Biology subjects

Kim, M. S.

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

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Forward genetic screening in engineered colorectal cancer organoids identifies novel regulators of metastasis

Cancer is the second leading cause of death globally, due primarily to metastatic dissemination and colonization of distal sites. Recurrent genetic drivers of metastasis are elusive, suggesting that, unlike the stereotyped mutations promoting primary tumor development, drivers of metastasis may be variable. Here, we interrogate pathways governing metastasis through CRISPR/Cas9-based forward genetic screening in a genetically defined colorectal adenocarcinoma tumor organoid (tumoroid) model using ex vivo invasion screens and orthotopic, in vivo screens for gain of metastatic potential. We identify Ctnna1 and Bcl2l13 as bona fide metastasis suppressors. CTNNA1 loss promotes carcinoma cell invasion and migration through an atypical EMT-like mechanism, whereas BCL2L13 loss promotes cell survival after extracellular matrix detachment and non-cell-autonomous macrophage polarization. Ultimately, this study provides a proof-of-principle that high-content forward genetic screening can be performed in tumor-organoid models in vivo and identifies novel regulators of colon cancer metastasis.

cancer biology↗

bioRxiv2022

Gli2 and Gli3 Regulate Horizontal Basal Cell-Mediated Regeneration of the Olfactory Epithelium

The olfactory epithelium (OE) is a specialized neuroepithelium that is replenished by two stem cell populations: globose basal cells (GBCs) and horizontal basal cells (HBCs). Previous work indicated that HBCs contain primary cilia, organelles that mediate Hedgehog (HH) pathway activity. However, a role for HH signaling in HBCs has not been investigated. We find that GLI2 and GLI3, transcriptional effectors of the HH pathway, are expressed in HBCs in the adult OE and that their expression expands following injury. Further, Gli2-expressing descendants contribute to all major OE cell types during OE regeneration. HBC-specific expression of constitutively active GLI2 drives inappropriate HBC proliferation, alters HBC identity, and culminates in a failure of HBCs to differentiate into olfactory sensory neurons (OSNs) following injury. HBC- specific deletion of endogenous Gli2 and Gli3 results in decreased HBCs and OSNs following OE injury. These data identify GLI2 and GLI3 as key regulators of HBC-mediated OE regeneration.

cell biology↗

bioRxiv2022

Kinetic Characterization and Computational Modeling of the Escherichia coli Heptosyltransferase II: Exploring the Role of Protein Dynamics in Catalysis for a GT-B Glycosyltransferase

Glycosyltransferases (GTs) are enzymes that are uniquely adapted to promote the formation of a glycosidic bond between a sugar molecule and a wide variety of substrates. Heptosyltransferase II (HepII) is a GT involved in the lipopolysaccharide (LPS) biosynthetic pathway that transfers the seven-carbon sugar (L-glycero-D-manno-heptose; Hep) onto a lipid anchored glycopolymer (heptosylated Kdo2-Lipid A, Hep-Kdo2-Lipid A or HLA). LPS plays a key role in Gram-negative bacterial sepsis as a stimulator of the human immune response and has been used as an adjuvant in vaccines. As such, ongoing efforts towards inhibition of LPS biosynthetic enzymes to aid development of novel antimicrobial therapeutics has driven significant effort towards the characterization of these enzymes. Three heptosyltransferases are involved in the inner-core biosynthesis, with E. coli HepII being the last to be quantitatively characterized in vivo, as described herein. HepII shares modest sequence similarity with heptosyltransferase I (HepI) while maintaining a high degree of structural homology. Here we report the first kinetic and biophysical characterization of HepII and demonstrate the properties of HepII that are shared by HepI to include sugar donor promiscuity, and sugar acceptor induced secondary structural changes which results in significant thermal stabilization. HepII also has an increased catalytic efficiency and a significantly tighter binding affinity for both of its substrates, with an insensitivity to the number of acyl chains on the sugar acceptor. Additionally, a structural model of the HepII ternary complex, refined by molecular dynamics simulations, was developed to probe potentially important substrate-protein contacts and revealed the potential of Tryptophan (Trp) residues responsible for reporting on ligand binding. As was previously described for HepI, Tryptophan fluorescence in HepII allowed observation of substrate induced changes in Trp fluorescence intensity which enabled determination of substrate dissociation constants. Combined, these efforts meaningfully enhance our understanding of the Heptosyltransferase family of enzymes and will aid in future efforts to design novel, potent and specific inhibitors for this family of enzymes.

