Biology subjects

Yoon, S.

Publications and source records attributed to Yoon, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

Human T follicular helper clones seed the germinal center-resident regulatory pool

How FOXP3+ T follicular regulatory (Tfr) cells simultaneously steer antibody formation toward microbe/vaccine recognition and away from self-reactivity remains unsettled. To explore human Tfr cell provenance, function and location heterogeneity, we used paired TCRVA/TCRVB sequencing to distinguish tonsillar Tfr cells clonally related to natural Tregs (nTfr) from those likely induced from Tfh cells (iTfr). The proteins iTfr and nTfr cells differentially expressed were utilized to pinpoint their in situ locations via multi-plex microscopy and establish divergent functional roles. In-silico and tonsil organoid tracking models corroborated the existence of separate Treg-to-nTfr and Tfh-to-iTfr developmental trajectories. In total, we have identified human iTfr cells as a distinct CD38-expressing, GC-resident, Tfh-descended subset that gains suppressive function while retaining capacities for B-cell help whereas CD38- nTfr cells are elite suppressors primarily localized to follicular mantles. Interventions differentially targeting Tfr subsets may provide therapeutic opportunities to boost immunity or more precisely treat autoimmune diseases. One sentence summaryHuman tonsillar Tfr clones descend from either Treg or Tfh lineages and provenance predicts their TCR repertoires, locations and functional characteristics.

immunology↗

bioRxiv2022

Adaptive Optical Coherent Raman Imaging of Axons through Mouse Cranial Bone

Coherent Raman scattering imaging has provided inherent chemical information of biomolecules without the need for any external labels.1-3 However, its working depth in deep-tissue imaging is extremely shallow because both the intrinsic scattering cross-section and image contrast are so small that even weak perturbation of the pump and Stokes beam focusing by the complex tissue causes the loss of the resolving power.4,5 Here, we propose a deep-tissue coherent Raman scattering (CRS) microscopy equipped with an advanced adaptive optics (AO) system measuring complex tissue aberration from elastic backscattering. Using this label-free AO-CRS microscopy, we demonstrate the vibrational imaging of lipid-rich substances such as myelin inside the mouse brain even through the thick and opaque cranial bones.

bioengineering↗

bioRxiv2022

Hierarchical cell-type identifier accurately distinguishes immune-cell subtypes enabling precise profiling of tissue microenvironment with single-cell RNA-sequencing

AO_SCPLOWBSTRACTC_SCPLOWSingle-cell RNA-seq enabled in-depth study on tissue micro-environment and immune-profiling, where a crucial step is to annotate cell identity. Immune cells play key roles in many diseases while their activities are hard to track due to diverse and highly variable nature. Existing cell-type identifiers had limited performance for this purpose. We present HiCAT, a hierarchical, marker-based cell-type identifier utilizing gene set analysis for statistical scoring for given markers. It features successive identification of major-type, minor-type and subsets utilizing subset markers structured in a three-level taxonomy tree. Comparison with manual annotation and pairwise match test showed HiCAT outperforms others in major- and minor-type identification. For subsets, we qualitatively evaluated marker expression profile demonstrating that HiCAT provide most clear immune cell landscape. HiCAT was also used for immune cell profiling in ulcerative colitis and discovered distinct features of the disease in macrophage and T cell subsets that could not be identified previously.

bioinformatics↗

bioRxiv2022

Computational conjugate adaptive optics for longitudinal through-skull imaging of cortical myelin

We present a 1.3-m reflection matrix microscope and computational conjugate adaptive optics algorithm for label-free longitudinal imaging of cortical myelin through an intact mouse skull. The myelination processes of the same mice were observed from 3 to 10 postnatal weeks down to cortical layer 4 with a near-diffraction-limited resolution of 0.79 {micro}m. Our system will expedite the investigations on the role of myelination in learning, memory, and brain disorders.

neuroscience↗

bioRxiv2022

HOPX governs a molecular and physiological switch between cardiomyocyte progenitor and maturation gene programs

