Biology subjects

Tao, X.

Publications and source records attributed to Tao, X..

31 records · Page 2Linked to original sources

bioRxiv2021

A CRISPR-Cas assisted shotgun mutagenesis method for evolutionary genome engineering

Genome mutagenesis drives the evolution of organisms. Here, we developed a CRISPR-Cas assisted random mutation (CARM) technology for whole genome mutagenesis. The method leverages an entirely random gRNA library and SpCas9-NG to randomly damage genomes in a controllable shotgun-like manner that then triggers diverse and abundant mutations via low-fidelity repair. As a proof-of-principle, CARM was applied to evolve the capacity of Saccharomyces cerevisiae BY4741 to produce {beta}-carotene. After seven rounds of iterative evolution over two months, a {beta}-carotene hyper-producing strain, C7-143, was isolated with a 10.5-fold increase in {beta}-carotene production and 857 diverse genomic mutants that comprised indels, duplications, inversions, and chromosomal rearrangements. Transcriptomic analysis revealed that the expression of 2,541 genes of strain C7-143 were significantly altered, suggesting that the metabolic landscape of the strain was deeply reconstructed. In addition, CARM was applied to evolve the industrially relevant Saccharomyces cerevisiae CEN.PK2-1C, the S-adenosyl-L-methionine production of which was increased to 2.28 times after just one round. Thus, CARM is a user-friendly and practical strategy for genetic remodeling and reverse engineering to investigate complicated organismal metabolism.

evolutionary biology↗

bioRxiv2021

Time Evolution of the Stroke Symptom-Herb Networks Based on TCM Prescriptions

Traditional Chinese Medicine (TCM) has its origins in distant antiquity and has piled up over a long time with much knowledge about diseases, especially stroke. Different combinations of symptom variables yield different combinations of herbs to form a myriad of prescriptions, and they have undergone repeated confirmation and are worthy objects of excavation and analysis. Herbal studies on stroke have developed from genomics to transcriptomics, proteomics and metabolomics, yet more thought is needed on putting time in a wider perspective of the symptoms and herbs for stroke in Chinese medicine. Due to this, we studied the dynamic structure of TCM prescriptions on stroke, using over 270 TCM prescription books containing 2231 prescriptions related to stroke recorded from 341 to 2000 CE. We labeled the functions of the prescriptions with the symptoms based on subject terms in MESH Neurologic Manifestations, then standardized the herbs in the prescriptions, and finally connected the co-occurring symptoms and herbs in the prescriptions to build an undirected complex network. The Stroke Symptom-Herb Networks (SSHNs) can be seen from its network characteristics that it is not a random network and has small-world characteristics. It has experienced two peaks in its nearly 1700-year history, during the Song dynasty, the Ming and Qing dynasties. From 600 years onwards, the core herb cluster has been initially formed. The comparison of sub-network similarities allowed us to identify several symptoms with similar herb clusters. We divided the community based on modularity, and by analyzing the community evolution, we found a more fixed historical evolutionary trend with Hemiplegia and Sialorrhea nodes and their associated symptom and herb nodes. In the time series analysis, we found many symptom-herb combinations that were consistently closely related to historic time depends on assessing the similarity between the symptoms and the herbs. The complex network provides a distinctive perspective for understanding the symptom-herb relationships embedded in TCM prescriptions in remote antiquity.

scientific communication and education↗

bioRxiv2021

Analysis of the Mechanosensor Channel Functionality of TACAN

Mechanosensitive ion channels mediate transmembrane ion currents activated by mechanical forces. A mechanosensitive ion channel called TACAN was recently reported. We began to study TACAN with the intent to understand how it senses mechanical forces and functions as an ion channel. Using cellular patch-recording methods we failed to identify mechanosensitive ion channel activity. Using membrane reconstitution methods we found that TACAN, at high protein concentrations, produces non-selective, heterogeneous conduction levels that are not mechanosensitive and are most consistent with disruptions of the lipid bilayer. We determined the structure of TACAN using single particle cryo-EM and observe that it forms a symmetrical dimeric transmembrane protein. Each protomer contains an intracellular-facing cleft with a coenzyme-A co-factor, confirmed by mass spectrometry. The TACAN protomers are related in 3-dimensional structure to a fatty acid elongase, ELOVL. Whilst its physiological function remains unclear, we anticipate that TACAN is not a mechanosensitive ion channel.

