Biology subjects

Lee, S.-H.

Publications and source records attributed to Lee, S.-H..

126 records · Page 7Linked to original sources

bioRxiv2020

Multiscale Characterization of Complex Binding Interactions of Cellulolytic Enzymes Highlights Limitations of Classical Approaches

Cellulolytic microorganisms, like Trichoderma reesei or Clostridium thermocellum, frequently have non-catalytic carbohydrate-binding modules (CBMs) associated with secreted or cell surface bound multidomain carbohydrate-active enzymes (CAZymes) like cellulases. Mostly type-A family CBMs are known to promote cellulose deconstruction by increasing the substrate-bound concentration of cognate cellulase catalytic domains. However, due to the interfacial nature of cellulose hydrolysis and the structural heterogeneity of cellulose, it has been challenging to fully understand the role of CBMs on cellulase activity using classical protein-ligand binding assays. Here, we report a single-molecule CAZyme assay for an industrially relevant processive cellulase Cel7A (from T. reesei) to reveal how subtle CBM1 binding differences can drastically impact cellulase motility/velocity and commitment to initial processive motion for deconstruction of two well-studied crystalline cellulose allomorphs (namely cellulose I and III). We take a multifaceted approach to characterize the complex binding interactions of all major type-A family representative CBMs including CBM1, using an optical-tweezers based single-molecule CBM-cellulose bond rupture assay to complement several classical bulk ensemble protein-ligand binding characterization methods. While our work provides a basis for the cautious use of Langmuir-type adsorption models to characterize classical protein-ligand binding assay data, we highlight the critical limitations of using such overly simplistic models to gain a truly molecular-level understanding of interfacial protein binding interactions at heterogeneous solid-liquid interfaces. Finally, molecular dynamics simulations provided a theoretical basis for the complex binding behavior seen for CBM1 towards two distinct cellulose allomorphs reconciling experimental findings from multiscale analytical methods. Significance StatementMultimodal biomolecular binding interactions involving carbohydrate polymers (e.g., cellulose, starch, chitin, glycosaminoglycans) are fundamental molecular processes relevant to the recognition, biosynthesis, and degradation of all major terrestrial and aquatic biomass. Protein-carbohydrate binding interactions are also critical to industrial biotechnology operations such as enzymatically-catalyzed bioconversion of starch and lignocellulose into biochemicals like ethanol. However, despite the ubiquitous importance of such interfacial processes, we have a poor molecular-level understanding of protein-polysaccharide binding interactions. Here, we provide a comprehensive experimental and theoretical analysis of bulk ensemble versus single-molecule binding interactions of enzyme motors and associated non-catalytic binding domains with cellulosic polysaccharides to highlight the critical limitations of applying classical biochemical assay techniques alone to understanding protein adsorption or biological activity at solid-liquid interfaces.

biochemistry↗

bioRxiv2020

CDC50A dependent phosphatidylserine exposure induces inhibitory post-synapse elimination by microglia

Glia contribute to synapse elimination through phagocytosis in the central nervous system. Despite important roles during development and neurological disorders, the "eat-me" signal that initiates glia-mediated phagocytosis of synapses remains largely elusive. Here, by generating inducible conditional knockout mice of Cdc50a, we induced stable exposure of phosphatidylserine in the neuronal outer membrane. Surprisingly, acute Cdc50a deletion in neurons causes specific loss of inhibitory post-synapses without affecting other synapses, thereby generating excessive excitability with appearance of seizure. Ablating microglia or deleting microglial Mertk rescues the loss of inhibitory post-synapses, indicating that microglial phagocytosis is responsible for inhibitory post-synapse elimination. Moreover, inhibitory post-synapses in normal juvenile brains also use phosphatidylserine for synapse pruning by microglia, suggesting that phosphatidylserine may serve as a general "eat-me" signal for inhibitory post-synapse elimination. One Sentence SummaryCdc50a dependent phosphatidylserine exposure functions as an "eat-me" signal for microglia-dependent inhibitory post-synapse elimination

neuroscience↗

bioRxiv2020

Successful Correction of ALD Patient-derived iPSCs Using CRISPR/Cas9

X-linked adrenoleukodystrophy (ALD) caused by the ABCD1 mutation, is the most common inherited peroxisomal disease. It is characterized by three phenotypes: inflammatory cerebral demyelination, progressive myelopathy, and adrenal insufficiency, but there is no genotype-phenotype correlation. Hematopoietic stem cell transplantation can only be used in a few patients in the early phase of cerebral inflammation; therefore, most affected patients have no curative option. Previously, we reported the generation of an ALD patient-derived iPSC model and its differentiation to oligodendrocytes. In this study, we have performed the first genome editing of ALD patient-derived iPSCs using homology-directed repair (HDR). The mutation site, c.1534G>A [GenBank: NM_000033.4], was corrected by introducing ssODN and the CRISPR/Cas9 system. The cell line exhibited normal ALD protein expression following genome editing. We differentiated the intermediate oligodendrocytes from mutation-corrected iPSCs and the metabolic derangement of ALD tended to correct but was not statistically significant. Mutation-corrected iPSCs from ALD patient can be used in research into the pathophysiology of and therapeutics for ALD.

