Biology subjects

Lee, Y.

Publications and source records attributed to Lee, Y..

At least 217 records · Page 12Linked to original sources

bioRxiv2022

Tendon-like cellular and matrix maturation in scaffold-free three-dimensional tendon cell culture using mouse tendon cells

Tendons transmit mechanical forces between muscle and bone. Their biomechanical function requires high tensile strength provided by highly organized collagen fibers. Tenocytes mainly drive tendon growth via extracellular matrix (ECM) production and organization. The biological mechanisms regulating tenocyte differentiation and morphological maturation have not been well-established, partly due to the lack of reliable in vitro systems that produce highly aligned collagenous tissues. In this study, we developed a scaffold-free, three-dimensional (3D) tendon culture system using mouse tendon cells and a differentially adherent growth channel. TGF{beta} treatment promoted tendon-like structure in the peripheral layer of the constructs with decreased cell density, decreased cell proliferation, increased thickness, and more elongated cells within highly aligned extracellular matrix. The constructs were used to understand the function of TGF{beta} signaling in tenogenic differentiation, collagen fibrillogenesis, and biomechanical properties. This scaffold-free 3D constructs system can serve as a reliable in vitro system to study underlying biological mechanisms that regulate cellular and matrix maturation in tendon development and growth.

bioengineering↗

bioRxiv2022

Circadian Control of Pulmonary Endothelial Signaling occurs via NADPH oxidase 2-NLRP3 pathway

Circadian rhythms are endogenous oscillations that occur with a 24-hr periodicity. These rhythms are ubiquitous and thus, vascular endothelial cells that line the vascular bed are also subjected to circadian regulation. While the circadian control of vascular function has been demonstrated in the context of various pathologies, the relevance and functional implication of clock control over pulmonary vasculature has never been investigated. As the pulmonary endothelium is a crucial site for the hosts inflammatory response to a lung specific pathogen, we investigated the role of the circadian clock in mediation the response of the pulmonary endothelium to inflammation. We hypothesized that the pulmonary endothelium is under circadian control and that the clock serves to curb inflammatory signaling. MethodsCircadian rhythms were monitored in pulmonary artery segments and endothelial cells isolated from mPer2luciferase transgenic mice in the presence of an inflammatory stimuli (LPS). Reactive oxygen species (ROS) production in LPS treated cells was measured by fluorescence microscopy using the cell permeant dye CellROX Green. NLRP3 inflammasome was monitored post-mortem (0-72 h post LPS instillation) by measuring the expression of the NLRP3 subunit in wild type and Bmal1-/- and Cry1/2-/- mice. Inflammation was quantified in these mice by measuring PMN adherence and intercellular adhesion molecule (ICAM-1). ResultsWe observed that the circadian rhythm of the pulmonary vasculature was altered LPS. LPS also led to ROS production in these cells; ROS increased 3 h post LPS treatment, peaked by 36 h and returned to baseline values by 72 h. ROS were inhibited by pretreating the cells with the NADPH oxidase 2 (NOX2) inhibitor dipheneylene iodonium (DPI). Addition of DPI, prior to LPS pretreatment also restored the circadian rhythmicity of the pulmonary endothelium. The increase in NLRP3 along the vessel wall (post LPS treatment) was resolved by 72 h in lungs of wild type mice but not in Bmal1-/- and Cry1-/-Cry2-/- lungs. Inflammation (ICAM-1 and PMN) was also resolved in wild type but not in mice wherein the circadian clock had been disrupted genetically. ConclusionOur data indicate that pro-inflammatory stimuli reprogram circadian rhythms in the pulmonary endothelium via ROS via the NOX2-NLRP3 pathway. Disruption of the clock mediates a sustained increase in ROS via this Nox2-NLRP3 pathway in endothelial cells, thus offering a novel mechanism for mitigating the effects of clock disruption.

physiology↗

bioRxiv2022

Global statistical models of protein coevolution reveal higher-order sectors beyond those obtained from structure alone

