Biology subjects

Wan, J.

Publications and source records attributed to Wan, J..

At least 73 records · Page 4Linked to original sources

bioRxiv2023

Targeting Ovarian Cancer Stem Cells by Dual Inhibition of the Long Noncoding RNA HOTAIR and Lysine Methyltransferase EZH2

Persistence of cancer stem cells (CSC) is believed to contribute to resistance to platinum-based chemotherapy and disease relapse in ovarian cancer (OC), the fifth leading cause of cancer- related death among US women. HOXC transcript antisense RNA (HOTAIR) is a long noncoding RNA (lncRNA) overexpressed in high-grade serous OC (HGSOC) and linked to chemoresistance. However, HOTAIR impacts chromatin dynamics in OCSC and how this oncogenic lncRNA contributes to drug resistant disease are incompletely understood. Here we generated HOTAIR knock-out (KO) HGSOC cell lines using paired CRISPR guide RNA design to investigate the function of HOTAIR. We show that loss of HOTAIR function re-sensitized OC cells to platinum treatment and decreased the population of OCSC. Furthermore, HOTAIR KO inhibited the development of stemness-related phenotypes, including spheroid formation ability, as well as expression of key stemness-associated genes ALDH1A1, Notch3, Sox9, and PROM1. HOTAIR KO altered both the cellular transcriptome and chromatin accessibility landscape of multiple oncogenic-associated genes and pathways, including the NF-kB pathway. HOTAIR functions as an oncogene by recruiting enhancer of zeste 2 (EZH2) to catalyze H3K27 tri-methylation to suppress downstream tumor suppressor genes, and it was of interest to inhibit both HOTAIR and EZH2. In vivo, combining a HOTAIR inhibitor with an EZH2 inhibitor and platinum chemotherapy decreased tumor formation and increased survival. These results suggest a key role for HOTAIR in OCSC and malignant potential. Targeting HOTAIR in combination with epigenetic therapies may represent a therapeutic strategy to ameliorate OC progression and resistance to platinum-based chemotherapy.

cancer biology↗

bioRxiv2023

Molecular imaging with aquaporin-based reporter genes: quantitative considerations from Monte Carlo diffusion simulations

Aquaporins provide a new class of genetic tools for imaging molecular activity in deep tissues by increasing the rate of cellular water diffusion, which generates magnetic resonance contrast. However, distinguishing aquaporin contrast from the tissue background is challenging because water diffusion is also influenced by structural factors such as cell size and packing density. Here, we developed and experimentally validated a Monte Carlo model to analyze how cell radius and intracellular volume fraction quantitatively affect aquaporin signals. We demonstrated that a differential imaging approach based on time-dependent changes in diffusivity can improve specificity by unambiguously isolating aquaporin-driven contrast from the tissue background. Finally, we used Monte Carlo simulations to analyze the connection between diffusivity and the percentage of cells engineered to express aquaporin, and established a simple mapping that accurately determined the volume fraction of aquaporin-expressing cells in mixed populations. This study creates a framework for broad applications of aquaporins, particularly in biomedicine and in vivo synthetic biology, where quantitative methods to measure the location and performance of genetic devices in whole vertebrates are necessary.

bioengineering↗

bioRxiv2023

Engineering ligand stabilized aquaporin reporters for magnetic resonance imaging

Imaging transgene expression in live tissues requires reporters that are detectable with deeply penetrant modalities, such as magnetic resonance imaging (MRI). Here, we show that LSAqp1, a water channel engineered from aquaporin-1, can be used to create background-free, drug-gated, and multiplex images of gene expression using MRI. LSAqp1 is a fusion protein composed of aquaporin-1 and a degradation tag that is sensitive to a cell-permeable ligand, which allows for dynamic small molecule modulation of MRI signals. LSAqp1 improves specificity for imaging gene expression by allowing reporter signals to be conditionally activated and distinguished from the tissue background by difference imaging. In addition, by engineering destabilized aquaporin-1 variants with different ligand requirements, it is possible to image distinct cell types simultaneously. Finally, we expressed LSAqp1 in a tumor model and showed successful in vivo imaging of gene expression without background activity. LSAqp1 provides a conceptually unique approach to accurately measure gene expression in living organisms by combining the physics of water diffusion and biotechnology tools to control protein stability.