biophysics↗

bioRxiv2021

NMNAT promotes glioma growth through regulating post-translational modifications of p53 to inhibit apoptosis

Gliomas are highly malignant brain tumors with poor prognosis and short survival. NAD+ has been shown to impact multiple processes that are dysregulated in cancer; however, anti-cancer therapies targeting NAD+ synthesis have been unsuccessful due to insufficient mechanistic understanding. Here we adapted a Drosophila glial neoplasia model and discovered the genetic requirement for NAD+ synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) in glioma progression in vivo and in human glioma cells. Overexpressing enzymatically active NMNAT significantly promotes glial neoplasia growth and reduces animal viability. Mechanistic analysis suggests that NMNAT interferes with DNA damage-p53-caspase-3 apoptosis signaling pathway by enhancing NAD+-dependent posttranslational modifications (PTMs) poly(ADP-ribosyl)ation (PARylation) and deacetylation of p53. Interestingly, NMNAT forms a complex with p53 and PTM enzyme PARP1 to facilitate PARylation. As PARylation and deacetylation reduce p53 pro-apoptotic activity, our results demonstrate that NMNAT promotes glioma progression through regulating p53 post-translational modifications. Our findings reveal a novel tumorigenic mechanism involving protein complex formation of p53 with NAD+ synthetic enzyme NMNAT and NAD+-dependent PTM enzymes that regulates glioma growth.

cancer biology↗

bioRxiv2021

Development of a mini-replicon-based reverse-genetics system for rice stripe tenuivirus

Negative-stranded RNA (NSR) viruses include both animal- and plant-infecting viruses that often cause serious diseases in human and livestock, and in agronomic crops. Rice stripe tenuivirus (RSV), a plant NSR virus with four negative-stranded/ambisense RNA segments, is one of the most destructive rice pathogens in many Asian countries. Due to the lack of a reliable reverse-genetics technology, molecular studies of RSV gene functions and its interaction with host plants are severely hampered. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV gene functional analysis in Nicotiana benthamiana. We first developed a mini-replicon system expressing RSV genomic RNA3 eGFP reporter (MR3(-)eGFP), a nucleocapsid (NP), and a codon usage optimized RNA-dependent RNA polymerase (RdRpopt), respectively. Using this mini-replicon system we determined that RSV NP and RdRpopt are indispensable for the eGFP expression from MR3(-)eGFP. The expression of eGFP from MR3(-)eGFP can be significantly enhanced in the presence of NSs and P19-HcPro-{gamma}b. In addition, NSvc4, the movement protein of RSV, facilitated eGFP trafficking between cells. We also developed an antigenomic RNA3-based replicon in N. benthamiana. However, we found that the RSV NS3 coding sequence acts as a cis-element to regulate viral RNA expression. Finally, we made mini-replicons representing all four RSV genomic RNAs. This is the first mini-replicon-based reverse-genetics system for monocot-infecting tenuivirus. We believe that this mini-replicon system described here will allow the studies of RSV replication, transcription, cell-to-cell movement and host machinery underpinning RSV infection in plants. IMPORTANCEPlant-infecting segmented negative-stranded RNA (NSR) viruses are grouped into 3 genera: Orthotospovirus, Tenuivirus and Emaravirus. The reverse-genetics systems have been established for members in the genera Orthotospovirus and Emaravirus, respectively. However, there is still no reverse-genetics system available for Tenuivirus. Rice stripe virus (RSV) is a monocot-infecting tenuivirus with four negative-stranded/ambisense RNA segments. It is one of the most destructive rice pathogens and causes significant damages to rice industry in Asian countries. Due to the lack of a reliable reverse-genetics system, molecular characterizations of RSV gene functions and the host machinery underpinning RSV infection in plants are extremely difficult. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV in Nicotiana benthamiana. This is the first mini-replicon-based reverse-genetics system for tenuivirus. We consider that this system will provide researchers a new working platform to elucidate the molecular mechanisms dictating segmented tenuivirus infections in plant.

pathology↗

bioRxiv2021

Correlation between structure and function in phosphatidylinositol lipid-dependent Kir2.2 gating

