Biology subjects

Lee, J. S.

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

At least 37 records · Page 2Linked to original sources

bioRxiv2024

A Third COVID-19 Vaccine Dose in Kidney Transplant Recipients Induces Antibody Response to Vaccine and Omicron Variants but Shows Limited Ig Subclass Switching

Solid organ transplant recipients (SOTRs) suffer more frequent and more severe infections due to their compromised immune responses resulting from immunosuppressive treatments designed to prevent organ rejection. Pharmacological immunosuppression can adversely affect immune responses to vaccination. A cohort of kidney transplant recipients (KTRs) received their third dose of ancestral, monovalent COVID-19 vaccine in the context of a clinical trial and antibody responses to the vaccine strain, as well as to Omicron variants BA.1 and BA.5 were investigated and compared with healthy controls. Total IgG and live virus neutralizing antibody titers were reduced in KTRs compared to controls for all variants. KTRs displayed altered IgG subclass switching, with significantly lower IgG3 antibodies. Responses in KTRs were also very heterogeneous, with some individuals showing strong responses but a significant number showing no Omicron-specific neutralizing antibodies. Taken together, immune responses after COVID-19 vaccination in KTRs were not only lower than healthy controls but highly variable, indicating that simply increasing the number of vaccine doses alone may not be sufficient to provide greater protection in this population. ImportanceThis study addresses the challenges faced by kidney transplant recipients (KTRs) in mounting effective immune responses against COVID-19. By evaluating the antibody responses to a third dose of monovalent mRNA COVID-19 vaccine and its effectiveness against Omicron subvariants (BA.1 and BA.5), this study reveals significant reductions in both binding and neutralizing antibodies in KTRs compared to healthy controls. The research highlights altered IgG subclass switching and heterogeneous responses within the KTR population. Reduced recognition of variants, coupled with differences in IgG subclasses, decreases both the quality and quantity of protective antibodies after vaccination in KTRs. These findings underscore the need for tailored vaccination strategies for immunosuppressed populations such as KTRs. Alternative formulations and doses of COVID-19 vaccines should be considered for people with severely compromised immune systems, as more frequent vaccinations may not significantly improve the response, especially regarding neutralizing antibodies.

immunology↗

bioRxiv2024

Characterization and analysis of neuronal signaling using microelectrode array combined with rapid and localized cooling device for cryo-neuromodulation

Cryoanesthesia--a purely physical anesthesia treatment that freezes tissue and attenuate nerve activity--can provide fast treatment through freezing and thawing of cryo-machine and is inexpensive compared to other anesthetics. However, cryoanesthesia has not been widely adopted because securing safe and effective conditions requires quantitative measurement and analysis of neuronal signaling during freezing and recovery, for which research tools are limited. A lack of rapid and localized cooling technologies for quantitative cellular level analysis, in particular, hinders research on not only the optimal cryo-modulation of neuronal activities but also its influence to neighboring cells via cellular networks. Here, we introduce a novel cryo-neuromodulation platform, a high-speed precision probe-type cooling device ([~]20{degrees}C/s at cooling) that provides localized cooling combined with a microelectrode array (MEA) system. We explored the temperature conditions for efficient silencing and recovery of neuronal activities without cell damage. We found that electrical activities of neurons were fully recovered within 1 minute of cooling duration with the maximum cooling speed, which was also confirmed with calcium imaging. The impact of silenced neurons on the neighboring neural network was explored using the localized cooling and we perceived that its influence can be transmitted if the neuronal network is well organized. Our new cryo-device provides rapid and reversible control of neural activities, which allows not just quantitative analysis of the network dynamics, but also new applications in clinical settings.

neuroscience↗

bioRxiv2024

Identification of targetable vulnerabilities of PLK1-overexpressing cancers by synthetic dosage lethality

