Biology subjects

Zhu, X.

Publications and source records attributed to Zhu, X..

At least 325 records · Page 18Linked to original sources

bioRxiv2020

A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody

Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that can cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential for cross-neutralizing epitopes on SARS-like viruses.

microbiology↗

bioRxiv2020

The Wnt effector TCF7l2 promotes oligodendroglial differentiation by repressing autocrine BMP4-mediated signaling

Promoting oligodendrocyte differentiation represents a promising option for remyelination therapy for treating the demyelinating disease multiple sclerosis (MS). The Wnt effector TCF7l2 was upregulated in MS lesions and had been proposed to inhibit oligodendrocyte differentiation. Recent data suggest the opposite yet underlying mechanisms remain elusive. Here we unravel a previously unappreciated function of TCF7l2 in controlling autocrine bone morphogenetic protein (BMP4)-mediated signaling. Disrupting TCF7l2 results in oligodendroglial-specific BMP4 upregulation and canonical BMP4 signaling activation in vivo. Mechanistically, TCF7l2 binds to Bmp4 gene regulatory element and directly represses its transcriptional activity. Functionally, enforced TCF7l2 expression promotes oligodendrocyte differentiation by reducing autocrine BMP4 secretion and dampening BMP4 signaling. Importantly, compound genetic disruption demonstrates that oligodendroglial-specific BMP4 deletion rescues arrested oligodendrocyte differentiation elicited by TCF7l2 disruption in vivo. Collectively, our study reveals a novel connection between TCF7l2 and BMP4 in oligodendroglial lineage and provides new insights into augmenting TCF7l2 for promoting remyelination in demyelinating disorders such as MS. Significance StatementIncomplete or failed myelin repairs, primarily resulting from the arrested differentiation of myelin-forming oligodendrocytes from oligodendroglial progenitor cells, is one of the major reasons for neurological progression in people affected by multiple sclerosis (MS). Using in vitro culture systems and in vivo animal models, this study unraveled a previously unrecognized autocrine regulation of BMP4-mediated signaling by the Wnt effector TCF7l2. We showed for the first time that TCF7l2 promotes oligodendroglial differentiation by repressing BMP4-mediated activity, which is dysregulated in MS lesions. Our study suggests that elevating TCF7l2 expression may be possible in overcoming arrested oligodendroglial differentiation as observed in MS patients.

neuroscience↗

bioRxiv2020

Genome-Wide Mapping of Human DNA Replication by Optical Replication Mapping Supports a Stochastic Model of Eukaryotic Replication Timing

DNA replication is regulated by the location and timing of replication initiation. Therefore, much effort has been invested in identifying and analyzing the sites of human replication initiation. However, the heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual initiation site utilization in metazoans has made mapping the location and timing of replication initiation in human cells difficult. A potential solution to the problem of human replication mapping is single-molecule analysis. However, current approaches do not provide the throughput required for genome-wide experiments. To address this challenge, we have developed Optical Replication Mapping (ORM), a high-throughput single-molecule approach to map newly replicated DNA, and used it to map early initiation events in human cells. The single-molecule nature of our data, and a total of more than 2000-fold coverage of the human genome on 27 million fibers averaging ~300 kb in length, allow us to identify initiation sites and their firing probability with high confidence. In particular, for the first time, we are able to measure genome-wide the absolute efficiency of human replication initiation. We find that the distribution of human replication initiation is consistent with inefficient, stochastic initiation of heterogeneously distributed potential initiation complexes enriched in accessible chromatin. In particular, we find sites of human replication initiation are not confined to well-defined replication origins but are instead distributed across broad initiation zones consisting of many initiation sites. Furthermore, we find no correlation of initiation events between neighboring initiation zones. Although most early initiation events occur in early-replicating regions of the genome, a significant number occur in late-replicating regions. The fact that initiation sites in typically late-replicating regions have some probability of firing in early S phase suggests that the major difference between initiation events in early and late replicating regions is their intrinsic probability of firing, as opposed to a qualitative difference in their firing-time distributions. Moreover, modeling of replication kinetics demonstrates that measuring the efficiency of initiation-zone firing in early S phase suffices to predict the average firing time of such initiation zones throughout S phase, further suggesting that the differences between the firing times of early and late initiation zones are quantitative, rather than qualitative. These observations are consistent with stochastic models of initiation-timing regulation and suggest that stochastic regulation of replication kinetics is a fundamental feature of eukaryotic replication, conserved from yeast to humans.

