Biology subjects

Song, W.

Publications and source records attributed to Song, W..

At least 145 records · Page 8Linked to original sources

bioRxiv2021

Natural variations in the P-type ATPase heavy metal transporter ZmCd1 controlling cadmium accumulation in maize grains

Cadmium (Cd) accumulation in maize grains is detrimental to human health. Developing maize varieties with low-Cd contents via marker-assisted selection is important for ensuring the production of maize grains safe for consumption. However, the key gene controlling maize grain Cd accumulation has not been cloned. In this study, we identified two major loci for maize grain Cd accumulation (qCd1 and qCd2) on chromosome 2 during a genome-wide association study (GWAS). The qCd1 locus was analyzed by bulked segregant RNA-seq and fine mapping with a biparental segregating population of Jing724 (low-Cd line) and Mo17 (high-Cd line). The ZmCd1 candidate gene in the qCd1 locus encodes a vacuolar membrane-localized heavy metal P-type ATPase transporter, ZmHMA3, which is orthologous to the tonoplast Cd transporter OsHMA3. Genomic DNA sequence and transcript analyses suggested that a transposon in intron 1 of ZmCd1 is responsible for the abnormal amino acid sequence in Mo17. An EMS mutant analysis and an allelism test confirmed ZmCd1 influences maize grain Cd accumulation. The natural variations in ZmCd1 were used to develop four PCR-based molecular markers, which revealed five ZmCd1 haplotypes in the GWAS population. The molecular markers were also used to predict the grain Cd contents in commonly cultivated maize germplasms in China. The predicted Cd contents for 36 inbred lines and 13 hybrids were consistent with the measured Cd contents. Furthermore, several low-Cd elite inbred lines and hybrids were identified, including Jing2416, MC01, Jingnonke728, and Jingke968. Therefore, the molecular markers developed in this study are applicable for molecular breeding and developing maize varieties with low grain Cd contents.

plant biology↗

bioRxiv2021

Sustained IL-15 response signature predicts RhCMV/SIV vaccine efficacy

Simian immunodeficiency virus (SIV) challenge of rhesus macaques (RMs) vaccinated with Rhesus Cytomegalovirus (RhCMV) vectors expressing SIV proteins (RhCMV/SIV) results in a binary outcome: stringent control and subsequent clearance of highly pathogenic SIV in ~55% of vaccinated RMs with no protection in the remaining 45%. Although previous work suggests that unconventionally restricted, SIV-specific, effector-memory (EM)-biased CD8+ T cell responses are necessary for efficacy, the magnitude of these responses does not predict efficacy, and the basis of protection vs. non-protection in RhCMV/SIV vector-vaccinated RMs has not been elucidated. Here, we report that RhCMV/SIV vector administration strikingly alters the whole blood transcriptome of vaccinated RMs, with the sustained induction of specific immune-related pathways, including non-canonical T cell receptor (TCR), toll-lie receptor (TLR), inflammasome/cell death, and interleukin-15 (IL-15) signaling, significantly predicting protection. The IL-15 gene expression signature was further evaluated in an independent RM IL-15 treatment cohort, revealing that in whole blood the response to IL-15 is inclusive of innate and adaptive immune gene expression networks that link with RhCMV/SIV vaccine efficacy. We also show that this IL-15 response signature similarly tracks with vaccine protection in an independent RhCMV/SIV vaccination/SIV challenge RM validation cohort. Thus, the RhCMV/SIV vaccine imparts a coordinated and persistent induction of innate and adaptive immune pathways featuring IL-15, a known regulator of CD8+ T cell function, that enable vaccine-elicited CD8+ T cells to mediate protection against highly pathogenic SIV challenge. Author SummarySIV insert-expressing vaccine vectors based on strain 68-1 RhCMV elicit robust, highly effector-memory-biased T cell responses that are associated with an unprecedented level of SIV control after challenge (replication arrest leading to clearance) in slightly over half of vaccinated monkeys. Since efficacy is not predicted by standard measures of immunogenicity, we used functional genomics analysis of RhCMV/SIV vaccinated monkeys with known challenge outcomes to identify immune correlates of protection. We found that arrest of viral replication after challenge significantly correlates with a vaccine-induced response to IL-15 that includes modulation of T cell, inflammation, TLR signaling, and cell death programming. These data suggest that RhCMV/SIV efficacy is not based on chance, but rather, results from a coordinated and sustained vaccine-mediated induction of innate and adaptive immune pathways featuring IL-15, a known regulator of CD8+ effector-memory T cell function, that enable vaccine-elicited CD8+ T cells to mediate efficacy.

