Biology subjects

Kim, J. Y.

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

49 records · Page 3Linked to original sources

bioRxiv2021

Respiratory syncytial virus matrix protein assembles as a lattice with local and extended order that coordinates the position of the fusion glycoprotein

Respiratory syncytial virus (RSV) is a significant cause of respiratory illness in young children and adults worldwide. There is currently no vaccine or targeted antiviral for RSV. RSV is an enveloped, filamentous, negative-strand RNA virus. Individual virions vary in both diameter and length, with an average diameter of [~]130 nm and ranging from [~]500 nm to over 10 m in length. The RSV matrix (M) protein is peripherally associated with the interior of the viral membrane. Though the general arrangement of structural proteins within the virion is known, the molecular organization of M and other structural proteins was previously unknown. Here, using whole-cell cryo-electron tomography and sub-tomogram averaging, we show that M is arranged in a packed helical-like lattice of M-dimers ordered at an angle of [~]47{degrees} to the viral long axis. Sub-tomogram averages including F and M indicate that the position of F on the viral surface is correlated with the underlying M lattice. Finally, we report that RSV F is frequently observed as pairs, with the F trimers oriented in an anti-parallel conformation to support potential interaction between trimers. These results provide insight into RSV assembly and virion organization and may aid in the identification and development of RSV vaccines and anti-viral targets.

biophysics↗

bioRxiv2021

Reduced production of the major allergen Bla g 1 and Bla g 2 in Blattella germanica after antibiotic treatment

PurposeAllergens present in the feces or frass of cockroaches can cause allergic sensitization in humans. The use of fecal and frass extracts for immunotherapy has been previously investigated but has not yet been fully standardized. Here, we treated cockroaches with ampicillin to produce extracts with reduced amounts of total bacteria. MethodsWe performed targeted high-throughput sequencing of 16S rDNA to compare the microbiomes of ampicillin-treated and untreated (control) cockroaches. RNA-seq was performed to identify differentially expressed genes (DEGs) in ampicillin-treated cockroaches. ResultsAnalysis of the microbiome revealed that alpha diversity was lower in the ampicillin-treated group than in the control group. Beta diversity analysis indicated that ampicillin treatment altered bacterial composition in the microbiome of cockroaches. Quantitative polymerase chain reaction revealed that almost all bacteria were removed from ampicillin-treated cockroaches. RNA-seq analysis revealed 1,236 DEGs in ampicillin-treated cockroaches (compared to untreated cockroaches). Unlike bacterial composition, the DEGs varied between the two groups. Among major allergens, the expression of Bla g 2 decreased significantly in ampicillin-treated cockroaches (compared to untreated group). ConclusionsIn this study, the reduced level of allergens observed in cockroaches may be related to lower amounts of total bacteria caused by treatment with antibiotics. It is possible to make a protein extract with few bacteria for use in immunotherapy.

bioinformatics↗

bioRxiv2021

Whole-cell cryo-electron tomography of cultured and primary eukaryotic cells on micropatterned TEM grids

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technique that can provide nanometer-level resolution of biological structures within the cellular context and in a near-native frozen-hydrated state. It remains a challenge to culture or adhere cells on TEM grids in a manner that is suitable for tomography while preserving the physiological state of the cells. Here, we demonstrate the versatility of micropatterning to direct and promote growth of both cultured and primary eukaryotic cells on TEM grids. We show that micropatterning is compatible with and can be used to enhance studies of host-pathogen interactions using respiratory syncytial virus infected BEAS-2B cells as an example. We demonstrate the ability to use whole-cell tomography of primary Drosophila neuronal cells to identify organelles and cytoskeletal stuctures in cellular axons and the potential for micropatterning to dramatically increase throughput for these studies. During micropatterning, cell growth is targeted by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design make micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (FIB-SEM).

cell biology↗

bioRxiv2021

JAG1 intracellular domain acts as a transcriptional cofactor that forms an oncogenic transcriptional complex with DDX17/SMAD3/TGIF2

Jagged1 (JAG1) is a Notch ligand that contact-dependently activates Notch receptors and regulates cancer progression. The JAG1 intracellular domain (JICD1) is generated from JAG1, such as the formation of NOTCH1 intracellular domain (NICD1), however, the role of JICD1 in tumorigenicity has not been comprehensively elucidated. Herein, we revealed that JICD1 induced astrocytes to acquire several cancer stem cell properties, including tumor formation, invasiveness, stemness, and resistance to chemotherapy and radiotherapy. The transcriptome, ChIP-sequencing, and proteomic analyses revealed that JICD1 increased SOX2 expression by forming a transcriptional complex with DDX17, SMAD3, and TGIF2. Furthermore, JICD1-driven tumorigenicity was directly regulated by SOX2. Therefore, our results demonstrated that, like NICD1, JICD1 acts as a transcriptional cofactor in the formation of the DDX17/SMAD3/TGIF2 transcriptional complex, leading to oncogenic transformation.