This study establishes the homeodomain only protein, HOPX, as a determinant controlling the molecular switch between cardiomyocyte progenitor and maturation gene programs. Time-course single-cell gene expression with genome-wide footprinting reveal that HOPX interacts with and controls core cardiac networks by regulating the activity of mutually exclusive developmental gene programs. Upstream hypertrophy and proliferation pathways compete to regulate HOPX transcription. Mitogenic signals override hypertrophic growth signals to suppress HOPX and maintain cardiomyocyte progenitor gene programs. Physiological studies show HOPX directly governs genetic control of cardiomyocyte cell stress responses, electro-mechanical coupling, proliferation, and contractility. We use human genome-wide association studies (GWAS) to show that genetic variation in the HOPX-regulome is significantly associated with complex traits affecting cardiac structure and function. Collectively, this study provides a mechanistic link situating HOPX between competing upstream pathways where HOPX acts as a molecular switch controlling gene regulatory programs underpinning metabolic, signaling, and functional maturation of cardiomyocytes.

developmental biology↗

bioRxiv2022

High-throughput Oligopaint screen identifies druggable regulators of genome folding

Although the molecular rules governing genome organization are being quickly elucidated, relatively few proteins regulating this process have been identified. To address this gap, we developed a fully automated imaging pipeline, called HiDRO (high-throughput DNA or RNA labeling with optimized Oligopaints), that permits quantitative measurement of chromatin interactions across a large number of samples. Using HiDRO, we screened the human druggable genome and identified >300 factors that regulate chromatin folding during interphase, including 43 validated hits that either increase or decrease interactions between topological associating domains (TADs). We discovered that genetic or chemical inhibition of the ubiquitous kinase GSK3A enhances long-range interactions by dysregulating cohesin-mediated chromatin looping. Collectively, these results highlight a noncanonical role for GSK3A signaling in nuclear architecture and underscore the broader utility of HiDRO-based screening to identify novel mechanisms that drive the spatial organization of the genome.

genetics↗

bioRxiv2022

Effects of low intensity pulsed ultrasound stimulation on metabolic lipolysis of adipocytes

Obesity is closely related to several metabolic diseases along with abnormal increase in fat cells. Reducing size and number of fat cells, a procedure known as lipolysis, may be used to prevent obesity as a potential therapy, which also requires fundamental understanding of the mechanisms of lipolysis at molecular level upon different types of stimulations. Here, we used low intensity pulsed ultrasound (LIPUS) stimulation to investigate underlying mechanisms of the activation of lipolysis and autophagy related genes and signaling pathways of adipocytes differentiated from 3T3-L1 cells. LIPUS with the center frequency of 2 MHz was applied for 10 minutes per day for three days. After LIPUS stimulation, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot were used to determine the regulation of lipolytic factors such as adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoacylglycerol lipase (MGL). At RNA level, all three factors were upregulated while only MGL was upregulated at protein level, which presents slightly different activation pattern of lipolysis compared to widely used chemical stimulation. These results demonstrate that LIPUS stimulation can promote the lipolytic capacity of adipocytes in the differentiated state. The differences between transcriptional genes and metabolites were analyzed by transcript analysis and metabolomic profiling experiments. Cellular RNA-sequencing (RNA-Seq) showed an increase in lipolysis and immune-related genes and autophagy related genes after LIPUS stimulation. This study may provide an important experimental basis for the clinical applications and a fundamental understanding of the mechanisms of lipolysis using LIPUS stimulation.

bioengineering↗

bioRxiv2022

Additive genetic effects in interacting species jointly determine the outcome of caterpillar herbivory

Plant-insect interactions are common and important in basic and applied biology. Trait and genetic variation can affect the outcome and evolution of these interactions, but the relative contributions of plant and insect genetic variation and how these interact remain unclear and are rarely subject to assessment in the same experimental context. Here we address this knowledge gap using a recent host range expansion onto alfalfa by the Melissa blue butterfly. Common garden rearing experiments and genomic data show that caterpillar performance depends on plant and insect genetic variation, with insect genetics contributing to performance earlier in development and plant genetics later. Our models of performance based on caterpillar genetics retained predictive power when applied to a second common garden. Much of the plant genetic effect could be explained by heritable variation in plant phytochemicals, especially saponins, peptides, and phosphatidyl cholines, providing a mechanistic understanding of variation in the species interaction. We find evidence of polygenic, mostly additive effects within and between species, with consistent effects of plant genotype on growth and development across multiple butterfly species. Our results inform theories of plant-insect coevolution and the evolution of diet breadth in herbivorous insects and other host-specific parasites. Teaser summaryThe combined, additive effects of plant and insect genetic variation explain Melissa blue caterpillar growth and development on alfalfa plants.

