Biology subjects

Zeng, C.

Publications and source records attributed to Zeng, C..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

Reconstruct a eukaryotic chromosome arm by de novo design and synthesis

The genome of an organism is inherited from its ancestor and keeps evolving over time, however, how much the current version could be altered remains unknown. Here, we use the left arm of chromosome XII (chrXIIL) as an example to probe the genome plasticity in Saccharomyces cerevisiae. A neochromosome was designed to harbor originally dispersed genes. The essentiality of sequences in chrXIIL was dissected by targeted DNA removal, chromosome truncation and random deletion. Notably, 12 genes were sufficient for survival, while 25 genes are required to retain robust fitness. Next, we demonstrated these genes could be reconstructed using synthetic regulatory sequences and recoded open-reading frames with "one-amino-acid-one-codon" strategy. Finally, we built a neochromsome, which could substitute for chrXIIL for cell viability, with these reconstructed genes. Our work not only highlights the high plasticity of yeast genome, but also illustrates the possibility of making functional chromosomes with completely artificial sequences. HIGHLIGHTSO_LIA neochromosome was designed to facilitate the assembly of exogenous DNA for stable expression in yeast C_LIO_LIThe left arm of chrXII could be minimized to just 12 genes to maintain viability, but additional genes were required to retain robust fitness C_LIO_LIComprehensive recoding and transcriptional refactoring using artificial regulatory sequences produced a functional chromosome arm C_LIO_LIA completely reconstructed neochromosome could replace the chrXIIL to maintain comparable fitness C_LI

synthetic biology↗

bioRxiv2022

Protection from Omicron and other VOCs by Bivalent S-Trimer COVID-19 Vaccine

The Omicron variant of SARS-COV-2 (GISAID GRA clade [B.1.1.529, BA.1 and BA.2]) is now the single dominant Variant of Concern (VOC). The high number of mutations in the Omicron Spike (S) protein promotes humoral immunological escape. Although a third homologous boost with S, derived from the ancestral strain, was able to increase neutralizing antibody titers and breadth including to Omicron, the magnitude of virus neutralization could benefit from further optimization. Moreover, combining SARS-COV-2 strains as additional valences may address the current antigenicity range occupied by VOCs. Using Trimer-Tag platform we have previously demonstrated phase 3 efficacy and safety of a prototypic vaccine SCB-2019 in the SPECTRA trial and have submitted applications for licensure. Here, we successfully generated a bivalent vaccine candidate including both Ancestor and Omicron variant S-proteins. Preclinical studies demonstrate this SARS-CoV-2 bivalent S-Trimer subunit vaccine elicits high titers of neutralizing antibodies against all VOCs, with markedly enhanced Omicron specific neutralizing antibody responses.

immunology↗

bioRxiv2022

Prime-pull immunization of mice with a BcfA-adjuvanted vaccine elicits mucosal immunity and prevents SARS CoV-2 infection and pathology

Vaccines against SARS-CoV-2 that induce mucosal immunity capable of preventing infection and disease remain urgently needed. We show that intramuscular priming of mice with an alum and BcfA-adjuvanted Spike subunit vaccine, followed by a BcfA-adjuvanted mucosal booster, generated Th17 polarized tissue resident CD4+ T cells, and mucosal and serum antibodies. The serum antibodies efficiently neutralized SARS-CoV-2 and its Delta variant, suggesting cross-protection against a recent variant of concern (VOC). Immunization with this heterologous vaccine prevented weight loss following challenge with mouse-adapted SARS-CoV-2 and reduced viral replication in the nose and lungs. Histopathology showed a strong leukocyte and polymorphonuclear (PMN) cell infiltrate without epithelial damage in mice immunized with BcfA-containing vaccines. In contrast, viral load was not reduced in the upper respiratory tract of IL-17 knockout mice immunized with the same formulation, suggesting that the Th17 polarized T cell responses are critical for protection. We show that vaccines adjuvanted with alum and BcfA, delivered through a heterologous prime-pull regimen, protect against SARS-CoV-2 infection without causing enhanced respiratory disease. SIGNIFICANCEThere remains a need for SARS CoV-2 booster vaccines that generate mucosal immunity and prevent transmission. We show that systemic priming followed by a mucosal booster with a BcfA-adjuvanted subunit vaccine generates neutralizing antibodies and Th17 polarized systemic and tissue-resident immune responses that provide sterilizing immunity against wildtype SARS CoV-2, and a variant of concern. Importantly, in contrast to alum alone, the addition of BcfA prevents respiratory pathology. These results suggest that a BcfA-adjuvanted mucosal booster may elicit mucosal immunity in individuals previously immunized systemically with approved vaccines. This foundational study in mice sets the stage for testing our vaccine regimen in larger animal models as a booster vaccine.

