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Oh, S.

Publications and source records attributed to Oh, S..

At least 91 records · Page 5Linked to original sources

Differential ion dehydration energetics explains selectivity in the non-canonical lysosomal K+ channel TMEM175

Structures of the human lysosomal K+ channel TMEM175 in open and closed states revealed a novel architecture lacking the canonical K+ selectivity filter motif present in previously known K+ channel structures. A hydrophobic constriction composed of four isoleucine residues was resolved in the pore and proposed to serve as the gate in the closed state, and to confer ion selectivity in the open state. Here, we achieve higher-resolution structures of the open and closed states and employ molecular dynamics simulations to analyze the conducting properties of the putative open state, demonstrating that it is capable of permeating K+ ions at the expected rate. Monovalent cations must dehydrate significantly to penetrate the narrow hydrophobic constriction, but ion flow is assisted by a favorable electrostatic field generated by the protein that spans the length of the pore. The balance of these opposing energetic factors explains why permeation is feasible, and why TMEM175 is selective for K+ over Na+, despite the absence of the canonical selectivity filter. Accordingly, mutagenesis experiments reveal an exquisite sensitivity of the channel to perturbations that mitigate the constriction. Together, these data reveal a novel mechanism for selective permeation of ions by TMEM175 that is unlike that of other K+ channels.

biophysics↗

DUSP6 mediates resistance to JAK2 inhibition and drives leukemic progression

Chronic myeloproliferative neoplasms (MPNs) exhibit a propensity for transformation to secondary acute myeloid leukemia (sAML), for which the underlying mechanisms remain poorly understood, resulting in limited treatment options and dismal clinical outcomes. Here, we performed bulk transcriptome profiling accompanied by single cell RNA-sequencing on CD34+ stem/progenitor cells from serial patient samples obtained at the chronic MPN and sAML phases, and identified aberrantly increased expression of dual-specificity phosphatase 6 (DUSP6) underlying disease transformation. Genetic and pharmacologic targeting of DUSP6 led to inhibition of S6 and JAK/STAT signaling, resulting in potent suppression of cell proliferation, while also reducing inflammatory cytokine production in primary samples. Furthermore, ectopic DUSP6 expression augmented proliferation and mediated JAK2 inhibitor resistance, while DUSP6 inhibition reduced colony-forming potential of JAK2 inhibitor-persistent patient cells. Mechanistically, DUSP6 perturbation dampened S6 signaling via inhibition of RSK1, which we identified as a second indispensable candidate associated with poor clinical outcome. Lastly, DUSP6 inhibition potently suppressed disease development across Jak2 V617F and MPL W515L MPN mouse models, and sAML patient-derived xenografts. These findings underscore DUSP6 in driving disease transformation and therapeutic resistance, and highlight the DUSP6-RSK1 axis as a novel, druggable pathway in myeloid malignancies.

cancer biology↗

GenomicSuperSignature: interpretation of RNA-seq experiments through robust, efficient comparison to public databases