1Recent methods have shown promise in using pairwise sequence coevolution predictions to illuminate physical interactions and functional relationships between pairs of protein residues. As a result, there has been an increased interest in identifying higher-order correlations between sequence positions in an effort to further understand how the multiple sequence alignment (MSA) encodes the conserved biological properties of a protein family. To this end, we propose a robust and generalizable spectral clustering model that can extract interconnected networks of coevolving residues - termed "protein sectors" - using pairwise sequence coevolution predicted by a global statistical model. We assess the statistical and evolutionary origins for protein sectors extracted from the MSA for 120 protein families. We show that protein sectors are extracted from a subset of densely connected components in the sequence coevolution matrix, many of which are not present in the pairwise residue contact graph that is constructed from the protein crystal structure, revealing the existence of networks of paired residues that are not necessarily in direct physical contact but are nonetheless evolutionarily coupled. We found that protein sectors form structurally connected entities in three-dimensional space, despite sector identification being independent of protein crystal structure. Interestingly, protein families with high structural similarity do not share similar protein sectors, suggesting that nuances in the sequence coevolution matrix can differentiate between the evolutionary histories of structurally-related protein families.

bioinformatics↗

bioRxiv2022

An in silico genome-wide screen for circadian clock strength in human samples

Years of time-series gene expression studies have built a strong understanding of clock-controlled pathways across species. However, comparatively little is known about how non-clock pathways influence clock function. We developed a new computational approach to explore candidate pathways coupled to the clock in human tissues. This method, termed LTM, is an in silico screen to infer genetic influences on circadian clock function. LTM uses natural variation in gene expression in human data and directly links gene expression variation to clock strength independent of longitudinal data. We applied LTM to three human skin and one melanoma datasets and found that the cell cycle is the top candidate clock-coupled pathway in healthy skin. In addition, we applied LTM to thousands of tumor samples from 11 cancer types in the TCGA database and found that extracellular matrix organization-related pathways are tightly associated with the clock strength in humans. Further analysis shows that clock strength in tumor samples are correlated with the proportion of cancer-associated fibroblasts and endothelial cells. Therefore, we show both the power of LTM in predicting clock-coupled pathways and classify factors associated with clock strength in human tissues. LTM is available on GitHub to facilitate its use.

bioinformatics↗

bioRxiv2022

Topology of gene regulatory compartments relative to the nuclear matrix

Summary ParagraphInside the nucleus, there are special compartments with characterized functions, some of which are involved in gene expression1. These compartments include transcriptional condensates and nuclear speckles, which contain factors required for transcription and splicing, respectively. While the characteristics of these intranuclear compartments were extensively investigated, spatial relationship between them is yet unclear. Meanwhile, RNA-protein structural network named the nuclear matrix has been observed inside the nucleus and suggested as a framework for the spatial segregation of the intranuclear space2-6. However, concept of the nuclear matrix was not widely accepted due to its lack of in vivo evidence7. Here, we report visualization of the nuclear matrix and intranuclear compartments using super-resolution fluorescence microscopy. We found the nuclear matrix is dynamic in live cells and easily disrupted upon transcription inhibition. Remarkably, we observed an orderly layered distribution of transcriptional condensates and nuclear speckles relative to the nuclear matrix. We also observed a separation of chromosome territories from transcriptional condensates and nuclear speckles by the nuclear matrix. Based on our findings, we propose a topological model for the regulation of transcription across the nuclear matrix.

cell biology↗

bioRxiv2022

Hypoxia promotes an inflammatory phenotype of fibroblasts in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extensive fibroinflammatory stroma and often experiences conditions of insufficient oxygen availability, or hypoxia. Cancer-associated fibroblasts (CAF) are a predominant and heterogeneous population of stromal cells within the pancreatic tumor microenvironment. Here, we uncover a previously unrecognized role for hypoxia in driving an inflammatory phenotype in PDAC CAFs. We identify hypoxia as a strong inducer of tumor IL1 expression, which is required for inflammatory CAF (iCAF) formation. Notably, iCAFs preferentially reside in hypoxic regions of PDAC. Our data implicate hypoxia as a critical regulator of CAF heterogeneity in PDAC.

