Biology subjects

Liu, L.

Publications and source records attributed to Liu, L..

At least 775 records · Page 43Linked to original sources

bioRxiv2020

Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes

Biotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from [~]0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike-ectodomain probes.

immunology↗

bioRxiv2020

Absence of heme at the catalytic site of heme oxygenase-2 triggers its lysosomal degradation

Heme oxygenase-2 (HO2) and -1 (HO1) catalyze heme degradation to biliverdin, CO, and iron, forming an essential link in the heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining heme homeostasis. While transcriptional control of HO1 expression has been well-studied, how the cellular levels and activity of HO2 are regulated remains unclear. Here, the mechanism of post-translational regulation of cellular HO2 level by heme is elucidated. Under heme deficient conditions, HO2 is destabilized and targeted for degradation. In HO2, three heme binding sites are potential targets of heme-dependent regulation: one at its catalytic site; the others at its two heme regulatory motifs (HRMs). We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of heme binding to the HRMs. Rather, under heme deficiency, loss of heme binding to the catalytic site destabilizes HO2. Consistently, a HO2 catalytic site variant that is unable to bind heme exhibits a constant low protein level and an enhanced protein degradation rate compared to the wild-type HO2. However, cellular heme overload does not affect HO2 stability. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel aspect of HO2 regulation and deepen our understanding of HO2s role in maintaining heme homeostasis, paving the way for future investigation into HO2s pathophysiological role in heme deficiency response.

biochemistry↗

bioRxiv2020

Ten Genetic Loci Identified for Milk Yield, Fat, and Protein in Holstein Cattle

High-yield and high-quality of milk are the primary goals of dairy production. Understanding the genetic architecture underlying these milk production traits is beneficial so that genetic variants can be targeted toward the genetic improvement. In this study, we measured five milk production traits in Holstein cattle population from China. These traits included milk yield, protein yield, fat yields; fat percentage and protein percentages. We used the estimated breeding values as dependent variables to conduct the genome-wide association studies (GWAS). Breeding values were estimated through pedigree relationships by using a mixed linear model for individuals with and without phenotypic data. Genotyping was carried out on the individuals with phenotypes by using the Illumina BovineSNP150 BeadChip. The association analyses were conducted by using the Fixed and random model Circulating Probability Unification (FarmCPU) method. A total of ten SNPs was detected above the genome-wide significant threshold, including six located in previously reported QTL regions. We found eight candidate genes within distances of 120 kb upstream or downstream to the associated SNPs. The most significant SNP is on DGAT1 gene affecting milk fat and protein percentage. These genetic variants and candidate genes would be valuable resources to enhance dairy cattle breeding.

genomics↗

bioRxiv2020

SARS-CoV-2 Neutralizing Antibody Responses Are More Robust in Patients with Severe Disease

We studied plasma antibody responses of 35 patients about 1 month after SARS-CoV-2 infection. Titers of antibodies binding to the viral nucleocapsid and spike proteins were significantly higher in patients with severe disease. Likewise, mean antibody neutralization titers against SARS-CoV-2 pseudovirus and live virus were higher in the sicker patients, by ~5-fold and ~7-fold, respectively. These findings have important implications for those pursuing plasma therapy, isolation of neutralizing monoclonal antibodies, and determinants of immunity.

microbiology↗

bioRxiv2020

Impact of the radiated brain microenvironment on a panel of human patient-derived xenografts.