bioengineering↗

bioRxiv2023

Dual-color GRAB sensors for monitoring spatiotemporal serotonin release in vivo

The serotonergic system plays important roles in both physiological and pathological processes, and is a widely used therapeutic target for many psychiatric disorders. Although several genetically encoded GFP-based serotonin (5-HT) sensors were recently developed, their sensitivities and spectral profiles are relatively limited. To overcome these limitations, we optimized green fluorescent G-protein-coupled receptor (GPCR)-activation-based 5-HT (GRAB5-HT) sensors and developed a new red fluorescent GRAB5-HT sensor. These sensors have excellent cell surface trafficking, high specificity, sensitivity, and spatiotemporal resolution, making them suitable for monitoring 5-HT dynamics in vivo. Besides recording subcortical 5-HT release in freely moving mice, we observed both uniform and gradient 5-HT release in the mouse dorsal cortex with mesoscopic imaging. Finally, we performed dual-color imaging and observed seizure-induced waves of 5-HT release throughout the cortex following calcium and endocannabinoid waves. In summary, these 5-HT sensors can offer valuable insights regarding the serotonergic system in both physiological and pathological states.

neuroscience↗

bioRxiv2023

The Prolonged Terminal Phase of Human Life Induces Survival Response in the Skin Transcriptome

Human death marks the end of organismal life under conditions such that the components of the human body continue to be alive. Such postmortem cellular survival depends on the nature (Hardy scale of slow-fast death) of human death. Slow and expected death typically results from terminal illnesses and includes a prolonged terminal phase of life. As such organismal death process unfolds, do cells of the human body adapt for postmortem cellular survival? Organs with low energy cost-of-living, such as the skin, are better suited for postmortem cellular survival. In this work, the effect of different durations of terminal phase of human life on postmortem changes in cellular gene expression was investigated using RNA sequencing data of 701 human skin samples from the Genotype-Tissue Expression (GTEx) database. Longer terminal phase (slow-death) was associated with a more robust induction of survival pathways (PI3K-Akt signaling) in postmortem skin. Such cellular survival response was associated with the upregulation of embryonic developmental transcription factors such as FOXO1, FOXO3, ATF4 and CEBPD. Upregulation of PI3K-Akt signaling was independent of sex or duration of death-related tissue ischemia. Analysis of single nucleus RNA-seq of post-mortem skin tissue specifically identified the dermal fibroblast compartment to be most resilient as marked by adaptive induction of PI3K-Akt signaling. In addition, slow death also induced angiogenic pathways in the dermal endothelial cell compartment of postmortem human skin. In contrast, specific pathways supporting functional properties of the skin as an organ were downregulated following slow death. Such pathways included melanogenesis and those representing the skin extracellular matrix (collagen expression and metabolism). Efforts to understand the significance of death as a biological variable (DABV) in influencing the transcriptomic composition of surviving component tissues has far-reaching implications including rigorous interpretation of experimental data collected from the dead and mechanisms involved in transplant-tissue obtained from dead donors.

