Biology subjects

Kim, H. S.

Publications and source records attributed to Kim, H. S..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Ampk activation by glycogen expenditure primes the exit of naive pluripotency

Embryonic and epiblast stem cells in pre-and post-implantation embryos are characterized by their naive and primed states, respectively, which represent distinct phases of pluripotency. Thus, the cellular transition from naive to primed pluripotency recapitulates a drastic metabolic and cellular remodeling after implantation to adapt to changes in extracellular conditions. Here, we found that inhibition of Ampk occurred during naive transition with two conventional inhibitors (2i) of the Mek1 and Gsk3{beta} pathways. The accumulation of glycogen due to the inhibition of Gsk3{beta} was responsible for Ampk inhibition, which accounted for high de novo fatty acid synthesis in naive embryonic stem cells (ESCs). The knockout of glycogen synthase 1 (Gys1) in naive ESCs (GKO), resulting in a drastic glycogen loss, led to a robust Ampk activation and lowered the level of fatty acids. GKO lost the cellular characteristics of naive ESCs and rapidly transitioned to a primed state, as evidenced by a decrease in pluripotency markers in teratoma from GKO. The characteristics of GKO were restored by the simultaneous knockout of Ampk. These findings suggest that glycogen in naive ESCs within the blastocyst may act as a signaling molecule for the timely activation of Ampk, thus ultimately contributing to the transition to the epiblast stage. Graphical Summary O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/541467v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@118ea81org.highwire.dtl.DTLVardef@cbe4bforg.highwire.dtl.DTLVardef@16d5c1org.highwire.dtl.DTLVardef@12b825c_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2022

Haploid-resolved and chromosome-scale genome assembly in hexa-autoploid sweetpotato (Ipomoea batatas (L.) Lam)

Sweetpotato (Ipomoea batatas (L.) Lam) is the worlds seventh most important food crop by production quantity. Cultivated sweetpotato is a hexaploid (2n = 6x = 90), and its genome (B1B1B2B2B2B2) is quite complex due to polyploidy, self-incompatibility, and high heterozygosity. Here we established a haploid-resolved and chromosome-scale de novo assembly of autohexaploid sweetpotato genome sequences. Before constructing the genome, we created chromosome-scale genome sequences in I. trifida using a highly homozygous accession, Mx23Hm, with PacBio RSII and Hi-C reads. Haploid-resolved genome assembly was performed for a sweetpotato cultivar, Xushu18 by hybrid assembly with Illumina paired-end (PE) and mate-pair (MP) reads, 10X genomics reads, and PacBio RSII reads. Then, 90 chromosome-scale pseudomolecules were generated by aligning the scaffolds onto a sweetpotato linkage map. De novo assemblies were also performed for chloroplast and mitochondrial genomes in I. trifida and sweetpotato. In total, 34,386 and 175,633 genes were identified on the assembled nucleic genomes of I. trifida and sweetpotato, respectively. Functional gene annotation and RNA-Seq analysis revealed locations of starch, anthocyanin, and carotenoid pathway genes on the sweetpotato genome. This is the first report of chromosome-scale de novo assembly of the sweetpotato genome. The results are expected to contribute to genomic and genetic analyses of sweetpotato.

genomics↗

bioRxiv2022

Adaptive nanopore sequencing for single cell characterization of cancer mutations and gene rearrangements

In this proof-of-concept study, we developed a single cell method that identifies somatic alterations found in coding regions of mRNAs and integrates these mutation genotypes with their matching cell transcriptomes. We used nanopore adaptive sampling on single cell cDNA libraries, generated long read sequences from target gene transcripts and identified coding variants among individual cells. Short-read single cell transcriptomes characterized the cell types with mutations. We delineated CRISPR edits from a cancer cell line. From primary cancer samples, we targeted hundreds of cancer genes, identified somatic coding mutations and a gene rearrangement among individual tumor cells.

genomics↗

bioRxiv2022

Single cell CRISPR base editor engineering and transcriptional characterization of cancer mutations