cancer biology↗

bioRxiv2022

Huntingtin turnover: Modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550

Huntingtons disease (HD) is a neurodegerative disorder caused by an inherited unstable HTT CAG repeat that expands further, thereby eliciting a disease process that may be initiated by polyglutamine-expanded huntingtin or a short polyglutamine-product. Phosphorylation of selected candidate residues is reported to mediate polyglutamine-fragment degradation and toxicity. Here to support the discovery of phospho-sites involved in the life-cycle of (full-length) huntingtin, we employed mass spectrometry-based phosphoproteomics to systematically identify sites in purified huntingtin and in the endogenous protein, by proteomic and phospho-proteomic analyses of members of an HD neuronal progenitor cell panel. Our results bring total huntingtin phospho-sites to 95, with more located in the N-HEAT domain relative to numbers in the Bridge and C-HEAT domains. Moreover, phosphorylation of C-HEAT Ser2550 by cAMP-dependent protein kinase (PKA), the top hit in kinase activity screens, was found to hasten huntingtin degradation, such that levels of the catalytic subunit (PRKACA) were inversely related to huntingtin levels. Taken together these findings highlight categories of phospho-sites that merit further study and provide a phospho-site kinase pair (pSer2550-PKA) with which to investigate the biological processes that regulate huntingtin degradation and thereby influence the steady state levels of huntingtin in HD cells.

biochemistry↗

bioRxiv2022

Elevated HLA-E and NKG2A as a consequence of chronic immune activation defines resistance to M. bovis BCG immunotherapy in Non-Muscle-Invasive Bladder Cancer

Mycobacterium bovis Bacillus Calmette-Guerin (BCG), the first-line treatment for non-muscle invasive bladder cancer (NMIBC), promotes the production of inflammatory cytokines, particularly interferon (IFN)-{gamma}. Prolonged inflammation and IFN-{gamma} exposure are known to cause an adaptive immune response, enabling immune escape and proliferation by tumor cells. We investigated HLA-E and NKG2A, a novel T and NK cell checkpoint pathway, as a driver of adaptive resistance in BCG unresponsive NMIBC. We observed ubiquitous inflammation in all patients after BCG immunotherapy, regardless of recurrence status. IFN-{gamma} was shown to drive tumor expression of HLA-E and PD-L1. Further, NKG2A-expressing NK and CD8 T cells were enriched in BCG unresponsive tumors and with enhanced capacity for cytolytic functions. Strikingly, in situ spatial analyses revealed that HLA-EHIGH tumors are activated to recruit NK and T cells via chemokine production, potentially sparing HLA-ELOW tumors that would otherwise be susceptible to lysis. Finally, blood-derived NK cells retained anti-tumor functions at the time of tumor recurrence. These data support combined NKG2A and PD-L1 blockade for BCG unresponsive disease.

immunology↗

bioRxiv2022

Activating ligands of Uncoupling protein 1 identified by rapid membrane protein thermostability shift analysis

Uncoupling protein 1 (UCP1) catalyzes mitochondrial proton leak in brown adipose tissue for heat production, and may combat metabolic disease if activated in humans. During the adrenergic stimulation of brown adipocytes, free fatty acids generated from lipolysis activate UCP1 via an unclear interaction. Here, we have utilized membrane protein thermostability shift analysis to characterize the interaction of activating molecules with purified UCP1. We reveal that activators influence the protein through a specific destabilizing interaction, behaving as transport substrates that shift UCP1 to a less stable conformation of a transport cycle. Through the detection of specific stability shifts in screens, we identify novel activators, including the drug ibuprofen, where ligand analysis indicates a relatively wide structural specificity for interacting molecules. Ibuprofen induces UCP1 activity in liposomes and isolated brown fat mitochondria, but not in cultured brown adipocytes. Though the drug does induce activity in UCP1-expressing HEK293 cells, demonstrating that the targeting of UCP1 in cells by approved drugs is in principle achievable as a therapeutic avenue, but requires variants with more effective delivery in brown adipocytes.

