Biology subjects

Singer, R.

Publications and source records attributed to Singer, R..

11 recordsLinked to original sources

bioRxiv2026

Ultra-high field microstructural MRI of living cortical organoids

Quantitative microstructural magnetic resonance imaging (MRI) can noninvasively characterize tissue configuration at micrometer scales, but clinical uptake is limited by validation and optimization in human-relevant scenarios. Organoids are powerful human-relevant tissue models, yet translation is hampered by lack of non-destructive, longitudinal microstructural assessment. Bridging these gaps, microstructural MRI of living organoids can accelerate MRI biomarker and organoid development and validation. Here, we address key obstacles to enable organoid microstructural MRI. First, we use a unique 28.2 T MRI system to achieve spatial resolution with adequate signal-to-noise ratio and feasible scan times. Second, we implement flexible acquisitions with fast readouts to expand multivariate experimental capacity. Third, we develop a workflow combining 3D MRI and 3D lightsheet microscopy for cross-modality anatomical comparison beyond 2D. Using this platform, we demonstrate microstructural MRI of cortical organoids with resolutions down to (20 {micro}m)3, revealing anisotropy, heterogeneity, maturation-dependent differences, and temporal changes in cortical organoids. Correlative lightsheet microscopy confirms correspondence to axonal and nuclear architecture. This platform enables live-organoid MRI as a complementary tool to human- and animal imaging for robust microstructural assessment.

biophysics↗

bioRxiv2026

Optimized Methods for Measuring Extracellular ATP from Human Airway Epithelial Cells and Bronchoalveolar Lavage Fluid

Extracellular adenosine triphosphate (eATP) is a mediator of purinergic signalling in the airways, implicated in mucociliary function, inflammation, and cough via activation of P2X3 receptors. Elevated airway eATP has been associated with multiple respiratory diseases, yet reliable measurement of eATP remains challenging due to its rapid enzymatic degradation and confounding contributions from intracellular ATP. Here, we describe an optimized, microwell plate-based luminescence method for quantifying eATP from human airway epithelial cell cultures and bronchoalveolar lavage (BAL) fluid with enhanced signal stability. Using a commercially available ATP detection assay with a prolonged luminescence half-life, we introduced a simple 0.45 {micro}m syringe filtration step to remove cells and thereby isolate extracellular ATP. This approach demonstrated ATP specificity via apyrase degradation, and provided a linear detection range from 5 nM to 5 {micro}M. Addition of ATP stabilization buffer preserved ATP levels in cell culture media for at least 4 hours at 4 {degrees}C and in human BAL samples for at least 6 weeks at -80{degrees}C. Applying this method to primary human bronchial epithelial cells revealed detectable eATP release, with preferential secretion at the apical surface under air-liquid interface conditions. Collectively, this optimized assay enables robust, high-throughput, and time-flexible quantification of eATP in both experimental and clinical airway samples. These methods support improved investigation of purinergic signalling in airway health and disease and may facilitate biomarker development relevant to eATP in the airways.

cell biology↗

bioRxiv2025

Identification and classification of abundant RNA-binding proteins in the mouse lens and interactions of Carhsp1, Igf2bp1/ZBP1, and Ybx1 with crystallin and β-actin mRNAs

RNA-binding proteins (RBPs) are critical regulators of mRNAs controlling all processes such as RNA transcription, transport, localization, translation, mRNA:ncRNA interactions, and decay. Cellular differentiation is driven by tissue-specific and/or tissue-preferred expression of proteins needed for the optimal function of mature cells, tissues and organs. Lens fiber cell differentiation is marked by high levels of expression of crystallin genes encoding critical proteins for lens transparency and light refraction. Herein we performed proteomic and transcriptomic analyses of RBPs in differentiating mouse lenses to identify the most abundant RBPs and establish dynamic changes of their expression in differentiating lens. Expression analyses include highly abundant RBPs, including Carhsp1, Igf2bp1/ZBP1, Ybx1, Pabpc1, Ddx39, and Rbm38. Binding sites of Carhsp1, Ybx1, and Igf2bp1/ZBP1 were predicted in various crystallin and {beta}-actin mRNAs. Immunoprecipitations using antibodies against Carhsp1, Igf2bp1/ZBP1, and Ybx1 confirmed their interactions with A-, B-, and {gamma}A-crystallin mRNAs. A combination of single molecule RNA FISH (smFISH) and immunofluorescence was used to probe in vivo interactions of these RBPs with A-, B-crystallin, and {beta}-actin mRNAs in cytoplasm and nucleoplasm of cultured mouse lens epithelial cells. Together, these results open new avenues to perform comprehensive genetic, cell, and molecular biology studies of individual RBPs in the lens.