Tumor heterogeneity poses a significant challenge in combating treatment resistance. Despite Polo-like kinase 1 (PLK1) being universally overexpressed in cancers and contributing to chromosomal instability (CIN), direct PLK1 inhibition hasnt yielded clinical progress. To address this, we utilized the synthetic dosage lethality (SDL) approach, targeting PLK1s genetic interactions for selective killing of overexpressed tumor cells while mitigating heterogeneity-associated challenges. Employing computational methods, we conducted a genome-wide shRNA screen, identifying 105 SDL candidates. Further in vivo CRISPR screening in a breast cancer xenograft model and in vitro CRISPR analysis validated these candidates. Employing Perturb-seq revealed IGF2BP2/IMP2 as a key SDL hit eliminating PLK1-overexpressing cells. Suppression of IGF2BP2, genetically or pharmacologically, downregulated PLK1 and limited tumor growth. Our findings strongly propose targeting PLK1s genetic interactions as a promising therapeutic approach, holding broad implications across multiple cancers where PLK1 is overexpressed.

cancer biology↗

bioRxiv2024

Inhibition of nitric oxide synthase transforms carotid occlusion-mediated benign oligemia into de novo large cerebral infarction

It remains unclear why unilateral proximal carotid artery occlusion (UCAO) causes benign oligemia, without progressing to cerebral infarction, in mice, yet leads to a wide variety of outcomes (ranging from asymptomatic to death) in humans. We hypothesized that inhibition of NOS both transforms UCAO-mediated oligemia into full infarction and expands pre-existing infarction. In support, intraperitoneal administration of N{omega}-nitro-L-arginine methyl ester (L-NAME) followed by UCAO induced large-arterial infarction in mice, unlike UCAO alone. Six-hour laser-speckle-contrast imaging detected spreading ischemia in mice with infarction as assessed at 24h. In agreement with vasoconstriction/microthrombus formation shown by intravital microscopy, the NO-donor, molsidomine and the endothelial-NOS- activating antiplatelet, cilostazol, attenuated or prevented progression to infarction. Moreover, UCAO without L-NAME caused infarction in mice with hyperglycemia and hyperlipidemia, which, in turn, were associated with greater symmetric dimethylarginine (SDMA) levels. Further, increased levels of glucose and cholesterol associated with significantly larger infarct volumes in 438 consecutive patients with UCAO-mediated infarction. Lastly, Mendelian randomization identified a causative role of NOS inhibition, particularly in elevated SDMA concentration, in ischemic stroke risk. Therefore, NOS activity is a key factor determining the fate of hypoperfused brain following acute carotid occlusion, where SDMA could be a potential risk predictor.

neuroscience↗

bioRxiv2024

FlowPacker: Protein side-chain packing with torsional flow matching

Accurate prediction of protein side-chain conformations is necessary to understand protein folding, proteinprotein interactions and facilitate de novo protein design. Here we apply torsional flow matching and equivariant graph attention to develop FlowPacker, a fast and performant model to predict protein sidechain conformations conditioned on the protein sequence and backbone. We show that FlowPacker outperforms previous state-of-the-art baselines across most metrics with improved runtime. We further show that FlowPacker can be used to inpaint missing side-chain coordinates and also for multimeric targets, and exhibits strong performance on a test set of antibody-antigen complexes. Code is available at https://gitlab.com/mjslee0921/flowpacker.

bioinformatics↗

bioRxiv2024

Detection of changes in membrane potential by magnetic resonance imaging

Membrane potential plays a crucial role in various cellular functions. However, existing techniques for measuring membrane potential are often invasive or have limited recording depth. In contrast, magnetic resonance imaging (MRI) offers noninvasive imaging with desirable spatial resolution over large areas. This study investigates the feasibility of utilizing MRI to detect responses of cultured cells and in vivo rat cortex to membrane potential-modulating ionic solutions by measuring magnetic resonance parameters. Our findings reveal that depolarizing (or hyperpolarizing) ionic solutions increase (or decrease) the T2 relaxation time, while the ratio of bound to free water proton shows the opposite trend. These findings also suggest a potential approach to noninvasively detect changes in membrane potential using MRI.