Millions of transcriptomic profiles have been deposited in public archives, yet remain underused for the interpretation of new experiments. We present a novel method for interpreting new transcriptomic datasets through near-instantaneous comparison to public archives without high-performance computing requirements. We performed Principal Component Analysis on 536 studies comprising 44,890 RNA sequencing profiles. Sufficiently similar loading vectors were aggregated to form Replicable Axes of Variation (RAV). RAVs were annotated with metadata of originating studies and samples and by gene set enrichment analysis. Functionality to associate new datasets with RAVs, extract interpretable annotations, and provide intuitive visualization are implemented as the GenomicSuperSignature R/Bioconductor package. We demonstrated the efficient and coherent database searching, robustness to batch effects and heterogeneous training data, and transfer learning capacity of our method using TCGA and rare diseases datasets. GenomicSuperSignature will aid analyzing new gene expression data in the context of existing databases using minimal computing resources. PURPOSEMillions of transcriptomic profiles have been deposited in public archives, yet remain underused for the interpretation of new experiments. Existing methods for leveraging these public resources have focused on the reanalysis of existing data or analysis of new datasets independently. We present a novel approach to interpreting new transcriptomic datasets by near-instantaneous comparison to public archives without high-performance computing requirements. METHODSTo identify replicable and interpretable axes of variation in any given gene expression dataset, we performed Principal Component Analysis (PCA) on 536 studies comprising 44,890 RNA sequencing profiles. Sufficiently similar loading vectors, when compared across studies, were aggregated to form Replicable Axes of Variation (RAV). RAVs were annotated with metadata of originating studies and samples and by gene set enrichment analysis. Functionality to associate new datasets with RAVs, extract interpretable annotations, and provide intuitive visualization are implemented as the GenomicSuperSignature R/Bioconductor package. RESULTSRAVs are robust to batch effects and the presence of low-quality or irrelevant studies, and identify signals that can be lost by merging samples across the training datasets. The GenomicSuperSignature package allows instantaneous matching of PCA axes in new datasets to pre-computed RAVs, cutting down the analysis time from days to the order of seconds on an ordinary laptop. We demonstrate that RAVs associated with a phenotype can provide insight into weak or indirectly measured biological attributes in a new study by leveraging accumulated data from published datasets. Benchmarking against complementary previous works demonstrates that the RAV index 1) identifies colorectal carcinoma transcriptome subtypes that are similar to but more correlated with clinicopathological characteristics than previous disease-specific efforts and 2) can estimate neutrophil counts through transfer learning on new data comparably to the previous efforts despite major differences in training datasets and model building processes with the additional benefits of flexibility and scalability of the model application. CONCLUSIONGenomicSuperSignature establishes an information resource and software tools to interrogate it. Prior knowledge databases are coherently linked, enabling researchers to analyze new gene expression data in the context of existing databases using minimal computing resources. The robustness of GenomicSuperSignature suggests that we can expand this approach beyond human gene expression profiles, such as single-cell RNA-seq, microbiome abundance, and different species transcriptomics datasets.

bioinformatics↗

Escherichia coli Cryptic Prophages Sense Nutrients to Control Persister Cell Resuscitation

We determined previously that some cryptic prophages are not genomic junk but instead enable cells to combat myriad stresses as part of an active stress response. However, how these phage fossils affect the extreme stress response of dormancy; i.e., how cryptic prophages affect persister cell formation and resuscitation, has not been fully explored. Persister cells form as a result of stresses such as starvation, antibiotics, and oxidative conditions, and resuscitation of these persister cells likely causes recurring infections such as those associated with tuberculosis, cystic fibrosis, and Lyme disease. Unlike for the active stress response, here we find that deletion of each of the nine Escherichia coli cryptic prophages has no effect on persister cell formation. Strikingly, elimination of each cryptic prophage results in an increase in persister cell resuscitation with a dramatic increase in resuscitation upon deleting all nine prophages. This increased resuscitation includes eliminating the need for a carbon source and is due to activation of the phosphate import system as a result of inactivating transcriptional regulator AlpA of the CP4-57 cryptic prophage, since we found {Delta}alpA increases persister resuscitation, and AlpA represses phosphate regulator PhoR. Therefore, we report a novel cellular stress mechanism controlled by cryptic prophages: regulation of phosphate uptake which controls the exit of the cell from dormancy and prevents premature resuscitation in the absence of nutrients.

microbiology↗

Functional inactivation of pulmonary MAIT cells following 5-OP-RU treatment of non-human primates

Targeting MAIT cells holds promise for the treatment of different diseases and infections. We previously showed that treatment of Mycobacterium tuberculosis infected mice with 5-OP-RU, a major antigen for MAIT cells, expands MAIT cells and enhances bacterial control. Here we treated M. tuberculosis infected rhesus macaques with 5-OP-RU intratracheally but found no clinical or microbiological benefit in M. tuberculosis infected macaques. In fact, after 5-OP-RU treatment MAIT cells did not expand, but rather upregulated PD-1 and lost the ability to produce multiple cytokines, a phenotype resembling T cell exhaustion. Furthermore, we show that vaccination of uninfected macaques with 5-OP-RU+CpG instillation into the lungs also drives MAIT cell dysfunction, and PD-1 blockade during vaccination partly prevents the loss of MAIT cell function without facilitating their expansion. Thus, in rhesus macaques MAIT cells are prone to the loss of effector functions rather than expansion after TCR stimulation in vivo, representing a significant barrier to therapeutically targeting these cells.

