Biology subjects

Hong, S.

Publications and source records attributed to Hong, S..

At least 91 records · Page 5Linked to original sources

bioRxiv2021

From COVID-19 to the Common Cold: Novel Host-Targeted, Pan-Respiratory Antiviral Small Molecule Therapeutics

We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for modulation of viral capsid assembly. Activity of PAV-431, a representative compound from the series, has been validated against infectious virus in multiple cell culture models for all six families of viruses causing most respiratory disease in humans. In animals this chemotype has been demonstrated efficacious for Porcine Epidemic Diarrhea Virus (a coronavirus) and Respiratory Syncytial Virus (a paramyxovirus). PAV-431 is shown to bind to the protein 14-3-3, a known allosteric modulator. However, it only appears to target the small subset of 14-3-3 which is present in a dynamic multi-protein complex whose components include proteins implicated in viral lifecycles and in innate immunity. The composition of this target multi-protein complex appears to be modified upon viral infection and largely restored by PAV-431 treatment. Our findings suggest a new paradigm for understanding, and drugging, the host-virus interface, which leads to a new clinical therapeutic strategy for treatment of respiratory viral disease.

biochemistry↗

bioRxiv2021

Topoisomerase 2b induces DNA breaks to regulate human papillomavirus replication

Topoisomerases regulate higher order chromatin structures through the transient breaking and re-ligating of one or both strands of the phosphodiester backbone of duplex DNA. TOP2{beta} is a type II topoisomerase that induces double strand DNA breaks at topological-associated domains (TADS) to relieve torsional stress arising during transcription or replication. TADS are anchored by CTCF and SMC1 cohesin proteins in complexes with TOP2{beta}. Upon DNA cleavage a covalent intermediate DNA-TOP2{beta} (TOP2{beta}cc) is transiently generated to allow for strand passage. The tyrosyl-DNA phosphodiesterase TDP2 can resolve TOP2{beta}cc but failure to do so quickly can lead to long-lasting DNA breaks. Given the role of CTCF/SMC1 proteins in the HPV life cycle we investigated if TOP2{beta} proteins contribute to HPV pathogenesis. Our studies demonstrated that levels of both TOP2{beta} and TDP2 were substantially increased in cells with high risk HPV genomes and this correlated with high amounts of DNA breaks. Knockdown of TOP2{beta} with shRNAs reduced DNA breaks by over 50% as determined through COMET assays. Furthermore this correlated with substantially reduced formation of repair foci such as {gamma}H2AX, pCHK1 and pSMC1 indicative of impaired activation of DNA damage repair pathways. Importantly, knockdown of TOP2{beta} also blocked HPV genome replication. Our previous studies demonstrated that CTCF /SMC1 factors associate with HPV genomes at sites in the late regions of HPV31 and these correspond to regions that also bind TOP2{beta}. This study identifies TOP2{beta} as responsible for enhanced levels of DNA breaks in HPV positive cells and as a regulator of viral replication.

microbiology↗

bioRxiv2021

A low-cost mouse cage warming system provides improved intra-ischemic and post-ischemic body temperature control &ndash: application for reducing variability in experimental stroke studies

Experimental guidelines have been proposed to improve the rigor and reproducibility of experimental stroke studies in rodents. As brain temperature is a strong determinant of ischemic injury, tight management of brain or body temperature (Tcore) during the experimental protocol is highly recommended. However, little guidance is provided regarding how or for how long temperature support should be provided. We compared a commonly used heat support method (cage on heating pad) with a low-cost custom built warm ambient air cage (WAAC) system. Both heat support systems were evaluated for the middle cerebral artery occlusion (MCAo) model in mice. The WAAC system provided improved temperature control (more normothermic Tcore and less Tcore variation) during the intra-ischemic period (60 min) and post-ischemic period (3 hrs). Neurologic deficit score showed significantly less variance at post-stroke day 1 (PSD1) in WAAC system mice. Mean infarct volume was not statistically different by heat support system, however, standard deviation was 54% lower in the WAAC system group. In summary, we provide a simple low-cost heat support system that provides superior Tcore management in mice during the intra-ischemic and post-ischemic periods, which results in reduced variability of experimental outcomes. HighlightsO_LIWe describe the fabrication of a low-cost mouse cage warming system (warmed ambient air cage; WAAC system) that can be assembled and applied in any stroke laboratory. C_LIO_LIThe WAAC system provides more precise control of post-stroke mouse body temperature compared with traditional heating pad warming system. C_LIO_LIThe more precise control of post-stroke core temperature reduces variability in some experimental measures in more severely injured mice. C_LI

