Biology subjects

Gu, C.

Publications and source records attributed to Gu, C..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

Capillary electrophoresis mass spectrometry identifies new isomers of inositol pyrophosphates in mammalian tissues

Technical challenges have to date prevented a complete profiling of the levels of myo-inositol phosphates (InsPs) and pyrophosphates (PP-InsPs) in mammalian tissues. Here, we have deployed capillary electrophoresis mass spectrometry to identify and record the levels of InsPs and PP-InsPs in several tissues obtained from wild type mice and a newly-created PPIP5K2 knockout strain. We observe that the mouse colon harbours unusually high levels of InsPs and PP-InsPs. Additionally, the PP-InsP profile is considerably more complex than previously reported for animal cells: using chemically synthesized internal stable isotope references, and high-resolution mass spectra, we characterize two new PP-InsP isomers as 4/6-PP-InsP5 and 2-PP-InsP5. The latter has not previously been described in Nature. Analysis of feces and the commercial mouse diet suggest the latter is one potential source of noncanonical isomers in the colon. However, we also identify both molecules in the heart, indicating unknown synthesis pathways in mammals. We also demonstrate that the CE-MS method is sensitive enough to measure PP-InsPs from patient samples such as colon biopsies and peripheral blood mononuclear cells (PBMCs). Strikingly, PBMCs also contain 4/6-PP-InsP5 and 2-PP-InsP5. In summary, our study substantially expands PP-InsP biology in mammals.

biochemistry↗

bioRxiv2022

Rapid Development of a Mucosal Nanoparticle Flu Vaccine by Genetic Engineering of Bacteriophage T4 using CRISPR-Cas

Mucosal vaccines that can induce local mucosal immune responses and combat the pathogens at entry sites are considered to be the most effective way to prevent infection. A universal platform that can be customized for development of mucosal vaccines against any given pathogen is therefore highly desired. Here, we demonstrate an efficient approach to develop nasal mucosal vaccines through genetic engineering of T4 phage to generate antigen-decorated nanoparticles. The antigen coding sequence was inserted into T4 genome in-frame at the C terminus of Soc (small outer capsid protein) using the CRISPR-Cas phage editing technology. During the propagation of recombinant T4 phages in E. coli, the Soc-antigen fusion proteins self-assemble on T4 capsids to form antigen-decorated nanoparticles that have intrinsic adjuvant activity and mucosal adhesive property. As a proof of concept, we showed that intranasal immunization with Flu viral M2e-decorated T4 nanoparticles efficiently induced local mucosal as well as systemic immune responses and provided complete protections against divergent influenza viruses in a mouse model. Potentially, our platform can be customized for any respiratory pathogen to rapidly generate mucosal vaccines against future emerging epidemics and pandemics.

immunology↗

bioRxiv2022

Truncated suPAR simultaneously causes kidney disease and autoimmune diabetes mellitus

Soluble urokinase-type plasminogen activator receptor (suPAR) is a risk factor for kidney diseases. Here we report the presence of C-terminal suPAR fragment, D2D3, in patients with diabetic nephropathy. D2D3-positive human sera inhibited glucose-stimulated insulin release in human islets and were associated with patients requiring insulin therapy. D2D3 transgenic mice presented kidney disease and diabetes marked by decreased levels of insulin and C-peptide, impaired glucose-stimulated insulin secretion, decreased pancreatic {beta}-cell mass, and high fasting glucose. D2D3 fragment dysregulated glucose-induced cytoskeletal dynamics, impaired maturation and trafficking of insulin granules, and inhibited bioenergetics of {beta}-cells in culture. An anti-uPAR antibody restored {beta}-cell function in D2D3 transgenic mice. We show that the D2D3 fragment injures the kidney and pancreas, offering a unique dual therapeutic approach for kidney diseases and insulin-dependent diabetes. SummaryProteolytic suPAR fragment, D2D3, simultaneously injures two organs, the kidney and pancreas, thus causing a dual organ disease.

physiology↗

bioRxiv2022

Structural and biochemical mechanism for increased infectivity and immune evasion of Omicron BA.1 and BA.2 variants and their mouse origins

