Biology subjects

Ji, H.

Publications and source records attributed to Ji, H..

At least 91 records · Page 5Linked to original sources

bioRxiv2022

DNA methylation entropy is associated with DNA sequence featuresand developmental epigenetic divergence

Epigenetic information defines tissue identity and is largely inherited in development through DNA methylation. While studied mostly for mean differences, methylation also encodes stochastic change, defined as entropy in information theory. Analyzing allelespecific methylation in 48 human tissue sample datasets, we find that methylation entropy is associated with specific DNA binding motifs, regulatory DNA, and CpG density. Then applying information theory to 42 mouse embryo methylation datasets, we find that time- and tissue-specific patterns of development are more strongly correlated with methylation entropy than with mean, and methylation entropy is associated with sequence and chromatin features conserved with human. Moreover, methylation entropy is directly related to gene expression variability in development, suggesting a role for epigenetic entropy in developmental plasticity.

genomics↗

bioRxiv2022

Lung tumor-infiltrating Treg have divergent transcriptional profiles and function linked to checkpoint blockade response

Regulatory T cells (Treg) are conventionally viewed to suppress endogenous and therapyinduced anti-tumor immunity; however, their role in modulating responses to immune checkpoint blockade (ICB) is unclear. In this study, we integrated single-cell RNAseq/TCRseq of >73,000 tumor-infiltrating Treg (TIL-Treg) from anti-PD-1-treated and treatment naive non-small cell lung cancers (NSCLC) with single cell analysis of tumor-associated antigen (TAA)-specific Treg derived from a murine tumor model. We identified 10 subsets of human TIL-Treg, most of which have high concordance with murine TIL-Treg subsets. Notably, one subset selectively expresses high levels of OX40 and GITR, whose engangement by cognate ligand mediated proliferative programs and NF-kB activation, as well as multiple genes involved in Treg suppression, in particular LAG3. Functionally, the OX40hiGITRhi subset in the most highly suppressive ex vivo and Treg expression of OX40, GITR and LAG3, correlated with resistance to PD-1 blockade. Surprisingly, in the murine tumor model, we found that virtually all TIL-Treg expressing T cell receptors that are specific for TAA fully develop a distinct Th1-like signature over a two-week period after entry into the tumor, down-regulating FoxP3 and up-regulating expression of TBX21 (Tbet), IFN{gamma} and certain pro-inflammatory granzymes. Application of a gene score from the murine TAA-specific Th1-like Treg subset to the human single-cell dataset revealed a highly analogous subcluster that was enriched in anti-PD-1 responding tumors. These findings demonstrate that TIL-Treg partition into multiple distinct transcriptionally-defined subsets with potentially opposing effects on ICB-induced anti-tumor immunity and suggest that TAA-specific TIL-Treg may positively contribute to anti-tumor responses. One-Sentence SummaryWe define 10 subsets of lung cancer-infiltrating regulatory T cells, one of which is highly suppressive and enriched in anti-PD-1 non-responders and the other is Th1-like and is enriched in PD-1 responders.

immunology↗

bioRxiv2022

A robotic system for automated genetic manipulation and analysis of Caenorhabditis elegans on agar media

