Biology subjects

Yeung, J.

Publications and source records attributed to Yeung, J..

36 records · Page 2Linked to original sources

bioRxiv2023

A single-dose of intranasal vaccination with a live-attenuated SARS-CoV-2 vaccine candidate promotes protective mucosal and systemic immunity

An attenuated SARS-CoV-2 virus with modified viral transcriptional regulatory sequences and deletion of open-reading frames 3, 6, 7 and 8 ({triangleup}3678) was previously reported to protect hamsters from SARS-CoV-2 infection and transmission. Here we report that a single-dose intranasal vaccination of {triangleup}3678 protects K18-hACE2 mice from wild-type or variant SARS-CoV-2 challenge. Compared with wild-type virus infection, the {triangleup}3678 vaccination induces equivalent or higher levels of lung and systemic T cell, B cell, IgA, and IgG responses. The results suggest {triangleup}3678 as an attractive mucosal vaccine candidate to boost pulmonary immunity against SARS-CoV-2.

microbiology↗

bioRxiv2023

Single-cell phenomics reveals behavioural and mechanical heterogeneities underpinning collective migration during mouse anterior patterning

Distal Visceral Endoderm (DVE) cells show a stereotypic unidirectional migration essential for correct orientation of the anterior-posterior axis. They migrate within a simple epithelium, the Visceral Endoderm (VE). It is unknown how DVE cells negotiate their way amongst the surrounding VE cells, what determines the bounds of DVE migration within the VE, and the relative contributions of different cell behaviours to this migration. To address these questions, we used lightsheet microscopy to generate a multi-embryo, singlecell resolution, longitudinal dataset of cell behaviour and morphology. We developed a machine learning based pipeline to segment cells and a data-informed systematic computational framework to extract and compare select morphological, behavioural and molecular parameters of all VE cells in a unified coordinate space. Unbiased clustering of this single-cell phenomic dataset reveals considerable patterned phenotypic heterogeneity within the VE and a previously unknown sub-grouping within the DVE. While migrating, DVE cells retain regular morphology, do not exchange neighbours and are crowded, all hallmarks of the jammed state. In contrast, VE cells immediately ahead of them deform and undergo neighbour exchange. We show that DVE cells are characterised by higher levels of apical F-actin and elevated tension relative to the VE cells immediately ahead of them through which they migrate, but stop migrating upon reaching a region of the VE with matching elevated tension. Lefty1 mutants, known to show abnormal over-migration of DVE cells, show disruption to this patterned tension in the VE. Our findings provide novel insights into the control of cell behaviour during the remodelling of curved epithelia, indicating that the collective migration of sub-sets of cells can be circumscribed by modulating the mechanical properties of surrounding cells and that migrating cells in this context remain as a jammed solid flock, with surrounding cells facilitating their movement by becoming unjammed. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/534937v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@10a7153org.highwire.dtl.DTLVardef@963a1forg.highwire.dtl.DTLVardef@c1fcd0org.highwire.dtl.DTLVardef@1bcdded_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2022

Intertumoral lineage diversity and immunosuppressive transcriptional programs in well-differentiated gastroenteropancreatic neuroendocrine tumors

Neuroendocrine tumors (NETs) are rare cancers that may arise in the gastrointestinal tract and pancreas. The fundamental mechanisms driving gastroenteropancreatic (GEP) NET growth remain incompletely elucidated; however, the heterogeneous clinical behavior of GEP-NETs suggests that both cellular lineage dynamics and tumor microenvironment influence tumor pathophysiology. Here, we investigated the single-cell transcriptomes of tumor and immune cells from patients with gastroenteropancreatic NETs. Malignant GEP-NET cells expressed genes and regulons associated with normal, gastrointestinal endocrine cell differentiation and fate determination stages. While tumor and lymphoid compartments sparsely expressed immunosuppressive targets, infiltrating myeloid cells were enriched for alternative immunotherapy pathways including VSIR, Tim3/Gal9, and SIGLEC10. Finally, analysis of paired primary and metastatic tissue specimens from small intestinal NETs demonstrated transcriptional transformation between the primary tumor and its distant metastasis. Our findings highlight the transcriptomic heterogeneity that distinguishes the cellular landscapes of GEP-NET anatomic subtypes and reveal potential avenues for future precision medicine therapeutics.

