Biology subjects

Tan, W.

Publications and source records attributed to Tan, W..

At least 73 records · Page 4Linked to original sources

bioRxiv2021

A tandem-repeat dimeric RBD protein-based COVID-19 vaccine ZF2001 protects mice and nonhuman primates

A safe, efficacious and deployable vaccine is urgently needed to control COVID-19 pandemic. We report here the preclinical development of a COVID-19 vaccine candidate, ZF2001, which contains tandem-repeat dimeric receptor-binding domain (RBD) protein with alum-based adjuvant. We assessed vaccine immunogenicity and efficacy in both mice and non-human primates (NHPs). ZF2001 induced high levels of RBD-binding and SARS-CoV-2 neutralizing antibody in both mice and NHPs, and also elicited balanced TH1/TH2 cellular responses in NHPs. Two doses of ZF2001 protected Ad-hACE2-transduced mice against SARS-CoV-2 infection, as detected by reduced viral RNA and relieved lung injuries. In NHPs, vaccination of either 25 g or 50 g ZF2001 prevented infection with SARS-CoV-2 in lung, trachea and bronchi, with milder lung lesions. No evidence of disease enhancement is observed in both models. ZF2001 is being evaluated in the ongoing international multi-center Phase 3 trials (NCT04646590) and has been approved for emergency use in Uzbekistan.

immunology↗

bioRxiv2021

Tumor protein D52 (TPD52) affects cancer cell metabolism by negatively regulating AMPK.

The AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis, with deregulation leading to cancer and other diseases. When intracellular ATP levels decrease during energy stress, AMPK is phosphorylated and activated through AMP binding. However, how this pathway is dysregulated in cancer remains unclear. Here, we find that tumor protein D52 (TPD52), initially identified to be overexpressed in many human cancers, forms a stable complex with AMPK in cancer cells. TPD52 directly interacts with AMPK and inhibits AMPK kinase activity in vitro and in vivo. We generated TPD52 transgenic mice, and found that overexpression of TPD52 leads to AMPK inhibition and multiple metabolic defects in mice. Together, our results shed new light on AMPK regulation and on our understanding of the etiology of cancers with TPD52 overexpression.

cell biology↗

bioRxiv2021

Thiophosphopeptides Instantly Targeting Golgi Apparatus and Selectively Killing Cancer Cells

Golgi apparatus is emerging as a key signaling hub of cells, but there are few approaches for targeting Golgi and selectively killing cancer cells. Here we show an unexpected result that changing an oxygen atom of the phosphoester bond in phospho-peptides by a sulfur atom enables instantly targeting Golgi apparatus (GA) and selectively killing cancer cells by enzymatic self-assembly. Specifically, conjugating cysteamine S-phosphate to the C-terminal of a self-assembling peptide generates a thiophospho-peptide. Being a substrate of alkaline phosphatase (ALP), the thiophosphopeptide undergoes rapid ALP-catalyzed dephosphorylation to form a thiopeptide that self-assembles. The thiophosphopeptide enters cells via caveolin-mediated endocytosis and macropinocytosis and instantly accumulates in GA because of dephosphorylation and formation of disulfide bonds in Golgi. Moreover, the thiophosphopeptide, targeting Golgi, potently and selectively inhibits cancer cells (e.g., HeLa) with the IC50 (about 3 M), which is an order of magnitude more potent than that of the parent phosphopeptide. This work, as the first report of thiophospho-peptide for targeting Golgi, illustrates a new molecular platform for designing enzyme responsive molecules that target subcellular compartment for functions.

biochemistry↗

bioRxiv2021

Neutralization of SARS-CoV-2 VOC 501Y.V2 by human antisera elicited by both inactivated BBIBP-CorV and recombinant dimeric RBD ZF2001 vaccines