genomics↗

bioRxiv2020

A SARS-CoV-2 neutralizing antibody protects from lung pathology in a COVID-19 hamster model

The emergence of SARS-CoV-2 led to pandemic spread of coronavirus disease 2019 (COVID-19), manifesting with respiratory symptoms and multi-organ dysfunction. Detailed characterization of virus-neutralizing antibodies and target epitopes is needed to understand COVID-19 pathophysiology and guide immunization strategies. Among 598 human monoclonal antibodies (mAbs) from ten COVID-19 patients, we identified 40 strongly neutralizing mAbs. The most potent mAb CV07-209 neutralized authentic SARS-CoV-2 with IC50 of 3.1 ng/ml. Crystal structures of two mAbs in complex with the SARS-CoV-2 receptor-binding domain at 2.55 and 2.70 [A] revealed a direct block of ACE2 attachment. Interestingly, some of the near-germline SARS-CoV-2 neutralizing mAbs reacted with mammalian self-antigens. Prophylactic and therapeutic application of CV07-209 protected hamsters from SARS-CoV-2 infection, weight loss and lung pathology. Our results show that non-self-reactive virus-neutralizing mAbs elicited during SARS-CoV-2 infection are a promising therapeutic strategy.

immunology↗

bioRxiv2020

PRG2 and AQPEP are misexpressed in fetal membranes in placenta previa and percreta

The obstetrical conditions placenta accreta spectrum (PAS) and placenta previa are a significant source of pregnancy-associated morbidity and mortality, yet the specific molecular and cellular underpinnings of these conditions are not known. In this study, we identified misregulated gene expression patterns in tissues from placenta previa and percreta (the most extreme form of PAS) compared with control cases. By comparing this gene set with existing placental single-cell and bulk RNA-Seq datasets, we show that the upregulated genes predominantly mark extravillous trophoblasts. We performed immunofluorescence on several candidate molecules and found that PRG2 and AQPEP protein levels are upregulated in both the fetal membranes and the placental disk in both conditions. While this increased AQPEP expression remains restricted to trophoblasts, PRG2 is mislocalized and is found throughout the fetal membranes. Using a larger patient cohort with a diverse set of gestationally aged-matched controls, we validated PRG2 as a marker for both previa and PAS and AQPEP as a marker for only previa in the fetal membranes membranes. Our findings suggest that the extraembryonic tissues surrounding the conceptus, including both the fetal membranes membranes and the placental disk, harbor a signature of previa and PAS that reflects increased trophoblast invasiveness. Summary sentence3SEQ and immunofluorescence reveal that extravillous trophoblast factors, most notably PRG2 and AQPEP, define the diseases placenta previa and placenta accreta spectrum (PAS) in both the chorioamniotic membranes and the placental disk.

pathology↗

bioRxiv2020

SRSF protein kinases 1 and 2 are essential host factors for human coronaviruses including SARS-CoV-2

