Biology subjects

Kim, Y. J.

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

At least 73 records · Page 4Linked to original sources

bioRxiv2021

Exon-Skipping Antisense Oligonucleotides for Cystic Fibrosis Therapy

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), and the CFTR-W1282X nonsense mutation causes a severe form of CF. Although Trikafta and other CFTR-modulation therapies benefit most CF patients, targeted therapy for patients with the W1282X mutation is lacking. The CFTR-W1282X protein has residual activity, but is expressed at a very low level due to nonsense-mediated mRNA decay (NMD). NMD-suppression therapy and read-through therapy are actively being researched for CFTR nonsense mutants. NMD suppression could increase the mutant CFTR mRNA, and read-through therapies may increase the levels of full-length CFTR protein. However, these approaches have limitations and potential side effects: because the NMD machinery also regulates the expression of many normal mRNAs, broad inhibition of the pathway is not desirable; and read-through drugs are inefficient, partly because the mutant mRNA template is subject to NMD. To bypass these issues, we pursued an exon-skipping antisense oligonucleotide (ASO) strategy to achieve gene-specific NMD evasion. A cocktail of two splice-site-targeting ASOs induced the expression of CFTR mRNA without the PTC-containing exon 23 (CFTR-{Delta}ex23), which is an in-frame exon. Treatment of human bronchial epithelial cells with this cocktail of ASOs that target the splice sites flanking exon 23 results in efficient skipping of exon 23 and an increase in CFTR-{Delta}ex23 protein. The splice-switching ASO cocktail increases the CFTR-mediated chloride current in human bronchial epithelial cells. Our results set the stage for developing an allele-specific therapy for CF caused by the W1282X mutation.

molecular biology↗

bioRxiv2021

Predictive modeling reveals that higher-order cooperativity drives transcriptional repression in a synthetic developmental enhancer

A challenge in quantitative biology is to predict output patterns of gene expression from knowledge of input transcription factor patterns and from the arrangement of binding sites for these transcription factors on regulatory DNA. We tested whether widespread thermodynamic models could be used to infer parameters describing simple regulatory architectures that inform parameter-free predictions of more complex enhancers in the context of transcriptional repression by Runt in the early fruit 2y embryo. By modulating the number and placement of Runt binding sites within an enhancer, and quantifying the resulting transcriptional activity using live imaging, we discovered that thermodynamic models call for higher-order cooperativity between multiple molecular players. This higher-order cooperativity capture the combinatorial complexity underlying eukaryotic transcriptional regulation and cannot be determined from simpler regulatory architectures, highlighting the challenges in reaching a predictive understanding of transcriptional regulation in eukaryotes and calling for approaches that quantitatively dissect their molecular nature.

biophysics↗

bioRxiv2021

Gene-Specific Nonsense-Mediated mRNA Decay Targeting for Cystic Fibrosis Therapy

Low CFTR mRNA expression due to nonsense-mediated mRNA decay (NMD) is a major hurdle in developing a therapy for cystic fibrosis (CF) caused by the W1282X mutation in the CFTR gene. CFTR-W1282X truncated protein retains partial function, so increasing its levels by inhibiting NMD of its mRNA will likely be beneficial. Because NMD regulates the normal expression of many genes, gene-specific stabilization of CFTR-W1282X mRNA expression is more desirable than general NMD inhibition. Synthetic antisense oligonucleotides (ASOs) designed to prevent binding of exon junction complexes (EJC) downstream of premature termination codons (PTCs) attenuate NMD in a gene-specific manner. We developed a cocktail of three ASOs that specifically increases the expression of CFTR W1282X mRNA and CFTR protein in ASO-transfected human bronchial epithelial cells. This treatment increased the CFTR-mediated chloride current. These results set the stage for clinical development of an allele-specific therapy for CF caused by the W1282X mutation.

