Biology subjects

Kim, G.

Publications and source records attributed to Kim, G..

At least 91 records · Page 5Linked to original sources

bioRxiv2023

Chiral Graphene Quantum Dots Enhanced Drug Loading into Exosomes

As nanoscale extracellular vesicles secreted by cells, exosomes have enormous potential as safe and effective vehicles to deliver drugs into lesion locations. Despite promising advances with exosome-based drug delivery systems, there are still challenges to drug loading into exosome, which hinder the clinical applications of exosomes. Herein, we report an exogenous drug-agnostic chiral graphene quantum dots (GQDs) exosome-loading platform, based on chirality matching with the exosome lipid bilayer. Both hydrophobic and hydrophilic chemical and biological drugs can be functionalized or adsorbed onto GQDs by {pi}-{pi} stacking and van der Waals interactions. By tuning the ligands and GQD size to optimize its chirality, we demonstrate drug loading efficiency of 66.3% and 64.1% for Doxorubicin and siRNA, which is significantly higher than other reported exosome loading techniques.

bioengineering↗

bioRxiv2023

Structural insights into ubiquitin chain cleavage by Legionella ovarian tumor deubiquitinases

Although ubiquitin is found only in eukaryotes, several pathogenic bacteria and viruses possess proteins that hinder the host ubiquitin system. Legionella, a gram-negative intracellular bacterium, possesses an ovarian tumor (OTU) family of deubiquitinases (Lot DUBs). Herein, we describe the molecular characteristics of Lot DUBs. We elucidated the structure of LotA OTU1 domain and revealed that entire Lot DUBs possess a characteristic extended helical lobe (EHL) that is not found in other OTU-DUBs. The structural topology of EHL is the same throughout the Lot family, and it provides an S1' ubiquitin-binding site. Moreover, the catalytic triads of Lot DUBs resemble those of the A20-type OTU-DUBs. Furthermore, we revealed a unique mechanism by which LotA OTU domains cooperate together to distinguish the length of the chain and preferentially cleaves longer K48-linked polyubiquitin chains. The LotA OTU1 domain itself cleaves K6-linked ubiquitin chains, while it is also essential for assisting the cleavage of longer K48-linked polyubiquitin chains by the OTU2 domain. Thus, this study provides novel insights into the structure and mechanism of action of Lot DUBs.

biochemistry↗

bioRxiv2022

Global analysis of the yeast knock-out phenome

Genome-wide phenotypic screens in the budding yeast Saccharomyces cerevisiae have produced the largest, richest and most systematic phenotypic description of any organism. Such an achievement was enabled by the development of highly scalable phenotypic assays and construction of the yeast knock-out (YKO) collection, comprising ~5,000 isogenic strains each deleted for exactly one open reading frame. Systematic screening of the YKO collection led to ~500 publications describing ~14,500 phenotypes capturing nearly every aspect of yeast biology. Yet, integrative analyses of this rich data source have been virtually impossible due to the lack of a central repository and consistent meta-data annotations. Here, we describe the aggregation, harmonization and analysis of all published phenotypic screens of the YKO collection, which we refer to as the Yeast Phenome (www.yeastphenome.org). To demonstrate the power of data integration and illustrate how much it facilitates the generation of testable hypotheses, we present three discoveries uniquely enabled by Yeast Phenome. First, we use the variation in the number of phenotypes per gene to identify tryptophan homeostasis as a central point of vulnerability to a wide range of chemical compounds, including FDA-approved drugs. Second, using phenotypic profiles as a tool for predicting gene function, we identify and validate the role of YHR045W as a novel regulator of ergosterol biosynthesis and DNA damage response, and YGL117W as a new member of the aromatic amino acid biosynthesis pathway. Finally, we describe a surprising exponential relationship between phenotypic similarity and intergenic distance in both yeast and human genomes. This relationship, which stretches as far as 380 kb in yeast and 100 Mb in humans, suggests that gene positions are optimized for function to a much greater extent than appreciated previously. Overall, we show that Yeast Phenome enables systematic enquiries into the nature of gene-gene and gene-phenotype relationships and is an important new resource for systems biology.