genomics↗

bioRxiv2022

Impaired OTUD7A-dependent Ankyrin regulation mediates neuronal dysfunction in mouse and human models of the 15q13.3 microdeletion syndrome

Copy number variations (CNV) are associated with psychiatric and neurodevelopmental disorders (NDDs), and most, including the recurrent 15q13.3 microdeletion disorder, have unknown disease mechanisms. We used a heterozygous 15q13.3 microdeletion mouse model and patient iPSC-derived neurons to reveal developmental defects in neuronal maturation and network activity. To identify the underlying molecular dysfunction, we developed a neuron-specific proximity-labeling proteomics (BioID2) pipeline, combined with patient mutations, to target the 15q13.3 CNV genetic driver OTUD7A. OTUD7A is an emerging independent NDD risk gene with no known function in the brain, but has putative deubiquitinase (DUB) function. The OTUD7A protein-protein interaction (PPI) network revealed interactions with synaptic, axonal, and cytoskeletal proteins and was enriched for known ASD and epilepsy risk genes. The interactions between OTUD7A and the NDD risk genes Ankyrin-G (Ank3) and Ankyrin-B (Ank2) were disrupted by an epilepsy-associated OTUD7A L233F variant. Further investigation of Ankyrin-G in mouse and human 15q13.3 microdeletion and OTUD7AL233F/L233F models revealed protein instability, increased polyubiquitination, and decreased levels in the axon initial segment (AIS), while structured illumination microscopy identified reduced Ankyrin-G nanodomains in dendritic spines. Functional analysis of human 15q13.3 microdeletion and OTUD7AL233F/L233F models revealed shared and distinct impairments to axonal growth and intrinsic excitability. Importantly, restoring OTUD7A or Ankyrin-G expression in 15q13.3 microdeletion neurons led to a reversal of abnormalities. These data reveal a critical OTUD7A-Ankyrin pathway in neuronal development, which is impaired in the 15q13.3 microdeletion syndrome, leading to neuronal dysfunction. Further, our study highlights the utility of targeting CNV genes using cell-type specific proteomics to identify shared and unexplored disease mechanisms across NDDs.

neuroscience↗

bioRxiv2021

Quantitative Mapping of Keratin Networks in 3D

Mechanobiology requires precise quantitative information on processes taking place in specific 3D microenvironments. Connecting the abundance of microscopical, molecular, biochemical and cell mechanical data with defined topologies has turned out to be extremely difficult. Establishing such structural and functional 3D maps needed for biophysical modeling is a particular challenge for the cytoskeleton, which consists of long and interwoven filamentous polymers coordinating subcellular processes and interactions of cells with their environment. To date, useful tools are available for the segmentation and modeling of actin filaments and microtubules but comprehensive tools for the mapping of intermediate filament organization are still lacking. In this work, we describe a workflow to model and examine the complete 3D arrangement of the keratin intermediate filament cytoskeleton in epithelial cells both in vitro and in vivo. Numerical models are derived from super resolution 3D imaging of fluorescence-tagged keratin filaments. They are interrogated and annotated at different length scales using different modes of visualization including immersive virtual reality. In this way, information is provided on network organization at the subcellular level including mesh arrangement, density and isotropic configuration as well as details on filament morphology such as bundling, curvature and orientation. We show that the comparison of these parameters helps to identify, in quantitative terms, similarities and differences of keratin network organization in epithelial cell types defining subcellular domains, notably basal, apical, lateral and perinuclear systems. The described approach and the presented data are pivotal for generating mechanobiological models that can be experimentally tested.

cell biology↗

bioRxiv2021

Multiplexed ultrasound imaging using clustered gas vesicles and spectral imaging

Current advances in ultrasound imaging techniques including super-resolution ultrasound imaging allows us to visualize microvasculature in biological specimens using microbubbles. However, microbubbles diffuse in blood stream limiting imaging acquisition and frame subtraction scheme of super-resolution ultrasound imaging cannot improve spatial resolution without moving microbubbles. Fluorescent proteins revolutionized to understand molecular and cellular functions in biological systems. Here, we devised a panel of gas vesicles to realize multiplexed ultrasound imaging to uniquely visualize locations of different species of gas vesicles. Mid-band fit spectral imaging technique demonstrated that stationary gas vesicles were efficiently localized in gel phantom and murine liver specimens by visualizing three-dimensional vessel structures. Clustered and unclustered gas vesicles were phagocytosed by murine macrophages to serve as carriers and beacons for the proposed multiplexed and single cell level imaging technique. The spatial distribution of macrophages containing clustered and unclustered gas vesicles were reconstructed by mid-band fit spectral imaging with pseudo-coloring scheme.