The Omicron BA.2 variant has become a dominant infective strain worldwide. Receptor binding studies reveal that the Omicron BA.2 spike trimer have 11-fold and 2-fold higher potency to human ACE2 than the spike trimer from the wildtype (WT) and Omicron BA.1 strains. The structure of the BA.2 spike trimer complexed with human ACE2 reveals that all three receptor-binding domains (RBDs) in the spike trimer are in open conformation, ready for ACE2 binding, thus providing a basis for the increased infectivity of the BA.2 strain. JMB2002, a therapeutic antibody that was shown to have efficient inhibition of Omicron BA.1, also shows potent neutralization activities against Omicron BA.2. In addition, both BA.1 and BA.2 spike trimers are able to bind to mouse ACE2 with high potency. In contrast, the WT spike trimer binds well to cat ACE2 but not to mouse ACE2. The structures of both BA.1 and BA.2 spike trimer bound to mouse ACE2 reveal the basis for their high affinity interactions. Together, these results suggest a possible evolution pathway for Omicron BA.1 and BA.2 variants from human-cat-mouse-human circle, which could have important implications in establishing an effective strategy in combating viral infection.

biophysics↗

bioRxiv2022

Genomic and phylogenetic analysis of the first myovirus isolated from Oceanospirillaceae, representing a novel viral cluster prevalent in polar oceans

The marine bacterial family Oceanospirillaceae, which is abundant in the deep-seas and polar oceans, is closely associated with algal blooms and petroleum hydrocarbons degradation. However, only a few Oceanospirillaceae-infecting phages have so far been reported. Here we report on a novel Oceanospirillum phage, vB_OsaM_PD0307, which is the first myovirus to be found that infects Oceanospirillaceae. vB_OsaM_PD0307 with a 44,421 bp linear dsDNA genome. Phylogenetic analysis and average nucleotide sequence identities suggest that vB_OsaM_PD0307 is different from other phage isolates and represents a novel genus-level myoviral cluster with two high-quality uncultured viral genomes, designed as Oceanospimyovirus. Additionally, the biogeographical distribution of the vB_OsaM_PD0307 cluster suggests that they are widespread in the oceans and abundant in polar areas. In summary, our findings expand the current understanding of the phylogenetic diversity, genomic characteristic and function of Oceanospimyovirus phages, and highlight the role of the vB_OsaM_PD0307 phage as a major ecological agent that can infect certain key bacterial groups associated with polar algal blooms. ImportanceOceanospirillumphage vB_OsaM_PD0307 is the first myovirus found to infect Oceanospirillaceae and represents a novel viral genus, Oceanospimyovirus. This study provides insights into the genomic, phylogenetic, and ecological characteristics of myoviruses infecting Oceanospirillaceae and improves our understanding of the interactions between Oceanospirillaceae and their phages in the oceans.

genomics↗

bioRxiv2022

The bladder microbiome, metabolome, cytokines, and phenotypes in patients with systemic lupus erythematosus

Background and aimsEmerging studies reveal a unique bacterial community in the human bladder, with alteration of composition associated to disease states. Systemic lupus erythematosus (SLE) is a complex autoimmune disease that is characterized by frequent impairment of the kidney. Here, we explored the bladder microbiome, metabolome, and cytokine profiles in SLE patients, as well as correlations between microbiome and metabolome, cytokines, and disease profiles. Methods and materialsWe recruited a cohort of 50 SLE patients and 50 individually matched asymptomatic controls. We used transurethral catheterization to collect urine samples, 16S rRNA gene sequencing to profile bladder microbiomes, and LC-MS/MS to perform untargeted metabolomic profiling. ResultsCompared to controls, SLE patients possessed a unique bladder microbial community and increased alpha diversity. These differences were accompanied by differences in urinary metabolomes, cytokines, and patients disease profiles. The SLE-enriched genera, including Bacteroides, were positively correlated with several SLE-enriched metabolites, including olopatadine. The SLE-depleted genera, such as Pseudomonas, were negatively correlated to SLE-depleted cytokines, including IL-8. Alteration of the bladder microbiome was associated with disease profile. For example, the genera Megamonas and Phocaeicola were negatively correlated with serum complement C3, and Streptococcus was positively correlated with IgG. ConclusionsOur present study reveals associations between the bladder microbiome and the urinary metabolome, cytokines, and disease phenotypes. Our results could help identify biomarkers for SLE.