molecular biology↗

bioRxiv2021

Community Evaluation of Glycoproteomics Informatics Solutions Reveals High-Performance Search Strategies of Glycopeptide Data

Glycoproteome profiling (glycoproteomics) is a powerful yet analytically challenging research tool. The complex tandem mass spectra generated from glycopeptide mixtures require sophisticated analysis pipelines for structural determination. Diverse software aiding the process have appeared, but their relative performance remains untested. Conducted through the HUPO Human Proteome Project - Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates the performance of informatics solutions for system-wide glycopeptide analysis. Mass spectrometry-based glycoproteomics datasets from human serum were shared with all teams. The relative team performance for N- and O-glycopeptide data analysis was comprehensively established and validated through orthogonal performance tests. Excitingly, several high-performance glycoproteomics informatics solutions were identified. While the study illustrated that significant informatics challenges remain, as indicated by a high discordance between annotated glycopeptides, lists of high-confidence (consensus) glycopeptides were compiled from the standardised team reports. Deep analysis of the performance data revealed key performance-associated search variables and led to recommendations for improved "high coverage" and "high accuracy" glycoproteomics search strategies. This study concludes that diverse software for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies, and specifies key variables that may guide future software developments and assist informatics decision-making in glycoproteomics.

biochemistry↗

bioRxiv2021

Nephrotoxicity of the BRAF-kinase inhibitor Vemurafenib isdriven by off-target Ferrochelatase inhibition

A multitude of disease and therapy related factors drive the frequent development of renal disorders in cancer patients. Along with chemotherapy, the newer targeted therapeutics can also cause renal dysfunction through on and off-target mechanisms. Interestingly, among the small-molecule inhibitors approved for the treatment of cancers that harbor BRAF-kinase activating mutations, vemurafenib can trigger tubular damage and acute kidney injury (AKI). To investigate the underlying mechanisms, here, we have developed cell culture and mouse models of vemurafenib nephrotoxicity. Our studies show that at clinically relevant concentrations vemurafenib induces cell-death in transformed and primary murine and human renal tubular epithelial cells (RTEC). In mice, two weeks of daily vemurafenib treatment causes moderate AKI with histopathological characteristics of RTEC injury. Importantly, RTEC-specific BRAF gene deletion did not influence renal function under normal conditions or alter the severity of vemurafenib-associated renal impairment. Instead, we found that inhibition of ferrochelatase (FECH), an enzyme involved in heme biosynthesis contributes to vemurafenib nephrotoxicity. FECH overexpression protected RTECs and conversely FECH knockdown increased the sensitivity to vemurafenib nephrotoxicity. Collectively, these studies suggest that vemurafenib-associated RTEC dysfunction and nephrotoxicity is BRAF-independent and caused in part by off-target FECH inhibition. Translational StatementBRAF is the most frequently mutated protein kinase and a critical oncogenic driver in human cancers. In melanoma and other cancers with BRAF activating mutations, BRAF targeted small-molecule therapeutics such as vemurafenib, and dabrafenib have shown remarkable clinical benefits. However, recent clinical studies have shown that a significant number of patients that receive vemurafenib develop AKI through mechanisms that remain unknown. The present study describes the development of novel experimental models of vemurafenib nephrotoxicity and reveals the underlying off-target mechanisms that contribute to renal injury.

pharmacology and toxicology↗

bioRxiv2020

SOX9 promotes stress-responsive transcription of VGF nerve growth factor inducible gene in kidney epithelial cells

Acute kidney injury (AKI) is a common clinical condition associated with diverse etiologies and abrupt loss of renal function. In patients with sepsis, rhabdomyolysis, cancer, as well as cardiovascular disorders, the underlying disease or associated therapeutic interventions can cause hypoxic, cytotoxic, and inflammatory insults to renal tubular epithelial cells (RTECs) resulting in the onset of AKI. To uncover stress-responsive disease-modifying genes, here we have carried out renal transcriptome profiling in three distinct murine models of AKI. We find that Vgf nerve growth factor inducible gene upregulation is a common transcriptional stress response in RTECs to ischemia, cisplatin, and rhabdomyolysis-associated renal injury. The Vgf gene encodes a secretory peptide precursor protein that has critical neuro-endocrine functions; however, its role in the kidneys remains unknown. Our functional studies show that RTEC-specific Vgf gene ablation exacerbates ischemia, cisplatin, and rhabdomyolysis-associated AKI in vivo and cisplatin-induced RTEC cell death in vitro. Importantly, addback experiments showed that aggravation of cisplatin-induced renal injury caused by Vgf gene ablation is partly reversed by TLQP-21, a Vgf-derived peptide. Finally, in vitro and in vivo mechanistic studies showed that injury-induced Vgf upregulation in RTECs is driven by the transcriptional regulator Sox9. These findings reveal a crucial downstream target of the Sox9-directed transcriptional program and identify Vgf as a stress-responsive protective gene in kidney epithelial cells.AbbreviationsAKIAcute kidney injuryRTECrenal tubular epithelial cellsBUNblood urea nitrogenChIPchromatin immunoprecipitationSox9SRY-Box transcription factor 9.View Full Text

cell biology↗

bioRxiv2020

Spatiotemporal contribution of neuromesodermal progenitor-derived neural cells in the elongation of developing mouse spinal cord