Inward rectifier K+(Kir) channels regulate cell membrane potential. Different Kir channels respond to unique ligands, but all are regulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Using planar lipid bilayers we show that Kir2.2 exhibits bursts of openings separated by long quiescent inter-burst periods. Increasing PI(4,5)P2 concentration shortens the Kir2.2 inter-burst duration and lengthens the burst duration without affecting dwell times within a burst. From this, we propose that burst and inter-burst durations correspond to the CTD-docked and CTD-undocked conformations observed in the presence and absence of PI(4,5)P2 in atomic structures. We also studied the effect of different phosphatidylinositol lipids on Kir2.2 activation and conclude that the 5 phosphate is essential to Kir2.2 pore opening. Other phosphatidylinositol lipids can compete with PI(4,5)P2 but cannot activate Kir2.2 without the 5 phosphate. PI(4)P, which is directly interconvertible to and from PI(4,5)P2, might thus be a regulator of Kir channels in the plasma membrane.

biophysics↗

bioRxiv2021

STING suppresses cancer pain via immune and neuronal modulation

Agonists of the innate immune regulator stimulator of interferon genes (STING) have shown great efficacy in promoting antitumor immunity in preclinical models, leading to their exploration in cancer immunotherapy trials. Patients with advanced stage cancers frequently suffer from severe pain as a result of bone metastasis and bone destruction, for which there is no efficacious treatment. Here, using multiple mouse models of metastatic bone cancer, we report that STING agonists confer remarkable protection against cancer pain, bone destruction, and local tumor burden. Repeated systemic administration of STING agonists robustly attenuated bone cancer-induced pain symptoms and improved locomotor function. Interestingly, STING agonists provided acute pain relief through direct neuronal modulation, as ex vivo incubation of STING agonists reduced excitability of pain-sensing nociceptive neurons from tumor-bearing mice. In addition, STING agonists protected local bone destruction and reduced local tumor burden through modulation of osteoclast and immune cell function in the tumor microenvironment, providing long-term cancer pain relief. Finally, these in vivo effects were dependent on host-intrinsic STING and Ifnar1. Overall, STING activation provides unique advantages in controlling metastatic bone cancer pain through distinct and synergistic actions on nociceptors, immune cells, and osteoclasts.

cancer biology↗

bioRxiv2020

Cytoplasmic and nuclear Sw-5b synergistically induce plant immune responses to inhibit different viral infection steps

O_LIPlant intracellular nucleotide binding-leucine-rich repeat (NLR) receptors play critical roles in mediating host immunity to pathogen attack. We use tomato Sw-5b::tospovirus as a model system to study the specific role of the compartmentalized plant NLR in dictating host defense against virus at different infection steps. C_LIO_LIWe demonstrated here that tomato NLR Sw-5b translocates to cytoplasm and nucleus, respectively, to play different roles in inducing host resistances against Tomato spotted wilt tospovirus (TSWV) infection. The cytoplasmic Sw-5b functions to induce a strong cell death response to inhibit TSWV replication. This host response is, however, insufficient to block viral intercellular and long-distance movement. The nucleus-localized Sw-5b triggers a host defense that weakly inhibits viral replication but strongly impedes virus intercellular and systemic movement. Furthermore, the cytoplasmic and nuclear Sw-5b act synergistically to dictate full host defense to TSWV infection. C_LIO_LIWe further demonstrated that the extended N-terminal Solanaceae domain (SD) of Sw-5b plays critical roles in cytoplasm/nucleus partitioning. Sw-5b nucleotide-binding leucine-rich repeat (NB-LRR) controls its cytoplasm localization. Strikingly, the SD but not coil-coil (CC) domain is crucial for Sw-5b receptor to translocate from cytoplasm to nucleus to trigger the immunity. The SD was found to interact with importins. Silencing both importin and {beta} expression disrupted Sw-5b nucleus translocation and host immunity against TSWV systemic infection. C_LIO_LICollectively, our findings suggest that Sw-5b bifurcates disease resistances by cytoplasm/nucleus partitioning to block different infection steps of TSWV. The findings also identified a new regulatory role of extra domain of a plant NLR in mediating host innate immunity. C_LI

plant biology↗

bioRxiv2020

Distinct mechanisms for TMPRSS2 expression explain organ-specific inhibition of SARS-CoV-2 infection by enzalutamide