biochemistry↗

bioRxiv2022

Autophagic state prospectively identifies facultative stem cells in the intestinal epithelium

The intestinal epithelium exhibits a rapid and efficient regenerative response to injury. Emerging evidence supports a model where plasticity of differentiated cells, particularly those in the secretory lineages, contributes to epithelial regeneration upon ablation of injury-sensitive stem cells. However, such facultative stem cell activity is rare within secretory populations. Here we ask whether specific functional properties predict facultative stem cell activity. We utilize in vivo labeling combined with ex vivo organoid formation assays to evaluate how cell age and autophagic state contribute to facultative stem cell activity within secretory lineages. Strikingly, we find that cell age (time elapsed since cell cycle exit) does not correlate with secretory cell plasticity. Instead, high autophagic activity predicts plasticity and resistance to DNA damaging injury independently of cell lineage. Our findings indicate that autophagic status prior to injury serves as a lineageagnostic marker for the prospective identification of facultative stem cells.

cell biology↗

bioRxiv2022

Targeted Down Regulation Of Core Mitochondrial Genes During SARS-CoV-2 Infection

Defects in mitochondrial oxidative phosphorylation (OXPHOS) have been reported in COVID-19 patients, but the timing and organs affected vary among reports. Here, we reveal the dynamics of COVID-19 through transcription profiles in nasopharyngeal and autopsy samples from patients and infected rodent models. While mitochondrial bioenergetics is repressed in the viral nasopharyngeal portal of entry, it is up regulated in autopsy lung tissues from deceased patients. In most disease stages and organs, discrete OXPHOS functions are blocked by the virus, and this is countered by the host broadly up regulating unblocked OXPHOS functions. No such rebound is seen in autopsy heart, results in severe repression of genes across all OXPHOS modules. Hence, targeted enhancement of mitochondrial gene expression may mitigate the pathogenesis of COVID-19. One-Sentence SummaryCovid-19 is associated with targeted inhibition of mitochondrial gene transcription.

molecular biology↗

bioRxiv2022

Plasma proteomic biomarkers identify non-responders and reveal biological insights about the tumor microenvironment in melanoma patients after PD1 blockade

Most patients treated with immune checkpoint blockade (ICB) do not have durable treatment responses. Therefore, there is a critical need to identify early non-invasive biomarkers of response. We performed plasma proteomic analysis (>700 proteins) at three timepoints on 174 metastatic melanoma patients treated with ICB. We leverage independent training and testing cohorts to build a predictor of immunotherapy response that outperforms several tissue-based approaches. We found 217 differentially expressed proteins between ICB responders (R) and non-responders (NR), including a co-regulated module of proteins enriched in certain NR patients. By analyzing single-cell RNA-sequencing data of tumor biopsies from 32 patients, we dissected the relative contribution of cells in the tumor to proteins in circulation. The majority of proteins in the co-regulated NR module derived from tumor and myeloid cells. Amongst myeloid cells, we identified a subset of tumor-associated macrophages (TAMs) with a suppressive phenotype that expressed high levels of the co-regulated NR module, thus suggesting they are key drivers of non-response signatures. Together, our data demonstrates the utility of plasma proteomics in biomarker discovery and in understanding the biology of host response to tumors.

cancer biology↗

bioRxiv2021

Patient induced pluripotent stem cell-derived hepatostellate organoids establish a basis for liver pathologies in telomeropathies