genomics↗

bioRxiv2020

A novel high-accuracy genome assembly method utilizing a high-throughput workflow

Across domains of biological research using genome sequence data, high-quality reference genome sequences are essential for characterizing genetic variation and understanding the genetic basis of phenotypes. However, the construction of genome assemblies for various species is often hampered by complexities of genome organization, especially repetitive and complex sequences, leading to mis-assembly and missing regions. Here, we describe a high-throughput gold standard genome assembly workflow using a large-scale bacterial artificial chromosome (BAC) library with a refined two-step pooling strategy and the Lamp assembler algorithm. This strategy minimizes the laborious processes of physical map construction and clone-by-clone sequencing, enabling inexpensive sequencing of several thousand BAC clones. By applying this strategy with a minimum tiling path BAC clone library for the short arm of chromosome 2D (2DS) of bread wheat, 98% of BAC sequences, covering 92.7% of the 2DS chromosome, were assembled correctly for this species with a highly complex and repetitive genome. We also identified 48 large mis-assemblies in the reference wheat genome assembly (IWGSC RefSeq v1.0) and corrected these large mis-assemblies in addition to filling 92.2% of the gaps in RefSeq v1.0. Our 2DS assembly represents a new benchmark for the assembly of complex genomes with both high accuracy and efficiency.

genomics↗

bioRxiv2020

CRISPR/Cas9 targeting Ttc30a mimics ciliary chondrodysplasia with polycystic kidney disease.

Skeletal ciliopathies (e.g. Jeune syndrome, short rib polydactyly syndrome, Sensenbrenner syndrome) are frequently associated with cystic kidney disease and other organ manifestations, but a common molecular mechanism has remained elusive. We established two models for skeletal ciliopathies (ift80 and ift172) in Xenopus tropicalis, which exhibited severe limb deformities, polydactyly, cystic kidneys, and ciliogenesis defects, closely matching the phenotype of affected patients. Employing data-mining and an in silico screen we identified candidate genes with similar molecular properties to genetically validated skeletal ciliopathy genes. Among four genes experimentally validated, CRISPR/Cas9 targeting of ttc30a replicated all aspects of the phenotypes observed in the models of genetically confirmed disease genes, including ciliary defects, limb deformations and cystic kidney disease. Our findings establish three new models for skeletal ciliopathies (ift80, ift172, ttc30a) and identify TTC30A/B as an essential node in the network of ciliary chondrodysplasia and nephronophthisis-like disease proteins implicating post-translational tubulin modifications in its pathogenesis.

developmental biology↗

bioRxiv2020

Characterization of LC-MS based urine metabolomics in healthy subjects across the life span

Previous studies reported that gender and age could influence urine metabolomics, which should be considered in biomarker discovery. As a consequence, for the baseline of urine metabolomics characteristics, it becomes critical to avoid confounding effects in clinical cohort studies. In this study, we provided a comprehensive lifespan characterization of urine metabolomics in a cohort of 348 healthy children and 315 adults aged 1 to 70 years using liquid chromatography coupled with high resolution mass spectrometry. Our results suggested that gender-dependent urine metabolites are much greater in adults than in children. The pantothenate and CoA biosynthesis and alanine metabolism pathways were enriched in early life. Androgen and estrogen metabolism showed high activity during adolescence and youth stages. Pyrimidine metabolism was enriched in the old stage. Based on the above analysis, metabolomic characteristics of each age stage were provided. This work could help us understand the baseline of urine metabolism characteristics and contribute to further studies of clinical disease biomarker discovery.

biochemistry↗

bioRxiv2020

A bacterial artificial chromosome (BAC)-vectored noninfectious replicon of SARS-CoV-2

