Biology subjects

Li, D.

Publications and source records attributed to Li, D..

At least 559 records · Page 31Linked to original sources

bioRxiv2020

CRISPR-Cas9 integrates exogeneous sorting new recombinant DNA in the model tree Populus trichocarpa

CRISPR-mediated genome editing has become a powerful tool for genetic modification of biological traits. However, developing an efficient, site-specific, gene knock-in system based on homology-directed DNA repair (HDR) remains a significant challenge in plants, especially in woody species like poplar. Here, we show that simultaneous inhibition of non-homologous end joining (NHEJ) recombination cofactor XRCC4 and overexpression of HDR enhancer factors CtIP and MRE11 can improve the HDR efficiency for gene knock-in. Using this approach, the BleoR gene was integrated onto the 3' end of the MKK2 MAP Kinase gene to generate a BleoR-MKK2 fusion protein. Based on exogenous BleoR expression, the HDR-mediated knock-in efficiency was up to [~]40-fold greater when using a XRCC4 silencing incorporated with a combination of CtIP and MRE11 overexpression compared to no HDR enhancement or NHEJ silencing. Furthermore, this corporation of HDR enhancer overexpression and NHEJ repression also resulted in 7-fold fewer CRISPR-induced Insertions and Deletions (InDels), resulting in no functional effects on MKK2-based salt stress responses in poplar. Therefore, this approach may be useful not only in poplar and plants or crops but also in mammalians for improving CRISPR-mediated gene knock-in efficiency.

plant biology↗

bioRxiv2020

Fungi ligE-type glutathione S-transferases horizontally transferred into plants and a protozoan

Horizontal gene transfers (HGT) were considered as common evolution approaches for organisms. However, most HGT especially those HGT among distant species, like microbes to plants, were over-estimated because they were just identified based on the criteria of Blast searches and gene tree-species tree reconciliation. Recently, a ligE-type glutathione S-transferase (GST), Fhb7-GST was considered as an HGT from Epichloe to Thinopyrum elongatum. To confirm and clarify the occurring patterns of this HGT, homologue searches were conducted. Although TeFhb7-GST was not found in other plants, ligE-GSTs were found in not only plants but also an ameba protozoan. Additionally, ligE-GSTs were likely to horizontally transfer from fungi to other organisms. LigE-GSTs evolve in various fungi, but they only exist in some liverworts and green algae. Interestingly, all these ligE-GST genes in these plants share more than 90% similarities with that from fungus Coniosporium apollinis. More than that, the protozoan homologue from Acanthamoeba castellanii have 94.9% similiarity with that from C. apollinis. Actually, only a few substitutions were found in two homologues except a 111-bp lost in A. castellanii ligE-GST. All these results suggested HGT is an important evolutionary method for all organisms. Notably, natural HGT remind us to reevaluate the transgenic crops.

evolutionary biology↗

bioRxiv2020

A yeast BiFC-seq method for genome-wide interactome mapping

Genome-wide physical protein-protein interaction (PPI) mapping remains a major challenge for current technologies. Here, we report a high-efficiency yeast bimolecular fluorescence complementation method coupled with next-generation DNA sequencing (BiFC-seq) for interactome mapping. We applied this technology to systematically investigate an intraviral network of Ebola virus (EBOV). Two-thirds (9/13) of known interactions of EBOV were recaptured and five novel interactions were discovered. Next, we used BiFC-seq method to map the interactome of the tumor protein p53. We identified 97 interactors of p53 with more than three quarters are novel. Furthermore, in more complex background, we screened potential interactors by pooling two BiFC-libraries together, and revealing a network of 229 interactions among 205 proteins. These results show that BiFC-seq is a highly sensitive, rapid and economical method in genome-wide interactome mapping.

genomics↗

bioRxiv2020

Potent synthetic nanobodies against SARS-CoV-2 and molecular basis for neutralization

