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Kim, K.

Publications and source records attributed to Kim, K..

At least 271 records · Page 15Linked to original sources
bioRxiv2021

Quantitative imaging of Caenorhabditis elegans dauer larvae during cryptobiotic transition using optical diffraction tomography

Upon starvation or overcrowding, the nematode Caenorhabditis elegans enters diapause by forming a dauer larva. This larva can further transit into an anhydrobiotic state and survive harsh desiccation. We previously identified the genetic and biochemical pathways essential for survival -- but without an accompanying physical model, the mechanistic understanding of this amazing phenomenon will remain inadequate. Neither microscopic investigation of structural changes upon entry into anhydrobiosis nor the most basic quantitative characterization of material properties of living desiccated larvae, however, have been feasible, due to lack of appropriate techniques. Here, we employed optical diffraction tomography (ODT) to quantitatively assess the internal mass density distribution of living larvae in the reproductive and diapause stages. More importantly, ODT allowed for the first time physical analysis of desiccated dauer larvae: their mass density was significantly increased in the anhydrobiotic state. We also applied ODT on different mutants that are sensitive to desiccation. Remarkably, one of them displayed structural abnormalities in the anhydrobiotic stage that could not be observed either by conventional light or electron microscopy. Our advance opens a door to quantitatively assessing fine differences in material properties and structure necessary to fully understanding an organism on the verge of life and death.

biophysics↗
bioRxiv2021

OpenCell: proteome-scale endogenous tagging enables the cartography of human cellular organization

Elucidating the wiring diagram of the human cell is a central goal of the post-genomic era. We combined genome engineering, confocal live-cell imaging, mass spectrometry and data science to systematically map the localization and interactions of human proteins. Our approach provides a data-driven description of the molecular and spatial networks that organize the proteome. Unsupervised clustering of these networks delineates functional communities that facilitate biological discovery, and uncovers that RNA-binding proteins form a specific sub-group defined by unique interaction and localization properties. Furthermore, we discover that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions. Paired with a fully interactive website opencell.czbiohub.org, we provide a resource for the quantitative cartography of human cellular organization.

cell biology↗
bioRxiv2021

High resolution structure of the membrane embedded skeletal muscle ryanodine receptor

The type 1 ryanodine receptor (RyR1)/calcium release channel on the sarcoplasmic reticulum (SR) is required for skeletal muscle excitation-contraction coupling and is the largest known ion channel, comprised of four 565 kDa protomers. Cryogenic electron microscopy (cryoEM) studies of the RyR have primarily used detergent to solubilize the channel, though a recent study resolved the structure with limited resolution in nanodiscs1. In the present study we have used cryoEM to solve high-resolution structures of the channel in liposomes using a gel-filtration approach with on-column detergent removal to form liposomes and incorporate the channel simultaneously, a method that improved the incorporation rate by more than 20-fold compared to a dialysis-based approach. In conjunction with new direct-detection cameras, this allowed us to resolve the structure of the channel in the closed and open states at 3.36 and 3.98 [A], respectively. This method offers validation for detergent-based structures of the RyR and lays the groundwork for studies utilizing an electrochemical gradient mimicking the native environment, such as that of the SR, where Ca2+ concentrations are millimolar in the lumen and nanomolar in the cytosol of the cell at rest.

biophysics↗
bioRxiv2021

Novel Paju Apodemus Paramyxovirus 1 and 2, Harbored by Apodemus agrarius in The Republic of Korea