Vaccines and antiviral agents are in urgent need to stop the COVID-19 pandemic. To facilitate antiviral screening against SARS-CoV-2 without requirement for high biosafety level facility, we developed a bacterial artificial chromosome (BAC)-vectored replicon of SARS-CoV-2, nCoV-SH01 strain, in which secreted Gaussia luciferase (sGluc) was encoded in viral subgenomic mRNA as a reporter gene. The replicon was devoid of structural genes spike (S), membrane (M), and envelope (E). Upon transfection, the replicon RNA replicated in various cell lines, and was sensitive to interferon alpha (IFN-), remdesivir, but was resistant to hepatitis C virus inhibitors daclatasvir and sofosbuvir. Replication of the replicon was also sensitive overexpression of zinc-finger antiviral protein (ZAP). We also constructed a four-plasmid in-vitro ligation system that is compatible with the BAC system, which makes it easy to introduce desired mutations into the assembly plasmids for in-vitro ligation. This replicon system would be helpful for performing antiviral screening and dissecting virus-host interactions.

microbiology↗

bioRxiv2020

The oriental fruit moth genome provides insights into evolutionary adaptation of tortricid pests

Moths of the family Tortricidae (Insecta: Lepidoptera) usually distribute in temperate and tropical high upland regions. The oriental fruit moth (OFM) Grapholita molesta is a globally important pest of stone and pome fruit from this family. In this study, we assembled a chromosome-level genome for the OFM and conducted a comparative genomic analysis with other lepidopterans. This genome was assembled to 28 chromosomes with a size of 517.71 Mb, an N50 of 19.5 Mb, and a BUSCO completeness of 97.1%. In total, 19968 protein-coding genes were predicted, among which 15269 were functionally annotated. We manually annotated ten gene families for 13 representative genomes from the Lepidopteran. In general, two tortricid moths have a moderate number of detoxification and receptor genes and the lowest number of HSP genes, congruent with their polyphagous dietary and pattern of distribution. However, the OFM has the highest number of P450, I Rs and ORs among all species. Compared to the other tortricid species of the codling moth Cydia pomonella, the OFM has more genes in all gene families. Our results indicate that the high number of some detoxification and receptor genes may be related to the strong adaptation of OFM as a global pest. The high-quality genome of OFM provides an invaluable resource for understanding the ecology, genetics, and evolution of tortricid moths.

genomics↗

bioRxiv2020

Gonadectomy and blood sampling procedures in small size teleost models

Sex steroids, produced by the gonads, play an essential role in the neuroendocrine control of reproduction in all vertebrates by providing feedback to the brain and pituitary. Sex steroids also play an important role in tissue plasticity by regulating cell proliferation in several tissues including the brain and the pituitary. Therefore, investigating the role of sex steroids and mechanisms by which they act is crucial to better understand both feedback mechanism and tissue plasticity. Teleost fish, which possess a higher degree of tissue plasticity and variations in reproduction strategies compared to mammals, appear to be useful models to investigate these questions. The removal of the main source of sex steroid production using gonadectomy together with blood sampling to measure steroid levels, have been well-established and fairly feasible in bigger fish and are powerful techniques to investigate the role and effects of sex steroids. However, small fish such as zebrafish and medaka, which are particularly good model organisms considering the well-developed genetic toolkit and the numerous protocols available to investigate their biology and physiology, raise challenges for applying such protocols due to their small size. Here, we demonstrate the step-by-step procedure of gonadectomy in both males and females followed by blood sampling in a small sized teleost model, the Japanese medaka (Oryzias latipes). The use of these procedures combined with the other advantages of using these small teleost models will greatly improve our understanding of feedback mechanisms in the neuroendocrine control of reproduction and tissue plasticity provided by sex steroids in vertebrates. SUMMARYThe article describes a quick protocol to gonadectomize and sample blood from small teleost fish, using medaka (Oryzias latipes) as a model, to investigate the role of sex steroids in animal physiology.

physiology↗

bioRxiv2020

Pathogenicity, immunogenicity, and protective ability of an attenuated SARS-CoV-2 variant with a deletion at the S1/S2 junction of the spike protein