ObjectiveRadiotherapy, combined with surgical resection and chemotherapy, remains a first-line treatment for infiltrative gliomas. However, these tumor are not surgically curable, and often recur, even within the prior radiation field, and may demonstrate a more aggressive phenotype. We recently demonstrated that the radiated brain tumor microenvironment promotes tumor aggressiveness in an orthotopic patient-derived xenograft (PDX) model of glioblastoma (Mayo GBM 143). Importantly, high grade gliomas display diverse molecular phenotypes, and whether this genetic variability leads to divergent behaviour in the radiated tumor microenvironment is unknown. Herein, we characterize the effects of the irradiated brain microenvinroment on nine additional unique GBM cell lines to better understand the nuances of how tumor molecular phenotypes influence cellular dynamics. MethodsFemale athymic nude mice were randomly divided into cranial radiation (15 Gy) and non-radiated groups. Mice then underwent intracranial implantation with one of the selected PDX GBM cell lines (GBM 6, 10, 12, 39, 46, 76, 123, 164, 196; total n=8-15, per group, per line). GBM 6 cells were additionally implanted 6 months after completion of fractionated radiation (4Gy x 10 fractions or 2Gy x 30 fractions) vs sham radiation. Kaplan-Meyer (K-M) and log-rank tests were performed to compare the survival between irradiated and non-irradiated groups. ResultOf nine previously untested human GBM lines, we found that five demonstrated shorter survival in the pre-radiated brain (GBM 6, 46, 76, 164, 196); similar to previous observations with GBM 143. GBM 6 was also evaluated 6 months after fractionated radiation yielding similar results. However, two lines yielded prolonged survival in the pre-radiated brain (GBM 10, 12); GBM12 and 10 demonstrated the fastest baseline growth in the non-radiated brain; GBM 39, 123 whose rate of growth was not impacted by the radiated brain, demonstrated a an intermediate baseline growth rate between that of those positively and negatively impacted by the radiated brain microenvironment. No other clinical or molecular phenotype was found to consistently correlate with response to the radiated microenvironment. ConclusionAmong a total of 10 total human GBM lines evaluated to date, 60% induce faster mortality in a radiated microenvironment, and 20% induce slower mortality. These results highlight the likely critical impact of the irradiated microenvironment on tumor behaviour, yet illustrate that different tumors may exhibit opposing responses. Although further evaluation will be needed to understand mechanisms of divergent behavior, our data suggest the increased rate of growth in the radiated microenvironment may not apply to the fastest-growing tumor lines, which could instead demonstrate a paradoxical response.

cancer biology↗

bioRxiv2020

TreeMap: A Structured Approach to Fine Mapping of eQTL Variants

MotivationExpression quantitative trait loci (eQTL) harbor genetic variants modulating gene transcription. Fine mapping of regulatory variants at these loci is a daunting task due to the juxtaposition of causal and linked variants at a locus as well as the likelihood of interactions among multiple variants. This problem is exacerbated in genes with multiple cis-acting eQTL, where superimposed effects of adjacent loci further distort the association signals. ResultsWe developed a novel algorithm, TreeMap, that identifies putative causal variants in cis-eQTL accounting for multisite effects and genetic linkage at a locus. Guided by the hierarchical structure of linkage disequilibrium, TreeMap performs an organized search for individual and multiple causal variants. Via extensive simulations, we show that TreeMap detects co-regulating variants more accurately than current methods. Furthermore, its high computational efficiency enables genome-wide analysis of long-range eQTL. We applied TreeMap to GTEx data of brain hippocampus samples and transverse colon samples to search for eQTL in gene bodies and in 4 Mbps gene-flanking regions, discovering numerous distal eQTL. Furthermore, we found concordant distal eQTL that were present in both brain and colon samples, implying long-range regulation of gene expression. AvailabilityTreeMap is available as an R package enabled for parallel processing at https://github.com/liliulab/treemap.

bioinformatics↗

bioRxiv2020

NF45/NF90-mediated rDNA transcription provides a novel target for immunosuppressant development

Herein, we demonstrate that NFAT, a key regulator of the immune response, translocates from cytoplasm to nucleolus and interacts with NF45/NF90 complex to collaboratively promote rDNA transcription via triggering the directly binding of NF45/NF90 to the ARRE2-like sequences in rDNA promoter upon T cell activation in vitro. The elevated pre-rRNA level of T cells is also observed in both mouse heart or skin transplantation models, and in kidney transplanted patients. Importantly, T cell activation can be significantly suppressed by inhibiting NF45/NF90-dependent rDNA transcription. Amazingly, CX5461, a rDNA transcription specific inhibitor, outperformed FK506, the most commonly used immunosuppressant, both in terms of potency and off-target activity (i.e. toxicity), as demonstrated by a series of skin and heart allograft models. Collectively, this reveals NF45/NF90-mediated rDNA transcription as a novel signaling pathway essential for T cell activation and as a new target for the development of safe and effective immunosuppressants.

molecular biology↗

bioRxiv2020

The Perfect Storm: Gene Tree Estimation Error, Incomplete Lineage Sorting, and Ancient Gene Flow Explain the Most Recalcitrant Ancient Angiosperm Clade, Malpighiales

