Biology subjects

Zhu, J. J.

Publications and source records attributed to Zhu, J. J..

At least 19 recordsLinked to original sources

bioRxiv2024

Mechanism of an Intrinsic Oscillation in Rat Geniculate Interneurons

Depolarizing current injections produced a rhythmic bursting of action potentials - a bursting oscillation - in a set of local interneurons in the lateral geniculate nucleus (LGN) of rats. The current dynamics underlying this firing pattern have not been determined, though this cell type constitutes an important cellular component of thalamocortical circuitry, and contributes to both pathologic and non-pathologic brain states. We thus investigated the source of the bursting oscillation using pharmacological manipulations in LGN slices in vitro and in silico. 1. Selective blockade of calcium channel subtypes revealed that high-threshold calcium currents IL and IP contributed strongly to the oscillation. 2. Increased extracellular K+ concentration (decreased K+ currents) eliminated the oscillation. 3. Selective blockade of K+ channel subtypes demonstrated that the calcium-sensitive potassium current (IAHP ) was of primary importance. A morphologically simplified, multicompartment model of the thalamic interneuron characterized the oscillation as follows: 1. The low-threshold calcium current (IT ) provided the strong initial burst characteristic of the oscillation. 2. Alternating fluxes through high-threshold calcium channels and IAHP then provided the continuing oscillations burst and interburst periods respectively. This interplay between IL and IAHP contrasts with the current dynamics underlying oscillations in thalamocortical and reticularis neurons, which primarily involve IT and IH, or IT and IAHP respectively. These findings thus point to a novel electrophysiological mechanism for generating intrinsic oscillations in a major thalamic cell type. Because local interneurons can sculpt the behavior of thalamocortical circuits, these results suggest new targets for the manipulation of ascending thalamocortical network activity.

neuroscience↗

bioRxiv2022

GESIAP: A Versatile Genetically Encoded Sensor-based Image Analysis Program

Intercellular communication mediated by a large number of neuromodulators diversifies physiological actions, yet neuromodulation remains poorly understood despite the recent upsurge of genetically encoded transmitter sensors. Here, we report the development of a versatile genetically encoded sensor-based image analysis program (GESIAP) that utilizes MATLAB-based algorithms to achieve high-throughput, high-resolution processing of sensor-based functional imaging data. GESIAP enables delineation of fundamental properties (e.g., transmitter spatial diffusion extent, quantal size, quantal content, release probability, pool size, and refilling rate at single release sites) of transmission mediated by various transmitters (i.e., monoamines, acetylcholine, neuropeptides, and glutamate) at various cell types (i.e., neurons, astrocytes, and other non-neuronal cells) of various animal species (i.e., mouse, rat, and human). Our analysis appraises a dozen of newly developed transmitter sensors, validates a conserved model of restricted non-volume neuromodulatory synaptic transmission, and accentuates a broad spectrum of presynaptic release properties that variegate neuromodulation.

neuroscience↗

bioRxiv2022

Plasticity and lineage commitment of individual Th1 cells are determined by stable T-bet expression quantities

T helper 1 (Th1) cell identity is defined by the expression of the lineage-defining transcription factor T-bet. Here, we examine the influence of T-bet expression heterogeneity on subset plasticity by leveraging cell sorting of distinct in vivo-differentiated Th1 cells based on their quantitative expression of T-bet and interferon-{gamma}. Heterogeneous T-bet expression states were regulated by virus-induced type-I interferons and were stably maintained even after secondary viral infection. Exposed to Th2-polarizing conditions, the sorted subpopulations exhibited graded levels of plasticity: T-bet quantities were inversely correlated with the ability to express the Th2 lineage-specifying transcription factor GATA-3 and Th2 cytokines. Reprogramed Th1 cells acquired graded, but stable mixed Th1+2 phenotypes with a hybrid epigenetic landscape. Continuous presence of T-bet in differentiated Th1 cells was essential to ensure Th1 cell stability. Thus, innate cytokine signals regulate Th1 cell plasticity via an individual cell-intrinsic rheostat to enable T cell subset adaptation to subsequent challenges. HIGHLIGHTSO_LIType-I interferons triggered by infection determine T-bet expression states in Th1 cells C_LIO_LIT-bet and IFN-{gamma} expression states indicate the plasticity of individual Th1 cells C_LIO_LIIndividual T-bet expression states and plasticity persist after secondary infection C_LIO_LIReprogramming yields stable Th1+2 phenotypes and a mixed epigenetic landscape C_LI