While vaccines are vital for preventing COVID-19 infections, it is critical to develop new therapies to treat patients who become infected. Pharmacological targeting of a host factor required for viral replication can suppress viral spread with a low probability of viral mutation leading to resistance. In particular, host kinases are highly druggable targets and a number of conserved coronavirus proteins, notably the nucleoprotein (N), require phosphorylation for full functionality. In order to understand how targeting kinases could be used to compromise viral replication, we used a combination of phosphoproteomics and bioinformatics as well as genetic and pharmacological kinase inhibition to define the enzymes important for SARS-CoV-2 N protein phosphorylation and viral replication. From these data, we propose a model whereby SRPK1/2 initiates phosphorylation of the N protein, which primes for further phosphorylation by GSK-3/{beta} and CK1 to achieve extensive phosphorylation of the N protein SR-rich domain. Importantly, we were able to leverage our data to identify an FDA-approved kinase inhibitor, Alectinib, that suppresses N phosphorylation by SRPK1/2 and limits SARS-CoV-2 replication. Together, these data suggest that repurposing or developing novel host-kinase directed therapies may be an efficacious strategy to prevent or treat COVID-19 and other coronavirus-mediated diseases.

microbiology↗

bioRxiv2020

Cross-neutralization of a SARS-CoV-2 antibody to a functionally conserved site is mediated by avidity

Most antibodies isolated from COVID-19 patients are specific to SARS-CoV-2. COVA1-16 is a relatively rare antibody that also cross-neutralizes SARS-CoV. Here we determined a crystal structure of COVA1-16 Fab with the SARS-CoV-2 RBD, and a negative-stain EM reconstruction with the spike glycoprotein trimer, to elucidate the structural basis of its cross-reactivity. COVA1-16 binds a highly conserved epitope on the SARS-CoV-2 RBD, mainly through a long CDR H3, and competes with ACE2 binding due to steric hindrance rather than epitope overlap. COVA1-16 binds to a flexible up conformation of the RBD on the spike and relies on antibody avidity for neutralization. These findings, along with structural and functional rationale for the epitope conservation, provide a blueprint for development of more universal SARS-like coronavirus vaccines and therapies.

microbiology↗

bioRxiv2020

An alternative binding mode of IGHV3-53 antibodies to the SARS-CoV-2 receptor binding domain

IGHV3-53-encoded neutralizing antibodies are commonly elicited during SARS-CoV-2 infection and target the receptor-binding domain (RBD) of the spike (S) protein. Such IGHV3-53 antibodies generally have a short CDR H3 due to structural constraints in binding the RBD (mode A). However, a small subset of IGHV3-53 antibodies to the RBD contain a longer CDR H3. Crystal structures of two IGHV3-53 neutralizing antibodies here demonstrate that a longer CDR H3 can be accommodated in a different binding mode (mode B). These two classes of IGHV3-53 antibodies both target the ACE2 receptor binding site, but with very different angles of approach and molecular interactions. Overall, these findings emphasize the versatility of IGHV3-53 in this common antibody response to SARS-CoV-2, where conserved IGHV3-53 germline-encoded features can be combined with very different CDR H3 lengths and light chains for SARS-CoV-2 RBD recognition and virus neutralization.

microbiology↗

bioRxiv2020

Top-down engineering of complex communities by directed evolution

Directed evolution has been used for decades to engineer biological systems from the top-down. Generally, it has been applied at or below the organismal level, by iteratively sampling the mutational landscape in a guided search for genetic variants of higher function. Above the organismal level, a small number of studies have attempted to artificially select microbial communities and ecosystems, with uneven and generally modest success. Our theoretical understanding of artificial ecosystem selection is still limited, particularly for large assemblages of asexual organisms, and we know little about designing efficient methods to direct their evolution. To address this issue, we have developed a flexible modeling framework that allows us to systematically probe any arbitrary selection strategy on any arbitrary set of communities and selected functions, in a wide range of ecological conditions. By artificially selecting hundreds of in-silico microbial metacommunities under identical conditions, we examine the fundamental limits of the two main breeding methods used so far, and prescribe modifications that significantly increase their power. We identify a range of directed evolution strategies that, particularly when applied in combination, are better suited for the top-down engineering of large, diverse, and stable microbial consortia. Our results emphasize that directed evolution allows an ecological structure-function landscape to be navigated in search for dynamically stable and ecologically and functionally resilient high-functioning communities.