Paramyxoviruses, negative-sense single-stranded RNA viruses, pose a potential threat to public health. Currently, 78 species and 17 genera of paramyxoviruses are classified and harbored by multiple natural reservoirs, including rodents, bats, birds, reptiles, and fish. Jeilongvirus has been proposed as a novel paramyxovirus genus containing J-, Beilong, and Tailam viruses, found in wild rodents. Using RT-PCR, 824 Apodemus agrarius individuals were examined for the prevalence of paramyxovirus infections. Paramyxovirus RNA was detected in 108 (13.1%) rodents captured at 14 trapping sites in Korea. We first present two genetically distinct novel paramyxoviruses (genus Jeilongvirus), Paju Apodemus paramyxoviruses 1 (PAPV-1) and 2 (PAPV-2), from A. agrarius. Six PAPV strains were completely sequenced using next-generation and Sanger sequencing. PAPV-1 genome comprised 19,716 nucleotides, with eight genes (3'-N-P/V/C-M-F-SH-TM-G-L-5'), whereas PAPV-2 genome contained 17,475 nucleotides, with seven genes (3'-N-P/V/C-M-F-TM-G-L-5'). The disparity between PAPV-1 and -2 revealed the presence of the SH gene and length of the G gene in the genome organization. The phylogenies of PAPV-1 and -2 belong to distinct genetic lineages of Jeilongvirus despite being from the same natural host. PAPV-1 clustered with Beilong and Tailam viruses, while PAPV-2 formed a genetic lineage with Mount Mabu Lophuromys virus-1. PAPV-1 infected human epithelial and endothelial cells, facilitating the induction of type I/III interferons, interferon-stimulated genes, and proinflammatory cytokines. Therefore, this study provides profound insights into the molecular epidemiology, virus-host interactions, and zoonotic potential of novel rodent-borne paramyxoviruses. ImportanceParamyxoviruses are a critical public health and socio-economic burden to humans. Rodents play a crucial role in transmitting pathogens to humans. In the last decade, novel paramyxoviruses have been discovered in different rodents. Here, we found that Apodemus agrarius harbored two distinct genotypes of the novel paramyxoviruses, Paju Apodemus paramyxovirues 1 (PAPV-1) and 2 (PAPV-2), possessing unique genome structures that are responsible for encoding TM and G proteins of different sizes. In addition, PAPV-1 infected human epithelial and endothelial cells, facilitating the induction of type I/III IFNs, ISGs, and proinflammatory cytokines. Thus, this study provides significant insights into molecular prevalence, virus-host interactions of paramyxoviruses. These observations raise the awareness of physicians and scientists about the emergence of new rodent-borne paramyxoviruses.

microbiology↗
bioRxiv2021

Perception of biological motion in point-light displays by jumping spiders

Over the last 50 years, point-light displays have been successfully used to explore how animals respond to dynamic visual stimuli--specifically, differentiation of the biological from the non-biological. These stimuli are designed to preserve movement patterns while minimizing static detail, with single dots representing each of the main joints of a moving animal. Imposed by their internal skeleton, vertebrate movements follow a specific semi-rigid dynamic pattern, termed "biological-motion", which can be used to distinguish animate from inanimate objects. Although biological motion detection has not been studied in invertebrates, rigid exoskeletons force many species to also follow semi-rigid movement principles. Due to their highly developed visual system and complex visual behaviors, we investigated the capability of jumping spiders to discriminate biological from non-biological motion using point-light display stimuli. By constraining spiders so that they could rotate but not move directionally, we simultaneously presented two point-light display stimuli with specific dynamic traits and registered their preference by observing which pattern they turned towards. Jumping spiders clearly demonstrated the ability to discriminate between stimuli. However, spiders showed no preference when both stimuli presented patterns with semi-rigid movements, results that are directly comparable to responses in vertebrate systems. This represents the first demonstration of biological motion recognition in an invertebrate, posing crucial questions about the evolutionary history of this ability and complex visual processing in non-vertebrate systems.

animal behavior and cognition↗
bioRxiv2021

A unique GCN5 histone acetyltransferase complex controls erythrocyte invasion and virulence in the malaria parasite Plasmodium falciparum