Patients with dyskeratosis congenita (DC) and related telomeropathies resulting from premature telomere dysfunction suffer from multi-organ failure. In the liver, DC patients present with nodular hyperplasia, steatosis, inflammation, and cirrhosis. We model DC liver pathologies using isogenic human induced pluripotent stem (iPS) cells harboring a causal DC mutation in DKC1, or a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-corrected control allele. Differentiation of these iPS cells into hepatocytes or hepatic stellate cells followed by generation of genotype-admixed hepatostellate organoids revealed a dominant phenotype in the parenchyma, with DC hepatocytes eliciting a pathogenic hyperplastic response in stellate cells independent of stellate cell genotype. Pathogenic phenotypes could be rescued via suppression of AKT activity, a central regulator of MYC-driven hyperplasia downstream of DKC1 mutation. Thus, isogenic iPS-derived admixed hepatostellate organoids offer insight into the liver pathologies in telomeropathies and provide a framework for evaluating emerging therapies.

cell biology↗

bioRxiv2021

Metabolic and Proteomic Defects in Human Hypertrophic Cardiomyopathy

RationaleImpaired cardiac energetics in hypertrophic cardiomyopathy (HCM) is thought to result from increased ATP utilization at the sarcomere and is believed to be central to pathophysiology. However, the precise defects in cardiac metabolism and substrate availability in human HCM have not been defined. ObjectiveThe purpose of this study is to define major disease pathways and determine the pool sizes of intermediary metabolites in human HCM. Methods and ResultsWe conducted paired proteomic and metabolomic analyses of septal myectomy samples from patients with HCM and compared results to non-failing control human hearts. Increased abundance of extracellular matrix and intermediate filament / Z-disc proteins, and decreased abundance of proteins involved in fatty acid oxidation and cardiac energetics was evident in HCM compared to controls. Acyl carnitines, byproducts of fatty acid oxidation, were markedly depleted in HCM samples. Conversely, the ketone body 3-hydroxybutyrate, lactate, and the 3 branched chain amino acids, were all significantly increased in HCM hearts, suggesting that they may serve as alternate fuel sources for the production of ATP. ATP, nicotinamide adenine dinucleotide (NADH), NADP and NADPH, and acetyl CoA were also severely depleted in HCM hearts. Based on measurements from human skinned muscle fibers, the magnitude of observed reduction in ATP content in the HCM hearts would be expected to decrease the rate of cross-bridge detachment, implying a direct effect of energy depletion on myofilament function that could contribute to diastolic dysfunction. ConclusionsHCM hearts display profound deficits in cardiac energetics, marked by depletion of fatty acid derivatives and compensatory increases in other metabolites that could serve as alternate fuel sources. These results lend support to the paradigm that energy depletion contributes to the pathophysiology of HCM and also have important therapeutic implications for the future design of metabolic modulators to treat HCM.

physiology↗

bioRxiv2021

β-adrenergic stimulation synchronizes a broad spectrum of action potential firing rates of cardiac pacemaker cells towards a higher population average

The heartbeat is initiated by pacemaker cells residing in the sinoatrial node (SAN). SAN cells generate spontaneous action potentials (APs), i.e. normal automaticity. The sympathetic nervous system increases heart rate commensurate with cardiac output demand via stimulation of SAN {beta}-adrenergic receptors ({beta}AR). While SAN cells reportedly represent a highly heterogeneous cell population, the current dogma is that in response to {beta}AR stimulation all cells increase their spontaneous AP firing rate in a similar fashion. The aim of the present study was to investigate cell-to-cell variability in the responses of a large population of SAN cells. We measured {beta}AR responses among 166 single SAN cells isolated from 33 guinea pig hearts. In contrast to the current dogma, the SAN cell responses to {beta}AR stimulation substantially varied. In each cell, changes in AP cycle length highly correlated (R2=0.97) with the AP cycle length before {beta}AR stimulation. While, as expected, on average the cells increased their pacemaker rate, greater responses were observed in cells with slower basal rates, and vice versa, cells with higher basal rates showed smaller responses, no responses, or even decreased their rate. Thus, {beta}AR stimulation synchronizes the operation of the SAN cell population towards a higher average rate, rather than uniformly shifting the rate in each cell, creating a new paradigm of {beta}AR-driven fight-or-flight response among individual pacemaker cells.

