Biology subjects

Fung, C.

Publications and source records attributed to Fung, C..

14 recordsLinked to original sources

bioRxiv2026

Bidirectional communication between neurons in the mesentery and ileal myenteric neurons

The intestinal mesentery has been recently classified as a new organ and contains various cell types including adipocytes, preadipocytes, endothelial cells, and immune cells. In addition, neuronal cell bodies are found in the small intestinal mesentery and are situated either individually or clustered together with glial cells in small ganglion structures close to the gut wall. However, little is known about the origin or function of these extra-intestinal mesenteric neurons. The aim of this study was to better these characterize mesenteric neurons and to examine their connectivity with the ENS using calcium imaging in adult mouse ileum with the mesentery attached. Here we show that neurons in the mesentery express typical ENS neurochemical markers, respond to 5-HT, ATP and the nicotinic agonist DMPP, and receive nicotinic synaptic inputs. Furthermore, using labeling with the neuronal tracer DiI, some mesenteric neurons were found to project into the gut wall and can provide functional excitatory inputs to myenteric neurons. By contrast, we did not find evidence for mesenteric neurons providing inputs to other extrinsic neuronal targets, suggesting that they preferentially interact with the ENS. We also demonstrate that mesenteric neurons can be activated by intestinal distension and that the mesentery provides a source of inhibition to the myenteric plexus. Taken together, we show that the ENS not only interacts with vagal and spinal afferents, and sympathetic and parasympathetic nerves, but also neurons situated in the mesentery. Finally, our data suggest that these neurons may provide a form of negative feedback to the myenteric plexus such as in the event of intestinal distension. These findings have important implications for the regulation of intestinal motility in physiological and pathophysiological conditions.

neuroscience↗

bioRxiv2026

Diffractive scanning live volumetric two-photon microscopy within the contracting mouse intestine

Obtaining structural information from the enteric nervous system (ENS) within intact intestinal tissue requires microscopy systems capable of imaging through multiple tissue layers and during ongoing physiological motion. Tissue opacity, three-dimensional geometry, and spontaneous contractions strongly constrain volumetric imaging, limiting the applicability of most conventional linear optical techniques to imaging in either dissected, stretched or pharmacologically suppressed tissues. We apply Spectro-temporal Laser Imaging by Diffracted Excitation (SLIDE) microscopy, a diffraction-based scanning approach enabling fast volumetric two-photon imaging, to record the ENS in an intact ex vivo intestinal preparation from a transgenic mouse line expressing the red fluorescent protein TdTomato in peripheral and enteric neurons and glia. We achieved fast volumetric imaging during spontaneous contractions, capable of resolving micrometer-scale displacements in three dimensions, without inducing observable photodamage or compromising tissue viability over the experimental timescale. This work establishes 4D-SLIDE microscopy as a robust experimental framework for visualizing enteric neural structures within their native three-dimensional context during physiological motion, with direct relevance for conditions involving altered intestinal mechanics.

bioengineering↗

bioRxiv2026

Ca2+-mediated protein citrullination regulates proliferation in the regenerating and malignant CNS

Unlike most adult mammals, regenerative vertebrates can awaken dormant neural stem cells in response to injury. Understanding how this process is regulated could guide strategies to activate stem cells for tissue repair and limit aberrant proliferation in cancers of the CNS. Here, using zebrafish injury models and high-speed live imaging, we identify hydrodynamically-activated Ca{superscript 2} signalling as a key driver of neural stem cell activation. Local injury-associated changes in CSF flow activate mechanoreceptors at the site of spinal cord lesions, triggering pulsatile Ca{superscript 2} activity and progenitor proliferation. We identify Ca{superscript 2}-regulated peptidylarginine deiminase enzymes (PADs) as key downstream effectors that citrullinate intracellular targets in a Ca{superscript 2}-dependent manner to drive progenitor activation. Finally, we show that PAD inhibitors suppress the growth of aggressive medulloblastoma cells in preclinical laboratory models. Together, these findings uncover a novel mechanism of proliferation control in the vertebrate CNS and highlight the value of regenerative studies for identifying therapeutic targets. Graphical Summary O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/701650v1_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@3b7a56org.highwire.dtl.DTLVardef@dcc81forg.highwire.dtl.DTLVardef@d6a208org.highwire.dtl.DTLVardef@127de86_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDamage signals trigger changes in cilia activity and CSF flow to create a transient hydrodynamic niche at the site of spinal cord injuries. C_LIO_LISpecialised CSF-contacting neurons detect altered CSF circulation and initiate a signalling relay that culminates in elevated Ca{superscript 2} activity within dormant neural progenitors. C_LIO_LICa{superscript 2}-activated PAD enzymes link Ca{superscript 2} signalling to cell cycle progression by citrullinating intracellular targets in a Ca{superscript 2}-dependent manner. C_LIO_LIPAD inhibition suppresses medulloblastoma growth in preclinical laboratory models, revealing a conserved regulatory mechanism with translational potential. C_LI