systems biology↗

bioRxiv2022

Metabolic consequences of polyphosphate synthesis and imminent phosphate limitation

Cells stabilize intracellular inorganic phosphate (Pi) to compromise between large biosynthetic needs and detrimental bioenergetic effects of Pi. Pi homeostasis in eukaryotes employs SPXs domains, which are receptors for inositol pyrophosphates. We explored how polymerization and storage of Pi in acidocalcisome-like vacuoles supports S. cerevisiae metabolism and how these cells recognize Pi scarcity. Whereas Pi starvation affects numerous metabolic pathways, beginning Pi scarcity affects few metabolites. These include inositol pyrophosphates and ATP, a low-affinity substrate for inositol pyrophosphate-synthesizing kinases. Declining ATP and inositol pyrophosphates may thus be indicators of impending Pi limitation. Actual Pi starvation triggers accumulation of the purine synthesis intermediate 5- aminoimidazole-4-carboxamide ribonucleotide (AICAR), which activates Pi-dependent transcription factors. Cells lacking polyphosphate show Pi starvation features already under Pi-replete conditions, suggesting that vacuolar polyphosphate supplies Pi for metabolism even when Pi is abundant. However, polyphosphate deficiency also generates unique metabolic changes that are not observed in starving wildtype cells. Polyphosphate in acidocalcisome-like vacuoles may hence be more than a global phosphate reserve and channel Pi to preferred cellular processes. Abstract importanceCells must strike a delicate balance between the high demand of inorganic phosphate (Pi) for synthesizing nucleic acids and phospholipids, and its detrimental bioenergetic effects by reducing the free energy of nucleotide hydrolysis. The latter may stall metabolism. Therefore, microorganisms manage the import and export of phosphate, its conversion into osmotically inactive inorganic polyphosphates, and their storage in dedicated organelles, acidocalcisomes. Here, we provide novel insights into metabolic changes that cells may use to signal declining phosphate availability in the cytosol and differentiate it from actual phosphate starvation. We also analyze the role of acidocalcisome-like organelles in phosphate homeostasis. This uncovers an unexpected role of the polyphosphate pool in these organelles under phosphate-rich conditions, indicating that its metabolic roles go beyond that of a phosphate reserve for surviving starvation.

cell biology↗

bioRxiv2022

Statistically unbiased prediction enables accurate denoising of voltage imaging data

Here we report SUPPORT (Statistically Unbiased Prediction utilizing sPatiOtempoRal information in imaging daTa), a self-supervised learning method for removing Poisson-Gaussian noise in voltage imaging data. SUPPORT is based on the insight that a pixel value in voltage imaging data is highly dependent on its spatially neighboring pixels in the same time frame, even when its temporally adjacent frames do not provide useful information for statistical prediction. Such spatiotemporal dependency is captured and utilized to accurately denoise voltage imaging data in which the existence of the action potential in a time frame cannot be inferred by the information in other frames. Through simulation and experiments, we show that SUPPORT enables precise denoising of voltage imaging data while preserving the underlying dynamics in the scene.

