Biology subjects

Lee, J.-H.

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

At least 73 records · Page 4Linked to original sources

bioRxiv2022

Glucocorticoids Mediate Transcriptome-wide Alternative Polyadenylation: Mechanisms and Functional Implications

Alternative polyadenylation (APA) is a common genetic regulatory mechanism that generates distinct 3' ends for RNA transcripts. Changes in APA have been associated with multiple biological processes and disease phenotypes. However, the role of hormones and their drug analogs in APA remains largely unknown. In this study, we investigated transcriptome-wide the impact of glucocorticoids on APA in 30 human B-lymphoblastoid cell lines. We found that glucocorticoids could regulate APA of a subset of genes, possibly by changing the expression of 142 RNA-binding proteins, some with known APA-regulating properties. Interestingly, genes with glucocorticoid-mediated APA were enriched in viral translation-related pathways, while genes with glucocorticoid-mediated expression were enriched in interferon and interleukin pathways, suggesting that glucocorticoid-mediated APA might result in functional consequences distinct from gene expression. Glucocorticoid-mediated APA was also cell-type-specific, suggesting an action of glucocorticoids that may be unique to immune regulation. We also observed evidence for genotype-dependent glucocorticoid-mediated APA, providing potential functional mechanisms for a series of common genetic variants that had previously been associated with immune disorders, but without a clear mechanism. In summary, this study reports a series of novel observations regarding the impact of glucocorticoids on APA, suggesting novel avenues for mechanistic studies of hormonal biology.

genomics↗

bioRxiv2022

Extended longevity geometrically-inverted proximal tubule organoids for protein uptake studies

While some in vitro platforms have been adapted to study proteinuric kidney disease, organoids have been challenging to study the disease. This is because apical access is historically difficult, and this is the surface on which megalin (LRP2), an endocytic receptor responsible for tubular reabsorption of filtered plasma proteins, resides. Based on our previous geometrically-inverted organoids, this study established high-throughput basal-in and apical-out proximal tubule organoids to study proteinuric kidney disease in a more physiologically consistent manner. Organoids successfully formed around a minimal Matrigel scaffold, and were maintained in culture for 90+ days, the longest reported hanging drop culture to date. The proximal tubule organoids exhibited good polarization, showed upregulation of maturity markers, such as aquaporin-1 and megalin, and experienced less epithelial-to-mesenchymal (EMT) transition compared to 2D cells. To assess protein uptake, fluorescent albumin was placed in the surrounding media, facing the apical surface, and organoids demonstrated functional protein uptake even at 90 days. To mimic proteinuric conditions, organoids were exposed to human serum albumin and released kidney injury molecule-1 (KIM-1), a common biomarker for kidney injury, in both dose- and time-dependent manners. While this study focuses on applications for modeling proteinuric kidney disease conditions, these organoids are envisioned to have broad utility where apical proximal tubule cell access is required.

bioengineering↗

bioRxiv2022

Slow-cycling cells in glioblastoma: a specific population in the cellular mosaic of cancer stem cells

AO_SCPLOWBSTRACTC_SCPLOWGlioblastoma (GBM) exhibits populations of cells that drive tumorigenesis, treatment resistance, and disease progression. Cells with such properties have been described to express specific surface and intracellular markers or exhibit specific functional features including being slow-cycling or quiescent with the ability to generate proliferative progenies. In GBM, each of these cellular fractions was shown to harbor cardinal features of cancer stem cells 1-7. In this study we focus on the comparison of these cells and present evidence of great phenotypic and functional heterogeneity in brain cancer cell populations with stemness properties, especially between slow-cycling cells and cells phenotypically defined based on the expression of markers commonly used to enrich for cancer stem cells (CSCs). Our data support the cancer stem cell mosaicism model with slow-cycling cells representing critical tesserae.

cancer biology↗

bioRxiv2021

Glucocorticoids Unmask Silent Non-Coding Genetic Risk Variants for Common Diseases