neuroscience↗

bioRxiv2023

Tuft cell-derived acetylcholine regulates epithelial fluid secretion

Tuft cells are solitary chemosensory epithelial cells that can sense lumenal stimuli at mucosal barriers and secrete effector molecules to regulate the physiology and immune state of their surrounding tissue. In the small intestine, tuft cells detect parasitic worms (helminths) and microbe-derived succinate, and signal to immune cells to trigger a Type 2 immune response that leads to extensive epithelial remodeling spanning several days. Acetylcholine (ACh) from airway tuft cells has been shown to stimulate acute changes in breathing and mucocilliary clearance, but its function in the intestine is unknown. Here we show that tuft cell chemosensing in the intestine leads to release of ACh, but that this does not contribute to immune cell activation or associated tissue remodeling. Instead, tuft cell-derived ACh triggers immediate fluid secretion from neighboring epithelial cells into the intestinal lumen. This tuft cell-regulated fluid secretion is amplified during Type 2 inflammation, and helminth clearance is delayed in mice lacking tuft cell ACh. The coupling of the chemosensory function of tuft cells with fluid secretion creates an epithelium-intrinsic response unit that effects a physiological change within seconds of activation. This response mechanism is shared by tuft cells across tissues, and serves to regulate the epithelial secretion that is both a hallmark of Type 2 immunity and an essential component of homeostatic maintenance at mucosal barriers.

immunology↗

bioRxiv2022

Tuft cells mediate commensal remodeling of the small intestinal antimicrobial landscape

Succinate produced by the commensal protist Tritrichomonas musculis (T. mu) stimulates chemosensory tuft cells, resulting in intestinal type 2 immunity. Tuft cells express the succinate receptor SUCNR1, yet this receptor does not mediate anti-helminth immunity nor alter protist colonization. Here, we report that microbial-derived succinate increases Paneth cell numbers and profoundly alters the antimicrobial peptide (AMP) landscape in the small intestine. Succinate was sufficient to drive this epithelial remodeling, but not in mice lacking tuft cell chemosensory components required to detect this metabolite. Tuft cells respond to succinate by stimulating type 2 immunity, leading to interleukin-13-mediated epithelial and AMP expression changes. Moreover, type 2 immunity decreases the total number of mucosa-associated bacteria and alters the small intestinal microbiota composition. These findings demonstrate that a single metabolite produced by commensals, like T. mu, can markedly shift the intestinal AMP profile and suggest that tuft cells utilize SUCNR1 to modulate bacterial homeostasis.

microbiology↗

bioRxiv2021

High confidence identification of intra-host single nucleotide variants for person-to-person influenza transmission tracking in congregate settings

Influenza within-host viral populations are the source of all global influenza diversity and play an important role in driving the evolution and escape of the influenza virus from human immune responses, antiviral treatment, and vaccines, and have been used in precision tracking of influenza transmission chains. Next Generation Sequencing (NGS) has greatly improved our ability to study these populations, however, major challenges remain, such as accurate identification of intra-host single nucleotide variants (iSNVs) that represent within-host viral diversity of influenza virus. In order to investigate the sources and the frequency of called iSNVs in influenza samples, we used a set of longitudinal influenza patient samples collected within a University of Maryland (UMD) cohort of college students in a living learning community. Our results indicate that technical replicates aid in removal of random RT-PCR, PCR, and platform sequencing errors, while the use of clonal plasmids for removal of systematic errors is more important in samples of low RNA abundance. We show that the choice of reference for read mapping affects the frequency of called iSNVs, with the sample self-reference resulting in the lowest amount of iSNV noise. The importance of variant caller choice is also highlighted in our study, as we observe differential sensitivity of variant callers to the mapping reference choice, as well as the poor overlap of their called iSNVs. Based on this, we develop an approach for identification of highly probable iSNVs by removal of sequencing and bioinformatics algorithm-associated errors, which we implement in phylogenetic analyses of the UMD samples for a greater resolution of transmission links. In addition to identifying closely related transmission connections supported by the presence of highly confident shared iSNVs between patients, our results also indicate that the rate of minor variant turnover within a host may be a limiting factor for utilization of iSNVs to determine patient epidemiological links.