During vertebrate development, the posterior end of the embryo progressively elongates in a head-to-tail direction to form the body plan. Recent lineage tracing experiments revealed that bi-potent progenitors, called neuromesodermal progenitors (NMPs), produce caudal neural and mesodermal tissues during axial elongation. However, their precise location and contribution to spinal cord development remain elusive. Here we used NMP-specific markers (Sox2 and BraT) and a genetic lineage tracing system to localize NMP progeny in vivo. NMPs were initially located at the tail tip, but were later found in the caudal neural tube, which is a unique feature of mouse development. In the neural tube, they produced neural stem cells (NSCs) and contributed to the spinal cord gradually along the AP axis during axial elongation. Interestingly, NMP-derived NSCs preferentially contributed to the ventral side first and later to the dorsal side at the lumbar spinal cord level, which may be associated with atypical junctional neurulation in mice. Our current observations detail the contribution of NMP progeny to spinal cord elongation and provide insights into how different species uniquely execute caudal morphogenesis.

developmental biology↗

bioRxiv2020

Calsyntenin-3 directly interacts with neurexins to orchestrate excitatory synapse development in the hippocampus

Calsyntenin-3 (Clstn3) is a postsynaptic adhesion molecule that induces presynaptic differentiation via presynaptic neurexins (Nrxns), but whether Nrxns directly bind to Clstn3 has been a matter of debate. Here, we show that {beta}-Nrxns directly interact via their LNS domain with Clstn3 and Clstn3 cadherin domains. Expression of splice site 4 (SS4) insert-positive {beta}-Nrxn variants, but not insert-negative variants, reversed the impaired Clstn3 synaptogenic activity observed in Nrxn-deficient neurons. Consistently, Clstn3 selectively formed complexes with SS4-positive Nrxns in vivo. Neuron-specific Clstn3 deletion caused significant reductions in number of excitatory synaptic inputs, and moderate impairment of light-induced anxiety-like behaviors in mice. Moreover, expression of Clstn3 cadherin domains in CA1 neurons of Clstn3 conditional knockout mice rescued structural deficits in excitatory synapses, especially within the stratum radiatum layer. Collectively, our results suggest that Clstn3 links to SS4-positive Nrxns to induce presynaptic differentiation and orchestrate excitatory synapse development in specific hippocampal neural circuits.

neuroscience↗

bioRxiv2019

Comparative microbiomes of three species of laboratory-reared Periplaneta cockroaches

Cockroaches are the most primitive insects, and are often used as the basic insect model in many studies. Three species of Periplaneta cockroaches were raised in the laboratory for many generations under the same conditions. We conducted 16S rRNA-targeted high-throughput sequencing to evaluate the overall bacterial composition in the microbiomes of three species of cockroaches. The number of operational taxonomic units (OTUs) was not significantly different between the three cockroach species. With respect to the Shannon and Pielou indexes, the microbiome of Periplaneta americana presented higher values than that of either P. japonica or P. fulginosa. In terms of species composition, endosymbionts accounted for over half of all the bacterial species in P. japonica and P. fulginosa. The beta diversity analysis showed that P. japonica and P. fulginosa exhibit a similar microbiome composition, which is different from that of P. americana. However, we also identified that P. japonica and P. fulginosa are hosts to distinct bacterial species. Thus, although the composition of the microbiome may vary based on multiple conditions, it is possible to identify distinct compositions of the microbiome among the different Periplaneta cockroach species even when individuals are reared under the same conditions.\n\nImportanceCockroaches inhabit various habitats--which are known to be related to their microbiome--and exhibit different features depending on the species. It is expected that their microbiomes would vary according to species depending on these features. Cockroach microbiomes are known to vary based on the diet or environmental shifts. In this study, we compare the diversity of bacteria in the three cockroach species under conditions of reduced diet and environment shifts. This study can establish the basic microbiomes of three Periplaneta species and can be the basic data for cockroach research.

microbiology↗

bioRxiv2019

Identification of peptide coatings that enhance diffusive transport of nanoparticles through the tumor microenvironment