genomics↗

bioRxiv2022

GESIAP: A Versatile Genetically Encoded Sensor-based Image Analysis Program

Intercellular communication mediated by a large number of neuromodulators diversifies physiological actions, yet neuromodulation remains poorly understood despite the recent upsurge of genetically encoded transmitter sensors. Here, we report the development of a versatile genetically encoded sensor-based image analysis program (GESIAP) that utilizes MATLAB-based algorithms to achieve high-throughput, high-resolution processing of sensor-based functional imaging data. GESIAP enables delineation of fundamental properties (e.g., transmitter spatial diffusion extent, quantal size, quantal content, release probability, pool size, and refilling rate at single release sites) of transmission mediated by various transmitters (i.e., monoamines, acetylcholine, neuropeptides, and glutamate) at various cell types (i.e., neurons, astrocytes, and other non-neuronal cells) of various animal species (i.e., mouse, rat, and human). Our analysis appraises a dozen of newly developed transmitter sensors, validates a conserved model of restricted non-volume neuromodulatory synaptic transmission, and accentuates a broad spectrum of presynaptic release properties that variegate neuromodulation.

neuroscience↗

bioRxiv2022

Asparagine starvation suppresses histone demethylation through iron depletion

Nutrient availability can impact epigenome to modify gene expression and dictate cell fate decision (Etchegaray and Mostoslavsky, 2016; Kinnaird et al., 2016). -ketoglutarate is an indispensable substrate for the Jumonji family of histone demethylases (JHDMs) mediating most of the cellular demethylation reactions on histone subunits (Schvartzman et al., 2018). Since -ketoglutarate is an intermediate of the tricarboxylic acid (TCA) cycle and a product of transamination, its intracellular levels are regulated by the metabolism of several amino acids (Baksh et al., 2020; Carey et al., 2015; Raffel et al., 2017; Vardhana et al., 2019). Here we show that asparagine starvation suppresses global histone demethylation. This process is neither due to the change of expression of histone modifying enzymes, nor due to the change of intracellular level of -ketoglutarate. Rather, asparagine starvation reduces intracellular pool of labile iron (Fe2+), which is a key cofactor for the JHDMs to function. Mechanistically, asparagine starvation post-transcriptionally suppresses the expression of iron responsive element binding protein 2 (IREB2), an iron sensing protein, which then reduces the mRNA expression of the transferrin receptor (TFRC), a major carrier for iron uptake (Hentze et al., 2010). Furthermore, iron supplementation to the culture medium restores histone demethylation and alters global gene expression to accelerate cell death under conditions of asparagine starvation. Collectively, our results uncover that suppression of iron-dependent histone demethylation is part of the cellular adaptive response to asparagine starvation.

cell biology↗

bioRxiv2022

Single Cell Cortical Bone Transcriptomics Defines Novel Osteolineage Gene Sets Altered in Chronic Kidney Disease

Due to a lack of spatial-temporal resolution at the single cell level, the etiologies of the bone dysfunction caused by diseases such as normal aging, osteoporosis, and the metabolic bone disease associated with chronic kidney disease (CKD) remain largely unknown. To this end, flow cytometry and scRNAseq were performed on long bone cells from Sost-cre/Ai9+ mice, and pure osteolineage transcriptomes were identified, including novel osteocyte-specific gene sets. Clustering analysis isolated osteoblast precursors that expressed Tnc, Mmp13, and Spp1, and a mature osteoblast population defined by Smpd3, Col1a1, and Col11a1. Osteocytes were demarcated by Cd109, Ptprz1, Ramp1, Bambi, Adamts14, Spns2, Bmp2, WasI, and Phex. We validated our in vivo scRNAseq using integrative in vitro promoter occupancy via ATACseq coupled with transcriptomic analyses of a conditional, temporally differentiated MSC cell line. Further, trajectory analyses predicted osteoblast-to-osteocyte transitions via defined pathways associated with a distinct metabolic shift as determined by single-cell flux estimation analysis (scFEA). Using the adenine mouse model of CKD, at a time point prior to major skeletal alterations, we found that gene expression within all stages of the osteolineage was disturbed. In sum, distinct populations of osteoblasts/osteocytes were defined at the single cell level. Using this roadmap of gene assembly, we demonstrated unrealized molecular defects across multiple bone cell populations in a mouse model of CKD, and our collective results suggest a potentially earlier and more broad bone pathology in this disease than previously recognized.