genomics↗

bioRxiv2021

An infection-activated redox switch promotes tumor growth

Oxidative stress is a defining feature of most cancers, including those that stem from carcinogenic infections1. Reactive oxygen species (ROS) can drive tumor formation2-4, yet the molecular oxidation events that contribute to tumorigenesis are largely unknown. Here we show that inactivation of a single, redox-sensitive cysteine in the host protease legumain, which is oxidized during infection with the gastric cancer-causing bacterium Helicobacter pylori, accelerates tumor growth. By using chemical proteomics to map cysteine reactivity in human gastric cells, we determined that H. pylori infection induces oxidation of legumain at Cys219. Legumain oxidation, which is enhanced by the ROS-promoting bacterial oncoprotein CagA, dysregulates intracellular legumain processing and localization and decreases legumain activity in H. pylori-infected cells. We further show that the site-specific loss of Cys219 reactivity increases tumor growth and mortality in a xenograft model. Our findings establish a link between the precise oxidation of a host protein and tumorigenic signaling during bacterial infection and demonstrate the importance of oxidative post-translational modifications in tumor growth.

microbiology↗

bioRxiv2021

Spatiotemporal heterogeneity of glioblastoma is dictated by microenvironmental interference

Glioblastomas are highly malignant tumors of the central nervous system. Evidence suggests that these tumors display large intra- and inter-patient heterogeneity hallmarked by subclonal diversity and dynamic adaptation amid developmental hierarchies1-3. However, the source for dynamic reorganization of cellular states within their spatial context remains elusive. Here, we in-depth characterized glioblastomas by spatially resolved transcriptomics, metabolomics and proteomics. By deciphering exclusive and shared transcriptional programs across patients, we inferred that glioblastomas develop along defined neural lineages and adapt to inflammatory or metabolic stimuli reminiscent of reactive transformation in mature astrocytes. Metabolic profiling and imaging mass cytometry supported the assumption that tumor heterogeneity is dictated by microenvironmental alterations. Analysis of copy number variation (CNV) revealed a spatially cohesive organization of subclones associated with reactive transcriptional programs, confirming that environmental stress gives rise to selection pressure. Deconvolution of age-dependent transcriptional programs in malignant and non-malignant specimens identified the aging environment as the major driver of inflammatory transformation in GBM, suggesting that tumor cells adopt transcriptional programs similar to inflammatory transformation in astrocytes. Glioblastoma stem cells implanted into human neocortical slices of varying age levels, independently confirmed that the ageing environment dynamically shapes the intratumoral heterogeneity towards reactive transcriptional programs. Our findings provide insights into the spatial architecture of glioblastoma, suggesting that both locally inherent tumor as well as global alterations of the tumor microenvironment shape its transcriptional heterogeneity. Global age-related inflammation in the human brain is driving distinct transcriptional transformation in glioblastomas, which requires an adjustment of the currently prevailing glioma models.

cancer biology↗

bioRxiv2021

Luminal nutrients activate distinct patterns in submucosal and myenteric neurons in the mouse small intestine