SARS-CoV-2 contains a PRRA polybasic cleavage motif considered critical for efficient infection and transmission in humans. We previously reported that virus variants with spike protein S1/S2 junction deletions spanning this motif are attenuated. Here we characterize a further cell-adapted SARS-CoV-2 variant, Ca-DelMut. Ca-DelMut replicates more efficiently than wild type or parental virus in cells, but causes no apparent disease in hamsters, despite replicating in respiratory tissues. Unlike wild type virus, Ca-DelMut does not induce proinflammatory cytokines in hamster infections, but still triggers a strong neutralizing antibody response. Ca-DelMut-immunized hamsters challenged with wild type SARS-CoV-2 are fully protected, demonstrating sterilizing immunity.

microbiology↗

bioRxiv2020

Single-cell cytometry via multiplexed fluorescence prediction by label-free reflectance microscopy

Traditional imaging cytometry uses fluorescence markers to identify specific structures, but is limited in throughput by the labeling process. Here we develop a label-free technique that alleviates the physical staining and provides highly multiplexed readouts via a deep learning-augmented digital labeling method. We leverage the rich structural information and superior sensitivity in reflectance microscopy and show that digital labeling predicts highly accurate subcellular features after training on immunofluorescence images. We demonstrate up to 3x improvement in the prediction accuracy over the state-of-the-art. Beyond fluorescence prediction, we demonstrate that single-cell level structural phenotypes of cell cycles are correctly reproduced by the digital multiplexed images, including Golgi twins, Golgi haze during mitosis and DNA synthesis. We further show that the multiplexed readouts enable accurate multi-parametric single-cell profiling across a large cell population. Our method can dramatically improve the throughput for imaging cytometry toward applications for phenotyping, pathology, and high-content screening.

bioengineering↗

bioRxiv2020

Surface-bound antigen induces B-cell permeabilization and lysosome exocytosis facilitating antigen uptake and presentation to T-cells

B-cell receptor (BCR)-mediated antigen internalization and presentation are essential for humoral memory immune responses. Antigen encountered by B-cells is often tightly associated with the surface of pathogens and/or antigen-presenting cells. Internalization of such antigens requires myosin-mediated traction forces and extracellular release of lysosomal enzymes, but the mechanism triggering lysosomal exocytosis is unknown. Here we show that BCR-mediated recognition of antigen tethered to beads, to planar lipid-bilayers or expressed on cell surfaces causes localized plasma membrane (PM) permeabilization, a process that requires BCR signaling and non-muscle myosin II activity. B-cell permeabilization triggers PM repair responses involving lysosomal exocytosis, and B-cells permeabilized by surface-associated antigen internalize more antigen than cells that remain intact. Higher affinity antigens cause more B-cell permeabilization and lysosomal exocytosis and are more efficiently presented to T-cells. Thus, PM permeabilization by surface-associated antigen triggers a lysosome-mediated B-cell resealing response, providing the extracellular hydrolases that facilitate antigen internalization and presentation.

cell biology↗

bioRxiv2020

GPCR Oligomerisation Modulation by Conformational State and Lipid Interactions Revealed by MD Simulations and Markov Models

G protein-coupled receptors (GPCRs) play key roles in cellular signalling. GPCRs are suggested to form dimers and higher order oligomers in response to activation. However, we do not fully understand GPCR activation at larger scales and in an in vivo context. We have characterised oligomeric configurations of the adenosine 2a receptor (A2aR) by combining large-scale molecular dynamics simulations with Markov state models. Receptor activation results in enhanced oligomerisation, more diverse oligomer populations, and a more connected oligomerisation network. The active state conformation of the A2aR shifts protein-protein association interfaces to those involving intracellular loop ICL3 and transmembrane helix TM6. Binding of PIP2 to A2aR stabilises protein-protein interactions via PIP2-mediated association interfaces. These results indicate that A2aR oligomerisation is responsive to the local membrane lipid environment. This in turn suggests a modulatory effect on A2aR whereby a given oligomerisation profile favours the dynamic formation of specific supra-molecular signalling complexes.

