Biology subjects

Lee, Y. J.

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

35 records · Page 2Linked to original sources

bioRxiv2022

A conserved enzyme of smut fungi facilitates cell-to-cell movement in the plant bundle sheath

The smut fungi are one of the largest groups of fungal plant pathogens, causing disease in all cereal crops. They directly penetrate their hosts and establish a biotrophic interaction. During colonization of the plant, smut fungi secrete a wide range of effector proteins, which suppress plant immunity and modulate cellular functions as well as development of the host, thereby determining the pathogens life-style and virulence potential. The conserved effector Erc1 (enzyme required for cell-to-cell movement) contributes to virulence of the corn smut Ustilago maydis in maize leaves, but not on the tassel. Erc1 binds to host cell wall components and has a 1,3-{beta}-glucanase activity, which is required to attenuate {beta}-glucan-induced defense responses in host leaves. Confocal microscopy revealed that Erc1 has a cell type-specific virulence function, being necessary for fungal cell-to-cell movement in the plant bundle sheath. This cell type-specific virulence function of Erc1 is fully conserved in the barley pathogen Ustilago hordei, which has a functionally conserved Erc1 orthologue. Thus, Erc1 is an enzymatically active core virulence factor with a cell type-specific virulence function in different hosts, which is important for cell-to-cell movement during host colonization of pathogenic smut fungi.

plant biology↗

bioRxiv2022

The molecular mechanism and physiological role of silent nociceptor activation

Silent nociceptors are sensory afferents that are insensitive to noxious mechanical stimuli under normal conditions but become sensitized to such stimuli during inflammation. Using RNA-sequencing and quantitative RT-PCR we demonstrate that inflammation selectively upregulates the expression of the transmembrane protein TMEM100 in silent nociceptors and electrophysiology revealed that over-expression of TMEM100 is required and sufficient to un-silence silent nociceptors. Moreover, we show that mice lacking TMEM100 do not develop secondary allodynia - i.e. pain hypersensitivity that spreads beyond the site of inflammation - in a mouse model of knee joint inflammation and that AAV-mediated overexpression of TMEM100 in articular afferents in the absence of inflammation is sufficient to induce allodynia in remote skin regions without causing knee joint pain. Thus, our work identifies TMEM100 as a key regulator of silent nociceptor un-silencing and reveals a physiological role for this hitherto enigmatic afferent subclass in triggering spatially remote secondary allodynia during inflammation.

neuroscience↗

bioRxiv2022

Analysis of altered pre-mRNA splicing patterns caused by a mutation in the RNA binding protein hnRNPA1 linked to amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease characterized by loss of motor neurons. Human genetic studies have linked mutations in RNA binding proteins as causative for this disease. The hnRNPA1 protein, a known pre-mRNA splicing factor, is mutated in a number of ALS patients. Here, we generate two cell models to investigate how a mutation in the C-terminal low complexity domain of hnRNPA1 affects global pre-mRNA splicing patterns and RNA binding. We show that a single amino acid change in the C-terminal low complexity domain (D262V) leads to changes in splicing of thousands of transcripts whose genes are linked to the DNA damage response, cilia organization and translation. We also show that there are changes in RNA binding of the mutant hnRNPA1 protein to transcripts whose splicing patterns change. Finally, we show that cells expressing the hnRNPA1 D262V mutation exhibit an aggregation phenotype, markedly reduced growth rates and changes in stress granules. This study shows that global changes in pre-mRNA splicing patterns caused by a single mutation in the hnRNPA1 protein lead to phenotypes related to ALS and that specific cellular pathways are affected.

biochemistry↗

bioRxiv2021

Cell cycle stage classification using phase imaging with computational specificity

Traditional methods for cell cycle stage classification rely heavily on fluorescence microscopy to monitor nuclear dynamics. These methods inevitably face the typical phototoxicity and photobleaching limitations of fluorescence imaging. Here, we present a cell cycle detection workflow using the principle of phase imaging with computational specificity (PICS). The proposed method uses neural networks to extract cell cycle-dependent features from quantitative phase imaging (QPI) measurements directly. Our results indicate that this approach attains very good accuracy in classifying live cells into G1, S, and G2/M stages, respectively. We also demonstrate that the proposed method can be applied to study single-cell dynamics within the cell cycle as well as cell population distribution across different stages of the cell cycle. We envision that the proposed method can become a nondestructive tool to analyze cell cycle progression in fields ranging from cell biology to biopharma applications. TeaserWe present a non-destructive, high-throughput method for cell cycle detection combining label-free imaging and deep learning.

