Biology subjects

Shin, J.

Publications and source records attributed to Shin, J..

At least 109 records · Page 6Linked to original sources

bioRxiv2021

Integrated single-cell transcriptomic and epigenetic analyses of cell-state transition and lineage commitment in the embryonic mouse cerebellum

Recent studies using single-cell RNA-seq have revealed cellular heterogeneity in the developing mammalian cerebellum, yet the regulatory logic underlying this cellular diversity remains to be elucidated. Using integrated single-cell RNA and ATAC analyses, we resolved developmental trajectories of cerebellar progenitors and identified putative trans- and cis- elements that control cell state transition. We reverse-engineered gene regulatory networks (GRNs) of each cerebellar cell type. Through in silico simulations and in vivo experiments, we validated the efficacy of GRN analyses and uncovered the molecular control of a newly identified stem zone, the posterior transitory zone (PTZ), which contains multipotent progenitors for granule neurons, Bergmann glia, and choroid plexus epithelium. Importantly, we showed that perturbing cell fate specification of PTZ progenitors causes posterior cerebellar vermis hypoplasia, the most common cerebellar birth defect in humans. Our study provides a foundation for comprehensive studies of developmental programs of the mammalian cerebellum.

neuroscience↗

bioRxiv2021

Inhibition of Ras1-MAPK pathways for hypha formation by novel drug candidates in Candida albicans

The opportunistic human fungal pathogen Candida albicans has morphogenesis as a virulence factor. The morphogenesis of C. albicans is closely related to pathogenicity (1). Ras1 in C. albicans is an important switch in the MAPK pathway for morphogenesis (2, 3). The MAPK pathway is important for the virulence, such as cell growth, morphogenesis, and biofilm formation (4, 5). Ume6 is a well-known transcriptional factor for hyphal-specific genes (6). Despite numerous studies, as a recent issue, it is necessary to develop a new drug that uses a different pathway mechanism to inhibit resistant C. albicans strains caused by chronic prescription of azole or echinocandin drugs, which are mainly used. Here, we show that the small carbazole derivatives attenuated the pathogenicity of C. albicans through inhibition of the Ras1/MAPK pathway. We found that the small molecules inhibit morphogenesis through repressing protein and RNA levels in Ras/MAPK related genes including UME6 and NRG1. Furthermore, we found the antifungal effect of the small molecules in vivo using a candidiasis murine model. We anticipate our findings are that the small molecules are the promising compounds for the development of new antifungal agents for the treatment of systemic candidiasis and possibly for other fungal diseases. Author summaryThe infection by the opportunistic human fungal pathogen Candida albicans occurs mainly in immunocompetent and immunocompromised humans, such as AIDS patients, immunosuppressant-treated organ transplant patients, and recent COVID-19 patients. Morphogenesis which the ability to switch between yeast and hyphal growth forms is one of the representative virulence factors of C. albicans. Here, we describe novel small molecules that show antifungal effects such as the inhibition of the morphogenesis and the biofilm formation, and maintenance of biofilm. Moreover, we found that these small molecules had antifungal activity in mouse experiments, and confirmed that they were also effective in drug-resistant C. albicans strains. Studies of some small molecules with structures similar to ours have already been reported to exhibit growth inhibitory activity against bacteria and Candida species. However, the mechanism of action of these molecules has not been elucidated. In this study, we demonstrated, for the first time, the mechanism by which these two small molecules inhibit C. albicans pathogenicity through inhibition of specific pathways. Our study, through the research of the mechanism of action of novel small molecules, provides new insights into the development of drug candidates not only for wild-type C. albicans, but also for strains resistant to existing drugs.

microbiology↗

bioRxiv2021

Identifying strengths and weaknesses of methods for computational network inference from single cell RNA-seq data

Single-cell RNA-sequencing (scRNA-seq) offers unparalleled insight into the transcriptional programs of different cellular states by measuring the transcriptome of thousands of individual cells. An emerging problem in the analysis of scRNA-seq is the inference of transcriptional gene regulatory networks and a number of methods with different learning frameworks have been developed to address this problem. Here, we present an expanded benchmarking study of eleven recent network inference methods on six published scRNA-seq datasets in human, mouse, and yeast considering different types of gold standard networks and evaluation metrics. We evaluate methods based on their computing requirements as well as on their ability to recover the network structure. We find that, while most methods have a modest recovery of experimentally derived interactions based on global metrics such as Area Under the Precision Recall curve, methods are able to capture targets of regulators that are relevant to the system under study. Among the top performing methods that use only expression were SCENIC, PIDC, MERLIN or Correlation. Addition of prior biological knowledge and the estimation of transcription factor activities resulted in the best overall performance with the Inferelator and MERLIN methods that use prior knowledge outperforming methods that use expression alone. We found little to no benefit from imputation for network inference, which is further dataset-dependent. Comparisons of inferred networks for comparable bulk conditions showed that the networks inferred from scRNA-seq datasets are often better or at par with the networks inferred from bulk datasets. Our analysis should be beneficial in selecting methods for network inference. At the same time, this highlights the need for improved methods and better gold standards for regulatory network inference from scRNAseq datasets.

