Biology subjects

Kang, B.

Publications and source records attributed to Kang, B..

29 records · Page 2Linked to original sources

bioRxiv2023

Highly multiplexed mRNA quantitation with CRISPR-Cas13

RNA quantitation tools are often either high-throughput or cost-effective, but rarely are they both. Existing methods can profile the transcriptome at great expense or are limited to quantifying a handful of genes by labor constraints. A technique that permits more throughput at a reduced cost could enable multi-gene kinetic studies, gene regulatory network analysis, and combinatorial genetic screens. Here, we introduce quantitative Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (qCARMEN): an RNA quantitation technique which leverages the programmable RNA-targeting capabilities of CRISPR-Cas13 to address this challenge by quantifying over 4,500 gene-sample pairs in a single experiment. Using qCARMEN, we studied the response profiles of interferon-stimulated genes (ISGs) during interferon (IFN) stimulation and flavivirus infection. Additionally, we observed isoform switching kinetics during epithelial-mesenchymal transition. qCARMEN is a simple and inexpensive technique that greatly enhances the scalability of RNA quantitation for novel applications with performance similar to gold-standard methods.

molecular biology↗

bioRxiv2023

Distinct prefrontal projections conversely orchestrate social competition and hierarchy

Social animals compete for limited resources, resulting in a social hierarchy. Although different neuronal subpopulations in the medial prefrontal cortex (mPFC), a hub brain region of social hierarchy, encode distinct social competition behaviors, their identities and associated molecular underpinnings have not yet been identified. In this study, we found that mPFC neurons projecting to the nucleus accumbens (mPFC-NAc) encode social winning behavior, whereas mPFC neurons projecting to the ventral tegmental area (mPFC-VTA) encode social losing behavior by monitoring and manipulating neural circuit activity. High-throughput single-cell transcriptomic analysis and projection-specific genetic manipulation revealed that the expression level of POU domain, class 3, transcription factor 1 (Pou3f1) in mPFC-VTA neurons controls social hierarchy. Optogenetic activation of mPFC-VTA neurons increases Pou3f1 expression and lowers social rank. Together, these data demonstrate that discrete activity and gene expression in separate mPFC projections oppositely orchestrate social competition and hierarchy.

neuroscience↗

bioRxiv2022

NF kappa B Regulator Bcl3 Controls Development and Function of Classical Dendritic Cells in Toxoplasma gondii

The atypical I{kappa}B family member Bcl3 associates with p50/NF-{kappa}B1 or p52/NF-{kappa}B2 homodimers in the nucleus, and positively or negatively modulates transcription in a context-dependent manner. In mice lacking Bcl3 globally or specifically in CD11c+ cells, Toxoplasma gondii infection is uniformly fatal and is associated with an impaired Th1 immune response. Since Bcl3 expression in dendritic cells (DC) is pivotal for antigen presentation and since classical DCs (cDC) are major antigen presenting cells, we investigated the role of Bcl3 specifically in cDCs in T. gondii infection in vivo by crossing Zbtb46 cre mice with Bcl3flx/flx mice. The conditional cDC Bcl3 KO was as susceptible to lethal T. gondii infection as the total Bcl3 KO and generated poor Th1 responses. Splenocyte single cell RNA seq in the model revealed defective Bcl3-dependent expression of genes involved in antigen processing. Consistent with this, soluble toxoplasma antigen presentation was impaired in Bcl3-deficient cDCs, and tetramer staining demonstrated defective T. gondii antigen-specific splenic CD4+ and CD8+ T cell responses in infected cDC Bcl3-/- mice. In vitro differentiation of bone marrow progenitors from wildtype and cDC Bcl3-/- mice using Flt3L, NOTCH and IFN-{gamma} stimulation recapitulated the defective Bcl3-dependent cDC antigen-presentation activity observed in vivo. Splenocyte single cell RNA seq also revealed the existence of a unique subpopulation of Zbtb46+LysM+ DC which exhibited Bcl3-dependent expansion after infection. We also detected cDCs coexpressing the monocytic markers CD64 and Ly6C (designated icDC1 and icDC2) mainly in infected spleen, which were less abundant in Bcl3flx/flx Zbtb46 cre mice. Together, our results indicate that Bcl3 in classical DCs is a major determinant of protective T cell responses and survival in T. gondii-infected mice, and shapes DC ontogeny. Author SummaryDendritic cells initiate immune responses against invading pathogens. As professional antigen presenting cells they process and present antigen via the major histocompatibility complex to T cells and thus activate them. Bcl3, an atypical member of the I{kappa}B family regulates the APC function of dendritic cells. In this study we show that expression of Bcl3 specifically in classical DCs is critical for host protection against a protozoan parasite, Toxoplasma gondii. Host protective proinflammatory mechanisms are compromised in mice deficient in Bcl3 in classical DCs leading to an elevated organ parasite load and eventually death of the infected animals. We also found the emergence of Bcl3-dependent hybrid DCs upon T. gondii infection, which have mixed phenotypic markers from DCs and monocytes. Antigen processing genes are significantly downregulated in Bcl3-deficient cDCs, which may account for defective cross presentation of T. gondii antigens. In an in vitro differentiation model, we showed that development of XCR1+cross presenting cDC1s is critically regulated by Bcl3. Overall, this study reveals the complexity of dendritic cell ontogeny and the role of Bcl3 in classical DC function in the context of Toxoplasma infection.

