Biology subjects

Lee, H. J.

Publications and source records attributed to Lee, H. J..

At least 73 records · Page 4Linked to original sources

bioRxiv2021

Hormonal contraceptives alter amphetamine place preference and responsivity in the intact female rat.

Hormonal contraceptives (HCs) are commonly used among reproductive aged women and alter the physiological state of the user by interfering with endogenous hormone concentrations and their actions on the reproductive tract. As hormones such as estradiol and progesterone modulate the incidence of substance abuse disorders in women, it is important to consider the influence HCs have on the female brain and behavior. This experiment explores how female sex steroid hormonal states associated with the rat estrous cycle, and modulating those states with HCs, influences measures of drug preference and responsivity. First, rats underwent food-light Pavlovian conditioning to measure conditioned orienting, a known predictor of amphetamine (AMP) place preference. Then, rats were conditioned and tested for AMP place preference with either an HC-implant or during estrous cycle stages associated with different ovarian hormone levels (i.e., proestrus (P) or metestrus/diestrus (M/D) while recording ultrasonic vocalizations (USVs) as an index of hedonic responsivity. Because of dopamines (DA) role in modulation of AMP actions, DA cell activity and availability were examined using tyrosine hydroxylase and FOS immunohistochemistry after final AMP challenge. Conditioned orienting did not differ between cycling and HC-implanted. P rats emitted more USVs during conditioning, showed higher AMP place preference throughout testing, and had higher DA cell activity in the substantia nigra compared to M/D and HC-implanted rats. Sex steroid hormone serum concentration and uterine horn thickness predicted some but not all of these measures. This experiment suggests ovarian hormones affect drug preference and responsivity, while providing novel insight into how hormone-altering contraceptives may reduce these measures.

animal behavior and cognition↗

bioRxiv2021

Expression patterns of Arc mRNA after renewal of appetitive behavior in female rats.

Renewal of appetitive behavior depends on the gonadal hormonal state of the female rat. In this experiment the effect of female rat estrous cycle stage on renewal of appetitive behaviors is replicated and extended upon to understand how endogenous hormonal states around the estrous cycle drive renewal at the neuronal population level. Estrous cycle stage (i.e., proestrus (P, high hormone) or metestrus/diestrus (M/D, low hormone)) was considered during two important learning and behavioral expression windows: at extinction training and during LTM/renewal testing. First, rats in P during context-dependent extinction training but in some other stage of the estrous cycle during long-term memory and renewal testing (Different) were shown to exhibit more renewal of conditioned foodcup (but not conditioned orienting) behavior compared to rats in other estrous cycle groups. Next, cellular compartment analysis of temporal activity using fluorescence in situ hybridization (catFISH) was used to examine immediate early gene activity of Arc mRNA in neuronal populations after distinct context-stimulus exposures (i.e., extinction and acquisition test contexts). Arc mRNA expression patterns were examined in the prefrontal cortex (PFC), amygdala, hippocampus (HPC), and paraventricular nucleus of the thalamus. P-different rats showed differential neuronal population activity in the infralimbic cortex of the PFC, the lateral amygdaloid nucleus, and both CA1 and CA3 regions of the dorsal HPC. In each region P-different rats exhibited more co-expression and less specificity of Arc mRNA compared to other hormonal groups, indicating that renewal of appetitive foodcup behavior induces Arc mRNA in overlapping neuronal populations in female rats.

animal behavior and cognition↗

bioRxiv2021

Comparative transcriptomic analysis reveals translationally relevant processes in mouse models of malaria

Recent initiatives to improve translation of findings from animal models to human disease have focussed on reproducibility but quantifying the relevance of animal models remains a challenge. Here we use comparative transcriptomics of blood to evaluate the systemic host response and its concordance between humans with different clinical manifestations of malaria and five commonly used mouse models. Plasmodium yoelii 17XL infection of mice most closely reproduces the profile of gene expression changes seen in the major human severe malaria syndromes, accompanied by high parasite biomass, severe anemia, hyperlactatemia, and cerebral microvascular pathology. However, there is also considerable discordance of changes in gene expression between species and across all models, indicating that the relevance of biological mechanisms of interest in each model should be assessed before conducting experiments. Our data will aid selection of appropriate models for translational malaria research, and the approach is generalizable to other disease models.