bioinformatics↗

bioRxiv2021

CRISPRpas: Programmable regulation of alternative polyadenylation by dCas9

Well over half of human mRNA genes produce alternative polyadenylation (APA) isoforms that differ in mRNA metabolism due to 3 UTR size changes or have variable coding potentials when coupled with alternative splicing. Aberrant APA is implicated in a growing number of human diseases. A programmable tool for APA regulation, hence, would be instrumental for understanding how APA events impact biological processes. Here, using a catalytically dead Cas9 (dCas9), we developed a method, named CRISPRpas, to alter cleavage and polyadenylation site (PAS) usage in 3 UTRs or introns. We present key features that facilitate CRISPRpas, including targeting DNA strand, distance between PAS and targeting sequence, and strength of the PAS. For intronic PAS, we additionally analyze strengths of 5 splice site and target location in intron. Our analyses implicate a dynamic competition between PAS usage and nascent RNA decay when RNA polymerase II elongation is blocked. We show modulation of APA of multiple endogenous genes including a gene that contains a single nucleotide polymorphism (SNP) that affects APA in the human population. CRISPRpas expands the CRISPR toolkit for perturbation of gene expression.

molecular biology↗

bioRxiv2021

Effect of chronic JUUL aerosol inhalation on inflammatory states of the brain, lung, heart and colon in mice

While health effects of conventional tobacco are well defined, data on vaping devices, including the most popular e-cigarette JUUL, are less established. Prior acute e-cigarette studies demonstrated inflammatory and cardiopulmonary physiology changes while chronic studies demonstrated extra-pulmonary effects, including neurotransmitter alterations in reward pathways. In this study we investigated effects of chronic flavored JUUL aerosol inhalation on inflammatory markers in brain, lung, heart, and colon. JUUL induced upregulation of cytokine and chemokine gene expression and increased HMGB1 and RAGE in the nucleus accumbens. Inflammatory gene expression increased in colon, and cardiopulmonary inflammatory responses to acute lung injury with lipopolysaccharide were exacerbated in the heart. Flavor-dependent changes in several responses were also observed. Our findings raise concerns regarding long-term risks of e-cigarette use as neuroinflammation may contribute to behavioral changes and mood disorders, while gut inflammation has been tied to poor systemic health and cardiac inflammation to development of heart disease. One Sentence SummaryChronic, daily inhalation of pod-based e-cigarette aerosols alters the inflammatory state across multiple organ systems in mice.

immunology↗

bioRxiv2020

Genetic variants for head size share genes and pathways with cancer

The size of the human head is determined by growth in the first years of life, while the rest of the body typically grows until early adulthood1. Such complex developmental processes are regulated by various genes and growth pathways2. Rare genetic syndromes have revealed genes that affect head size3, but the genetic drivers of variation in head size within the general population remain largely unknown. To elucidate biological pathways underlying the growth of the human head, we performed the largest genome-wide association study on human head size to date (N = 79,107). We identified 67 genetic loci, 50 of which are novel, and found that these loci are preferentially associated with head size and mostly independent from height. In subsequent neuroimaging analyses, the majority of genetic variants demonstrated widespread effects on the brain, whereas the effects of 17 variants could be localized to one or two specific brain regions. Through hypothesis-free approaches, we find a strong overlap of head size variants with both cancer pathways and cancer genes. Gene set analyses showed enrichment for different types of cancer and the p53, Wnt and ErbB signalling pathway. Genes overlapping or close to lead variants - such as TP53, PTEN and APC - were enriched for genes involved in macrocephaly syndromes (up to 37-fold) and high-fidelity cancer genes (up to 9-fold), whereas this enrichment was not seen for human height variants. This indicates that genes regulating early brain and cranial growth are associated with a propensity to neoplasia later in life, irrespective of height. Our results warrant further investigations of the link between head size and cancer, as well as its clinical implications in the general population.