neuroscience↗

bioRxiv2020

Single Cell Sequencing Reveals Glial Specific Responses to Tissue Processing & Enzymatic Dissociation in Mice and Humans

A key aspect of nearly all single cell experiments is the necessity to dissociate intact tissues into single cell suspensions for processing. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that dissociation can have on ex vivo gene expression changes during this process. Microglia, the brains resident macrophages, are a highly dynamic population that are extremely sensitive to their microenvironment and have been shown to dramatically alter their transcriptome upon stimulation. We demonstrate that use of enzymatic dissociation methods on mouse central nervous system (CNS) tissue induces an aberrant gene expression signature in microglia that can significantly confound downstream analysis. To minimize this issue, we developed a flexible protocol, that can be used with existing enzymatic protocols for fresh tissue, to eliminate artifactual gene expression while allowing for increased cell type diversity and yield. We demonstrate efficacy of this protocol in analysis of diverse CNS cell types and sorted myeloid populations while using enzymatic dissociation. Generation of new and reanalysis of previously published human brain single nucleus RNAseq (snRNA-seq) datasets reveal that a similar signature is also present in post-mortem tissue. Through novel snRNA-seq analysis of acutely-resected neurosurgical tissue we demonstrate that this signature can be induced in human tissue due to technical differences in sample processing. These results provide key insight into the potential confounds of enzymatic digestion and provide a solution to allow for enzymatic digestion for scRNA-seq while avoiding ex vivo transcriptional artifacts. Analysis of human tissue reveals potential for artifacts in current and future snRNA-seq datasets that will require deeper analysis and careful consideration to separate true biology from artifacts related to post-mortem processes.

neuroscience↗

bioRxiv2020

Deep learning enables rapid and robust analysis of fluorescence lifetime imaging in photon-starved conditions

Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to quantify molecular compositions and study the molecular states in the complex cellular environment as the lifetime readings are not biased by the fluorophore concentration or the excitation power. However, the current methods to generate FLIM images are either computationally intensive or unreliable when the number of photons acquired at each pixel is low. Here we introduce a new deep learning-based method termed flimGANE (fluorescence lifetime imaging based on Generative Adversarial Network Estimation) that can rapidly generate accurate and high-quality FLIM images even in the photon-starved conditions. We demonstrated our model is not only 258 times faster than the most popular time-domain least-square estimation (TD_LSE) method but also provide more accurate analysis in barcode identification, cellular structure visualization, Forster resonance energy transfer characterization, and metabolic state analysis. With its advantages in speed and reliability, flimGANE is particularly useful in fundamental biological research and clinical applications, where ultrafast analysis is critical.

cell biology↗

bioRxiv2020

Hsp90 and its co-chaperone Sti1 control TDP-43 misfolding and toxicity

Protein misfolding is a central feature of most neurodegenerative diseases. Molecular chaperones can modulate the toxicity associated with protein misfolding, but it remains elusive which molecular chaperones and co-chaperones interact with specific misfolded proteins. TDP-43 misfolding and inclusion formation is a hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. Using yeast and mammalian neuronal cells we find that Hsp90 and its co-chaperones have a strong capacity to alter TDP-43 misfolding, inclusion formation, aggregation, and cellular toxicity. Our data also demonstrate that impaired Hsp90 function sensitizes cells to TDP-43 toxicity. We further show that the co-chaperone Sti1 specifically interacts with and modulates TDP-43 toxicity in a dose-dependent manner. Our study thus uncovers a previously unrecognized tie between Hsp90, Sti1, TDP-43 misfolding, and its cellular toxicity.