neuroscience↗

bioRxiv2022

Triangulation reduces the polygon of error for the history of Transeurasian

In a recent study we used an interdisciplinary approach combining linguistics, archaeology and genetics to analyse the Transeurasian languages1. Our analysis concluded that the early dispersals of these languages were driven by agriculture. A preprint published on this server presents objections to the Transeurasian hypothesis and its association with farming dispersals2. However, close inspection of that text reveals numerous misinterpretations and inconsistencies. In the interest of furthering scientific debate over Transeurasian language and population history, we address the critiques, revising datasets and fine-tuning approaches. The linguistic critique questions the quantity and quality of our datasets. Here we show that the number of surviving cognate sets for Transeurasian is in line with that for well-established language families. In addition, we find that Tian et al.s failure to reject a core of regularly corresponding cognates in the basic vocabulary creates ground for a consensus about the genealogical relatability of the Transeurasian languages. The archaeological critique attempts a re-analysis of one Bayesian test using re-scored data only for northern China. Over half of the suggested re-scorings contain inconsistencies and it is not explained why the re-analysis retains the original data for sites outside northern China, comprising almost 60% of the total. More importantly, the sweeping claim that there is no evidence supporting the prehistoric migrations analysed in our study is not backed by any discussion of the archaeological record. With respect to genetics, the preprint claims a re-analysis showing that the data do not conclusively support the farming-driven dispersal of Turkic, Mongolian, and Tungusic, nor the two-wave spread of farming to Korea. In fact, the only genetic re-analysis presented is limited to samples from Korea and Japan and does not contradict our original conservative modelling of Neolithic individuals with Hongshan and our Bronze Age ones with Upper Xiajiadian. In sum, in bringing multiple lines of evidence together through triangulation, we gained a more balanced and richer understanding of Transeurasian dispersals than each discipline could provide individually. Our research doubtless leaves room for improvement but we remain confident that triangulation did not fail, but rather brought us a step closer to understanding the history of the Transeurasian languages.

evolutionary biology↗

bioRxiv2022

High sensitivity single cell RNA sequencing with split pool barcoding

Single cell RNA sequencing (scRNA-seq) has become a core tool for researchers to understand biology. As scRNA-seq has become more ubiquitous, many applications demand higher scalability and sensitivity. Split-pool combinatorial barcoding makes it possible to scale projects to hundreds of samples and millions of cells, overcoming limitations of previous droplet based technologies. However, there is still a need for increased sensitivity for both droplet and combinatorial barcoding based scRNA-seq technologies. To meet this need, here we introduce an updated combinatorial barcoding method for scRNA-seq with dramatically improved sensitivity. To assess performance, we profile a variety of sample types, including cell lines, human peripheral blood mononuclear cells (PBMCs), mouse brain nuclei, and mouse liver nuclei. When compared to the previously best performing approach, we find up to a 2.6-fold increase in unique transcripts detected per cell and up to a 1.8-fold increase in genes detected per cell. These improvements to transcript and gene detection increase the resolution of the resulting data, making it easier to distinguish cell types and states in heterogeneous samples. Split-pool combinatorial barcoding already enables scaling to millions of cells, the ability to perform scRNA-seq on previously fixed and frozen samples, and access to scRNA-seq without the need to purchase specialized lab equipment. Our hope is that by combining these previous advantages with the dramatic improvements to sensitivity presented here, we will elevate the standards and capabilities of scRNA-seq for the broader community.

genomics↗

bioRxiv2022

Indoxyl Sulfate, a Uremic Toxin, Induces Trained Immunity of Monocytes through the AhR-Dependent Arachidonic Acid Pathway

Trained immunity is the long-term functional reprogramming of innate immune cells, which results in altered responses toward a secondary challenge. Despite indoxyl sulfate (IS) being a potent stimulus associated with chronic kidney disease (CKD)-related inflammation, its impact on trained immunity has not been explored. Here, we demonstrate that IS induces trained immunity in monocytes via epigenetic and metabolic reprogramming, resulting in augmented cytokine production. Mechanistically, the aryl hydrocarbon receptor (AhR) contributes to IS-trained immunity by enhancing the expression of arachidonic acid (AA) metabolism-related genes such as Arachidonate 5-Lipoxygenase (ALOX5) and ALOX5 Activating Protein (ALOX5AP). Inhibition of AhR during IS training suppresses the induction of IS-trained immunity. Monocytes from end-stage renal disease (ESRD) patients have increased ALOX5 expression and after 6-day training, they exhibit enhanced TNF- and IL-6 production to LPS. Furthermore, healthy control-derived monocytes trained with uremic sera from ESRD patients exhibit increased production of TNF- and IL-6. Consistently, IS-trained mice and their splenic myeloid cells had increased production of TNF- after in vivo and ex vivo LPS stimulation compared to that of control mice. These results provide insight into the role of IS in the induction of trained immunity, which is critical during inflammatory immune responses in CKD patients.