biophysics↗

bioRxiv2021

Stable Isotope Tracing In Vivo Reveals A Metabolic Bridge Linking The Microbiota To Host Histone Acetylation

The gut microbiota influences host epigenetics by fermenting dietary fiber into butyrate. Although butyrate could promote histone acetylation by inhibiting histone deacetylases, it may also undergo oxidation to acetyl-CoA, a necessary cofactor for histone acetyltransferases. Here, we find that epithelial cells from germ-free mice harbor a loss of histone H4 acetylation across the genome except at promoter regions. Using stable isotope tracing in vivo with 13C-labeled fiber, we demonstrate that the microbiota supplies carbon for histone acetylation. Subsequent metabolomic profiling revealed hundreds of labeled molecules and supported a microbial contribution to host fatty acid metabolism, which declined in response to colitis and correlated with reduced expression of genes involved in fatty acid oxidation. These results illuminate the flow of carbon from the diet to the host via the microbiota, disruptions to which may affect energy homeostasis in the distal gut and contribute to the development of colitis.

biochemistry↗

bioRxiv2021

Enteric coronavirus infection and treatment modeled with an immunocompetent human intestine-on-a-chip

Many patients infected with coronaviruses, such as SARS-CoV-2 and NL63 that use ACE2 receptors to infect cells, exhibit gastrointestinal symptoms and viral proteins are found in the human gastrointestinal tract, yet little is known about the inflammatory and pathological effects of coronavirus infection on the human intestine. Here, we used a human intestine-on-a-chip (Intestine Chip) microfluidic culture device lined by patient organoid-derived intestinal epithelium interfaced with human vascular endothelium to study host cellular and inflammatory responses to infection with NL63 coronavirus. These organoid-derived intestinal epithelial cells dramatically increased their ACE2 protein levels when cultured under flow in the presence of peristalsis-like mechanical deformations in the Intestine Chips compared to when cultured statically as organoids or in Transwell inserts. Infection of the intestinal epithelium with NL63 on-chip led to inflammation of the endothelium as demonstrated by loss of barrier function, increased cytokine production, and recruitment of circulating peripheral blood mononuclear cells (PMBCs). Treatment of NL63 infected chips with the approved protease inhibitor drug, nafamostat, inhibited viral entry and resulted in a reduction in both viral load and cytokine secretion, whereas remdesivir, one of the few drugs approved for COVID19 patients, was not found to be effective and it also was toxic to the endothelium. This model of intestinal infection was also used to test the effects of other drugs that have been proposed for potential repurposing against SARS-CoV-2. Taken together, these data suggest that the human Intestine Chip might be useful as a human preclinical model for studying coronavirus related pathology as well as for testing of potential anti-viral or anti-inflammatory therapeutics.

bioengineering↗

bioRxiv2021

Distinct function of Mediator subunits in fungal development, stress response and secondary metabolism in fungal pathogen Fusarium verticillioides

Mediator is a conserved, nucleus-localized, multi-subunit complex highly conserved across eukaryotes. It interacts with RNA polymerase II transcription machinery as well as various transcription factors to regulate gene expression. However, systematic characterization of Mediator complex has not be thoroughly performed in filamentous fungi. In current study, our aim was to investigate key biological functions of Mediator subunits in a mycotoxigentic plant pathogen Fusarium verticillioides. While we recognized some level of divergence in subunits constituent, overall structure remained conserved between Saccharomyces cerevisiae and F. verticillioides. We were able to generate 11 Mediator subunit deletion mutants and characterize serveal important phenotypes associated with fungal development, environmental stress, nutrient utilization, virulence as well as secondary metabolism. While each Mediator subunit deletion mutant showed deficiencies in at least three of the phenotypes tested, it is important to note that different modules or subunits showed unique regulatory role in different cellular processes. Significantly, the deletion of FvMed1 led to increased FB1 production, and we were able to confirm that FvMed1 is transported from the nucleus to the cytoplasm under fumonisin-producing conditions. Taken together, our study characterized the different biological functions of Mediator subunits in plant pathogen F. verticillioides and the possibility of select subunits unique cytoplasmic function independent of the core Mediator. IMPORTANCEMediator is a highly conserved component of RNA polymerase II pre-initiation complex (PIC) in eukaryotes. Studies in yeast have demonstrated the important roles of Mediator subunits, but we lack deeper understanding in filamentous fungi. Here, we selected Fusarium verticillioides, a major fungal pathogen of maize worldwide, to study transcriptional profile followed by mutational analyses of all Mediator genes. Each Mediator subunit deletion mutant showed some shared deficiencies but it is also important that different modules or subunits showed unique regulatory role in different cellular processes. Significantly, FvMed1 deletion mutant showed elevated levels of FB1 production, and we were able to confirm that FvMed1 translocates from the nucleus to cytoplasmic organelles under fumonisin-producing conditions. This is a first study demonstrating that a Mediator subunit regulate cellular functions while not directly associating with PIC in filamentous fungi.