cell biology↗

bioRxiv2020

Sex-specific lesion topographies explain outcomesafter acute ischemic stroke

Acute ischemic stroke affects men and women differently in many ways. In particular, women are oftentimes reported to experience a higher acute stroke severity than men. Here, we derived a low-dimensional representation of anatomical stroke lesions and designed a sex-aware Bayesian hierarchical modelling framework for a large-scale, well phenotyped stroke cohort. This framework was tailored to carefully estimate possible sex differences in lesion patterns explaining acute stroke severity (NIHSS) in 1,058 patients (39% female). Anatomical regions known to subserve motor and language functions emerged as relevant regions for both men and women. Female patients, however, presented a more widespread pattern of stroke severity-relevant lesions than male patients. Furthermore, particularly lesions in the posterior circulation of the left hemisphere underlay a higher stroke severity exclusively in women. These sex-sensitive lesion pattern effects could be discovered and subsequently robustly replicated in two large independent, multisite lesion datasets. The constellation of findings has several important conceptual and clinical implications: 1) suggesting sex-specific functional cerebral asymmetries, and 2) motivating a sex-stratified approach to management of acute ischemic stroke. To go beyond sex-averaged stroke research, future studies should explicitly test whether acute therapies administered on the basis of sex-specific cutoff volumes of salvageable tissue will lead to improved outcomes in women after acute ischemic stroke.

neuroscience↗

bioRxiv2020

Structural insights into mRNA reading frame regulation by tRNA modification and slippery codon-anticodon pairing

Modifications in the tRNA anticodon, adjacent to the three-nucleotide anticodon, influence translation fidelity by stabilizing the tRNA to allow for accurate reading of the mRNA genetic code. One example is the N1-methylguaonosine modification at guanine nucleotide 37 (m1G37) located in the anticodon loop, immediately adjacent to the anticodon nucleotides 34-36. The absence of m1G37 in tRNAPro causes +1 frameshifting on polynucleotide, slippery codons. Here, we report structures of the bacterial ribosome containing tRNAPro bound to either cognate or slippery codons to determine how the m1G37 modification prevents mRNA frameshifting. The structures reveal that certain codon-anticodon contexts and m1G37 destabilize interactions of tRNAPro with the peptidyl site, causing large conformational changes typically only seen during EF-G mediated translocation of the mRNA-tRNA pairs. These studies provide molecular insights into how m1G37 stabilizes the interactions of tRNAPro with the ribosome and the influence of slippery codons on the mRNA reading frame. IMPACT STATEMENTChemical modifications near the tRNA anticodon and specific mRNA-tRNA pairs combine to control the ribosomal three-nucleotide mRNA reading frame, essential for the sequential addition of amino acids into polypeptide chains. Data depositionCrystallography, atomic coordinates, and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB codes 6NTA, 6NSH, 6NUO, 6NWY, 6O3M, 6OSI)

biochemistry↗

bioRxiv2020

Multiomic Immunophenotyping of COVID-19 Patients Reveals Early Infection Trajectories

Host immune responses play central roles in controlling SARS-CoV2 infection, yet remain incompletely characterized and understood. Here, we present a comprehensive immune response map spanning 454 proteins and 847 metabolites in plasma integrated with single-cell multi-omic assays of PBMCs in which whole transcriptome, 192 surface proteins, and T and B cell receptor sequence were co-analyzed within the context of clinical measures from 50 COVID19 patient samples. Our study reveals novel cellular subpopulations, such as proliferative exhausted CD8+ and CD4+ T cells, and cytotoxic CD4+ T cells, that may be features of severe COVID-19 infection. We condensed over 1 million immune features into a single immune response axis that independently aligns with many clinical features and is also strongly associated with disease severity. Our study represents an important resource towards understanding the heterogeneous immune responses of COVID-19 patients and may provide key information for informing therapeutic development.