Recently, the emerged and rapidly spreading SARS-CoV-2 variant of concern (VOC) 501Y.V2 with 10 amino acids in spike protein were found to escape host immunity induced by infection or vaccination. Global concerns have been raised for its potential to affect vaccine efficacy. Here, we evaluated the neutralization activities of two vaccines developed in China against 501Y.V2. One is licensed inactivated vaccine BBIBP-CorV and the other one is recombinant dimeric receptor-binding domain (RBD) vaccine ZF2001. Encouragingly, both vaccines largely preserved neutralizing titres, with slightly reduction, against 501Y.V2 authentic virus compare to their titres against both original SARS-CoV-2 and the currently circulating D614G virus. These data indicated that 501Y.V2 variant will not escape the immunity induced by vaccines targeting whole virus or RBD.

microbiology↗

bioRxiv2020

Ferritin nanoparticle based SARS-CoV-2 RBD vaccine induces persistent antibody response and long-term memory in mice

Since the outbreak of COVID-19, over 200 vaccine candidates have been documented and some of them have advanced to clinical trials with encouraging results. However, the antibody persistence over 3 months post immunization and the long-term memory have been rarely reported. Here, we report that a ferritin nanoparticle based SARS-CoV-2 RBD vaccine induced in mice an efficient antibody response which lasts for at least 7 months post immunization. Significantly higher number of memory B cells were maintained and a significantly higher level of recall response was induced upon antigen challenge. Thus, we believe our current study provide the first information about the long-term antibody persistence and memory response of a COVID-19 vaccine. This information would be also timely useful for the development and evaluation of other vaccines.

immunology↗

bioRxiv2020

Spike Protein of SARS-CoV-2 Activates Macrophages and Contributes to Induction of Acute Lung Inflammations in Mice

BackgroundCoronavirus disease 2019 (COVID-19) patients exhibit multiple organ malfunctions with a primary manifestation of acute and diffuse lung injuries. The Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to mediate viral entry into host cells; however, whether it can be cellularly pathogenic and contribute to pulmonary hyper-inflammations in COVID-19 is not well known. Methods and FindingsIn this study, we developed a Spike protein-pseudotyped (Spp) lentivirus with the proper tropism of SARS-CoV-2 Spike protein on the surface and tracked down the fate of Spp in wild type C57BL/6J mice receiving intravenous injection of the virus. A lentivirus with vesicular stomatitis virus glycoprotein (VSV-G) was used as the control. Two hours post-infection (hpi), Spp showed more than 27-75 times more viral burden in the lungs than other organs; it also exhibited about 3-5 times more viral burden than VSV-G lentivirus in the lungs, liver, kidney and spleen. Acute pneumonia was evident in animals 24 hpi. Spp lentivirus was mainly found in LDLR+ macrophages and pneumocytes in the lungs, but not in MARC1+ macrophages. IL6, IL10, CD80 and PPAR-{gamma} were quickly upregulated in response to infection of Spp lentivirus in the lungs in vivo as well as in macrophage-like RAW264.7 cells in vitro. We further confirmed that forced expression of the Spike protein in RAW264.7 cells could significantly increase the mRNA levels of the same panel of inflammatory factors. ConclusionsOur results demonstrate that the Spike protein of SARS-CoV-2 alone can induce cellular pathology, e.g. activating macrophages and contributing to induction of acute inflammatory responses.

pathology↗

bioRxiv2020

QuantifyPolarity, a new tool-kit for measuring planar polarized protein distributions and cell properties in developing tissues

The coordination of cells or structures within the plane of a tissue is known as planar polarization. It is often governed by the asymmetric distribution of planar polarity proteins within cells. A number of quantitative methods have been developed to provide a readout of planar polarization of protein distributions. However, the quantification of planar polarization can be affected by different cell geometries. Hence, we developed a novel planar polarity quantification method based on Principal Component Analysis (PCA) that is shape insensitive. Here, we compare this method with other state-of-the-art methods on simulated models and biological datasets. We found that the PCA method performs robustly in quantifying planar polarity independently of variation in cell geometry. Furthermore, we designed a user-friendly graphical user interface called QuantifyPolarity, equipped with three different methods for automated quantification of polarity. QuantifyPolarity also provides image analysis tools to quantify cell morphology and packing geometry, allowing the relationship of these characteristics to planar polarization to be investigated. This all-in-one tool enables experimentalists with no prior computational expertise to perform high-throughput cell polarity and shape analysis automatically and efficiently.