Understanding the function of non-coding genetic variants represents a formidable challenge for biomedicine. We previously identified genetic variants that influence gene expression only after exposure to a hormone or drug. Using glucocorticoid signaling as a model system, we have now demonstrated, in a genome-wide manner, that exposure to glucocorticoids triggered disease risk variants with previously unclear function to influence the expression of genes involved in autoimmunity, metabolic and mood disorders, osteoporosis and cancer. Integrating a series of pharmacogenomic and pharmacoepigenomic datasets, we identified the cis-regulatory elements and 3-dimensional interactions underlying the ligand-dependent associations between those genetic variants and distant risk genes. These observations increase our understanding of mechanisms of non-coding genetic variant-chemical environment interactions and advance the fine-mapping of disease risk and pharmacogenomic loci.

genomics↗

bioRxiv2021

A new protocol for speed vernalisation of winter cereals

There are many challenges facing the development of high-yielding, nutritious crops for future environments. One limiting factor is generation time, which prolongs research and plant breeding timelines. Recent advances in speed breeding protocols have dramatically reduced generation time for many short-day and long-day species by optimising light and temperature conditions during plant growth. However, winter crops with a vernalisation requirement still require up to 6-10 weeks in low-temperature conditions before transition to reproductive development. Here, we tested a suite of environmental conditions and protocols to investigate if vernalisation can be satisfied more efficiently. We identified a vernalisation method consisting of exposing seeds at the soil surface to an extended photoperiod of 22 h day:2 h night at 10{degrees}C with transfer to speed breeding conditions that dramatically reduces generation time in both winter wheat (Triticum aestivum) and winter barley (Hordeum vulgare). Implementation of this protocol achieved up to five generations per year for winter wheat or barley, instead of the two typically obtained under standard vernalisation and plant growth conditions. The protocol has great potential to enhance training and to accelerate research, pre-breeding, and breeding outcomes focussed on winter crop improvement.

plant biology↗

bioRxiv2021

Temperature-responsive structural reversibility of FGF21 and structure-based design of its variant with enhanced potency

Fibroblast growth factor 21 (FGF21) has pharmaceutical potential against obesity-related metabolic disorders, including non-alcoholic fatty liver disease. Since thermal stability is a desirable factor for therapeutic proteins, we investigated the thermal behavior of human FGF21. FGF21 remained soluble after heating; thus, we examined its temperature-induced structural changes using circular dichroism (CD). FGF21 showed inter-convertible temperature-specific CD spectra. The CD spectrum at 100 {degrees}C returned to that at 20 {degrees}C when the heated FGF21 solution was cooled. Through loop swapping, the connecting loop between {beta}10 and {beta}12 in FGF21 was revealed to be associated with the unique thermal behavior of FGF21. According to in vitro cell-based assays and model high-fat diet (HFD)-induced obesity studies, heated FGF21 maintained biological activities that were comparable to those of non-heated and commercial FGF21s. Based on sequence comparison and structural analysis, five point-mutations were introduced into FGF21. Compared with the wild type, the heated FGF21 variant displayed improved therapeutic potential in terms of body weight loss, the levels of hepatic triglycerides and lipids, and the degree of vacuolization of liver in HFD-fed mice.