immunology↗

bioRxiv2022

Near-atomic Structure of the Cytoplasmic Ring of the Xenopus laevis Nuclear Pore Complex

The nuclear pore complex (NPC) mediates nucleocytoplasmic shuttling. Here we present single-particle cryo-EM structure of the cytoplasmic ring (CR) from the Xenopus laevis NPC at 4.1-4.7 [A] resolutions. The structure of an N-terminal domain of Nup358 was resolved at 3.0 [A], facilitating identification of five Nup358 molecules in each CR subunit. Aside from unveiling the assembly details of the two Y-shaped multicomponent complexes (Y complexes) in each CR subunit, the improved resolutions reveal the C-terminal fragment of Nup160 to be an organizing center at the vertex of each Y complex. Our structures show that the scaffold of a CR subunit comprises five Nup358, two Nup205 and two Nup93 molecules in addition to the previously characterized Y complexes. One-Sentence SummaryImproved resolutions of the cytoplasmic ring (CR) of the Xenopus laevis nuclear pore complex reveal that five Nup358 molecules, together with two copies of interweaved Nup205, Nup93 and Y complexes, constitute the scaffold of each CR subunit.

biophysics↗

bioRxiv2021

Loss of Neutralizing Antibody Response to mRNA Vaccination against SARS-CoV-2 Variants: Differing Kinetics and Strong Boosting by Breakthrough Infection

The waning efficacy of SARS-CoV-2 vaccines combined with the continued emergence of variants resistant to vaccine-induced immunity has reignited debate over the need for booster vaccines. To address this, we examined the neutralizing antibody (nAb) response against four major SARS-CoV-2 variants--D614G, Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2)--in health care workers (HCWs) at pre-vaccination, post-first and post-second mRNA vaccine dose, and six months post-second mRNA vaccine dose. Neutralizing antibody titers against all variants, especially the Delta variant, declined dramatically from four weeks to six months post-second mRNA vaccine dose. Notably, SARS-CoV-2 infection enhanced vaccine durability, and mRNA-1273 vaccinated HCWs also exhibited ~2-fold higher nAb titers than BNT162b2 vaccinated HCWs. Together these results demonstrate possible waning of protection from infection against SARS-CoV-2 Delta variant based on decreased nAb titers, dependent on COVID-19 status and the mRNA vaccine received.

microbiology↗

bioRxiv2021

Cryo-EM structure of the Inner Ring from Xenopus laevis Nuclear Pore Complex

Nuclear pore complex (NPC) mediates nucleocytoplasmic shuttling. Here we present single-particle cryo-electron microscopy (cryo-EM) structure of the inner ring (IR) subunit from Xenopus laevis NPC at an average resolution of 4.4 [A]. The symmetric IR subunit comprises a cytoplasmic half and a nuclear half. A homo-dimer of Nup205 resides at the center of the IR subunit, flanked by two molecules of Nup188. Four molecules of Nup93 each places an extended helix into the axial groove of Nup205 or Nup188, together constituting the central scaffold. The channel nucleoporin heterotrimer (CNT) of Nup54/58/62 is anchored on the central scaffold. Six Nup155 molecules interact with the central scaffold and together with the NDC1-ALADIN hetero-dimers anchor the IR subunit to the nuclear envelope and to outer rings. The scarce inter-subunit contacts may allow sufficient latitude in conformation and diameter of the IR. Our structure of vertebrate IR reveals key insights that are functionally important.

biophysics↗

bioRxiv2021

Lung-selective Cas13d-based nanotherapy inhibits lethal SARS-CoV-2 infection by targeting host protease Ctsl