immunology↗

APOBEC3A drives acquired resistance to targeted therapies in non-small cell lung cancer

Acquired drug resistance to even the most effective anti-cancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified1-6, the underlying molecular mechanisms shaping tumor evolution during treatment are incompletely understood. The extent to which therapy actively drives tumor evolution by promoting mutagenic processes7 or simply provides the selective pressure necessary for the outgrowth of drug-resistant clones8 remains an open question. Here, we report that lung cancer targeted therapies commonly used in the clinic induce the expression of cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Induction of A3A facilitated the formation of double-strand DNA breaks (DSBs) in cycling drug-treated cells, and fully resistant clones that evolved from drug-tolerant intermediates exhibited an elevated burden of chromosomal aberrations such as copy number alterations and structural variations. Preventing therapy-induced A3A mutagenesis either by gene deletion or RNAi-mediated suppression delayed the emergence of drug resistance. Finally, we observed accumulation of A3A mutations in lung cancer patients who developed drug resistance after treatment with sequential targeted therapies. These data suggest that induction of A3A mutagenesis in response to targeted therapy treatment may facilitate the development of acquired resistance in non-small-cell lung cancer. Thus, suppressing expression or enzymatic activity of A3A may represent a potential therapeutic strategy to prevent or delay acquired resistance to lung cancer targeted therapy.

cancer biology↗

Hybrid Clustering of single-cell gene-expression and cell spatial information via integrated NMF and k-means

MotivationRecent advances in single cell transcriptomics have allowed us to examine the identity of single cells, which has led to the discovery of new cell types and high resolution maps of cell type composition in tissues. Technologies that measure multiple modalities of single cell data provide a more comprehensive picture of a cell, but they also create challenges for data integration tasks. ResultsIn our work, we jointly consider the spatial location and gene expression profiles of cells to determine their identity. Specifically, we have developed scHybridNMF (single-cell Hybrid Nonnegative Matrix Factorization), which performs cell type identification by incorporating single cell gene expression data with cell location data. We combined nonnegative matrix factorization (NMF) with k-means clustering to cohesively represent high-dimensional gene expression data and low-dimensional location data, respectively. We show that scHybridNMF can utilize location data to improve cell type clustering. In particular, we show that under multiple scenarios, including the cases where there is a small number of genes profiled and the location data is noisy, scHybridNMF outperforms sparse NMF, k-means, and an existing method (HMRF) that also uses cell location and gene expression data for cell type identification. Availabilityhttps://github.com/soobleck/scHybridNMF Contacthpark@cc.gatech.edu, xiuwei.zhang@gatech.edu

bioinformatics↗

Yeast Nuak1 phosphorylates histone H3 threonine 11 in low glucose stress conditions by the cooperation of AMPK and CK2 signaling

Changes in available nutrients are inevitable events for most living organisms. Upon nutritional stress, several signaling pathways cooperate to change the transcription program through chromatin regulation to rewire cellular metabolism. In budding yeast, histone H3 threonine 11 phosphorylation (H3pT11) acts as a marker of low glucose stress and regulates the transcription of nutritional stress responsive genes. Understanding how this histone modification senses external glucose changes remains elusive. Here, we show that Tda1, the yeast orthologue of human Nuak1, is a direct kinase for H3pT11 upon low glucose stress. Yeast AMPK directly phosphorylates Tda1 to govern Tda1 activity, while CK2 regulates Tda1 nuclear localization. Collectively, AMPK and CK2 signaling converge on histone kinase Tda1 to link external low glucose stress to chromatin regulation.

cell biology↗

HGNChelper: Identification and correction of invalid gene symbols for human and mouse

