Biology subjects

Shi, K.

Publications and source records attributed to Shi, K..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

HIV-2 Immature Particle Morphology Provides Insights into Gag Lattice Stability and Virus Maturation

Retrovirus immature particle morphology consists of a membrane enclosed, pleomorphic, spherical and incomplete lattice of Gag hexamers. Previously, we demonstrated that human immunodeficiency virus type 2 (HIV-2) immature particles possess a distinct and extensive Gag lattice morphology. To better understand the nature of the continuously curved hexagonal Gag lattice, we have used single particle cryo-electron microscopy with a retrovirus to determine the HIV-2 Gag lattice structure for immature virions. The reconstruction map at 5.5 [A] resolution revealed a stable, wineglass-shaped Gag hexamer structure with structural features consistent with other lentiviral immature Gag structures. Cryo-electron tomography provided evidence for nearly complete ordered Gag lattice structures in HIV-2 immature particles. We also solved a 1.98 [A] resolution crystal structure of the carboxyl-terminal domain (CTD) of the HIV-2 capsid (CA) protein that identified a structured helix 12 supported via an interaction of helix 10 in the absence of the SP1 region of Gag. Residues at the helix 10-12 interface proved critical in maintaining HIV-2 particle release and infectivity. Taken together, our findings provide the first 3D organization of HIV-2 immature Gag lattice and important insights into both HIV Gag lattice stabilization and virus maturation.

microbiology↗

bioRxiv2022

Single cell profiling of γδ hepatosplenic T-cell lymphoma unravels tumor cell heterogeneity associated with disease progression

Hepatosplenic T-cell lymphoma (HSTCL), mostly derived from {gamma}{delta} T cells, is a rare but very aggressive lymphoma with poor outcomes. The molecular pathogenesis driving HSTCL is largely unknown while only limited treatment options are available. In this study, by performing paired single cell RNA-seq and T cell receptor (TCR) sequencing on biopsies from a HSTCL patient pre- and post-chemotherapy treatments, we characterized unique gene expressing signatures of malignant {gamma}{delta} T cells, with a set of marker genes were newly identified in HSTCL (AREG, PLEKHA5, VCAM1 etc.). Although the malignant {gamma}{delta} T cells were expanded from a single TCR clonotype according to their TCR identities, they evolved into two transcriptional distinct tumor subtypes during the disease progression. The Tumor_1 subtype was dominant in pre-treatment samples with highly aggressive phenotypes. While the Tumor_2 had relative mild cancer hallmark signatures but expressed genes associated with tumor survival signal and drug resistance (IL32, TOX2, AIF1, AKAP12 etc.), and finally became the main tumor subtype post-treatment. We further dissected the tumor microenvironment of the HSTCL and noticed that CD8 memory T cells were clonal expanded post-treatment. In addition, we discovered dynamically rewiring cell-cell interaction networks during the treatment. The tumor cells had reduced communications with the microenvironment post-treatment. Our study reveals heterogenous and dynamic tumor and microenvironment underlying pathogenesis of HSTCL and may contribute to identify novel targets for diagnosis and cure of HSTCL in the future.

cancer biology↗

bioRxiv2021

Drosulfakinin signaling modulates female sexual receptivity in Drosophila

Female sexual behavior as an innate behavior is of prominent biological importance for survival and reproduction. However, molecular and circuit mechanisms underlying female sexual behavior is not well understood. Here, we identify the Cholecystokinin-like peptide Drosulfakinin (DSK) promotes female sexual behavior in Drosophila. Manipulation both Dsk and DSK neuronal activity impact female sexual receptivity. In addition, we reveal that Dsk-expressing neurons receive input signal from R71G01GAL4 neurons to promote female sexual receptivity. Based on intersectional technique, we further found the regulation of female sexual behavior relies mainly on medial DSK neurons rather than lateral DSK neurons, and medial DSK neurons modulate female sexual behavior by acting on its receptor CCKLR-17D3. Thus, we characterized DSK/CCKLR-17D3 as R71G01GAL4 neurons downstream signaling to regulate female sexual behavior.

neuroscience↗

bioRxiv2021

Metformin modulates microbiota-derived inosine and ameliorates methamphetamine-induced anxiety and depression-like withdrawal symptoms in mice