The COVID-19 pandemic persists as a global health crisis for which curative treatment has been elusive. Development of effective and safe anti-SARS-CoV-2 therapies remains an urgent need. SARS-CoV-2 entry into cells requires specific host proteases including TMPRSS2 and Cathepsin L (Ctsl)1-3, but there has been no reported success in inhibiting host proteases for treatment of SARS-CoV-2 pathogenesis in vivo. Here we have developed a lung Ctsl mRNA-targeted, CRISPR/Cas13d-based nanoparticle therapy to curb fatal SARS-CoV-2 infection in a mouse model. We show that this nanotherapy can decrease lung Ctsl expression in normal mice efficiently, specifically, and safely. Importantly, this lung-selective Ctsl-targeted nanotherapy significantly extended the survival of lethally SARS-CoV-2 infected mice by decreasing lung virus burden, reducing expression of pro-inflammatory cytokines/chemokines, and diminishing the severity of pulmonary interstitial inflammation. Additional in vitro analyses demonstrated that Cas13d-mediated Ctsl knockdown inhibited infection mediated by the spike protein of SARS-CoV-1, SARS-CoV-2, and more importantly, the authentic SARS-CoV-2 B.1.617.2 Delta variant, regardless of TMPRSS2 expression status. Our results demonstrate the efficacy and safety of a lung-selective, Ctsl-targeted nanotherapy against infection by SARS-CoV-2 and likely other emerging coronaviruses, forming a basis for investigation of this approach in clinical trials.

microbiology↗

bioRxiv2021

A missense KCNQ1 Mutation Impairs Insulin Secretion in Neonatal Diabetes

KCNQ1/Kv7 is a voltage-gated K+ channel that regulates heart rhythm, glucose and salt homeostasis. Mutations of KCNQ1 are primarily associated with long-QT syndrome and type 2 diabetes; however, thus far KCNQ1 mutations have not been associated with monogenetic diabetes. Here, we identified a homozygous KCNQ1 missense mutation (R397W) in an individual with permanent neonatal diabetes (PND). To identify the mechanisms that link the mutation to the disease, we introduced the mutation into human embryonic stem cells (hESCs), and used them to derived pancreatic {beta}-like cells (hESC-{beta} cell). In early {beta}-like cells, we observed atypical membrane electrical activity, increased levels of cytoplasmic Ca2+, and a hypersecretion of insulin. Upon extended culture, their insulin secretion decreased and the number of apoptotic cells increased, resulting in a reduction in the numbers of {beta}-like cells. Late-stage {beta}-like cells exhibited a decrease in the expression of metabolic genes, e.g. HNF4, PDX1 and GLUT1, providing a possible mechanism for {beta}-cell dysfunction. Our study identifies KCNQ1 as a novel candidate gene of monogenetic diabetes and shows that KCNQ1 regulates {beta}-cell function and survival.

cell biology↗

bioRxiv2021

NSUN2-mediated m5C methylation of IRF3 mRNA negatively regulates type I interferon responses

5-Methylcytosine (m5C) is a widespread post-transcriptional RNA modification and is reported to be involved in manifold cellular responses and biological processes through regulating RNA metabolism. However, its regulatory role in antiviral innate immunity has not yet been elucidated. Here, we report that NSUN2, a typical m5C methyltransferase, can negatively regulate type I interferon responses during viral infection. NSUN2 specifically mediates m5C methylation of IRF3 mRNA and accelerates its degradation, resulting in low levels of IRF3 and downstream IFN-{beta} production. Knockout or knockdown of NSUN2 could enhance type I interferon responses and downstream ISG expression after viral infection in vitro. And in vivo, the antiviral innate responses is more dramatically enhanced in Nsun2+/- mice than in Nsun2+/+ mice. Four highly m5C methylated cytosines in IRF3 mRNA were identified, and their mutation could enhance the cellular IRF3 mRNA levels. Moreover, infection with Sendai virus (SeV), vesicular stomatitis virus (VSV), herpes simplex virus 1 (HSV-1), Zika virus (ZIKV), or especially SARS-CoV-2 resulted in a reduction in endogenous levels of NSUN2. Together, our findings reveal that NSUN2 serves as a negative regulator of interferon response by accelerating the fast turnover of IRF3 mRNA, while endogenous NSUN2 levels decrease after viral infection to boost antiviral responses for the effective elimination of viruses. Our results suggest a paradigm of innate antiviral immune responses ingeniously involving NSUN2-mediated m5C modification.

immunology↗

bioRxiv2021

Binding patterns of RNA binding proteins to repeat-derived RNA sequences reveal putative functional RNA elements