bioengineering↗

bioRxiv2021

Targeting mitochondrial double-stranded RNAs ameliorates autoimmune characteristics of Sjogrens syndrome

ObjectiveSjogrens syndrome (SS) is a systemic autoimmune disease that targets the exocrine glands, resulting in impaired saliva and tear secretion. To date, type I interferons (I-IFNs) are increasingly recognized as pivotal mediators in SS, but their endogenous drivers have not been elucidated. This study investigates the role of mitochondrial double-stranded RNAs (mt-dsRNAs) in regulating I-IFN response in SS. MethodsSaliva and tear from SS patients and controls (n=73 for saliva and n=16 for tear), the salivary glands of the SS-prone-non-obese-diabetic mouse, and primary human salivary glandular cells were screened for mt-dsRNAs by RT-qPCR. The human salivary cell line (NS-SV-AC) grown as three-dimensional spheroids were subject to dsRNA stress to measure mt-dsRNA induction and recapitulation of SS glandular inflammatory features. Acetylcholine, SS-IgG, upadacitinib (JAK1 inhibitor), or 2-C'-methyladenosine (mitochondrial transcription inhibitor) were applied to characterize the roles of mt-dsRNAs. To identify endogenous dsRNA-sensor and confirm the mitochondrial origin of cytoplasmic dsRNAs, the immunoprecipitation of dsRNAs was performed. Resultsmt-dsRNAs were elevated in the SS specimens with salivary ND5 and tear CYTB1 being statistically associated with secretory dysfunction/inflammation and corneal/conjunctival damage, respectively. Stimulation of the spheroids with dsRNA stress of poly I:C induced mt-dsRNAs, p-PKR, and I-IFNS via the JAK1/STAT pathway whereas the inhibition of mt-RNA synthesis or JAK1 attenuated the glandular signature. The inhibitory effect of acetylcholine on mt-dsRNAs and I-IFNS induction was reversed by SS-IgG. Conclusionmt-dsRNAs amplify the impact of dsRNA stress on SS glandular signatures in vitro, potentially propagating a pseudo-viral signal in the SS target tissue. SummaryMitochondrial double-stranded RNA levels were elevated in the tear and saliva of SS patients, which was associated with secretory dysfunction and tissue inflammation. These RNAs amplified type I interferon signature as well as glandular phenotypes reported in SS. Inhibitors of mitochondrial RNA transcription or JAK1 in salivary gland acinar cell spheroids attenuated the mitochondrial RNA-mediated changes.

molecular biology↗

bioRxiv2021

Cerebello-cerebral Functional Connectivity Networks in Major Depressive Disorder: A CAN-BIND-1 Study Report

ObjectiveNeuroimaging studies have demonstrated aberrant structure and function of the "cognitive-affective cerebellum" in Major Depressive Disorder (MDD), although the specific role of the cerebello-cerebral circuitry in this population remains largely uninvestigated. The objective of this study was to delineate the role of cerebellar functional networks in depression. MethodsA total of 308 unmedicated participants completed resting-state functional magnetic resonance imaging scans, of which 247 (148 MDD; 99 Healthy Controls, HC) were suitable for this study. Seed-based resting-state functional connectivity (RsFc) analysis was performed using three cerebellar regions of interest (ROIs): ROI1 corresponded to default mode network (DMN) / inattentive processing; ROI2 corresponded to attentional networks including frontoparietal, dorsal attention, and ventral attention; ROI3 corresponded to motor processing. These ROIs were delineated based on prior functional gradient analyses of the cerebellum. A general linear model was used to perform within-group and between-group comparisons. ResultsIn comparison to HC, participants with MDD displayed increased RsFc within the cerebello-cerebral DMN (ROI1) and significantly elevated RsFc between the cerebellar ROI1 and bilateral angular gyrus at a voxel threshold (p < 0.001, two-tailed) and at a cluster level (p < 0.05, FDR-corrected). Group differences were non-significant for ROI2 and ROI3. ConclusionsThese results contribute to the development of a systems neuroscience approach to the diagnosis and treatment of MDD. Specifically, our findings confirm previously reported associations between MDD, DMN, and cerebellum, and highlight the promising role of these functional and anatomical locations for the development of novel imaging-based biomarkers and targets for neuromodulation therapies.

neuroscience↗

bioRxiv2021

TRPC3 and NALCN channels drive pacemaking in substantia nigra dopaminergic neurons