ObjectiveMetformin exhibits therapeutic potential in behavioural deficits induced by methamphetamine (METH) in rats. Emerging studies suggest gut microbiota may impact psychiatric symptoms, but there is no direct evidence supporting metformins participation in the pathophysiology of withdrawal symptoms via modulation of gut microbiota. MehodsIn order to define the functional contributions by gut microbiota and metformin to the behavioural deficits during METH withdrawal, we utilized a combination of fecal microbiota transplantation (FMT), high-throughput sequencing, and untargeted metabolomics technologies. ResultsFirst, METH addicts exhibited higher diversity and distinct microbial structures compared to heathy controls. In particular, the relative abundance of Rikenellaceae was positively correlated with the severity of anxiety and depression. Second, both human-to-mouse and mouse-to-mouse FMTs confirmed that METH-altered-microbiota transplantation is sufficient to promote anxiety and depression-like behaviours in recipient germ-free mice, and these behavioural disturbances could be ameliorated by metformin. In-depth analysis revealed that METH significantly altered the bacterial composition and structure as well as relative abundance of several bacterial taxa and metabolites, including Rikenellaceae and inosine, respectively, whereas add-on metformin could remodel these alterations. Finally, the inosine complementation successfully restored METH-induced anxiety and depression-like behaviours in mice. DiscussionThis study demonstrates that METH withdrawal-induced anxiety and depression-like behaviours are convertible and transmissible via gut microbiota in a mouse model. The therapeutic effects of metformin on psychiatric manifestations are associated with microbiota-derived metabolites, highlighting the role of the gut microbiota in substance use disorders and the pathophysiology of withdrawal symptoms. Study HighlightsO_ST_ABSWhat is known?C_ST_ABSO_LIThere are no targeted therapies for substance withdrawal syndrome, but there is considerable evidence that withdrawal-associated psychiatric manifestations contribute to the poor adherence to rehabilitation treatment as well as the relapse rates. C_LIO_LIMetformin has shown its therapeutic potential against METH-induced neurobehavioural changes and neurodegeneration in rats through CREB/BDNF and Akt/GSK3 signaling pathways in the anxiety-related brain nuclei. C_LI What is new here?O_LIMETH withdrawal-induced anxiety and depression-like behaviours are convertible and transmissible via gut microbiota in a mouse model. C_LIO_LIThe therapeutic effects of metformin on psychiatric manifestations are associated with microbiota derived metabolites. C_LIO_LIInosine complementation could restore METH withdrawal-induced anxiety and depression-like behaviours. C_LI

animal behavior and cognition↗

bioRxiv2021

CryoEM structure of the EBV ribonucleotide reductase BORF2 and mechanism of APOBEC3B inhibition

Viruses use a plethora of mechanisms to evade immune responses. A new example is neutralization of the nuclear DNA cytosine deaminase APOBEC3B by the Epstein-Barr virus (EBV) ribonucleotide reductase subunit BORF2. Cryo-EM studies of APOBEC3B-BORF2 complexes reveal a large >1000 [A]2 binding surface comprised of multiple structural elements from each protein, which effectively blocks the APOBEC3B active site from accessing single-stranded DNA substrates. Evolutionary optimization is suggested by unique insertions in BORF2 absent from other ribonucleotide reductases and preferential binding to APOBEC3B relative to the highly related APOBEC3A and APOBEC3G enzymes. An atomic understanding of this novel pathogen-host interaction may contribute to the development of drugs that block the interaction and liberate the natural antiviral activity of APOBEC3B. One-Sentence SummaryThese studies show how a conserved viral nucleotide metabolism protein is repurposed to inhibit a potent antiviral factor.

microbiology↗

bioRxiv2021

Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in non-structural protein 14 (nsp14), which excises nucleotides including antiviral drugs mis-incorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here we determined a 1.6-[A] resolution crystal structure of SARS-CoV-2 ExoN in complex with its essential co-factor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. Molecular dynamics simulations further show remarkable flexibility of multi-domain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA-binding to support its exoribonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anti-coronaviral drugs or strategies to attenuate the viral virulence.

biochemistry↗

bioRxiv2021

A novel type of IRES having variable numbers of eIF4E-binding site and synergistic effect in RNA2 of WYMV isolates