immunology↗

bioRxiv2020

Modulate Siglec-7 Signaling via in situ Created High-affinity cis-Ligands

Sialic acid-binding immunoglobulin-like lectins, also known as Siglecs, have recently been designated as glyco-immune checkpoints. Through their interactions with sialylated glycan epitopes overexpressed on tumor cells, inhibitory Siglecs on innate and adaptive immune cells modulate signaling cascades to restrain anti-tumor immune responses. However, the mechanisms underlying these processes are just starting to be elucidated. We discover that when human natural killer (NK) cells attack tumor cells, glycan remodeling occurs on the target cells at the immunological synapse. This remodeling occurs through both transfer of sialylated glycans from NK cells to target tumor cells and accelerated de novo synthesis of sialosides on the tumor cell themselves. The functionalization of NK cells with a high-affinity ligand of Siglec-7 leads to multifaceted consequences in modulating Siglec-7-regulated NK-activation. At high levels, the added Siglec-7 ligand suppresses NK-cytotoxicity through the recruitment of Siglec-7, whereas at low levels the same ligand triggers the release of Siglec-7 from the cell surface into the culture medium, preventing Siglec-7-mediated inhibition of NK cytotoxicity. These results suggest that glycan engineering of NK cells may provide a means to boost NK effector functions.

biochemistry↗

bioRxiv2020

Ultra-Fast Homomorphic Encryption Models enable Secure Outsourcing of Genotype Imputation

ABSTRACTGenotype imputation is a fundamental step in genomic data analysis such as GWAS, where missing variant genotypes are predicted using the existing genotypes of nearby ‘tag’ variants. Imputation greatly decreases the genotyping cost and provides high-quality estimates of common variant genotypes. As population panels increase, e.g., the TOPMED Project, genotype imputation is becoming more accurate, but it requires high computational power. Although researchers can outsource genotype imputation, privacy concerns may prohibit genetic data sharing with an untrusted imputation service. To address this problem, we developed the first fully secure genotype imputation by utilizing ultra-fast homomorphic encryption (HE) techniques that can evaluate millions of imputation models in seconds. In HE-based methods, the genotype data is end-to-end encrypted, i.e., encrypted in transit, at rest, and, most importantly, in analysis, and can be decrypted only by the data owner. We compared secure imputation with three other state-of-the-art non-secure methods under different settings. We found that HE-based methods provide full genetic data security with comparable or slightly lower accuracy. In addition, HE-based methods have time and memory requirements that are comparable and even lower than the non-secure methods. We provide five different implementations and workflows that make use of three cutting-edge HE schemes (BFV, CKKS, TFHE) developed by the top contestants of the iDASH19 Genome Privacy Challenge. Our results provide strong evidence that HE-based methods can practically perform resource-intensive computations for high throughput genetic data analysis. In addition, the publicly available codebases provide a reference for the development of secure genomic data analysis methods.Competing Interest StatementThe authors have declared no competing interest.View Full Text

bioinformatics↗

bioRxiv2020

Accommodating individual travel history, global mobility, and unsampled diversity in phylogeography: a SARS-CoV-2 case study.

Spatiotemporal bias in genome sequence sampling can severely confound phylogeographic inference based on discrete trait ancestral reconstruction. This has impeded our ability to accurately track the emergence and spread of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Despite the availability of unprecedented numbers of SARS-CoV-2 genomes on a global scale, evolutionary reconstructions are hindered by the slow accumulation of sequence divergence over its relatively short transmission history. When confronted with these issues, incorporating additional contextual data may critically inform phylodynamic reconstructions. Here, we present a new approach to integrate individual travel history data in Bayesian phylogeographic inference and apply it to the early spread of SARS-CoV-2, while also including global air transportation data. We demonstrate that including travel history data for each SARS-CoV-2 genome yields more realistic reconstructions of virus spread, particularly when travelers from undersampled locations are included to mitigate sampling bias. We further explore methods to ameliorate the impact of sampling bias by augmenting the phylogeographic analysis with lineages from undersampled locations in the analyses. Our reconstructions reinforce specific transmission hypotheses suggested by the inclusion of travel history data, but also suggest alternative routes of virus migration that are plausible within the epidemiological context but are not apparent with current sampling efforts. Although further research is needed to fully examine the performance of our travel-aware phylogeographic analyses with unsampled diversity and to further improve them, they represent multiple new avenues for directly addressing the colossal issue of sample bias in phylogeographic inference.

evolutionary biology↗

bioRxiv2020

TMEM106B and CPOX are genetic determinants of cerebrospinal fluid Alzheimer's disease biomarker levels