cancer biology↗

bioRxiv2022

Cross-neutralization and viral fitness of SARS-CoV-2 Omicron sublineages

The rapid evolution of SARS-CoV-2 Omicron sublineages mandates a better understanding of viral replication and cross-neutralization among these sublineages. Here we used K18-hACE2 mice and primary human airway cultures to examine the viral fitness and antigenic relationship among Omicron sublineages. In both K18-hACE2 mice and human airway cultures, Omicron sublineages exhibited a replication order of BA.5 [≥] BA.2 [≥] BA.2.12.1 > BA.1; no difference in body weight loss was observed among different sublineage-infected mice. The BA.1-, BA.2-, BA.2.12.1-, and BA.5-infected mice developed distinguisable cross-neutralizations against Omicron sublineages, but exhibited little neutralizations against the index virus (i.e., USA-WA1/2020) or the Delta variant. Surprisingly, the BA.5-infected mice developed higher neutralization activity against heterologous BA.2 and BA.2.12.1 than that against homologous BA.5; serum neutralizing titers did not always correlate with viral replication levels in infected animals. Our results revealed a distinct antigenic cartography of Omicron sublineages and support the bivalent vaccine approach.

microbiology↗

bioRxiv2022

ViReMaShiny: An Interactive Application for Analysis of Viral Recombination Data

Recombination is an essential driver of virus evolution and adaption, giving rise to new chimeric viruses, structural variants, sub-genomic RNAs, and Defective-RNAs. Next-Generation Sequencing of virus samples, either from experimental or clinical settings, has revealed a complex distribution of recombination events that contributes to the intrahost diversity. We and others have previously developed alignment tools to discover and map these diverse recombination events in NGS data. However, there is no standard for data visualization to contextualize events of interest and downstream analysis often requires bespoke coding. To address this, we present ViReMaShiny, a web-based application built using the R Shiny framework to allow interactive exploration and point-and-click visualization of viral recombination data provided in BED format generated by computational pipelines such as ViReMa (Viral-Recombination-Mapper).

bioinformatics↗

bioRxiv2021

Temporal analysis of enhancers during mouse brain development reveals dynamic regulatory function and identifies novel regulators of cerebellar development.

In this study, we identified active enhancers in the mouse cerebellum at embryonic and postnatal stages establishing the first catalog of enhancers active during embryonic cerebellum development. The majority of cerebellar enhancers have dynamic activity between embryonic and postnatal development. Cerebellar enhancers were enriched for neural transcription factor binding sites with temporally specific expression. Putative gene targets displayed spatially restricted expression patterns, indicating cell-type specific expression regulation. Functional analysis of target genes indicated that enhancers regulate processes spanning several developmental epochs such as specification, differentiation and maturation. We use these analyses to discover one novel regulator and one novel marker of cerebellar development: Bhlhe22 and Pax3, respectively. We identified an enrichment of de novo mutations and variants associated with autism spectrum disorder in cerebellar enhancers. Our study provides insight into the dynamics of gene expression regulation by enhancers in the developing brain and delivers a rich resource of novel gene-enhancer associations providing a basis for future in-depth studies in the cerebellum.

neuroscience↗

bioRxiv2021

Interferon-stimulated and metallothionein-expressing macrophages are associated with acute and chronic allograft dysfunction after lung transplantation