immunology↗

bioRxiv2022

Cellular and Molecular Organization of the Rhesus Macaque Dorsal Horn with a Comparison to Mouse

Key mechanisms underlying chronic pain occur within the neural circuitry of the dorsal horn. Recent genome-wide association studies (GWAS) have identified genetic variants associated with the predisposition to chronic pain. However, most of these variants lie in regulatory non-coding regions that have so far not been linked to spinal cord function. Here, we take a multi-species approach to determine whether chronic pain variants impact regulatory elements of dorsal horn neurons. We first built a more comprehensive single cell atlas; filling gaps by generating a high-quality Rhesus macaque atlas and integrating it with human and mouse. With cellular-resolution spatial transcriptomics, we mapped the laminar distributions of the resulting species-conserved neuron subtypes, uncovering an unexpected organization. Lastly, we generated a mouse single-nucleus open chromatin atlas to partition the heritability of chronic pain traits. From this, we identified strong, selective associations between specific, conserved neuron subtypes and major forms of chronic pain.

neuroscience↗

bioRxiv2021

The in vitro and in vivo potency of CT-P59 against Delta and its associated variants of SARS-CoV-2

The Delta variant originally from India is rapidly spreading across the world and causes to resurge infections of SARS-CoV-2. We previously reported that CT-P59 presented its in vivo potency against Beta and Gamma variants, despite its reduced activity in cell experiments. Yet, it remains uncertain to exert the antiviral effect of CT-P59 on the Delta and its associated variants (L452R). To tackle this question, we carried out cell tests and animal study. CT-P59 showed reduced antiviral activity but enabled neutralization against Delta, Epsilon, and Kappa variants in cells. In line with in vitro results, the mouse challenge experiment with the Delta variant substantiated in vivo potency of CT-P59 showing symptom remission and virus abrogation in the respiratory tract. Collectively, cell and animal studies showed that CT-P59 is effective against the Delta variant infection, hinting that CT-P59 has therapeutic potency for patients infected with Delta and its associated variants. HighlightsO_LICT-P59 exerts the antiviral effect on authentic Delta, Epsilon and Kappa variants in cell-based experiments. C_LIO_LICT-P59 showed neutralizing potency against variants including Delta, Epsilon, Kappa, L452R, T478K and P681H pseudovirus variants. C_LIO_LIThe administration of clinically relevant dose of CT-P59 showed in vivo C_LIO_LIprotection against Delta variants in animal challenge experiment. C_LI

microbiology↗

bioRxiv2020

Object recognition under occlusion revisited: elucidating algorithmic advantages of recurrent computation

Despite the ubiquity of recurrent connections in the brain, their role in visual processing is less understood than that of feedforward connections. Occluded object recognition, an ethologically critical cognitive capacity, is thought to rely on recurrent processing of visual information, but it remains unclear whether and how recurrent processing improves recognition of occluded objects. Using convolutional models of the visual system, we demonstrate how a distinct form of computation arises in recurrent-but not feedforward- networks that leverages information about the occluder to "explain-away" the occlusion-- i.e., recognition of the occluder provides an account for missing or altered features, potentially rescuing recognition of occluded objects. This occurs without any constraint placed on the computation and is observed both across a systematic architecture sweep of convolutional models and in a model explicitly constructed to approximate the primate visual system. Building on these computational results, we conduct a behavioral experiment to study explaining-away in humans, and find evidence consistent with explaining-away. Finally, to demonstrate a specific mechanism for explaining-away, we develop an experimentally inspired recurrent model that recovers fine-grained features of occluded stimuli by explaining-away. Recurrent connections capability to explain away may extend to more general cases where undoing context-dependent changes in representations could benefit perception.

neuroscience↗

bioRxiv2020

Regulation of specific cell clusters in TCR-T cells responding to differential expression of tumor PD-L1

PD-L1 signaling is important in regulating T cell function and keeping balance of tumor microenvironment, but its role in modifying TCR-T cell cytotoxicity remains unknown. MART-1-specific TCR-T cells (TCR-TMART-1) were stimulated by MEL-526 tumor cells expressing different proportions of PD-L1 and used to perform cytotoxicity assays and single-cell RNA sequencing. Percentage changes of different specific cell clusters were analyzed. The percentage of cluster HLA-DR+CD38+CD8+ was upregulated after antigen stimulation and tumor PD-L1 modified TCR-T cell function through downregulating the percentages of clusters HLA-DR+CD28+CD8+ and HLA-DR+CD38+CD8+ which were higher in TCR-TMART-1 than in Tnull.