microbiology↗

bioRxiv2020

Tumor edge architecture in glioblastoma is constructed by inter-cellular signals from vascular endothelial cells

One of the hallmarks of glioblastoma (GBM) is extensive neovascularization. In addition to supplying blood and nutrients, vascular endothelial (VE) cells provide trophic support to GBM cells via paracrine signaling, the precise mechanisms of which are being unraveled. Here, using patient-derived GBM and VE cells as well as orthotopic GBM mouse models, we report that Endocan (ESM1), an endothelial-secreted proteoglycan, confers enhanced proliferative, migratory, and angiogenic properties to GBM cells and regulates their spatial identity. Mechanistically, Endocan exerts at least part of its functions via direct binding and activation of the PDGFRA receptor. Subsequent downstream signaling enhances chromatin accessibility of the Myc promoter and upregulates Myc expression inducing highly stable phenotypic changes in GBM cells. Furthermore, Endocan confers a radioprotection phenotype in GBM cells, both in vitro and in vivo. Inhibition of Endocan-PDGFRA signaling with ponatinib increases survival in the Esm1 wild-type but not in the Esm1 knock-out mouse GBM model. Our findings identify Endocan and its downstream signaling axis as a potential target to subdue the recurrence of GBM and further highlight the importance of vascular to tumor cell signaling for GBM biology. Significance statementIdentification of the Endocan/PDGFRA/Myc axis demonstrates an important role of VE cells in GBM malignancy. The contribution of Endocan to the development of GBM cell populations with different phenotypes reveal an additional pathway underlying the origin of GBM intratumoral heterogeneity. Targeting Endocan-mediated crosstalk may enhance the efficacy of GBM treatment.

cancer biology↗

bioRxiv2020

The instantaneous multi-pronged defense system of latex against general plant enemies

Based on the hypothesis that variation of the metabolomes of latex is a response to selective pressure and should thus be affected differently from other organs, their variation could provide insight into the defensive chemical selection of plants. Metabolic profiling was utilized to compare tissues of Euphorbia species collected in various regions. The metabolic variation of latexes was much more limited than that of other organs. In all of the species, the levels of polyisoprenes and terpenoids were found to be much higher in latexes than in leaves and roots. Polyisoprenes were also observed to physically delay the contact and growth of pathogens with plant tissues. A secondary barrier composed of terpenes and, in particular, 24-methylenecycloartanol, exhibited antifungal activity. These results, together with the known roles of the enzymes also present in latexes, demonstrate that they are part of a cooperative defense system that comprises both biochemical and physical elements.

ecology↗

bioRxiv2020

Digit ratio (2D:4D) and congenital adrenal hyperplasia (CAH): Systematic literature review and meta-analysis