Lung transplant (LT) recipients experience episodes of immune-mediated acute lung allograft dysfunction (ALAD). ALAD episodes are a risk factor for chronic lung allograft dysfunction (CLAD), the major cause of death after LT. We have applied single-cell RNA sequencing (scRNAseq) to bronchoalveolar lavage (BAL) cells from stable and ALAD patients and to cells from explanted CLAD lung tissue to determine key cellular elements in dysfunctional lung allografts, with a focus on macrophages. We identified two alveolar macrophage (AM) subsets uniquely represented in ALAD. Using pathway analysis and differentially expressed genes, we annotated these as pro-inflammatory interferon-stimulated gene (ISG) and metallothionein-mediated inflammatory (MT) AMs. Functional analysis of an independent set of AMs in vitro revealed that ALAD AMs exhibited a higher expression of CXCL10, a marker of ISG AMs, and increased secretion of pro-inflammatory cytokines compared to AMs from stable patients. Using publicly available BAL scRNAseq datasets, we found that ISG and MT AMs are associated with more severe inflammation in COVID-19 patients. Analysis of cells from four explanted CLAD lungs revealed similar macrophage populations. Using a single nucleotide variation calling algorithm, we also demonstrated contributions of donor and recipient cells to all AM subsets early post-transplant, with loss of donor-derived cells over time. Our data reveals extensive heterogeneity among lung macrophages after LT and indicates that specific sub-populations may be associated with allograft dysfunction, raising the possibility that these cells may represent important therapeutic targets.

immunology↗

bioRxiv2021

Hierarchical chromatin regulation during blood formation uncovered by single cell sortChIC

Post-translational histone modifications modulate chromatin packing to regulate gene expression. How chromatin states, at euchromatic and heterochromatic regions, underlie cell fate decisions in single cells is relatively unexplored. We develop sort assisted single-cell chromatin immunocleavage (sortChIC) and map active (H3K4me1 and H3K4me3) and repressive (H3K27me3 and H3K9me3) histone modifications in hematopoietic stem and progenitor cells (HSPCs), and mature blood cells in the mouse bone marrow. During differentiation, HSPCs acquire distinct active chromatin states that depend on the specific cell fate, mediated by cell type-specifying transcription factors. By contrast, most regions that gain or lose repressive marks during differentiation do so independent of cell fate. Joint profiling of H3K4me1 and H3K9me3 demonstrates that cell types within the myeloid lineage have distinct active chromatin but share similar myeloid-specific heterochromatin-repressed states. This suggests hierarchical chromatin regulation during hematopoiesis: heterochromatin dynamics define differentiation trajectories and lineages, while euchromatin dynamics establish cell types within lineages.

genomics↗

bioRxiv2021

Deconvolving multiplexed histone modifications in single cells

Recent advances have enabled mapping of histone modifications in single cells1-12, but current methods are constrained to profile only one histone modification per cell. Here we present an integrated experimental and computational framework, scChIX (single-cell chromatin immunocleavage and unmixing), to map multiple histone modifications in single cells. scChIX multiplexes two histone modification profiles together in single cells, then computationally deconvolves the signal using training data from respective histone modification profiles. We first validate this method using purified blood cells and show that although the two repressive marks, H3K27me3 and H3K9me3, are generally mutually exclusive, the transitions between the two regions can vary between cell types. Next we apply scChIX to a heterogenous cell population from mouse bone marrow to generate linked maps of active (H3K4me1) and repressive (H3K27me3) chromatin landscapes in single cells, where coordinates in the active modification map correspond to coordinates in the repressive map. Linked analysis reveals that immunoglobulin genes in the Igkv region are in a repressed chromatin state in pro-B cells, but become activated in B cells. Overall, scChIX unlocks systematic interrogation of the interplay between histone modifications in single cells.

genomics↗

bioRxiv2021

Pro-inflammatory alveolar macrophages associated with allograft dysfunction after lung transplantation