The genomic revolution offers renewed hope of resolving rapid radiations in the Tree of Life. The development of the multispecies coalescent (MSC) model and improved gene tree estimation methods can better accommodate gene tree heterogeneity caused by incomplete lineage sorting (ILS) and gene tree estimation error stemming from the short internal branches. However, the relative influence of these factors in species tree inference is not well understood. Using anchored hybrid enrichment, we generated a data set including 423 single-copy loci from 64 taxa representing 39 families to infer the species tree of the flowering plant order Malpighiales. This order alone includes nine of the top ten most unstable nodes in angiosperms, and the recalcitrant relationships along the backbone of the order have been hypothesized to arise from the rapid radiation during the Cretaceous. Here, we show that coalescent-based methods do not resolve the backbone of Malpighiales and concatenation methods yield inconsistent estimations, providing evidence that gene tree heterogeneity is high in this clade. Despite high levels of ILS and gene tree estimation error, our simulations demonstrate that these two factors alone are insufficient to explain the lack of resolution in this order. To explore this further, we examined triplet frequencies among empirical gene trees and discovered some of them deviated significantly from those attributed to ILS and estimation error, suggesting gene flow as an additional and previously unappreciated phenomenon promoting gene tree variation in Malpighiales. Finally, we applied a novel method to quantify the relative contribution of these three primary sources of gene tree heterogeneity and demonstrated that ILS, gene tree estimation error, and gene flow contributed to 15%, 52%, and 32% of the variation, respectively. Together, our results suggest that a perfect storm of factors likely influence this lack of resolution, and further indicate that recalcitrant phylogenetic relationships like the backbone of Malpighiales may be better represented as phylogenetic networks. Thus, reducing such groups solely to existing models that adhere strictly to bifurcating trees greatly oversimplifies reality, and obscures our ability to more clearly discern the process of evolution.

evolutionary biology↗

bioRxiv2020

Altered N-glycan composition impacts flagella mediated adhesion in Chlamydomonas reinhardtii

For the unicellular alga Chlamydomonas reinhardtii, the presence of N-glycosylated proteins on the surface of two flagella is crucial for both cell-cell interaction during mating and flagellar surface adhesion. It is unknown whether the composition of N-glycans attached to respective proteins is important for these processes. To this end, we examined several C. reinhardtii insertional mutants and a CRIPSR/Cas9 knockout mutant of xylosyltransferase 1A, all possessing altered N-glycan compositions. Taking advantage of atomic force microscopy and micropipette force measurements, our data revealed that reduction in N-glycan complexity impedes the adhesion force required for binding the flagella to surfaces. In addition, polystyrene bead binding and transport is impaired. Notably, assembly, Intraflagellar Transport and FMG-1B transport into flagella are not affected by altered N-glycosylation. Thus, we conclude that proper N-glycosylation of flagellar proteins is crucial for adhering C. reinhardtii cells onto surfaces, indicating that N-glycans mediate surface adhesion via direct surface contact.

plant biology↗

bioRxiv2020

Massively parallel quantification of CRISPR editing in cells by TRAP-seq enables better design of Cas9, ABE, CBE gRNAs of high efficiency and accuracy

The CRISPR RNA-guided endonucleases Cas9, and Cas9-derived adenine/cytosine base editors (ABE/CBE), have been used in both research and therapeutic applications. However, broader use of this gene editing toolbox is hampered by the great variability of efficiency among different target sites. Here we present TRAP-seq, a versatile and scalable approach in which the CRISPR gRNA expression cassette and the corresponding surrogate site are captured by Targeted Reporter Anchored Positional Sequencing in cells. TRAP-seq can faithfully recapitulate the CRISPR gene editing outcomes introduced to the corresponding endogenous genome site and most importantly enables massively parallel quantification of CRISPR gene editing in cells. We demonstrate the utility of this technology for high-throughput quantification of SpCas9 editing efficiency and indel outcomes for 12,000 gRNAs in human embryonic kidney cells. Using this approach, we also showed that TRAP-seq enables high throughput quantification of both ABE and CBE efficiency at 12,000 sites in cells. This rich amount of ABE/CBE outcome data enable us to reveal several novel nucleotide features (e.g. preference of flanking bases, nucleotide motifs, STOP recoding types) affecting base editing efficiency, as well as designing improved machine learning-based prediction tools for designing SpCas9, ABE and CBE gRNAs of high efficiency and accuracy (>70%). We have integrated all the 12,000 CRISPR gene editing outcomes for SpCas9, ABE and CBE into a CRISPR-centered portal: The Human CRISPR Atlas. This study extends our knowledge on CRISPR gene and base editing, and will facilitate the application and development of CRISPR in both research and therapy.