immunology↗

bioRxiv2020

Adult newborn granule cells confer emotional-state-dependent plasticity in memory retrieval

Achieving optimal behavior requires animals to flexibly retrieve prior knowledge. Here we show that adult newborn granule cells (anbGCs) mediate emotional-state-dependent plasticity of memory retrieval. We find that acute social reward (aSR) enhances memory retrieval by increasing the reactivation of engram cells, while acute social stress (aSS) weakens retrieval and reduces the reactivation. Such bidirectional regulation relies on the activation of distinct populations of anbGCs by aSR and aSS, triggering opposing modifications of dDG activity, which is sufficient to regulate and predict the performance of memory retrieval. Concordantly, in emotional disorder models, aSR-dependent memory plasticity is impaired, while the effect of aSS remains intact. Together, our data revealed that anbGCs mediate plasticity of memory retrieval, allowing animals to flexibly retrieve memory according to the current emotional state, and suggested the essential roles of anbGCs in translating emotional information to the regulation of memory expression.

neuroscience↗

bioRxiv2019

Selective targeting of an oncogenic KRAS mutant allele by CRISPR/Cas9 induces efficient tumor regression

BackgroundKRAS is one of the most frequently mutated oncogenes in human cancers, but its activating mutations have remained undruggable due to its picomolar affinity for GTP/GDP and its smooth protein structure resulting in the absence of known allosteric regulatory sites. ResultsWith the goal of treating mutated KRAS-driven cancers, two CRISPR systems, CRISPR-SpCas9 genome-editing system and transcription-regulating system dCas9-KRAB, were developed to directly deplete KRAS mutant allele or to repress its transcription in cancer cells, respectively, through guide RNA specifically targeting the mutant but not wild-type allele. The effect of in vitro proliferation and cell cycle on cancer cells as well as in vivo tumor growth was examined after delivery of Cas9 system. SpCas9 and dCas9-KRAB systems with sgRNA targeting the mutant allele both blocked the expression of mutant KRAS gene, leading to an inhibition of cancer cell proliferation. Local adenoviral injections using SpCas9 and dCas9-KRAB systems both suppressed tumor growth in vivo. The gene-depletion system (SpCas9) performed more effectively than the transcription-suppressing system (dCas9-KRAB) on tumor inhibition. Application of both Cas9 systems to wild-type KRAS tumor cells did not affect cell proliferation in vitro and in vivo. Furthermore, through bioinformatic analysis of 31555 SNP mutations of the top 20 cancer driver genes, we showed that our mutant-specific editing strategy could be extended to a list of oncogenic mutations with high editing potentials, and this pipeline can be applied to analyze the distribution of PAM sequence in the genome to survey the best targets for other editing purpose. ConclusionsWe successfully developed both gene-depletion and transcription-suppressing systems to specifically target an oncogenic mutant allele of KRAS which led to significant tumor regression. It provides a promising strategy for the treatment of tumors with driver gene mutations.

genetics↗

bioRxiv2019

A Dual-sensing DNA Nanostructure with an Ultra-broad Detection Range

Despite considerable interest in the development of biosensors that can measure analyte concentrations with a dynamic range spanning many orders of magnitude, this goal has proven difficult to achieve. We describe here a modular biosensor architecture that integrates two different readout mechanisms into a single-molecule construct that can achieve target detection across an extraordinarily broad dynamic range. Our dual-mode readout DNA biosensor (DMRD) combines an aptamer and a DNAzyme to quantify ATP with two different mechanisms, which respond to low (micromolar) and high (millimolar) concentrations by generating distinct readouts based on changes in fluorescence and absorbance, respectively. Importantly, we have also devised regulatory strategies to finely tune the target detection range of each sensor module by controlling the target-sensitivity of each readout mechanism. Using this strategy, we report the detection of ATP at a dynamic range spanning 1-500,000 M--more than five orders of magnitude, representing the largest dynamic range reported to date with a single biosensor construct.

bioengineering↗

bioRxiv2019

SciBet: An ultra-fast classifier for cell type identification using single cell RNA sequencing data

Fast, robust and technology-independent computational methods are needed for supervised cell type annotation of single-cell RNA sequencing data. We present SciBet, a Bayesian classifier that accurately predicts cell identity for newly sequenced cells or cell clusters. We enable web client deployment of SciBet for rapid local computation without uploading local data to the server. This user-friendly and cross-platform tool can be widely useful for single cell type identification.

bioinformatics↗