genomics↗

bioRxiv2021

Atmospheric plasma jet device for versatile electron microscope grid treatment

Atmospheric pressure plasmas have been widely applied in surface modification and biomedical treatment due to its ability to generate highly reactive radicals and charged particles. In negative-stain electron microscopy (Neg-EM) and cryogenic electron microscopy (cryo-EM), plasmas have been used in eliminating the surface contaminants as well as generating the hydrophilic surface to embed the specimen on grids. Plasma treatment is a prerequisite for negative stain and quantifoil grids, which are coated with hydrophobic carbon on the grid surface. Here we introduce a non-thermal atmospheric plasma jet system as an alternative new tool for surface treatment. Unlike the conventional glow discharger, we found that the plasma jet system successfully cleans the grid surface and introduces hydrophilicity on grids in the ambient environment without introducing a vacuum. Therefore, we anticipate the plasma jet system will be beneficial in many aspects, such as cost-effective, convenient, versatile, and potential applications in surface modification for both negative stain and cryo-EM grid treatment.

biochemistry↗

bioRxiv2021

SINEultaneous profiling of epigenetic heterogeneity and transcriptome in single cells

Global changes in DNA methylation are observed in developmental and disease contexts, and singlecell analyses are highlighting the heterogeneous regulation of these processes. However, technical challenges associated with single-cell analysis of DNA methylation limit these studies. We present single-cell transposable element methylation sequencing (scTEM-seq) for cost-effective estimation of global DNA methylation levels. By targeting high-copy LINE-1 and SINE Alu elements, we achieve amplicon bisulphite sequencing with thousands of loci covered in each library. Parallel transcriptome analysis is also performed to link global DNA methylation heterogeneity with gene expression. We apply scTEM-seq to KG1a acute myeloid leukaemia (AML) cells, and primary AML cells. Decitabine treatment of KG1a cells induces global DNA methylation heterogeneity associated with altered expression of immune process genes. We also compare global levels of DNA methylation to expression of transposable elements and find a predominance of negative correlations in both the KG1a and patient cells. Finally, we observe co-ordinated upregulation of many transposable elements in a sub-set of decitabine treated cells. By linking global DNA methylation heterogeneity with transcription, scTEM-seq will refine our understanding of epigenetic regulation in cancer and beyond.

genomics↗

bioRxiv2021

The intrinsic chaperone network of Arabidopsis stem cells confers protection against proteotoxic stress

The biological purpose of plant stem cells is to maintain themselves while providing new pools of differentiated cells that form organs and rejuvenate or replace damaged tissues1-3. Protein homeostasis, or proteostasis, is required for cell function and viability4-7. However, the link between proteostasis and plant stem cell identity remains unknown. In contrast to their differentiated counterparts, we find that root stem cells can prevent the accumulation of aggregated proteins even under proteotoxic stress conditions such as heat stress or proteasome inhibition. Notably, root stem cells exhibit enhanced expression of distinct chaperones that maintain proteome integrity. Particularly, intrinsic high levels of the TRiC/CCT chaperonin determine stem cell maintenance and their remarkable ability to suppress protein aggregation. Overexpression of CCT8, a key activator of TRiC/CCT assembly8, is sufficient to ameliorate protein aggregation in differentiated cells and confer resistance to proteotoxic stress in plants. Taken together, our results indicate that enhanced proteostasis mechanisms in stem cells could be an important requirement for plants to persist under extreme environmental conditions and reach extreme long ages. Thus, proteostasis of stem cells could provide insights to design and breed plants tolerant to environmental challenges caused by the climate change.

plant biology↗

bioRxiv2020

Pre-conditioning Modifies the Tumor Microenvironment to Enhance Solid Tumor CAR T Cell Efficacy and Endogenous Immunity