immunology↗

bioRxiv2022

Topology of gene regulatory compartments relative to the nuclear matrix

Summary ParagraphInside the nucleus, there are special compartments with characterized functions, some of which are involved in gene expression1. These compartments include transcriptional condensates and nuclear speckles, which contain factors required for transcription and splicing, respectively. While the characteristics of these intranuclear compartments were extensively investigated, spatial relationship between them is yet unclear. Meanwhile, RNA-protein structural network named the nuclear matrix has been observed inside the nucleus and suggested as a framework for the spatial segregation of the intranuclear space2-6. However, concept of the nuclear matrix was not widely accepted due to its lack of in vivo evidence7. Here, we report visualization of the nuclear matrix and intranuclear compartments using super-resolution fluorescence microscopy. We found the nuclear matrix is dynamic in live cells and easily disrupted upon transcription inhibition. Remarkably, we observed an orderly layered distribution of transcriptional condensates and nuclear speckles relative to the nuclear matrix. We also observed a separation of chromosome territories from transcriptional condensates and nuclear speckles by the nuclear matrix. Based on our findings, we propose a topological model for the regulation of transcription across the nuclear matrix.

cell biology↗

bioRxiv2022

Systematic Characterization of Double Emulsion Droplets for Biological Applications

Double emulsion droplets (DEs) are water/oil/water droplets that can be sorted via Fluorescence-Activated Cell Sorting (FACS), allowing for new opportunities in high-throughput cellular analysis, enzymatic screening, and synthetic biology. These applications require stable, uniform droplets with predictable microreactor volumes. However, predicting DE droplet size, shell thickness, and stability as a function of flow rate has remained challenging for monodisperse single core droplets and those containing biologically-relevant buffers, which influence bulk and interfacial properties. As a result, developing novel DE-based bioassays has typically required extensive initial optimization of flow rates to find conditions that produce stable droplets of the desired size and shell thickness. To address this challenge, we conducted systematic size parameterization quantifying how differences in flow rates and buffer properties (viscosity and interfacial tension at water/oil interfaces) alter droplet size and stability, across 6 inner aqueous buffers used across applications such as cellular lysis, microbial growth, and drug delivery, quantifying the size and shell thickness of >22,000 droplets overall. We restricted our study to stable single core droplets generated in a 2-step dripping-dripping formation regime in a straightforward PDMS device. Using data from 138 unique conditions (flow rates and buffer composition), we also demonstrated that a recent physically-derived size law of Wang et al1 can accurately predict double emulsion shell thickness for >95% of observations. Finally, we validated the utility of this size law by using it to accurately predict droplet sizes for a novel bioassay that requires encapsulating growth media for bacteria in droplets. This work has the potential to enable new screening-based biological applications by simplifying novel DE bioassay development.

bioengineering↗

bioRxiv2021

Targeting RTN4/NoGo-Receptor reduces levels of ALS protein ataxin-2

Gene-based therapeutic strategies to lower ataxin-2 levels are emerging for neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type 2 (SCA2). To identify additional ways of reducing ataxin-2 levels, we performed a genome-wide screen in human cells for regulators of ataxin-2 and identified RTN4R, the gene encoding the RTN4/NoGo-Receptor, as a top hit. RTN4R knockdown, or treatment with a peptide inhibitor, was sufficient to lower ataxin-2 protein levels in mouse and human neurons in vitro and Rtn4r knockout mice have reduced ataxin-2 levels in vivo. Remarkably, we observed that ataxin-2 shares a role with the RTN4/NoGo-Receptor in limiting axonal regeneration. Reduction of either protein increases axonal regrowth following axotomy. These data define the RTN4/NoGo-Receptor as a novel therapeutic target for ALS and SCA2 and implicate the targeting of ataxin-2 as a potential treatment following nerve injury.

neuroscience↗

bioRxiv2021

Small molecule v-ATPase inhibitor Etidronate lowers levels of ALS protein ataxin-2