Some viral proteins were translated in cap-independent manner via internal ribosome entry site (IRES), which ever maintained conservative characteristic among different isolates of same species of virus. However, IRES activity presented 7-fold of variance in RNA2 of wheat yellow mosaic virus (WYMV) HC and LYJN isolates. Based on RNA structure probing and mutagenesis assay, the loosened middle stem of H1 and hepta-nucleotide top loop of H2 in LYJN isolate synergistically ensured the higher IRES activity than that in HC isolate. In addition, the conserved top loop of H1 ensured basic IRES activity in HC and LYJN isolates. RNA2 5'-UTR specifically interacted with the wheat eIF4E, which was accomplished by the top loop of H1 in HC isolate or the top loop of H1 and H2 in LYJN isolate. Different IRES activity of WYMV RNA2 was regulated by different numbers of eIF4E-binding site and their synergistic effect, which was accomplished by the proximity of H1 and H2 due to the flexibility of middle stem in H1. It is represented a novel evolution pattern of IRES.

microbiology↗

bioRxiv2020

Phase separation of Mer2 organizes the meiotic loop-axis structure of chromatin during meiosis I

Sexually reproducing organisms acquire genetic diversity through meiotic recombination during meiosis I, which is initiated via programmed DNA double-strand breaks (DSBs) induced by Spo11-containing machinery in each meiotic cell. The combination of programmed DSB sites in each meiotic cell must be diverse, which requires a certain degree of randomness in the distribution of DSBs. The formation of programmed DSBs requires a preestablished loop-axis structure of chromatin. Here, we demonstrate that the axial element protein Mer2 undergoes liquid-liquid phase separation in vitro and in vivo through its intrinsically disordered C-terminal domain. A DNA binding motif within its central domain is responsible for bringing DNA into Mer2 liquid droplets and Mer2-DNA complex could assemble into filamentous structures extending from the droplets. These results suggest that phase separation of Mer2 drives the formation of a droplet-loop structure of meiotic chromatin to facilitate and to diversify programmed DSB formation.

biophysics↗

bioRxiv2020

The Development of a Novel Nanobody Therapeutic for SARS-CoV-2

Combating the COVID-19 pandemic requires potent and low-cost therapeutics. We identified a novel series of single-domain antibodies (i.e., nanobody), Nanosota-1, from a camelid nanobody phage display library. Structural data showed that Nanosota-1 bound to the oft-hidden receptor-binding domain (RBD) of SARS-CoV-2 spike protein, blocking out viral receptor ACE2. The lead drug possessing an Fc tag (Nanosota-1C-Fc) bound to SARS-CoV-2 RBD with a Kd of 15.7picomolar ([~]3000 times more tightly than ACE2 did) and inhibited SARS-CoV-2 infection with an ND50 of 0.16microgram/milliliter ([~]6000 times more potently than ACE2 did). Administered at a single dose, Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy in hamsters subjected to SARS-CoV-2 infection. Unlike conventional antibody drugs, Nanosota-1C-Fc was produced at high yields in bacteria and had exceptional thermostability. Pharmacokinetic analysis of Nanosota-1C-Fc documented a greater than 10-day in vivo half-life efficacy and high tissue bioavailability. Nanosota-1C-Fc is a potentially effective and realistic solution to the COVID-19 pandemic. Impact statementPotent and low-cost Nanosota-1 drugs block SARS-CoV-2 infections both in vitro and in vivo and act both preventively and therapeutically.

microbiology↗

bioRxiv2020

Reduction of neuronal activity mediated by blood-vessel regression in the brain

The brain vasculature supplies neurons with glucose and oxygen, but little is known about how vascular plasticity contributes to brain function. Using longitudinal in vivo imaging, we reported that a substantial proportion of blood vessels in the adult brain sporadically occluded and regressed. Their regression proceeded through sequential stages of blood-flow occlusion, endothelial cell collapse, relocation or loss of pericytes, and retraction of glial endfeet. Regressing vessels were found to be widespread in mouse, monkey and human brains. Both brief occlusions of the middle cerebral artery and lipopolysaccharide-mediated inflammation induced an increase of vessel regression. Blockage of leukocyte adhesion to endothelial cells alleviated LPS-induced vessel regression. We further revealed that blood vessel regression caused a reduction of neuronal activity due to a dysfunction in mitochondrial metabolism and glutamate production. Our results elucidate the mechanism of vessel regression and its role in neuronal function in the adult brain.

neuroscience↗

bioRxiv2020

Molecular underpinnings of ssDNA specificity by Rep HUH endonucleases and implications for HUH-tag multiplexing and engineering

Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and for anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay, termed HUH-seq, to interrogate subtleties in Rep specificity and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed engineering of only four amino acids in a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications.

biophysics↗

bioRxiv2020

Full-length annotation with multi-strategy RNA-seq uncovers transcriptional regulation of lncRNAs in diploid cotton G. arboreum

Long noncoding RNAs (lncRNAs) are crucial factors during plant development and environmental responses. High-throughput and accurate identification of lncRNAs is still lacking in plants. To build an accurate atlas of lncRNA in cotton, we combined Isoform-sequencing (Iso-seq), strand-specific RNA-seq (ssRNA-seq), cap analysis gene expression (CAGE-seq) with PolyA-seq and compiled a pipeline named plant full-length lncRNA (PULL) to integrate multi-omics data. A total of 9240 lncRNAs from 21 tissue samples of the diploid cotton Gossypium arboreum were identified. We revealed that alternative usage of transcription start site (TSS) and transcription end site (TES) of lncRNAs occurs pervasively during plant growth and responses to stress. We identified the lncRNAs which co-expressed or be linked to the protein coding genes (PCGs) or GWAS studied SNPs associated with ovule and fiber development. We also mapped the genome-wide binding sites of two lncRNAs with chromatin isolation by RNA purification sequencing (ChIRP-seq) and validated the trans transcriptional regulation of lnc-Ga13g0352 via virus induced gene suppression (VIGS) assay. These findings provide valuable research resources for plant community and broaden our understandings of biogenesis and regulation function of plant lncRNAs. One sentence summaryThe full-length annotation and transcriptional regulation of long noncoding RNAs in cotton.

plant biology↗

bioRxiv2020

Ptr1 evolved convergently with RPS2 and Mr5 to mediate recognition of AvrRpt2 in diverse solanaceous species

The Ptr1 (Pseudomonas tomato race 1) locus in Solanum lycopersicoides confers resistance to strains of Pseudomonas syringae pv. tomato expressing AvrRpt2 and Ralstonia pseudosolanacearum expressing RipBN. Here we describe the identification and phylogenetic analysis of the Ptr1 gene. A single recombinant among 585 F2 plants segregating for the Ptr1 locus was discovered that narrowed the Ptr1 candidates to eight nucleotide-binding leucine-rich repeat protein (NLR)-encoding genes. From analysis of the gene models in the S. lycopersicoides genome sequence and RNA-Seq data, two of the eight genes emerged as the strongest candidates for Ptr1. One of these two candidates was found to encode Ptr1 based on its ability to mediate recognition of AvrRpt2 and RipBN when it was transiently expressed with these effectors in leaves of Nicotiana glutinosa. The ortholog of Ptr1 in tomato and in Solanum pennellii is a pseudogene. However, a functional Ptr1 ortholog exists in N. benthamiana and potato and both mediate recognition of AvrRpt2 and RipBN. In apple and Arabidopsis, recognition of AvrRpt2 is mediated by the Mr5 and RPS2 proteins, respectively. Phylogenetic analysis places Ptr1 in a distinct clade compared to Mr5 and RPS2 and it therefore appears to have arisen by convergent evolution for recognition of AvrRpt2.

plant biology↗

bioRxiv2020

A Rainbow Reporter Tracks Single Cells and Reveals Heterogeneous Cellular Dynamics among Pluripotent Stem Cells and their Differentiated Derivatives

Recent single cell analyses have found molecular heterogeneities within populations of pluripotent stem cells (PSCs). A tool that tracks single cell lineages and their phenotypes longitudinally would reveal whether heterogeneity extends beyond molecular identity. Hence, we generated a stable Cre-inducible rainbow reporter human PSC line that provides up to 18 unique membrane-targeted fluorescent barcodes. These barcodes enable repeated assessments of single cells as they clonally expand, change morphology, and migrate. Owing to the cellular resolution of this reporter, we identified subsets of PSCs with enhanced clonal expansion, synchronized cell divisions, and persistent localization to colony edges. Reporter expression was stably maintained throughout directed differentiation into cardiac myocytes, cortical neurons, and hepatoblasts. Repeated examination of neural differentiation revealed self-assembled cortical tissues derive from clonally dominant progenitors. Collectively, these findings demonstrate the broad utility and easy implementation of this reporter line for tracking single cell behavior.

