Biology subjects

Kim, S.

Publications and source records attributed to Kim, S..

At least 595 records · Page 33Linked to original sources

bioRxiv2020

Epigenetic engineering of yeast reveals dynamic molecular adaptation to methylation stress and genetic modulators of specific DNMT3 family members

Cytosine methylation is a ubiquitous modification in mammalian DNA generated and maintained by several DNA methyltransferases (DNMTs) with partially overlapping functions and genomic targets. To systematically dissect the factors specifying each DNMTs activity, we engineered combinatorial knock-in of human DNMT genes in Komagataella phaffii, a yeast species lacking endogenous DNA methylation. Time-course expression measurements captured dynamic network-level adaptation of cells to DNMT3B1-induced DNA methylation stress and showed that coordinately modulating the availability of S-adenosyl methionine (SAM), the essential metabolite for DNMT-catalyzed methylation, is an evolutionarily conserved epigenetic stress response, also implicated in several human diseases. Convolutional neural networks trained on genome-wide CpG-methylation data learned distinct sequence preferences of DNMT3 family members. A simulated annealing interpretation method resolved these preferences into individual flanking nucleotides and periodic poly(A) tracts that rotationally position highly methylated cytosines relative to phased nucleosomes. Furthermore, the nucleosome repeat length defined the spatial unit of methylation spreading. Gene methylation patterns were similar to those in mammals, and hypo- and hypermethylation were predictive of increased and decreased transcription relative to control, respectively, in the absence of mammalian readers of DNA methylation. Introducing controlled epigenetic perturbations in yeast thus enabled characterization of fundamental genomic features directing specific DNMT3 proteins.

genomics↗

bioRxiv2020

Continuous detection of glucose and insulin in live animals

Real-time biosensors that can continuously measure circulating biomolecules in vivo would provide valuable insights into a patients health status and their response to therapeutics even when there is considerable variability in pharmacokinetics and pharmacodynamics across patient populations. Unfortunately, current real-time biosensors are limited to a handful of analytes (e.g. glucose and blood oxygen) and are limited in sensitivity (high nanomolar). In this work, we describe a general approach for continuously and simultaneously measuring multiple analytes with picomolar sensitivity and sub-second temporal resolution. As exemplars, we report the simultaneous detection of glucose and insulin at picomolar concentrations in live diabetic rats. Using our system, we demonstrate the capacity to resolve inter-individual differences in the pharmacokinetic responses to insulin and discriminate profiles from different insulin formulations at a high temporal resolution. Critically, our approach is general and could be readily modified to continuously and simultaneously measure other circulating analytes in vivo by swapping the affinity reagents, thus making it a versatile tool for biomedical research.

bioengineering↗

bioRxiv2020

AAV Ablates Neurogenesis in the Adult Murine Hippocampus

Recombinant adeno-associated virus (rAAV) has been widely used as a viral vector across mammalian biology and has been shown to be safe and effective in human gene therapy. We demonstrate that neural progenitor cells (NPCs) and immature dentate granule cells (DGCs) within the adult murine hippocampus are particularly sensitive to rAAV-induced cell death. Cell loss is dose dependent and nearly complete at experimentally relevant viral titers. rAAV-induced cell death is rapid and persistent, with loss of BrdU-labeled cells within 18 hours post-injection and no evidence of recovery of adult neurogenesis at 3 months post-injection. The remaining mature DGCs appear hyperactive 4 weeks post-injection based on immediate early gene expression, consistent with previous studies investigating the effects of attenuating adult neurogenesis. In vitro application of AAV or electroporation of AAV2 inverted terminal repeats (ITRs) is sufficient to induce cell death. Efficient transduction of the dentate gyrus (DG)--without ablating adult neurogenesis--can be achieved by injection of rAAV2-retro serotyped virus into CA3. rAAV2-retro results in efficient retrograde labeling of mature DGCs and permits in vivo 2-photon calcium imaging of dentate activity while leaving adult neurogenesis intact. These findings expand on recent reports implicating rAAV-linked toxicity in stem cells and other cell types and suggest that future work using rAAV as an experimental tool in the DG and as a gene therapy for diseases of the central nervous system (CNS) should be carefully evaluated.

neuroscience↗

bioRxiv2020

Complement and coagulation cascades are potentially involved in dopaminergic neurodegeneration in alpha-synuclein-based mouse models of Parkinson's disease