biochemistry↗

bioRxiv2021

Mutual Regulation between PFKP and VEGF Promotes Glioblastoma Tumor Growth

Glioblastoma (GBM) is highly vascular malignant brain tumor that overexpresses vascular endothelial growth factor (VEGF) and phosphofructokinase 1 platelet isoform (PFKP), which catalyzes a rate-limiting reaction in glycolysis. However, it remains unknown whether PFKP and VEGF are reciprocally regulated during GBM tumor growth. Here, we show that PFKP promotes EGFR activation-induced VEGF expression in HIF-1-dependent and -independent manners in GBM cells. Importantly, we demonstrate that EGFR-phosphorylated PFKP Y64 has critical roles in the AKT/SP1-mediated transcriptional expression of HIF-1 and in the AKT-mediated {beta}-catenin S552 phosphorylation, to fully enhance VEGF transcription and subsequent blood vessel formation and brain tumor growth. The levels of PFKP Y64 phosphorylation in human GBM specimens positively correlate with HIF-1 expression, {beta}-catenin S552 phosphorylation, and VEGF expression. Conversely, VEGF upregulates PFKP expression in a PFKP S386 phosphorylation-dependent manner, leading to increased PFK enzyme activity, aerobic glycolysis, and proliferation in GBM cells. These findings highlight a novel mechanism underlying the mutual regulation that occurs between PFKP and VEGF for promoting GBM tumor growth and provide the therapeutic potential of targeting the PFKP/VEGF regulatory loop for GBM patients.

cancer biology↗

bioRxiv2021

Symmetry breaking of hPSCs in micropattern generates a polarized spinal cord-like organoid (pSCO) with dorsoventral organization

Brain organoid research is advancing, but generation of organoids with proper axis formation, which could lead to spatially ordered structures for complex brain structure and function, still remains a challenge. Axis formation and related spatial cell organization in the CNS are initiated by the symmetry breaking during the early embryo development. It has been demonstrated that the geometrically confined culture of human pluripotent stem cells (hPSCs) can be used to induce symmetry breaking and regionalized cell differentiation. In this study, we generated a polarized spinal cord organoid with a self-organized dorsoventral (DV) organization, using 2D cell patterning by geometric confinement. Initially, the application of caudalization signals to hPSCs promoted the regionalized cell differentiation along the radial axis and sprouting-like protrusion morphogenesis in cell colonies confined to ECM protein micropatterns. Detachment of colonies turned them into extended spinal cord-like organoids which maintained center- and edge-derived two poles. Further analyses including single cell RNA sequencing and spatial transcriptome analysis unveiled that these organoids contained rich repertoire of developing spinal cord cells and exhibited the spatially ordered DV domain formation along the long axis without external organizing signals. Modulation of BMP and Shh signaling can control the extent of DV coverage in organoids following the principles of embryo patterning. Our study provides a simple, and precisely controllable method to generate spatially-ordered organoids for understanding of biological principles of cell patterning and axis formation during neural development.

developmental biology↗

bioRxiv2021

Development of a machine learning model to estimate biotic ligand model-based predicted no-effect concentrations for copper in freshwater

The copper biotic ligand model (BLM) has been used for environmental risk assessment by taking into account the bioavailability of copper in freshwater. However, the BLM-based environmental risk of copper has been assessed only in Europe and North America, with monitoring datasets containing all of the BLM input variables. For other areas, it is necessary to apply surrogate tools with reduced data requirements to estimate the BLM-based predicted no-effect concentration (PNEC) from commonly available monitoring datasets. To develop an optimized PNEC estimation model based on an available monitoring dataset, an initial model that considers all BLM variables, a second model that requires variables excluding alkalinity, and a third model using electrical conductivity as a surrogate of the major cations and alkalinity have been proposed. Furthermore, deep neural network (DNN) models have been used to predict the nonlinear relationships between the PNEC (outcome variable) and the required input variables (explanatory variables). The predictive capacity of DNN models in this study was compared with the results of other existing PNEC estimation tools using a look-up table and multiple linear and multivariate polynomial regression methods. Three DNN models, using different input variables, provided better predictions of the copper PNECs compared with the existing tools for four test datasets, i.e., Korean, United States, Swedish, and Belgian freshwaters. The adjusted r2 values in all DNN models were higher than 0.95 in the test datasets, except for the Swedish dataset (adjusted r2 > 0.87). Consequently, the most applicable model among the three DNN models could be selected according to the data availability in the collected monitoring database. Because the most simplified DNN model required only three water quality variables (pH, dissolved organic carbon, and electrical conductivity) as input variables, it is expected that the copper BLM-based risk assessment can be applied to monitoring datasets worldwide.