SARS-CoV-2, the causative agent of COVID-191, recognizes host cells by attaching its receptor-binding domain (RBD) to the host receptor ACE22-7. Neutralizing antibodies that block RBD-ACE2 interaction have been a major focus for therapeutic development8-18. Llama-derived single-domain antibodies (nanobodies, [~]15 kDa) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization19, which are attractive features for bio-drugs against the global respiratory disease. Here, we generated 99 synthetic nanobodies (sybodies) by in vitro selection using three libraries. The best sybody, MR3 bound to RBD with high affinity (KD = 1.0 nM) and showed high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.40 g mL-1). Structural, biochemical, and biological characterization of sybodies suggest a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency were generated by structure-based design, biparatopic construction, and divalent engineering. Among these, a divalent MR3 conjugated with the albumin-binding domain for prolonged half-life displayed highest potency (IC50 = 12 ng mL-1) and protected mice from live SARS-CoV-2 challenge. Our results pave the way to the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid responses for future outbreaks.

biochemistry↗

bioRxiv2020

The nuclear localization sequence mediates hnRNPA1 amyloid fibril formation revealed by cryoEM structure

Human heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) serves as a key regulating protein in RNA metabolism. Malfunction of hnRNPA1 in nucleo-cytoplasmic transport or dynamic phase separation leads to abnormal amyloid aggregation and neurodegeneration. The low complexity (LC) domain of hnRNPA1 drives both dynamic phase separation and amyloid aggregation. Here, we use cryo-electron microscopy to determine the amyloid fibril structure formed by hnRNPA1 LC domain. Remarkably, the structure reveals that the nuclear localization sequence of hnRNPA1 (termed PY-NLS), which is initially known to mediate the nucleo-cytoplamic transport of hnRNPA1 through binding with karyopherin-{beta}2 (Kap{beta}2), represents the major component of the fibril core. The residues that contribute to the binding of PY-NLS with Kap{beta}2 also exert key molecular interactions to stabilize the fibril structure. Notably, hnRNPA1 mutations found in familial amyotrophic lateral sclerosis (ALS) and multisystem proteinopathoy (MSP) are all involved in the fibril core and contribute to fibril stability. Our work illuminate structural understandings on the pathological amyloid aggregation of hnRNPA1 and the amyloid disaggregase activity of Kap{beta}2, and highlights the multiple roles of PY-NLS in hnRNPA1 homeostasis.

biophysics↗

bioRxiv2020

The Atr-Chek1 pathway inhibits axon regeneration in response to Piezo-dependent mechanosensation

Atr is a serine/threonine kinase, known to sense single-stranded DNA breaks and activate the DNA damage checkpoint by phosphorylating Chek1, which inhibits Cdc25, causing cell cycle arrest. This pathway has not been implicated in neuroregeneration. We show that in Drosophila sensory neurons, removing Atr or Chek1, or overexpressing Cdc25 promotes regeneration, whereas Atr or Chek1 overexpression, or Cdc25 knockdown impedes regeneration. Inhibiting the Atr-associated checkpoint complex in neurons promotes regeneration and improves synapse/behavioral recovery after CNS injury. Independent of DNA damage, Atr responds to the mechanical stimulus elicited during regeneration, via the mechanosensitive ion channel Piezo and its downstream NO signaling. Sensory neuron-specific knockout of Atr in adult mice, or pharmacological inhibition of Atr-Chek1 in mammalian neurons in vitro and in flies in vivo enhance regeneration. Our findings reveal the Piezo-Atr-Chek1-Cdc25 axis as an evolutionarily conserved inhibitory mechanism for regeneration, and identify potential therapeutic targets for treating nervous system trauma.