genomics↗

bioRxiv2020

GxEsum: genotype-by-environment interaction model based on summary statistics

Genetic variation in response to the environment is fundamental in the biology of complex traits and diseases, i.e. genotype-by-environment interaction (GxE). However, existing methods are computationally demanding and infeasible to handle biobank-scale data. Here we introduce GxEsum, a method for estimating the phenotypic variance explained by genome-wide GxE based on GWAS summary statistics. Through comprehensive simulations and analysis of UK Biobank with 288,837 individuals, we show that GxEsum can handle a large-scale biobank dataset with controlled type I error rates and unbiased GxE estimates, and its computational efficiency can be hundreds of times higher than existing GxE methods.

genetics↗

bioRxiv2020

Thermal fluctuations assist mechanical signal propagation in coiled-coil proteins

Recently it has been shown that the long coiled-coil membrane tether protein Early Endosome Antigen 1 (EEA1) switches from a rigid to a flexible conformation upon binding of a signaling protein to its free end. This flexibility switch represents a novel motor-like activity, allowing EEA1 to generate a force that moves vesicles closer to the membrane they will fuse with. To elucidate how binding of a single signaling protein can globally change the stiffness of a 200 nm long chain, we propose a simplified description of the coiled-coil as a one-dimensional Frenkel-Kontorova chain. Using numerical simulations, we find that an initial perturbation of the chain can propagate along its whole length in the presence of thermal fluctuations, changing the configuration of the entire molecule and thereby affecting its stiffness. Our work sheds light onto intramolecular communication and force generation in long coiled-coil proteins.

biophysics↗

bioRxiv2020

Impact of metastatic prostate cancer on human bone marrow

Bone metastases are devastating complications of cancer. They are particularly common in prostate cancer, represent incurable disease and are refractory to immunotherapy. We sought to define distinct features of the bone marrow microenvironment by analyzing single cells from prostate cancer patients involved bone, uninvolved bone and distant bone sites as well as bone from cancer-free, orthopedic patients and healthy individuals. Metastatic prostate cancer was associated with multifaceted immune distortion, specifically exhaustion of distinct T cell subsets, appearance of macrophages with states specific to prostate cancer bone metastases. The chemokine CCL20 was notably overexpressed by myeloid cells, as was its cognate CCR6 receptor on T cells. Disruption of the CCL20-CCR6 axis in mice with syngeneic prostate bone metastases restored T cell reactivity and significantly prolonged animal survival. Comparative high resolution analysis of prostate cancer bone metastasis shows a targeted approach for relieving local immunosuppression for therapeutic effect.

cancer biology↗

bioRxiv2020

Presynaptic PTPσ organizes neurotransmitter release machinery at excitatory synapses

Leukocyte common antigen-related receptor tyrosine phosphatases (LAR-RPTPs) are evolutionarily conserved presynaptic organizers. The synaptic role of vertebrate LAR-RPTPs in vivo, however, remains unclear. This study systematically analyzed the effects of genetic deletions of LAR-RPTP genes by generating single conditional knockout (cKO) mice targeting PTP{sigma} and PTP{delta}. Although the numbers of synapses were reduced in cultured neurons deficient in individual PTPs, abnormalities in synaptic transmission, synaptic ultrastructures, and vesicle localization were observed only in PTP{sigma}-deficient neurons. Strikingly, loss of presynaptic PTP{sigma} reduced neurotransmitter release prominently at excitatory synapses, concomitant with drastic reductions in excitatory innervations onto postsynaptic target areas in vivo. However, postsynaptic PTP{sigma} deletion had no effect on excitatory synaptic strength. Furthermore, conditional deletion of PTP{sigma} in ventral CA1 specifically altered anxiety-like behaviors. Taken together, these results demonstrate that PTP{sigma} is a bona fide presynaptic adhesion molecule that controls neurotransmitter release and excitatory inputs.

neuroscience↗

bioRxiv2020

Behavioral evidence for memory replay of video episodes in macaque monkeys

Humans recall the past by replaying fragments of events temporally. Here, we demonstrate a similar effect in macaques. We trained six rhesus monkeys with a temporal-order judgement (TOJ) task and collected 5000 TOJ trials. In each trial, they watched a naturalistic video of about 10 s comprising two across-context clips, and after a 2-s delay, performed TOJ between two frames from the video. The monkeys apply a non-linear forward, time-compressed replay mechanism during the temporal-order judgement. In contrast with humans, such compression of replay is however not sophisticated enough to allow them to skip over irrelevant information by compressing the encoded video globally. We also reveal that the monkeys detect event contextual boundaries and such detection facilitates recall by an increased rate of information accumulation. Demonstration of a time-compressed, forward replay like pattern in the macaque monkeys provides insights into the evolution of episodic memory in our lineage. Impact StatementMacaque monkeys temporally compress past experiences and use a forward-replay mechanism during judgment of temporal-order between episodes.