systems biology↗

bioRxiv2020

Seesaw Conformations of Npl4 in the Human p97 Complex and the Inhibitory Mechanism of a Disulfiram Derivative

p97, also known as valosin-containing protein (VCP) or Cdc48, plays a central role in cellular protein homeostasis1. Human p97 mutations are associated with several neurodegenerative diseases2,3. Targeting p97 and its cofactors is a strategy for cancer drug development4. Despite significant structural insights into the fungal homolog Cdc485-7, little is known about how human p97 interacts with its cofactors. Recently, the anti-alcohol abuse drug disulfiram was found to target cancer through Npl4, a cofactor of p978, but the molecular mechanism remains elusive. Here, using single-particle cryo-electron microscopy (cryo-EM), we uncovered three Npl4 conformational states in complex with human p97 before ATP hydrolysis. The motion of Npl4 results from its zinc finger motifs interacting with the N domain of p97, which is essential for the unfolding activity of p97. In vitro and cell-based assays showed that under oxidative conditions, the disulfiram derivative bis-(diethyldithiocarbamate)-copper (CuET) inhibits p97 function by releasing cupric ions, which disrupt the zinc finger motifs of Npl4, locking the essential conformational switch of the complex.

biophysics↗

bioRxiv2020

Single-cell regulatory landscape and disease vulnerability map of adult Macaque cortex

Single cell approaches have increased our knowledge about the cell type composition of the non-human primate (NHP), but a detailed characterization of area-specific regulatory features remains outstanding. We generated single-cell chromatin accessibility (single-cell ATAC) and transcriptomic data of 358,237 cells from prefrontal cortex (PFC), primary motor cortex (M1) and primary visual cortex (V1) of adult cynomolgus monkey brain, and integrated this dataset with Stereo-seq (Spatio-Temporal Enhanced REsolution Omics-sequencing) of the corresponding cortical areas to assign topographic information to molecular states. We identified area-specific chromatin accessible sites and their targeted genes, including the cell type-specific transcriptional regulatory network associated with excitatory neurons heterogeneity. We reveal calcium ion transport and axon guidance genes related to specialized functions of PFC and M1, identified the similarities and differences between adult macaque and human oligodendrocyte trajectories, and mapped the genetic variants and gene perturbations of human diseases to NHP cortical cells. This resource establishes a transcriptomic and chromatin accessibility combinatory regulatory landscape at a single-cell and spatially resolved resolution in NHP cortex.

neuroscience↗

bioRxiv2020

Eating to dare - Nutrition impacts human risk decision and related brain function

Macronutrient composition modulates plasma amino acids that are precursors of neurotransmitters and can impact brain function and decisions. Neurotransmitter serotonin has been shown to regulate not only food intake, but also economic decisions. We investigated whether an acute nutrition-manipulation inducing plasma tryptophan fluctuation affects brain function, thereby affecting risky decisions. Breakfasts differing in carbohydrate/protein ratios were offered to test changes in risky decision making while metabolic and neural dynamics were tracked. We identified that a high-carbohydrate/protein meal increased plasma tryptophan which mapped to individual risk propensity changes. Moreover, the meal-driven fluctuation in tryptophan and risk propensity changes were modulated by individual difference in body fat mass. Using fMRI, we further identified activation in the parietal lobule during risk-processing, of which activities 1) were correlated with the risk propensity changes in decision making, 2) were sensitive to the tryptophan fluctuation, and 3) were modulated by individuals body fat mass. Furthermore, the activity in the parietal lobule positively mediated the tryptophan-fluctuation to risk-propensity-changes relationship. Our results provide evidence for a personalized nutrition-driven modulation on human risky decisions and its metabolic and neural mechanisms.

neuroscience↗

bioRxiv2020

Identifying a supramodal language network in human brain with individual fingerprint

Where is human language processed in the brain independent of its form? We addressed this issue by analyzing the cortical responses to spoken, written and signed sentences at the level of individual subjects. By applying a novel fingerprinting method based on the distributed pattern of brain activity, we identified a left-lateralized network composed by the superior temporal gyrus/sulcus (STG/STS), inferior frontal gyrus (IFG), precentral gyrus/sulcus (PCG/PCS), and supplementary motor area (SMA). In these regions, the local distributed activity pattern induced by any of the three language modalities can predict the activity pattern induced by the other two modalities, and such cross-modal prediction is individual-specific. The prediction is successful for speech-sign bilinguals across all possible modality pairs, but fails for monolinguals across sign-involved pairs. In comparison, conventional group-mean focused analysis detects shared cortical activations across modalities only in the STG, PCG/PCS and SMA, and the shared activations were found in two groups. This study reveals the core language system in the brain that is shared by spoken, written and signed language, and demonstrates that it is possible and desirable to utilize the information of individual differences for functional brain mapping.

neuroscience↗

bioRxiv2020

The winning methods for predicting cellular position in the DREAM single cell transcriptomics challenge