immunology↗

bioRxiv2022

cAMP-EPAC-PKCε-RIM1α signaling regulates presynaptic long-term potentiation and motor learning

The cerebellum is involved in learning of fine motor skills, yet whether presynaptic plasticity contributes to such learning remains elusive. Here we report that the EPAC-PKC{varepsilon} module has a critical role in a presynaptic form of long-term potentiation in the cerebellum and motor behavior. Presynaptic cAMP-EPAC-PKC{varepsilon} signaling cascade induces a previously unidentified threonine phosphorylation of RIM1, and thereby initiates the assembly of the Rab3A-RIM1-Munc13-1 tripartite complex that facilitates docking and release of synaptic vesicles. Granule cell-specific blocking of EPAC-PKC{varepsilon} signaling abolishes presynaptic long-term potentiation at the parallel fiber to Purkinje cell synapses and impairs basic performance and learning of cerebellar motor behavior. These results unveil a functional relevance of presynaptic plasticity that is regulated through a novel signaling cascade, thereby enriching the spectrum of cerebellar learning mechanisms.

neuroscience↗

bioRxiv2022

Targeted directional kilobase sequence insertion by combining prime editing with recombinases or integrases

Targeted insertion of exogenous sequences to genomes is useful for therapeutics and biological research. While CRISPR/Cas technologies have been very efficient in gene knockouts by double-strand breaks (DSBs) followed by indel formation through non-homologous end-joining (NHEJ) repair pathway, the precise introduction of new sequences mainly rely on inefficient homology directed repair (HDR) pathways following Cas9-induced DSBs and are restricted to dividing cells. The recent invention of Prime Editing allows short sequences to be precisely inserted at target sites without DSBs. Here, we combine Prime Editing and sequence-specific recombinases and integrases to insert kilobase sequences directionally at target sites. This technique, called PRIMAS for Prime editing, Recombinase, Integrase-mediated Addition of Sequence, will expand our genome editing toolbox for targeted insertion of long sequences up to kilobases and beyond.

bioengineering↗

bioRxiv2022

Local recruitment of DNA repair proteins enhances CRISPR-ssODN-HDR editing

CRISPR-Cas technologies enable precise editing of genomic sequences. One major way to introduce precise editing is through homology directed repair (HDR) of DNA double strand breaks (DSB) templated by exogenously supplied single-stranded oligodeoxyribonucleotides (ssODN). Competing pathways determine the outcome of edits. Non-homologous end-joining pathways produce destructive insertions/deletions (indels) at target sites and are dominant over the precise homology directed repair pathways. In this study, we aim to favor HDR and use two strategies to recruit DNA repair proteins (DRPs) to Cas9 cut site, the Casilio-DRP approach that recruits RNA-binding protein-tethered DRPs to target site via aptamers appended to guide RNA; and the 53BP1-DRP approach that recruits DRPs to DSBs via DSB-sensing activity of 53BP1. We conducted two screens using these approaches and identified DRPs such as FANCF and BRCA1 that when recruited to Cas9 cut site, enhance ssODN-templated HDR and increase the proportion of precise edits. This study provides not only new constructs for enhanced CRISPR-ssODN-HDR but also a collection of DRP fusions for studying DNA repair processes.

