a generic problem of aging and likely to be an important target for strategies designed to maintain physiological functions in later stages of life.

cell biology↗

bioRxiv2018

Fast fluorescence lifetime imaging reveals the maturation process of α-synuclein aggregates in ageing Caenorhabditis elegans

The nematode worm Caenorhabditis elegans has emerged as an important model organism to study the molecular mechanisms of protein misfolding diseases associated with amyloid formation because of its small size, ease of genetic manipulation and optical transparency. Obtaining a reliable and quantitative read-out of protein aggregation in this system, however, remains a challenge. To address this problem, we here present a fast time-gated fluorescence lifetime imaging (TG-FLIM) method and show that it provides functional insights into the process of protein aggregation in living animals by enabling the rapid characterisation of different types of aggregates. More specifically, in longitudinal studies of C. elegans models of Parkinsons and Huntingtons diseases, we observed marked differences in the aggregation kinetics and the nature of the protein inclusions formed by -synuclein and polyglutamine. In particular, we found that -synuclein inclusions do not display amyloid-like features until late in the life of the worms, whereas polyglutamine forms amyloid characteristics rapidly in early adulthood. Furthermore, we show that the TG-FLIM method is capable of imaging live and non-anaesthetised worms moving in specially designed agarose micro-chambers. Taken together, our results show that the TG-FLIM method enables high-throughput functional imaging of living C. elegans that can be used to study in vivo mechanisms of aggregation and that has the potential to aid the search for therapeutic modifiers of protein aggregation and toxicity.

biophysics↗