Chimeric antigen receptor (CAR) T cell therapy has led to impressive clinical responses in patients with hematological malignancies; however, its utility in patients with solid tumors has been limited. While CAR T cells for the treatment of advanced prostate cancer are being clinically evaluated and are anticipated to show bioactivity, their safety and the impact of the immunosuppressive tumor microenvironment (TME) have not been faithfully explored preclinically. Using a novel human prostate stem cell antigen knock-in (hPSCA-KI) immunocompetent mouse model and syngeneic murine PSCA CAR T cells, we performed analyses of normal and tumor tissues by flow cytometry, immunohistochemistry, and/or RNA sequencing. We further assessed the beneficial impact of cyclophosphamide (Cy) pre-conditioning on modifications to the immunosuppressive TME and impact on PSCA-CAR T cell safety and efficacy. We observed an in vivo requirement of Cy pre-conditioning in uncovering the efficacy of PSCA-CAR T cells in prostate and pancreas cancer models, with no observed toxicities in normal tissues with endogenous PSCA expression. This combination also dampened the immunosuppressive TME, generated pro-inflammatory myeloid and T cell signatures in tumors, and enhanced the recruitment of antigen-presenting cells, and endogenous as well as adoptively-transferred CAR T cells, resulting in long-term anti-tumor immunity.

immunology↗

bioRxiv2020

Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT enhances the effector function of CD19-targeting CAR T cells and promotes an early-memory phenotype

CD19-targeting chimeric antigen receptor (CAR) T cells have become an important therapeutic option for patients with relapsed and refractory B cell malignancies. However, recent clinical data indicate that a significant portion of patients still do not benefit from the therapy owing to various resistance mechanisms, including high expression of multiple inhibitory immune checkpoint receptors on activated CAR T cells. Here, we report a lentiviral two-in-one CAR T approach in which two checkpoint receptors are downregulated simultaneously by a dual short-hairpin RNA (shRNA) cassette integrated into a CAR vector. Using this system, we evaluated CD19-targeting CAR T cells in the context of four different checkpoint combinations--PD-1/TIM-3, PD-1/LAG-3, PD-1/CTLA-4 and PD-1/TIGIT--and found that CAR T cells with PD-1/TIGIT downregulation uniquely exerted synergistic antitumor effects in mouse xenograft models compared with PD-1 single downregulation, and maintained cytolytic and proliferative capacity upon repeated antigen exposure. Importantly, functional and phenotypic analyses of CAR T cells as well as analyses of transcriptomic profiles suggested that downregulation of PD-1 enhances short-term effector function, whereas downregulation of TIGIT is primarily responsible for maintaining a less-differentiated/exhausted state, providing a potential mechanism for the observed synergy. The PD-1/TIGIT-downregulated CAR T cells generated from diffuse large B-cell lymphoma patient-derived T cells using a clinically applicable manufacturing process also showed robust antitumor activity and significantly improved persistence in vivo compared with conventional CD19-targeting CAR T cells. Overall, our results demonstrate that the cell-intrinsic PD-1/TIGIT dual downregulation strategy may prove effective in overcoming immune checkpoint-mediated resistance in CAR T therapy.

immunology↗

bioRxiv2020

Not out of the box: phylogeny of the broadly sampled Buxaceae

The Buxaceae constitute a morphologically diverse phylogenetic lineage of six genera, which includes about 140 species. The most well-known genera are Buxus, Sarcococca, and Pachysandra. Few species of woody Styloceras grow on mid-elevations in the Andes mountains region. Didymeles, with three species endemic to Madagascar, and the monotypic Haptanthus from Honduras, are the most unusual members of the group. The infra-familial classification of Buxaceae is controversial, and molecular data about many species, especially Old World, is still lacking. We used broad taxonomic sampling and molecular data from four chloroplast markers, and the nuclear ribosomal ITS to estimate their phylogeny. These data provide phylogenetic placements of 50 species and enabled better estimates of boundaries in Buxaceae. We described two subfamilies, two monotypic genera, two Buxus subgenera, and one new species of Didymeles from Madagascar.

plant biology↗

bioRxiv2020

Size-dependent protein segregation creates a spatial switch for Notch and APP signaling