pharmacology and toxicology↗

bioRxiv2021

SARS-CoV-2 Restructures the Host Chromatin Architecture

SARS-CoV-2 has made >190-million infections worldwide, thus it is pivotal to understand the viral impacts on host cells. Many viruses can significantly alter host chromatin1, but such roles of SARS-CoV-2 are largely unknown. Here, we characterized the three-dimensional (3D) genome architecture and epigenome landscapes in human cells after SARS-CoV-2 infection, revealing remarkable restructuring of host chromatin architecture. High-resolution Hi-C 3.0 uncovered widespread A compartmental weakening and A-B mixing, together with a global reduction of intra-TAD chromatin contacts. The cohesin complex, a central organizer of the 3D genome, was significantly depleted from intra-TAD regions, supporting that SARS-CoV-2 disrupts cohesin loop extrusion. Calibrated ChIP-Seq verified chromatin restructuring by SARS-CoV-2 that is particularly manifested by a pervasive reduction of euchromatin modifications. Built on the rewired 3D genome/epigenome maps, a modified activity-by-contact model2 highlights the transcriptional weakening of antiviral interferon response genes or virus sensors (e.g., DDX58) incurred by SARS-CoV-2. In contrast, pro-inflammatory genes (e.g. IL-6) high in severe infections were uniquely regulated by augmented H3K4me3 at their promoters. These findings illustrate how SARS-CoV-2 rewires host chromatin architecture to confer immunological gene deregulation, laying a foundation to characterize the long-term epigenomic impacts of this virus.

molecular biology↗

bioRxiv2021

Release of Notch activity coordinated by IL-1β signalling confers differentiation plasticity of airway progenitors via Fosl2 during alveolar regeneration

While the acquisition of cellular plasticity in adult stem cells is essential for rapid regeneration after tissue injury, little is known about the underlying molecular mechanisms governing this process. Our data reveal the coordination of airway progenitor differentiation plasticity by inflammatory signals during alveolar regeneration. Upon damage, IL-1{beta} signalling-dependent modulation of Jag1/2 expression in ciliated cells results in the inhibition of Notch signalling in secretory cells, which drives reprogramming and acquisition of differentiation plasticity. We identify a core role for the transcription factor Fosl2/Fra2 in secretory cell fate conversion to alveolar type 2 (AT2) cells retaining the distinct genetic and epigenetic signatures of secretory lineages. We furthermore reveal that KDR/FLK-1+ human secretory cells display a conserved capacity to generate AT2 cells via Notch inhibition. Our results demonstrate the functional role of a IL-1{beta}-Notch-Fosl2 axis for the fate decision of secretory cells during injury repair, proposing a new potential therapeutic target for human lung alveolar regeneration.

cell biology↗

bioRxiv2021

FXR inhibition reduces ACE2 expression, SARS-CoV-2 infection and may improve COVID-19 outcome

Prevention of SARS-CoV-2 entry in cells through the modulation of viral host receptors, such as ACE2, could represent a new therapeutic approach complementing vaccination. However, the mechanisms controlling ACE2 expression remain elusive. Here, we identify the farnesoid X receptor (FXR) as a direct regulator of ACE2 transcription in multiple COVID19-affected tissues, including the gastrointestinal and respiratory systems. We demonstrate that FXR antagonists, including the over-the-counter compound z-guggulsterone (ZGG) and the off-patent drug ursodeoxycholic acid (UDCA), downregulate ACE2 levels, and reduce susceptibility to SARS-CoV-2 infection in lung, cholangiocyte and gut organoids. We then show that therapeutic levels of UDCA downregulate ACE2 in human organs perfused ex situ and reduce SARS-CoV-2 infection ex vivo. Finally, we perform a retrospective study using registry data and identify a correlation between UDCA treatment and positive clinical outcomes following SARS-CoV-2 infection, including hospitalisation, ICU admission and death. In conclusion, we identify a novel function of FXR in controlling ACE2 expression and provide evidence that this approach could be beneficial for reducing SARS-CoV-2 infection, thereby paving the road for future clinical trials.