cell biology↗

bioRxiv2022

Targeting Chromatin Effector Pygo2 to Enhance Immunotherapy in Prostate Cancer

The noninflamed microenvironment in prostate cancer represents a barrier to immunotherapy. Genetic alterations underlying cancer cell-intrinsic oncogenic signaling have emerging roles in shaping the immune landscape. Recently, we identified Pygopus 2 (PYGO2) as the driver oncogene for the amplicon at 1q21.3. Here, using transgenic mouse models of metastatic prostate adenocarcinoma, we found that Pygo2 deletion decelerated tumor progression, diminished metastases, and extended survival. Pygo2 loss augmented the infiltration of cytotoxic T lymphocytes (CTLs) and sensitized tumor cells to T cell killing. Mechanistically, Pygo2 orchestrated a p53/Sp1/Kit/Ido1 signaling network to foster a microenvironment hostile to CTLs. Genetic or pharmacological inhibition of Pygo2 enhanced the anti-tumor efficacy of immunotherapies using immune checkpoint blockade, adoptive cell transfer, or myeloid-derived suppressor cell inhibitors. In human prostate cancer samples, Pygo2 expression was inversely correlated with CD8+ T cells. Our results highlight a promising path to improving immunotherapy with targeted therapy for lethal prostate cancer.

cancer biology↗

bioRxiv2022

Single nuclei transcriptomics of muscle reveals intra-muscular cell dynamics linked to dystrophin loss and rescue

In Duchenne muscular dystrophy, dystrophin loss leads to chronic muscle damage, dysregulation of repair, fibro-fatty replacement, and weakness. We develop methodology to efficiently isolate individual nuclei from frozen skeletal muscle, allowing single nuclei sequencing of irreplaceable archival samples from small samples. We apply this method to identify cell and gene expression dynamics within human DMD and mdx mouse muscle, characterizing treatment effects of dystrophin rescue by exon skipping therapy at single nuclei resolution. DMD exon 23 skipping events are directly observed and increased in myonuclei from treated mice. We describe partial rescue of type IIa and IIx myofibers, expansion of a novel MDSC-like myeloid population, recovery of repair/remodeling M2-macrophage, and repression of inflammatory POSTN1+ fibroblasts in response to exon skipping and partial dystrophin restoration. Use of this method enables exploration of cellular and transcriptomic mechanisms of dystrophin loss and repair.

genetics↗

bioRxiv2022

McaA and McaB control the dynamic positioning of a bacterial magnetic organelle

Magnetotactic bacteria (MTB) are a diverse group of microorganisms that use intracellular chains of ferrimagnetic nanocrystals, produced within their magnetosome organelles, to align and navigate along the geomagnetic field. The cell biological and biochemical properties of magnetosomes make them a powerful model for studying the molecular mechanisms of biomineralization and compartmentalization in bacteria. While several conserved magnetosome formation genes have been described, the evolutionary strategies for their species-specific diversification remain unknown. Here, we demonstrate that the fragmented nature of magnetosome chains in Magnetospirillum magneticum AMB-1 is controlled by two genes named mcaA and mcaB. McaA recognizes the positive curvature of the inner cell membrane while McaB localises to magnetosomes. Along with the MamK actin-like cytoskeleton, they create space for addition of new magnetosomes in between pre-existing magnetosomes. Phylogenetic analyses suggest that McaAB homologs are widespread and may represent an ancient strategy for organelle positioning in MTB.

microbiology↗

bioRxiv2022

Local 5-HT signal bi-directionally regulates the coincidence time window of associative learning