Antisense oligonucleotide therapy targeting ATXN2--a gene in which mutations cause neurodegenerative diseases spinocerebellar ataxia type 2 and amyotrophic lateral sclerosis--has entered clinical trials in humans. Additional methods to lower ataxin-2 levels would be beneficial not only in uncovering potentially cheaper or less invasive therapies, but also in gaining greater mechanistic insight into how ataxin-2 is normally regulated. We performed a genome-wide fluorescence activated cell sorting (FACS)-based CRISPR screen in human cells and identified multiple subunits of the lysosomal vacuolar ATPase (v-ATPase) as regulators of ataxin-2 levels. We demonstrate that Etidronate--a U.S. Food and Drug Administration (FDA)-approved drug that inhibits the v-ATPase--lowers ataxin-2 protein levels in mouse and human neurons. Moreover, oral administration of the drug to mice in their water supply and food is sufficient to lower ataxin-2 levels in the brain. Thus, we uncover Etidronate as a safe and inexpensive compound for lowering ataxin-2 levels and demonstrate the utility of FACS-based screens for identifying targets to modulate levels of human disease proteins.

neuroscience↗

bioRxiv2021

Spontaneous emergence of music detectors in a deep neural network trained for natural sound recognition

Music exists in almost every society, has universal acoustic features, and is processed by distinct neural circuits in humans even with no experience of musical training. These characteristics suggest an innateness of the sense of music in our brain, but it is unclear how this innateness emerges and what functions it has. Here, using an artificial deep neural network that models the auditory information processing of the brain, we show that units tuned to music can spontaneously emerge by learning natural sound detection, even without learning music. By simulating the responses of network units to 35,487 natural sounds in 527 categories, we found that various subclasses of music are strongly clustered in the embedding space, and that this clustering arises from the music-selective response of the network units. The music-selective units encoded the temporal structure of music in multiple timescales, following the population-level response characteristics observed in the brain. We confirmed that the process of generalization is critical for the emergence of music-selectivity and that music-selectivity can work as a functional basis for the generalization of natural sound, thereby elucidating its origin. These findings suggest that our sense of music can be innate, universally shaped by evolutionary adaptation to process natural sound. One-sentence summaryMusic-selectivity can arise spontaneously in deep neural networks trained for natural sound detection without learning music.

neuroscience↗

bioRxiv2021

Poly(A)-binding protein is an ataxin-2 chaperone that emulsifies biomolecular condensates.

Biomolecular condensation underlies the biogenesis of an expanding array of membraneless assemblies, including stress granules (SGs) which form under a variety of cellular stresses. Advances have been made in understanding the molecular grammar that dictates the behavior of a few key scaffold proteins that make up these phases but how the partitioning of hundreds of other SG proteins is regulated remains largely unresolved. While investigating the rules that govern the condensation of ataxin-2, a SG protein implicated in neurodegenerative disease, we unexpectedly identified a short 14aa sequence that acts as an ataxin-2 condensation switch and is conserved across the eukaryote lineage. We identify poly(A)-binding proteins as unconventional RNA-dependent chaperones that control this regulatory switch. Our results uncover a hierarchy of cis and trans interactions that fine-tune ataxin-2 condensation and reveal a new molecular function for ancient poly(A)-binding proteins as emulsifiers of biomolecular condensate proteins. These findings may inspire novel approaches to therapeutically target aberrant phases in disease.

cell biology↗

bioRxiv2021

Daily oscillation of the excitation/inhibition ratio is disrupted in two mouse models of autism