We developed a multiplexed single cell technology to genome engineer mutations, directly delineate their genotype among individual cells and determine each mutations transcriptional phenotype. This approach uses CRISPR base editors to introduce predesignated variants into a target gene. Long-read sequencing of the target genes transcript identifies the engineered mutations among individual cells. Simultaneously, we analyzed the transcriptome profile from the same set of cells by short-read sequencing. By integrating the two types of data, we determined the mutations genotype and expression phenotype at single cell resolution. Using cell lines, we engineered and evaluated the phenotype of more than 100 TP53 mutations. Based on the single cell gene expression, we classified the mutations as having a functionally significant phenotype versus the wild-type state. We validated these results on a subset of mutations using isolated clones analyzed with RNA-seq. Overall, we successfully demonstrated single cell mutation engineering and phenotypic assessment.

genomics↗

bioRxiv2022

Fever temperatures modulate intraprotein dynamics and enhance the binding affinity between monoclonal antibodies and the Spike protein from SARS-CoV-2

Fever is a typical symptom of most infectious diseases. While prolonged fever may be clinically undesirable, mild reversible fever (< 39{degrees}C, 312K) can potentiate the immune responses against pathogens. Here, using molecular dynamics, we investigated the effect of febrile temperatures (38{degrees}C to 40{degrees}C, 311K to 313K) on the immune complexes formed by the SARS-CoV-2 spike protein with two neutralizing antibodies. We found that, at mild fever temperatures (311-312K), the binding affinities of the two antibodies improve when compared to the physiological body temperature (37{degrees}C, 310K). Furthermore, only at 312K, antibodies exert distinct mechanical effects on the receptor binding domains of the spike protein that may hinder SARS-CoV-2 infectivity. Enhanced antibody binding affinity may thus be obtained using appropriate temperature conditions.

bioinformatics↗

bioRxiv2022

Altered CD8+ T cell associated aging gene signature in the peripheral blood of patients with Alzheimer's disease.

INTRODUCTIONEffector memory (EM) CD8+ T cells have been associated with poor cognition in Alzheimers disease (AD). Our lab recently discovered an age-associated gene expression signature of IL-7 receptor alpha (IL-7R)low EM CD8+ T cells. We hypothesized that individuals with AD have altered levels of this IL-7Rlow aging gene expression. METHODSForty genes associated with IL-7Rlow EM CD8+ T cells, AD, or memory, were analyzed in peripheral blood of participants with normal cognition, mild cognitive impairment, and dementia by qPCR. RESULTSOf the eight genes that were found to be differentially expressed based on clinical diagnosis, 5 genes (62.5%) were IL-7Rlow aging genes. Principal component analysis revealed 3 clusters of participants with dementia which had distinct expression levels of IL-7Rlow aging genes and cognitive function. DISCUSSIONOur findings support the possible relationship of the IL-7Rlow EM CD8+ T cell aging signature with cognition in individuals with dementia due to AD.

immunology↗

bioRxiv2022

Two Interaction Surfaces between XPA and RPA Organize the Preincision Complex in Nucleotide Excision Repair

The XPA and RPA proteins fulfill essential roles in the assembly of the preincision complex in the nucleotide excision repair pathway. We have previously characterized the two interaction surfaces between XPA and RPA, with the RPA32 and RPA70AB subunits. Here we show that the mutations in the two individual interaction surfaces reduce NER activity in biochemical and cellular systems, and that combining mutations in two domains leads to an additive inhibition of NER, suggesting that they fulfill distinct roles. Our data suggest that the interaction between XPA and RPA32 is important for the initial association of XPA with NER complexes, while the interaction between XPA and RPA70 is needed for structural organization of the complex to license the dual incision reaction. SAXS analysis of complexes of XPA and RPA bound to ss/dsDNA junction substrates reveals the architecture of XPA and RPA in the preincision complex and shows that the two interaction domains between RPA and XPA are located at opposite sides of the two molecules. We propose a structure for the overall NER preincision complex that shows that the two strands of the NER bubble assume a U-shape with the two ss/dsDNA junctions localized in close proximity, with the interaction between XPA and RPA70 as one of the key organizing elements.

biochemistry↗

bioRxiv2022

Emergence of glycogen synthase kinase-3 interaction domain enhances phosphorylation of SARS-CoV-2 nucleocapsid protein