BackgroundNeurofilament light (NF-L), chitinase-3-like protein 1 (YKL-40), and neurogranin (Ng) are utilized as biomarkers for Alzheimers disease (AD), to monitor axonal damage, astroglial activation, and synaptic degeneration, respectively. Here we performed genome-wide association study (GWAS) analyses using all three biomarkers as outcome. MethodsDNA and cerebrospinal fluid (CSF) samples originated from the European Medical Information Framework AD Multimodal Biomarker Discovery (EMIF-AD MBD) study. Overlapping genotype/phenotype data were available for n=671 (NF-L), 677 (YKL-40), and 672 (Ng) individuals. GWAS analyses applied linear regression models adjusting for relevant covariates. FindingsWe identify novel genome-wide significant associations with markers in TMEM106B and CSF levels of NF-L. Additional novel signals were observed with DNA variants in CPOX and CSF levels of YKL-40. Lastly, we confirmed previous work suggesting that YKL-40 levels are regulated by cis protein quantitative trait loci (pQTL) in CHI3L1. InterpretationOur study provides important new insights into the genetic architecture underlying inter-individual variation in all three tested AD-related CSF biomarkers. In particular, our data shed light on the sequence of events regarding the initiation and progression of neuropathological processes relevant in AD.

genomics↗

bioRxiv2020

Glycoengineering of NK cells with glycan ligands of CD22 and selectins for B-cell lymphoma therapy

CD22, a member of Siglec family of sialic acid binding proteins, has restricted expression on B cells. Antibody-based agents targeting CD22 or CD20 (Rituxan) on B lymphoma and leukemia cells exhibit clinical efficacy for treating these malignancies, but also attack normal B cells leading to immune deficiency. Here, we report a chemoenzymatic glycocalyx editing strategy to introduce high-affinity and specific CD22 ligands onto NK-92MI and cytokine-induced killer (CIK) cells to achieve tumor-specific CD22 targeting. These CD22-ligand modified cells exhibited significantly enhanced tumor cell binding and killing in vitro without harming healthy B cells. For effective lymphoma cell killing in vivo we further functionalized CD22 ligand-modified NK-92MI cells with the E-selectin ligand sialyl Lewis X to promote trafficking to bone marrow. The cells containing the ligands of both CD22 and selectins resulted in the efficient suppression of B lymphoma in a xenograft model. Our results suggest that NK cells modified with glycan ligands to CD22 and selectins promote both targeted killing of B lymphoma cells and improved trafficking to sites where the cancer cells reside, respectively.

bioengineering↗

bioRxiv2020

Cerebellar Purkinje cells can differentially modulate coherence between sensory and motor cortex depending on region and behavior

Coherence among sensory and motor cortices is indicative of binding of critical functions in perception, motor planning, action and sleep. Evidence is emerging that the cerebellum can impose coherence between cortical areas, but how and when it does so is unclear. Here, we studied coherence between primary somatosensory (S1) and motor (M1) cortices during sensory stimulation of the whiskers in the presence and absence of optogenetic stimulation of cerebellar Purkinje cells in awake mice. Purkinje cell activation enhanced and reduced sensory-induced S1-M1 coherence in the theta and gamma bands, respectively. This impact only occurred when Purkinje cell stimulation was given simultaneously with sensory stimulation; a 20 ms delay was sufficient to alleviate its impact, suggesting the existence of a fast, cerebellar sensory pathway to S1 and M1. The suppression of gamma band coherence upon Purkinje cell stimulation was significantly stronger during trials with relatively large whisker movements, whereas the theta band changes did not show this correlation. In line with the anatomical distribution of the simple spike and complex spike responses to whisker stimulation, this suppression also occurred following focal stimulation of medial crus 2, but not of lateral crus 1. Granger causality analyses and computational modeling of the involved networks suggest that Purkinje cells control S1-M1 coherence most prominently via the ventrolateral thalamus and M1. Our results indicate that coherences between sensory and motor cortices in different frequency ranges can be dynamically modulated by cerebellar input, and that the modulation depends on the behavioral context and is site-specific. Significance StatementCoherent activity between sensory and motor areas is essential in sensorimotor integration. We show here that the cerebellum can differentially affect cortical theta and gamma band coherences evoked by whisker stimulation via a fast ascending and predictive pathway. In line with the functional heterogeneity of its modular organization, the impact of the cerebellum is region-specific and tuned to ongoing motor responses. These data highlight site-specific and context-dependent interactions between the cerebellum and the cerebral cortex that can come into play during a plethora of sensorimotor functions.