bioengineering↗

bioRxiv2021

Ratiometric RNA labeling allows dynamic multiplexed analysis of gene circuits in single cells

Biological processes are highly dynamic and are regulated by genes that connect with one and another, forming regulatory circuits and networks. Understanding how gene regulatory circuits operate dynamically requires monitoring the expression of multiple genes in the same cell. However, it is limited by the relatively few distinguishable fluorescent proteins. Here, we developed a multiplexed real-time transcriptional imaging method based on two RNA stem-loop binding proteins, and employed it to analyze the temporal dynamics of synthetic gene circuits. By incorporating different ratios of MS2 and PP7 stem-loops, we were able to monitor the real-time nascent transcriptional activities of up to five genes in the same cell using only two fluorescent proteins. Applying this multiplexing capability to synthetic linear or branched gene regulatory cascades revealed that propagation of transcriptional dynamics is enhanced by non-stationary dynamics and is dictated by the slowest regulatory branch in the presence of combinatorial regulation. Mathematical modeling provided further insight into temporal multi-gene interactions and helped to understand potential challenges in regulatory inference using snapshot single-cell data. Ratiometric multiplexing should scale exponentially with additional labelling channels, providing a way to track the dynamics of larger circuits.

systems biology↗

bioRxiv2021

Meiosis-Specific Cohesin Complexes Display Distinct and Essential Roles in Mitotic ESC Chromosomes

Cohesin is a chromosome-associated SMC-kleisin complex that mediates sister chromatid cohesion, recombination, and most chromosomal processes during mitosis and meiosis. Through high-resolution 3D-structured illumination microscopy and functional analyses, we report multiple biological processes associated with the meiosis-specific cohesin components, REC8 and STAG3, and the distinct loss of function of meiotic cohesin during the cell cycle of embryonic stem cells (ESCs). First, we show that REC8 is translocated into the nucleus in a STAG3-dependent manner. REC8/STAG3-containing cohesin regulates chromosome topological properties and specifically maintains centromeric cohesion. Second, REC8 and mitotic cohesin RAD21 are located at adjacent sites but predominantly at nonoverlapping sites on ESC chromosomes, implying that REC8 can function independent of RAD21 in ESCs. Third, knockdown of REC8-cohesin not only leads to higher rates of premature centromere separation and stalled replication forks, which can cause proliferation and developmental defects, but also enhances compaction of the chromosome structure by hyperloading of retinoblastoma protein-condensin complexes from prophase onward. We propose that the delicate balance between mitotic and meiotic cohesins may regulate ESC- specific chromosomal organization and mitotic program.

molecular biology↗

bioRxiv2021

Stripenn detects architectural stripes from chromatin conformation data using computer vision

Architectural stripes tend to form at genomic regions harboring genes with salient roles in cell identity and function. Therefore, the accurate identification and quantification of these features is essential for the understanding of lineage-specific gene regulation. Here, we present Stripenn, an algorithm rooted in computer vision to systematically detect and quantitate architectural stripes from chromatin conformation measurements of various technologies. We demonstrate that Stripenn outperforms existing methods, highlight its biological applications in the context of B and T lymphocytes, and examine the role of sequence variation on architectural stripes by studying the conservation of these features in inbred strains of mice. In summary, Stripenn is a computational method which borrows concepts from widely used image processing techniques for demarcation and quantification of architectural stripes.

bioinformatics↗

bioRxiv2021

Single cell trajectory modeling identifies a primitive trophoblast state defined by BCAM enrichment