Midbrain dopamine (DA) neurons are slow pacemakers that maintain extracellular DA levels. During the interspike intervals, subthreshold slow depolarization underlies autonomous pacemaking and determines its rate. However, the ion channels that determine slow depolarization are unknown. Here we show that TRPC3 and NALCN channels together form sustained inward currents responsible for the slow depolarization of nigral DA neurons. Specific TRPC3 channel blockade completely blocked DA neuron pacemaking, but the pacemaking activity in TRPC3 knock-out (KO) mice was perfectly normal, suggesting the presence of compensating ion channels. Blocking NALCN channels abolished pacemaking in both TRPC3 KO and wild-type mice. The NALCN current and mRNA and protein expression are increased in TRPC3 KO mice, indicating that NALCN compensates for TRPC3 currents. In normal conditions, TRPC3 and NALCN contribute equally to slow depolarization. Therefore, we conclude that TRPC3 and NALCN are two major leak channels that drive robust pacemaking in nigral DA neurons.

neuroscience↗

bioRxiv2021

Distinct developmental mechanisms influence sexual dimorphisms in the milkweed bug Oncopeltus fasciatus

Sexual dimorphism is common in animals. The most complete model of sex determination comes from Drosophila melanogaster, where the relative dosage of autosomes and X chromosomes leads indirectly to sex-specific transcripts of doublesex. Female Dsx interacts with a mediator complex protein encoded by intersex to activate female development. In males the transcription factor encoded by fruitless promotes male-specific behavior. The genetics of sex determination have been examined in a small number of other insects, yet several questions remain about the pleisomorphic state. Is doublesex required for female and male development? Is fruitless conserved in male behavior or morphology? Are other components such as intersex functionally conserved? To address these questions, we report expression and functional tests of doublesex, intersex and fruitless in the hemipteran Oncopeltus fasciatus, characterizing three sexual dimorphisms. doublesex prevents intersex phenotypes in all sexes and dimorphic traits in the milkweed bug. intersex and fruitless are expressed across the body, in females and males. fruitless and intersex also affect the genitalia of both sexes, but have effects limited to different dimorphic structures in different sexes. These results reveal roles for intersex and fruitless distinct from other insects, and demonstrate distinct development mechanisms in different sexually dimorphic structures.

developmental biology↗

bioRxiv2020

LOCAL REGULATION AND FUNCTION OF IMPORTIN-β1 DURING TRANSCRIPTION-DEPENDENT PLASTICITY

Activity-dependent transcription is critical for the encoding of long-term memories. Regulated nuclear entry of soluble proteins is one method to relay synaptic signals to the nucleus to couple neuronal excitation with transcription. To date, the role of importin-{beta}1 in nuclear shuttling of proteins during activity-dependent transcription has always been inferred but not directly investigated. In this study, we demonstrate activity-dependent nuclear accumulation of importin-{beta}1 from the soma and the synapto-dendritic compartments. Importantly, inhibition of importin-{beta}1 mediated nuclear import during synaptic stimulation impairs long-term plasticity. We show evidence that importin-{beta}1 mRNA-ribosome complex is distributed throughout the synapto-dendritic compartment and synaptic stimulation induces importin-{beta}1 local protein synthesis. Finally, we identified candidate proteins that associate with importin-{beta}1 at the synapse and characterize NDRG1 as an importin-{beta}1 interactor that undergoes activity-dependent translocation into the nucleus. Collectively, our results highlight the crucial role of importin-{beta}1 in the nuclear import of soluble proteins during long-term plasticity.

neuroscience↗

bioRxiv2020

Label-free cell viability assay using phase imaging with computational specificity

Existing approaches to evaluate cell viability involve cell staining with chemical reagents. However, this step of exogenous staining makes these methods undesirable for rapid, nondestructive and long-term investigation. Here, we present instantaneous viability assessment of unlabeled cells using phase imaging with computation specificity (PICS). This new concept utilizes deep learning techniques to compute viability markers associated with the specimen measured by label-free quantitative phase imaging. Demonstrated on different live cell cultures, the proposed method reports approximately 95% accuracy in identifying live and dead cells. The evolution of the cell dry mass and projected area for the labelled and unlabeled populations reveal that the viability reagents decrease viability. The nondestructive approach presented here may find a broad range of applications, from monitoring the production of biopharmaceuticals, to assessing the effectiveness of cancer treatments.

bioengineering↗

bioRxiv2020

Newly developed silicone airway stent (GINA stent): Mechanical characteristics and performance evaluation in pigs