The histone acetyltransferase GCN5-associated SAGA complex is evolutionarily conserved from yeast to human and functions as a general transcription co-activator in global gene regulation. In this study, we identified a divergent GCN5 complex in Plasmodium falciparum, which contains two plant homeodomain (PHD) proteins (PfPHD1 and PfPHD2) and a plant apetela2 (AP2)-domain transcription factor (PfAP2-LT). To dissect the functions of the PfGCN5 complex, we generated parasites with the bromodomain deletion in PfGCN5 and the PHD domain deletion in PfPHD1. The two deletion mutants closely phenocopied each other, exhibiting significantly reduced merozoite invasion of erythrocytes and elevated sexual conversion. These domain deletions caused dramatic decreases not only in histone H3K9 acetylation but also in H3K4 trimethylation, indicating synergistic crosstalk between the two euchromatin marks. Domain deletion in either PfGCN5 or PfPHD1 profoundly disturbed the global transcription pattern, causing altered expression of more than 60% of the genes. At the schizont stage, these domain deletions were linked to specific downregulation of merozoite genes involved in erythrocyte invasion, many of which harbor the DNA-binding motifs for AP2-LT and/or AP2-I, suggesting targeted recruitment of the PfGCN5 complex to the invasion genes by these specific transcription factors. Conversely, at the ring stage, PfGCN5 or PfPHD1 domain deletions disrupted the mutually exclusive expression pattern of the entire var gene family, which encodes the virulent factor PfEMP1. Correlation analysis between the chromatin state and alteration of gene expression demonstrated that up- and down-regulated genes in these mutants are highly correlated with the silenct and active chromatin states in the wild-type parasite, respectively. Collectively, the PfGCN5 complex represents a novel HAT complex with a unique subunit composition including the AP2 transcription factor, which signifies a new paradigm for targeting the co-activator complex to regulate general and parasite-specific cellular processes in this low-branching parasitic protist. Author SummaryEpigenetic regulation of gene expression plays essential roles in orchestrating the general and parasite-specific cellular pathways in the malaria parasite Plasmodium falciparum. Using tandem affinity purification and proteomic characterization, we identified a divergent transcription co-activator - the histone acetyltransferase GCN5-associated complex in P. falciparum, which contains nine core components, including two PHD domain proteins (PfPHD1 and PfPHD2) and a plant apetela2-domain transcription factor. To understand the functions of the PfGCN5 complex, we performed gene disruption in two subunits of this complex, PfGCN5 and PfPHD1. We found that the two deletion mutants displayed very similar growth phenotypes, including significantly reduced merozoite invasion rates and elevated sexual conversion. These two mutants were associated with dramatic decreases in histone H3K9 acetylation and H3K4 trimethylation, which led to global changes in chromatin states and gene expression. Genes significantly affected by the PfGCN5 and PfPHD1 gene disruption include those participating in parasite-specific pathways such as invasion, virulence, and sexual development. In conclusion, this study presents a new model of the PfGCN5 complex for targeting the co-activator complex to regulate general and parasite-specific cellular processes in this low-branching parasitic protist.

microbiology↗
bioRxiv2021

Quantitative coupling of cell volume and membrane tension during osmotic shocks

During osmotic changes of their environment, cells actively regulate their volume and plasma membrane tension that can passively change through osmosis. How tension and volume are coupled during osmotic adaptation remains unknown, as a quantitative characterization is lacking. Here, we performed dynamic membrane tension and cell volume measurements during osmotic shocks. During the first few seconds following the shock, cell volume varied to equilibrate osmotic pressures inside and outside the cell, and membrane tension dynamically followed these changes. A theoretical model based on the passive, reversible unfolding of the membrane as it detaches from the actin cortex during volume increase, quantitatively describes our data. After the initial response, tension and volume recovered from hypoosmotic shocks but not from hyperosmotic shocks. During these asymmetric recoveries, tension and volume remained coupled. Pharmacological disruption of the cytoskeleton and functional inhibition of ion channels and mTOR all affected tension and volume responses, proving that a passive mechanism is necessary and critical for the cell to adapt fast. The coupling between them was, nonetheless, maintained for a few exceptions suggesting that volume and tension regulations are independent from the regulation of their coupling.