developmental biology↗

bioRxiv2024

Open-source 3D printed manifolds for exposure studies using human airway epithelial cells

RationaleInhalation of airborne stimuli can damage the airway epithelium, increasing the risk of developing respiratory or systemic diseases. In vitro studies using air-liquid interface cell cultures enable controlled investigation of cellular responses to relevant exposures. Commercial in vitro exposure systems provide precise and reproducible dosage but require significant capital investment and are not amenable to customization. Research groups interested in respiratory exposure science may benefit from a more accessible alternative open-source exposure system. We present 3D printed manifolds for applying a range of airborne exposures uniformly across standard, commercially available 6- and 24-well plates with air-liquid interface culture inserts. MethodsA simple chamber-style exposure system and the manifolds were evaluated for exposure uniformity via computational fluid dynamics simulations and deposition of nebulized FITC-labelled dextran. The chamber and manifolds were manufactured using a stereolithography 3D printer. Cannabis concentrate vapor was generated from 3 different vaporizers and applied to well plates using the manifold system. Calu-3 cells were cultured on Transwell inserts and exposed to whole tobacco smoke or room air. ResultsThe manifolds produced less variation in simulated air velocities and physical deposition of FITC-dextran aerosol deposition across well plates compared to those of the chamber-style exposure system. Distinct doses of cannabis concentrate vapour were delivered to well plates with low variation among wells. Whole tobacco smoke exposure using the manifold system induced functional changes in Calu-3 airway epithelial cell barrier function, cytokine production (IL-6 and IL-8), and cell membrane potential. ConclusionsCollectively, our data demonstrate the feasibility and the validity of our open-source 3D printed manifolds for use in studying various respiratory exposures and position our designs as more accessible options in parallel with commercially available systems. All article content is licensed under a Creative Commons Attribution (CC BY-NC 4.0) license (https://creativecommons.org/licenses/by-nc/4.0/).

bioengineering↗

bioRxiv2023

Common genetic variation impacts molecular stress response in the brain

To explain why individuals exposed to identical stressors experience divergent clinical outcomes, we determine how molecular encoding of stress modifies genetic risk for brain disorders. Analysis of post-mortem brain (n=304) revealed 8557 stress-interactive expression quantitative trait loci (eQTLs) that dysregulate expression of 915 eGenes in response to stress, and lie in stress-related transcription factor binding sites. Response to stress is robust across experimental paradigms: up to 50% of stress-interactive eGenes validate in glucocorticoid treated hiPSC-derived neurons (n=39 donors). Stress-interactive eGenes show brain region- and cell type-specificity, and, in post-mortem brain, implicate glial and endothelial mechanisms. Stress dysregulates long-term expression of disorder risk genes in a genotype-dependent manner; stress-interactive transcriptomic imputation uncovered 139 novel genes conferring brain disorder risk only in the context of traumatic stress. Molecular stress-encoding explains individualized responses to traumatic stress; incorporating trauma into genomic studies of brain disorders is likely to improve diagnosis, prognosis, and drug discovery. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/573459v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1e07fa9org.highwire.dtl.DTLVardef@1dd530forg.highwire.dtl.DTLVardef@17822a5org.highwire.dtl.DTLVardef@2a889f_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2023

Irreversible PDMS bonding using flame activation of adhesives for fabrication of microfluidic and organ-on-chip devices

Polydimethylsiloxane (PDMS) is widely used for microfluidics fabrication in many disciplines due to its ease of use in soft lithography and its ability to bond liquid-tight seals. A variety of PDMS-PDMS bonding methods exist, but each may have limitations for some applications. For example, chemical bonding via oxygen plasma treatment is reliable but requires expensive equipment and specialized training. Here we present a rapid, low-cost, and accessible method for irreversible PDMS bonding in which flame treatment activates PDMS and Nitto 5302A adhesive surfaces. Using this technique, PDMS microchannels can be fabricated with a bonding integrity of up to 325 kPa burst pressure. This technique is suitable for fabricating organ-on-chip devices for cell line and primary cell culture, supporting cell viability and establishment of key cellular features in apical and basal compartments, while maintaining bonding integrity.