A structural protein of SARS-CoV-2, nucleocapsid (N) protein is abundantly expressed during viral replication. The N protein is phosphorylated by glycogen synthase kinase (GSK)-3 on the serine/arginine (SR) rich motif located in disordered regions. Although phosphorylation by GSK-3{beta} constitutes a critical event for viral replication, the molecular mechanism underlying N phosphorylation is not well understood. In this study, we found the putative alpha-helix L/FxxxL/AxxRL motif known as the GSK-3 interacting domain (GID), commonly found in many endogenous GSK-3{beta} binding proteins, such as Axins, FRATs, WWOX and GSKIP. Indeed, N interacts with GSK-3{beta} similarly to Axin, and Leu to Glu substitution of the GID abolished the interaction, with loss of N phosphorylation. Unlike with endogenous GID proteins, the N interaction neither disturbs endogenous GSK-3 activity nor regulates subsequent canonical Wnt activity and the Snail-EMT program. Notably, N abundance in SARS-CoV-2 is incomparably high compared to other coronaviruses, such as 229E, OC43 and HKU1. Compared to other coronaviruses, N harbors a CDK1 primed phosphorylation site and Gly-rich linker for enhanced phosphorylation by GSK-3{beta}. Furthermore, we found that the S202R mutant found in Delta and R203K/G204R mutant found in the Omicron variant allows increased abundance and hyper-phosphorylation of N. Our observations suggest that the emergence of GID and mutations for increased phosphorylation in N may have contributed to the emergence of SARS-CoV-2 and evolution of variants, respectively. Further study, especially in a BSL3-equipped facility, is required to elucidate the functional importance of GID and N phosphorylation in SARS-CoV-2 and variants.

cell biology↗

bioRxiv2021

Oncomodulin derived from regeneration-associated macrophages in dorsal root ganglia promotes axon regeneration in the spinal cord

Preconditioning nerve injury drives pro-regenerative perineuronal macrophage activation in dorsal root ganglia (DRG). The present study reports that oncomodulin (ONCM) is produced from the regeneration-associated macrophages (RAMs) and strongly influences regeneration of DRG sensory axons. Preconditioning injury upregulated ONCM in DRG macrophages in a CCR2 dependent manner. ONCM in macrophages was necessary to produce RAMs in the in vitro model of neuron-macrophage interaction and played an essential role in for preconditioning or CCL2-induced neurite outgrowth. ONCM potently increased neurite outgrowth in cultured DRG neurons by activating a distinct gene set, particularly neuropeptide-related genes. Increasing extracellularly secreted ONCM in DRGs sufficiently enhanced capacity of neurite outgrowth. To achieve sustained ONCM activity in vivo, recombinant ONCM was encapsulated by a reducible epsilon-poly(L-lysine)-nanogel (REPL-NG) system based on electrostatic interaction. Localized injection of REPL-NG/ONCM complex into DRGs achieved a remarkable long-range axonal regeneration beyond spinal cord lesion, surpassing the extent of the preconditioning effects.

neuroscience↗

bioRxiv2021

In vitro validation of neoantigen prediction algorithm for developing personalized cancer vaccine therapy