biophysics↗
bioRxiv2020

Dopamine receptors in obesity: a meta-analysis

The brain plays a major role in controlling the desire to eat. This meta-analysis aimed to assess the association between dopamine receptor (DR) availability, measured using positron emission tomography, and obesity. We performed a systematic search of MEDLINE (from inception to June 2019) and EMBASE (from inception to June 2019) for articles published in English using the keywords "dopamine receptor," "obesity," and "neuroimaging." Fitting a locally weighted estimated scatterplot smoothing curve for binding potential (BPND) of the ventral striatum measured by 11C-raclopride was performed. Five studies involving 119 subjects were included in this analysis. 11C-raclopride was used in four studies, regardless of the ROIs included. The curve of BPND of the ventral striatum went up and down at BMI of 20-30 kg/m2 and became flat after a BMI of >40 kg/m2. Linear regression analysis was performed with BPND of the striatum from two studies (n = 20). The BPND of the striatum was negatively associated with BMI. In conclusion, dopamine plays a major role in the reward system with regard to obesity. Compared to normal-weight subjects, overweight and obese subjects had decreased DR availability. However, this tendency should be interpreted carefully with regard to radiopharmaceuticals and ROIs.

neuroscience↗
bioRxiv2020

CRP immunodeposition and proteomic analysis in abdominal aortic aneurysm

ObjectiveThe molecular mechanisms of the degeneration of the aortic wall in abdominal aortic aneurysm (AAA) are poorly understood. The monomeric form of C-reactive protein (mCRP) is deposited in damaged cardiovascular organs and aggravates the prognosis; however, it is unknown whether mCRP is deposited in the degenerated aorta of abdominal aortic aneurysm (AAA). We investigated whether mCRP is deposited in AAA and examined the associated pathogenic signaling pathways. MethodsTwenty-four cases of AAA were analyzed and their histological features were compared according to the level of serum CRP and the degree of mCRP deposition. Proteomic analysis was performed in AAA cases with strong and diffuse CRP immunopositivity (n=7) and those with weak, focal, and junctional CRP immunopositivity (n=3). ResultsmCRP was deposited in the aortic specimens of AAA in a characteristic pattern that coincided with the lesion of the diminished elastic layer of the aortic wall. High serum CRP level was associated with stronger mCRP immunopositivity and a larger maximal diameter of aortic aneurysm. Proteomic analysis in AAA showed that multiple proteins were differentially expressed according to mCRP immunopositivity. Also, ingenuity pathway analysis showed that pathways associated with atherosclerosis, acute phase response, complement system, immune system, and coagulation were enriched in AAA cases with high mCRP immunopositivity. ConclusionsAAA showed a characteristic deposition of mCRP, and multiple potentially pathologic signaling pathways were upregulated in AAA cases with strong CRP immunopositivity. mCRP and the aforementioned pathological pathways may serve as targets for managing the progression of AAA.

pathology↗
bioRxiv2020

Open-top axially swept light sheet microscopy

Open-top light sheet microscopy (OT-LSM) is a specialized microscopy for the high throughput cellular imaging of large tissues including optically cleared tissues by having all the optical setup below the sample stage. Current OT-LSM systems had relatively low axial resolutions by using weakly focused light sheets to cover the imaging field of view (FOV). In this report, open-top axially swept LSM (OTAS-LSM) was developed for high-throughput cellular imaging with the improved axial resolution. OTAS-LSM swept the tightly focused excitation light sheet across the imaging FOV by using an electro tunable lens (ETL) and collected emission light at the focus of light sheet with a camera in the rolling shutter mode. OTAS-LSM was developed by using air objective lenses and a water prism for simplicity and it had on-axis optical aberration associated with the mismatch of refractive indices between air and immersion medium. The effects of optical aberration were analyzed by both simulation and experiment. The image resolutions were 1.5-1.6m, and approximately 140% of the aberration-free theoretical values. The newly developed OTAS-LSM was applied to the imaging of optically cleared mouse brain and small intestine and it could visualize neuronal networks in the single cell level. OTAS-LSM might be useful for the high-throughput cellular examination of optically cleared large tissues.