The nematode Caenorhabditis elegans is one of the most widely studied organisms in biology due to its small size, rapid life cycle, and manipulable genetics. Research with C. elegans depends on labor-intensive and time-consuming manual procedures, imposing a major bottleneck for many studies, especially those involving large numbers of animals. Here we describe the first general-purpose tool, WormPicker, a robotic system capable of performing complex genetic manipulations and other tasks by imaging, phenotyping, and transferring C. elegans on standard agar media. Our system uses a motorized stage to move an imaging system and a robotic arm over an array of plates. Machine vision tools identify animals and assay developmental stage, morphology, sex, expression of fluorescent reporters, and other phenotypes. Based on the results of these assays the robotic arm selectively transfers individual animals using an electrically self-sterilized wire loop, with the aid of machine vision and electrical capacitance sensing. Automated C. elegans manipulation shows reliability and throughput comparable to standard manual methods. We developed software to enable the system to autonomously carry out complex protocols. To validate the effectiveness and versatility of our methods we used the system to perform a collection of common C. elegans procedures, including genetic crossing, genetic mapping, and genomic integration of a transgene. Our robotic system will accelerate C. elegans research and opens possibilities for performing genetic and pharmacological screens that would be impractical using manual methods. Significance StatementThe nematode Caenorhabditis elegans is a powerful genetic model organism in life sciences due to its compact anatomy, short life cycle, and optical transparency. Current methods for worm genetics rely on laborious, time-consuming, and error-prone manual work. Here, we describe the first general-purpose automated tool capable of genetically manipulating C. elegans. Our robotic system will accelerate a broad variety of C. elegans research and opens possibilities for performing genetic and pharmacological screens that would be impractical using manual methods.

bioengineering↗

bioRxiv2022

Limited Defense of Liver Energy Status in Rats Susceptible to Diet-induced Obesity

Rats that are susceptible to diet-induced obesity have a preexisting reduced capacity for hepatic fatty acid oxidation compared with those resistant to diet-induced obesity. The eating response to administration of a fatty acid oxidation inhibitor is more closely associated with low liver energy status than it is with reduced hepatic fatty acid oxidation, a finding consistent with studies showing that lowered liver energy status stimulates food intake. To evaluate whether susceptibility to diet-induced obesity is associated with a preexisting impairment in liver energy status, we conducted two experiments in obesity-prone (OP) and -resistant (OR) outbred rats. In one experiment, OP rats increased food intake more than did OR rats during refeeding after a 24 h fast. When fasted and refed again, liver energy status (i.e., liver ATP content, ATP:ADP ratio and phosphorylation index) was lower in OP rats after a fast. When OP animals were refed fixed rations of food, liver energy status increased more slowly during refeeding than it did in OR rats. The delay in restoration of liver energy status during refeeding corresponded to the interval during which OP rats ate significantly more food in the intake test. In a second experiment, liver energy status was lower in OP than it was in OR rats after injection of the fructose analogue, 2,5-anhydro-D-mannitol, which depletes liver ATP. These results suggest that liver energy status is more vulnerable in rats susceptible to diet-induced obesity.

physiology↗

bioRxiv2022

A proprioceptive feedback circuit controls Caenorhabditis elegans locomotor amplitude through dopamine signaling

An animal adapts its motor behavior to navigate the external environment. This adaptation depends on proprioception, which provides feedback on an animals body postures. How proprioception mechanisms interact with motor circuits and contribute to locomotor adaptation remains unclear. Here we describe and characterize proprioception-mediated homeostatic control of undulatory movement in the roundworm Caenorhabditis elegans. We found the worm responds to optogenetically or mechanically induced decreases in midbody bending amplitude by increasing its anterior amplitude. Conversely, it responds to increased midbody amplitude by decreasing the anterior amplitude. Using genetics, microfluidic and optogenetic perturbation response analyses, and optical neurophysiology, we elucidated the neural circuit underlying this compensatory postural response. The dopaminergic PDE neurons proprioceptively sense midbody bending and signal to AVK interneurons via the D2-like dopamine receptor DOP-3. The FMRFamide-like neuropeptide FLP-1, released by AVK, regulates SMB head motor neurons to modulate anterior bending. We propose that this homeostatic behavioral control optimizes locomotor efficiency. Our findings demonstrate a mechanism in which proprioception works with dopamine and neuropeptide signaling to mediate motor control, a motif that may be conserved in other animals.

neuroscience↗

bioRxiv2022

Single-cell allele-specific expression analysis reveals dynamic and cell-type-specific regulatory effects