The ratio of length between the second and fourth fingers (2D:4D) is commonly used as an indicator of prenatal sex hormone exposure. Several approaches have been used to try to validate the measure, including examining 2D:4D in people with congenital adrenal hyperplasia (CAH), a suite of conditions characterised by elevated adrenal androgen production secondary to defective steroidogenesis. We present here a systematic review that examines the relationship between these two variables. Twelve articles relating to nine CAH cohorts were identified, and 2D:4D comparisons have been made between cases and controls in eight of these cohorts. Altogether, at least one 2D:4D variable has been compared between n=251 females with CAH and n=358 unaffected females, and between n=108 males with CAH and n=204 unaffected males. A previous meta-analysis (Honekopp & Watson, 2010) reported lower right hand (R2D:4D) and left hand (L2D:4D) digit ratios in patients with CAH relative to sex-matched controls. Our meta-analysis showed the same direction of results; however, the effects were only statistically significant for R2D:4D in males and L2D:4D in females (R2D:4D: females, p = 0.072, g = 0.591; males, p = 0.019, g = 0.513; L2D:4D: females, p = 0.020, g = 0.245; males, p = 0.334, g = 0.218), and the average effect size had reduced by 46.70%. We also found no evidence to suggest the right-left difference in 2D:4D (D[R-L]) is associated with prenatal sex hormone exposure.

developmental biology↗

bioRxiv2020

Engineering multiple levels of specificity in an RNA viral vector

Synthetic molecular circuits could provide powerful therapeutic capabilities, but delivering them to specific cell types and controlling them remains challenging. An ideal “smart” viral delivery system would enable controlled release of viral vectors from “sender” cells, conditional entry into target cells based on cell-surface proteins, conditional replication specifically in target cells based on their intracellular protein content, and an evolutionarily robust system that allows viral elimination with drugs. Here, combining diverse technologies and components, including pseudotyping, engineered bridge proteins, degrons, and proteases, we demonstrate each of these control modes in a model system based on the rabies virus. This work shows how viral and protein engineering can enable delivery systems with multiple levels of control to maximize therapeutic specificity.Competing Interest StatementX.J.G, L.S.C., M.S.K., and M.B.E. are inventors on a U.S. patent provisional application related to this work.View Full Text

synthetic biology↗

bioRxiv2020

No Difference in 2D:4D Ratio between Youth with Elevated Prenatal Androgen Exposure due to Congenital Adrenal Hyperplasia and Controls

The second-to-fourth digit ratio (2D:4D) has been associated with sexual dimorphism, with a lower 2D:4D in males. A large body of research has relied on the 2D:4D as a proxy for prenatal androgen exposure, and includes reports of relationships between 2D:4D and a wide range of human traits. Here, we examine the validity of the 2D:4D proxy by studying the association between 2D:4D and classical Congenital Adrenal Hyperplasia (CAH) due to 21-hydroxylase deficiency, a condition characterized by excessive prenatal exposure to androgens during most of the gestational period. To this end, we retrospectively examine 513 serial radiographs of the left hand obtained clinically in 90 youth with classical CAH (45 female) and 70 control youth (31 female). Replicating previous reports, we observe associations of the 2D:4D with sex (lower 2D:4D in males) and age (increase of 2D:4D through development). However, we find no evidence for differences in 2D:4D between CAH and controls (full sample: {square} = -0.001 (-0.008, 0.006)]; females: {square} = -0.004 [-0.015, 0.007]; males: {square} = 0.001, [-0.008, 0.011]). Although our findings do not rule out a small association between the 2D:4D and CAH, they cast doubt on the usefulness of the 2D:4D as a biomarker for prenatal androgen exposure in behavioral research.

developmental biology↗

bioRxiv2020

Modeling metabolic variation with single-cell expression data

Cellular metabolism encompasses the biochemical reactions and transportation of various metabolites in cells and their surroundings, which are integrated at all levels of cellular functions. We developed a method to systematically simulate cellular metabolism using single-cell RNA-seq (scRNA-seq) data through constraint-based context specific metabolic modeling. We simulated the NAD+ biosynthesis activity in 7 different mouse tissues, and the simulated NAD+ biosynthesis flux levels showed significant linear correlation with experimental measurements in previous research. We also show that the simulated NAD+ biosynthesis fluxes are reproducible using two additional scRNA-seq datasets.

systems biology↗