neuroscience↗
bioRxiv2020

Human brain organoids reveal accelerated development of cortical neuron classes as a shared feature of autism risk genes

Genetic risk for autism spectrum disorder (ASD) has been associated with hundreds of genes spanning a wide range of biological functions. The phenotypic alterations in the human brain resulting from mutations in ASD risk genes remain unclear, and the level at which these alterations converge on shared disease pathology is poorly understood. Here, we leveraged reproducible organoid models of the human cerebral cortex to identify cell type-specific developmental abnormalities associated with haploinsufficiency in three ASD risk genes, SUV420H1 (KMT5B), PTEN, and CHD8. We performed comprehensive single-cell RNA-sequencing (scRNA-seq) of over 400,000 cells, and proteomic analysis on individual organoids sampled at different developmental stages to investigate phenotypic convergence among these genes. We find that within a defined period of early cortical development, each of the three mutations demonstrates accelerated development of cortical neurons. Notably, they do so by affecting different neuronal populations: excitatory deep layer (SUV420H1) and callosal (PTEN) neurons, and inhibitory interneurons (CHD8). This work shows that haploinsufficiency in ASD risk genes converge on early developmental defects in the generation of neurons of the cortical microcircuit.

developmental biology↗
bioRxiv2020

Human reference gut microbiome comprising 5,414 prokaryotic species, including newly assembled genomes from under-represented Asian metagenomes

Metagenome sampling bias for geographical location and lifestyle is partially responsible for the incomplete catalog of reference genomes of gut microbial species. Here, we present a substantially expanded microbiome catalog, the Human Reference Gut Microbiome (HRGM). Incorporating newly assembled 29,082 genomes from 845 fecal samples collected from three under-represented Asian countries--Korea, India, and Japan--the HRGM contains 232,098 non-redundant genomes of 5,414 representative prokaryotic species, >103 million unique proteins, and >274 million single-nucleotide variants. This is an over 10% increase from the largest reference database. The newly assembled genomes were enriched for members of the Bacteroidaceae family, including species associated with high-fiber and seaweed-rich diet. Single-nucleotide variant density was positively associated with the speciation rate of gut commensals. Ultra-deep sequencing facilitated the assembly of genomes of low-abundance taxa, and deep sequencing (>20 million read pairs) was needed for the profiling of low-abundance taxa. Importantly, the HRGM greatly improved the taxonomic and functional classification of sequencing reads from fecal samples. Finally, mapping homologous sequences for human auto-antigens onto the HRGM genomes revealed the association of commensal bacteria with high cross-reactivity potential with autoimmunity. The HRGM (www.mbiomenet.org/HRGM/) will facilitate the identification and functional analysis of disease-associated gut microbiota.

microbiology↗
bioRxiv2020

Combined fluorescence, optical diffraction tomography and Brillouin microscopy

Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples -- so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epi-fluorescence imaging for explicitly measuring the Brillouin shift, RI and absolute density with specificity to fuorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample -- a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.

biophysics↗
bioRxiv2020

Quantitative phase microscopy enables precise and efficient determination of biomolecular condensate composition

Biomolecular condensates are membrane-less organelles made of multiple components, often including several distinct proteins and nucleic acids. However, current tools to measure condensate composition are limited and cannot capture this complexity quantitatively, as they either require fluorescent labels, which we show can perturb composition, or can distinguish only 1-2 components. Here, we describe a label-free method based on quantitative phase microscopy to measure the composition of condensates with an arbitrarily large number of components. We first validate the method empirically in binary mixtures, revealing sequence-encoded density variation and complex aging dynamics for condensates composed of full-length proteins. In simplified multi-component protein/RNA condensates, we uncover a regime of constant condensate density and a large range of protein:RNA stoichiometry when varying average composition. The unexpected decoupling of density and composition highlights the need to determine molecular stoichiometry in multi-component condensates. We foresee this approach enabling the study of compositional regulation of condensate properties and function.

biophysics↗
bioRxiv2020

Aggregation of Cohorts for Histopathological Diagnosis with Deep Morphological Analysis