Alterations to the balance between excitation and inhibition (E/I ratio) are postulated to underlie behavioral phenotypes in autism spectrum disorder (ASD) patients and mouse models. However, in wild type mice the E/I ratio is not constant, but instead oscillates across the 24h day. Therefore, we tested whether the E/I oscillation, rather than the overall E/I ratio, is disrupted in two ASD-related mouse lines: Fmr1 KO and BTBR, models of syndromic and idiopathic ASD, respectively. The E/I ratio is dysregulated in both models, but in different ways: the oscillation is lost in Fmr1 KO and reversed in BTBR mice. In both models these phenotypes associate with differences the timing of excitatory and inhibitory synaptic transmission and endocannabinoid signaling compared to wild type mice, but not with altered sleep. These findings raise the possibility that ASD-related phenotypes may be produced by a mismatch of E/I to the appropriate behavioral state, rather than alterations to overall E/I levels per se.

neuroscience↗

bioRxiv2021

Dendrimer-like supramolecular assembly of proteins with a tunable size and valency through stepwise iterative growth

The assembly of proteins in a programmable manner provides insight into the underlying mechanisms of protein self-assembly in nature as well as the creation of novel functional nanomaterials for practical applications. Despite many advances, however, a rational protein assembly with an easy scalability in terms of size and valency remains a challenging task. Here, we present a simple bottom-up approach to the supramolecular protein assembly with a tunable size and valency in a programmable manner. The dendrimer-like protein assembly, called a "prodrimer," was constructed using a total of three monomeric proteins: a core and two building-block proteins. The prodrimer generations were grown by a stepwise and alternate addition of a building block using two pairs of orthogonal protein-peptide interactions, leading to a higher-generation prodrimer with a mega-dalton size and multi-valency. The valency of the prodrimers at the periphery was tunable with the generation, enabling a single-step functionalization. A second-generation prodrimer functionalized with a target-specific protein binder showed a three-order of magnitude increase in binding affinity compared to a monomeric counterpart due to the avidity. The prodrimers functionalized with a targeting moiety and a cytotoxic protein cargo exhibited a highly enhanced cellular cytotoxicity, exemplifying their utility as a protein delivery platform. The present approach can be effectively used in the creation of protein architectures with new functions for biotechnological and medical applications.

bioengineering↗

bioRxiv2021

Widespread occurrence of hybrid internal-terminal exons in human transcriptomes

Alternative RNA processing is a major mechanism for diversifying the human transcriptome. Messenger RNA isoform differences are predominantly driven by alternative first exons, cassette internal exons and alternative last exons. Despite the importance of classifying exons to understand isoform structure, there is a lack of tools to look at isoform-specific exon usage using RNA-sequencing data. We recently observed that alternative transcription start sites often arise near annotated internal exons, creating "hybrid" exons that can be used as both first or internal exons. To investigate the creation of hybrid exons, we built the HIT (Hybrid-Internal-Terminal) exon pipeline that systematically classifies exons depending on their isoform-specific usage. Using a combination of junction reads coverage and probabilistic modeling, the HIT index identified thousands of hybrid first-internal and internal-last exons that were previously misclassified. Hybrid exons are enriched in long genes with at least ten internal exons, have longer flanking introns and strong splice sites. The usage of hybrid exons varies considerably across human tissues, but they are predominantly used in brain, testis and colon cells. Notably, genes involved in RNA splicing have the highest fraction of intra-tissue hybrid exons. Further, we found more than 100,000 inter-tissue hybrid exons that changed from internal to terminal exons across tissues. By developing the first method that can classify exons according to their isoform contexts, our findings demonstrate the existence of hybrid exons, expand the repertoire of tissue-specific terminal exons and uncover unexpected complexities of the human transcriptome.

bioinformatics↗

bioRxiv2021

TDP-43 represses cryptic exon inclusion in FTD/ALS gene UNC13A

A hallmark pathological feature of neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing. Single nucleotide polymorphisms (SNPs) in UNC13A are among the strongest genome-wide association study (GWAS) hits associated with FTD/ALS in humans, but how those variants increase risk for disease is unknown. Here we show that TDP-43 represses a cryptic exon splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines, and iPSC-derived motor neurons resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. Remarkably, the top variants associated with FTD/ALS risk in humans are located in the cryptic exon harboring intron itself and we show that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD/ALS (UNC13A genetic variants) and loss of TDP-43 function.

neuroscience↗