biophysics↗

bioRxiv2024

Ribonanza: deep learning of RNA structure through dual crowdsourcing

Prediction of RNA structure from sequence remains an unsolved problem, and progress has been slowed by a paucity of experimental data. Here, we present Ribonanza, a dataset of chemical mapping measurements on two million diverse RNA sequences collected through Eterna and other crowdsourced initiatives. Ribonanza measurements enabled solicitation, training, and prospective evaluation of diverse deep neural networks through a Kaggle challenge, followed by distillation into a single, self-contained model called RibonanzaNet. When fine tuned on auxiliary datasets, RibonanzaNet achieves state-of-the-art performance in modeling experimental sequence dropout, RNA hydrolytic degradation, and RNA secondary structure, with implications for modeling RNA tertiary structure.

biophysics↗

bioRxiv2023

Whole-genome sequences reveal zygotic composition in chimeric twins

The monochorionic placenta in dizygotic twins allows in utero exchange of embryonic cells, resulting in chimerism in the twins. In practice, this chimerism is incidentally identified on mixed ABO blood types or in the presence of cells with a discordant sex chromosome. Here, we applied whole-genome sequencing to one triplet and one twin families to precisely understand their zygotic compositions, using millions of genomic variants as barcodes of zygotic origins. Peripheral blood showed asymmetrical contributions from two sister zygotes, where one of the zygotes was the major clone in both twins. Single-cell RNA sequencing of peripheral blood tissues further showed differential contributions from the two sister zygotes across blood cell types. In contrast, buccal tissues were pure in genetic composition, suggesting that in utero cellular exchanges were confined to the blood tissues. Our study illustrates the cellular history of twinning during human development, which is critical for managing the health of chimeric individuals in the era of genomic medicine.

genomics↗

bioRxiv2023

Evolutionary arms race between SARS-CoV-2 and interferon signaling via dynamic interaction with autophagy.

SARS-CoV-2 emerged, and is evolving to efficiently infect humans worldwide. SARS-CoV-2 evades early innate recognition, interferon signaling activated only in bystander cells. This balance of innate activation and viral evasion has important consequences, but the pathways involved are incompletely understood. Here we find that autophagy genes regulate innate immune signaling, impacting the basal set point of interferons, and thus permissivity to infection. Mechanistically, autophagy genes negatively regulate MAVS, and this low basal level of MAVS is efficiently antagonized by SARS-CoV-2 ORF9b, blocking interferon activation in infected cells. However, upon loss of autophagy increased MAVS overcomes ORF9b-mediated antagonism suppressing infection. This has led to the evolution of SARS-CoV-2 variants to express higher levels of ORF9b, allowing SARS-CoV-2 to replicate under conditions of increased MAVS signaling. Altogether, we find a critical role of autophagy in the regulation of innate immunity and uncover an evolutionary trajectory of SARS-CoV-2 ORF9b to overcome host defenses.

microbiology↗

bioRxiv2023

Genome-wide CRISPR screen identifies KEAP1 as a genetic dependency of ARID1A in an ovarian clear cell carcinoma model

ARID1A is the core DNA binding subunit of the BAF chromatin remodeling complex and is mutated in about [~]8% of all cancers. The frequency of ARID1A loss varies between cancer subtypes, with clear cell ovarian carcinoma (CCOC) presenting the highest incidence at >50% of cases. Despite a growing understanding of the consequences of ARID1A-loss in cancer, there remains limited targeted therapeutic options for ARID1A-deficient cancers. Using a genome-wide CRISPR screening approach, we identify KEAP1 as a genetic dependency of ARID1A in CCOC. Depletion or chemical perturbation of KEAP1 results in selective growth inhibition of ARID1A-KO cell lines and edited primary endometrial epithelial cells. While we confirm that KEAP1-NRF2 signalling is dysregulated in ARID1A-KO cells, we suggest that this synthetic lethality is not due to aberrant NRF2 signalling. Rather, we find that KEAP1 perturbation exacerbates genome instability phenotypes associated with ARID1A-deficiency. Together, our findings identify a potentially novel synthetic lethal interaction of wARID1A-deficient cells.