molecular biology↗

bioRxiv2021

Role of Gasdermins in the Biogenesis of Apoptotic Cell-Derived Exosomes

The gasdermins, GSDMA, GSDMB, GSDMC, GSDMD, DFNA5, and DFNB59, are a family of pore-forming proteins that has recently been suggested to play a central role in the pyroptosis and the release of inflammatory cytokines. Here, we describe the novel roles of gasdermins in the biogenesis of apoptotic cell-derived exosomes. In apoptotic cells, GADMA, GSDMC, GSDMD, and DFNA5 increased the release of ApoExos, and both their full-length and cleaved forms were localized in the exosomal membrane. GSDMB and DFNB59, on the other hand, negatively affected the release of ApoExos. The caspase-mediated cleavage of gasdermins, especially DFNA5, is suggested to enable cytosolic Ca2+ to flow through endosomal pores and thus increase the biogenesis of ApoExos. In addition, the DFNA5-meidiated biogenesis of ApoExos depended on the ESCRT-III complex and endosomal recruitment of Ca2+-dependent proteins: annexins A2 and A7, the PEF domain family proteins sorcin and grancalcin, and the Bro1 domain protein HD-PTP. Therefore, we propose that the biogenesis of ApoExos begins when gasdermin-mediated endosomal pores increase cytosolic Ca2+, continues through the recruitment of annexin-sorcin/grancalcin-HD-PTP, and is completed when the ESCRT-III complex synthesizes intraluminal vesicles in the multivesicular bodies of dying cells. Finally, we found that Dfna5-bearing tumors released ApoExos to induce inflammatory responses in the in vivo 4T1 orthotropic model of breast cancer. The data presented in this study indicate that the switch from apoptosis to pyroptosis could drive the transfer of mass signals to nearby or distant living cells and tissues by way of extracellular vesicles, and that gasdermins play critical roles in that process.

cell biology↗

bioRxiv2021

Cryo-EM structure of the yeast TREX complex and coordination with the SR-like protein Gbp2

The evolutionarily conserved TREX complex plays central roles during mRNP (messenger ribonucleoprotein) maturation and export from the nucleus to the cytoplasm. In yeast, TREX is composed of the THO sub-complex (Tho2, Hpr1, Tex1, Mft1, and Thp2), the DEAD box ATPase Sub2, and Yra1. Here we present a 3.7 [A] cryo-EM structure of the yeast THO*Sub2 complex. The structure reveals the intimate assembly of THO revolving around its largest subunit Tho2. THO stabilizes a semi-open conformation of the Sub2 ATPase via interactions with Tho2. We show that THO interacts with the SR-like protein Gbp2 through both the N-terminal domain and RRM domains of Gbp2. Crosslinking mass spectrometry analysis supports the extensive interactions between THO and Gbp2, further revealing that RRM domains of Gbp2 are in close proximity to the C-terminal domain of Tho2. We propose that THO serves as a landing pad to configure Gbp2 to facilitate its loading onto mRNP.

biophysics↗

bioRxiv2020

3D8, a nucleic acid-hydrolyzing scFv, confers antiviral activity against SARS-CoV-2 and multiple coronaviruses in vitro

BackgroundThe current pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the etiology of Coronavirus-induced disease 19 (COVID-19) and poses a critical public health threat worldwide. Effective therapeutics and vaccines against multiple coronaviruses remain unavailable. Single chain variable fragment (scFv), a recombinant antibody exhibits broad-spectrum antiviral activity against DNA and RNA viruses owing to its nucleic acid-hydrolyzing property. ObjectiveThis study is aimed to investigate an antiviral activity of 3D8 scFv against SARS-CoV-2 and other coronaviruses. Methods3D8, a recombinant scFv antibody was evaluated for antiviral activity against SARS-CoV-2, HCoV-OC43 and PEDV in Vero E6 cell cultures. Viral growth was quantified with quantitative RT-qPCR and plaque assay. Nucleic acid hydrolyzing activity of 3D8 was assessed through abzyme assays of in vitro viral transcripts and cell viability was determined by MTT assay. Results3D8 inhibited the replication of SARS-CoV-2, human coronavirus OC43 (HCoV-OC43), and porcine epidemic diarrhea virus (PEDV). Our results revealed the prophylactic and therapeutic effects of 3D8 scFv against SARS-CoV-2 in Vero E6 cells. Immunoblot and plaque assays showed the reduction of coronavirus nucleoproteins and infectious particles respectively in 3D8 scFv-treated cells. ConclusionsThis data demonstrates the broad-spectrum antiviral activity of 3D8 against SARS-CoV-2 and other coronaviruses. Thus, it could be considered a potential antiviral countermeasure against SARS-CoV-2 and zoonotic coronaviruses.