BackgroundThe development of personalized neoantigen-based therapeutic cancer vaccines relies on computational algorithm-based pipelines. One of the critical issues in the pipeline is obtaining higher positive predictive value (PPV) performance, i.e., how many are immunogenic when selecting the top 5 to 20 candidate neoepitopes for the vaccination. We attempted to test the PPV of a neoepitope prediction algorithm Neopepsee. MethodsSix breast cancer patients and patient-derived xenografts from three lung cancer patients and their paired peripheral blood samples were subjected to whole-exome and RNA sequencing. Neoantigen was predicted using two different algorithms (Neopepsee and pVACseq). Response of induced memory T cells to neopeptide candidates was evaluated by IFN-{gamma} Enzyme-linked immune absorbent spot (ELISpot) assays of peripheral blood mononuclear cell (PBMC) from three HLA-matched donors. Positive ELISpot response to a candidate peptide in at least 2 of 3 donor PBMC was regarded as an immunogenic response. ResultsNeopepsee predicted 159 HLA-A matched neoepitope candidates out of 898 somatic mutations in nine patients (six breast and three lung cancer patients), whereas pVACseq predicted 84 HLA-A matched candidates. A total of 26 neopeptide candidates overlapped between the two predicted candidate pools. Among the candidates, 28 (20%, 28/ 137) and 15 (20%, 15/ 75) were positive by ELISpot assay, respectively. Among 26 overlapped candidates, 20 could be tested, and 7 of them (35%) were validated by ELISpot. Neopepsee identified at least one neoepitope in 7 of 9 patients (range 0-6), compared to 6 by pVACseq (range 0-5). ConclusionAs suggested by Tumor Neoantigen Selection Alliance (TESLA), our results demonstrate low PPV of individual prediction models as well as the complementary nature of the Neopepsee and pVACseq and may help design neoepitope targeted cancer vaccines. Our data contribute a significant addition to the database of tested neoepitope candidates that can be utilized to further train and improve the prediction algorithms.

immunology↗

bioRxiv2021

Comparison of Plaque Size, Thermal Stability, and Replication Rate among SARS-CoV-2 Variants of Concern

SARS-CoV-2, like other RNA viruses, has a propensity for genetic evolution owing to the low fidelity of its viral polymerase. This evolution results in the emergence of novel variants with different characteristics than their ancestral strain. Several recent reports have described a series of novel SARS-CoV-2 variants. Some of these have been identified as variants of concern (VOCs), including alpha (B.1.1.7, Clade GRY), beta (B.1.351, Clade GH), gamma (P.1, Clade GR), and delta (B.1.617.2, Clade G). VOCs are likely to have some effect on transmissibility, antibody evasion, and changes in therapeutic or vaccine effectiveness. However, the physiological and virological understanding of these variants remains poor. We demonstrated that these four VOCs exhibited differences in plaque size, thermal stability at physiological temperature, and replication rates. The mean plaque size of beta was the largest, followed by those of gamma, delta, and alpha. Thermal stability, evaluated by measuring infectivity and half-life after prolonged incubation at physiological temperature, was correlated with plaque size in all variants except alpha. However, despite its relatively high thermal stability, alphas small plaque size resulted in lower replication rates and fewer progeny viruses. Our findings may inform further virological studies of SARS-CoV-2 variant characteristics, VOCs, and variants of interest. These studies are important for the effective management of the COVID-19 pandemic. IMPORTANCEThe global pandemic caused by SARS-CoV-2 continues to persist, due in part to mutations that have resulted in the emergence of different variants. Many of these variants have become more virulent and infectious than their ancestral strain, resulting in an ever-increasing spread. However, our virological understanding of these variants remains poor. Here, we directly compared the plaque size, stability, and replication kinetics of four SARS-CoV-2 variants of concern following prolonged incubation at physiological temperatures. Our observations may help to characterize each variant in terms of their interactions with host factors and responses to environmental conditions. We also believe that our evaluations will improve understanding of the emergence of new variants and contribute to controlling their spread.

microbiology↗

bioRxiv2021

Establishment of reference standards for multifaceted mosaic variant analysis

Detection of somatic mosaicism in non-proliferative cells is a new challenge in genome research, however, the accuracy of current detection strategies remains uncertain due to the lack of a ground truth. Herein, we sought to present a set of reference standards based on a total of 386,613 mosaic single-nucleotide variants (SNVs) and insertion-deletion mutations (INDELs) with variant allele frequencies (VAFs) ranging from 0.5% to 56%, as well as 35,113,417 non-variant and 19,936 germline variant sites as a negative control. The whole reference standard set mimics the cumulative aspect of mosaic variant acquisition in the early developmental stage owing to the progressive mixing of cell lines with established genotypes, ultimately unveiling 741 possible inter-sample relationships with respect to variant sharing and asymmetry in VAFs. We expect that our reference standards will be essential for optimizing the current use of mosaic variant detection strategies and for developing algorithms to enable future improvements.