biochemistry↗

bioRxiv2022

Existence of blood circulating immune-cell clusters (CICs) comprising antigen-presenting cells and B cells

Cell-to-cell physical interactions are involved in almost every physiological processes in multicellular organisms. Although the dynamics of these interactions could be highly diverse and complex in many circumstances, certain cell-to-cell interactions among immune cells have been well studied due importance in understanding disease pathogenesis and immune therapy development1. Dendritic cells (DCs) and B cells are directly involved in adaptive immune response against pathogens. Interaction mechanism between these two celltypes is well-known to occur in germinal centers either indirectly via helper T (Th) cells or directly via cell contact. However, there are animal in vitro and in vivo evidence that such direct DC-to-B cell contact can occur outside germinal centers like in peripheral blood or collagen matrix and display antiviral immune-related activity2,3. Here, we provide evidence that certain types of antigen presenting cells (APCs) can form robust cell clusters with B cells and circulate in blood. From healthy human blood immune single cell RNA-seq datasets, we detected APC subpopulations (0.34{+/-}0.19% of total PBMCs) that were also enriched with well-known naive B cell markers. We visually observed DC:B doublets and multiplets ([~]0.69% of total live PBMCs) in wildtype mouse blood using flow cytometry and microscopic imaging, thus proving the existence of circulating immune-cell clusters (CICs) composed of APCs and B cells. BCR repertoire of these healthy mouse CICs were similar to circulating B cells. Noticeably, frequency of these APC:B CICs were higher COVID-19 patients than healthy donors and their B cell subtype composition (e.g. naive, plasmablast, IgM+, IgG+) varied with disease severity.

immunology↗

bioRxiv2022

Ion transfer mechanisms in Mrp-type antiporters from high resolution cryoEM and molecular dynamics simulations

Multiple resistance and pH adaptation (Mrp) cation/proton antiporters are essential for growth of a variety of halophilic and alkaliphilic bacteria under stress conditions. Mrp-type antiporters are closely related to the membrane domain of respiratory complex I. We determined the structure of the Mrp antiporter from Bacillus pseudofirmus by electron cryo-microscopy at 2.2 [A] resolution. The structure resolves more than 99% of the sidechains of the seven membrane subunits MrpA to MrpG plus 360 water molecules, including [~]70 in putative ion translocation pathways. Molecular dynamics simulations based on the high-resolution structure revealed details of the antiport mechanism. We find that switching the position of a histidine residue between three hydrated pathways in the MrpA subunit is critical for proton transfer that drives gated transmembrane sodium translocation. Several lines of evidence indicate that the same histidine-switch mechanism operates in respiratory complex I.

biochemistry↗

bioRxiv2021

MSD2, an apoplastic Mn-SOD, contributes to root skotomorphogenic growth by modulating ROS distribution in Arabidopsis

Reactive oxygen species (ROS) play essential roles as a second messenger in various physiological processes in plants. Due to their oxidative nature, ROS can also be harmful. Thus, the generation and homeostasis of ROS are tightly controlled by multiple enzymes. Membrane-localized NADPH oxidases are well known to generate ROS during developmental and stress responses, but the metabolic pathways of the superoxide (O2{middle dot}-) generated by them in the apoplast are poorly understood, and the identity of the apoplastic superoxide dismutase (SOD) is unknown in Arabidopsis. Here, we show that a putative manganese SOD, MSD2 is secreted and possesses a SOD activity that can be inhibited by nitration at tyrosine 68. The expression of MSD2 in roots is light condition-dependent, suggesting that MSD2 may act on ROS metabolism in roots during the light-to-dark transition. Root architecture is governed by ROS distribution that exhibits opposite gradient of H2O2 and O2{middle dot}-, which is indeed altered in etiolated msd2 mutants and accompanied by changes in the onset of differentiation. These results provide a missing link in our understanding of ROS metabolism and suggest that MSD2 plays a role in root skotomorphogenesis by regulating ROS distribution, thereby playing a pivotal role in plant growth and development.