cancer biology↗

bioRxiv2023

2D and 3D multiplexed subcellular profiling of nuclear instability in human cancer

Nuclear atypia, including altered nuclear size, contour, and chromatin organization, is ubiquitous in cancer cells. Atypical primary nuclei and micronuclei can rupture during interphase; however, the frequency, causes, and consequences of nuclear rupture are unknown in most cancers. We demonstrate that nuclear envelope rupture is surprisingly common in many human cancers, particularly glioblastoma. Using highly-multiplexed 2D and super-resolution 3D-imaging of glioblastoma tissues and patient-derived xenografts and cells, we link primary nuclear rupture with reduced lamin A/C and micronuclear rupture with reduced lamin B1. Moreover, ruptured glioblastoma cells activate cGAS-STING-signaling involved in innate immunity. We observe that local patterning of cell states influences tumor spatial organization and is linked to both lamin expression and rupture frequency, with neural-progenitor-cell-like states exhibiting the lowest lamin A/C levels and greatest susceptibility to primary nuclear rupture. Our study reveals that nuclear instability is a core feature of cancer, and links nuclear integrity, cell state, and immune signaling.

cancer biology↗

bioRxiv2023

ACE Configurator for ELISpot (ACE): Optimizing Combinatorial Design of Pooled ELISpot Assays with an Epitope Similarity Model

The ELISpot assay is a powerful in vitro immunoassay that enables cost-effective quantification of antigen-specific T-cell reactivity. It is widely used in the context of cancer and infectious diseases to validate the immunogenicity of epitopes. While technological advances in hardware and software have kept pace with the need for increased throughput, assay design and deconvolution methodology have largely remained stagnant. Current methods for designing multiplexed ELISpot assays are restricted to preset configurations, lack support for high-throughput scenarios, and ignore peptide identity during pool assignment. We introduce the ACE Configurator for ELISpot (ACE) to address these gaps. ACE generates optimized peptide-pool assignments from highly customizable user inputs and handles positive peptide deconvolution using assay readouts. We present a novel sequence-aware pooling strategy, powered by a fine-tuned ESM-2 deep sequence model to identify immunologically similar peptides, reducing the number of false positives and subsequent confirmatory assays. To validate the performance of ACE using real-world datasets, we conducted a comprehensive benchmark study against various design heuristics, deconvolution methods, and experimental conditions, contextualizing design parameter choices with their impact on precision and number of total pools. Our results demonstrate ACEs capacity to further increase precision of identified immunogenic peptides, maximizing experimental efficiency at the bench-side. ACE is freely available as an executable with a graphical user interface and command-line interfaces at https://github.com/pirl-unc/ace.

bioinformatics↗

bioRxiv2023

A tryptophan metabolite modulates the host response to bacterial infection via kainate receptors

Bacterial infection involves a complex interaction between the pathogen and host where the outcome of infection is not solely determined by pathogen eradication. To identify small molecules that promote host survival by altering the host-pathogen dynamic, we conducted an in vivo chemical screen using zebrafish embryos and found that treatment with 3-hydroxy-kynurenine protects from lethal gram-negative bacterial infection. 3-hydroxy-kynurenine, a metabolite produced through host tryptophan metabolism, has no direct antibacterial activity but enhances host survival by restricting bacterial expansion in macrophages by targeting kainate-sensitive glutamate receptors. These findings reveal new mechanisms by which tryptophan metabolism and kainate-sensitive glutamate receptors function and interact to modulate immunity, with significant implications for the coordination between the immune and nervous systems in pathological conditions.

immunology↗

bioRxiv2023

Generalizing deep variant callers via domain adaptation and semi-supervised learning