Gene symbols are recognizable identifiers for gene names but are unstable and error-prone due to aliasing, manual entry, and unintentional conversion by spreadsheets to date format. Official gene symbol resources such as HUGO Gene Nomenclature Committee (HGNC) for human genes and the Mouse Genome Informatics project (MGI) for mouse genes provide authoritative sources of valid, aliased, and outdated symbols, but lack a programmatic interface and correction of symbols converted by spreadsheets. We present HGNChelper, an R package that identifies known aliases and outdated gene symbols based on the HGNC human and MGI mouse gene symbol databases, in addition to common mislabeling introduced by spreadsheets, and provides corrections where possible. HGNChelper identified invalid gene symbols in the most recent Molecular Signatures Database (mSigDB 7.0) and in platform annotation files of the Gene Expression Omnibus, with prevalence ranging from [~]3% in recent platforms to 30-40% in the earliest platforms from 2002-03. HGNChelper is installable from CRAN, with open development and issue tracking on GitHub and an associated pkgdown site https://waldronlab.io/HGNChelper/.

bioinformatics↗

Plant NLR targets P-type ATPase for executing plasma membrane depolarization leading to calcium influx and cell death

Hypersensitive response (HR) is a robust immune response mediated by plant nucleotide-binding and leucine-rich repeat receptor (NLR). However, the early molecular event linking NLR to cell death is obscure. Here we demonstrate that NLR targets plasma membrane H+-ATPases (PMA) generating electrochemical potential across the membrane. CCA309, an autoactive N-terminal domain of pepper coiled-coil NLR (CNL), associates with PMAs and its autoactivity is affected by silencing or overexpression of PMA. CCA309-induced extracellular alkalization accompanied with membrane depolarization is followed by calcium influx and cell death. CCA309 interacts with C-terminal regulatory domain of PMA and 14-3-3 negatively affects CCA309-induced cell death. Moreover, pharmacological experiments with fusicoccin, an irreversible PMA activator, confirmed that CC- and CNL-mediated cell death occurred through inhibiting PMA. We propose PMAs as the primary target of plasma membrane-associated CNL to disrupt electrochemical homeostasis leading to HR cell death.

plant biology↗

Rapid and Efficient BAC Recombineering: Gain & Loss Screening System

Recombineering has been developed to modify bacterial artificial chromosome (BAC) via homologous recombination. Nevertheless, as a screening strategy to identify the correct clone was not properly developed, it was difficult to obtain a correct clone within a limited time period. To address these issues, we developed a new screening method (a gain & loss screening system) that enables the efficient identification of the recombineered clone. Simple inoculation of cells into LB medium with appropriate antibiotics visually revealed the positive clones within 24 h. DNA sequencing confirmed 100% accuracy of this screening method by showing that all positive clones exhibited recombinant sequences. Furthermore, our new method allowed us to complete the entire procedure consisting of 1st recombineering, flip-out and 2nd recombineering in just 13 days. Overall, our new strategy may provide a new avenue for BAC recombeerining, as evidenced by markedly increased accuracy and subsequently shortened recombineering duration.View Full Text

bioengineering↗

The plasticity of the pyruvate dehydrogenase complex confers a labile structure that is associated with its catalytic activity

The pyruvate dehydrogenase complex (PDC) is a multienzyme complex that plays a key role in energy metabolism by converting pyruvate to acetyl-CoA. An increase of nuclear PDC has been shown to be correlated with an increase of histone acetylation that requires acetyl-CoA. PDC has been reported to form a ~ 10 MDa macromolecular machine that is proficient in performing sequential catalytic reactions via its three components. In this study, we show that the PDC displays size versatility in an ionic strength-dependent manner using size exclusion chromatography of yeast cell extracts. Biochemical analysis in combination with mass spectrometry indicates that yeast PDC (yPDC) is a salt-labile complex that dissociates into sub-megadalton individual components even under physiological ionic strength. Interestingly, we find that each oligomeric component of yPDC displays a larger size than previously believed. In addition, we show that the mammalian PDC also displays this uncommon characteristic of salt-lability, although it has a somewhat different profile compared to yeast. We show that the activity of yPDC is reduced in higher ionic strength. Our results indicate that the structure of PDC may not always maintain its ~ 10 MDa organization, but is rather variable. We propose that the flexible nature of PDC may allow modulation of its activity.