plant biology↗

bioRxiv2021

HEAT SHOCK FACTOR BINDING PROTEIN limits meiotic crossovers by repressing HEI10 transcription

The number of meiotic crossovers is tightly controlled and most depend on pro-crossover ZMM proteins, such as the E3 ligase HEI10. Despite the importance of HEI10 dosage for crossover formation, how HEI10 transcription is controlled remains unexplored. In a forward genetic screen using a sensitive fluorescent seed crossover reporter in Arabidopsis thaliana we identify heat shock factor binding protein (HSBP) as a repressor of HEI10 transcription and crossover numbers. Using genome-wide crossover mapping and cytogenetics, we show that hsbp mutations or meiotic HSBP knockdowns increase ZMM-dependent crossovers towards the telomeres, mirroring the effects of HEI10 overexpression. Through RNA sequencing, DNA methylome and chromatin immunoprecipitation analysis, we reveal that HSBP directly represses HEI10 transcription by binding with heat shock factors (HSFs) at the HEI10 promoter and maintaining DNA methylation over the HEI10 5' untranslated region. Our findings provide insights into how the temperature response regulator HSBP restricts meiotic HEI10 transcription and crossover number by attenuating HSF activity.

plant biology↗

bioRxiv2021

Exhausted mature dendritic cells exhibit a slower and less persistent random motility but retain chemotaxis against CCL19

Dendritic cells (DCs), which are immune sentinels in the peripheral tissues, play a number of roles, including patrolling for pathogens, internalising antigens, transporting antigens to the lymph nodes (LNs), interacting with T cells, and secreting cytokines. The well-coordinated migration of DCs under various immunological or inflammatory conditions is therefore essential to ensure an effective immune response. Upon maturation, DCs migrate faster and more persistently than immature DCs (iDCs), which is believed to facilitate CCR7-dependent chemotaxis. It has been reported that lipopolysaccharide-activated DCs produce IL-12 only transiently, and become resistant to further stimulation through exhaustion. However, little is known about the influence of DC exhaustion on cellular motility. Here, we studied the cellular migration of exhausted DCs in tissue-mimicked confined environments. We found that the speed of exhausted matured DCs (xmDCs) decreased significantly compared to active matured DCs (amDCs) and iDCs. In contrast, the speed fluctuation increased compared to that of amDCs and was similar to that of iDCs. In addition, the diffusivity of the xmDCs was significantly lower than that of the amDCs, which implies that DC exhaustion reduces the space exploration ability. Interestingly, CCR7-dependent chemotaxis against CCL19 in xmDCs was not considerably different from that observed in amDCs. Taken together, we report a unique intrinsic cell migration behavior of xmDCs, which exhibit a slower, less persistent, and less diffusive random motility, which results in the DCs remaining at the site of infection, although a well-preserved CCR7-dependent chemotactic motility is maintained.

cell biology↗

bioRxiv2021

pH-dependent polymorphism of the structure of SARS-CoV-2 nsp7

The solution structure of SARS-CoV-2 nonstructural protein 7 (nsp7) at pH 7.0 has been determined by NMR spectroscopy. nsp7 is conserved in the coronavirinae subfamily and is an essential co-factor of the viral RNA-dependent RNA polymerase for active and processive replication. Similar to the previously deposited structures of SARS-CoV-1 nsp7 at acidic and basic conditions, SARS-CoV-2 nsp7 has a helical bundle folding at neutral pH. Remarkably, the 4 helix shows gradual dislocation from the core 2-3 structure as pH increases from 6.5 to 7.5. The protonation state of residue H36 contributes to the change of nsp7s intramolecular interactions, and thus, to the structural variation near-neutral pH. Spin-relaxation results revealed that all three loop regions in nsp7 possess dynamic properties associated with this structural variation.