OBJECTIVESCentral airway obstruction (CAO) is caused by various malignant and benign processes. Surgery is a preferred option for CAO, but if not possible, bronchoscopic treatment could be performed. Recently, bronchoscopic treatments have been improved. Particularly in airway stents, new attempts are being made to overcome the existing shortcomings of stents (migration, mucostasis, and granulation tissue formation). We recently developed a new silicone airway stent (GINA stent). The GINA stent has anti-migration design, dynamic structure enabling reduction of stent cross-sectional area, and radiopaqueness. We sought to evaluate mechanical characteristics and performance of our novel GINA stent in a pig tracheal stenosis model. METHODSAll tests were performed by comparing GINA stent [outer diameter (OD, mm) 14, length (L, mm) 55] with Dumon stent (OD14L50). Mechanical tests were done using digital force gage to determine the anti-migration force, expansion force, and flexibility. Short-term (3 weeks) performance was evaluated after stent implantations [GINA (n = 4) vs. Dumon (n = 3)] in a pig model of tracheal stenosis. RESULTSMechanical properties outcomes for GINA vs. Dumon: anti-migration force [18.4 vs. 12.8 Newton (N)]; expansion force (11.9 vs. 14.5 N); flexibility (3.1 vs. 4.5 N). Short-term (3 weeks) GINA vs. Dumon performances: mucus retention (0/4 vs. 0/3); granulation tissue formation (0/4 vs. 0/3); migration (1/4 vs. 2/3). CONCLUSIONSGINA stent demonstrated better mechanical properties than Dumon stent with a stent performance not inferior to Dumon stent.

physiology↗

bioRxiv2020

Agromonas: a rapid disease assay for Pseudomonas syringae growth in agroinfiltrated leaves

The lengthy process to generate transformed plants is a limitation in current research on the interactions of the model plant pathogen Pseudomonas syringae with plant hosts. Here we present an easy method called agromonas, where we quantify P. syringae growth in agroinfiltrated leaves of Nicotiana benthamiana using a cocktail of antibiotics to select P. syringae on plates. As a proof of concept, we demonstrate that transient expression of PAMP receptors reduces bacterial growth and that transient depletion of a host immune gene and transient expression of a T3 effector increase P. syringae growth in agromonas assays. We show that we can rapidly achieve structure-function analysis of immune components and test the function of immune hydrolases. The agromonas method is easy, fast and robust for routine disease assays with various Pseudomonas strains without transforming plants or bacteria. The agromonas assay offers reliable opportunity for further comprehensive analysis of plant immunity. One sentence summaryAgromonas is a rapid and robust disease assay to monitor Pseudomonas syringae growth in agroinfiltrated leaves expressing immune components and their suppressors.

plant biology↗

bioRxiv2020

Mitochondrial dsRNAs activate PKR and TLR3 to promote chondrocyte degeneration in osteoarthritis

Protein kinase R (PKR) is an immune response protein that becomes activated by long double-stranded RNAs (dsRNAs). Several studies reported the misactivation of PKR in patients of degenerative diseases including primary osteoarthritis (OA). However, the molecular identity of PKR-activating dsRNAs remains unknown. Here, we investigate the role of mitochondrial dsRNAs (mt-dsRNAs) in the development of OA. We find that in response to OA-mimicking stressors, cytosolic efflux of mt-dsRNAs is increased, leading to PKR activation and subsequent induction of inflammatory cytokines and apoptosis. Moreover, mt-dsRNAs are exported to the extracellular space where they activate toll-like receptor 3. Elevated expression of mt-dsRNAs in the synovial fluids of OA patients further supports our data. Lastly, we show that autophagy protects chondrocytes from mitochondrial dysfunction partly by removing cytosolic mt-dsRNAs. Together, these findings establish the PKR-mt-dsRNA as a critical regulatory axis in OA development and suggest mt-dsRNAs as a potential target in fighting OA.

molecular biology↗

bioRxiv2019

rMSIcleanup: An open-source tool for matrix-related peak annotation in mass spectrometry imaging and its application to silver-assisted laser desorption/ionization