MotivationPredicting cell locations is important since with the understanding of cell locations, we may estimate the function of cells and their integration with the spatial environment. Thus, the DREAM Challenge on Single Cell Transcriptomics required participants to predict the locations of single cells in the Drosophila embryo using single cell transcriptomic data. ResultsWe have developed over 50 pipelines by combining different ways of pre-processing the RNA-seq data, selecting the genes, predicting the cell locations, and validating predicted cell locations, resulting in the winning methods for two out of three sub-challenges in the competition. In this paper, we present an R package, SCTCwhatateam, which includes all the methods we developed and the Shiny web-application to facilitate the research on single cell spatial reconstruction. All the data and the example use cases are available in the Supplementary material. AvailabilityThe scripts of the package are available at https://github.com/thanhbuu04/SCTCwhatateam and the Shiny application is available at https://github.com/pvvhoang/SCTCwhatateam-ShinyApp ContactThuc.Le@unisa.edu.au Supplementary informationSupplementary data are available at Briefings in Bioinformatics online.

systems biology↗

bioRxiv2020

SARS-CoV-2 spike protein binds heparan sulfate in a length- and sequence-dependent manner

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is causing an unprecedented global pandemic demanding the urgent development of therapeutic strategies. Microarray binding experiments using an extensive heparan sulfate (HS) oligosaccharide library showed that the receptor binding domain (RBD) of the spike of SARS-CoV-2 can bind HS in a length-and sequence-dependent manner. Hexa- and octa-saccharides composed of IdoA2S-GlcNS6S repeating units were identified as optimal ligands. Surface plasma resonance (SPR) showed the SARS-CoV-2 spike protein binds with much higher affinity to heparin (KD = 55 nM) compared to the RBD (KD = 1 M) alone. We also found that heparin does not interfere in angiotensin-converting enzyme 2 (ACE2) binding or proteolytic processing of the spike. Our data supports a model in which HS functions as the point of initial attachment for SARS-CoV-2 infection. Tissue staining studies using biologically relevant tissues indicate that heparan sulfate proteoglycan (HSPG) is a critical attachment factor for the virus. Collectively, our results highlight the potential of using HS oligosaccharides as a therapeutic agent by inhibiting SARS-CoV-2 binding to target cells.

biochemistry↗

bioRxiv2020

Skin Delivery of Modified Vaccinia Ankara Viral Vectors Generates Superior T Cell Immunity Against a Respiratory Viral Challenge

Modified Vaccinia Ankara (MVA) was recently approved as a Smallpox vaccine. Transmission of Variola is by respiratory droplets, and MVA delivered by skin scarification (s.s.) protected mice far more effectively against lethal respiratory challenge with VACV than any other route of delivery, and at much lower doses. Comparisons of s.s. with intradermal, subcutaneous or intramuscular routes showed that MVAOVA s.s.-generated T cells were both more abundant and transcriptionally distinct. MVAOVA s.s. produced greater numbers of lung Ova-specific CD8+ TRM and was superior in protecting mice against lethal VACVOVA respiratory challenge. Nearly as many lung TRM were generated with MVAOVA s.s. compared to direct pulmonary immunization with MVAOVA, and both routes vaccination protected mice against lethal pulmonary challenge with VACVOVA. Strikingly, MVAOVA s.s.-generated effector T cells exhibited overlapping gene transcriptional profiles to those generated via direct pulmonary immunization. Overall, our data suggest that heterologous MVA vectors delivered via s.s. are uniquely well-suited as vaccine vectors for respiratory pathogens like COVID-19. In addition, MVA delivered via s.s. could represent a more effective dose-sparing smallpox vaccine.

immunology↗

bioRxiv2020

De novo synthesis and salvage pathway coordinately regulates polyamine homeostasis and determines T cell proliferation and function.

Robust and effective T cell-mediated immune responses require proper allocation of metabolic resources through metabolic pathways to sustain the energetically costly immune response. As an essential class of polycationic metabolites ubiquitously present in all living organisms, the polyamine pool is tightly regulated by biosynthesis and salvage pathway. We demonstrated that arginine is a major carbon donor and glutamine is a minor carbon donor for polyamine biosynthesis in T cells. Accordingly, the dependence of T cells can be partially relieved by replenishing the polyamine pool. In response to the blockage of de novo synthesis, T cells can rapidly restore the polyamine pool through a compensatory increase in polyamine uptake from the environment, indicating a layer of metabolic plasticity. Simultaneously blocking synthesis and uptake depletes the intracellular PA pool, inhibits T cell proliferation, suppresses T cell inflammation, indicating the potential therapeutic value of targeting the polyamine for managing inflammatory and autoimmune diseases.

cell biology↗