evolutionary biology↗

bioRxiv2020

Partial inhibition of mitochondrial complex I attenuates neurodegeneration and restores energy homeostasis and synaptic function in a symptomatic Alzheimers mouse model

We demonstrate that mitochondrial respiratory chain complex I is an important small molecule druggable target in Alzheimers Disease (AD). Partial inhibition of complex I triggers the AMP-activated protein kinase-dependent signaling network leading to neuroprotection in symptomatic APP/PS1 mice, a translational model of AD. Treatment of APP/PS1 mice with complex I inhibitor after the onset of AD-like neuropathology improved energy homeostasis, synaptic activity, long-term potentiation, dendritic spine maturation, cognitive function and proteostasis, and reduced oxidative stress and inflammation in brain and periphery, ultimately blocking the ongoing neurodegeneration. Therapeutic efficacy in vivo was monitored using translational biomarkers FDG-PET, 31P NMR, and metabolomics. Cross-validation of the mouse and the human AMP-AD transcriptomic data demonstrated that pathways improved by the treatment in APP/PS1 mice, including the immune system response and neurotransmission, represent mechanisms essential for therapeutic efficacy in AD patients.

neuroscience↗

bioRxiv2020

Decoding herbal materials of representative TCM preparations with the multi-barcoding approach

With the rapid development of high-throughput sequencing (HTS) technology, the techniques for the assessment of biological ingredients in Traditional Chinese Medicine (TCM) preparations have also advanced. By using HTS together with the multi-barcoding approach, all biological ingredients could be identified from TCM preparations in theory, as long as their DNA is present. The biological ingredients of a handful of classical TCM preparations were analyzed successfully based on this approach in previous studies. However, the universality, sensitivity and reliability of this approach used on TCM preparations remain unclear. Here, four representative TCM preparations, namely Bazhen Yimu Wan, Da Huoluo Wan, Niuhuang Jiangya Wan and You Gui Wan, were selected for concrete assessment of this approach. We have successfully detected from 77.8% to 100% prescribed herbal materials based on both ITS2 and trnL biomarkers. The results based on ITS2 have also shown a higher level of reliability than those of trnL at species level, and the integration of both biomarkers could provide higher sensitivity and reliability. In the omics big-data era, this study has undoubtedly made one step forward for the multi-barcoding approach for prescribed herbal materials analysis of TCM preparation, towards better digitization and modernization of drug quality control.

bioinformatics↗

bioRxiv2020

Microtubules regulate pancreatic beta cell heterogeneity via spatiotemporal control of insulin secretion hot spots

Heterogeneity of glucose-stimulated insulin secretion (GSIS) in pancreatic islets is physiologically important but poorly understood. Here, we utilize whole mouse islets to determine how microtubules affect secretion toward the vascular extracellular matrix. Our data indicate that microtubule stability in the {beta}-cell population is heterogenous, and that cells with more stable microtubules secrete less in response to a stimulus. Consistently, microtubule hyper-stabilization prevents, and microtubule depolymerization promotes {beta}-cell activation. Analysis of spatiotemporal patterns of secretion events shows that microtubule depolymerization activates otherwise dormant {beta}-cells via initiation of secretion clusters (hot spots). Microtubule depolymerization also enhances secretion from individual cells, introducing both additional clusters and scattered events. Interestingly, without microtubules, the timing of clustered secretion is dysregulated, extending the first phase of GSIS. Our findings uncover a novel microtubule function in tuning insulin secretion hot spots, which leads to accurately measured and timed response to glucose stimuli and promotes functional {beta}-cell heterogeneity.