biophysics↗

bioRxiv2020

Membrane Insertion of MoS2 Nanosheets: Fresh vs. Aged

Fresh two-dimensional (2D) molybdenum disulfide (MoS2) can absorb the hydrocarbon contamination from the ambient air and cause surface aging. Thus, understanding how the surface aging process of MoS2 affects the interaction with biomolecules is crucial for its applications in the biomedical field. Here, we employed atomistic molecular dynamics simulations to investigate the interactions of fresh and aged MoS2 nanosheets with POPE lipid membranes. Our results show that even though both the fresh and aged MoS2 nanosheets are capable of spontaneous insertion into the POPE bilayer membrane, the fresh MoS2 nanosheet displays significantly more robust interaction than the aged one. The potential mean force (PMF) calculations further confirm that the fresh MoS2 nanosheet is more energetically favorable than the aged one in penetrating into the POPE lipid membranes, with the former having ~17 kJ/mol stronger binding affinity than the later. This work provides a deeper understanding of the surface-aging-dependent interaction of MoS2 nanosheet with biomolecules, which might help the design of better MoS2-based nanodevices with appropriate surface properties.

biophysics↗

bioRxiv2020

Thiamine pyrophosphokinase deficiency induces Alzheimer's pathology

BackgroundThiamine diphosphate (TDP) reduction plays an important role in cerebral glucose hypometabolism, the neurodegenerative indicator, in Alzheimers disease (AD). The mechanism underlying TDP reduction remains elusive. Thus, it is critical to define the mechanism and its effect on neurodegeneration, the pathological basis of the disease occurrence and progression. MethodsThe mRNA levels of all known genes associated with thiamine metabolism, including thiamine pyrophosphokinase (TPK), Solute Carrier Family 19 Member 2 (SLC19A2), SLC19A3, and SLC25A19, in brain samples of patients with AD and other neurodegenerative disorders in multiple independent datasets were analyzed. TPK protein levels were further examined in the brain tissues of AD patients and control subjects. A mouse model with conditional knockout (cKO) of TPK gene in the excitatory neurons of adult brain was established. ResultsThe brain TPK mRNA level was markedly lower in AD patients, but not in other neurodegenerative disorders. The brain TPK protein level was also significantly decreased in AD patients. TPK gene knockout in the mice caused cerebral glucose hypometabolism, {beta}-amyloid deposition, Tau hyperphosphorylation, neuroinflammation, and neuronal loss and brain atrophy. Cross-species correlation analysis revealed the similar changes of gene profiling between the cKO mice and AD patients. ConclusionsThe deficiency of brain TPK, a key enzyme for TDP synthesis, is specific to AD. The cKO mice show AD-associated phenotypes and could serve as a new mouse model for AD studies. Our study provides a novel insight into the critical role of TPK in AD pathogenesis and its potential for the disease treatment.

neuroscience↗

bioRxiv2020

Tumor-specific EphA2 receptor tyrosine kinase inhibits anti-tumor immunity by recruiting suppressive myeloid populations in NSCLC

Given the success of both targeted and immunotherapies against cancer, there is increasing utility for identifying targeted agents that also promote anti-tumor immunity. EphA2 is a receptor tyrosine kinase that contributes to tumor growth and metastasis and has been identified as a viable target for many solid cancers. Investigating EphA2s impact on the host immune system may advance our understanding of tumor immune evasion and the consequences of targeting EphA2 on the tumor microenvironment. Here, we examine how tumor-specific EphA2 affects the activation and infiltration of immune cell populations and the cytokine and chemokine milieu in non-small cell lung cancer (NSCLC). Effects of EphA2 overexpression in murine NSCLC cells were evaluated in both in vitro cell viability assays and in vivo tumor models. Tumor immune infiltrate was assessed by flow cytometry. Cytokine and chemokine expression was evaluated using NanoString technology and qRT-PCR. Although EphA2 overexpression in NSCLC cells did not display proliferative advantage in vitro, it conferred a growth advantage in vivo. Analysis of lung tumor infiltrate revealed decreased natural killer and T cells in the EphA2-overexpressing tumors, as well as increased myeloid populations, including tumor-associated macrophages (TAMs). T cell activation, particularly in CD8 T cells, was decreased, while PD-1 expression was increased. These changes were accompanied by increased monocyte-attracting chemokines, specifically CCL2, CCL7, CCL8, and CCL12, and immunosuppressive proteins TGF-{beta} and arginase 1. Our studies suggest EphA2 on tumor cells recruits monocytes and promotes their differentiation into TAMs that likely inhibit activation and infiltration of cytotoxic lymphocytes, promoting tumor immune escape. Further studies are needed to determine the molecular mechanisms by which EphA2 affects the recruitment of these cell types and to test the function of these myeloid cells.