cell biology↗

bioRxiv2019

MetroNome - a visual data exploration platform for integrating human genotypic and phenotypic data across diseases

MetroNome is a web-based visual data exploration platform which integrates de-identified genomic, transcriptomic, and phenotypic data sets. Users can define and compare cohorts constructed from multimodal data and share the data and analyses with outside tools. MetroNomes interactive visualization and analysis tools allow researchers to quickly form and explore novel hypotheses. The deidentified data is linked back to the source biosample inventories in multiple biobanks, enabling researchers to further investigate new ideas using the most relevant samples.

bioinformatics↗

bioRxiv2019

Natural variation for unusual host responses and flagellin-mediated immunity against Pseudomonas syringae in genetically diverse tomato accessions

The interaction between tomato and Pseudomonas syringae pv. tomato (Pst) is a well-developed model for investigating the molecular basis of the plant immune system. There is extensive natural variation in Solanum lycopersicum (tomato) but it has not been fully leveraged to enhance our understanding of the tomato-Pst pathosystem. We screened 216 genetically diverse accessions of cultivated tomato and a wild tomato species for natural variation in their response to three strains of Pst. The screen uncovered a broad range of previously unseen host symptoms in response to Pst, and one of these, stem galls, was found to be simply inherited. The screen also identified tomato accessions that showed enhanced responses to flagellin in bacterial population assays and in reactive oxygen species assays upon exposure to flagellin-derived peptides, flg22 and flgII-28. Reporter genes confirmed that the host responses were due primarily to pattern recognition receptor-immunity. This study revealed extensive natural variation in tomato for susceptibility and resistance to Pst and will enable elucidation of the molecular mechanisms underlying these host responses.

plant biology↗

bioRxiv2019

Active site plasticity and possible modes of chemical inhibition of the human DNA deaminase APOBEC3B

The single-stranded DNA cytosine deaminase APOBEC3B (A3B) functions in innate immunity against viruses, but is also strongly implicated in eliciting mutations in cancer genomes. Because of the critical role of A3B in promoting virus and tumor evolution, small molecule inhibitors are desirable. However, there is no reported structure for any of the APOBEC3-family enzymes in complex with a small molecule bound in the active site, which hampers the development of small molecules targeting A3B. Here we report high-resolution structures of an active A3B catalytic domain chimera with loop 7 residues exchanged with those from the corresponding region of APOBEC3G (A3G). The structures reveal novel open conformations lacking the catalytically essential zinc ion, with the highly conserved active site residues extensively rearranged. These inactive conformations are stabilized by 2-pyrimidone or an iodide ion bound in the active site. Molecular dynamics simulations corroborate the remarkable plasticity of the engineered active site and identify key interactions that stabilize the native A3B active site. These data provide insights into A3B active site dynamics and suggest possible modes of its inhibition by small molecules, which would aid in rational design of selective A3B inhibitors for constraining virus and tumor evolution.

biochemistry↗

bioRxiv2017

The Antiviral and Cancer Genomic DNA Deaminase APOBEC3H Is Regulated by a RNA-Mediated Dimerization Mechanism

Human APOBEC3H and homologous single-stranded DNA cytosine deaminases are unique to mammals. These DNA editing enzymes function in innate immunity by restricting the replication of viruses and transposons. Misregulated APOBEC3H also contributes to cancer mutagenesis. Here we address the role of RNA in APOBEC3H regulation. APOBEC3H co-purifies with RNA as an inactive protein, and RNase A treatment yields enzyme preparations with stronger DNA deaminase activity. RNA binding-defective mutants are DNA hypermutators. Chromatography profiles and crystallographic data demonstrate a mechanism in which double-stranded RNA mediates enzyme dimerization. RNA binding is required for APOBEC3H cytoplasmic localization and for packaging into HIV-1 particles and antiviral activity. Related DNA deaminases including other APOBEC3 family members and the antibody gene diversification enzyme AID also bind RNA and are predicted to have a similar RNA binding motif suggesting mechanistic conservation and relevance to innate and adaptive immunity and to multiple diseases.\n\nHIGHLIGHTSRNA inhibits human APOBEC3H DNA cytosine deaminase activity\n\nRNA binding mutants are DNA hypermutators\n\nX-ray structure demonstrates an RNA duplex-mediated APOBEC3H dimerization mechanism\n\nRNA binding is required for packaging into HIV-1 particles and antiviral activity

microbiology↗