neuroscience↗

bioRxiv2020

Whole exome precision oncology targeting synthetic lethal vulnerabilities across the tumor transcriptome

Precision oncology has made significant advances in the last few years, mainly by targeting actionable mutations in cancer driver genes. However, the proportion of patients whose tumors can be targeted therapeutically remains limited. Recent studies have begun to explore the benefit of analyzing tumor transcriptomics data to guide patient treatment, raising the need for new approaches for systematically accomplishing that. Here we show that computationally derived genetic interactions can successfully predict patient response. Assembling a broad repertoire of 32 datasets spanning more than 1,500 patients and including both tumor transcriptomics and response data, we predicted the response in 17 out of 21 targeted and 8 out of 11 checkpoint therapy datasets across 8 different cancer types with considerable accuracy, without ever training on these datasets. Analyzing the recently published multi-arm WINTHER trial, we show that the fraction of patients benefitting from transcriptomic-based treatments could potentially be markedly increased from 15% to about 85% by targeting synthetic lethal vulnerabilities in their tumors. In summary, this is the first computational approach to obtain considerable predictive performance across many different targeted and immunotherapy datasets, providing a promising new way for guiding cancer treatment based on the tumor transcriptomics of cancer patients.

cancer biology↗

bioRxiv2019

A new formalism for perceptual classification with normative inference of internal criteria

Classification, one of the key ingredients for human cognition, entails establishing a criterion that splits a given feature space into mutually exclusive subspaces. In classification tasks performed in daily life, however, a criterion is often not provided explicitly but instead needs to be guessed from past samples of a feature space. For example, we judge todays temperature to be "cold" or "warm" by implicitly comparing it against a "typical" seasonal temperature. In such situations, establishing an optimal criterion is challenging for cognitive agents with bounded memory because it requires retrieving an entire set of past episodes with precision. As a computational account for how humans carry out this challenging operation, we developed a normative Bayesian model of classification (NBMC), in which Bayesian agents, whose working-memory precision decays as episodes elapse, continuously update their criterion as they perform a binary perceptual classification task on sequentially presented stimuli. We drew a set of specific implications regarding key properties of classification from the NBMC, and demonstrated the correspondence between the NBMC and human observers in classification behavior for each of those implications. Furthermore, in the functional magnetic resonance imaging responses acquired concurrently with behavioral data, we identified an ensemble of brain activities that coherently represent the latent variables, including the inferred criterion, of the NBMC. Given these results, we believe that the NBMC is worth being considered as a useful computational model that guides behavioral and neural studies on perceptual classification, especially for agents with bounded memory representation of past sensory events. Significant StatementAlthough classification--assigning events into mutually exclusive classes--requires a criterion, people often have to perform various classification tasks without explicit criteria. In such situations, forming a criterion based on past experience is quite challenging because peoples memory of past events deteriorates quickly over time. Here, we provided a computational model for how a memory-bounded yet normative agent infers the criterion from past episodes to maximally perform a binary perceptual classification task. This model successfully captured several key properties of human classification behavior, and the neural signals representing its latent variables were identified in the classifying human brains. By offering a rational account for memory-bonded agents classification, our model can guide future behavioral and neural studies on perceptual classification.

neuroscience↗

bioRxiv2019

Long-term functional culture and in vitro manipulation of adult mouse cardiomyocytes

Primary adult cardiomyocyte (aCM) culture is challenged by poor survival and loss of phenotype, rendering extended in vitro experiments unfeasible. Here, we establish murine aCM culture methods that enhance survival and maintain sarcomeric structure and Ca2+ cycling to enable physiologically-relevant contractile force measurements. We also demonstrate genetic and small-molecule manipulations that probe mechanisms underlying myocyte functional performance.

cell biology↗

bioRxiv2019

Dynamic healing and remodeling of mandibular ramus in segmental mobility and cortical overlap after intraoral vertical osteotomy