PurposeLung transplant (LT) recipients experience episodes of immune-mediated acute lung allograft dysfunction (ALAD). We have applied single-cell RNA sequencing (scRNAseq) to bronchoalveolar lavage (BAL) cells of stable and ALAD patients to determine key cellular elements in dysfunctional lung allografts. Our particular focus here is on studying alveolar macrophages (AMs) as scRNAseq enables us to elucidate their heterogeneity and possible association with ALAD where our knowledge from cytometry-based assays is very limited. MethodsFresh bronchoalveolar lavage (BAL) cells from 6 LT patients, 3 with stable lung function (3044 {+/-} 1519 cells) and 3 undergoing an episode of ALAD (2593 {+/-} 904 cells) were used for scRNAseq. R Bioconductor and Seurat were used to perform QC, dimensionality reduction, annotation, pathway analysis, and trajectory. Donor and recipient deconvolution was performed using single nucleotide variations. ResultsOur data revealed that AMs are highly heterogeneous (12 transcriptionally distinct subsets in stable). We identified two AM subsets uniquely represented in ALAD. Based on pathway analysis and the top differentially expressed genes in BAL we annotated them as pro-inflammatory interferon-stimulated genes (ISG) and metallothioneins-mediated inflammatory (MT). Pseudotime analysis suggested that ISG AMs represent an earlier stage of differentiation which may suggest them as monocyte drive macrophages. Our functional analysis on an independent set of BAL samples shows that ALAD samples have significantly higher expression of CXCL10, a marker of ISG AM, as we as higher secretion of pro-inflammatory cytokines. Single nucleotide variation calling algorithm has allowed us to identify macrophages of donor origin and demonstrated that donor AMs are lost with time post-transplant. ConclusionUsing scRNAseq, we observed AMs heterogeneity and identified specific subsets that may be associated with allograft dysfunction. Further exploration with scRNAseq will shed light on LT immunobiology and the role of AMs in allograft injury and dysfunction.

immunology↗

bioRxiv2021

Multicellular immune hubs and their organization in MMRd and MMRp colorectal cancer

Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd patients. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across patient tumors and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage, and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T-cell-attracting chemokines. By identifying interacting cellular programs, we thus reveal the logic underlying spatially organized immune-malignant cell networks.

cancer biology↗

bioRxiv2020

PHAIR, A biosensor for pH measurement in air-liquid interface of human airway epithelial cells

1In many biological systems, pH can be used as a parameter to understand and study cell dynamics. However, measuring pH in live cell culture is limited by the sensor ion specificity, proximity to the cell surface, and scalability. Commercially available pH sensors are difficult to integrate into a small-scale cell culture system due to their size and are not cost-effective for disposable use. We made PHAIR - a new pH sensor that uses a micro-wire format to measure pH in vitro human airway cell culture. Tungsten micro-wires were used as the working electrodes, and silver micro-wires with a silver/silver chloride coating were used as a pseudo reference electrode. pH sensitivity, in a wide and narrow range, and stability of these sensors were tested in common standard buffer solutions as well as in culture media of human airway epithelial cells grown at the air-liquid interface in a 24 well cell culture plate. When measuring the pH of cells grown under basal and challenging conditions using PHAIR, cell viability and cytokine responses were not affected. Our results confirm that micro-wires-based sensors have the capacity for miniaturization, and detection of diverse ions while maintaining sensitivity. This suggests the broad application of PHAIR in various biological experimental settings.

bioengineering↗

bioRxiv2020

The circadian oscillator analysed at the single-transcript level

The circadian clock is an endogenous and self-sustained oscillator that anticipates daily environmental cycles and coordinates physiology accordingly. While rhythmic gene expression of circadian genes is well-described in populations of cells, the single-cell mRNA dynamics of multiple core-clock genes remain largely unknown. Here we use single molecule fluorescence in-situ hybridization (smFISH) at multiple time points to measure pairs of core-clock transcripts, Rev-erb (Nr1d1), Cry1 and Bmal1, in mouse fibroblasts at single-molecule resolution. The mean mRNA level oscillates over 24 hours for all three genes, but mRNA numbers show considerable spread between cells. While transcript number scales with cell size for all genes, gene-to-gene correlations of mRNA number depends on the gene pair. To account for these features of the data, we develop a probabilistic model for multivariate smFISH mRNA counts that quantifies changes in transcriptional bursting across genes and over circadian time. We identify a mixture model of negative binomials as the preferred model of the mRNA count distributions, which accounts for cell-to-cell heterogeneity, notably in cell size. The paired count data and modelling allows the decomposition of mRNA variability into distinct noise sources, showing that circadian clock time contributes only a small fraction of the total variability in mRNA number between cells. Thus, our results highlight the intrinsic biological challenges in estimating circadian phase from single-cell mRNA counts and suggest that circadian phase in single cells is encoded post-transcriptionally.