developmental biology↗

bioRxiv2020

Molecular hallmarks of heterochronic parabiosis at single cell resolution

Slowing or reversing biological ageing would have major implications for mitigating disease risk and maintaining vitality. While an increasing number of interventions show promise for rejuvenation, the effectiveness on disparate cell types across the body and the molecular pathways susceptible to rejuvenation remain largely unexplored. We performed single-cell RNA-sequencing on 13 organs to reveal cell type specific responses to young or aged blood in heterochronic parabiosis. Adipose mesenchymal stromal cells, hematopoietic stem cells, hepatocytes, and endothelial cells from multiple tissues appear especially responsive. On the pathway level, young blood invokes novel gene sets in addition to reversing established ageing patterns, with the global rescue of genes encoding electron transport chain subunits pinpointing a prominent role of mitochondrial function in parabiosis-mediated rejuvenation. Intriguingly, we observed an almost universal loss of gene expression with age that is largely mimicked by parabiosis: aged blood reduces global gene expression, and young blood restores it. Altogether, these data lay the groundwork for a systemic understanding of the interplay between blood-borne factors and cellular integrity.

genomics↗

bioRxiv2020

Evaluation of azithromycin or hydroxychloroquine plus azithromycin combination therapy on cardiac conduction and function in guinea pigs

BackgroundIn the early stages of the coronavirus disease pandemic, the anti-malarial drug hydroxychloroquine (HCQ) and the antibiotic drug azithromycin (AZM) were widely used as emerging treatments. However, controversial cardiac toxicity results obtained from clinical trials and epidemic studies suggest that the cardiotoxicity of these two drugs should be re-evaluated. In the present study, we aimed to assess the impact of a short course of AZM or HCQ + AZM combination treatment on ECG and cardiac function in healthy guinea pigs. MethodsThirty-two male guinea pigs were randomly divided into four groups: control; AZM; HCQ; and HCQ + AZM groups. At 3, 6, and 9 days after treatment, electrocardiograms (ECGs) and echocardiographic techniques were used to determine important ECG parameters and cardiac functional parameters of the left ventricle (including posterior wall thickness, end systolic/end diastolic volume, ejection fraction, and fractional shortening). ResultsAlthough AZM decreased the heart rates of guinea pigs on day 9 (under anesthetized conditions), HCQ + AZM decreased heart rates on days 3, 6, and 9. The corrected QT intervals of guinea pigs after AZM and HCQ + AZM treatments were significantly increased, compared with CON and HCQ treatment respectively, on days 3, 6, and 9. However, QRS complex durations were not significantly different between the groups. AZM significantly decreased left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) on days 3, 6, and 9, whereas HCQ + AZM only decreased LVEF and LVFS on day 9. Posterior wall thickness and of the left ventricle in the diastolic and systolic states were not significantly different between these groups. In addition, compared with CON, AZM and HCQ decreased the EDV. And, in comparison with HCQ treatment, HCQ + AZM treatment increased ESV on day 9. ConclusionsAccording to our study, AZM significantly prolongs the QT interval and damages cardiac function. Moreover, HCQ + AZM treatment increased the risk of cardiac dysfunction compared with HCQ treatment.

physiology↗

bioRxiv2020

Duple extinguishment of COVID-19: single compound synergized inhibition of SARS-CoV-2 replication and direct suppression of inflammatory cytokines in vitro/vivo

We firstly disclose single compound yields better therapeutic outcome than Remdesivir in COVID-19 hamster treatments as it is armed with direct inhibition viral replication and intrinsic suppression inflammatory cytokines expression. Crystal data reveals that Au (I), released from Au22Glutathione18 (GA), covalently binds thiolate of Cys145 of SARS-CoV-2 Mpro. GA directly decreases SARS-CoV-2 viral replication (EC50: ~0.24 M) and intrinsically down-regulates NF{kappa}B pathway therefore significantly inhibiting expression of inflammatory cytokines in cells. The lung viral load and inflammatory cytokines in GA-treated COVID-19 transgenic mice are found to be significantly lower than that of control mice. When COVID-19 golden hamsters are treated by GA, the lung inflammatory cytokines levels are significantly lower than that of Remdesivir while their lung viral load are decreased to same level. The pathological results show that GA treatment significantly reduce lung inflammatory injuries when compared to that of Remdesivir-treated COVID-19 golden hamsters. One Sentence SummaryWe found that gold cluster molecule directly inhibits SARS-CoV-2 replication and intrinsically suppresses inflammatory cytokines expression in COVID-19 transgenic mouse and golden hamster model, gold cluster providing a better lung injury protection than Remdesivir in COVID-19 golden hamsters via intranasally dropping administration.