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly become a global public health threat due to the lack of effective drugs or vaccines against SARS-CoV-2. The efficacy of several repurposed drugs has been evaluated in clinical trials. Among these drugs, a relatively new antiandrogen agent, enzalutamide, was proposed because it reduces the expression of transmembrane serine protease 2 (TMPRSS2), a key component mediating SARS-CoV-2-driven entry into host cells, in prostate cancer cells. However, definitive evidence for the therapeutic efficacy of enzalutamide in COVID-19 is lacking. Here, we evaluated the antiviral efficacy of enzalutamide in prostate cancer cells, lung cancer cells, human lung organoids and SARS-CoV-2-infected Ad-ACE2-transduced Tmprss2 knockout (Tmprss2-KO) and wild-type (WT) mice. TMPRSS2 knockout significantly inhibited SARS-CoV-2 infection in vivo. Enzalutamide effectively inhibited SARS-CoV-2 infection in human prostate cancer cells (LNCaP) but not in human lung cancer cells or patient-derived lung organoids. Although Tmprss2 knockout effectively blocked SARS-CoV-2 infection in ACE2-transduced mice, enzalutamide showed no antiviral activity due to the AR independence of TMPRSS2 expression in mouse and human lung epithelial cells. Moreover, we observed distinct AR binding patterns between prostate cells and lung cells and a lack of direct binding of AR to TMPRSS2 in human lung cells. Thus, our findings do not support the postulated protective role of enzalutamide in treating COVID-19.

microbiology↗

bioRxiv2020

CGMTSA: An R package for continuous glucose monitoring time series data analysis

The R package CGMTSA was developed to facilitate investigations that examine the continuous glucose monitoring (CGM) data as a time series. Accordingly, novel time series functions were introduced to: 1) enable more accurate missing data imputation and outlier identification; 2) calculate recommended CGM metrics as well as key time series parameters; 3) plot interactive and 3D graphs that allow direct visualizations of temporal CGM data and time series model optimization. The software was designed to accommodate all popular CGM devices and support all common data processing steps.Competing Interest StatementThe authors have declared no competing interest.View Full Text

bioinformatics↗

bioRxiv2020

Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1) Formulated with Alum Induces Protective Immunity and Reduces Immune Enhancement

We developed a severe acute respiratory syndrome (SARS) subunit recombinant protein vaccine candidate based on a high-yielding, yeast-engineered, receptor-binding domain (RBD219-N1) of the SARS beta-coronavirus (SARS-CoV) spike (S) protein. When formulated with Alhydrogel®, RBD219-N1 induced high-level neutralizing antibodies against both pseudotyped virus and a clinical (mouse-adapted) isolate of SARS-CoV. Here, we report that mice immunized with RBD219-N1/Alhydrogel® were fully protected from lethal SARS-CoV challenge (0% mortality), compared to ∼ 30% mortality in mice when immunized with the SARS S protein formulated with Alhydrogel®, and 100% mortality in negative controls. An RBD219-N1 formulation Alhydrogel® was also superior to the S protein, unadjuvanted RBD, and AddaVax (MF59-like adjuvant)-formulated RBD in inducing specific antibodies and preventing cellular infiltrates in the lungs upon SARS-CoV challenge. Specifically, a formulation with a 1:25 ratio of RBD219-N1 to Alhydrogel® provided high neutralizing antibody titers, 100% protection with non-detectable viral loads with minimal or no eosinophilic pulmonary infiltrates. As a result, this vaccine formulation is under consideration for further development against SARS-CoV and potentially other emerging and re-emerging beta-CoVs such as SARS-CoV-2.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗

bioRxiv2019

Rescue of Tomato spotted wilt tospovirus entirely from cDNA clones, establishment of the first reverse genetics system for a segmented (-)RNA plant virus

