Biology subjects

Liu, P.

Publications and source records attributed to Liu, P..

At least 253 records · Page 14Linked to original sources

bioRxiv2022

Cell culture model system utilizing engineered A549 cells to express high levels of ACE2 and TMPRSS2 for investigating SARS-CoV-2 infection and antivirals

Novel pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose an imminent global threat since its initial outbreak in December 2019. A simple in vitro model system using cell lines highly susceptible to SARS-CoV-2 infection are critical to facilitate the study of the virus cycle and to discover effective antivirals against the virus. Human lung alveolar A549 cells are regarded as a useful and valuable model for respiratory virus infection. However, SARS-CoV-2 uses the ACE2 as receptor for viral entry and the TMPRSS2 to prime the Spike protein, both of which are negligibly expressed in A549 cells. Here, we report the generation of a robust human lung epithelial cell-based model by transducing ACE2 and TMPRSS2 into A549 cells and show that the ACE2 enriched A549ACE2/TMPRSS2 cells (ACE2plus) and its single-cell-derived subclone (ACE2plusC3) are highly susceptible to SARS-CoV-2 infection. These engineered ACE2plus showed higher ACE2 and TMPRSS2 mRNA expression levels than currently used Calu3 and commercial A549ACE2/TMPRSS2 cells. ACE2 and TMPRSS2 proteins were also highly and ubiquitously expressed in ACE2plusC3 cells. Additionally, antiviral drugs like Camostat mesylate, EIDD-1931, and Remdesivir strongly inhibited SARS-CoV-2 replication. Notably, multinucleated syncytia, a clinical feature commonly observed in severe COVID-19 patients was induced in ACE2plusC3 cells either by virus infection or by overexpressing the Spike proteins of different variants of SARS-CoV-2. Syncytial process was effectively blocked by the furin protease inhibitor, Decanoyl-RVKR-CMK. Taken together, we have developed a robust human A549 lung epithelial cell-based model that can be applied to probe SARS-CoV-2 replication and to facilitate the discovery of SARS-CoV-2 inhibitors.

cell biology↗

bioRxiv2021

Structural and functional characterizations of altered infectivity and immune evasion of SARS-CoV-2 Omicron variant

The SARS-CoV-2 Omicron with increased fitness is spreading rapidly worldwide. Analysis of cryo-EM structures of the Spike (S) from Omicron reveals amino acid substitutions forging new interactions that stably maintain an "active" conformation for receptor recognition. The relatively more compact domain organization confers improved stability and enhances attachment but compromises the efficiency of viral fusion step. Alterations in local conformation, charge and hydrophobic microenvironments underpin the modulation of the epitopes such that they are not recognized by most NTD- and RBD-antibodies, facilitating viral immune escape. Apart from already existing mutations, we have identified three new immune escape sites: 1) Q493R, 2) G446S and 3) S371L/S373P/S375F that confers greater resistance to five of the six classes of RBD-antibodies. Structure of the Omicron S bound with human ACE2, together with analysis of sequence conservation in ACE2 binding region of 25 sarbecovirus members as well as heatmaps of the immunogenic sites and their corresponding mutational frequencies sheds light on conserved and structurally restrained regions that can be used for the development of broad-spectrum vaccines and therapeutics.

biophysics↗

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

A subset of Memory B-derived antibody repertoire from 3-dose vaccinees is ultrapotent against diverse and highly transmissible SARS-CoV-2 variants, including Omicron

Omicron, the most heavily mutated SARS-CoV-2 variant so far, is highly resistant to neutralizing antibodies, raising unprecedented concerns about the effectiveness of antibody therapies and vaccines. We examined whether sera from individuals who received two or three doses of inactivated vaccine, could neutralize authentic Omicron. The seroconversion rates of neutralizing antibodies were 3.3% (2/60) and 95% (57/60) for 2- and 3-dose vaccinees, respectively. For three-dose recipients, the geometric mean neutralization antibody titer (GMT) of Omicron was 15, 16.5-fold lower than that of the ancestral virus (254). We isolated 323 human monoclonal antibodies derived from memory B cells in 3-dose vaccinees, half of which recognize the receptor binding domain (RBD) and show that a subset of them (24/163) neutralize all SARS-CoV-2 variants of concern (VOCs), including Omicron, potently. Therapeutic treatments with representative broadly neutralizing mAbs individually or antibody cocktails were highly protective against SARS-CoV-2 Beta infection in mice. Atomic structures of the Omicron S in complex with three types of all five VOC-reactive antibodies defined the binding and neutralizing determinants and revealed a key antibody escape site, G446S, that confers greater resistance to one major class of antibodies bound at the right shoulder of RBD through altering local conformation at the binding interface. Our results rationalize the use of 3-dose immunization regimens and suggest that the fundamental epitopes revealed by these broadly ultrapotent antibodies are a rational target for a universal sarbecovirus vaccine. One sentence summaryA sub-set of antibodies derived from memory B cells of volunteers vaccinated with 3 doses of an inactivated SARS-CoV-2 vaccine work individually as well as synergistically to keep variants, including Omicron, at bay.