microbiology↗

bioRxiv2021

Structures of the Omicron Spike trimer with ACE2 and an anti-Omicron antibody

The Omicron variant of SARS-CoV-2 has rapidly become the dominant infective strain and the focus efforts against the ongoing COVID-19 pandemic. Here we report an extensive set of structures of the Omicron spike trimer by its own or in complex with ACE2 and an anti-Omicron antibody. These structures reveal that most Omicron mutations are located on the surface of the spike protein, which confer stronger ACE2 binding by nearly 10 folds but become inactive epitopes resistant to many therapeutic antibodies. Importantly, both RBD and the closed conformation of the Omicron spike trimer are thermodynamically unstable, with the melting temperature of the Omicron RBD decreased by as much as 7{degrees}C, making the spiker trimer prone to random open conformations. An unusual RBD-RBD interaction in the ACE2-spike complex unique to Omicron is observed to support the open conformation and ACE2 binding, serving the basis for the higher infectivity of Omicron. A broad-spectrum therapeutic antibody JMB2002, which has completed Phase 1 clinical trial, is found to interact with the same two RBDs to inhibit ACE2 binding, in a mode that is distinguished from all previous antibodies, thus providing the structural basis for the potent inhibition of Omicron by this antibody. Together with biochemical data, our structures provide crucial insights into higher infectivity, antibody evasion and inhibition of Omicron.

biophysics↗

bioRxiv2021

The secreted neuronal signal Spock1 regulates the blood-brain barrier

The blood-brain barrier (BBB) is a unique set of properties of the brain vasculature which severely restricts its permeability to proteins and small molecules. Classic chick-quail chimera studies showed that these properties are not intrinsic to the brain vasculature but rather are induced by surrounding neural tissue. Here we identify Spock1 as a candidate neuronal signal for regulating BBB permeability in zebrafish and mice. Mosaic genetic analysis shows that neuronally-expressed Spock1 is cell non-autonomously required for a functional BBB. Leakage in spock1 mutants is associated with altered extracellular matrix (ECM), increased endothelial transcytosis, and altered pericyte-endothelial interactions. Furthermore, a single dose of recombinant SPOCK1 partially restores BBB function in spock1 mutants by quenching gelatinase activity and restoring vascular expression of BBB genes including mcamb. These analyses support a model in which neuronally secreted Spock1 induces BBB properties by altering the ECM, thereby regulating pericyte-endothelial interactions and downstream vascular gene expression. One-Sentence SummarySpock1 is a signal secreted by neurons that induces barrier properties in the brain vasculature

neuroscience↗

bioRxiv2021

Bacteriophage T4 Vaccine Platform for Next-generation Influenza Vaccine Development

Developing influenza vaccines that protect against a broad range of viruses is a public health priority, and several conserved viral proteins or domains have been identified as promising targets for such vaccine development. However, none of the targets is immunogenic, and vaccine platforms that can incorporate multiple antigens with enhanced immunogenicity are desperately needed. In this study, we provided proof-of-concept for the development of next-generation influenza vaccine using T4 phage virus-like particle (VLP) platform. With extracellular domain of influenza matrix protein 2 (M2e) as a readout, we showed that more than 1,280 M2e molecules can be assembled on a 120x90 nanometer phage capsid to form T4-M2e VLPs, which are highly immunogenic and induced complete protection against influenza virus challenge without any addition adjuvant. Potentially, additional conserved antigens or molecular adjuvants could be incorporated into the T4-M2e VLPs to customize influenza vaccines to address different issues. All the components of T4 VLP vaccines can be mass-produced in E. coli in a short time, therefore, providing a rapid approach to deal with the potential influenza pandemic.

microbiology↗

bioRxiv2021

Vascular and perivascular cell profiling reveals the molecular and cellular bases of blood-brain barrier heterogeneity