microbiology↗

bioRxiv2020

CDK-Mediated Phosphorylation of FANCD2 Promotes Mitotic Fidelity

Fanconi anemia (FA) is a rare genetic disease characterized by increased risk for bone marrow failure and cancer. The FA proteins function together to repair damaged DNA. A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins under conditions of cellular stress and during S-phase of the cell cycle. The regulatory mechanisms governing S-phase monoubiquitination, in particular, are poorly understood. In this study, we have identified a CDK regulatory phospho-site (S592) proximal to the site of FANCD2 monoubiquitination. FANCD2 S592 phosphorylation was detected by LC-MS/MS and by immunoblotting with a S592 phospho-specific antibody. Mutation of S592 leads to abrogated monoubiquitination of FANCD2 during S-phase. Furthermore, FA-D2 (FANCD2-/-) patient cells expressing S592 mutants display reduced proliferation under conditions of replication stress and increased mitotic aberrations, including micronuclei and multinucleated cells. Our findings describe a novel cell cycle-specific regulatory mechanism for the FANCD2 protein that promotes mitotic fidelity. Author SummaryFanconi anemia (FA) is a rare genetic disease characterized by high risk for bone marrow failure and cancer. FA has strong genetic and biochemical links to hereditary breast and ovarian cancer. The FA proteins function to repair DNA damage and to maintain genome stability. The FANCD2 protein functions at a critical stage of the FA pathway and its posttranslational modification is defective in >90% of FA patients. However, the function, and regulation of FANCD2, particularly under unperturbed cellular conditions, remains remarkably poorly characterized. In this study, we describe a novel mechanism of regulation of the FANCD2 protein during S-phase of the cell cycle. CDK-mediated phosphorylation of FANCD2 on S592 promotes the ubiquitination of FANCD2 during S-phase. Disruption of this phospho-regulatory mechanism results in compromised mitotic fidelity and an increase in mitotic chromosome instability.

biochemistry↗

bioRxiv2020

SARS-CoV-2 Quasispecies provides insight into its genetic dynamics during infection

A novel coronavirus disease (COVID-19) caused by SARS-CoV-2 has been pandemic worldwide. The genetic dynamics of quasispecies afford RNA viruses a great fitness on cell tropism and host range. However, no quasispecies data of SARS-CoV-2 have been reported yet. To explore quasispecies haplotypes and its transmission characteristics, we carried out single-molecule real-time (SMRT) sequencing of the full-length of SARS-CoV-2 spike gene within 14 RNA samples from 2 infection clusters, covering first-to third-generation infected-patients. We observed a special quasispecies structure of SARS-CoV-2 (modeled as One-King): one dominant haplotype (mean abundance ~70.15%) followed by numerous minor haplotypes (mean abundance < 0.10%). We not only discovered a novel dominant haplotype of F1040 but also realized that minor quasispecies were also worthy of attention. Notably, some minor haplotypes (like F1040 and currently pandemic one G614) could potentially reveal adaptive and converse into the dominant one. However, minor haplotypes exhibited a high transmission bottleneck (~6% could be stably transmitted), and the new adaptive/dominant haplotypes were likely originated from genetic variations within a host rather than transmission. The evolutionary rate was estimated as 2.68-3.86 x 10-3 per site per year, which was larger than the estimation at consensus genome level. The One-King model and conversion event expanded our understanding of the genetic dynamics of SARS-CoV-2, and explained the incomprehensible phenomenon at the consensus genome level, such as limited cumulative mutations and low evolutionary rate. Moreover, our findings suggested the epidemic strains may be multi-host origin and future traceability would face huge difficulties.