microbiology↗

bioRxiv2021

Replication is the key barrier during the dual-host adaption of mosquito-borne flaviviruses

Mosquito-borne flaviviruses (MBFs) adapt to a dual-host transmission circle between mosquitoes and vertebrates. Dual-host affiliated insect-specific flaviviruses (dISFs), discovered from mosquitoes, are phylogenetically similar to MBFs but do not infect vertebrates. Thus, dISF-MBF chimeras could be an ideal model to study the dual-host adaption of MBFs. Using the pseudo-infectious reporter virus particle and reverse genetics systems, we found dISFs entered vertebrate cells as efficiently as the MBFs, but failed to initiate replication. Exchange of the un-translational regions (UTRs) of Donggang virus (DONV), an dISF, with those from Zika virus (ZIKV) rescued DONV replication in vertebrate cells and critical secondary RNA structures were further mapped. Essential UTR-binding host factors were screened for ZIKV replication in vertebrate cells, displaying different binding patterns. Therefore, our data demonstrate a post-entry cross-species transmission mechanism of MBFs, while UTR-host interaction is critical for dual-host adaption. Significance StatementMost viruses have a relatively narrow host range. In contrast, vector-borne flaviviruses, such as dengue virus and Zika virus, maintain their transmission cycle between arthropods and vertebrates, belonging to different phyla. How do these viruses adapt to the distinct cellular environments of two phyla? By comparing the single-host insect specific flavivirus and dual-host Zika virus, we identified three key molecular factors that determine MBF host tropism. This study will greatly increase the understanding of entry, replication, and cross-species evolution of mosquito-borne flaviviruses.

microbiology↗

bioRxiv2021

Adenine Base Editing in vivo with a Single Adeno-Associated Virus Vector

Base editors (BEs) have opened new avenues for the treatment of genetic diseases. However, advances in delivery approaches are needed to enable disease targeting of a broad range of tissues and cell types. Adeno-associated virus (AAV) vectors remain one of the most promising delivery vehicles for gene therapies. Currently, most BE/guide combinations and their promoters exceed the packaging limit (~5 kb) of AAVs. Dual-AAV delivery strategies often require high viral doses that impose safety concerns. In this study, we engineered an adenine base editor using a compact Cas9 from Neisseria meningitidis (Nme2Cas9). Compared to the well-characterized Streptococcus pyogenes Cas9-containing ABEs, Nme2-ABE possesses a distinct PAM (N4CC) and editing window, exhibits fewer off-target effects, and can efficiently install therapeutically relevant mutations in both human and mouse genomes. Importantly, we show that in vivo delivery of Nme2-ABE and its guide RNA by a single-AAV vector can efficiently edit mouse genomic loci and revert the disease mutation and phenotype in an adult mouse model of tyrosinemia. We anticipate that Nme2-ABE, by virtue of its compact size and broad targeting range, will enable a range of therapeutic applications with improved safety and efficacy due in part to packaging in a single-vector system.

molecular biology↗

bioRxiv2021

Developmental cell death of cortical projection neurons is controlled by a Bcl11a/Bcl6-dependent pathway

Developmental neuron death plays a pivotal role in refining organization and wiring during neocortex formation. Aberrant regulation of this process results in neurodevelopmental disorders including impaired learning and memory. Underlying molecular pathways are incompletely determined. Loss of Bcl11a in cortical projection neurons induces pronounced cell death in upper-layer cortical projection neurons during postnatal corticogenesis. We used this genetic model to explore genetic mechanisms by which developmental neuron death is controlled. Unexpectedly, we found Bcl6, previously shown to be involved in transition of cortical neurons from progenitor to postmitotic differentiation state to provide a major check point regulating neuron survival during late cortical development. We show that Bcl11a is a direct transcriptional regulator of Bcl6. Deletion of Bcl6 exerts death of cortical projection neurons. In turn, reintroduction of Bcl6 into Bcl11a mutants prevents induction of cell death in these neurons. Together, our data identify a novel Bcl11a/Bcl6-dependent molecular pathway in regulation of developmental cell death during corticogenesis.