microbiology↗

bioRxiv2020

Combined assessment of MHC binding and antigen expression improves T cell epitope predictions

MHC class I antigen processing consists of multiple steps that result in the presentation of MHC bound peptides that can be recognized as T cell epitopes. Many of the pathway steps can be predicted using computational methods, but one is often neglected: mRNA expression of the epitope source proteins. In this study, we improve epitope prediction by taking into account both peptide-MHC binding affinities and expression levels of the peptides source protein. Specifically, we utilized biophysical principles and existing MHC binding prediction tools in concert with RNA expression to derive a function that estimates the likelihood of a peptide being presented on a given MHC class I molecule. Our combined model of Antigen eXpression based Epitope Likelihood-Function (AXEL-F) outperformed predictions based only on binding or based only on antigen expression for discriminating eluted ligands from random background peptides as well as in predicting neoantigens that are recognized by T cells. We also showed that in cases where cancer patient-specific RNA-Seq data is not available, cancer-type matched expression data from TCGA can be used to accurately estimate patient-specific gene expression. Using AXEL-F together with TGCA expression data we were able to more accurately predict neoantigens that are recognized by T cells. The method is available in the IEDB Analysis Resource and free to use for the academic community. Significance statementEpitope prediction tools have been used to call epitopes in viruses and other pathogens for almost 30 years, and more recently, to call cancer neoantigens. Several such tools have been developed, however most of them ignore the mRNA expression of the epitope source proteins. In the present study, we have, to our knowledge for the first time, developed a biophysically motivated model to combine peptide-MHC binding and abundance of the peptides source protein to improve epitope predictions. Our novel tool AXEL-F is freely available on the IEDB and presents a clear opportunity for predicting and selecting epitopes more efficiently.

bioinformatics↗

bioRxiv2020

Ultra-fast Prediction of Somatic Structural Variations by Reduced Read Mapping via Pan-Genome k-mer Sets

Genome rearrangements often result in copy number alterations of cancer-related genes and cause the formation of cancer-related fusion genes. Current structural variation (SV) callers, however, still produce massive numbers of false positives (FPs) and require high computational costs. Here, we introduce an ultra-fast and high-performing somatic SV detector, called ETCHING, that significantly reduces the mapping cost by filtering reads matched to pan-genome and normal k-mer sets. To reduce the number of FPs, ETCHING takes advantage of a Random Forest classifier that utilizes six breakend-related features. We systematically benchmarked ETCHING with other SV callers on reference SV materials, validated SV biomarkers, tumor and matched-normal whole genomes, and tumor-only targeted sequencing datasets. For all datasets, our SV caller was much faster ([≥]15X) than other tools without compromising performance or memory use. Our approach would provide not only the fastest method for largescale genome projects but also an accurate clinically practical means for real-time precision medicine.

bioinformatics↗

bioRxiv2020

High-throughput phenotypic characterization of zebrafish CRISPR mutants of DNA repair genes

A systematic knowledge of the roles of DNA repair genes at the level of the organism has been limited due to the lack of appropriate experimental techniques. Here, we generated zebrafish loss-of-function mutants for 32 DNA repair and replication genes through multiplexed CRISPR/Cas9-mediated mutagenesis. High-throughput phenotypic characterization of our mutant collection revealed that three genes (atad5a, ddb1, pcna) are essential for proper embryonic development and hematopoiesis; seven genes (apex1, atrip, ino80, mre11a, shfm1, telo2, wrn) are required for growth and development during juvenile stage and six genes (blm, brca2, fanci, rad51, rad54l, rtel1) play critical roles in sex development. Furthermore, mutation in six genes (atad5a, brca2, polk, rad51, shfm1, xrcc1) displayed hypersensitivity to DNA damage agents. Further characterization of atad5a-/- mutants demonstrate that Atad5a is required for normal brain development and hematopoiesis. Our zebrafish mutant collection provides a unique resource for understanding of the roles of DNA repair genes at the organismal level.