Mass spectrometry imaging (MSI) has become a mature, widespread analytical technique to perform non-targeted spatial metabolomics. However, the compounds used to promote desorption and ionization of the analyte during acquisition cause spectral interferences in the low mass range that hinder downstream data processing in metabolomics applications. Thus, it is advisable to annotate and remove matrix-related peaks to reduce the number of redundant and non-biologically-relevant variables in the dataset. We have developed rMSIcleanup, an open-source R package to annotate and remove matrix-related signals based on its chemical formula and the spatial distribution of its ions. To validate the annotation method, rMSIcleanup was challenged with several images acquired using silver-assisted laser desorption ionization MSI (AgLDI MSI). The algorithm was able to correctly classify m/z signals related to silver clusters. Visual exploration of the data using Principal Component Analysis (PCA) demonstrated that annotation and removal of matrix-related signals improved spectral data post-processing. The results highlight the need for including matrix-related peak annotation tools such as rMSIcleanup in MSI workflows. Resources availabilityThe R package presented in this publication is freely available under the terms of the GNU General Public License v3.0 at https://github.com/gbaquer/rMSIcleanup. The datasets used in the experiments can be accessed upon request to the corresponding author.

bioinformatics↗

bioRxiv2019

A resource of targeted mutant mouse lines for 5,061 genes

The International Mouse Phenotyping Consortium reports the generation of new mouse mutant strains for over 5,000 genes from targeted embryonic stem cells on the C57BL/6N genetic background. This includes 2,850 null alleles for which no equivalent mutant mouse line exists, 2,987 novel conditional-ready alleles, and 4,433 novel reporter alleles. This nearly triples the number of genes with reporter alleles and almost doubles the number of conditional alleles available to the scientific community. When combined with more than 30 years of community effort, the total mutant allele mouse resource covers more than half of the genome. The extensively validated collection is archived and distributed through public repositories, facilitating availability to the worldwide biomedical research community, and expanding our understanding of gene function and human disease.

genetics↗

bioRxiv2019

High-phytate/low-calcium diet is a risk factor for crystal nephropathies, renal phosphate wasting, and bone loss

Phosphate overload contributes to mineral bone disorders associated with crystal nephropathies. Phytate, the major form of phosphorus in plant seeds, is known as an indigestible and negligible in humans. However, the mechanism and adverse effects of high-phytate intake on Ca2+ and phosphate absorption and homeostasis are unknown. Here we show that excessive intake of phytate with a low-Ca2+ diet fed to rats contributed to the development of crystal nephropathies, renal phosphate wasting, and bone loss through tubular dysfunction secondary to dysregulation of intestinal calcium and phosphate absorption. Moreover, Ca2+ supplementation alleviated the detrimental effects of excess dietary phytate on bone and kidney through excretion of undigested Ca2+-phytate, which prevented a vicious cycle of intestinal phosphate overload and renal phosphate wasting while improving intestinal Ca2+ bioavailability. Thus, we demonstrate that phytate is digestible without a high-Ca2+ diet and a risk factor for phosphate overloading and developing crystal nephropathies and bone disease.

physiology↗

bioRxiv2019

Transcriptome responses associated with carbon and energy flows under high salinity stress suggest the overflow of acetyl-CoA from glycolysis and NADPH co-factor induces high lipid accumulation and halotolerance in Chlorella sp. HS2

Previously, we isolated Chlorella sp. HS2 (referred hereupon HS2) from a local tidal rock pool and demonstrated its halotolerance and relatively high biomass productivity under different salinity conditions. To further understand acclimation responses of this alga against high salinity stress, we performed transcriptome analysis of triplicated culture samples grown in freshwater and marine conditions at both exponential and stationary growth phases. De novo assembly followed by differential expression analysis identified 5907 and 6783 differentially expressed genes (DEGs) respectively at exponential and stationary phases from a total of 52770 transcripts, and the functional enrichment of DEGs with KEGG database resulted in 1445 KEGG Orthology (KO) groups with a defined differential expression. Specifically, the transcripts involved in photosynthesis, TCA and Calvin cycles were downregulated, whereas the upregulation of DNA repair mechanisms and an ABCB subfamily of eukaryotic type ABC transporter was observed at high salinity condition. In addition, while key enzymes associated with glycolysis pathway and triacylglycerol (TAG) synthesis were determined to be upregulated from early growth phase, salinity stress seemed to reduce the carbohydrate content of harvested biomass from 45.6 dw% to 14.7 dw% and nearly triple the total lipid content from 26.0 dw% to 62.0 dw%. These results suggest that the reallocation of storage carbon toward lipids played a significant role in conferring the viability of this alga under high salinity stress, most notably by remediating high level of cellular stress partially caused by ROS generated in oxygen-evolving thylakoids.\n\nSummary StatementRedirection of storage carbon towards the synthesis of lipids played a critical role in conferring the halotolerance of a Chlorella isolate by remediating excess oxidative stress experienced in photosystems.

microbiology↗