neuroscience↗

bioRxiv2021

Stem cell-derived macrophages as a new platform for studying host-pathogen interactions in livestock

Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. We explored how pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages from livestock. Porcine and bovine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages. Pig PSCdMs were productively infected by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and African Swine Fever Virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, Pig PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied in parental stem cells, or directly by lentiviral vector transduction. PSCs and differentiated derivatives therefore provide a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in livestock.

immunology↗

bioRxiv2021

The gut microbial signature of gestational diabetes mellitus and the association with diet intervention

Gestational diabetes mellitus (GDM) is a high-risk pregnancy complication that is associated with metabolic disorder phenotypes, such as abnormal blood glucose and obesity. The link between microbiota and diet management contributes to metabolic homeostasis in GDM. Therefore, it is crucial to understand the structure of the gut microbiota in GDM and to explore the effect of dietary management on the microbiota structure. In this study, we analyzed the composition of the gut microbiota between 27 GDM and 30 healthy subjects at two time points using Illumina HiSeq 2500 platform. The taxonomy analyses suggested that the overall bacteria clustered by diabetes status, rather than diet intervention. Of particular interest, the phylum Acidobacteria in GDM was significantly increased, and positively correlated with blood glucose levels. Moreover, Partial least-squares discriminant analysis (PLS-DA) revealed that certain genera in the phyla Firmicutes, Bacteroidetes, Proteobacteria, and Lentisphaerae characterized the GDM gut microbiota. Correlation analysis indicated that blood glucose levels and BMI index were correlated with the relative abundance of SCFAS-producing genera. Through the comparison between the GDM and healthy samples with or without diet intervention, we discovered that the role of short-term diet management in GDM processes is associated with the change in the Firmicutes/Bacteroidetes ratio and some specific taxa, rather than an alternative gut microbial pattern. Our study have important implications for understanding the beneficial effects of diet intervention on the specific gut microbiota and thus possibly their metabolism in pregnant women with GDM. ImportanceUnderstanding the composition and dynamics of the gut microbiota in GDM women under diet intervention is important because there may be opportunities for preventive strategies. We examined the relationships between GDM gut microbiota at two times before and after the diet intervention during second trimester of pregnancy and clinical characteristics in cohort of GDM women. We found that short-term diet management in GDM processes is associated with changes in the Firmicutes/Bacteroidetes ratio and some specific taxa rather than an alternative gut microbial pattern. Our study highlights the importance of considering diet intervention as the rescue of microbial dysfunction of GDM disease and can serve as a strategy for early prevention in future study.

microbiology↗

bioRxiv2021

Development of a flexible split prime editor using truncated reverse transcriptase

Prime Editor (PE) has tremendous promise for gene therapy. However, it remains a challenge to deliver PE (>6.3 kb) in vivo. Although PE can be split into two fragments and delivered using dual adeno-associated viruses (AAVs), choice of split sites within Cas9 - which affects editing efficiency - is limited due to the large size of PE. Furthermore, the potential effect of overexpressing RT in mammalian cells is largely unknown. Here, we developed a compact PE with complete deletion of the RNase H domain of reverse transcriptase (RT), which showed comparable editing to full-length PE. Using compact PE, we tested the effect of 4 different Cas9 split sites and found that the Glu 573 split site supports robust editing (up to 93% of full-length PE). The compact PE, but not PE2, abolished its binding to eRF1 and showed minimal effect on stop codon readthrough, which therefore might reduce the effects on protein biosynthesis. This study identifies a safe and efficient compact PE2 that enables flexible split-PE design to facilitate efficient delivery in vivo and advance the utility of prime editing.

genetics↗

bioRxiv2021

A universal microfluidic approach for quantitative study of bacterial biofilms

Bacteria usually live in densely packed communities called biofilms, where interactions between the bacteria give rise to complex properties. Quantitative analysis is indispensable in understanding those properties. However, current biofilm culturing approaches impose various limitations to these types of analysis. Here, we developed a microfluidic approach for quantitative study of biofilms, which is universal and can be used to culture biofilms of various bacterial species. To demonstrate the advantages of this approach, we present two examples, both of which revealed new biological insights. In the first example, we explored the response of Escherichia coli biofilms to exogenous hydrogen peroxide; We found the biofilms gained resistance to H2O2, but their growth was slowed down due to the metabolic cost of maintaining the resistance; However, under oxygen limitation, H2O2 can anti-intuitively boost biofilm growth. In the second example, we explored resource retention by Pseudomonas aeruginosa biofilms; We observed a fluorescent substance within the biofilm and identified it as the siderophore pyoverdine; We further showed that the extracellular matrix component Psl acted as a retention barrier for pyoverdine, minimizing its loss into the environment and therefore potentially promoting sharing of pyoverdine within the biofilm.

microbiology↗

bioRxiv2021

Identification and targeting of a pan-genotypic influenza A virus RNA structure that mediates packaging and disease.