developmental biology↗

bioRxiv2020

Nonlinear decoding of natural images from large-scale primate retinal ganglion recordings

Decoding sensory stimuli from neural activity can provide insight into how the nervous system might interpret the physical environment, and facilitates the development of brain-machine interfaces. Nevertheless, the neural decoding problem remains a significant open challenge. Here, we present an efficient nonlinear decoding approach for inferring natural scene stimuli from the spiking activities of retinal ganglion cells (RGCs). Our approach uses neural networks to improve upon existing decoders in both accuracy and scalability. Trained and validated on real retinal spike data from > 1000 simultaneously recorded macaque RGC units, the decoder demonstrates the necessity of nonlinear computations for accurate decoding of the fine structures of visual stimuli. Specifically, high-pass spatial features of natural images can only be decoded using nonlinear techniques, while low-pass features can be extracted equally well by linear and nonlinear methods. Together, these results advance the state of the art in decoding natural stimuli from large populations of neurons. Author summaryNeural decoding is a fundamental problem in computational and statistical neuroscience. There is an enormous literature on this problem, applied to a wide variety of brain areas and nervous systems. Here we focus on the problem of decoding visual information from the retina. The bulk of previous work here has focused on simple linear decoders, applied to modest numbers of simultaneously recorded cells, to decode artificial stimuli. In contrast, here we develop a scalable nonlinear decoding method to decode natural images from the responses of over a thousand simultaneously recorded units, and show that this decoder significantly improves on the state of the art.

neuroscience↗

bioRxiv2020

Quantitative characterization of the eukaryotic transcription cycle using live imaging and statistical inference

The eukaryotic transcription cycle consists of three main steps: initiation, elongation, and cleavage of the nascent RNA transcript. Although each of these steps can be regulated as well as coupled with each other, their in vivo dissection has remained challenging because available experimental readouts lack sufficient spatiotemporal resolution to separate the contributions from each of these steps. Here, we describe a novel application of Bayesian inference techniques to simultaneously infer the effective parameters of the transcription cycle in real time and at the single-cell level using a two-color MS2/PP7 reporter gene and the developing fruit fly embryo as a case study. Our method enables detailed investigations into cell-to-cell variability in transcription-cycle parameters as well as single-cell correlations between these parameters. These measurements, combined with theoretical modeling, suggest a substantial variability in the elongation rate of individual RNA polymerase molecules. We further illustrate the power of this technique by uncovering a novel mechanistic connection between RNA polymerase density and nascent RNA cleavage efficiency. Thus, our approach makes it possible to shed light on the regulatory mechanisms in play during each step of the transcription cycle in individual, living cells at high spatiotemporal resolution.

biophysics↗

bioRxiv2020

COVID-19 patients upregulate toll-like receptor 4-mediated inflammatory signaling that mimics bacterial sepsis

Observational studies of the ongoing coronavirus disease 2019 (COVID-19) outbreak suggest that a cytokine storm is involved in the pathogenesis of severe illness. However, the molecular mechanisms underlying the altered pathological inflammation in COVID-19 are largely unknown. We report here that toll-like receptor (TLR) 4-mediated inflammatory signaling molecules are upregulated in peripheral blood mononuclear cells (PBMCs) from COVID-19 patients, compared with healthy controls. Among the most highly increased inflammatory mediators in severe/critically ill patients, S100A9, an alarmin and TLR4 ligand, was found as a noteworthy biomarker, because it inversely correlated with the serum albumin levels. These data support a link between TLR4 signaling and pathological inflammation during COVID-19 and contribute to develop therapeutic approaches through targeting TLR4-mediated inflammation.

immunology↗

bioRxiv2020

YASS: Yet Another Spike Sorter applied to large-scale multi-electrode array recordings in primate retina