systems biology↗

bioRxiv2020

Oscillating and stable genome topologies underlie hepatic physiological rhythms during the circadian cycle

The circadian clock drives extensive temporal gene expression programs controlling daily changes in behavior and physiology. In mouse liver, transcription factors dynamics, chromatin modifications, and RNA Polymerase II (PolII) activity oscillate throughout the 24-hour (24h) day, regulating the rhythmic synthesis of thousands of transcripts. Also, 24h rhythms in gene promoter-enhancer chromatin looping accompany rhythmic mRNA synthesis. However, how chromatin organization impinges on temporal transcription and liver physiology remains unclear. Here, we applied time-resolved chromosome conformation capture (4C-seq) in livers of WT and arrhythmic Bmal1 knockout mice. In WT, we observed 24h oscillations in promoter-enhancer contact at multiple loci including the core-clock genes Period1, Period2 and Bmal1. In addition, we detected rhythmic PolII activity, chromatin modifications and transcription involving stable chromatin loops at gene promoters representing key liver function such as glucose and lipid metabolism and detoxification. Intriguingly, these contacts persisted in clock-impaired mice in which both PolII activity and chromatin marks no longer oscillated. Finally, we observed chromatin interaction hubs connecting neighbouring genes showing coherent transcription regulation across genotypes. Thus, both clock-controlled and clock-independent chromatin topology underlie rhythmic regulation of liver physiology.

genomics↗

bioRxiv2020

The transcription factor Pou3f1 provides a new map to the glutamatergic neurons of the cerebellar nuclei

Pou3f1 is a transcription factor involved in early neural differentiation. Cap Analysis Gene Expression (5-CAGE) analysis reveals that Pou3f1 transcript is highly enriched in the developing cerebellum. Between embryonic (E) days E10.5 and E12.5, Pou3f1 expression is present prominently along the subpial stream (SS), suggesting that Pou3f1+ cells are glutamatergic cerebellar nuclear (CN) neurons. This finding was confirmed by immunofluorescent (IF) co-labeling of Pou3f1 and Atoh1, the master regulator of cells from the rhombic lip (RL) that are destined for neurons of the glutamatergic lineage, as well as in Atoh1-null tissues, in which Pou3f1 expression is absent. Interestingly, the expression of Pax6, another key molecule for CN neuron survival, does not co-localize with that of Pou3f1. In the Pax6-null Small Eye (Sey) mutant, which is characterized by a loss of many glutamatergic CN neurons, Pou3f1+ CN neurons are still present. Furthermore, Pou3f1-labeled cells do not co-express Tbr1, a well-established marker of glutamatergic CN neurons. These results highlight that Pou3f1+ cells are a distinct and previously unrecognized subtype of glutamatergic CN neurons that do not have the "canonical" sequence of Atoh1[->]Pax6[->]Tbr1 expressions. Instead, they express Atoh1, Pou3f1, and other markers of CN neurons, Brn2 and Irx3. These findings illustrate that glutamatergic CN neurons that arise from the RL are composed of molecularly heterogeneous subpopulations that are determined by at least two distinct transcriptional programs. Significance StatementThe present work has identified Pou3f1 as a marker for a previously unidentified subtype of glutamatergic cerebellar nuclear neurons, the principal output neurons of the cerebellum. The classical model of glutamatergic CN neurons development follows the sequential expression of transcription factors Atoh1[->]Pax6[->]Tbr1. However, we found that the development of Pou3f1+ neurons requires Atoh1 but not Pax6. Moreover, Pou3f1+ neurons do not express Tbr1, but instead express two other transcription factors, Brn2 and Irx3. Anatomically, Pou3f1+ CN neurons populate the interposed and dentate nuclei, while the Tbr1+ CN neurons populate the fastigial nucleus. These findings reveal the heterogeneity of CN neuron populations and the diversity of molecular programming that lead to different CN neuron subtypes.

neuroscience↗

bioRxiv2019

Expression of heat shock protein 70 is insufficient to extend Drosophila melanogaster longevity