Deep learning-based variant callers (DVCs) offer state-of-the-art perfor-mance in small variant detection from DNA sequencing data. However, their reliance on supervised learning and the subsequent need for exten-sive labeled data pose a potential hurdle to their generalizability across diverse sequencing methods with varying error profiles. Indeed, even minor discrepancies in error profiles can compromise the robustness of DVCs and impair the variant calling accuracy in the target sequencing method. To mitigate these challenges, we propose RUN-DVC, the first semi-supervised training approach for DVCs that presents two complemen-tary training techniques to the conventional supervised training approach. RUN-DVC leverages semi-supervised learning techniques to learn error profiles from unlabeled datasets of the target sequencing method as well as a domain adaptation technique to aid semi-supervised learning by reducing the domain discrepancy due to different error profiles. We ana-lyze and contrast RUN-DVC against the supervised training approach under various generalization scenarios using nine sequencing methods from Illumina, BGI, PacBio, and Oxford Nanopore sequencing platforms. Remarkably, RUN-DVC significantly improves the variant calling accu-racy of DVC in the target sequencing method even with purely unlabeled datasets in the target domain and enables label-efficient generalization when partially labeled datasets are available. Our results suggest RUN-DVC is a promising semi-supervised training method for DVCs with the potential to broaden the use of DVC across diverse sequencing methods.

bioinformatics↗

bioRxiv2023

Targeted Amplification and Genetic Sequencing of the Severe Acute Respiratory Syndrome Coronavirus 2 Surface Glycoprotein

The SARS-CoV-2 spike protein is a highly immunogenic and mutable protein that is the target of vaccine prevention and antibody therapeutics. This makes the encoding S-gene an important sequencing target. The SARS-CoV-2 sequencing community overwhelmingly adopted tiling amplicon-based strategies for sequencing the entire genome. As the virus evolved, primer mismatches inevitably led to amplicon drop-out. Given the exposure of the spike protein to host antibodies, mutation occurred here most rapidly, leading to amplicon failure over the most insightful region of the genome. To mitigate this, we developed SpikeSeq, a targeted method to amplify and sequence the S-gene. We evaluated 20 distinct primer designs through iterative in silico and in vitro testing to select the optimal primer pairs and run conditions. Once selected, periodic in silico analysis monitor primer conservation as SARS-CoV-2 evolves. Despite being designed during the Beta wave, the selected primers remain > 99% conserved through Omicron as of 2023-04-14. To validate the final design, we compared SpikeSeq data and National SARS-CoV-2 Strain Surveillance whole-genome data for 321 matching samples. Consensus sequences for the two methods were highly identical (99.998%) across the S-gene. SpikeSeq can serve as a complement to whole-genome surveillance or be leveraged where only S-gene sequencing is of interest. While SpikeSeq is adaptable to other sequencing platforms, the Nanopore platform validated here is compatible with low to moderate throughputs, and its simplicity better enables users to achieve accurate results, even in low resource settings.

genomics↗

bioRxiv2023

Trained immunity in human monocyte enhances myeloid-T-cell pathogenic crosstalk in Ankylosing Spondylitis

Recent studies in infectious, cardiovascular and neurodegenerative diseases have established the presence of memory in innate immune cells. This "trained immunity (TI)" leads to an enhanced response to a second challenge. Monocytes in Ankylosing Spondylitis (AS), a common form of inflammatory arthritis, are known to be hyper-responsive to microbial stimulus lipopolysaccharide (LPS). We asked if TI is present in AS monocytes and, if so, how it contributes to disease pathology. Using Single-cell RNA sequencing (scRNA-seq), flow cytometry and enzyme-linked immunosorbent assays (ELISA), we identify a subset of monocytes from AS patients exhibiting features of trained immunity and being hyperresponsive to LPS stimulation. Surprisingly, both trained monocytes in AS and {beta}-glucan-trained monocytes from healthy donors are hyper-responsive to T-cell-induced activation. scRNA-seq of AS synovial mononuclear cells shows enrichment of a monocyte population with these/analogous features. Additionally, T cell-stimulated monocytes act back on T-cells to support Th17 responses (of established pathology in AS). Lastly, using genetic and chemical perturbations we show that ERN1, an AS risk gene enriched in this trained monocyte population, contributes to T-cell-induced monocyte activation. Our data provide strong evidence for the first time for the key role of TI in common human inflammatory arthritis.