genomics↗

bioRxiv2021

BRCA mutational status shapes the stromal microenvironment of pancreatic cancer linking CLU+ CAF expression with HSF1 signaling

Cancer-associated fibroblasts (CAFs) give rise to desmoplastic stroma, which supports tumor progression and metastasis, and comprises up to 90% of the tumor mass in pancreatic cancer. Recent work by us and others has shown that CAFs are transcriptionally rewired by adjacent cancer cells to form heterogeneous subtypes. Whether this rewiring is differentially affected by different driver mutations in cancer cells is largely unknown. Here we address this question by dissecting and comparing the stromal landscape of BRCA-mutated and BRCA Wild-type (WT) pancreatic ductal adenocarcinoma (PDAC). We comprehensively analyze PDAC samples from a cohort of 42 patients by laser-capture microdissection, RNA-sequencing and multiplexed immunofluorescence, revealing different CAF subtype compositions in germline BRCA-mutated vs. BRCA-WT tumors. In particular, we detect an increase in a subset of Clusterin (CLU)-positive CAFs in BRCA-mutated tumors. We further unravel a network of stress responses upregulated in BRCA-mutated tumors. Using cancer organoids and cell co-cultures, we show that the transcriptional shift of pancreatic stellate cells into CLU+ CAFs is mediated through activation of heat-shock factor 1 (HSF1), the transcriptional regulator of Clu. Our findings unravel a new dimension of stromal heterogeneity, influenced by germline mutations in cancer cells, with direct translational implications for clinical research. SignificanceBRCA1/2 mutations initiate some of the deadliest cancers, yet the fibroblastic microenvironment of BRCA-mutated cancers remains uncharted. Our work addresses a major unsolved question - to what extent is the tumor microenvironment determined by cancer mutations? We find that BRCA mutations in the cancer cells affect the composition of CAFs in PDAC. These findings have direct implications for diagnosis and for efforts to exploit CAFs for therapy.

cancer biology↗

bioRxiv2021

Single cell characterization of CRISPR-modified transcript isoforms with nanopore sequencing

Transcript isoforms are mRNAs that arise from alternative splicing events. During RNA processing, different combinations of a gene's exons lead to a diverse set of isoforms. Polymorphisms or mutations at splice junctions can generate alternative splicing events. Various splicing factors also impact the representation of a gene's transcript isoforms. To assess how these two features contribute to alternative splicing, we developed a single cell approach to introduce CRISPR edits that modify mRNA transcript structure. Our method combines (1) long-read sequencing to characterize the expressed transcripts and identify the edit at single cell resolution; (2) short-read sequencing to match the single cell gene expression profiles of the cells with the altered isoform. First, we modify target exon-intron segments with CRISPR-Cas9. Second, using cDNAs with cell barcodes, we use long read sequencing to directly identify the changes in transcript isoforms from the targeted CRISPR edits. As a variation on this approach, we also determined how modifying specific splicing factors influence isoform expression and structure. Overall, we demonstrate how the integration of single cell long read analysis and CRISPR engineering can be used to directly confirm transcript isoform and target genomic edits at single cell resolution. This approach will improve our understanding of the role of alternative splicing in transcriptional regulation.

genomics↗

bioRxiv2021

Chronic opioid treatment arrests neurodevelopment and alters synaptic activity in human midbrain organoids

The impact of long-term opioid exposure on the embryonic brain is crucial to healthcare due to the surging number of pregnant mothers with an opioid dependency. Current studies on the neuronal effects are limited due to human brain inaccessibility and cross-species differences among animal models. Here, we report a model to assess cell-type specific responses to acute and chronic fentanyl treatment, as well as fentanyl withdrawal, using human induced pluripotent stem cell (hiPSC)-derived midbrain organoids. Single cell mRNA sequencing (25,510 single cells in total) results suggest that chronic fentanyl treatment arrests neuronal subtype specification during early midbrain development and alters the pathways associated with synaptic activities and neuron projection. Acute fentanyl treatment, however, increases dopamine release but does not induce significant changes in gene expressions of cell lineage development. To date, our study is the first unbiased examination of midbrain transcriptomics with synthetic opioid treatment at the single cell level.