Nutrient signals sensed by enteroendocrine cells are conveyed to the enteric nervous system (ENS) to initiate intestinal reflexes. We addressed whether there are specific enteric pathways dedicated to detecting different luminal nutrients. Calcium imaging was performed on intact jejunal preparations from Wnt1-cre;R26R-GCaMP3 and Villin-cre;R26R-GCaMP3 mice which express a fluorescent calcium indicator in their ENS or intestinal epithelium, respectively. Glucose, acetate, and L-phenylalanine were perfused onto the mucosa whilst imaging underlying enteric neurons. Nutrient transport or diffusion across the mucosa was mimicked by applying nutrients onto sensory nerve endings in a villus, or onto myenteric ganglia. The nutrients perfused onto the mucosa each elicited Ca2+ transients in submucosal neurons and in distinct patterns of myenteric neurons. Notably, the neurochemical subtypes of myenteric neurons that responded differed between the nutrients, while submucosal responders were predominantly cholinergic. Nutrients applied into villi or onto ganglia did not elicit specific neuronal responses but did stimulate Ca2+ signaling in the mucosal epithelium. These data suggest that nutrients are first detected at the level of the epithelium and that the ENS is capable of discriminating between different compositions of luminal content. Furthermore, our data show that responses to mucosal stimulation are primarily in the myenteric plexus and submucosal neurons respond secondarily.

neuroscience↗

bioRxiv2020

Vector and cell-culture passaging of dengue clinical samples for virus isolation and amplification does not significantly change genome consensus or frequencies of intra-host viral variants

Intra-host single nucleotide variants (iSNVs) have been increasingly used in genomic epidemiology to increase phylogenetic resolution and reconstruct fine-scale outbreak dynamics. These analyses are preferably done on sequence data from direct clinical samples, but in many cases due to low viral loads, there might not be enough genetic material for deep sequencing and iSNV determination. Isolation of the virus from clinical samples with low passage number increases viral load, but to date, no studies have investigated how dengue virus (DENV) culture isolation from a clinical sample impacts the consensus sequence, and there is no information on the intra-host virus population changes that may result from viral isolation. In this study, we investigate consensus and iSNV frequency differences between DENV sequenced directly from clinical samples and their corresponding low-passage isolates. Twenty five DENV1 and DENV2 positive sera and their corresponding viral isolates (T.splendens inoculation and C6/36 passage) were obtained from a prospective cohort study in the Philippines. These were sequenced on MiSeq with minimum nucleotide depth of coverage of 1000x, and iSNVs were detected using LoFreq. For both DENV1 and DENV2, we found that the nucleotide call concordance (including called iSNVs with variant cutoff at 5%) between direct sera sample and its cultured virus was on average 99.99%. There were a maximum of one consensus nucleotide difference between clinical sample and isolate. Interestingly, we found that iSNV frequencies were also largely preserved between the samples, with an average difference in minor variant frequency of 6.8% (95CI: 3.6%-10%) and 9.6% (95CI: 7%-12.2%) for DENV1 and DENV2, respectively. Furthermore, we found no significant differences in either DENV1 or DENV2 between the sample pairs (clinical sample and isolate) in their number of iSNV positions per genome, or in the difference in variant frequencies (p=0.36 and p=0.13, respectively, F-test). Our results show that low-passage DENV isolates may be used for identification of the majority of their human-derived within-host variant populations, which are increasingly being used for precision tracking of DENV and other RNA viruses.

evolutionary biology↗

bioRxiv2020

Atrophy associated with tau pathology precedes overt cell death in a mouse model of progressive tauopathy

In the early stages of Alzheimers disease (AD), tau pathology first develops in the entorhinal cortex (EC), then spreads to the hippocampus and at later stages, to the neocortex. Pathology in the neocortex correlates with impaired cognitive performance. Overall, tau pathology correlates well with neurodegeneration but the spatial and temporal association between tau pathology and overt volume loss is unclear. Using in vivo magnetic resonance imaging (MRI) with tensor-based morphometry (TBM) we mapped the spatio-temporal pattern of structural changes in a mouse model of AD-like progressive tauopathy. A novel, co-registered in vivo MRI atlas identified particular regions in the medial temporal lobe (MTL) that had significant volume reduction. The medial entorhinal cortex (MEC) and pre-/para-subiculum (PPS) had the most significant atrophy at the early stage, but atrophy then spread into the hippocampus, most notably, the CA1, dentate gyrus (DG) and subiculum (Sub). TBM-related atrophy in the DG and Sub preceded overt cell loss that has been reported in ex vivo studies in the same mouse model. By unifying an ex vivo 3D reconstruction of tau pathology with the TBM-MRI results we mapped the progression of atrophy in the MTL with the corresponding spread of tau pathology. Our study shows that there is an association between the spread of tau pathology and TBM-related atrophy from the EC to the hippocampus, but atrophy in the DG and Sub preceded overt cell loss. One Sentence SummarySpread of tau pathology in a mouse model of Alzheimers disease assessed by MRI was associated with reduced brain tissue volume but not neuronal loss.