neuroscience↗

bioRxiv2019

Selective metabolic redundancy of Gpi1 allows for specific inhibition of inflammatory Th17 cells

SummaryTargeting glycolysis has been considered therapeutically intractable owing to its essential housekeeping role. However, the context-dependent requirement for individual glycolytic steps has not been fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic redundancies can be exploited for selective targeting of disease processes.

immunology↗

bioRxiv2019

Transcriptome-wide transmission disequilibrium analysis identifies novel risk genes for autism spectrum disorder

Recent advances in consortium-scale genome-wide association studies (GWAS) have highlighted the involvement of common genetic variants in autism spectrum disorder (ASD), but our understanding of their etiologic roles, especially the interplay with rare variants, is incomplete. In this work, we introduce an analytical framework to quantify the transmission disequilibrium of genetically regulated gene expression from parents to offspring. We applied this framework to conduct a transcriptome-wide association study (TWAS) on 7,805 ASD proband-parent trios, and replicated our findings using 35,740 independent samples. We identified 31 associations at the transcriptome-wide significance level. In particular, we identified POU3F2 (p=2.1e-7), a transcription factor (TF) mainly expressed in developmental brain. TF targets regulated by POU3F2 showed a 2.1-fold enrichment for known ASD genes (p=4.6e-5) and a 2.7-fold enrichment for loss-of-function de novo mutations in ASD probands (p=7.1e-5). These results provide a clear example of the connection between ASD genes affected by very rare mutations and an unlinked key regulator affected by common genetic variations.

genetics↗

bioRxiv2019

Amygdalostriatal coupling underpins positive but not negative coloring of ambiguous affect

Humans routinely integrate affective information from multiple sources. For example, we rarely interpret an emotional facial expression devoid of context. Here, we describe the neural correlates of an affective computation that involves integrating multiple sources, by leveraging the ambiguity and subtle feature-based valence signals found in surprised faces. Using functional magnetic resonance imaging, participants reported the valence of surprised faces modulated by positive or negative sentences. Amygdala activity corresponded to the valence value assigned to each contextually modulated face, with greater activity reflecting more negative ratings. Amygdala activity did not track the valence of the faces or sentences per se. Moreover, the amygdala was functionally coupled with the nucleus accumbens only during face trials preceded by positive contextual cues. These data suggest 1) valence-related amygdala activity reflects the integrated valence values rather than the valence values of each individual component, and 2) amygdalostriatal coupling underpins positive but not negative coloring of ambiguous affect.

neuroscience↗

bioRxiv2019

Vaccination with single plasmid DNA encoding IL-12 and antigens of severe fever with thrombocytopenia syndrome virus elicits complete protection in IFNAR knockout mice

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by SFTS virus (SFTSV) infection. Despite a gradual increase of SFTS cases and high mortality in endemic regions, no specific viral therapy nor vaccine is available. Here, we developed a single recombinant plasmid DNA encoding SFTSV genes, Gn and Gc together with NP-NS fusion antigen, as a vaccine candidate. The viral antigens were fused with Fms-like tyrosine kinase-3 ligand (Flt3L) and IL-12 gene was incorporated into the plasmid to enhance cell-mediated immunity. Vaccination with the DNA provides complete protection of IFNAR KO mice upon lethal SFTSV challenge, whereas immunization with a plasmid without IL-12 gene resulted in partial protection. Since we failed to detect antibodies against surface glycoproteins, Gn and Gc, in the immunized mice, antigen-specific cellular immunity, as confirmed by enhanced antigen-specific T cell responses, might play major role in protection. Finally, we evaluated the degree of protective immunity provided by protein immunization of the individual glycoprotein, Gn or Gc. Although both protein antigens induced a significant level of neutralizing activity against SFTSV, Gn vaccination resulted in relatively higher neutralizing activity and better protection than Gc vaccination. However, both antigens failed to provide complete protection. Given that DNA vaccines have failed to induce sufficient immunogenicity in human trials when compared to protein vaccines, optimal combinations of DNA and protein elements, proper selection of target antigens, and incorporation of efficient adjuvant, need to be further investigated for SFTSV vaccine development.\n\nAuthor summarySevere fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne infection endemic to East Asia including China, Korea, and Japan. Gradual rise of disease incidence and relatively high mortality have become a serious public health problem in the endemic countries. In this study, we developed a recombinant plasmid DNA encoding four antigens, Gn, Gc, NP, and NS, of SFTS virus (SFTSV) as a vaccine candidate. In order to enhance cell-mediated immunity, the viral antigens were fused with Flt3L and IL-2 gene was incorporated into the plasmid. Immunization with the DNA vaccine provides complete protection against lethal SFTSV infection in IFNAR KO mice. Antigen-specific T cell responses might play a major role in the protection since we observed enhanced T cell responses specific to the viral antigens but failed to detect neutralizing antibody in the immunized mice. When we immunized with either viral glycoprotein, Gn protein induced relatively higher neutralizing activity and better protection against SFTSV infection than Gc antigen, but neither generated complete protection. Therefore, an optimal combination of DNA and protein elements, as well as proper selection of target antigens, might be required to produce an effective SFTSV vaccine.