neuroscience↗

bioRxiv2021

An ammonium transporter is a non-canonical olfactory receptor for ammonia

Two families of ligand-gated ion channels function as olfactory receptors in insects. Here, we show that these canonical olfactory receptors are not necessary for responses to ammonia, a key ecological odor that is attractive to many insects including disease vectors and agricultural pests. Instead, we show that a member of the ancient electrogenic ammonium transporter family, Amt, is a new type of olfactory receptor. We report two hitherto unidentified olfactory neuron populations that mediate neuronal and behavioral responses to ammonia. Their endogenous ammonia responses are Amt-dependent, and ectopic expression of either Drosophila or Anopheles Amt confers ammonia sensitivity. Amt is the first transporter known to function as an olfactory receptor in animals, and its role may be conserved across insect species.

neuroscience↗

bioRxiv2020

Comparison of everting sutures and the lateral tarsal strip with or without everting sutures for involutional lower eyelid entropion: A meta-analysis

There are three pathophysiologies of involutional entropion, vertical laxity (VL), horizontal laxity (HL), and overriding of the preseptal orbicularis. The effects of methods to correct VL only, HL only, or both VL and HL in patients with involutional entropion were compared using the published results of randomized controlled trials (RCTs). To find RCT studies that investigated methods to correct involutional entropion, a systematic search was performed from database inception to April 2020 in the Medline, EMBASE, and Cochrane databases. Two independent researchers conducted the literature selection and data extraction. Evaluation of the quality of the reports was performed using the Cochrane Collaboration tool for assessing the risk of bias (ROB 2.0). The data analysis was conducted according to the PRISMA guidelines using Review Manager 5.3. Two RCT studies were included in this meta-analysis. Surgery for involutional entropion was performed on a total of 109 eyes. Everting sutures (ES) were used on 57 eyes and lateral tarsal strips (LTS) or combined procedures (LTS + ES) were performed on 52 eyes. At the end of the follow-up periods, involutional entropion recurred in 18 eyes (31.6%) in the ES group and three eyes (5.8%) in the LTS +/- ES group. Analysis of the risk ratio showed that the LTS +/- ES method significantly lowered the recurrence rate compared to using ES only (P = 0.007). Performing LTS +/- ES effectively lowered the recurrence rate of involutional entropion compared to ES alone. However, some patients cannot tolerate more invasive corrections such as LTS. Therefore, sequential procedures, in which ES is performed first and then when entropion recurs LTS +/- ES is performed, or another methods depending upon the degree of HL may be used.

biophysics↗

bioRxiv2020

Accurate Microbiome Sequencing with Synthetic Long Read Sequencing

The microbiome plays a central role in biochemical cycling and nutrient turnover of most ecosystems. Because it can comprise myriad microbial prokaryotes, eukaryotes and viruses, microbiome characterization requires high-throughput sequencing to attain an accurate identification and quantification of such co-existing microbial populations. Short-read next-generation-sequencing (srNGS) revolutionized the study of microbiomes and remains the most widely used approach, yet read lengths spanning only a few of the nine hypervariable regions of the 16S rRNA gene limit phylogenetic resolution leading to misclassification or failure to classify in a high percentage of cases. Here we evaluate a synthetic long-read (SLR) NGS approach for full-length 16S rRNA gene sequencing that is high-throughput, highly accurate and low-cost. The sequencing approach is amenable to highly multiplexed sequencing and provides microbiome sequence data that surpasses existing short and long-read modalities in terms of accuracy and phylogenetic resolution. We validated this commercially-available technology, termed LoopSeq, by characterizing the microbial composition of well-established mock microbiome communities and diverse real-world samples. SLR sequencing revealed differences in aquatic community complexity associated with environmental gradients, resolved species-level community composition of uterine lavage from subjects with histories of misconception and accurately detected strain differences, multiple copies of the 16S rRNA in a single strains genome, as well as low-level contamination in soil cyanobacterial cultures. This approach has implications for widespread adoption of high-resolution, accurate long-read microbiome sequencing as it is generated on popular short read sequencing platforms without the need for additional infrastructure.

microbiology↗