cell biology↗

bioRxiv2020

Dichloroacetate improves hepatic and systemic energy metabolism during sepsis

Dramatic metabolic reprogramming between an anabolic resistance and catabolic tolerance state occurs within the immune system in response to systemic infection with the sepsis syndrome. While metabolic tissues such as the liver are subject to end-organ damage during sepsis and are the primary cause of sepsis death, how their metabolic and energy reprogramming during sepsis state ensures survival is unclear. Employing comprehensive metabolomic screening, targeted lipidomic screening, and transcriptional profiling in a mouse model of septic shock, we show that hepatocyte lipid metabolism, mitochondrial TCA energetics, and redox balance are significantly reprogramed after cecal ligation and puncture (CLP). We identify increases in TCA cycle metabolites citrate, cis-aconitate, and itaconate with reduced fumarate and triglyceride accumulation in septic hepatocytes. Transcription analysis of liver tissue supports and extends the hepatocyte findings. Strikingly, the administration of the pyruvate dehydrogenase kinase (PDK) inhibitor dichloroacetate (DCA) reverses dysregulated hepatocyte metabolism and mitochondrial dysfunction. Our data indicate sepsis promotes hepatic metabolic dysfunction. Furthermore, our data indicate that targeting the mitochondrial PDC/PDK energy home-ostat rebalances transcriptional and metabolic manifestations of sepsis within the liver.

immunology↗

bioRxiv2020

Structural basis of a public antibody response to SARS-CoV-2

Molecular-level understanding of human neutralizing antibody responses to SARS-CoV-2 could accelerate vaccine design and facilitate drug discovery. We analyzed 294 SARS-CoV-2 antibodies and found that IGHV3-53 is the most frequently used IGHV gene for targeting the receptor binding domain (RBD) of the spike (S) protein. We determined crystal structures of two IGHV3-53 neutralizing antibodies +/- Fab CR3022 ranging from 2.33 to 3.11 [A] resolution. The germline-encoded residues of IGHV3-53 dominate binding to the ACE2 binding site epitope with no overlap with the CR3022 epitope. Moreover, IGHV3-53 is used in combination with a very short CDR H3 and different light chains. Overall, IGHV3-53 represents a versatile public VH in neutralizing SARS-CoV-2 antibodies, where their specific germline features and minimal affinity maturation provide important insights for vaccine design and assessing outcomes.

microbiology↗

bioRxiv2020

Allergen-induced dendritic cell migration is controlled through Substance P release by sensory neurons

Dendritic cells (DCs) of the cDC2 lineage are necessary for the initiation of the allergic immune response and in the dermis are marked by their expression of CD301b. CD301b+ dermal DCs respond to allergens encountered in vivo, but not in vitro. This suggests that another cell in the dermis may sense allergens and relay that information to activate and induce the migration of CD301b+ DCs to the draining lymph node. Using a model of cutaneous allergen exposure, we show that allergens directly activate TRPV1+ sensory neurons leading to itch and pain behaviors. Allergen-activated sensory neurons release the neuropeptide Substance P, which stimulates proximally located CD301b+ DCs through MRGPRA1. Substance P induces CD301b+ DC migration to the draining lymph node where they initiate Th2 differentiation. Thus, sensory neurons act as primary sensors of allergens, linking exposure to activation of allergic-skewing DCs and the initiation of the allergic immune response.

immunology↗

bioRxiv2020

Structural insight into outer membrane asymmetry maintenance of Gram-negative bacteria by the phospholipid transporter MlaFEDB

The asymmetric phospholipid outer membrane (OM) of Gram-negative bacteria serves as the first line of defense against cytotoxic substances such as antibiotics. The Mla pathway is known to maintain the lipid asymmetry of the OM by transporting phospholipids between the inner and outer membranes. Six Mla proteins MlaFEDBCA are involved, with the ABC transporter MlaFEDB acts through a mechanism yet to be elucidated. Here we determine cryo-EM structures of MlaFEDB in apo, phospholipid-, ADP- or AMP-PNP-bound state to 3.3-3.75 [A] resolution and establish a proteoliposome-based transport system containing MlaFEDB, MlaC and MlaA/OmpF to reveal the transport direction of phospholipids. Mutagenetic in vitro transport assays and in vivo sensitivity assays reveal functional residues which recognize and transport phospholipids as well as regulate the activity and structural stability of the MlaFEDB complex. Our work provides molecular basis for understanding the mechanism of the Mla pathway which could be targeted for antimicrobial drug development.