Recent reports have revealed that repeat-derived sequences embedded in introns or long noncoding RNAs (lncRNAs) are targets of RNA binding proteins (RBPs) and contribute to biological processes such as RNA splicing or transcriptional regulation. These findings suggest that repeat-derived RNAs are important as scaffolds of RBPs and functional elements. However, the overall functional sequences of the repeat-derived RNAs are not fully understood. Here, we show the putative functional repeat-derived RNAs by analyzing the binding patterns of RBPs based on ENCODE eCLIP data. We mapped all eCLIP reads to repeat sequences and observed that 10.75 % and 7.04 % of reads on average were enriched (at least 2-fold over control) in the repeats in K562 and HepG2 cells, respectively. Using these data, we predicted functional RNA elements on the sense and antisense strands of long interspersed element 1 (LINE1) sequences. Furthermore, we found several new sets of RBPs on fragments derived from other transposable element (TE) families. Some of these fragments show specific and stable secondary structures and are found to be inserted into the introns of genes or lncRNAs. These results suggest that the repeat-derived RNA sequences are strong candidates for the functional RNA elements of endogenous noncoding RNAs.

genomics↗

bioRxiv2021

SARS-CoV-2 Spreads through Cell-to-Cell Transmission

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein we provide evidence that SARS-CoV-2 spreads through cell-cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than SARS-CoV spike, which reflects, in part, their differential cell-cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While ACE2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the variants of concern (VOC) B.1.1.7 and B.1.351 have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccine sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis.

microbiology↗

bioRxiv2021

Molecular basis for PICS-mediated piRNA biogenesis and cell division

By incorporating two mutually exclusive factors, PID-1 and TOST-1, C. elegans PICS complex plays important roles in piRNA biogenesis, chromosome segregation and cell division, respectively. We firstly mapped the interaction network between PICS subunits. By solving the several complex structures, including those of TOFU-6/PICS-1, ERH-2/PICS-1, and ERH-2/TOST-1, we uncover the mechanisms underlying the interactions between PICS subunits. Our biochemical experiment demonstrates that PICS exists as an octamer consisting of two copies of each subunits. Combining structural analyses with mutagenesis experiments, we identified residues of PICS subunits that are critical for maintaining intact PICS complex in vitro. Furthermore, using genetics, cell biology and imaging experiments, we found that those mutants impairing the in vitro interaction network within PICS, also lead to abnormal dysfunction PICS in vivo, including mislocalization of PICS, and reduced levels of piRNAs or abnormal chromosome segregation and cell division. Therefore, our work provides structural insights into understanding the PICS-mediated piRNA biogenesis and cell division.

molecular biology↗

bioRxiv2021

A multiple-causal-gene-cluster model underlying GWAS signals of Alzheimer's disease

Genome-wide association studies (GWASs) have identified dozens of genetic susceptibility loci for Alzheimers disease (AD). Nevertheless, the underlying causal variants and biological mechanisms remain elusive. Here, we systematically integrated AD GWAS with comprehensive multi-omics data, and distilled 304 potentially functional variants and 166 causal genes from 49 loci. Intriguingly, we found that most of AD GWAS loci contain multiple functional variants and causal genes. In vitro assays showed that one functional variant regulated multiple genes in the 11p11.2 locus (the CELF1/SPI1 locus) and alteration of these target genes contributed to AD-related molecular processes, supporting the co-existence of multiple functional variants and AD-relevant causal genes within a single locus. We thus proposed a multiple-causal-gene-cluster model that co-dysregulation of a cluster of genes within a single GWAS loci individually or synergistically contribute to AD development. This model provides a novel insight into the biological mechanisms underlying the GWAS loci of complex traits.

genetics↗

bioRxiv2021

Differential gene expression by RNA-Seq in Sigma-2 Receptor/TMEM97 knockout cells reveals its role in complement activation and SARS-CoV-2 viral uptake

Our lab has recently shown that the Sigma-2 Receptor/Transmembrane Protein 97 (sigma-2R/TMEM97) interacts with the low-density lipoprotein receptor (LDLR) and facilitates the enhanced uptake of various ligands including lipoproteins and intrinsically disordered proteins. TMEM97 has been recently been shown to interact with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins, highlighting its potential involvement with viral entry into the cell. We hypothesized that sigma-2R/TMEM97 may play a role in facilitating viral uptake, and with the regulation of inflammatory and thrombotic pathways that are involved with viral infection. In this study, we identified the top differentially expressed genes upon the knockout of sigma-2R/TMEM97, and analyzed the genes involved with the inflammatory and thrombotic cascades, effects that are observed in patients infected with SARS-CoV-2. We found that the ablation of sigma-2R/TMEM97 resulted in an increase in Complement Component 4 Binding Protein (C4BP) proteins, at both the translational and transcriptional levels. We also showed that sigma-2R/TMEM97 interacts with the cellular receptor for SARS-CoV-2, the human angiotensin-converting enzyme 2 (ACE2) receptor, forming a protein complex, and that disruption of this complex results in the inhibition of viral uptake. The results of this study suggest that sigma-2R/TMEM97 may be a novel therapeutic target to inhibit SARS-CoV-2 viral uptake, as well as to decrease inflammatory and thrombotic effects through the modulation of the complement cascade.