Parkinsons disease (PD) is the second most common neurodegenerative disorder that results in motor dysfunction and eventually, cognitive impairment. -Synuclein protein has been known to be the most culprit protein, but the underlying pathological mechanism still remains to be elucidated. As an effort to clarify the pathogenesis mechanism by -synuclein, various PD mouse models with -synuclein overexpression have been developed. However, the systemic analysis of protein abundance change by the overexpressed -synuclein in the whole proteome level has been still lacking. To address this issue, we established two sophisticated mouse models of PD by injecting -synuclein preformed fibrils (PFF) or by inducing overexpression of human A53T -synuclein to discover overlapping pathways, which could be altered in the two different types of PD mouse model. For more accurate quantification of mouse brain proteome, stable isotope labeling with amino acid in mammal-based quantification was implemented. As a result, we have successfully identified a total of 8,355 proteins from both of the mouse models; [~]6,800 and [~]7,200 proteins from -synuclein PFF injected mice and human A53T -synuclein transgenic mice, respectively. From the pathway analysis of the differentially expressed proteins in common, the complement and coagulation cascade pathway were determined as the most enriched ones. This is the first study that highlights the significance of the complement and coagulation pathway in the pathogenesis of PD through proteome analyses with two sophisticated mouse models of PD.

neuroscience↗

bioRxiv2020

Competitive interactions between motor and episodic memory systems during interleaved learning

Distinct motor and episodic memory systems are widely thought to compete during memory consolidation and retrieval, yet the nature of their interactions during learning is less clear. Motor learning is thought to depend on contributions from both systems, with the episodic system supporting rapid updating and the motor system supporting gradual tuning of responses by feedback. However, this competition has been identified when both systems are engaged in learning the same material (motor information), and so competition might be emphasized. We tested whether such competition also occurs when learning involved separate episodic-memory and motor information presented distinctly but yet in close temporal proximity. We measured behavioral and brain-activity correlates of motor-episodic competition during learning using a novel task with interleaved motor-adaptation and episodic-learning demands. Despite unrelated motor versus episodic information and temporal segregation, motor learning interfered with episodic learning and episodic learning interfered with motor learning. This reciprocal competition was tightly coupled to corresponding reductions of fMRI activity in motor versus episodic learning systems. These findings suggest that distinct motor and episodic learning systems compete even when they are engaged by system-specific demands in close temporal proximity during memory formation.

neuroscience↗

bioRxiv2020

Crowdsourced mapping of unexplored target space of kinase inhibitors

Despite decades of intensive search for compounds that modulate the activity of particular targets, there are currently small-molecules available only for a small proportion of the human proteome. Effective approaches are therefore required to map the massive space of unexplored compound-target interactions for novel and potent activities. Here, we carried out a crowdsourced benchmarking of predictive models for kinase inhibitor potencies across multiple kinase families using unpublished bioactivity data. The top-performing predictions were based on kernel learning, gradient boosting and deep learning, and their ensemble resulted in predictive accuracy exceeding that of kinase activity assays. We then made new experiments based on the model predictions, which further improved the accuracy of experimental mapping efforts and identified unexpected potencies even for under-studied kinases. The open-source algorithms together with the novel bioactivities between 95 compounds and 295 kinases provide a resource for benchmarking new prediction algorithms and for extending the druggable kinome.

biochemistry↗

bioRxiv2020

Label-free three-dimensional observations and quantitative characterisation of on-chip vasculogenesis using optical diffraction tomography

Label-free, three-dimensional (3D) quantitative observations of on-chip vasculogenesis were achieved using optical diffraction tomography. Exploiting 3D refractive index maps as an intrinsic imaging contrast, the vascular structures, multicellular activities, and subcellular organelles of endothelial cells were imaged and analysed throughout vasculogenesis to characterise mature vascular networks without exogenous labelling. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/892620v1_ufig1.gif" ALT="Figure 1"> View larger version (98K): org.highwire.dtl.DTLVardef@12a786eorg.highwire.dtl.DTLVardef@14892dforg.highwire.dtl.DTLVardef@1a28091org.highwire.dtl.DTLVardef@1a6e9df_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2019

Double dissociation of fMRI activity in the caudate nucleus supports de novo motor skill learning

Motor skill learning involves a complex process of generating novel movement patterns typically guided by evaluative feedback such as reward. Many studies have suggested that two separate circuits in the basal ganglia, rostral and caudal, are implicated in early goal-directed and later automatic stages of motor skill learning, respectively. However, there remains much to be elucidated about the respective involvement of the basal ganglia circuits in learning motor skills from scratch, which requires learning arbitrary action-outcome associations. To investigate this issue, we conducted a novel human fMRI experiment in which the participants learned to control a computer cursor on a screen by manipulating their right fingers. The experiment consisted of two fMRI sessions separated by five behavioral training sessions over multiple days. We discovered a double dissociation of fMRI activities in the rostral and caudal caudate nucleus, which were associated with skill performance in the early and late stages of learning. Moreover, we found that cognitive and sensorimotor cortico-caudate interactions distinctively predicted individual learning performance. In line with recent non-human primate studies, our results support the existence of parallel cortico-caudate networks involved in goal-directed and automatic stages of de novo motor skill learning.