biochemistry↗

Type 2 and interferon inflammation strongly regulate SARS-CoV-2 related gene expression in the airway epithelium

Coronavirus disease 2019 (COVID-19) outcomes vary from asymptomatic infection to death. This disparity may reflect different airway levels of the SARS-CoV-2 receptor, ACE2, and the spike protein activator, TMPRSS2. Here we explore the role of genetics and co-expression networks in regulating these genes in the airway, through the analysis of nasal airway transcriptome data from 695 children. We identify expression quantitative trait loci (eQTL) for both ACE2 and TMPRSS2, that vary in frequency across world populations. Importantly, we find TMPRSS2 is part of a mucus secretory network, highly upregulated by T2 inflammation through the action of interleukin-13, and that interferon response to respiratory viruses highly upregulates ACE2 expression. Finally, we define airway responses to coronavirus infections in children, finding that these infections upregulate IL6 while also stimulating a more pronounced cytotoxic immune response relative to other respiratory viruses. Our results reveal mechanisms likely influencing SARS-CoV-2 infectivity and COVID-19 clinical outcomes.

genomics↗

Is FAM19A5 an adipokine? Peripheral FAM19A5 in wild-type, FAM19A5 knock-out, and LacZ knock-in mice.

FAM19A5 is a novel secretory protein primarily expressed in the brain. However, a recent study reported that FAM19A5 is an adipocyte-derived adipokine that regulates vascular smooth muscle function through sphingosine-1-phosphate receptor 2 (S1PR2). In our study, we investigated FAM19A5 transcript and protein levels in peripheral tissues, including adipose tissues, from wild-type, FAM19A5 knockout, and FAM19A5 LacZ knockin mice. We found that FAM19A5 transcript levels in the central nervous system were much greater than those in any of the peripheral tissues, including adipose tissues. Furthermore, the FAM19A5 protein levels in adipose and reproductive tissues were below detectable limits for Western blot analysis. Additionally, we found that the FAM19A5 protein did not interact with S1PR2 in terms of G protein-mediated signal transduction, {beta}-arrestin recruitment, or ligand-mediated internalization. Taken together, our findings revealed basal levels of FAM19A5 transcripts and proteins in peripheral tissues, confirming its primary expression in the central nervous system and lack of significant interaction with S1PR2.

genetics↗

Genome-wide expression analysis of immune receptors in hot pepper reveals an autonomous NLR cluster in higher plants

Plants possess hundreds of intracellular immune receptors encoding nucleotide-binding domain and leucine-rich repeat (NLR) proteins. Autoactive NLRs, in some cases a specific NLR domain, induce plant cell death in the absence of pathogen infection. In this study, we identified a group of NLRs (G10) carrying autoactive coiled-coil (CC) domains in pepper (Capsicum annuum L. cv. CM334) by genome-wide transient expression analysis. The G10-CC-mediated cell death mimics hypersensitive response (HR) cell death triggered by interaction between NLR and effectors derived from pathogens. Sequence alignment and mutagenesis analyses revealed that the intact 1 helix of G10-CCs is critical for both G10-CC- and R gene-mediated HR cell death. The cell death induced by G10-CCs does not require known helper NLRs, suggesting G10-NLRs are novel singleton NLRs. We also found that G10-CCs localize in the plasma membrane as Arabidopsis singleton NLR ZAR1. Extended studies revealed that autoactive G10-CCs are well conserved in other Solanaceae plants, including tomato, potato, and tobacco, as well as a monocot plant, rice. Furthermore, G10-NLR is an ancient form of NLR that present in all tested seed plants (spermatophytes). Our studies not only uncover the autonomous NLR cluster in plants but also provide powerful resources for dissecting the underlying molecular mechanism of NLR-mediated cell death in plants.