bioengineering↗

bioRxiv2020

C11orf95-RELA Reprograms 3D Epigenome in Supratentorial Ependymoma

Supratentorial ependymoma (ST-EPN) is a type of malignant brain tumor mainly seen in children. Since 2014, it has been known that an intrachromosomal fusion C11orf95-RELA is an oncogenic driver in ST-EPN1 but the molecular mechanisms of oncogenesis are unclear. Here we show that the C11orf95 component of the fusion protein dictates DNA binding activity while the RELA component is required for driving the expression of ependymoma-associated genes. Epigenomic characterizations using ChIP-seq and HiChIP approaches reveal that C11orf95-RELA modulates chromatin states and mediates chromatin interactions, leading to transcriptional reprogramming in ependymoma cells. Our findings provide important characterization of the molecular underpinning of C11orf95-RELA fusion and shed light on potential therapeutic targets for C11orf95-RELA subtype ependymoma.

cancer biology↗

bioRxiv2020

CRISPR-mediated Multiplexed Live Cell Imaging of Nonrepetitive Genomic Loci

Three-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR- and TALE-based genomic labeling techniques have been hampered by laborious protocols and are ineffective in labeling non-repetitive sequences. Here, we report a versatile CRISPR/Casilio-based imaging method that allows for a nonrepetitive genomic locus to be labeled using one guide RNA. We construct Casilio dual-color probes to visualize the dynamic interactions of DNA elements in single live cells in the presence or absence of the cohesin subunit RAD21. Using a three-color palette, we track the dynamic 3D locations of multiple reference points along a chromatin loop. Casilio imaging reveals intercellular heterogeneity and interallelic asynchrony in chromatin interaction dynamics, underscoring the importance of studying genome structures in 4D.

genomics↗

bioRxiv2020

JACKIE: Fast enumeration of single- and multi-copy CRISPR binding sites

ZFP-, TALE-, and CRISPR-based methods for genome, epigenome editing and imaging have provided powerful tools to interrogate functions of genomes. Targeting sequence design is vital to the success of these experiments. While existing design software mainly focus on designing target sequence for specific elements, we report here the implementation of JACKIE (Jackie and Alberts Comprehensive K-mer Instances Enumerator), a suite of software for enumerating all single- and multi-copy sites in the genome that can be incorporated for genome-scale designs as well as loaded onto genome browsers alongside other tracks for convenient web-based graphic-user-interface (GUI)-enabled design. We also implement fast algorithms to identify sequence neighborhoods or off-target counts of targeting sequences so that designs with low probability of off-target can be identified among millions of design sequences in reasonable time. We demonstrate the application of JACKIE-designed CRISPR site clusters for genome imaging.

bioinformatics↗

bioRxiv2020

A genetically encoded GRAB sensor for measuring serotonin dynamics in vivo

Serotonin (5-HT) is a phylogenetically conserved monoamine neurotransmitter modulating a variety of processes in the brain. To directly visualize the dynamics of 5-HT, we developed a genetically encoded GPCR-Activation-Based 5-HT (GRAB5-HT) sensor with high sensitivity, selectivity, and spatiotemporal resolution. GRAB5-HT, detected 5-HT release in multiple physiological and pathological conditions in both flies and mice, and thus provides new insights into the dynamics and mechanisms of 5-HT signaling.

neuroscience↗

bioRxiv2020

A fast genetically encoded fluorescent sensor for faithful in vivo acetylcholine detection in mice, fish, worms and flies

Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and in vivo preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all animals.

neuroscience↗

bioRxiv2020

Targeting nuclear pore protein, NUP210, reduces metastasis through heterochromatin-mediated silencing of mechanosensitive genes

Mechanical signals from the extracellular microenvironment have been implicated in tumor and metastatic progression. Here, we identified nucleoporin NUP210 as a metastasis susceptibility gene for human estrogen receptor positive (ER+) breast cancer and a cellular mechanosensor. Nup210 depletion suppresses lung metastasis in mouse models of breast cancer. Mechanistically, NUP210 interacts with LINC complex protein SUN2 which connects the nucleus to the cytoskeleton. In addition, the NUP210/SUN2 complex interacts with chromatin via the short isoform of BRD4 and histone H3.1/H3.2 at the nuclear periphery. In Nup210 knockout cells, mechanosensitive genes accumulate H3K27me3 heterochromatin modification, mediated by the polycomb repressive complex 2 and differentially reposition within the nucleus. Transcriptional repression in Nup210 knockout cells results in defective mechanotransduction and focal adhesion necessary for their metastatic capacity. Our study provides a new insight into the role of nuclear pore protein in cellular mechanosensation and metastasis.