Temporal coincidence between the conditioned stimulus (CS) and unconditioned stimulus (US) is essential for associative learning across species. Despite its ubiquitous presence, the mechanism that may regulate this time window duration remains unclear yet. Using olfactory associative learning in Drosophila as a model, we find that suppressing or promoting serotonin (5-HT) signal could respectively shorten or prolong the coincidence time window of odor-shock associative learning and synaptic plasticity in mushroom body (MB) Kenyon cells (KCs). Capitalizing on GPCR-activation based (GRAB) sensors for 5-HT and acetylcholine (ACh), we characterized the in vivo 5-HT dynamics in MB lobes during odor and shock stimulations and further dissected this microcircuit. Interestingly, local KC-released ACh activates nicotinic receptors on the dorsal paired medial (DPM) neuron, and in turn the DPM neuron releases 5-HT to inhibit the ACh signal via the 5-HT1a receptor. Finally, we demonstrated that the DPM-mediated serotonergic feedback circuit is sufficient and necessary to regulate the coincidence time window. This work provides a model for studying the temporal contingency of environmental events and their causal relationship.

neuroscience↗

bioRxiv2022

SARS-CoV-2 Viral Genes Compromise Survival and Functions of Human Pluripotent Stem Cell-derived Cardiomyocytes via Reducing Cellular ATP Level

Cardiac manifestations are commonly observed in COVID-19 patients and prominently contributed to overall mortality. Human myocardium could be infected by SARS-CoV-2, and human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are susceptible to SARS-CoV-2 infection. However, molecular mechanisms of SARS-CoV-2 gene-induced injury and dysfunction of human CMs remain elusive. Here, we find overexpression of three SARS-CoV-2 coding genes, Nsp6, Nsp8 and M, could globally compromise transcriptome of hPSC-CMs. Integrated transcriptomic analyses of hPSC-CMs infected by SARS-CoV-2 with hPSC-CMs of Nsp6, Nsp8 or M overexpression identified concordantly activated genes enriched into apoptosis and immune/inflammation responses, whereas reduced genes related to heart contraction and functions. Further, Nsp6, Nsp8 or M overexpression induce prominent apoptosis and electrical dysfunctions of hPSC-CMs. Global interactome analysis find Nsp6, Nsp8 and M all interact with ATPase subunits, leading to significantly reduced cellular ATP level of hPSC-CMs. Finally, we find two FDA-approved drugs, ivermectin and meclizine, could enhance the ATP level, and ameliorate cell death and dysfunctions of hPSC-CMs overexpressing Nsp6, Nsp8 or M. Overall, we uncover the global detrimental impacts of SARS-CoV-2 genes Nsp6, Nsp8 and M on the whole transcriptome and interactome of hPSC-CMs, define the crucial role of ATP level reduced by SARS-CoV-2 genes in CM death and functional abnormalities, and explore the potentially pharmaceutical approaches to ameliorate SARS-CoV-2 genes-induced CM injury and abnormalities.

molecular biology↗

bioRxiv2022

Crystal Structure of Caskin1/CASK complex reveals the molecular basis of the binding specificity of CASK_CAMK domain and its binding partners

CASK is a unique scaffold protein in the synapse system. It links numerous proteins to the pre- or post-synaptic region and is critical to the homeostasis of synaptic vesicles. The N-terminus of CASK is a calcium/calmodulin-dependent protein kinase (CAMK) domain, which has diverse functions and interacts with downstream proteins to form a scaffold platform. Caskin1 is one of the brain-specific adaptor proteins of CASK. Previous studies showed that CASK_CAMK domain interacts with Caskin1 CID domain with relatively low affinity. In this study, we re-visit this interaction by remapping the interaction boundary and solving their complex structure. Based on the structure, we systematically compared the interactions between CASK_CAMK and other binding partners. Our results showed that CAMK domain occupies the CID peptide by using its C-lobe groove (between the 1 and 2) and there is a highly conserved signature motif ({zeta}-x-{psi}-W-{psi}-x-R) in the CID domain, where {zeta} is acidic side chain containing residues, x is any amino acid residue, {psi} is hydrophobic residues, W is for tryptophan, and R is arginine. These findings allowed us to identify several new potential cytoplasmic binding partners for CASK_CAMK.