neuroscience↗

bioRxiv2020

Recovery of Consciousness and Cognition after General Anesthesia in Humans

Understanding how consciousness and cognitive function return after a major perturbation is important clinically and neurobiologically. To address this question, we conducted a three-center study of 30 healthy humans receiving general anesthesia at clinically relevant doses for three hours. We administered a pre- and post-anesthetic battery of neurocognitive tests, recorded continuous electroencephalography to assess cortical dynamics, and monitored sleep-wake activity before and following anesthetic exposure. We hypothesized that cognitive reconstitution would be a process that evolved over time in the following sequence: attention, complex scanning and tracking, working memory, and executive function. Contrary to our hypothesis, executive function returned first and electroencephalographic analyses revealed that frontal cortical dynamics recovered faster than posterior cortical dynamics. Furthermore, actigraphy indicated normal sleep-wake patterns in the post-anesthetic period. These recovery patterns of higher cognitive function and arousal states suggest that the healthy human brain is resilient to the effects of deep general anesthesia.

neuroscience↗

bioRxiv2020

Mechanistic insights into ventricular arrhythmogenesis of hydroxychloroquine and azithromycin for the treatment of COVID-19

Background: Recent reports on the use of hydroxychloroquine (HCQ) alone, or combined with azithromycin (AZM) in the management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have raised cardiac safety concerns. Currently, there is limited mechanistic data evaluating cardiac safety with HCQ and AZM therapy. Methods: Using comprehensive In Vitro ProArrhythmia Assay (CiPA) Schema IC50 paradigms, we examined the cardiac electrophysiological effects of HCQ and HCQ/AZM. Molecular modelling explored HCQ and AZM binding properties to hERG. Langendorff-perfused guinea-pig hearts were electrically and optically mapped by multi-electrode array and voltage (RH237) and Ca2+ (Rhod-2 AM) dyes. Human action potential and ion current reconstructions were performed in silico. Results: HCQ blocked IKr and IK1 with IC50 concentrations (10+-0.6 and 34+-5.0 microM) within the therapeutic range observed clinically. HCQ also blocked INa and ICaL but at higher IC50, whilst Ito and IKs were unaffected. Contrastingly, AZM produced minor inhibition of INa, ICaL, IKs, and IKr,, with no effect on IK1 and Ito. HCQ + AZM combined inhibited IKr and IK1 with IC50s of 7.7 +- 0.8 microM and 30.4 +- 3.0 microM, but spared INa, ICaL and Ito,. Molecular modelling confirmed potential HCQ binding to hERG. Cardiac mapping and ECG studies in isolated hearts demonstrated that HCQ slowed heart rate and ventricular conduction with associated prolongation of PR, QRS and QT intervals. Optical mapping demonstrated, and prolonged, more heterogeneous, action potential durations and intracellular Ca2+ transients. These effects were accentuated with combined HCQ+AZM treatment, which elicited electrical alternans, re-entrant circuits and wave breaks. Reconstruction in a human in-silico model demonstrated that this is attributable to the integrated action of HCQ and AZM reducing IKr, IKs and IK1. Conclusions: These data provide an electrophysiological basis for recent FDA guidelines cautioning against combined HCQ/AZM administration for the treatment of Covid-19 on the grounds of potential cardiac safety. We would strongly recommend monitoring of electrocardiographic QT interval with the use of this combination of medications.Competing Interest StatementThe authors have declared no competing interest.

pharmacology and toxicology↗

bioRxiv2020

Label-free optical detection of bioelectric potentials using electrochromic thin films

Understanding how a network of interconnected neurons receives, stores, and processes information in the human brain is one of the outstanding scientific challenges of our time. The ability to reliably detect neuroelectric activities is essential to addressing this challenge. Optical recording using voltage-sensitive fluorescent probes has provided unprecedented flexibility for choosing regions of interest in recording neuronal activities. However, when recording at a high frame rate such as 500-1000 Hz, fluorescence-based voltage sensors often suffer from photobleaching and phototoxicity, which limit the recording duration. Here, we report a new approach, Electro-Chromic Optical REcording (ECORE), that achieves label-free optical recording of spontaneous neuroelectrical activities. ECORE utilizes the electrochromism of PEDOT:PSS thin films, whose optical absorption can be modulated by an applied voltage. Being based on optical reflection instead of fluorescence, ECORE offers the flexibility of an optical probe without suffering from photobleaching or phototoxicity. Using ECORE, we optically recorded spontaneous action potentials in cardiomyocytes, cultured hippocampal and dorsal root ganglion neurons, and brain slices. With minimal perturbation to cells, ECORE allows long-term optical recording over multiple days.