molecular biology↗

bioRxiv2023

L-type Ca2+ channels mediate regulation of glutamate release by subthreshold potential changes

Subthreshold depolarization enhances neurotransmitter release evoked by action potentials and plays a key role in modulating synaptic transmission by combining analog and digital signals. This process is known to be Ca2+-dependent. However, the underlying mechanism of how small changes in basal Ca2+ caused by subthreshold depolarization can regulate transmitter release triggered by a large increase in local Ca2+ is not well understood. This study aimed to investigate the source and signaling mechanisms of Ca2+ that couple subthreshold depolarization with the enhancement of glutamate release in hippocampal cultures and CA3 pyramidal neurons. Subthreshold depolarization increased presynaptic Ca2+ levels, the frequency of spontaneous release, and the amplitude of evoked release, all of which were abolished by blocking L-type Ca2+ channels. A high concentration of intracellular Ca2+ buffer or blockade of calmodulin and phospholipase C abolished depolarization induced increases in transmitter release. Estimation of the readily releasable pool size using hypertonic sucrose showed depolarization induced increases in readily releasable pool size, and this increase was abolished by blockade of calmodulin or phospholipase C. Our results provide mechanistic insights into the modulation of transmitter release by subthreshold potential change and highlight the role of L-type Ca2+ channels in coupling subthreshold depolarization to the activation of Ca2+-dependent signaling molecules that regulate transmitter release. SIGNIFICANCENeuronal activities are encoded by action potentials, but subthreshold changes in resting membrane potentials also play important roles in regulating neuronal functions including synaptic transmission. It is, however, poorly understood how small changes in basal Ca2+ induced by subthreshold depolarization regulate transmitter release triggered by a large increase in local Ca2+ in presynaptic terminals. We demonstrate that L-type Ca2+ channels are the major source of presynaptic Ca2+ influx at basal state and during subthreshold depolarization, resulting in the activation of signaling molecules such as calmodulin and phospholipase C, which facilitate transmitter release by increasing both release probability and the readily releasable pool size. Our results provide mechanistic insight into how subthreshold potential changes contribute to regulating transmitter release.

neuroscience↗

bioRxiv2022

Identification of hyperosmotic stress-responsive genes in Chinese hamster ovary cells via genome-wide virus-free CRISPR/Cas9 screening

Chinese hamster ovary (CHO) cells are the preferred mammalian host cells for therapeutic protein production that have been extensively engineered to possess the desired attributes for high-yield protein production. However, empirical approaches for identifying novel engineering targets are laborious and time-consuming. Here, we established a genome-wide CRISPR/Cas9 screening platform for CHO-K1 cells with 111,651 guide RNAs (gRNAs) targeting 21,585 genes using a virus-free recombinase-mediated cassette exchange-based gRNA integration method. Using this platform, we performed a positive selection screening under hyperosmotic stress conditions and identified 180 genes whose perturbations conferred resistance to hyperosmotic stress in CHO cells. Functional enrichment analysis identified hyperosmotic stress responsive gene clusters, such as tRNA wobble uridine modification and signaling pathways associated with cell cycle arrest. Furthermore, we validated 32 top-scoring candidates and observed a high rate of hit confirmation, demonstrating the potential of the screening platform. Knockout of the novel target genes, Zfr and Pnp, in monoclonal antibody (mAb)-producing recombinant CHO (rCHO) cells and bispecific antibody (bsAb)-producing rCHO cells enhanced their resistance to hyperosmotic stress, thereby improving mAb and bsAb production. Overall, the collective findings demonstrate the value of the screening platform as a powerful tool to investigate the functions of genes associated with hyperosmotic stress and to discover novel targets for rational cell engineering on a genome-wide scale in CHO cells.

bioengineering↗