neuroscience↗

bioRxiv2020

Obesity-linked PPARγ S273 phosphorylation promotes insulin resistance through Growth Differentiation Factor 3

Overnutrition and obesity promote adipose tissue dysfunction, often leading to systemic insulin resistance. The thiazolidinediones (TZDs) are a potent class of insulin-sensitizing drugs and ligands of PPAR{gamma} that improve insulin sensitivity, but their use is limited due to significant side effects. Recently, we demonstrated a mechanism by which TZDs improve insulin sensitivity distinct from receptor agonism and adipogenesis: reversal of obesity-linked phosphorylation of PPAR{gamma} at Serine 273. However, the role of this modification has not been tested genetically. Here we demonstrate that mice encoding an allele of PPAR{gamma} which cannot be phosphorylated at S273 are protected from insulin resistance, without exhibiting differences in body weight or TZD-associated side effects. Indeed, hyperinsulinemic-euglycemic clamp experiments confirm improved insulin sensitivity, as evidenced by increased whole-body glucose uptake. RNA-seq experiments reveal PPAR{gamma} S273 phosphorylation specifically enhances transcription of Gdf3, a BMP family member. Ectopic expression of Gdf3 is sufficient to induce insulin resistance in lean, healthy mice. We find that Gdf3 can impact metabolism by inhibition of BMP signaling. Together, these results highlight the diabetogenic role of PPAR{gamma} S273 phosphorylation and focuses attention on a putative target, Gdf3.

cell biology↗

bioRxiv2019

PULL-PUSH NEUROMODULATION OF CORTICAL PLASTICITY ENABLES RAPID BI-DIRECTIONAL SHIFTS IN OCULAR DOMINANCE

Neuromodulatory systems are essential for remodeling glutamatergic connectivity during experience-dependent cortical plasticity. This permissive/enabling function of neuromodulators has been associated with their capacity to facilitate the induction of Hebbian forms of long-term potentiation (LTP) and depression (LTD) by affecting cellular and network excitability. In vitro studies indicate that neuromodulators can also affect the expression of Hebbian plasticity in a pull-push manner: receptors coupled to the G-protein Gs promote the expression of LTP at the expense of LTD, and Gq-coupled receptors promote LTD at the expense of LTD. Here we show that the pull-push mechanism can be recruited in vivo by pairing brief monocular stimulation with pharmacological or chemogenetical activation of Gs- or Gq-coupled receptors to respectively enhance or reduce visual cortical responses. These changes were stable, can be induced in adults after the termination of the critical period for juvenile ocular dominance plasticity, and can rescue deficits induced by prolonged monocular deprivation.

neuroscience↗

bioRxiv2019

hFUT1-based live cell assay to profile α1-2-fucosides enhanced influenza A virus infection

Host cell-surface glycans play critical roles in influenza A virus (IAV) infection ranging from modulation of IAV attachment to membrane fusion and host tropism. Approaches for quick and sensitive profiling of the viral avidity towards a specific type of host-cell glycan can contribute to the understanding of tropism switching among different strains of IAV. In this study, we developed a method based on chemoenzymatic glycan engineering to investigate the possible involvement of 1-2-fucosides in IAV infections. Using a truncated human fucosyltransferase 1 (hFuT1), we were able to create 1-2-linked fucosides in situ on the host cell surface to assess their influence on the host cell binding to IAV hemagglutinin and the susceptibility of host cells toward IAV induced killing. We discovered that the newly added 1-2-fucosides on host cells enhanced the infection of several human pandemic IVA subtypes. These findings suggest that glycan epitopes other than sialic aicds should be taken into consideration for assessing the human pandemic risk of this viral pathogen.

biochemistry↗