Currently approved anti-influenza drugs target viral proteins, are subtype limited, and are challenged by rising antiviral resistance. To overcome these limitations, we sought to identify a conserved essential RNA secondary structure within the genomic RNA predicted to have greater constraints on mutation in response to therapeutics targeting this structure. Here, we identified and genetically validated an RNA stemloop structure we termed PSL2, which serves as a packaging signal for genome segment PB2 and is highly conserved across influenza A virus (IAV) isolates. RNA structural modeling rationalized known packaging-defective mutations and allowed for predictive mutagenesis tests. Disrupting and compensating mutations of PSL2s structure give striking attenuation and restoration, respectively, of in vitro virus packaging and mortality in mice. Antisense Locked Nucleic Acid oligonucleotides (LNAs) designed against PSL2 dramatically inhibit IAV in vitro against viruses of different strains and subtypes, possess a high barrier to the development of antiviral resistance, and are equally effective against oseltamivir carboxylate-resistant virus. A single dose of LNA administered 3 days after, or 14 days before, a lethal IAV inoculum provides 100% survival. Moreover, such treatment led to the development of strong immunity to rechallenge with a ten-fold lethal inoculum. Together, these results have exciting implications for the development of a versatile novel class of antiviral therapeutics capable of prophylaxis, post-exposure treatment, and "just-in-time" universal vaccination against all IAV strains, including drug-resistant pandemics. One Sentence SummaryTargeting a newly identified conserved RNA structure in the packaging signal region of influenza segment PB2 abrogates virus production in vitro and dramatically attenuates disease in vivo.

microbiology↗

bioRxiv2021

Structures of SARS-CoV-2 B.1.351 neutralizing antibodies provide insights into cocktail design against concerning variants

The spread of the SARS-CoV-2 variants could seriously dampen the global effort to tackle the COVID-19 pandemic. Recently, we investigated the humoral antibody responses of SARS-CoV-2 convalescent patients and vaccinees towards circulating variants, and identified a panel of monoclonal antibodies (mAbs) that could efficiently neutralize the B.1.351 (Beta) variant. Here we investigate how these mAbs target the B.1.351 spike protein using cryo-electron microscopy. In particular, we show that two superpotent mAbs, BD-812 and BD-836, have non-overlapping epitopes on the receptor-binding domain (RBD) of spike. Both block the interaction between RBD and the ACE2 receptor; and importantly, both remain fully efficacious towards the B.1.617.1 (Kappa) and B.1.617.2 (Delta) variants. The BD-812/BD-836 pair could thus serve as an ideal antibody cocktail against the SARS-CoV-2 VOCs.

biochemistry↗

bioRxiv2021

The generation of a Bcl11a lineage tracing mouse model

The transcription factor B-cell lymphoma/leukaemia 11A (BCL11A) has essential functions in physiological processes as well as haematological and solid malignancies, however, its contribution to tissue development and tumour progression is poorly understood. Here we show the generation of a Bcl11a lineage tracing mouse model to allow for in vivo tracking of Bcl11a-expressing cells and their progeny. We validate the model in the mammary gland by using flow cytometry and whole-tissue 3D imaging to locate labelled cells after induction of tracing in early development. We show that Bcl11a is predominantly expressed in long-lived luminal progenitors which populate mammary alveoli upon pregnancy, confirming bona fide labelling of Bcl11a cells. The Bcl11a lineage tracing mouse model therefore provides a powerful resource to study Bcl11a cells in development, homeostasis, and cancer.

developmental biology↗

bioRxiv2021

Human placental villi immune cells help maintain homeostasis in utero

Maintenance of healthy pregnancy is reliant on successful balance between the fetal and maternal immune systems. Although maternal mechanisms responsible have been well studied, those used by the fetal immune system remain poorly understood. Using suspension mass cytometry and various imaging modalities, we report a complex immune system within the mid-gestation (17-23 weeks) human placental villi (PV). Further, we identified immunosuppressive signatures in innate immune cells and antigen presenting cells that potentially maintain immune homeostasis in utero. Consistent with recent reports in other fetal organs, T cells with memory phenotypes were detected within the PV tissue and vasculature. Moreover, we determined PV T cells could be activated to upregulate CD69 and proliferate after T cell receptor (TCR) stimulation and when exposed to maternal uterine antigens. Finally, we report that cytokine production by PV T cells is sensitive to TCR stimulation and varies between mid-gestation, preterm (26-35 weeks) and term deliveries (37-40 weeks). Collectively, we elucidated the complexity and functional maturity of fetal immune cells within the PV and highlighted their immunosuppressive potential.