cancer biology↗

bioRxiv2019

Development of a highly effective African swine fever virus vaccine by deletion of the I177L gene results in sterile immunity against the current epidemic Eurasia strain

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The disease is devastating the swine industry in Central Europe and East Asia, with current outbreaks caused by circulating strains of ASFV derived from the 2007 Georgia isolate (ASFV-G), a genotype II ASFV. In the absence of any available vaccines, African Swine Fever (ASF) outbreak containment relies on control and culling of infected animals. Limited cross protection studies suggest that in order to ensure a vaccine is effective it must be derived from the current outbreak strain or at the very least from an isolate with the same genotype. Here we report the discovery that deletion of a previously uncharacterized gene, I177L, from the highly virulent ASFV-G produces complete virus attenuation in swine. Animals inoculated intramuscularly with the virus lacking the I177L gene, ASFV-G-{Delta}I177L, in a dose range of 102 to 106 HAD50 remained clinically normal during the 28 day observational period. All ASFV-G-{Delta}I177L-infected animals had low viremia titers, showed no virus shedding, developed a strong virus-specific antibody response and, importantly, they were protected when challenged with the virulent parental strain ASFV-G. ASFV-G-{Delta}I177L is one of the few experimental vaccine candidate virus strains reported to be able to induce protection against the ASFV Georgia isolate, and the first vaccine capable of inducing sterile immunity against the current ASFV strain responsible for recent outbreaks. ImportanceCurrently there is no commercially available vaccine against African swine fever. Outbreaks of this disease are devastating the swine industry from Central Europe to East Asia, and they are being caused by circulating strains of African swine fever virus derived from the Georgia2007 isolate. Here we report the discovery of a previously uncharacterized virus gene, which when deleted completely attenuates the Georgia isolate. Importantly, animals infected with this genetically modified virus were protected from developing ASF after challenge with the virulent parental virus. Interestingly, ASFV-G-{Delta}I177L confers protection even at low doses (102 HAD50) and remains completely attenuated when inoculated at high doses (106 HAD50), demonstrating its potential as a safe vaccine candidate. At medium doses (104 HAD50) sterile immunity is achieved. Therefore, ASFV-G-{Delta}I177L is a novel efficacious experimental ASF vaccine protecting pigs from the epidemiologically relevant ASFV Georgia isolate.

microbiology↗

bioRxiv2019

Revaluation of magnetic properties of Magneto, MagR and αGFP-TRPV1/GFP-ferritin

ABSTRACT AND INTRODUCTIONMagnetic control of neuronal activity offers many obvious advantages over electric, optogenetic and chemogenetic manipulations. A recent series of highly visible papers reported the development of magnetic actuators (i.e., Magneto, MagR and GFP-TRPV1/GFP-ferritin) that appeared to be effective in controlling neuronal firing1-3, yet their action mechanisms seem to conflict with the principles of physics4. We found that neurons expressing Magneto, MagR and GFP-TRPV1/GFP-ferritin did not respond to magnetic stimuli with any membrane depolarization (let alone action potential firing), although these neurons frequently generated spontaneous action potentials. Because the previous study did not establish the precise temporal correlation between magnetic stimuli and action potentials in recorded neurons1-3, the reported magnetically-evoked action potentials are likely to represent mismatched spontaneous firings.

neuroscience↗

bioRxiv2019

Selective Activation of TASK-3-containing K+ Channels Reveals Their Therapeutic Potentials in Analgesia