Aberrant cleavage of Notch by {gamma}-secretase is implicated in numerous diseases, but how cleavage is regulated in space and time is unclear. Here, we report that cadherin-based adherens junctions (cadAJs) are sites of high cell-surface {gamma}-secretase activity, as well as sites of constrained physical space that excludes {gamma}-secretase substrates having large extracellular domains (ECDs) like Notch. ECD shedding initiates drastic spatial relocalization of Notch to cadAJs, allowing enzyme-substrate interactions and downstream signaling. Spatial mutations by adjusting the ECD size or the physical constraint alter signaling. Dysregulation of this spatial switch promotes precocious differentiation of ventricular zone neural progenitor cells in vivo. We show the generality of this spatial switch for amyloid precursor protein proteolysis. Thus, cadAJs create spatially distinct biochemical compartments regulating cleavage events involving {gamma}-secretase and preventing aberrant activation of receptors. One Sentence SummaryNotch cleavage by {gamma}-secretase is regulated through dynamic spatial control of receptors, adhesion molecules, and activating proteases

cell biology↗

bioRxiv2020

Th2-like T-follicular helper cells promote functional antibody production during Plasmodium falciparum infection

The most advanced malaria vaccine only has approximately 30% efficacy in target populations, and avenues to improve next generation vaccines need to be identified. Functional antibodies are key effectors of both vaccine induced and naturally acquired immunity, with induction driven by T-follicular helper cells (TfH) CD4+ T cells. We assessed circulating TfH (cTfH) responses and functional antibody production in human volunteers experimentally infected with Plasmodium falciparum. Longitudinal single-cell RNA-sequencing of cTfH revealed peak transcriptional activation and clonal expansion of major cTfH subsets occurred at day 8 following infection and a population structure of cTfH capturing phenotypical subsets of Th1- and Th2-like cells. Among 40 volunteers, infection resulted in the emergence of activated ICOS+ cTfH cells. During peak infection, activation was restricted to Th2-like cTfH cells, while Th1-like cTfH cell activation occurred one week after treatment. To link cTfH activation to antibody induction, we assessed the magnitude and function of anti-malarial IgM and IgG after infection. The functional breadth and magnitude of parasite-specific antibodies was positively associated with Th2-cTfH activation. In contrast, Th1-cTfH activation was associated with the induction of plasma cells, which we have previously shown have a detrimental role in germinal cell formation and antibody development. Thus, we identified that during P. falciparum malaria infection in humans, the activation of Th2-cTfH but not other subsets correlates with the development of functional antibodies required for protective immunity. Data for the first time identify a specific cellular response that can be targeted by future malaria vaccines to improve antibody induction.

immunology↗

bioRxiv2020

A Thermodynamic Interpretation of the Stimulated Raman Spectroscopic Signature to Action Potential in Single Neurons

It has previously been suggested that the plasma membrane condenses and melts reversibly during an action potential in a neuron, analogous to an acoustic wave travelling in the compressive membrane region. If true it has fundamental consequences for our understanding of the regulation of biological functions during an action potential. It has long been known that the electrical dipoles in the neuronal membrane reorient during an action potential, observed through a variety of optical methods. However, this information has been insufficient to confirm if and how the collective thermodynamic state of the neuronal membrane changes during an action potential. Here, we show that hyperspectral stimulated Raman spectroscopy (SRS) can resolve the thermodynamic state of the neuronal membranes in a single neuron during an action potential. These measurements indicate that the system becomes ordered and compressed during the de-polarisation phase and disordered and expanded during hyper polarisation Therefore, the observation is consistent with the acoustic hypothesis and SRS provides a powerful tool to not only further validate the hypothesis in future, but also explore the role of membrane thermodynamics during an action potential.

biophysics↗

bioRxiv2020

Body mass index trajectories and adiposity rebound during the first 6 years in Korean children: based on the National Health Information Database, 2008-2015