biochemistry↗

bioRxiv2021

RORA regulates neutrophil migration and activation in zebrafish

Neutrophil migration and activation are essential for defense against pathogens. However, this process may also lead to collateral tissue injury. We used microRNA overexpression as a platform and discovered protein-coding genes that regulate neutrophil migration. Here we show that miR-99 decreased the chemotaxis of zebrafish neutrophils and human neutrophil-like cells. In zebrafish neutrophils, miR-99 directly targets the transcriptional factor RAR-related orphan receptor alpha (roraa). Inhibiting ROR, but not the closely related ROR{gamma}, reduced chemotaxis of zebrafish and primary human neutrophils without causing cell death, and increased susceptibility of zebrafish to bacterial infection. Expressing a dominant-negative form of Ror or disrupting the roraa locus specifically in zebrafish neutrophils reduced cell migration. At the transcriptional level, ROR regulates transmembrane signaling receptor activity and protein phosphorylation pathways. Our results, therefore, reveal previously unknown functions of miR- 99 and ROR in regulating neutrophil migration and anti-microbial defense.

cell biology↗

bioRxiv2021

MECP2pedia: A Comprehensive Transcriptome Portal for MECP2 Disease Research

Mutations in MeCP2 result in a crippling neurological disease, but we lack a lucid picture of MeCP2s molecular role. Focusing on individual transcriptomic studies yields inconsistent differentially expressed genes. We have aggregated and homogeneously processed modern public MeCP2 transcriptome data, which we present in a web portal. With this big data, we discovered a commonly perturbed core set of genes that transcends the limitations of any individual study. We then found distinct consistently up and downregulated subsets within these genes. We observe enrichment for this mouse core in other species MeCP2 models and see overlap between this core and ASD models. Analysis of signal to noise finds that many studies lack enough biological replicates. By integrating and examining transcriptomic data at scale, we have generated a valuable resource and insight on MeCP2 function.

bioinformatics↗

bioRxiv2021

Different mycorrhizal nutrient acquisition strategies shape tree species competition and coexistence dynamics

Mycorrhizal fungi with different nutrient acquisition strategies influence the functional separation among plant species. This might drive resource competition dynamics that cumulatively impact tree species coexistence, but few manipulative experiments have directly tested this. Combining surveys and experiments in a modern coexistence theory framework, we tested how variation in mycorrhizal strategies and nutrient conditions affect competitive outcomes between co-occurring ectomycorrhizal (EM) and arbuscular mycorrhizal (AM) tree species. The dependency on EM symbioses increased with latitude and nitrogen (N) limitation, matching global trends. Host-specific soil microbiome conditioning and N fertilization combined to qualitatively affect coexistence outcomes. Lower N conditions favored EM over AM trees, but N fertilization reversed this outcome for southern species, consistent with regional-scale forest mycorrhizal transitions. As the magnitude and outcome of microbially-mediated competition depends on mycorrhizal differentiation and soil nutrient availability, this strongly supports the importance of mycorrhizal symbioses in driving large-scale biogeographic patterns of tree species.

ecology↗

bioRxiv2021

A serotonergic axon-cilium synapse drives nuclear signaling to maintain chromatin accessibility

Chemical synapses between axons and dendrites mediate much of the brains intercellular communication. Here we describe a new kind of synapse - the axo-ciliary synapse - between axons and primary cilia. By employing enhanced focused ion beam - scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between the serotonergic axons arising from the brainstem, and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, 5-hydroxytryptamine receptor 6 (HTR6), whose mutation is associated with learning and memory defects. Using a newly developed cilia-targeted serotonin sensor, we show that optogenetic stimulation of serotonergic axons results in serotonin release onto cilia. Ciliary HTR6 stimulation activates a non-canonical Gq/11-RhoA pathway. Ablation of this pathway results in nuclear actin and chromatin accessibility changes in CA1 pyramidal neurons. Axo-ciliary synapses serve as a distinct mechanism for neuromodulators to program neuron transcription through privileged access to the nuclear compartment.

cell biology↗