cell biology↗

bioRxiv2020

Association analysis of repetitive elements and R-loop formation across species

Genomes are known to have a large number of repetitive elements. Emerging evidence suggests that these non-coding elements may play an important regulatory role. However, few studies have investigated the effect of repetitive elements on R-loop formation. In this study, we found different repetitive elements related to R-loop formation in various species. By controlling length and genomic distributions, we observed that satellites, long interspersed nuclear elements (LINEs), and DNAs were each specifically enriched for R-loops in humans, fruit flies, and Arabidopsis thaliana, respectively. R-loops also tended to arise in regions of low-complexity or simple repeats across species. We also found that the repetitive elements associated with R-loop formation differ according to developmental stage. For instance, LINEs and long terminal repeats (LTRs) are more likely to contain R-loops in embryos (fruit fly) and then turn out to be low-complexity and simple repeats in post-developmental S2 cells. Our results indicate that repetitive elements may have species-specific or development-specific regulatory effects on R-loop formation. This work advances our understanding of repetitive elements and R-loop biology.

genomics↗

bioRxiv2020

Leveraging mRNAs sequences to express SARS-CoV-2 antigens in vivo

SARS-CoV-2 has rapidly become a pandemic worldwide; therefore, an effective vaccine is urgently needed. Recently, messenger RNAs (mRNAs) have emerged as a promising platform for vaccination. Here, we systematically investigated the untranslated regions (UTRs) of mRNAs in order to enhance protein production. Through a comprehensive analysis of endogenous gene expression and de novo design of UTRs, we identified the optimal combination of 5 and 3 UTR, termed as NASAR, which was five to ten-fold more efficient than the tested endogenous UTRs. More importantly, NASAR mRNAs delivered by lipid-derived nanoparticles showed dramatic expression of potential SARS-CoV-2 antigens both in vitro and in vivo. These NASAR mRNAs merit further development as alternative SARS-CoV-2 vaccines.

bioengineering↗

bioRxiv2020

Molecular architecture of the luminal ring of the Xenopus laevis nuclear pore complex

Nuclear pore complex (NPC) mediates the flow of substances between the nucleus and cytoplasm in eukaryotic cells. Here we report the cryo-electron tomography (cryo-ET) structure of the luminal ring (LR) of the NPC from Xenopus laevis oocyte. The observed key structural features of the LR are independently confirmed by single-particle cryo-electron microscopy (cryo-EM) analysis. The LR comprises eight butterfly-shaped subunits, each containing two symmetric wings. Each wing consists of four elongated, tubular protomers. Within the LR subunit, the eight protomers form a Finger domain, which directly contacts the fusion between the inner and outer nuclear membranes, and a Grid domain, which serves as a rigid base for the Finger domain. Two neighbouring LR subunits interact with each other through the lateral edges of their wings to constitute a Bumper domain, which displays two major conformations and appears to cushion neighbouring NPCs. Our study reveals previously unknown features of the LR and potentially explains the elastic property of the NPC.

biophysics↗

bioRxiv2020

Structure of the Cytoplasmic Ring of the Xenopus laevis Nuclear Pore Complex

Nuclear pore complex (NPC) exhibits structural plasticity and has only been characterized at local resolutions of up to 15 [A] for the cytoplasmic ring (CR). Here we present a single-particle cryo-electron microscopy (cryo-EM) structure of the CR from Xenopus laevis NPC at average resolutions of 5.5-7.9 [A], with local resolutions reaching 4.5 [A]. Improved resolutions allow identification and placement of secondary structural elements in the majority of the CR components. The two Y complexes in each CR subunit interact with each other and associate with those from flanking subunits, forming a circular scaffold. Within each CR subunit, the Nup358-containing region wraps around the stems of both Y complexes, likely stabilizing the scaffold. Nup205 connects the short arms of the two Y complexes and associates with the stem of a neighbouring Y complex. The Nup214-containing region uses an extended coiled-coil to link Nup85 of the two Y complexes and protrudes into the axial pore of the NPC. These previously uncharacterized structural features reveal insights into NPC assembly.

biophysics↗