The paucity of selective agonists for TASK-3, a member of two-pore domain K+ (K2P) channels, has contributed to our limited understanding of its biological functions. By targeting a novel druggable transmembrane cavity using a structure-based drug design approach, we discovered a biguanide compound, CHET3, as a highly selective allosteric activator for TASK-3-containing K2P channels, including TASK-3 homomer and TASK-3/TASK-1 heteromer. CHET3 displayed unexpectedly potent analgesic effects in vivo in a variety of acute and chronic pain models in rodents that could be abolished by pharmacology or genetic ablation of TASK-3. We further found that TASK-3-containing channels anatomically define a unique subset population of small-sized, TRPM8, TRPV1 or tyrosine hydroxylase-positive nociceptive sensory neurons and functionally regulate their membrane excitability, supporting CHET3 analgesia in thermal hyperalgesia and mechanical allodynia under chronic pain. Overall, our proof-of-concept study reveals TASK-3-containing K2P channels as a novel druggable target for treating pain.\n\nOne Sentence SummaryIdentification of a novel drug target and its new hit compounds for developing new-generation non-opioid analgesics.

pharmacology and toxicology↗

bioRxiv2019

Super resolution imaging of a distinct chromatin loop in human lymphoblastoid cells

The three-dimensional genome structure plays a fundamental role in gene regulation and cellular functions. Recent studies in genomics based on sequencing technologies inferred the very basic functional chromatin folding structures of the genome known as chromatin loops, the long-range chromatin interactions that are often mediated by protein factors. To visualize the looping structure of chromatin we applied super-resolution microscopy iPALM to image a specific chromatin loop in GM12878 cells. Totally, we have generated six images of the target chromatin region at the single molecule resolution. To infer the chromatin structures from the captured images, we modeled them as looping conformations using different computational algorithms and then evaluated the models by comparing with Hi-C data to examine the concordance. The results showed a good correlation between the imaging data and sequencing data, suggesting the visualization of higher-order chromatin structures for the very short genomic segments can be realized by microscopic imaging.

molecular biology↗

bioRxiv2018

A genetically-encoded fluorescent acetylcholine indicator

Acetylcholine (ACh) regulates a diverse array of physiological processes throughout the body, yet cholinergic transmission in the majority of tissues/organs remains poorly understood due primarily to the limitations of available ACh-monitoring techniques. We developed a family of G-protein-coupled receptor activation-based ACh sensors (GACh) with sensitivity, specificity, signal-to-noise ratio, kinetics and photostability suitable for monitoring ACh signals in vitro and in vivo. GACh sensors were validated with transfection, viral and/or transgenic expression in a dozen types of neuronal and non-neuronal cells prepared from several animal species. In all preparations, GACh sensors selectively responded to exogenous and/or endogenous ACh with robust fluorescence signals that were captured by epifluorescent, confocal and/or two-photon microscopy. Moreover, analysis of endogenous ACh release revealed firing pattern-dependent release and restricted volume transmission, resolving two long-standing questions about central cholinergic transmission. Thus, GACh sensors provide a user-friendly, broadly applicable toolbox for monitoring cholinergic transmission underlying diverse biological processes.

neuroscience↗

bioRxiv2018

Multiplex Chromatin Interaction Analysis with Single-Molecule Precision

We describe a microfluidics-based strategy for genome-wide analysis of multiplex chromatin interactions with single-molecule precision. In multiplex chromatin interaction analysis (multi-ChIA), individual chromatin complexes are partitioned into droplets that contain a gel bead with unique DNA barcode, in which tethered chromatin DNA fragments are barcoded and amplified for sequencing and mapping to demarcate chromatin contacts. Thus, multi-ChIA has the unprecedented ability to uncover multiplex chromatin interactions at single-molecule level, which has been impossible using previous methods that rely on analyzing pairwise contacts via proximity ligation. We demonstrate that multiplex chromatin interactions predominantly contribute to topologically associated domains, and clusters of gene promoters and enhancers provide a fundamental topological framework for co-transcriptional regulation.

genomics↗