Allele-specific expression, which measures the expression of two alleles of a gene in a diploid individual, is a powerful signal to study cis-regulatory effects. Comparing ASE across conditions, or differential ASE, can reveal context-specific gene regulation. Recently, single-cell RNA sequencing (scRNA-seq) has allowed the measurement of ASE at the resolution of individual cells, but there is a lack of statistical methods to analyze such data. We develop DAESC, a statistical method for differential ASE analysis across any condition of interest using scRNA-seq data from multiple individuals. DAESC includes a baseline model based on beta-binomial regression with random effects accounting for multiple cells from the same individual (DAESC-BB), and an extended mixture model that incorporates implicit haplotype phasing (DAESC-Mix). We demonstrate through simulations that DAESC accurately captures differential ASE effects in a wide range of scenarios. Application to scRNA-seq data from 105 induced pluripotent stem cell lines identifies 657 genes that are dynamically regulated during endoderm differentiation. A second application identifies several genes that are differentially regulated in pancreatic endocrine cells between type 2 diabetes patients and controls. In conclusion, DAESC is a powerful method for single-cell differential ASE analysis and can facilitate the discovery of context-specific regulatory effects.

genomics↗

bioRxiv2022

Modulation effect of non-invasive transcranial ultrasound stimulation in an ADHD rat model

Previous studies have demonstrated that transcranial ultrasound stimulation (TUS) with noninvasive high penetration and high spatial resolution has an effective neuromodulatory effect on neurological diseases. Attention deficit hyperactivity disorder (ADHD) is a persistent neurodevelopmental disorder that severely affects child health. However, the neuromodulatory effects of TUS on ADHD have not been reported to date. To reveal the neuromodulatory effects, TUS was performed in ADHD model rats for 2 consecutive weeks, and the behavioral improvement of ADHD, neural activity of ADHD from neurons and neural oscillation levels, and the plasma membrane dopamine transporter (DAT) and brain-derived neurotrophic factor (BDNF) in the brains of ADHD rats were evaluated. The results showed that TUS can improve cognitive behavior in ADHD rats, and that TUS altered neuronal firing patterns and modulated the relative power and sample entropy of local field potentials in an ADHD rat model. In addition, TUS can also enhance BDNF expression in the brain tissue of the ADHD rat model. In conclusion, TUS has an effective neuromodulatory effect on ADHD and thus has the potential to clinically improve cognitive dysfunction in ADHD.

neuroscience↗

bioRxiv2022

Identification of regulatory elements in primary sensory neurons involved in neuropathic pain

Chronic pain is a significant public health issue that is often refractory to existing therapies. Here we use a multiomic approach to identify cis-regulatory elements that show differential chromatin accessibility, and reveal transcription factor (TF) binding motifs with functional regulation in the dorsal root ganglion (DRG), which contain cell bodies of primary sensory neurons, after nerve injury. We integrated RNA-seq to understand how differential chromatin accessibility after nerve injury may influence gene expression. Using TF protein arrays and chromatin immunoprecipitation-qPCR, we confirmed C/EBP{gamma} binding to a differentially accessible sequence and used RNA-seq to identify processes in which C/EBP{gamma} plays an important role. Our findings offer insights into TF motifs that are associated with chronic pain. These data show how interactions between chromatin landscapes and TF expression patterns may work together to determine gene expression programs in DRG neurons after nerve injury.