microbiology↗

bioRxiv2019

The histone chaperone FACT induces Cas9 multi-turnover behavior and modifies genome manipulation in human cells

Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Using a proximity labeling system for unbiased detection of transient protein interactions in cell-free Xenopus laevis egg extract, we identified the dimeric histone chaperone FACT as an interactor of substrate-bound Cas9. Immunodepletion of FACT subunits from extract potently inhibits Cas9 unloading and converts Cas9s activity from multi-turnover to single-turnover. In human cells, depletion of FACT delays genome editing and alters the balance between indel formation and homology directed repair. Depletion of FACT also increases epigenetic marking by dCas9-based transcriptional effectors with concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.

cell biology↗

bioRxiv2019

Pyrazinamide triggers degradation of its target aspartate decarboxylase

The introduction of pyrazinamide (PZA) in the tuberculosis drug regimen shortened treatment from 12 to 6 months 1. PZA is a prodrug that is activated by a Mycobacterium tuberculosis (Mtb) amidase to release its bioactive component pyrazinoic acid (POA) 2. Aspartate decarboxylase PanD, a proenzyme activated by autocatalytic cleavage (Supplementary Fig. 1A, 3) and required for Coenzyme A (CoA) biosynthesis, emerged as a target of POA 4-7. In vitro and in vivo screening to isolate spontaneous POA-resistant Mtb mutants identified missense mutations in either panD or the unfoldase clpC1, encoding a component of the caseinolytic protease ClpC1-ClpP 4,6-9. Overexpression and binding studies of PanD or ClpC1 pointed to PanD as the direct target of POA whereas clpC1 mutations appeared to indirectly cause resistance 4,5,7,9,10. Indeed, supplementing growth media with CoA precursors downstream of the PanD catalyzed step conferred POA resistance 4,7,11. Metabolomic analyses and biophysical studies using recombinant proteins confirmed targeting of PanD by POA 5. However, the exact molecular mechanism of PanD inhibition by POA remained unknown. While most drugs act by inhibiting protein function upon target binding, we show here that POA is not a bona fide enzyme inhibitor. Rather, POA binding to PanD triggers degradation of the protein by ClpC1-ClpP. Thus, the old tuberculosis drug PZA promotes degradation of its target. While novel for an antibacterial, drug-induced target degradation has recently emerged as a strategy in drug discovery across disease indications. Our findings provide the basis for the rational discovery of next generation PZA.

microbiology↗

bioRxiv2019

Species-specific InDel markers for authentication of the Korean herbs Zanthoxylum schinifolium and Zanthoxylum piperitum

ObjectiveZanthoxylum schinifolium and Zanthoxylum piperitum are the sources of the well-known traditional Korean herbal medicines \"sancho\" (prickly ash) and \"chopi\" (Korean pepper), respectively. Sancho and chopi are often indiscriminately mixed due to the similar appearance of the herbal materials when used as spices and herbal medicines. Moreover, commercial sancho and chopi products often contain adulterants, which could compromise drug efficacy and safety.\n\nMethodsIn this study, we developed hypervariable InDel markers to distinguish between sancho and chopi products by comparing the complete chloroplast genome sequences of four Zanthoxylum species deposited in NCBI GenBank.\n\nResultsComparative analyses of the nucleotide diversity (Pi) of these Zanthoxylum genomes revealed four hypervariable divergent sites (trnH-psbA, psbZ-trnG, trnfM-rps14, and trnF-ndhK) with Pi > 0.025 among 520 windows. Of these four regions, including two genic and two intergenic regions, only psbZ-trnG yielded accurate PCR amplification results between commercial sancho and chopi products from the Korean herbal medicine market. We therefore selected psbZ-trnG, an InDel-variable locus with high discriminatory powers, as a candidate DNA barcode locus.\n\nConclusionThis InDel marker could be used as a valuable, simple, and efficient tool for identifying these medicinal herbs, thereby increasing the safety of these spices and herbal materials in the commercial market.

bioinformatics↗