There have been substantial efforts in using deep learning (DL) to diagnose cancer from digital images of pathology slides. Existing algorithms typically operate by training deep neural networks (DNNs) either specialized in specific cohorts or an aggregate of all cohorts when there are only a few images available for the target cohort. A trade-off between decreasing the number of models and their cancer detection performance was evident in our experiments with The Cancer Genomic Atlas (TCGA) dataset, with the former approach achieving higher performance at the cost of having to acquire large datasets from the cohort of interest. Constructing annotated datasets for individual cohorts is extremely time-consuming, with the acquisition cost of such datasets growing linearly with the number of cohorts. Another issue associated with developing cohort-specific models is the difficulty of maintenance: all cohort-specific models may need to be adjusted when a new DL algorithm is to be used, where training even a single model may require a non-negligible amount of computation, or when more data is added to some cohorts. In resolving the sub-optimal behavior of a universal cancer detection model trained on an aggregate of cohorts, we investigated how cohorts can be grouped to augment a dataset without increasing the number of models linearly with the number of cohorts. This study introduces several metrics which measure the morphological similarities between cohort pairs and demonstrates how the metrics can be used to control the trade-off between performance and the number of models.

pathology↗
bioRxiv2020

Human FcRn expression and Type I Interferon signaling control Echovirus 11 pathogenesis in mice

Neonatal echovirus infections are characterized by severe hepatitis and neurological complications that can be fatal. Here, we show that expression of the human homologue of the neonatal Fc receptor (hFcRn), the primary receptor for echoviruses, and ablation of type I interferon (IFN) signaling are key host determinants involved in echovirus pathogenesis. We show that expression of hFcRn alone is insufficient to confer susceptibility to echovirus infections in mice. However, expression of hFcRn in mice deficient in type I interferon (IFN) signaling, hFcRn-IFNAR-/-, recapitulate the echovirus pathogenesis observed in humans. Luminex-based multianalyte profiling from E11 infected hFcRn-IFNAR-/- mice revealed a robust systemic immune response to infection, including the induction of type I IFNs. Furthermore, similar to the severe hepatitis observed in humans, E11 infection in hFcRn-IFNAR-/- mice caused profound liver damage. Our findings define the host factors involved in echovirus pathogenesis and establish in vivo models that recapitulate echovirus disease.

microbiology↗
bioRxiv2020

Effects of Healthy Aging on Right Ventricular Structure and Biomechanical Properties

Healthy aging has been associated with alterations in pulmonary vasculature and right ventricular (RV) hemodynamics, potentially leading to RV remodeling. Despite the current evidence suggesting an association between aging and alterations in RV function and higher prevalence of pulmonary hypertension in the elderly, limited data exist on age-related differences in RV structure and biomechanics. In this work we report our preliminary findings on the effects of healthy aging on RV structure, function, and biomechanical properties. Hemodynamic measurements, biaxial mechanical testing, constitutive modeling, and quantitative histological analysis were employed to study two groups of Sprague-Dawley rats: control (11 weeks) and aging (80 weeks). Aging was associated with increases in RV peak pressures ({approx}{uparrow}17%, p=0.017), RV contractility ({approx}{uparrow}52%, p= 0.004), and RV wall thickness ({approx}{uparrow}34%, p=0.002). Longitudinal realignment of RV collagen (16.4{degrees}, p=0.013) and myofibers (14.6{degrees}, p=0.017) were observed with aging, accompanied by transmural cardiomyocyte loss and fibrosis. A bimodal alteration in biomechanical properties was noted, resulting in increased myofiber stiffness ({approx}{uparrow}158%, p=0.0006) and decreased effective collagen fiber stiffness ({approx}{downarrow}67%, p=0.031). Our results demonstrate the potential of healthy aging to modulate RV remodeling via increased peak pressures, cardiomyocyte loss, fiber reorientation, and altered collagen/myofiber stiffness. Some similarities were observed between aging-induced remodeling patterns and those of RV remodeling in pressure overload.

bioengineering↗