SUMMARY The blood-brain barrier (BBB) is critical for protecting the brain and maintaining neuronal homeostasis. Although the BBB is a unique feature of the central nervous system (CNS) vasculature, not all brain regions have the same degree of impermeability. Differences in BBB permeability are important for controlling the local extracellular environment of specific brain regions to regulate the function and plasticity of particular neural circuits. However, how BBB heterogeneity occurs is poorly understood. Here, we demonstrate how regional specialization of the BBB is achieved. With unbiased cell profiling in small, defined brain regions, we compare the median eminence, which has a naturally leaky BBB, with the cortex, which has an impermeable BBB. We identify hundreds of molecular differences in endothelial cells (ECs) and demonstrate the existence of differences in perivascular astrocytes and pericytes in these regions, finding 3 previously unknown subtypes of astrocytes and several key differences in pericytes. By serial electron microscopy reconstruction and a novel, aqueous-based tissue clearing imaging method, we further reveal previously unknown anatomical specializations of these perivascular cells and their unique physical interactions with neighboring ECs. Finally, we identify ligand-receptor pairs between ECs and perivascular cells that may regulate regional BBB integrity in ECs. Using a bioinformatic approach we identified 26 and 26 ligand-receptor pairs underlying EC-pericyte and EC-astrocyte interactions, respectively. Our results demonstrate that differences in ECs, together with region-specific physical and molecular interactions with local perivascular cells, contribute to BBB functional heterogeneity. These regional cell inventories serve as a platform for further investigation of the dynamic and heterogeneous nature of the BBB in other brain regions. Identification of local BBB specializations provides insight into the function of different brain regions and will permit the development of region-specific drug delivery in the CNS.

neuroscience↗

bioRxiv2021

Pericyte-derived vitronectin regulates blood-CNS barrier function via integrin signaling

Endothelial cells of blood vessels of the central nervous system (CNS) constitute blood-CNS barriers. Barrier properties are not intrinsic to these cells; rather they are induced and maintained by CNS microenvironment. Notably, the abluminal surface of CNS capillaries are ensheathed by pericytes and astrocytes. However, extrinsic factors from these perivascular cells that regulate barrier integrity are largely unknown. Here, we establish vitronectin, an extracellular-matrix protein secreted by CNS pericytes, as a regulator of blood-CNS barrier function via interactions with its integrin receptor, 5 in endothelial cells. Genetic ablation of vitronectin or mutating vitronectin to prevent integrin binding as well as endothelial-specific deletion of integrin 5 causes barrier leakage. Furthermore, vitronectin-integrin 5 signaling maintains barrier integrity by actively inhibiting transcytosis in endothelial cells. These results demonstrate that signaling from perivascular cells to endothelial cells via ligand-receptor interactions is a key mechanism to regulate barrier permeability.

cell biology↗

bioRxiv2021

Ultrapotent SARS-CoV-2 neutralizing antibodies with protective efficacy against newly emerged mutational variants

Accumulating mutations in the SARS-CoV-2 Spike (S) protein can increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, 3 receptor binding domain (RBD) specific monoclonal antibodies (mAbs), 58G6, 510A5 and 13G9, with high neutralizing potency blocking authentic SARS-CoV-2 virus displayed remarkable efficacy against authentic B.1.351 virus. Each of these 3 mAbs in combination with one neutralizing Ab recognizing non-competing epitope exhibited synergistic effect against authentic SARS-CoV-2 virus. Surprisingly, structural analysis revealed that 58G6 and 13G9, encoded by the IGHV1-58 and the IGKV3-20 germline genes, both recognized the steric region S470-495 on the RBD, overlapping the E484K mutation presented in B.1.351. Also, 58G6 directly bound to another region S450-458 in the RBD. Significantly, 58G6 and 510A5 both demonstrated prophylactic efficacy against authentic SARS-CoV-2 and B.1.351 viruses in the transgenic mice expressing human ACE2 (hACE2), protecting weight loss and reducing virus loads. These 2 ultrapotent neutralizing Abs can be promising candidates to fulfill the urgent needs for the prolonged COVID-19 pandemic.