PurposeWe analyzed the nationwide longitudinal data to explore body mass index (BMI) growth trajectories and the time of adiposity rebound (AR). MethodsPersonal data of 84,005 subjects born between 2008 and 2012 were obtained from infant health check-ups which were performed at 5, 11, 21, 33, 45, 57, and 69 months. BMI 00trajectories of each subject were made according to sex and birth weight (Bwt) and the timing of AR was defined as the lowest BMI occurred. Subjects were divided according to Bwt and AR timing as follows: very low birth weight (VLBW), 0.5 kg [≤] Bwt [≤] 1.5 kg; low birth weight (LBW), 1.5 kg < Bwt [≤] 2.5 kg; non-LBW, 2.5 kg < Bwt [≤] 5.0 kg; early AR, before 45 months; moderate AR, at 57 months; and late AR, not until 69 months. Main resultsMedian time point of minimum BMI was 45 months, and the prevalence rates of early, moderate, and late AR were 63.0%, 16.6%, and 20.4%, respectively. BMI at the age of 57 months showed a strong correlation with AR timing after controlling for Bwt (P < 0.001). Sugar-sweetened beverage intake at 21 months (P = 0.02) and no-exercise habit at 57 months (P < 0.001) showed correlations with early AR. When VLBW and LBW subjects were analyzed, BMI at 57 months and breastfeeding at 11 months were correlated with rapid weight gain during the first 5 months (both P < 0.001). ConclusionsBased on this first longitudinal study, the majority of children showed AR before 57 months and the degree of obesity at the age of 57 months had a close correlation with early AR or early weight during infancy.

developmental biology↗

bioRxiv2020

Targeted Eicosanoid Profiling Reveals MicroRNA-155 Shifts PGE2/PGD2 Balance Towards Oncogenic State

microRNA-155 is a strong oncogenic microRNAs with multiple functions. To reveal its novel function in cancer metabolism, we performed a targeted metabolomic analysis of icosanoids, the key metabolites of inflammation-related carcinogenesis. We found miR-155-depleted cells expressed unbalanced prostaglandins, especially in PGE2 and PGD2. Subsequent analysis of primary cancer cells and 20 TNBC specimen indicated a positive correlation between miR-155 and PGE2/PGD2 ratio. Mechanistically, miR-155 controls the prostaglandin shift by up-regulating PGE2-producing enzymes PTGES/PTGES2 and down-regulating PGD2-producing enzyme PTGDS. Further analysis showed that the up-regulation of PTGES2 is driven by miR-155-cMYC axis whereas PTGES is transactivated by miR-155-KLF4 axis, indicating a dual-regulatory mode for the metabolic enzyme expression drives a shift in PGE2/PGD2 balance. Lastly, we show the miR-155-driven cellular proliferation is significantly restored by the siRNA of PTGES1/2, of which expression also correlates with patients survival. Taken altogether, our study reveals an unprecedented oncogenic function of miR-155 on prostaglandin regulation.

cancer biology↗

bioRxiv2020

Single-cell transcriptomics of allo-reactive CD4+ T cells over time reveals divergent fates during gut GVHD

Acute gastrointestinal Graft-versus-Host-Disease (GVHD) is a primary determinant of mortality after allogeneic hematopoietic stem-cell transplantation (alloSCT). It is mediated by alloreactive donor CD4+ T cells that differentiate into pathogenic subsets expressing IFN{gamma}, IL-17A or GM-CSF, and is regulated by subsets expressing IL-10 and/or Foxp3. Developmental relationships between T-helper states during priming in mesenteric lymph nodes (mLN) and effector function in the GI tract remain undefined at genome-scale. We used scRNA-seq and computational modelling to create an atlas of putative differentiation pathways during GVHD. Computational trajectory inference suggested emergence of pathogenic and regulatory states along a single developmental trajectory in mLN. Importantly, we identified an unexpected second trajectory, categorised by little proliferation or cytokine expression, reduced glycolysis, and high TCF1 expression. TCF1hi cells upregulated 4{beta}7 prior to gut migration and failed to express cytokines therein. Nevertheless, they demonstrated recall potential and plasticity following secondary transplantation, including cytokine or Foxp3 expression, but reduced TCF1. Thus, scRNA-seq revealed divergence of allo-reactive CD4+ T cells into quiescent and effector states during gut GVHD, reflecting putative heterogenous priming in vivo. These findings, the first at a single-cell level during GVHD over time, can now be used to interrogate T cell differentiation in patients undergoing alloSCT.