neuroscience↗

bioRxiv2022

Small cytosolic dsDNAs repress cGAS activation and induce autophagy

Cyclic GMP-AMP (cGAMP) synthase (cGAS), a major cytosolic DNA sensor, activates innate immune responses by producing cGAMP, which activates stimulator of interferon genes (STING)1. Cytosolic DNA induces autophagy in a cGAS-dependent manner to avoid persistent immune stimulation. Although dsDNAs < 20 bp can bind to cGAS, robust cGAS activation requires dsDNAs > 45 bp 2-4. However, whether cytosolic dsDNAs < 45 bp exist in mammalian cells remains unclear. Here, we identified a class of small cytosolic DNAs (scDNAs) of [~]20-40 bp in human and mouse cell lines. scDNAs competed with herring testis DNA (HT-DNA, [~]200-1500 bp) for binding to cGAS, and repressing HT-DNA-induced cGAS activation and the associated interferon {beta} (IFN{beta}) production. Moreover, scDNAs promoted cGAS and Beclin-1 interaction, triggering the release of Rubicon, a negative regulator of phosphatidylinositol 3-kinase class III (PI3KC3)5,6, from the Beclin-1-PI3KC3 complex, activating PI3KC3 and inducing autophagy. DNA damage decreased and autophagy inducers increased scDNA levels. scDNA transfection or autophagy induction attenuated DNA damage-induced cGAS-STING activation and IFN{beta} expression. Thus, scDNAs acted as molecular brakes of cGAS activation, preventing excessive inflammatory cytokine production following DNA damage. Our findings lay foundations for understanding the physiological and pathological functions of scDNAs.

molecular biology↗

bioRxiv2022

Probabilistic embedding and clustering with alignment for spatial transcriptomics data integration with PRECAST

Spatially resolved transcriptomics involves a set of emerging technologies that enable the transcriptomic profiling of tissues with the physical location of expressions. Although a variety of methods have been developed for data integration, most of them are for single-cell RNA-seq datasets without consideration of spatial information. Thus, methods that can integrate spatial transcriptomics data from multiple tissue slides, possibly from multiple individuals, are needed. Here, we present PRECAST, a data integration method for multiple spatial transcriptomics datasets with complex batch effects and/or biological effects between slides. PRECAST unifies spatial factor analysis simultaneously with spatial clustering and embedding alignment, while requiring only partially shared cell/domain clusters across datasets. Using both simulated and four real datasets, we show improved cell/domain detection with outstanding visualization, and the estimated aligned embeddings and cell/domain labels facilitate many downstream analyses. We demonstrate that PRECAST is computationally scalable and applicable to spatial transcriptomics datasets from different platforms.

genomics↗

bioRxiv2022

Spatial Transcriptomics-correlated Electron Microscopy

Current spatial transcriptomics methods identify cell states in a spatial context but lack morphological information. Scanning electron microscopy, in contrast, provides structural details at nanometer resolution but lacks molecular decoding of the diverse cellular states. To address this, we correlated MERFISH spatial transcriptomics with large area volume electron microscopy using adjacent tissue sections. We applied our technology to characterize the damage-associated microglial identities in mouse brain, allowing us, for the first time, to link the morphology of foamy microglia and interferon-response microglia with their transcriptional signatures.

neuroscience↗

bioRxiv2022

T cells induce interferon-responsive oligodendrocytes during white matter aging

A hallmark of nervous system aging is a decline of white matter volume and function, but the underlying mechanisms leading to white matter pathology are unknown. Here, we found age-related alterations of oligodendrocytes with a reduction of total oligodendrocyte density in the aging murine white matter. Using single-cell RNA sequencing, we identify interferon-responsive oligodendrocytes, which localize in proximity of CD8+ T cells in the aging white matter. Absence of functional lymphocytes decreased oligodendrocyte reactivity and rescued oligodendrocyte loss, while T-cell checkpoint inhibition worsened the aging affect. In summary, we provide evidence that T cells induced interferon-responsive oligodendrocytes are important modifiers of white matter aging.

neuroscience↗

bioRxiv2022

Chimeric mRNA based COVID-19 vaccine elicits potent neutralizing antibodies and protection against Omicron and Delta