It has been known for over 20 years that Drosophila melanogaster flies with twelve additional copies of the hsp70 gene encoding the 70 kDa heat shock protein lives longer after a non-lethal heat treatment. Since the heat treatment also induces the expression of additional heat shock proteins, the biological effect can be due either to HSP70 acting alone or in combination. This study used the UAS/GAL4 system to determine whether hsp70 is sufficient to affect the longevity and the resistance to thermal, oxidative or desiccation stresses of the whole organism. We observed that HSP70 expression in the nervous system or muscles has no effect on longevity or stress resistance but ubiquitous expression reduces the life span of males. We also observed that the down-regulation of Hsp70 using RNAi did not affect longevity.

genetics↗

bioRxiv2019

Simple and complex interactions between sleep-wake driven and circadian processes shape daily genome regulatory dynamics in the mouse

The timing and duration of sleep results from the interaction between a sleep-wake driven, or homeostatic, process (S) and a circadian process (C), and involves changes in gene expression and genomic regulation. Unraveling the respective contributions of S and C, and their interaction, to transcriptional and epigenomic regulatory dynamics requires sampling over time under unperturbed conditions and conditions of perturbed sleep. Here, we profiled mRNA expression and chromatin accessibility in the cerebral cortex of mice over a three-day period, including a 6-hour sleep deprivation (SD) on day two. Mathematical modeling established that a large proportion of rhythmic genes are actually governed by Process S with varying degrees of interaction with Process C, sometimes working in opposition. Remarkably, SD causes long-term effects on gene expression dynamics, outlasting phenotypic recovery, most strikingly illustrated by a dampening of the oscillation of most core clock genes, including Bmal1, suggesting that enforced wakefulness directly impacts the molecular clock machinery. Chromatin accessibility proved highly plastic and dynamically affected by SD. Distal regions, rather than promoters, display dynamics corresponding to gene transcription, implying that changes in mRNA expression result from constantly accessible promoters under the influence of distal enhancers or repressors. Srf was predicted as a transcriptional regulator driving immediate response, suggesting that Srf activity mirrors the build-up and release of sleep pressure. Our results demonstrate that a single, short SD has long-term aftereffects at the genomic regulatory level. Such effects might accumulate with repeated sleep restrictions, thereby contributing to their adverse health effects.\n\nSignificance statementWhen and how long we sleep is determined by the time-of-day and how long we have been awake, which are tracked molecularly by a circadian and a sleep-wake driven process, respectively. We measured the long-term consequences of a short-term sleep deprivation (SD) on gene expression and regulation in the mouse brain, and used mathematical models to determine the relative contributions of the circadian and sleep-wake driven processes. We find that many genes, including most of the genes that constitute the molecular circadian clock, are perturbed by SD long after the mice ceased showing behavioral signs of sleep loss. Our results have implications for human health, given the high prevalence of insufficient and poor quality sleep in our contemporary society.

genomics↗

bioRxiv2017

Transcription factor activity rhythms and tissue-specific chromatin interactions explain circadian gene expression across organs.

AbstractTemporal control of physiology requires the interplay between gene networks involved in daily timekeeping and tissue function across different organs. How the circadian clock interweaves with tissue-specific transcriptional programs is poorly understood. Here we dissected temporal and tissue-specific regulation at multiple gene regulatory layers by examining mouse tissues with an intact or disrupted clock over time. Integrated analysis uncovered two distinct regulatory modes underlying tissue-specific rhythms: tissue-specific oscillations in transcription factor (TF) activity, which were linked to feeding-fasting cycles in liver and sodium homeostasis in kidney; and co-localized binding of clock and tissue-specific transcription factors at distal enhancers. Chromosome conformation capture (4C-Seq) in liver and kidney identified liver-specific chromatin loops that recruited clock-bound enhancers to promoters to regulate liver-specific transcriptional rhythms. Furthermore, this looping was remarkably promoter-specific on the scale of less than ten kilobases. Enhancers can contact a rhythmic promoter while looping out nearby nonrhythmic alternative promoters, confining rhythmic enhancer activity to specific promoters. These findings suggest that chromatin folding enables the clock to regulate rhythmic transcription of specific promoters to output temporal transcriptional programs tailored to different tissues.

genomics↗