neuroscience↗

bioRxiv2019

Insights into the involvement of spliceosomal mutations in myelodysplastic disorders from an analysis of SACY-1/DDX41 in Caenorhabditis elegans

Mutations affecting spliceosomal proteins are frequently found in hematological malignancies, including myelodysplastic syndromes and acute myeloid leukemia. DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase found to be recurrently mutated in inherited myelodysplastic syndromes and in relapsing cases of acute myeloid leukemia. The genetic properties and genomic impacts of disease-causing missense mutations in DDX41 and other spliceosomal proteins have been uncertain. Here we conduct a comprehensive molecular genetic analysis of the C. elegans DDX41 ortholog, SACY-1. Our results reveal general essential functions for SACY-1 in both the germline and the soma, as well as specific functions affecting germline sex determination and cell cycle control. Certain sacy-1/DDX41 mutations, including the R525H human oncogenic variant, confer antimorphic activity, suggesting that they compromise the function of the spliceosome. Consistent with these findings, sacy-1 exhibits synthetic lethal interactions with several spliceosomal components, and biochemical analyses suggest that SACY-1 is a component of the C. elegans spliceosome. We used the auxin-inducible degradation system to analyze the impact of SACY-1 on the transcriptome using RNA sequencing. SACY-1 depletion impacts the transcriptome through splicing-independent and splicing-dependent mechanisms. The observed transcriptome changes suggest that disruption of spliceosomal function induces a stress response. Altered 3 splice site usage represents the predominant splicing defect observed upon SACY-1 depletion, consistent with a role for SACY-1 as a second-step splicing factor. Missplicing events appear more prevalent in the soma than the germline, suggesting that surveillance mechanisms protect the germline from aberrant splicing. Author SummaryMutations affecting spliceosomal proteins are frequently found in hematological malignancies. DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase recurrently mutated in inherited and relapsing myelodysplastic syndromes and acute myeloid leukemia. The genetic properties and genomic impacts of disease-causing mutations in spliceosomal proteins have been uncertain. Here we conduct a comprehensive molecular genetic analysis of the C. elegans DDX41 ortholog, SACY-1. Our results reveal that multiple sacy-1/DDX41 missense mutations, including the R525H human oncogenic variant, exhibit antimorphic activity that likely compromises the function of the spliceosome. The genomic consequences of SACY-1 depletion include splicing-splicing-independent and splicing-dependent alterations in the transcriptome.

genetics↗

bioRxiv2019

Natural scene statistics predict how humans pool information across space in the estimation of surface tilt

Visual systems estimate the three-dimensional (3D) structure of scenes from information in two-dimensional (2D) retinal images. Visual systems use multiple sources of information to improve the accuracy of these estimates, including statistical knowledge of the probable spatial arrangements of natural scenes. Here, we examine how 3D surface tilts are spatially related in real-world scenes, and show that humans pool information across space when estimating surface tilt in accordance with these spatial relationships. We develop a hierarchical model of surface tilt estimation that is grounded in the statistics of tilt in natural scenes and images. The model computes a global tilt estimate by pooling local tilt estimates within an adaptive spatial neighborhood. The spatial neighborhood in which local estimates are pooled changes according to the value of the local estimate at a target location. The hierarchical model provides more accurate estimates of groundtruth tilt in natural scenes and provides a better account of human performance than the local model. Taken together, the results imply that the human visual system pools information about surface tilt across space in accordance with natural scene statistics.

neuroscience↗

bioRxiv2019

MyD88-Syk axis is a critical determinant for inflammatory response in macrophages

Inhibition of Syk or MyD88 decreased generation of reactive oxygen species (ROS)/reactive nitrogen species (RNS) and consequent ROS/RNS-induced phagocytic activity in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Syk inhibition downregulated expression of ROS/RNS-generating enzymes by inhibiting the nuclear factor-kappa B (NF-kappa?B) signaling pathway and phagocytic activity by suppressing suppressor of cytokine signaling 1 (SOCS1) via its nitration in the LPS-stimulated RAW264.7 cells. Inhibition of ROS/RNS generation suppressed SOCS1 nitration, leading to a decrease in the phagocytic activity. Syk was activated by the interaction with MyD88, and the tyrosine 58 residue (Y58) in the hemi-immunoreceptor tyrosine-based activation motif (ITAM) of MyD88 was critical for interaction and consequent activation of Syk in macrophages. Src activated MyD88 by phosphorylation at Y58 via the Src kinase domain. Moreover, LPS-induced formation of filamentous actin (F-actin) and Ras-related C3 botulinum toxin substrate 1 (Rac1) activation induced Src activation. Conclusivley, these results suggest that the MyD88-Syk axis plays a pivotal role in macrophage-mediated inflammatory responses by inducing ROS/RNS generation and phagocytic activity via activation of Src and its upstream stimulators, F-actin and Rac1.