The group of negative strand RNA viruses (NSVs) includes not only dangerous pathogens of medical importance but also serious plant pathogens of agronomical importance. Tomato spotted wilt tospovirus (TSWV) is one of those plant NSVs that cause severe diseases on agronomic crops and pose major threats to global food security. Its negative-strand segmented RNA genome has, however, always posed a major obstacle to molecular genetic manipulation. In this study, we report the complete recovery of infectious TSWV entirely from cDNA clones, the first reverse genetics (RG) system for a segmented plant NSV. First, a replication and transcription competent mini-genome replication system was established based on 35S-driven constructs of the S(-)-genomic (g) or S(+)-antigenomic (ag) RNA template, flanked by a 5 Hammerhead and 3 Ribozyme sequence of Hepatitis Delta virus, a nucleocapsid (N) protein gene and codon-optimized viral RNA dependent RNA polymerase (RdRp) gene. Next, a movement competent mini-genome replication system was developed based on M(-)-gRNA, which was able to complement cell-to-cell and systemic movement of reconstituted ribonucleoprotein complexes (RNPs) of S RNA replicon. After further optimization, infectious TSWV and derivatives carrying eGFP reporters were successfully rescued in planta via simultaneous expression of full-length cDNA constructs coding for S(+)-agRNA, M(-)-gRNA and L(+)-agRNA. Viral rescue occurred in the additional presence of various viral suppressors of RNAi, but TSWV NSs interfered with the rescue of genomic RNA. The establishment of a RG system for TSWV now allows detailed molecular genetic analysis of all aspects of tospovirus life cycle and their pathogenicity.\n\nSignificanceFor many different animal-infecting segmented negative-strand viruses (NSVs), a reverse genetics system has been established that allows the generation of mutant viruses to study disease pathology and the role of cis- and trans-acting elements in the virus life cycle. In contrast to the relative ease to establish RG systems for animal-infecting NSVs, establishment of such system for the plant-infecting NSVs with a segmented RNA genome so far has not been successful. Here we report the first reverse genetics system for a segmented plant NSV, the Tomato spotted wilt tospovirus, a virus with a tripartite RNA genome. The establishment of this RG system now provides us with a new and powerful platform to study their disease pathology during a natural infection.

microbiology↗

bioRxiv2018

Tenuivirus uses a molecular bridge strategy to overcome insect midgut barriers for virus persistent transmission

Many persistent transmitted plant viruses, including Rice stripe tenuivirus (RSV), cause serious damages to crop productions in China and worldwide. Although many reports have indicated that successful insect-mediated virus transmission depends on proper virus-insect vector interactions, the mechanism(s) controlling interactions between viruses and insect vectors for virus persistent transmission remained poorly understood. In this study, we used RSV and its small brown planthopper (SBPH) vector as a working model to elucidate the molecular mechanism controlling RSV virion entrance into SBPH midgut for persistent transmission. We have now demonstrated that this non-enveloped Tenuivirus uses its non-structural glycoprotein NSvc2 as a helper component to bridge the specific interaction between virion and SBPH midgut cells, leading to overcome SBPH midgut barriers for virus persistent transmission. In the absence of this glycoprotein, purified RSV virion is not capable of entering SBPH midgut cells. In RSV-infected cells, glycoprotein NSvc2 is processed into two mature proteins: an amino-terminal protein NSvc2-N and a carboxyl-terminal protein NSvc2-C. We determined that NSvc2-N interacted with RSV virion and bound directly to midgut lumen surface via its N-glycosylation sites. Upon recognition by midgut cells, the midgut cells underwent endocytosis followed by compartmentalizing RSV virion and NSvc2 into early and then late endosomes. The acidic condition inside the late endosome triggered conformation change of NSvc2-C and caused cell membrane fusion via its highly conserved fusion loop motifs, leading to the release of RSV virion from endosome into cytosol. In summary, our results showed for the first time that a rice Tenuivirus uses a molecular bridge strategy to ensure proper interactions between virus and insect midgut for successful persistent transmission.\n\nAuthor summaryOver 75% of the known plant viruses are insect transmitted. Understanding how plant viruses interacted with their insect vectors during virus transmission is one of the key steps to manage virus diseases worldwide. Both the direct and indirect virus-insect vector interaction models have been proposed for virus non-persistent and semi-persistent transmission. However, the indirect virus-vector interaction mechanism during virus persistent transmission has not been reported previously. In this study, we developed a new reverse genetics technology and demonstrated that the circulative and propagative transmitted Rice stripe tenuivirus utilizes a glycoprotein NSvc2 as a helper component to ensure a specific interaction between Tenuivirus virion and midgut cells of small brown planthopper (SBPH), leading to conquering the midgut barrier of SBPH. This is the first report of a helper component mediated-molecular bridge mechanism for virus persistent transmission. These new findings and our new model on persistent transmission expand our understanding of molecular mechanism(s) controlling virus-insect vector interactions during virus transmission in nature.

pathology↗