Spike sorting is a critical first step in extracting neural signals from large-scale multi-electrode array (MEA) data. This manuscript presents several new techniques that make MEA spike sorting more robust and accurate. Our pipeline is based on an efficient multi-stage "triage-then-cluster-then-pursuit" approach that initially extracts only clean, high-quality waveforms from the electrophysiological time series by temporarily skipping noisy or "collided" events (representing two neurons firing synchronously). This is accomplished by developing a neural network detection and denoising method followed by efficient outlier triaging. The denoised spike waveforms are then used to infer the set of spike templates through nonparametric Bayesian clustering. We use a divide-and-conquer strategy to parallelize this clustering step. Finally, we recover collided waveforms with matching-pursuit deconvolution techniques, and perform further split-and-merge steps to estimate additional templates from the pool of recovered waveforms. We apply the new pipeline to data recorded in the primate retina, where high firing rates and highly-overlapping axonal units provide a challenging testbed for the deconvolution approach; in addition, the well-defined mosaic structure of receptive fields in this preparation provides a useful quality check on any spike sorting pipeline. We show that our pipeline improves on the state-of-the-art in spike sorting (and outperforms manual sorting) on both real and semi-simulated MEA data with > 500 electrodes; open source code can be found at https://github.com/paninski-lab/yass.

neuroscience↗

bioRxiv2019

Pinealectomy increases thermogenesis and decreases lipogenesis

This study was designed to determine the effects of pineal gland-derived melatonin on obesity by employing rat pinealectomy (Pnx) model. After 10 weeks of high-fat diet (HFD) feeding, rats received sham or Pnx surgery followed by 10 weeks normal chow diet (NCD) feeding. Pnx decreased expressions of melatonin receptors, MTNR1A and MTNR1B, in brown (BAT) and white adipose tissues (WAT). Pnx rats showed increased insulin sensitivity compared with those that received sham surgery. Leptin levels were significantly decreased in the serum of Pnx group. In addition, Pnx stimulated thermogenic genes in BAT whereas attenuated lipogenic genes in WAT and the liver. Histologic analyses revealed marked decreased in the size of lipid droplets and increased expressions of UCP1 in BAT and attenuated lipid droplets in the sized and the number in the liver of Pnx group. In conclusion, these results in the current study suggest that Pnx increases thermogenesis in BAT and decreases lipogenesis in WAT and the liver.

biochemistry↗

bioRxiv2019

Treatment with β3 adrenergic agonists reverses impaired cardiac myocyte Na+ export and improves severely decompensated heart failure; a clinical application of an experimental finding

BackgroundReported one- and two-year mortality for patients with advanced heart failure (HF) treated medically are ~75% and nearly 100%. In such patients, reversible cellular abnormalities are potential treatment targets and a raised cytosolic Na+ concentration that impairs their myocardial contractility is one potential target. {beta}3 adrenoceptor ({beta}3 AR) agonists stimulate the myocyte Na+-K+ pump. MethodsWe induced severe HF in rabbits by coronary ligation and measured indices of organ congestion after treatment with {beta}3 AR agonists. Na+-K+ pump current was measured in voltage-clamped myocytes isolated from non-infarct myocardium. To assess if {beta}3 ARs might add benefit to optimised guideline-directed medical treatment we report outcomes of giving the {beta}3 AR agonist mirabegron to patients hospitalized with advanced, treatment-refractory stage D HF. ResultsTreatment of rabbits after coronary ligation with {beta}3 AR agonist reversed a decreased myocyte Na+-K+ pump current and significantly reduced organ congestion and prevalence of ascites. Oral treatment with mirabegron rapidly improved signs and symptoms of 9 patients with advanced HF and improvement of [≥]1 NYHA Class was maintained early post-discharge with continued treatment. One patient died from HF at 16 months, 4 died from other causes at 2 - 30 months and 4 remain alive at 38 {+/-} 4 months with NYHA Class II symptoms. ConclusionsParallel {beta}3 AR agonist-induced reversal of Na+-K+ pump inhibition and severe HF in rabbits identify pump inhibition as a treatment target, and changed in-hospital clinical trajectory and post-discharge course more favorable than expected suggest efficacy of mirabegron in advanced human HF.