immunology↗

bioRxiv2020

Smchd1 is a maternal effect gene required for autosomal imprinting

Genomic imprinting establishes parental allele-biased expression of a suite of mammalian genes based on parent-of-origin specific epigenetic marks. These marks are under the control of maternal effect proteins supplied in the oocyte. Here we report the epigenetic repressor Smchd1 as a novel maternal effect gene that regulates imprinted expression of 16 genes. Most Smchd1-sensitive genes only show loss of imprinting post-implantation, indicating maternal Smchd1s long-lived epigenetic effect. Sm-chd1-sensitive genes include both those controlled by germline polycomb marks and germline DNA methylation imprints; however, Smchd1 differs to other maternal effect genes that regulate the latter group, as Smchd1 does not affect germline DNA methylation imprints. Instead, Smchd1-sensitive genes are united by their reliance on polycomb-mediated histone methylation marks as germline or secondary imprints. We propose that Smchd1 translates these imprints to establish a heritable chromatin state required for imprinted expression later in development, revealing a new mechanism for maternal effect genes.

genomics↗

bioRxiv2019

Transcriptome Dynamics Reveals Progressive Transition from Effector to Memory in CD4+ T cells.

CD4+ T cells are repositories of immune memory, conferring enhanced immunity to many infectious agents. Studies of acute viral and bacterial infection suggest that memory CD4+ T cells develop directly from effectors. However, delineating these dynamic developmental pathways has been challenging. Here, we used high-resolution single-cell RNA-seq and temporal mixture modelling to examine the fate of Th1 and Tfh effector cells during non-lethal Plasmodium infection in mice. We observed linear Th1 and Tfh pathways towards memory, characterized by progressive halving in the numbers of genes expressed, and partial transcriptomic coalescence. Low-level persisting infection diverted but did not block these pathways. We observed in the Th1-pathway a linear transition from Th1 through a Tr1 state to TEM cells, which were then poised for Th1 re-call. The Tfh-pathway exhibited a modest Th1-signature throughout, with little evidence of Tr1 development, and co-expression of TCM and memory Tfh markers. Thus, we present a high-resolution atlas of transcriptome dynamics for naive to memory transitions in CD4+ T cells. We also defined a subset of memory-associated genes, including transcription factors Id2 and Maf, whose expression increased progressively against the background of transcriptomic quiescence. Single-cell ATAC-seq revealed substantial heterogeneity in chromatin accessibility in single effectors, which was extensively, though incompletely reset and homogenized in memory. Our data reveal that linear transitions from effector to memory occur in a progressive manner over several weeks, suggesting opportunities for manipulating CD4+ T cell memory after primary infection.\n\nHighlightsO_LIscRNA-seq reveals progressive transition from effector to memory in CD4+ T cells.\nC_LIO_LITranscriptome dynamics suggest linear not branching models for memory development.\nC_LIO_LIA subset of genes associates with gradual onset of CD4+ T cell memory.\nC_LIO_LITh1/Tfh predisposition varies among clonotypes with identical antigen-specificity.\nC_LIO_LIscATAC-seq uncovers non-coding \"memory\" elements in the genome.\nC_LI

immunology↗

bioRxiv2019

Somatic mutation landscape reveals differential variability of cell-of-origin for primary liver cancer

BackgroundPrimary liver tissue cancers display consistent increase in global disease burden and mortality. Identification of cell-of-origins for primary liver cancers would be a necessity to expand options for designing relevant therapeutics and preventive medicine for these cancer types. Previous reports on cell-of-origin for primary liver cancers was mainly from animal studies, and integrative research utilizing human specimen data was poorly established.\n\nMethodsWe analyzed a whole-genome sequencing data set for a total of 363 tumor and progenitor tissues along with 423 normal tissue epigenomic marks to predict the cell-of-origin for primary liver cancer subtypes.\n\nResultsDespite the mixed histological features, the predicted cell-of-origin for mixed hepatocellular carcinoma / intrahepatic cholangiocarcinoma were uniformly predicted as a hepatocytic origin. Individual sample-level prediction revealed differential level of cell-of-origin heterogeneity depending on the primary liver cancer types, with more heterogeneity observed in intrahepatic cholangiocarcinomas. Additional analyses on the whole genome sequencing data of hepatic progenitor cells suggest these progenitor cells might not a direct cell-of-origin for liver cancers.\n\nConclusionsThese results provide novel insights on the heterogeneous nature and potential contributors of cell-of-origin predictions for primary liver cancers.

genomics↗