bioengineering↗

bioRxiv2023

mRNA Localization and Local Translation of the Microtubule Severing Enzyme, Fidgetin-Like 2, in Polarization, Migration and Outgrowth

Cell motility requires strict spatiotemporal control of protein expression. During cell migration, mRNA localization and local translation in subcellular areas like the leading edge and protrusions are particularly advantageous for regulating the reorganization of the cytoskeleton. Fidgetin-Like 2 (FL2), a microtubule severing enzyme (MSE) that restricts migration and outgrowth, localizes to the leading edge of protrusions where it severs dynamic microtubules. FL2 is primarily expressed during development but in adulthood, is spatially upregulated at the leading edge minutes after injury. Here, we show mRNA localization and local translation in protrusions of polarized cells are responsible for FL2 leading edge expression after injury. The data suggests that the RNA binding protein IMP1 is involved in the translational regulation and stabilization of FL2 mRNA, in competition with the miRNA let-7. These data exemplify the role of local translation in microtubule network reorganization during migration and elucidate an unexplored MSE protein localization mechanism. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/537087v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@139f4e5org.highwire.dtl.DTLVardef@1fd1a4eorg.highwire.dtl.DTLVardef@1ca17b4org.highwire.dtl.DTLVardef@da2b56_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIThe microtubule severing enzyme FL2 RNA is localized to the leading edge C_LIO_LIFL2 mRNA localization leads to FL2 translation within protrusions C_LIO_LIThe IMP family works in concert with Let-7 miRNA to regulate FL2 mRNA C_LI

cell biology↗

bioRxiv2022

Visualization of early RNA replication kinetics of SARS-CoV-2 by using single molecule RNA-FISH

SARS-CoV-2 infection has caused a major global burden. Despite intensive research, the mechanism and dynamics of early viral replication are not completely understood including the kinetics of formation of plus stranded genomic and subgenomic RNAs (gRNA and sgRNA) starting from the RNA from the first virus that enters the cell. We employed single-molecule RNA-fluorescence in situ hybridization (smRNA-FISH) to simultaneously detect viral gRNA and sgRNA in infected cells and carried out a time course analysis to determine the kinetics of their replication. We visualized the single molecules of gRNA within the cytoplasm of infected cells 30 minutes post-infection and detected the co-expression of gRNA and sgRNA within two hours post-infection. Furthermore, we observed the formation of a replication organelle (RO) from a single RNA, which led to the formation of multiple ROs within the same cells. Single molecule analysis indicated that while gRNA resided in the center of these ROs, the sgRNAs were found to radiate and migrate out of these structures. Our results also indicated that after the initial delay, there was a rapid but asynchronous replication, and the gRNA and sgRNAs dispersed throughout the cell within 4-5 hours post-infection forming multiple ROs that filled the entire cytoplasm. These results provide insight into the kinetics of early post-entry events of SARS-CoV-2 and the formation of RO, which will help to understand the molecular events associated with viral infection and facilitate the identification of new therapeutic targets that can curb the virus at a very early stage of replication to combat COVID-19. Author SummarySARS-CoV-2 infection continues to be a global burden. Soon after the entry, SARS-CoV-2 replicates by an elaborate process, producing genomic and subgenomic RNAs (gRNA and sgRNAs) within specialized structures called replication organelles (RO). Many questions including the timing of multiplication of gRNA and sgRNA, the generation, subcellular localization, and function of the ROs, and the mechanism of vRNA synthesis within ROs is not completely understood. Here, we have developed probes and methods to simultaneously detect the viral gRNA and a sgRNA at single cell single molecule resolution and have employed a method to scan thousands of cells to visualize the early kinetics of gRNA and sgRNA synthesis soon after the viral entry into the cell. Our results reveal that the replication is asynchronous and ROs are rapidly formed from a single RNA that enters the cell within 2 hours, which multiply to fill the entire cell cytoplasm within ~4 hours after infection. Furthermore, our studies provide a first glimpse of the gRNA and sgRNA synthesis within ROs at single molecule resolution. Our studies may facilitate the development of drugs that inhibit the virus at the earliest possible stages of replication to minimize the pathogenic impact of viral infection.