In solid tumors, increasing drug penetration promotes their regression and improves the therapeutic index of compounds. However, the heterogeneous extracellular matrix (ECM) acts a steric and interaction barrier that hinders effective transport of therapeutics, including nanomedicines. Specifically, the interactions between the ECM and surface physicochemical properties of nanomedicines (e.g. charge, hydrophobicity) impedes their diffusion and penetration. To address the challenges using existing surface chemistries, we used peptide-presenting phage libraries as a high-throughput approach to screen and identify peptides as coatings with desired physicochemical properties that improve diffusive transport through the tumor microenvironment. Through iterative screening against the ECM and identification by next-generation DNA sequencing and analysis, we selected individual clones and measured their transport by diffusion assays. Here, we identified a net-neutral charge, hydrophilic peptide P4 that facilitates significantly higher diffusive transport of phage than negative control through in vitro tumor ECM. Through alanine mutagenesis, we confirmed that the hydrophilicity, charge, and their spatial ordering impact diffusive transport. P4 phage clone exhibited almost 200-fold improved uptake in ex vivo pancreatic tumor xenografts compared to the negative control. Nanoparticles coated with P4 exhibited [~]40-fold improvement in diffusivity in pancreatic tumor tissues, and P4-coated particles demonstrated less hindered diffusivity through the ECM compared to particles functionalized with gold standard poly(ethylene) glycol or iRGD peptide ligand. By leveraging the power of molecular diversity using phage display, we can greatly expand the chemical space of surface chemistries that can improve the transport of nanomedicines through the complex tumor microenvironment to ultimately improve their efficacy.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC=\"FIGDIR/small/659524v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (23K):\norg.highwire.dtl.DTLVardef@18bc04forg.highwire.dtl.DTLVardef@1feb597org.highwire.dtl.DTLVardef@14403e5org.highwire.dtl.DTLVardef@5bbf6c_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2019

Longitudinal single cell profiling of regulatory T cells identifies IL-33 as a driver of tumor immunosuppression

Regulatory T cells (Tregs) can impair anti-tumor immune responses and are associated with poor prognosis in multiple cancer types. Tregs in human tumors span diverse transcriptional states distinct from those of peripheral Tregs, but their contribution to tumor development remains unknown. Here, we used single cell RNA-Seq to longitudinally profile conventional CD4+ T cells (Tconv) and Tregs in a genetic mouse model of lung adenocarcinoma. Tissue-infiltrating and peripheral CD4+ T cells differed, highlighting divergent pathways of activation during tumorigenesis. Longitudinal shifts in Treg heterogeneity suggested increased terminal differentiation and stabilization of an effector phenotype over time. In particular, effector Tregs had enhanced expression of the interleukin 33 receptor ST2. Treg-specific deletion of ST2 reduced effector Tregs, increased infiltration of CD8+ T cells into tumors, and decreased tumor burden. Our study shows that ST2 plays a critical role in Treg-mediated immunosuppression in cancer, highlighting new potential paths for therapeutic intervention.

cancer biology↗

bioRxiv2018

TNF-α induces reactivation of human cytomegalovirus independently of myeloid cell differentiation following post-transcriptional establishment of latency

We used the Kasumi-3 model to study HCMV latency and reactivation in myeloid progenitor cells. Kasumi-3 cells were infected with HCMV strain TB40/Ewt-GFP, flow sorted for GFP+ cells, and cultured for various times to monitor establishment of latency, as judged by repression of viral gene expression (RNA/DNA ratio) and loss of virus production. We found that, in the vast majority of cells, latency was established post-transcriptionally in the GFP+ infected cells: transcription was initially turned on, and then turned off. We also found that some of the GFP-cells were infected, suggesting that latency might be established in these cells at the outset of infection. We were not able to test this hypothesis because some GFP-cells expressed lytic genes, and thus, it was not possible to separate them from GFP-quiescent cells. In addition, we found that the pattern of expression of lytic genes that have been associated with latency, including UL138, US28, and RNA2.7, was the same as that of other lytic genes, indicating that there was no preferential expression of these genes once latency is established. We confirmed previous studies showing that TNF- induced reactivation of infectious virus, and by analyzing expression of the progenitor cell marker CD34 as well as myeloid cell differentiation markers in IE+ cells after treatment with TNF-, we showed that TNF- induced transcriptional reactivation of IE gene expression independently of differentiation. TNF--mediated reactivation in Kasumi-3 cells was correlated with activation of NF-{kappa}B, KAP-1 and ATM.\n\nIMPORTANCEHCMV is an important human pathogen that establishes lifelong latent infection in myeloid progenitor cells, and reactivates frequently to cause significant disease in immunocompromised people. Our observation that viral gene expression is first turned on, and then turned off to establish latency suggests that there is a host defense, which may be myeloid-specific, responsible for transcriptional silencing of viral gene expression. Our observation that TNF- induces reactivation independently of differentiation provides insight into molecular mechanisms that control reactivation.

microbiology↗