physiology↗

bioRxiv2019

Identification of type 2 diabetes loci in 433,540 East Asian individuals

Meta-analyses of genome-wide association studies (GWAS) have identified >240 loci associated with type 2 diabetes (T2D), however most loci have been identified in analyses of European-ancestry individuals. To examine T2D risk in East Asian individuals, we meta-analyzed GWAS data in 77,418 cases and 356,122 controls. In the main analysis, we identified 298 distinct association signals at 178 loci, and across T2D association models with and without consideration of body mass index and sex, we identified 56 loci newly implicated in T2D predisposition. Common variants associated with T2D in both East Asian and European populations exhibited strongly correlated effect sizes. New associations include signals in/near GDAP1, PTF1A, SIX3, ALDH2, a microRNA cluster, and genes that affect muscle and adipose differentiation. At another locus, eQTLs at two overlapping T2D signals act through two genes, NKX6-3 and ANK1, in different tissues. Association studies in diverse populations identify additional loci and elucidate disease genes, biology, and pathways.\n\nType 2 diabetes (T2D) is a common metabolic disease primarily caused by insufficient insulin production and/or secretion by the pancreatic {beta} cells and insulin resistance in peripheral tissues1. Most genetic loci associated with T2D have been identified in populations of European (EUR) ancestry, including a recent meta-analysis of genome-wide association studies (GWAS) of nearly 900,000 individuals of European ancestry that identified >240 loci influencing the risk of T2D2. Differences in allele frequency between ancestries affect the power to detect associations within a population, particularly among variants rare or monomorphic in one population but more frequent in another3,4. Although smaller than studies in European populations, a recent T2D meta-analysis in almost 200,000 Japanese individuals identified 28 additional loci4. The relative contributions of different pathways to the pathophysiology of T2D may also differ between ancestry groups. For example, in East Asian (EAS) populations, T2D prevalence is greater than in European populations among people of similar body mass index (BMI) or waist circumference5. We performed the largest meta-analysis of East Asian individuals to identify new genetic associations and provide insight into T2D pathogenesis.

genetics↗

bioRxiv2019

Defining the Subcellular Distribution and Metabolic Channeling of Phosphatidylinositol

Phosphatidylinositol (PtdIns) is an essential structural component of eukaryotic membranes that also serves as the common precursor for polyphosphoinositide (PPIn) lipids. Despite the recognized importance of PPIn species for signal transduction and membrane homeostasis, there is still a limited understanding of how the dynamic regulation of PtdIns synthesis and transport contributes to the turnover of PPIn pools. To address these shortcomings, we capitalized on the substrate selectivity of a bacterial enzyme, PtdIns-specific PLC, to establish a molecular toolbox for investigations of PtdIns distribution and availability within intact cells. In addition to its presence within the ER, our results reveal low steady-state levels of PtdIns within the plasma membrane (PM) and endosomes as well as a relative enrichment of PtdIns within the cytosolic leaflets of the Golgi complex, peroxisomes, and outer mitochondrial membranes. Kinetic studies also demonstrate the requirement for sustained PtdIns supply from the ER for the maintenance of monophosphorylated PPIn species within the PM, Golgi complex, and endosomal compartments.\n\nSummaryPemberton et al. characterize a molecular toolbox for the visualization and manipulation of phosphatidylinositol (PtdIns) within intact cells. Results using these approaches define the steady-state distribution of PtdIns across subcellular membrane compartments as well as provide new insights into the relationship between PtdIns availability and polyphosphoinositide turnover.

cell biology↗

bioRxiv2018

Bacterial clearance enhanced by α2, 3- and α2, 6-sialyllactose via receptor-mediated endocytosis and phagocytosis

Sialyllactose (SL) is a representative human milk oligosaccharide (HMO) of human breast milk. The roles of SL in infant brain development and immunity have been reported in previous studies. In this study, we identified the impact of SL on innate immunity. Our results showed that the administration of SL had significant efficacy on bacterial clearance in Pseudomonas aeruginosa K-infected mice. We also examined the role of SL in the human THP-1 macrophage-like cell line. SL effectively promoted receptor-mediated endocytosis and phagocytosis. Furthermore, SL accelerated the recruitment of Rac1 to the cell membrane, leading to the generation of reactive oxygen species for the elimination of phagocytosed bacteria. Our findings provide a new perspective on the role of SL in breast milk and suggest its potential application as a therapeutic agent to treat bacterial and viral infections.

immunology↗