plant biology↗

LESSeq: Local event-based analysis of alternative splicing using RNA-Seq data

Alternative splicing, which can be observed genome-wide by RNA-Seq, is important in cellular development and evolution. Comparative RNA-Seq experiments between different cellular conditions allow alternative splicing signatures to be detected. However, inferring alternative splicing signatures from short-read technology is unreliable and still presents many challenges before biologically significant signatures may be identified. To enable the robust discovery of differential alternative splicing, we developed the Local Event-based analysis of alternative Splicing using RNA-Seq (LESSeq) pipeline. LESSeq utilizes information of local splicing events (i.e., the partial structures in genes where transcript-splicing patterns diverge) to identify unambiguous alternative splicing. In addition, LESSeq quantifies the abundance of these alternative events using Maximum Likelihood Estimation (MLE) and provides their significance between different cellular conditions. The utility of LESSeq is demonstrated through two case studies relevant to human variation and evolution. Using an RNA-Seq data set of lymphoblastoid cell lines in two human populations, we examined within-species variation and discovered population-differential alternative splicing events. With an RNA-Seq data set of several tissues in human and rhesus macaque, we studied cross-species variation and identified lineage-differential alternative splicing events. LESSeq is implemented in C++ and R, and made publicly available on GitHub at: https://github.com/gersteinlab/LESSeq

bioinformatics↗

Many paths to the same goal: metaheuristic operation of brains during natural behavior

During self-guided behaviors animals rapidly identify the constraints of the problems they face and adaptively employ appropriate cognitive strategies and heuristics to solve these problems1,2. This ability is currently an area of active investigation in artificial intelligence3. Recent work in computer science has suggested that this type of flexible problem solving could be achievable with metaheuristic approaches in which specific algorithms are selected based upon the identified demands of the problem to be solved4,5,6,7. Investigating how animals employ such metaheuristics while solving self-guided natural problems is a fertile area for biologically inspired algorithm development. Here we show that animals adaptively shift cognitive resources between sensory and memory systems during natural behavior to optimize performance under uncertainty. We demonstrate this using a new, laboratory-based discovery method to define the strategies used to solve a difficult optimization scenario, the stochastic \"traveling salesman\" problem5,8,9. Using this system we precisely manipulated the strength of prior information available to animals as well as the complexity of the problem. We find that rats are capable of efficiently solving this problem, even under conditions in which prior information is unreliable and the space of possible solutions is large. We compared animal performance to a Bayesian search and found that performance is consistent with a metaheuristic approach that adaptively allocates cognitive resources between sensory processing and memory, enhancing sensory acuity and reducing memory load under conditions in which prior information is unreliable. Our findings set the foundation for new approaches to understand the neural substrates of natural behavior as well as the rational development of biologically inspired metaheuristic approaches for complex real-world optimization.

animal behavior and cognition↗

In situ measurement of absolute concentrations by Normalized Raman Imaging

Cell mass and its chemical composition are important aggregate cellular variables for physiological processes including growth control and tissue homeostasis. Despite their central importance, it has been difficult to quantitatively measure these quantities from single cells in intact tissue. Here, we introduce Normalized Raman Imaging (NoRI), a Stimulated Raman Scattering (SRS) microscopy method that provides the local concentrations of protein, lipid and water from live or fixed tissue samples with high spatial resolution. Using NoRI, we demonstrate that single cell protein, lipid and water concentrations are maintained in a tight range in cells under same physiological conditions and are altered in different physiological states such as cell cycle stages, attachment to substrates of different stiffness, or by entering senescence. In animal tissues, protein and lipid concentration varies with cell types, yet an unexpected cell-to-cell heterogeneity was found in cerebellar Purkinje cells. Protein and lipid concentration profile provides a new means to quantitatively compare disease-related pathology as demonstrated using models of Alzheimers disease. Our demonstration shows that NoRI is a broadly applicable tool for probing the biological regulation of protein mass, lipid mass and water in cellular and tissue growth, homeostasis, and disease.

bioengineering↗