immunology↗

bioRxiv2021

A network regularized linear model to infer spatial expression pattern for singlecells

Spatial gene-expression is a crucial determinant of cell fate and behavior. Recent imaging and sequencing-technology advancements have enabled scientists to develop new tools that use spatial information to measure gene-expression at close to single-cell levels. Yet, while Fluorescence In-situ Hybridization (FISH) can quantify transcript numbers at single-cell resolution, it is limited to a small number of genes. Similarly, slide-seq was designed to measure spatial-expression profiles at the single-cell level but has a relatively low gene-capture rate. And although single-cell RNA-seq enables deep cellular gene-expression profiling, it loses spatial information during sample-collection. These major limitations have stymied these methods broader application in the field. To overcome spatio-omics technologys limitations and better understand spatial patterns at single-cell resolution, we designed a computation algorithm that uses glmSMA to predict cell locations by integrating scRNA-seq data with a spatialomics reference atlas. We treated cell-mapping as a convex optimization problem by minimizing the differences between cellular-expression profiles and location-expression profiles with a L1 regularization and graph Laplacian based L2 regularization to ensure a sparse and smooth mapping. We validated the mapping results by reconstructing spatial-expression patterns of well-known marker genes in complex tissues, like the mouse cerebellum and hippocampus. We used the biological literature to verify that the reconstructed patterns can recapitulate cell-type and anatomy structures. Our work thus far shows that, together, we can use glmSMA to accurately assign single cells to their original reference-atlas locations.

bioinformatics↗

bioRxiv2021

A human antibody with blocking activity to RBD proteins of multiple SARS-CoV-2 variants including B.1.351 showed potent prophylactic and therapeutic efficacy against SARS-CoV-2 in rhesus macaques

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes coronavirus disease-2019 (COVID-19), interacts with the host cell receptor angiotensin-converting enzyme 2 (hACE2) via its spike 1 protein for infection. After the virus sequence was published, we identified two potent antibodies against SARS-CoV-2 RBD from antibody libraries using a phage-to-yeast (PtY) display platform in only 10 days. Our lead antibody JMB2002, now in a phase I clinical trial, showed broad-spectrum in vitro blocking activity against hACE2 binding to the RBD of multiple SARS-CoV-2 variants including B.1.351 that was reportedly much more resistant to neutralization by convalescent plasma, vaccine sera and some clinical stage neutralizing antibodies. Furthermore, JMB2002 has demonstrated complete prophylactic and potent therapeutic efficacy in a rhesus macaque disease model. Prophylactic and therapeutic countermeasure intervention of SARS-CoV-2 using JMB2002 would likely slow down the transmission of currently emerged SARS-CoV-2 variants and result in more efficient control of the COVID-19 pandemic.

microbiology↗

bioRxiv2020

A key linear epitope for a potent neutralizing antibody to SARS-CoV-2 S-RBD

The spread of SARS-CoV-2 confers a serious threat to the public health without effective intervention strategies1-3. Its variant carrying mutated Spike (S) protein D614G (SD614G) has become the most prevalent form in the current global pandemic4,5. We have identified a large panel of potential neutralizing antibodies (NAbs) targeting the receptor-binding domain (RBD) of SARS-CoV-2 S6. Here, we focused on the top 20 potential NAbs for the mechanism study. Of them, the top 4 NAbs could individually neutralize both authentic SARS-CoV-2 and SD614G pseudovirus efficiently. Our epitope mapping revealed that 16/20 potent NAbs overlapped the same steric epitope. Excitingly, we found that one of these potent NAbs (58G6) exclusively bound to a linear epitope on S-RBD (termed as 58G6e), and the interaction of 58G6e and the recombinant ACE2 could be blocked by 58G6. We confirmed that 58G6e represented a key site of vulnerability on S-RBD and it could positively react with COVID-19 convalescent patients plasma. We are the first, as far as we know, to provide direct evidences of a linear epitope that can be recognized by a potent NAb against SARS-CoV-2 S-RBD. This study paves the way for the applications of these NAbs and the potential safe and effective vaccine design.