microbiology↗

bioRxiv2021

Kasugamycin is a novel chitinase 1 inhibitor with strong antifibrotic effects on pulmonary fibrosis

RationalePulmonary fibrosis is a devastating lung disease with few therapeutic options. Chitinase 1 (CHIT1), an 18 glycosyl hydrolase family member, contributes to the pathogenesis of pulmonary fibrosis through regulation of Transforming Growth Factor (TGF)-{beta} signaling and effector function. Therefore, CHIT1 is a potential therapeutic target of pulmonary fibrosis. ObjectivesThis study aimed to identify and characterize a druggable CHIT1 inhibitor with strong antifibrotic activity and minimal toxicity for therapeutic application to pulmonary fibrosis. MethodsExtensive screening of small molecule libraries identified the aminoglycoside antibiotic Kasugamycin as a potent CHIT1 inhibitor. Measurements and Main ResultsElevated levels of CHIT1 were detected in the lungs of patients with pulmonary fibrosis. In vivo bleomycin- and TGF-{beta}-stimulated murine models of pulmonary fibrosis, Kasugamycin showed impressive anti-fibrotic effects in both preventive and therapeutic conditions. In vitro studies also demonstrated that Kasugamycin inhibits fibrotic macrophage activation, fibroblast proliferation and myofibroblast transformation. Null mutation of transforming growth factor beta associated protein 1 (TGFBRAP1), a recently identified CHIT1 interacting signaling molecule, phenocopied antifibrotic effects of Kasugamycin in in vivo lungs and in vitro fibroblasts responses. Kasugamycin inhibits physical association between CHIT1 and TGFBRAP1, suggesting that antifibrotic effect of Kasugamycin is mediated through regulation of TGFBRAP1, at least in part. ConclusionsThese studies demonstrate that Kasugamycin is a novel CHIT1 inhibitor with strong antifibrotic effect that can be further developed as an effective and safe therapeutic drug for pulmonary fibrosis.

immunology↗

bioRxiv2021

TPX2 Amplification-Driven Aberrant Mitosis in Long-Term Cultured Human Embryonic Stem Cells

Although human embryonic stem cells (hESCs) are equipped with highly effective machinery for the maintenance of genome integrity, the frequency of genetic aberrations during long-term in vitro hESC culture has been a serious issue that raises concerns over their safety in future clinical applications. By passaging hESCs over a broad range of timepoints, we found that mitotic aberrations, such as the delay of mitosis, multipolar centrosomes, and chromosome mis-segregation, were increased in the late-passaged hESCs (LP-hESCs) in parallel with polyploidy compared to early-passaged hESCs (EP-hESCs). Through high-resolution genome-wide approaches and by following transcriptome analysis, we found that LP-hESCs with a minimal amplicon in chromosome 20q11.21 highly expressed TPX2 (targeting protein for Xklp2), a key protein for governing spindle assembly and cancer malignancy. Consistent with these findings, the inducible expression of TPX2 in EP-hESCs reproduced aberrant mitotic events, such as the delay of mitotic progression, spindle stability, misaligned chromosomes, and polyploidy. This data suggests that the amplification and increased transcription of the TPX2 gene at 20q11.21 could contribute to an increase in aberrant mitosis due to altered spindle dynamics.

cell biology↗

bioRxiv2021

Quantitative 3D microscopy reveals a genetic network predicting the local activity of anti-Aβ compounds

Many efforts targeting amyloid-{beta} (A{beta}) plaques for the treatment of Alzheimers Disease thus far have resulted in failures during clinical trials. Regional and temporal heterogeneity of efficacy and dependence on plaque maturity may have contributed to these disappointing outcomes. In this study, we mapped the regional and temporal specificity of various anti-A{beta} treatments through high-resolution light-sheet imaging of electrophoretically-cleared brains. We assessed the effect on amyloid plaque formation and growth in Thy1-APP/PS1 mice subjected to {beta}-secretase inhibitors, polythiophenes, or anti-A{beta} antibodies. Each treatment showed unique spatiotemporal A{beta} clearance, with polythiophenes emerging as a potent anti-A{beta} compound. Furthermore, aligning with a spatial-transcriptomic atlas revealed transcripts that correlate with the efficacy of each A{beta} therapy. As observed in this study, there is a striking dependence of specific treatments on the location and maturity of A{beta} plaques. This may also contribute to the clinical trial failures of A{beta}-therapies, suggesting that combinatorial regimens may be significantly more effective in clearing amyloid deposition.