immunology↗

bioRxiv2019

Identification of conserved gene regulatory networks that integrate environmental sensing and growth in the root cambium

Cambium drives lateral growth of stems and roots, contributing to diverse plant growth forms. Root crop is one outstanding example of the cambium-driven growth. To understand its molecular basis, we used radish to generate a compendium of root tissue- and stage-specific transcriptomes from two contrasting inbred lines in root growth. Expression patterns of key cambium regulators and hormone signaling components were validated. Clustering and GO enrichment analyses of radish datasets followed by comparative analysis against the newly established Arabidopsis early cambium data revealed evolutionary conserved stress-response transcription factors that might intimately control the cambium. Indeed, in vivo network made of selected stress-response and cambium regulators indicated ERF-1 as a potential key checkpoint of cambial activities, explaining how the cambium-driven growth is altered in response to environmental changes. Together, this study provides rich information about dynamic gene expression changes along the cambium-driven root growth with future engineering schemes for crop yields.

developmental biology↗

bioRxiv2019

Metastatic function of METTL18 in breast cancer via actin methylation and Src

Recently, a SET domain containing 3 (SETD3) was identified as an actin histidine methyltransferase, functioning to control replication and pathogenesis in multiple mouse models for enterovirus infection as well as the regulation of smooth muscle contractility linked to primary dystocia. Here, in this study, we report another type of actin histidine methyltransferase, METTL18, that regulates the metastatic potential of breast cancer in human. Among methyltransferases, METTL18 was highly amplified in human breast cancer. In particular, poor prognosis was associated with high expression of METTL18 in HER2-negative breast cancer patients. This gene product was also found to be a critical component of metastatic responses. Loss of METTL18 expression significantly reduced metastatic responses of breast tumor cells both in vitro and in vivo. Mechanistically, it was observed that METTL18 increased actin polymerization, upregulated complex formation with HSP90AA1 and Src, enhanced the activity of an intermediate form of Src with tyrosine phosphorylation at both Y416 and Y527, and induced cellular metastatic responses, including morphological change, migration, and invasion of MDA-MB-231 cells in vitro and in mice. Methylated actin at His73 served as a critical site for interaction with HSP90AA1 and Src to activate p85/PI3K and STAT3. Our findings suggest that METTL18 plays critical roles in metastatic responses of HER2-negative breast cancer cells via actin polymerization and the generation of an intermediate form of Src.

cancer biology↗

bioRxiv2019

External and Internal Signal-to-noise Ratios Alter Timing and Location of Cortical Activities During Speech-in-noise Perception

Understanding speech in noise (SiN) is a complex task that recruits multiple cortical subsystems. There is a variance in individuals ability to understand SiN that cannot be explained by simple hearing profiles, which suggests that central factors may underlie the variance in SiN ability. Here, we elucidated a few cortical functions involved during a SiN task and their contributions to individual variance using both within- and across-subject approaches. Through our within-subject analysis of source-localized electroencephalography, we investigated how acoustic signal-to-noise ratio (SNR) alters cortical evoked responses to a target word across the speech recognition areas, finding stronger responses in left supramarginal gyrus (SMG, BA40 the dorsal lexicon area) with quieter noise. Through an individual differences approach, we found that listeners show different neural sensitivity to the background noise and target speech, reflected in the amplitude ratio of earlier auditory-cortical responses to speech and noise, named as an internal SNR. Listeners with better internal SNR showed better SiN performance. Further, we found that the post-speech time SMG activity explains a further amount of variance in SiN performance that is not accounted for by internal SNR. This result demonstrates that at least two cortical processes contribute to SiN performance independently: pre-target time processing to attenuate neural representation of background noise and post-target time processing to extract information from speech sounds.

neuroscience↗

bioRxiv2019

A model for the emergence of RNA from a prebiotically plausible mixture of ribonucleotides, arabinonucleotides and 2’-deoxynucleotides