immunology↗

bioRxiv2021

Comprehensive analysis of RNA-seq and whole genome sequencing data reveals no evidence for SARS-CoV-2 integrating into host genome

SARS-CoV-2, as the causation of severe epidemic of COVID-19, is one kind of positive single-stranded RNA virus with high transmissibility. However, whether or not SARS-CoV-2 can integrate into host genome needs thorough investigation. Here, we performed both RNA sequencing (RNA-seq) and whole genome sequencing on SARS-CoV-2 infected human and monkey cells, and investigated the presence of host-virus chimeric events. Through RNA-seq, we did detect the chimeric host-virus reads in the infected cells. But further analysis using mixed libraries of infected cells and uninfected zebrafish embryos demonstrated that these reads are falsely generated during library construction. In support, whole genome sequencing also didnt identify the existence of chimeric reads in their corresponding regions. Therefore, the evidence for SARS-CoV-2s integration into host genome is lacking. One-Sentence SummarySARS-CoV-2 does not integrate into host genome through whole genome sequencing.

genomics↗

bioRxiv2021

Protein phosphatase 1 regulatory inhibitor subunit 14C promotes triple-negative breast cancer progression via sustaining inactive glycogen synthase kinase 3 beta

The majority of clinical deaths among patients with triple-negative breast cancer (TNBC) are caused by uncontrolled cell proliferation and aggressive metastases, which is regulated by hyperactive glycogen synthase kinase 3 beta (GSK3{beta}); however, the underlying mechanisms remain largely unknown. In the present study, we found that protein phosphatase 1 regulatory inhibitor subunit 14C (PPP1R14C) was specifically upregulated in TNBC, compared with that in normal tissues and non-TNBC. High PPP1R14C expression correlated significantly with shorter 5-year relapse-free survival and overall survival in patients with TNBC. Overexpressing PPP1R14C promoted, while suppression of PPP1R14C decreased the cell proliferation and the aggressive phenotype of TNBC cells, in vitro and in vivo. Importantly, we revealed that PPP1R14C interacted with and inactivated type 1 Ser/Thr protein phosphatase (PP1) to sustain GSK3{beta} phosphorylation at S9, and induced the ubiquitylation and degradation of non-phosphorylated GSK3{beta} (S9A) via recruiting E3 ligase, TRIM25. Furthermore, treating with C2 ceramide (C2), which recovered kinase activity of GSK3{beta}, resulted in tumor growth inhibition in PPP1R14C-overexpressing TNBC cells. Our findings reveal a novel mechanism whereby PPP1R14C sustains inactive GSK3{beta}, which might lead to targeted therapy for TNBC.

cancer biology↗

bioRxiv2021

Increased signal to noise ratios within experimental field trials by regressing spatially distributed soil properties as principal components.

Environmental variability poses a major challenge to any field study. Researchers attempt to mitigate this challenge through replication. Thus, the ability to detect experimental signals is determined by the degree of replication and the amount of environmental variation, noise, within the experimental system. A major source of noise in field studies comes from the natural heterogeneity of soil properties which create micro-treatments throughout the field. To make matters worse, the variation within different soil properties is often non-randomly distributed across a field. We explore this challenge through a sorghum field trial dataset with accompanying plant, microbiome and soil property data. Diverse sorghum genotypes and two watering regimes were applied in a split-plot design. We describe a process of identifying, estimating, and controlling for the effects of spatially distributed soil properties on plant traits and microbial communities using minimal degrees of freedom. Importantly, this process provides a tool with which sources of environmental variation in field data can be identified and removed, improving our ability to resolve effects of interest and to quantify subtle phenotypes. IMPORTANCEData from field experiments are notoriously noisy. Proper field designs with high replication aid in mitigating this challenge, yet true biological correlations are still often masked by environmental variability. This work identifies soil property composition as a spatially distributed source of variance to three types of characteristics: plant phenotype, microbiome composition, and leaf traits. We show that once identified, spatial principal component regression was able to account for these effects so that more precise estimates of experimental factors were obtained. This generalizable method is applicable to diverse field experiments.

systems biology↗