biochemistry↗

bioRxiv2021

Highly specific chimeric DNA-RNA guided genome editing with enhanced CRISPR-Cas12a system

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system is composed of a Cas12a effector that acts as a deoxyribonucleic acid (DNA)-cleaving endonuclease and a crispr ribonucleic acid (crRNA) that guides the effector to the target DNA. It is considered a key molecule for inducing target-specific gene editing in various living systems. Here, we improved the efficiency and specificity of the CRISPR-Cas12a system through protein and crRNA engineering. In particular, to optimize the CRISPR-Cas12a system at the molecular level, we used a chimeric DNA-RNA guide chemically similar to crRNA to maximize target sequence specificity. Compared to the wild type (wt)-Cas12a system, when using enhanced Cas12a system (en-Cas12a), the efficiency and target specificity improved on average by 7.41 and 7.60 times respectively. In our study, when the chimeric DNA-RNA guided en-Cas12a effector was used, the gene editing efficiency and accuracy were simultaneously increased. These findings could contribute to highly accurate genome editing, such as human gene therapy, in the near future.

biochemistry↗

bioRxiv2021

Vaccinia virus-based vaccines confer protective immunity against SARS-CoV-2 virus in Syrian hamsters

COVID-19 in humans is caused by Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that belongs to the beta family of coronaviruses. SARS-CoV-2 causes severe respiratory illness in 10-15% of infected individuals and mortality in 2-3%. Vaccines are urgently needed to prevent infection and to contain viral spread. Although several mRNA- and adenovirus-based vaccines are highly effective, their dependence on the "cold chain" transportation makes global vaccination a difficult task. In this context, a stable lyophilized vaccine may present certain advantages. Accordingly, establishing additional vaccine platforms remains vital to tackle SARS- CoV-2 and any future variants that may arise. Vaccinia virus (VACV) has been used to eradicate smallpox disease, and several attenuated viral strains with enhanced safety for human applications have been developed. We have generated two candidate SARS-CoV-2 vaccines based on two vaccinia viral strains, MVA and v-NY, that express full-length SARS-CoV-2 spike protein. Whereas MVA is growth-restricted in mammalian cells, the v-NY strain is replication-competent. We demonstrate that both candidate recombinant vaccines induce high titers of neutralizing antibodies in C57BL/6 mice vaccinated according to prime-boost regimens. Furthermore, our vaccination regimens generated TH1-biased immune responses in mice. Most importantly, prime-boost vaccination of a Syrian hamster infection model with MVA-S and v-NY-S protected the hamsters against SARS-CoV-2 infection, supporting that these two vaccines are promising candidates for future development. Finally, our vaccination regimens generated neutralizing antibodies that partially cross-neutralized SARS-CoV-2 variants of concern.

immunology↗

bioRxiv2021

Genome-wide DNA methylation and multi-omics study of human chondrocyte ontogeny and an epigenetic clock analysis of adult chondrocytes

Articular chondrocytes undergo functional changes and their regenerative potential declines with age. Although the molecular mechanisms guiding articular cartilage aging is poorly understood, DNA methylation is known to play a mechanistic role in aging. However, our understanding of DNA methylation in chondrocyte development across human ontogeny is limited. To better understand DNA methylome changes, methylation profiling was performed in human chondrocytes. This study reveals association between methylation of specific CpG sites and chondrocyte age. We also determined the putative binding targets of STAT3, a key age-patterned transcription factor in fetal chondrocytes and genetic ablation of STAT3 induced a global genomic hypermethylation. Moreover, an epigenetic clock built for adult human chondrocytes revealed that exposure of aged adult human chondrocytes to STAT3 agonist, decreased epigenetic age. Taken together, this work will serve as a foundation to understand development and aging of chondrocytes with a new perspective for development of rejuvenation agents for synovial joints.

molecular biology↗