immunology↗

bioRxiv2020

Analysis of Inositol Phosphate Metabolism by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry (CE-ESI-MS)

The analysis of myo-inositol phosphates (InsPs) and myo-inositol pyrophosphates (PP-InsPs) is a daunting challenge due to the large number of possible isomers, the absence of a chromophore, the high charge density, the low abundance, and the instability of the esters and anhydrides. Given their importance in biology, an analytical approach to follow and understand this complex signaling hub is highly desirable. Here, capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESI-MS) is implemented to analyze complex mixtures of InsPs and PP-InsPs with high sensitivity. Stable isotope labeled (SIL) internal standards allow for matrix-independent quantitative assignment. The method is validated in wild-type and knockout mammalian cell lines and in model organisms. SIL-CE-ESI-MS enables for the first time the accurate monitoring of InsPs and PP-InsPs arising from compartmentalized cellular synthesis pathways, by feeding cells with either [13C6]-myo-inositol or [13C6]-D-glucose. In doing so, we uncover that there must be unknown inositol synthesis pathways in mammals, highlighting the unique potential of this method to dissect inositol phosphate metabolism and signalling.

biochemistry↗

bioRxiv2020

A rapid and efficient screening system for neutralizing antibodies and its application for the discovery of potent neutralizing antibodies to SARS-CoV-2 S-RBD

Neutralizing antibodies (Abs) have been considered as promising therapeutics for the prevention and treatment of pathogens. After the outbreak of COVID-19, potent neutralizing Abs to SARS-CoV-2 were promptly developed, and a few of those neutralizing Abs are being tested in clinical studies. However, there were few methodologies detailly reported on how to rapidly and efficiently generate neutralizing Abs of interest. Here, we present a strategically optimized method for precisive screening of neutralizing monoclonal antibodies (mAbs), which enabled us to identify SARS-CoV-2 receptor-binding domain (RBD) specific Abs within 4 days, followed by another 2 days for neutralization activity evaluation. By applying the screening system, we obtained 198 Abs against the RBD of SARS-CoV-2. Excitingly, we found that approximately 50% (96/198) of them were candidate neutralizing Abs in a preliminary screening of SARS-CoV-2 pseudovirus and 20 of these 96 neutralizing Abs were confirmed with high potency. Furthermore, 2 mAbs with the highest neutralizing potency were identified to block authentic SARS-CoV-2 with the half-maximal inhibitory concentration (IC50) at concentrations of 9.88 ng/ml and 11.13 ng/ml. In this report, we demonstrated that the optimized neutralizing Abs screening system is useful for the rapid and efficient discovery of potent neutralizing Abs against SARS-CoV-2. Our study provides a methodology for the generation of preventive and therapeutic antibody drugs for emerging infectious diseases.

immunology↗

bioRxiv2020

Immunization with the receptor-binding domain of SARS-CoV-2 elicits antibodies cross-neutralizing SARS-CoV-2 and SARS-CoV without antibody-dependent enhancement

Recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic. Currently, there is no vaccine available for preventing SARS-CoV-2 infection. Like closely related severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2 also uses its receptor-binding domain (RBD) on the spike (S) protein to engage the host receptor, human angiotensin-converting enzyme 2 (ACE2), facilitating subsequent viral entry. Here we report the immunogenicity and vaccine potential of SARS-CoV-2 RBD (SARS2-RBD)-based recombinant proteins. Immunization with SARS2-RBD recombinant proteins potently induced a multi-functional antibody response in mice. The resulting antisera could efficiently block the interaction between SARS2-RBD and ACE2, inhibit S-mediated cell-cell fusion, and neutralize both SARS-CoV-2 pseudovirus entry and authentic SARS-CoV-2 infection. In addition, the anti-RBD sera also exhibited cross binding, ACE2-blockade, and neutralization effects towards SARS-CoV. More importantly, we found that the anti-RBD sera did not promote antibody-dependent enhancement of either SARS-CoV-2 pseudovirus entry or authentic virus infection of Fc receptor-bearing cells. These findings provide a solid foundation for developing RBD-based subunit vaccines for SARS-CoV2.

microbiology↗