neuroscience↗

bioRxiv2020

Inhibition of 2-Hydroxyglutrate Elicits Metabolic-reprograming and Mutant IDH1 Glioma Immunity

Mutant isocitrate-dehydrogenase-1 (IDH1-R132H; mIDH1) is a hallmark of adult gliomas. Lower grade mIDH1 gliomas are classified into two molecular subgroups: (i) 1p/19q co-deletion/TERT-promoter mutations or (ii) inactivating mutations in -thalassemia/mental retardation syndrome X-linked (ATRX) and TP53. This work, relates to the gliomas subtype harboring mIDH1, TP53 and ATRX inactivation. IDH1-R132H is a gain-of-function mutation that converts -ketoglutarate into 2-hydroxyglutarate (D-2HG). The role of D-2HG within the tumor microenvironment of mIDH1/mATRX/mTP53 gliomas remains unexplored. Inhibition of 2HG, when used as monotherapy or in combination with radiation and temozolomide (IR/TMZ), led to increased median survival (MS) of mIDH1 glioma bearing mice. Also, 2HG inhibition elicited anti-mIDH1 glioma immunological memory. In response to 2HG inhibition, PD-L1 expression levels on mIDH1-glioma cells increased to similar levels as observed in wild-type-IDH1 gliomas. Thus, we combined 2HG inhibition/IR/TMZ with anti-PDL1 immune checkpoint-blockade and observed complete tumor regression in 60% of mIDH1 glioma bearing mice. This combination strategy reduced T-cell exhaustion and favored the generation of memory CD8+T-cells. Our findings demonstrate that metabolic reprogramming elicits anti-mIDH1 glioma immunity, leading to increased MS and immunological memory. Our preclinical data supports the testing of IDH-R132H inhibitors in combination with IR/TMZ and anti-PDL1 as targeted therapy for mIDH1/mATRX/mTP53 glioma patients. Brief SummaryInhibition of 2-Hydroxyglutrate in mutant-IDH1 glioma in the genetic context of ATRX and TP53 inactivation elicits metabolic-reprograming and anti-glioma immunity.

immunology↗

bioRxiv2020

Comment on 'Initiation of chromosome replication controls both division and replication cycles in E. coli through a double-adder mechanism'

Witz et al. recently performed single-cell mother machine experiments to track growth and the replication cycle in E. coli. They analyzed the correlation structure of selected parameters using both their data and published data, and concluded that E. coli cell-size control is implemented at replication initiation, which challenged the newly emerged division-centric mechanism of cell-size control in bacteria. We repeated Witz et al.s analysis, and performed additional experiments and analytical calculations. These results explain Witz et al.s observation and in fact support the division-centric model.

systems biology↗

bioRxiv2020

Endophilin A1 promotes Actin Polymerization in response to Ca2+/calmodulin to Initiate Structural Plasticity of Dendritic Spines

Dendritic spines of excitatory neurons undergo activity-dependent structural and functional plasticity, which are cellular correlates of learning and memory. However, mechanisms underlying the rapid morphological changes immediately after NMDAR-mediated Ca2+ influx into spines remain poorly understood. Here we report that endophilin A1, a neuronal N-BAR protein, orchestrates membrane dynamics with actin polymerization to initiate spine enlargement in the induction phase of long-term potentiation (LTP). Upon LTP induction, Ca2+/calmodulin enhances its binding to both membrane and p140Cap, a cytoskeleton regulator. As a result, endophilin A1 rapidly associates with the relaxed plasma membrane and promotes actin polymerization, leading to acute expansion of spine head. Moreover, not only the p140Cap-binding, but also calmodulin- and membrane-binding capacities of endophilin A1 are required for LTP and long-term memory. Thus, endophilin A1 functions as calmodulin effector to drive spine enlargement in response to Ca2+ influx in the initial phase of structural plasticity.