The abiotic synthesis of ribonucleotides is thought to have been an essential step towards the emergence of the RNA world. However, it is likely that the prebiotic synthesis of ribonucleotides was accompanied by the simultaneous synthesis of arabinonucleotides, 2'-deoxyribonucleotides, and other variations on the canonical nucleotides. In order to understand how relatively homogeneous RNA could have emerged from such complex mixtures, we have examined the properties of arabinonucleotides and 2'-deoxyribonucleotides in nonenzymatic template-directed primer extension reactions. We show that nonenzymatic primer extension with activated arabinonucleotides is much less efficient than with activated ribonucleotides, and furthermore that once an arabinonucleotide is incorporated, continued primer extension is strongly inhibited. As previously shown, 2'-deoxyribonucleotides are also less efficiently incorporated in primer extension reactions, but the difference is more modest. Experiments with mixtures of nucleotides suggest that the coexistence of ribo- and arabino-nucleotides does not impede the copying of RNA templates. Moreover, chimeric oligoribonucleotides containing 2'-deoxy- or arabino-nucleotides are effective templates for RNA synthesis. We propose that the initial genetic polymers were random sequence chimeric oligonucleotides formed by untemplated polymerization, but that template copying chemistry favored RNA synthesis; multiple rounds of replication may have led to pools of oligomers composed mainly of RNA.

biochemistry↗

bioRxiv2019

Plexin-Bs enhance their GAP activity with a novel activation switchloop

Plexins receive guidance cues from semaphorin ligands and transmit their signal through the plasma membrane. This family of proteins is unique amongst single-pass transmembrane receptors as their intracellular regions interact directly with several small GTPases, which regulate cytoskeletal dynamics and cell adhesion. Here, we characterize the GTPase Activating Protein (GAP) function of Plexin-B1 and find that a cooperative GAP activity towards the substrate GTPase, Rap1b, is associated with the N-terminal Juxtamembrane region of Plexin-B1. Importantly, we unveil an activation mechanism of Plexin-B1 by identifying a novel functional loop which partially blocks Rap1b entry to the plexin GAP domain. Consistent with the concept of allokairy developed for other systems, Plexin-B activity is increased by an apparent substrate mediated cooperative effect. Simulations and mutagenesis suggest the repositioned JM conformation is stabilized by the new activation switch loop when the active site is occupied, giving rise to faster enzymatic turnover and cooperative behavior. The biological implications, essentially of a threshold behavior for cell migration are discussed.

biophysics↗

bioRxiv2019

Phytoene Synthase 2 Can Compensate for the Absence of Psy1 in Pepper Fruit (Capsicum annuum)

Phytoene synthase 1 (PSY1) and Capsanthin-capsorubin synthase (CCS) are two major genes responsible for fruit color variation in pepper (Capsicum spp.), although fruit colors cannot be explained by variations in these two genes alone. Furthermore, the role of PSY2 in fruit color development in pepper is unknown. Here, we used a systemic approach to discover the genetic factors responsible for the yellow fruit color of C. annuum MicroPep Yellow (MY) and to reveal the role of PSY2 in fruit color. We detected a complete deletion of PSY1 and a retrotransposon insertion in CCS in MY. Despite the loss of PSY1 and CCS function, the MY and mutant F2 plants from a cross between MY and the MicroPep Red (MR) accumulated basal levels of carotenoids, indicating that other PSY genes may complement the loss of PSY1. A qRT-PCR analysis demonstrated that PSY2 is constitutively expressed in both MR and MY fruits, and a color complementation assay using Escherichia coli revealed that PSY2 is capable of biosynthesizing a carotenoid. Virus-induced gene silencing of PSY2 in MY resulted in white fruits. These findings suggest that PSY2 can compensate for the absence of PSY1 in fruit, resulting in the yellow color of MY fruits.\n\nHighlightWe reveal the novel function of PSY2 in the development of yellow pepper fruit coloration using a psy1 knockout mutant. This gene function was not previously identified in solanaceous crops.

plant biology↗

bioRxiv2019

A topological analysis of difference topology experiments of condensin with Topoisomerases II

An experimental technique called difference topology combined with the mathematics of tangle analysis has been used to unveil the structure of DNA bound by the Mu transpososome. However, difference topology experiments can be difficult and time-consuming. We discuss a modification that greatly simplifies this experimental technique. This simple experiment involves using a topoisomerase to trap DNA crossings bound by a protein complex and then running a gel to determine the crossing number of the knotted product(s). We develop the mathematics needed to analyze the results and apply these results to model the topology of DNA bound by 13S condensin and by the condensin MukB.\n\nSUMMARY STATEMENTTangles are used to model protein-DNA complexes: A 3-dimensional ball represents protein while strings embedded in this ball represent protein-bound DNA. We use this simple model to analyze experimental results.

molecular biology↗