biochemistry↗

bioRxiv2020

Light-Sheet Fluorescence Imaging Charts the Gastrula Origin of Vascular Endothelial Cells in Early Zebrafish Embryos

It remains challenging to construct a complete cell lineage map of the origin of vascular endothelial cells in any vertebrate embryo. Here, we report the application of in toto light-sheet fluorescence imaging of embryos to tracing the origin of vascular endothelial cells (ECs) at single-cell resolution in zebrafish. We first adapted a previously-reported method to mount embryos and light-sheet imaging, created an alignment, fusion, and extraction all-in-one software (AFEIO) for processing big data, and performed quantitative analysis of cell lineage relationships using commercially-available Imaris software. Our data revealed that vascular ECs originated from broad regions of the gastrula along the dorsal-ventral and anterior-posterior axes, of which the dorsal-anterior cells contributed to cerebral ECs, the dorsal-lateral cells to anterior trunk ECs, and the ventral-lateral cells to posterior trunk and tail ECs. Therefore, this work, to our knowledge, charts the first comprehensive map of the gastrula origin of vascular ECs in zebrafish, and has potential applications for studying the origin of any embryonic organs in zebrafish and other model organisms.

developmental biology↗

bioRxiv2020

Peptide YY: a novel Paneth cell antimicrobial peptide that maintains fungal commensalism

Perturbed interactions between the intestinal microbes and host correlate with emergence of fungal virulence. Here we report a previously unknown role for peptide YY (PYY), a described endocrine molecule, as an antimicrobial peptide (AMP) expressed by gut immune epithelial Paneth Cells (PC). PC-PYY differs from other AMPs, including lysozyme, because of limited antibacterial activity, packaging in discrete secretory granules, and selective antifungal activity to virulent hyphae, but not yeast forms of Candida albicans. The latter action is through binding of cationic PC-PYY to the anionic hyphal surface, resulting in membrane disruption and killing. PC-PYY is compartmentalized to surface mucus, which optimizes activity and prevents conversion to endocrine PYY by dipeptidyl peptidase-IV (DPP-IV). We conclude PC-PYY is a unique AMP with selective antifungal activity that maintains gut fungal commensalism. Compromised PC-PYY action from PC dysfunction and/or mucus depletion in ileal Crohns disease may initiate or contribute to disease via fungal pathogenesis. Highlights Paneth Cell PYY (PC-PYY) is an antimicrobial peptide that differs from endocrine-PYY PC-PYY is a selective anti-fungal peptide, targeting the virulent form of C. albicans PC-PYY is separately packaged, retained by mucus, and released by C. albicans hyphae PC-PYY is proposed as essential for maintenance of fungal commensalism in the gut Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Model for Paneth cell (PC) PYY action and regulation of fungal commensalisms and potential role in the pathogenesis of ileal Crohns Disease (iCD) (A) In a healthy ileum, commensal yeast reside and do not stimulate PYY1-36 release from PCs. (B) Increased virulent hyphae (purple hyphae) results in PYY1-36 release from crypt PCs into the mucus. Hyphae are targeted by PYY1-36 and killed (red hyphae) to manage the increased fungi community in gut. (C) In a diseased ileum such as iCD, hyphal load induces immune activation and increased inflammation through PC dysfunction (gray PCs) and decreased PYY1-36 release or mucus depletion and PC dysfunction. C_FIG_DISPLAY

molecular biology↗