pathology↗

bioRxiv2020

Comparative transcriptome analysis reveals the intensive early-stage responses of host cells to SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a widespread outbreak of highly pathogenic COVID-19. It is therefore important and timely to characterize interactions between the virus and host cell at the molecular level to understand its disease pathogenesis. To gain insights, we performed high-throughput sequencing that generated time-series data simultaneously for bioinformatics analysis of virus genomes and host transcriptomes implicated in SARS-CoV-2 infection. Our analysis results showed that the rapid growth of the virus was accompanied by an early intensive response of host genes. We also systematically compared the molecular footprints of the host cells in response to SARS-CoV-2, SARS-CoV and MERS-CoV. Upon infection, SARS-CoV-2 induced hundreds of up-regulated host genes hallmarked by a significant cytokine production followed by virus-specific host antiviral responses. While the cytokine and antiviral responses triggered by SARS-CoV and MERS-CoV were only observed during the late stage of infection, the host antiviral responses during the SARS-CoV-2 infection were gradually enhanced lagging behind the production of cytokine. The early rapid host responses were potentially attributed to the high efficiency of SARS-CoV-2 entry into host cells, underscored by evidence of a remarkably up-regulated gene expression of TPRMSS2 soon after infection. Taken together, our findings provide novel molecular insights into the mechanisms underlying the infectivity and pathogenicity of SARS-CoV-2.

microbiology↗

bioRxiv2020

In Silico Identification Of Novel PRFA Inhibitors To Fight Listeriosis: A Virtual Screening And Molecular Dynamics Studies

Listeria monocytogenes is considered to be one of the most dangerous foodborne pathogens as it can cause listeriosis, a life-threatening human disease. While the incidence of listeriosis is very low its fatality rate is exceptionally high. Because many multi-resistance Listeria monocytogenes strains that do not respond to conventional antibiotic therapy have been recently described, development of new antimicrobials to fight listeriosis is necessary. The positive regulatory factor A (PrfA) is a key homodimeric transcription factor that modulates the transcription of multiple virulence factors which are ultimately responsible of Listeria monocytogenes pathogenicity. In the present manuscript we describe several new potential PrfA inhibitors that were identified after performing ligand-based virtual screening followed by structure-based virtual screening against the wild-type PrfA structure. The three top-scored drug-likeness inhibitors bound to the wild-type PrfA structure were further assessed by Molecular Dynamics simulations. Besides, the three top-scored inhibitors were docked into a constitutive active apoPrfA mutant structure and the corresponding complexes were also simulated. According to the obtained data, PUBChem 87534955 (P875) and PUBChem 58473762 (P584) may not only bind and inhibit wild-type PrfA but the aforementioned apoPrfA mutant as well. Therefore, P875 and P584 might represent good starting points for the development of a completely new set of antimicrobial agents to treat listeriosis.

bioinformatics↗

bioRxiv2020

Scutellaria baicalensis extract and baicalein inhibit replication of SARS-CoV-2 and its 3C-like protease in vitro

COVID-19 has become a global pandemic that threatens millions of people worldwide. There is an urgent call for developing effective drugs against the virus (SARS-CoV-2) causing this disease. The main protease of SARS-CoV-2, 3C-like protease (3CLpro), is highly conserved across coronaviruses and is essential for the maturation process of viral polyprotein. Scutellariae radix (Huangqin in Chinese), the root of Scutellaria baicalensis has been widely used in traditional Chinese medicine to treat viral infection related symptoms. The extracts of S. baicalensis have exhibited broad spectrum antiviral activities. We studied the anti-SARS-CoV-2 activity of S. baicalensis and its ingredient compounds. We found that the ethanol extract of S. baicalensis inhibits SARS-CoV-2 3CLpro activity in vitro and the replication of SARS-CoV-2 in Vero cells with an EC50 of 0.74 g/ml. Among the major components of S. baicalensis, baicalein strongly inhibits SARS-CoV-2 3CLpro activity with an IC50 of 0.39 M. We further identified four baicalein analogue compounds from other herbs that inhibit SARS-CoV-2 3CLpro activity at microM concentration. Our study demonstrates that the extract of S. baicalensis has effective anti-SARS-CoV-2 activity and baicalein and analogue compounds are strong SARS-CoV-2 3CLpro inhibitors.