In early placental development, progenitor cytotrophoblasts (CTBs) differentiate along one of two cellular trajectories: the villous or extravillous pathways. CTBs committed to the villous pathway fuse with neighboring CTBs to form the outer multinucleated syncytiotrophoblast (SCT), while CTBs committed to the extravillous pathway differentiate into invasive extravillous trophoblasts (EVT). Unfortunately, little is known about the processes controlling human CTB progenitor maintenance and differentiation. To address this, we established a single cell RNA sequencing (scRNA-seq) dataset from first trimester placentas to identify cell states important in trophoblast progenitor establishment, renewal, and differentiation. Multiple distinct trophoblast states were identified, representing progenitor CTBs, column CTBs, SCT precursors, and EVT. Lineage trajectory analysis identified a progenitor origin that was reproduced in human trophoblast stem cell organoids. Heightened expression of basal cell adhesion molecule (BCAM) defined this primitive state, where BCAM enrichment or gene silencing resulted in enhanced or diminished organoid growth. Together, this work describes at high-resolution trophoblast heterogeneity within the first trimester, resolves gene networks within human CTB progenitors, and identifies BCAM as a primitive progenitor marker and possible regulator. Summary StatementLineage trajectory modeling identifies multiple human progenitor trophoblast states and defines trophoblast differentiation kinetics, where BCAM-expressing progenitors demonstrate enhanced regenerative ability.

developmental biology↗

bioRxiv2021

Measurements of acoustic radiation force of ultrahigh frequency ultrasonic transducers using model-based approach

Even though ultrahigh frequency ultrasonic transducers over 60 MHz have been used for single cell level manipulation such as intracellular delivery, acoustic tweezers, and stimulation to investigate cell phenotype and cell mechanics, no techniques have been available to measure actual acoustic radiation force (ARF) applied to target cells. Therefore, we have developed an approach to measure ARF of ultrahigh frequency ultrasonic transducers using theoretical model of the dynamics of a solid sphere in a gelatin phantom. To estimate ARF at the focus of 130 MHz transducer, we matched measured maximum displacements of a solid sphere with theoretical calculations. We selected appropriate ranges of input voltages and pulse durations for single cell applications and estimated ARF were in the range of tens of pN to nN. FRET live cell imaging was demonstrated to visualize calcium transport between cells after a target single cell was stimulated by the developed ultrasonic transducer.

bioengineering↗

bioRxiv2020

Ultra-fast Prediction of Somatic Structural Variations by Reduced Read Mapping via Pan-Genome k-mer Sets

Genome rearrangements often result in copy number alterations of cancer-related genes and cause the formation of cancer-related fusion genes. Current structural variation (SV) callers, however, still produce massive numbers of false positives (FPs) and require high computational costs. Here, we introduce an ultra-fast and high-performing somatic SV detector, called ETCHING, that significantly reduces the mapping cost by filtering reads matched to pan-genome and normal k-mer sets. To reduce the number of FPs, ETCHING takes advantage of a Random Forest classifier that utilizes six breakend-related features. We systematically benchmarked ETCHING with other SV callers on reference SV materials, validated SV biomarkers, tumor and matched-normal whole genomes, and tumor-only targeted sequencing datasets. For all datasets, our SV caller was much faster ([≥]15X) than other tools without compromising performance or memory use. Our approach would provide not only the fastest method for largescale genome projects but also an accurate clinically practical means for real-time precision medicine.

bioinformatics↗

bioRxiv2020

High-throughput phenotypic characterization of zebrafish CRISPR mutants of DNA repair genes

A systematic knowledge of the roles of DNA repair genes at the level of the organism has been limited due to the lack of appropriate experimental techniques. Here, we generated zebrafish loss-of-function mutants for 32 DNA repair and replication genes through multiplexed CRISPR/Cas9-mediated mutagenesis. High-throughput phenotypic characterization of our mutant collection revealed that three genes (atad5a, ddb1, pcna) are essential for proper embryonic development and hematopoiesis; seven genes (apex1, atrip, ino80, mre11a, shfm1, telo2, wrn) are required for growth and development during juvenile stage and six genes (blm, brca2, fanci, rad51, rad54l, rtel1) play critical roles in sex development. Furthermore, mutation in six genes (atad5a, brca2, polk, rad51, shfm1, xrcc1) displayed hypersensitivity to DNA damage agents. Further characterization of atad5a-/- mutants demonstrate that Atad5a is required for normal brain development and hematopoiesis. Our zebrafish mutant collection provides a unique resource for understanding of the roles of DNA repair genes at the organismal level.

developmental biology↗