microbiology↗

bioRxiv2022

Hidden species diversity in a living fossil vertebrate

Ancient, species-poor lineages persistently occur across the Tree of Life. These evolutionarily unique lineages are likely to contain unrecognized species diversity masked by the low rates of morphological evolution that characterize living fossils [1, 2]. Halecomorphi is a major clade of ray-finned fishes that diverged from its closest relatives over 200 million years ago [3, 4] yet is represented by only one recognized living species in eastern North America, the Bowfin Amia calva Linnaeus. Here, we use double digest restriction-site associated DNA (ddRAD) sequencing and high-resolution computed tomography to illuminate recent speciation in the bowfins. Our results support the resurrection of a second living species of Bowfin with the timing of diversification dating to the Pleistocene. In turn, we expand the species diversity of an ancient lineage that is integral to studies of vertebrate genomics and development [2, 3, 5], yet is facing growing conservation threats driven by the caviar fishery [6].

evolutionary biology↗

bioRxiv2019

MS2-TRIBE evaluates protein-RNA interactions and nuclear organization of transcription by RNA editing

Nearly every step of RNA regulation is mediated by binding proteins (RBPs). The most common method to identify specific RBP target transcripts in vivo is by crosslinking (\"CLIP\" and its variants), which rely on protein-RNA crosslinking and specific antibodies. Another recently introduced method exploits RNA editing, with the catalytic domain of ADAR covalently attached to a specific RBP (\"TRIBE\"). Both approaches suffer from difficulties in distinguishing real RNA targets from false negative and especially false positive signals. To critically evaluate this problem, we used fibroblasts from a mouse where every endogenous {beta}-actin mRNA molecule was tagged with the bacteriophage MS2 RNA stem loops; hence there is only a single bona fide target mRNA for the MS2 capsid protein (MCP). CLIP and TRIBE could both detect the single RNA target, albeit with some false positives (transcripts lacking the MS2 stem loops). Consistent false positive CLIP signals could be attributed to nonspecific antibody crosslinking. To our surprise, the supposed false positive TRIBE targets correlated with the location of genes spatially proximal to the {beta}-actin gene. This result indicates that MCP-ADAR bound to {beta}-actin mRNA contacted and edited nearby nascent transcripts, as evidenced by frequent intronic editing. Importantly, nascent transcripts on nearby chromosomes were also edited, agreeing with the interchromosomal contacts observed in chromosome paint and Hi-C. The identification of nascent RNA-RNA contacts imply that RNA-regulatory proteins such as splicing factors can associate with multiple nascent transcripts and thereby form domains of post-transcriptional activity, which increase their local concentrations. These results more generally indicate that TRIBE combined with the MS2 system, MS2-TRIBE, is a new tool to study nuclear RNA organization and regulation.

cell biology↗

bioRxiv2017

Associations Between Nutrition, Gut Microbiome, and Health in A Novel Nonhuman Primate Model

Red-shanked doucs (Pygathrix nemaeus) are endangered, foregut-fermenting colobine primates which are difficult to maintain in captivity. There are critical gaps in our understanding of their natural dietary habits including consumption of leaves, unripe fruit, flowers, seeds, and other plant parts. There is also a lack of understanding of enteric adaptations, including their unique microflora. To address these knowledge gaps, we used the douc as a model to study relationships between gastrointestinal microbial community structure, diet, and health. We analyzed published fecal samples as well as detailed dietary history from doucs with four distinct lifestyles (wild, semi-wild, semi-captive, and captive) and determined gastrointestinal bacterial microbiome composition using 16S rRNA sequencing. A clear gradient of microbiome composition was revealed along an axis of natural lifestyle disruption, including significant associations with diet, health, biodiversity, and microbial function. We identified potential microbial biomarkers of douc dysbiosis, including Bacteroides and Prevotella. Our results suggest a gradient-like shift in captivity causes an attendant shift to severe gut dysbiosis, thereby resulting in gastrointestinal issues.

microbiology↗