cell biology↗

bioRxiv2020

Development of a COVID-19 vaccine based on the receptor binding domain displayed on virus-like particles

The ongoing coronavirus COVID-19 pandemic is caused by a new coronavirus (SARS-CoV-2) with its origin in the city of Wuhan in China. From there it has been rapidly spreading to many cities inside and outside China. Nowadays more than 33 millions with deaths surpassing 1 million have been recorded worldwide thus representing a major health issue. Rapid development of a protective vaccine against COVID-19 is therefore of paramount importance. Here we demonstrated that recombinantly expressed receptor binding domain (RBD) of the spike protein homologous to SARS binds to ACE2, the viral receptor. Higly repetitive display of RBD on immunologically optimized virus-like particles derived from cucumber mosaic virus (CuMVTT) resulted in a vaccine candidate that induced high levels of specific antibodies in mice which were able to block binding of spike protein to ACE2 and potently neutralized COVID-19 virus in vitro.

immunology↗

bioRxiv2020

The qBED track: a novel genome browser visualization for point processes

SummaryTransposon calling cards is a genomic assay for identifying transcription factor binding sites in both bulk and single cell experiments. Here we describe the qBED format, an open, text-based standard for encoding and analyzing calling card data. In parallel, we introduce the qBED track on the WashU Epigenome Browser, a novel visualization that enables researchers to inspect calling card data in their genomic context. Finally, through examples, we demonstrate that qBED files can be used to visualize non-calling card datasets, such as CADD scores and GWAS/eQTL hits, and may have broad utility to the genomics community. Availability and ImplementationThe qBED track is available on the WashU Epigenome Browser (http://epigenomegateway.wustl.edu/browser), beginning with version 46. Source code for the WashU Epigenome Browser with qBED support is available on GitHub (http://github.com/arnavm/eg-react and http://github.com/lidaof/eg-react). We have also released a tutorial on how to upload qBED data to the browser (dx.doi.org/10.17504/protocols.io.bca8ishw).

bioinformatics↗

bioRxiv2020

HIV Vpr modulates the host DNA damage response at two independent steps to damage DNA and repress double-strand DNA break repair

The DNA damage response (DDR) is a signaling cascade that is vital to ensuring the fidelity of the host genome in the presence of genotoxic stress. Growing evidence has emphasized the importance of both activation and repression of the host DDR by diverse DNA and RNA viruses. Previous work has shown that HIV-1 is also capable of engaging the host DDR, primarily through the conserved accessory protein Vpr. However, the extent of this engagement has remained unclear. Here we show that HIV-1 and HIV-2 Vpr directly induce DNA damage and stall DNA replication, leading to the activation of several markers of double- and single-strand DNA breaks. Despite causing damage and activating the DDR, we found that Vpr repress the repair of double-strand breaks (DSB) by inhibiting homologous recombination (HR) and non-homologous end joining (NHEJ). Mutational analyses of Vpr revealed that DNA damage and DDR activation are independent from repression of HR and Vpr-mediated cell-cycle arrest. Moreover, we show that repression of HR does not require cell-cycle arrest but instead may precede this long-standing enigmatic Vpr phenotype. Together, our data uncover that Vpr globally modulates the host DDR at at least two independent steps; offering novel insight into the primary functions of lentiviral Vpr and the roles of the DNA damage response in lentiviral replication. IMPORTANCEThe DNA damage response (DDR) is a signaling cascade that safeguards the genome from genotoxic agents, including human pathogens. However, the DDR has also been utilized by many pathogens, such as Human Immunodeficiency Virus (HIV), to enhance infection. To properly treat HIV positive individuals, we must understand how the virus usurps our own cellular processes. Here, we have found that an important yet poorly-understood gene in HIV, Vpr, targets the DDR at two unique steps: it causes damage and activates DDR signaling, and it represses the ability of cells to repair this damage, which we hypothesize is central to the primary function of Vpr. In clarifying these important functions of Vpr, our work highlights the multiple ways human pathogens engage the DDR, and further suggests that modulation of the DDR may be a novel way to help in the fight against HIV.