cancer biology↗

bioRxiv2020

Structural, in silico, and functional analysis of a Disabled-2-derived peptide for recognition of sulfatides

Disabled-2 (Dab2) is an adaptor protein that regulates numerous cellular processes. Among them, Dab2 modulates the extent of platelet aggregation by two mechanisms. In the first mechanism, Dab2 intracellularly downregulates the integrin IIb{beta}3 receptor, converting it to a low affinity state for adhesion and aggregation processes. In the second mechanism, Dab2 is released extracellularly and interacts with both the integrin IIb{beta}3 receptor and sulfatides, both of which are known to be pro-aggregatory mediators, blocking their association to fibrinogen and P-selectin, respectively. Our previous research indicated that a 35-amino acid region within Dab2, which we refer to as the sulfatide-binding peptide (SBP), contains two potential sulfatide-binding motifs represented by two consecutive polybasic regions. Using a combined methodology including molecular docking, nuclear magnetic resonance, lipid-binding assays, and surface plasmon resonance, this work identifies the critical Dab2 residues within SBP that are responsible for sulfatide binding. A hydrophilic region, primarily mediated by R42, is responsible for the interaction with the sulfatide headgroup, whereas the C-terminal polybasic region contributes to interactions with the acyl chains. Furthermore, we demonstrated that, in Dab2 SBP, R42 significantly contributes to the inhibition of platelet P-selectin surface expression. The interacting Dab2 SBP residues with sulfatide resemble those described for sphingolipid-binding in other proteins, suggesting that sulfatide-binding proteins share common binding mechanisms.

biophysics↗

bioRxiv2020

Visible light optical coherence tomography angiography (vis-OCTA) and local microvascular retinal oximetry in human retina

We report herein the first visible light optical coherence tomography angiography (vis-OCTA) for human retinal imaging. Compared to the existing vis-OCT systems, we devised a spectrometer with a narrower bandwidth to increase the spectral power density for OCTA imaging, while retaining the major spectral contrast in the blood. We achieved a 100 kHz A-line rate, the fastest acquisition speed reported so far for human retinal vis-OCT. We rigorously optimized the imaging protocol such that a single acquisition takes <6 seconds with a field of view (FOV) of 3x7.8 mm2. The angiography enables accurate localization of microvasculature down to the capillary level and thus enables oximetry at vessels < 100 m in diameter. We demonstrated microvascular hemoglobin oxygen saturation (sO2) at the feeding and draining vessels at the perifoveal region. The longitudinal repeatability was assessed by <5% coefficient of variation (CV). The unique capabilities of our vis-OCTA system may allow studies on the role of microvascular oxygen in various retinal pathologies.

bioengineering↗

bioRxiv2020

Genome-wide identification, characterization and expression analysis of the monovalent cation-proton antiporter superfamily, and their function analysis in maize salt tolerance.

Na+, K+ and pH homeostasis are important for plant life and they are controlled by the monovalent cation proton antiporter (CPA) superfamily. The roles of ZmCPAs in salt tolerance are not fully elucidated. In this study, we identified 35 ZmCPAs comprising 13 Na+/H+ exchangers (ZmNHXs), 16 cation/H+ exchanger (ZmCHXs), and 6 K+ efflux antiporters (ZmKEAs). All ZmCPAs have transmembrane domains and maize protoplasts transient expression showed that ZmNHX5 and ZmCHX17 localized to cell membrane system. ZmCHXs were specifically highly expressed in anthers, while ZmNHXs and ZmKEAs showed high expression in various tissues. ZmNHX5 and ZmKEA2 were up-regulated in maize seedlings under both NaCl and KCl stresses. Yeast complementation experiments revealed the roles of ZmNHX5, ZmKEA2 in NaCl tolerance. Analyses of the maize mutants further validated the salt tolerance functions of ZmNHX5 and ZmKEA2. Our study highlights comprehensive information of ZmCPAs and provides new gene targets for salt tolerance maize breeding.

plant biology↗