pharmacology and toxicology↗

bioRxiv2020

Orthogonal genome-wide screenings in bat cells identify MTHFD1 as a target of broad antiviral therapy

Bats are responsible for the zoonotic transmission of several major viral diseases including the 2003 SARS outbreak and the ongoing COVID-19 pandemic. While bat genomic sequencing studies have revealed characteristic adaptations of the innate immune system, functional genomic studies are urgently needed to provide a foundation for the molecular dissection of the tolerance of viral infections in bats. Here we report the establishment and screening of genome-wide RNAi library and CRISPR library for the model megabat, Pteropus Alecto. We used the complementary RNAi and CRISPR libraries to interrogate Pteropus Alecto cells for infection with two different viruses, mumps virus and Influenza A virus, respectively. Screening results converged on the endocytosis pathway and the protein secretory pathway as required for both viral infections. Additionally, we revealed a general dependence of the C-1-tetrahydrofolate synthase gene, MTHFD1, for viral replication in bat cells as well as in human cells. MTHFD1 inhibitor carolacton potently blocked replication of several RNA viruses including SARS-CoV-2. Our studies provide a resource for systematic inquiry into the genetic underpinnings of bat biology and a potential target for developing broad spectrum antiviral therapy.

microbiology↗

bioRxiv2020

Ageing compromises mouse thymus function and remodels epithelial cell differentiation

Ageing is characterised by cellular senescence, leading to imbalanced tissue maintenance, cell death and compromised organ function. This is first observed in the thymus, the primary lymphoid organ that generates and selects T cells. However, the molecular and cellular mechanisms underpinning these ageing processes remain unclear. Here, we show that mouse ageing leads to less efficient T cell selection, decreased self-antigen representation and increased T cell receptor repertoire diversity. Using a combination of single-cell RNA-seq and lineage-tracing, we find that progenitor cells are the principal targets of ageing, whereas the function of mature thymic epithelial cells is compromised only modestly. Specifically, an early-life precursor cell population, retained in the mouse cortex postnatally, is virtually extinguished at puberty. Concomitantly, a medullary precursor cell quiesces, thereby impairing maintenance of the medullary epithelium. Thus, ageing disrupts thymic progenitor differentiation and impairs the core immunological functions of the thymus.

immunology↗

bioRxiv2020

Mutations, Recombination and Insertion in the Evolution of 2019-nCoV

BackgroundThe 2019 novel coronavirus (2019-nCoV or SARS-CoV-2) has spread more rapidly than any other betacoronavirus including SARS-CoV and MERS-CoV. However, the mechanisms responsible for infection and molecular evolution of this virus remained unclear. MethodsWe collected and analyzed 120 genomic sequences of 2019-nCoV including 11 novel genomes from patients in China. Through comprehensive analysis of the available genome sequences of 2019-nCoV strains, we have tracked multiple inheritable SNPs and determined the evolution of 2019-nCoV relative to other coronaviruses. ResultsSystematic analysis of 120 genomic sequences of 2019-nCoV revealed co-circulation of two genetic subgroups with distinct SNPs markers, which can be used to trace the 2019-nCoV spreading pathways to different regions and countries. Although 2019-nCoV, human and bat SARS-CoV share high homologous in overall genome structures, they evolved into two distinct groups with different receptor entry specificities through potential recombination in the receptor binding regions. In addition, 2019-nCoV has a unique four amino acid insertion between S1 and S2 domains of the spike protein, which created a potential furin or TMPRSS2 cleavage site. ConclusionsOur studies provided comprehensive insights into the evolution and spread of the 2019-nCoV. Our results provided evidence suggesting that 2019-nCoV may increase its infectivity through the receptor binding domain recombination and a cleavage site insertion. One Sentence SummaryNovel 2019-nCoV sequences revealed the evolution and specificity of betacoronavirus with possible mechanisms of enhanced infectivity.

microbiology↗