The ramus immediately after intraoral vertical ramus osteotomy (IVRO) shows segmental mobility, cortical overlap, and projection, which yield smooth-surfaced continuity and stability after one year. This study aimed to elucidate the three-dimensional (3D) morphological changes driven by regional healing and remodeling 12 months postoperatively (Post) and their relationships with the immediate postoperative (Imm) segmental overlapping pattern. We performed a retrospective study of the morphological change of mandibular ramus in terms of time and amount of cortical overlap after IVRO. 3D computed tomography data of 108 hemi-mandibles were analyzed by independent superimposition of Imm proximal and distal segment to Post mandible. Analysis showed extensive regional bone resorption and apposition resulting in a relatively flat-surfaced and morphologically intact Post ramus. The middle ramal remodeling was significantly associated with Imm overlapping patterns and Post mandibular movement, new bony projections of various shapes being observed at the posterior border of the Post ramus. The ramus after IVRO underwent dynamic morphological changes during healing/remodeling which were partly associated with Imm overlap. Moreover, the Post functional and smooth-surfaced ramus seems to have been facilitated by the close surgical approximation of segments and the postoperative mandibular functional rehabilitation, even with segmental positional changes.

pathology↗

bioRxiv2019

Kv4.1, a key ion channel for low frequency firing of dentate granule cells, is crucial for pattern separation

The dentate gyrus (DG) in the hippocampus may play key roles in remembering distinct episodes through pattern separation, which may be subserved by the sparse firing properties of granule cells (GCs) in the DG. Low intrinsic excitability is characteristic of mature GCs, but ion channel mechanisms are not fully understood. Here, we investigated ionic channel mechanisms for firing frequency regulation in GCs of the rat hippocampus, and identified Kv4.1 as a key player. Immunofluorescence analysis showed that Kv4.1 was preferentially expressed in the DG, and its expression level was higher at 8-week than 3-week-old mice, suggesting a developmental regulation of Kv4.1 expression. With respect to firing frequency, GCs are categorized into two distinctive groups: low frequency (LF) and high frequency (HF) firing GCs. Input resistance (Rin) of most LF-GCs is lower than 200 M{Omega}, suggesting that LF-GCs are fully mature GCs. Kv4.1 channel inhibition by intracellular perfusion of Kv4.1 antibody increased firing rates and gain of the input-output relationship selectively in LF-GCs with no significant effect on resting membrane potential and Rin, but had no effect in HF-GCs. Importantly, mature GCs from mice depleted of Kv4.1 transcripts in the DG showed increased firing frequency, and these mice showed an impairment in contextual discrimination task. Our findings suggest that Kv4.1 expression occurring at late stage of GC maturation is essential for low excitability of DG networks and thereby contributes to pattern separation.

neuroscience↗

bioRxiv2019

Broad Dengue Neutralization in Mosquitoes Expressing an Engineered Antibody

With dengue virus (DENV) becoming endemic in tropical and subtropical regions worldwide, there is a pressing global demand for effective strategies to control the mosquitoes that spread this disease. Recent advances in genetic engineering technologies have made it possible to create mosquitoes with reduced vector competence, limiting their ability to acquire and transmit pathogens. Here we describe the development of Aedes aegypti mosquitoes synthetically engineered to impede vector competence to DENV. These mosquitoes express a gene encoding an engineered single-chain variable fragment derived from a broadly neutralizing DENV human monoclonal antibody and have significantly reduced viral infection, dissemination, and transmission rates for all four major antigenically distinct DENV serotypes. Importantly, this is the first engineered approach that targets all DENV serotypes, which is crucial for effective disease suppression. These results provide a compelling route for developing effective genetic-based DENV control strategies, which could be extended to curtail other arboviruses.\n\nSignificance StatementHere we describe the development of Ae. aegypti mosquitoes synthetically engineered to impede vector competence to multiple serotypes of DENV. Given the global significance of DENV, these mosquitoes may be used to curtail Dengue transmission by this potent vector.

bioengineering↗

bioRxiv2019

The Ras family members follow the blood progesterone level during formation and regression in bovine corpus luteum

Ras family members regulate cellular differentiation, proliferation and survival. CL formation and regression are regulated by the blood P4 level. This study investigated the association between changes in Ras family members and the serum P4 level and determined protein interactions among Ras family members, hormone receptors, and angiogenetic and apoptotic factors during formation and regression of the bovine CL. RASAL3 and RASA3 were found using proteomics in CL and were significantly increased in the SPCL compared to the PPCL, whereas RasGEF1B was decreased in the PPCL. Hormone receptors and angiogenetic proteins expression was lower in the PPCL and SPCL than that in the RPCL, but apoptotic proteins were increased in the RPCL. The P4 and estrogen receptors positive correlated with RasGEF1B, R-Ras, and H-Ras through VEGFA, VEGFR2 and Tie2 in STRING database. RasGAP, H-Ras and R-Ras protein expression was increased in the PPCL compared to that in the SPCL, whereas RasGEF expression was decreased. In summary, Ras activation and angiogenesis in the CL were positively correlated with the blood P4 during estrous cylce. These results may increase understanding of Ras biological functions following stimulation of hormones and their receptors during tissue proliferation and degeneration.