microbiology↗

bioRxiv2020

Genes adapt to outsmart gene targeting strategies in mutant mouse strains by skipping exons to reinitiate transcription and translation

Gene disruption in mouse embryonic stem cells or zygotes is a conventional genetics approach to identify gene function in vivo. However, because different gene-disruption strategies use different mechanisms to disrupt genes, the strategies can result in diverse phenotypes in the resulting mouse model. To determine whether different gene-disruption strategies affect the phenotype of resulting mutant mice, we characterized Rhbdf1 mouse mutant strains generated by three commonly used strategies--definitive-null, targeted knockout (KO)-first, and CRISPR/Cas9. We find that Rhbdf1 responds differently to distinct KO strategies, for example, by skipping exons and reinitiating translation to potentially yield gain-of-function alleles rather than the expected null or severe hypomorphic alleles. Our analysis also revealed that at least 4% of mice generated using the KO-first strategy show conflicting phenotypes, suggesting that exon skipping is a widespread phenomenon occurring across the genome. Additionally, our study emphasizes that at least 35% of mouse and 45% of human protein-coding genes could be predisposed to targeted KO-first- and CRISPR/Cas9-mediated unexpected translation. Our findings have significant implications for the application of genome editing in both basic research and clinical practice.

genetics↗

bioRxiv2020

Parkinson's disease-related phosphorylation at Tyr39 rearranges α-synuclein amyloid fibril structure revealed by cryo-EM

Post-translational modifications (PTMs) of -synuclein (-syn), e.g. phosphorylation, play an important role in modulating -syn pathology in Parkinsons disease (PD) and -synucleinopathies. Accumulation of phosphorylated -syn fibrils in Lewy bodies and Lewy neurites is the histological hallmark of these diseases. However, it is unclear how phosphorylation relates to -syn pathology. Here, by combining chemical synthesis and bacterial expression, we obtained homogeneous -syn fibrils with site-specific phosphorylation at Y39, which exhibits enhanced neuronal pathology in rat primary cortical neurons. We determined the cryo-EM structure of pY39 -syn fibril, which reveals a new fold of -syn with pY39 in the center of the fibril core forming electrostatic interaction network with eight charged residues in the N-terminal region of -syn. This structure composed of residues 1-100 represents the largest -syn fibril core determined so far. This work provides structural understanding on the pathology of pY39 -syn fibril, and highlights the importance of PTMs in defining the polymorphism and pathology of amyloid fibrils in neurodegenerative diseases.

biophysics↗

bioRxiv2020

LAMP-Seq: Population-Scale COVID-19 Diagnostics Using a Compressed Barcode Space

The ongoing SARS-CoV-2 pandemic has already caused devastating losses. Exponential spread can be slowed by social distancing and population-wide isolation measures, but those place a tremendous burden on society, and, once lifted, exponential spread can re-emerge. Regular population-scale testing, combined with contact tracing and case isolation, should help break the cycle of transmission, but current detection strategies are not capable of such large-scale processing. Here we present a protocol for LAMP-Seq, a barcoded Reverse-Transcription Loop-mediated Isothermal Amplification (RT-LAMP) method that is highly scalable. Individual samples are stabilized, inactivated, and amplified in three isothermal heat steps, generating barcoded amplicons that can be pooled and analyzed en masse by sequencing. Using unique barcode combinations per sample from a compressed barcode space enables extensive pooling, potentially further reducing cost and simplifying logistics. We validated LAMP-Seq on 28 clinical samples, empirically optimized the protocol and barcode design, and performed initial safety evaluation. Relying on world-wide infrastructure for next-generation sequencing, and in the context of population-wide sample collection, LAMP-Seq could be scaled to analyze millions of samples per day.

molecular biology↗