The emerging SARS-CoV-2 variants of concern (VOCs) exhibit enhanced transmission and immune escape, reducing the efficacy and effectiveness of the two FDA-approved mRNA vaccines. Here, we explored various strategies to develop novel mRNAs vaccines to achieve safer and wider coverage of VOCs. Firstly, we constructed a cohort of mRNAs that feature a furin cleavage mutation in the spike (S) protein of predominant VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2). Not present in the mRNA vaccines currently in use, the mutation abolished the cleavage between the S1 and S2 subunits, potentially enhancing the safety profile of the immunogen. Secondly, we systematically evaluated the induction of neutralizing antibodies (nAb) in vaccinated mice, and discovered that individual VOC mRNAs elicited strong neutralizing activity in a VOC-specific manner. Thirdly, the IgG produced in mice immunized with Beta-Furin and Washington (WA)-Furin mRNAs showed potent cross-reactivity with other VOCs, which was further corroborated by challenging vaccinated mice with the live virus of VOCs. However, neither WA-Furin nor Beta-Furin mRNA elicited strong neutralizing activity against the Omicron variant. Hence, we further developed an Omicron-specific mRNA vaccine that restored protection against the original and the sublineages of Omicron variant. Finally, to broaden the protection spectrum of the new Omicron mRNA vaccine, we tested the concept of bivalent immunogen. Instead of just fusing two RBDs head-to-tail, we for the first time constructed an mRNA-based chimeric immunogen by introducing the RBD of Delta variant into the entire S antigen of Omicron. The resultant chimeric mRNA was capable of inducing potent and broadly acting nAb against Omicron (both BA.1 and BA.2) and Delta, which paves the way to develop new vaccine candidate to target emerging variants in the future.

immunology↗

bioRxiv2022

Quantitative fate mapping: Reconstructing progenitor field dynamics via retrospective lineage barcoding

Natural and induced somatic mutations that accumulate in the genome during development record the phylogenetic relationships of cells; however, whether these lineage barcodes can capture the dynamics of complex progenitor fields remains unclear. Here, we introduce quantitative fate mapping, an approach to simultaneously map the fate and quantify the commitment time, commitment bias, and population size of multiple progenitor groups during development based on a time-scaled phylogeny of their descendants. To reconstruct time-scaled phylogenies from lineage barcodes, we introduce Phylotime, a scalable maximum likelihood clustering approach based on a generalizable barcoding mutagenesis model. We validate these approaches using realistically-simulated barcoding results as well as experimental results from a barcoding stem cell line. We further establish criteria for the minimum number of cells that must be analyzed for robust quantitative fate mapping. Overall, this work demonstrates how lineage barcodes, natural or synthetic, can be used to obtain quantitative fate maps, thus enabling analysis of progenitor dynamics long after embryonic development in any organism.

developmental biology↗

bioRxiv2022

The Drosophila chemokine-like Orion bridges phosphatidylserine and Draper in phagocytosis of neurons

Phagocytic clearance of degenerating neurons is triggered by "eat-me" signals exposed on the neuronal surface. The conserved neuronal eat-me signal phosphatidylserine (PS) and the engulfment receptor Draper (Drpr) mediate phagocytosis of degenerating neurons in Drosophila. However, how PS is recognized by Drpr-expressing phagocytes in vivo remains poorly understood. Using multiple models of dendrite degeneration, we show that the Drosophila chemokine-like protein Orion can bind to PS and is responsible for detecting PS exposure on neurons; it is supplied cell-non-autonomously to coat PS-exposing dendrites and to mediate interactions between PS and Drpr, thus enabling phagocytosis. As a result, the accumulation of Orion on neurons and on phagocytes produces opposite outcomes by potentiating and suppressing phagocytosis, respectively. Moreover, the Orion dosage is a key determinant of the sensitivity of phagocytes to PS exposed on neurons. Lastly, mutagenesis analyses show that the sequence motifs shared between Orion and human immunomodulatory proteins are important for Orion function. Thus, our results uncover a missing link in PS-mediated phagocytosis in Drosophila and imply conserved mechanisms of phagocytosis of neurons. SIGNIFICANCE STATEMENTPhagocytes efficiently clear sick or damaged neuronal branches by engulfing them, while leaving healthy branches untouched. How phagocytes recognize degenerating neurites remains poorly understood. Here, we identified a key role for the secreted protein Orion in the detection and engulfment of degenerating neurites in Drosophila. Using multiple models of dendrite degeneration, we found that Orion acts as a bridging molecule between the neuronal "eat-me" signal phosphatidylserine and the engulfment receptor Draper on phagocytes, enabling phagocytosis. Our study reveals a missing link in phosphatidylserine-mediated phagocytosis in vivo, sheds light on factors determining the sensitivity of phagocytes, and implies the potential for manipulating the detection of neuronal "eat-me" signals in neurodegenerative diseases.