neuroscience↗

bioRxiv2020

Lineage and Spatial Mapping of Glioblastoma-associated Immunity

The diversity of molecular states and cellular plasticity of immune cells in the glioblastoma environment is still poorly understood. Here, we performed scRNA sequencing of the immune compartment and mapped potential cellular interactions leading to an immunosuppressive microenvironment and dysfunction of T cells. Through inferring the dynamic adaptation during T cell activation, we identified three different terminal states with unique transcriptional programs. Modeling of driver genes for terminal T cell fate identified IL-10 signaling alterations in a subpopulation of HAVCR2(+) T cells. To explore in depth cellular interactions, we established an in-silico model by the integration of spatial transcriptomic and scRNA-sequencing, and identified a subset of HMOX1+ myeloid cells defined by IL10 release leading to T cell exhaustion. We found a spatial overlap between HMOX(+) myeloid and HAVCR2(+) T cells, suggesting that myeloid-lymphoid interaction causes immunosuppression present in tumor regions with enriched mesenchymal gene expression. Using human neocortical GBM model, coupled with patient-derived T cells, we confirmed that the functional interaction between myeloid and lymphoid cells, leads to a dysfunctional state of T cells. This IL-10 driven T cell exhaustion was found to be rescued by JAK/STAT inhibition. A comprehensive understanding of the cellular states and plasticity of lymphoid cells in GBM will aid towards successful immunotherapeutic approaches.

cancer biology↗

bioRxiv2020

Analysis of cell-associated DENV RNA by oligo(dT) primed 5' capture scRNAseq

Dengue is one of the most widespread vector-borne viral diseases in the world. However, the size, heterogeneity, and temporal dynamics of the cell-associated viral reservoir during acute dengue virus (DENV) infection remains unclear. In this study, we analyzed cells infected in vitro with DENV and PBMC from an individual experiencing a natural DENV infection utilizing 5 capture single cell RNA sequencing (scRNAseq). Both positive- and negative-sense DENV RNA was detected in reactions containing either an oligo(dT) primer alone, or in reactions supplemented with a DENV-specific primer. The addition of a DENV-specific primer did not increase the total amount of DENV RNA captured or the fraction of cells identified as containing DENV RNA. However, inclusion of a DENV-specific cDNA primer did increase the viral genome coverage immediately 5 to the primer binding site. Furthermore, while the majority of intracellular DENV sequence captured in this analysis mapped to the 5 end of the viral genome, distinct patterns of enhanced coverage within the DENV polyprotein coding region were observed. The 5 capture scRNAseq analysis of PBMC not only recapitulated previously published reports by detecting virally infected memory and naive B cells, but also identified cell-associated genomic variants not observed in contemporaneous serum samples. These results demonstrate that oligo(dT) primed 5 capture scRNAseq can detect DENV RNA and quantify virus-infected cells in physiologically relevant conditions, and provides insight into viral sequence variability within infected cells. IMPORTANCEDengue is one of the most widespread vector-borne viral diseases in the world. However, it is still unclear which cells harbor virus during DENV infection, and how viral reservoirs in serum and infected cells are related. These results demonstrate for the first time that intracellular DENV RNA can be identified and infected cells quantified by 5 capture scRNAseq. This strategy provides a significantly greater throughput and higher sensitivity than previously published methods and has the potential to provide additional information on the genomic heterogeneity of intracellular DENV RNA.

microbiology↗

bioRxiv2017

BPG: Seamless, Automated and Interactive Visualization of Scientific Data

We introduce BPG, an easy-to-use framework for generating publication-quality, highly-customizable plots in the R statistical environment. This open-source package includes novel methods of displaying high-dimensional datasets and facilitates generation of complex multi-panel figures, making it ideal for complex datasets. A web-based interactive tool allows online figure customization, from which R code can be downloaded for seamless integration with computational pipelines. BPG is available at http://labs.oicr.on.ca/boutros-lab/software/bpg

bioinformatics↗