neuroscience↗

bioRxiv2021

Mutational impact and signature of ionizing radiation

Whole-genome sequencing (WGS) of human tumors and normal cells exposed to various carcinogens has revealed distinct mutational patterns that provide deep insights into the DNA damage and repair processes. Although ionizing radiation (IR) is conventionally known as a strong carcinogen, its genome-wide mutational impacts have not been comprehensively investigated at the single-nucleotide level. Here, we explored the mutational landscape of normal single-cells after exposure to the various levels of IR. On average, 1 Gy of IR exposure generated [~]16 mutational events with a spectrum consisting of predominantly small nucleotide deletions and a few characteristic structural variations. In [~]30% of the post-irradiated cells, complex genomic rearrangements, such as chromoplexy, chromothripsis, and breakage-fusion-bridge cycles, were resulted, indicating the stochastic and chaotic nature of DNA repair in the presence of the massive number of concurrent DNA double-strand breaks. These mutational signatures were confirmed in the genomes of 22 IR-induced secondary malignancies. With high-resolution genomic snapshots of irradiated cells, our findings provide deep insights into how IR-induced DNA damage and subsequent repair processes operate in mammalian cells.

genomics↗

bioRxiv2020

Human spinal cord organoids exhibiting neural tube morphogenesis for a quantifiable drug screening system of neural tube defects

The human spinal cord forms well-organized neural circuits for environment sensing and motor behavior. The three-dimensional (3D) induction of the spinal cord-like tissue from human pluripotent stem cells has been reported, but they often do not mimic morphological features of neurulation and their maturity is limited. Here, we report an advanced 3D culture system for the production of human spinal cord-like organoids (hSCOs) suitable for the scale-up and quantitative studies. The hSCOs exhibited many aspects of spinal cord development, including neurulation-like tube-forming morphogenesis, differentiation of the major spinal cord neurons and glial cells, and mature synaptic functional activities. We further demonstrated that hSCOs platform allowed quantitative and systematic high-throughput examination of the potential risk of neural tube defects induced by antiepileptic drugs. Thus, hSCOs can be used for understanding human spinal cord development, disease modeling, and toxicology screening.

cell biology↗

bioRxiv2020

Bioassemblying Macro-Scale, Lumnized Airway Tubes of Defined Shape via Multi-Organoid Patterning and Fusion

Epithelial, stem-cell derived organoids are ideal building blocks for tissue engineering, however, scalable and shape-controlled bioassembly of epithelial organoids into larger and anatomical structures has yet to be achieved. Here, a robust organoid engineering approach, Multi-Organoid Patterning and Fusion (MOrPF), is presented to assemble individual airway organoids of different sizes into upscaled, scaffold-free airway tubes with pre-defined shapes. Multi-Organoid Aggregates (MOAs) undergo accelerated fusion in a matrix-depleted, free-floating environment, possess a continuous lumen and maintain prescribed shapes without an exogenous scaffold interface. MOAs in the floating culture exhibit a well-defined three-stage process of inter-organoid surface integration, luminal material clearance and lumina connection. The observed shape stability of patterned MOAs is confirmed by theoretical modelling based on organoid morphology and the physical forces involved in organoid fusion. Immunofluorescent characterization shows that fused MOA tubes possess an unstratified epithelium consisting mainly of tracheal basal stem cells. By generating large, shape-controllable organ tubes, MOrPF enables upscaled organoid engineering towards integrated organoid-devices and structurally complex organ tubes.

bioengineering↗