neuroscience↗

bioRxiv2022

Why SARS-CoV-2 Omicron variant is milder? A single high-frequency mutation of structural envelope protein matters.

SARS-CoV-2 Omicron variant is highly transmissible and extensive morbidity, which has raised concerns for antiviral therapy. In addition, the molecular basis for the attenuated pathogenicity and replication capacity of Omicron remains elusive. Here, we report for the first time that a high-frequency mutation T9I on 2-E of SARS-CoV-2 variant Omicron forms a non-selective ion channel with abolished calcium permeability and reduced acid sensitivity compared to the WT channel. In addition, T9I caused less cell death and a weaker cytokine production. The channel property changes might be responsible for the Omicron variant releases less efficiently and induces a comparatively lower level of cell damage in the infected cells. Our study gives valuable insights into key features of the Omicron variant, further supporting 2-E is a promising drug target against SARS-CoV-2 and providing critical information for the COVID-19 treatment.

microbiology↗

bioRxiv2022

High-Throughput Screening for Myelination Promoting Compounds Using Human Stem Cell-derived Oligodendrocyte Progenitor Cells Identifies Novel Targets

Promoting myelination capacity of endogenous oligodendrocyte precursor cells (OPCs) is a promising therapeutic approach for central nervous system demyelinating disorders such as Multiple Sclerosis (MS). To aid in the discovery of myelination promoting compounds, we generated an advanced, genome engineered, human pluripotent stem cell (hPSC) line that consist of three reporters (identification-and-purification tag, GFP, and secreted NanoLuc) driven by the endogenous PDGFR, PLP1 and MBP genes, respectively. Based upon this line, we established a high-throughput drug screening platform and performed a small molecule screen with 2500 bioactive molecules. In addition to a number of previously known pathways, our screening effort identified new pathways whose inhibition enhance oligodendrocyte maturation and myelination. Although further genetic and molecular validation is required, the identified inhibitors could potentially be repurposed to develop remyelination therapy for MS and other demyelinating disorders.

neuroscience↗

bioRxiv2022

Hedgehog regulation of epithelial cell state and morphogenesis in the larynx

The larynx enables speech while regulating swallowing and respiration. Larynx function hinges on the laryngeal epithelium which originates as part of the anterior foregut and undergoes extensive remodeling to separate from the esophagus and form vocal folds that interface with the adjacent trachea. Here we find that Sonic hedgehog (SHH) is essential for epithelial integrity in the larynx as well as the anterior foregut. During larynx-esophageal separation, low Shh expression marks specific domains of actively remodeling epithelium that undergo an epithelial to mesenchymal transition (EMT) characterized by the induction of N-Cadherin and movement of cells out of the epithelial layer. Consistent with a role for SHH signaling in regulating this process, Shh mutants undergo an abnormal EMT throughout the anterior foregut and larynx, marked by a cadherin switch, movement out of the epithelial layer and cell death. Unexpectedly, Shh mutant epithelial cells are replaced by a new population of Pax-1 expressing cells that form a rudimentary epithelium. These findings have important implications for interpreting the etiology of HH- dependent birth defects within the foregut. We propose that SHH signaling has a default role in maintaining epithelial identity throughout the anterior foregut and that regionalized reductions in SHH trigger epithelial remodeling.

developmental biology↗