cell biology↗

bioRxiv2019

JuLI: accurate detection of DNA fusions in clinical sequencing for precision oncology

Accurate detection of genomic fusions by high-throughput sequencing in clinical samples with inadequate tumor purity and formalin-fixed paraffin embedded (FFPE) tissue is an essential task in precise oncology. We developed the fusion detection algorithm Junction Location Identifier (JuLI) for optimization of high-depth clinical sequencing. We implemented novel filtering steps to minimize false positives and a joint calling function to increase sensitivity in clinical setting. We comprehensively validated the algorithm using high-depth sequencing data from cancer cell lines and clinical samples and whole genome sequencing data from NA12878. We showed that JuLI outperformed state-of-the-art fusion callers in cases with high-depth clinical sequencing and rescued a driver fusion from false negative in plasma cell-free DNA. JuLI is freely available via GitHub (https://github.com/sgilab/JuLI).

bioinformatics↗

bioRxiv2018

Effects of individuation and grouping on face representations in the visual cortex

Top-down signals can influence our visual perception by providing guidance on information processing. Especially, top-down control between two basic frameworks, \"Individuation\" and \"grouping\", is critical for information processing during face perception. Individuation of faces supports identity recognition while grouping subserves higher category level face perception such as race or gender. However, it still remains elusive how top-down dependent control between individuation and grouping affects cortical representations during face perception. Here we performed an fMRI experiment to investigate whether representations across early and high-level visual areas can be altered by top-down control between individuation and grouping process during face perception. Focusing on neural response patterns across the early visual cortex (EVC) and the face-selective area (the fusiform face area (FFA)), we found that the discriminability of individual faces from the response patterns was strong in the FFA but weak in the EVC during the individuation task whereas the EVC but not the FFA showed significant face discrimination during the grouping tasks. Thus, these findings suggest that the representation of face information across the early and high-level visual cortex is flexible depending on the top-down control of the perceptual framework between individuation and grouping.

neuroscience↗

bioRxiv2018

Automated 3-D mapping of single neurons in the standard brain atlas using single brain slices

Recent breakthroughs in neuroanatomical tracing methods have helped unravel complicated neural connectivity in whole brain tissue at a single cellular resolution. However, analysis of brain images remains dependent on highly subjective manual processing. In the present study, we introduce AMaSiNe, a novel software for automated mapping of single neurons in the standard mouse brain atlas. The AMaSiNe automatically calibrates alignment angles of each brain slice to match the Allen Reference Atlas (ARA), locates labeled neurons from multiple brain samples in a common brain space, and achieves a standardized 3D-rendered brain. Due to the high fidelity and reliability of AMaSiNe, the retinotopic structures of neural projections to the primary visual cortex (VISp) were determined from single and dual injections of the rabies virus onto different visual areas. Our results demonstrate that distinct retinotopic organization of bottom-up and top-down projections could be precisely mapped using AMaSiNe.

neuroscience↗

bioRxiv2018

Pseudomonas syringae evades phagocytosis in animal cells through type III effector-mediated inhibition of the LIM kinase-cofilin system

Certain animal and plant pathogenic bacteria have developed virulence factors (including effector proteins) that enable them to overcome host immunity. A plant pathogen, Pseudomonas syringae pv. tomato (Pto), secretes a large repertoire of effectors into plant cells via a type III secretory apparatus, thereby suppressing plant immunity. Here, we show that exposure to Pto caused sepsis in mice. Surprisingly, the effector HopQ1 disrupted phagocytosis by inhibiting actin rearrangement via a direct interaction with the LIM domain of the animal target protein LIM kinase, a key regulator of actin polymerization. The results provide new insights into cross-kingdom pathogenicity of bacteria. The current studies demonstrate that certain plant pathogenic bacteria such as Pto can be fatal in animals due to cross-kingdom host immune suppression.

microbiology↗

bioRxiv2018

Stimulus-responsive self-assembly of enzymatic fractal structures by computational design

Fractal topologies, which are statistically self-similar over multiple length scales, are pervasive in nature. The recurrence of patterns at increasing length scales in fractal-shaped branched objects, e.g., trees, lungs, and sponges, results in high effective surface areas, and provides key functional advantages, e.g., for molecular trapping and exchange. Mimicking these topologies in designed protein-based assemblies will provide access to novel classes of functional biomaterials for wide ranging applications. Here, we describe a modular, multi-scale computational design method for the reversible self-assembly of proteins into tunable supramolecular fractal-like topologies in response to phosphorylation. Computationally-guided atomic-resolution modeling of fusions of symmetric, oligomeric proteins with Src homology 2 (SH2) binding domain and its phosphorylatable ligand peptide was used to design iterative branching leading to fractal-like assembly formation by enzymes of the atrazine degradation pathway. Structural characterization using various microscopy techniques and Cryo-electron tomography revealed a variety of dendritic, hyperbranched, and sponge-like topologies which are self-similar over three decades ([~]10nm-10m) of length scale, in agreement with models from multi-scale computational simulations. We demonstrate control over mesoscale topology (by linker design), formation dynamics, and functional enhancements due to dynamic multi-component assemblies constructed with three atrazine degradation pathway enzymes. The described design method should enable the construction of a variety of novel, spatiotemporally responsive catalytic biomaterials featuring fractal topologies.

biophysics↗

bioRxiv2018

Dendritic spikes in hippocampal granule cells are necessary for long-term potentiation at the perforant path synapse

Long-term potentiation (LTP) of synaptic responses is essential for hippocampal memory function. Perforant-path (PP) synapses on hippocampal granule cells (GCs) contribute to the formation of associative memories, which are considered the cellular correlates of memory engrams. However, the mechanisms of LTP at these synapses are not well understood. Due to sparse firing activity and the voltage attenuation in their dendrites, it remains unclear how associative LTP at distal synapses occurs. Here we show that NMDA receptor-dependent LTP can be induced at PP-GC synapses without backpropagating action potentials (bAPs) in acute rat brain slices. Dendritic recordings reveal substantial attenuation of bAPs as well as local dendritic Na+ -spike generation during PP-GC input. Inhibition of Na+ -spikes impairs LTP suggesting that LTP at PP-GC synapse requires local Na+ -spikes. Thus, dendritic spikes are essential for LTP induction at PP-GC synapse and may constitute a key cellular mechanism for memory formation in the dentate gyrus.

neuroscience↗

bioRxiv2017

Modulatory upregulation of an insulin peptide gene by different pathogens in C. elegans

When an animal is infected, its innate immune response needs to be tightly regulated across tissues and coordinated with other aspects of organismal physiology. Previous studies with Caenorhabditis elegans have demonstrated that insulin-like peptide genes are differentially expressed in response to different pathogens. They represent prime candidates for conveying signals between tissues upon infection. Here, we focused on one such gene, ins-11 and its potential role in mediating cross-tissue regulation of innate immune genes. While diverse bacterial intestinal infections can trigger the up-regulation of ins-11 in the intestine, we show that epidermal infection with the fungus Drechmeria coniospora triggers an upregulation of ins-11 in the epidermis. Using the Shigella virulence factor OpsF, a MAP kinase inhibitor, we found that in both cases, ins-11 expression is controlled cell autonomously by p38 MAPK, but via distinct transcription factors, STA-2/STAT in the epidermis and HLH-30/TFEB in the intestine. We established that ins-11, and the insulin signaling pathway more generally, are not involved in the regulation of antimicrobial peptide gene expression in the epidermis. The up-regulation of ins-11 in the epidermis does, however, affect intestinal gene expression in a complex manner, and has a deleterious effect on longevity. These results support a model in which insulin signaling, via ins-11, contributes to the coordination of the organismal response to infection, influencing the allocation of resources in an infected animal.

genetics↗

bioRxiv2017

Widespread intronic polyadenylation diversifies immune cell transcriptomes

Alternative cleavage and polyadenylation (ApA) can generate mRNA isoforms with differences in 3UTR length without changing the coding region (CDR). However, ApA can also recognize intronic polyadenylation (IpA) signals to generate transcripts that lose part or all of the CDR. We analyzed 46 3-seq and RNA-seq profiles from normal human tissues, primary immune cells, and multiple myeloma (MM) samples and created an atlas of 4,927 high confidence IpA events. Up to 16% of expressed genes in immune cells generate IpA isoforms, a majority of which are differentially used during B cell development or in different cellular environments, while MM cells display a striking loss of IpA isoforms expressed in plasma cells, their cell type of origin. IpA events can lead to truncated proteins lacking C-terminal functional domains. This can mimic ectodomain shedding through loss of transmembrane domains or alter the binding specificity of proteins with DNA-binding or protein-protein interaction domains, thus contributing to diversification of the transcriptome.

genomics↗