Biology subjects

Xu, J.

Publications and source records attributed to Xu, J..

At least 739 records · Page 41Linked to original sources

bioRxiv2020

I-KCKT allows dissection-free RNA profiling of adult Drosophila intestinal progenitor cells

The adult Drosophila intestinal epithelium is a model system for stem cell biology, but its utility is limited by current biochemical methods that lack cell type resolution. Here, we describe a new proximity-based profiling method that relies upon a GAL4 driver, termed intestinal-kickout-GAL4 (I-KCKT-GAL4), exclusively expressed in intestinal progenitor cells. This method used UV cross-linked whole animal frozen powder as its starting material to immunoprecipitate the RNA cargoes of transgenic epitope-tagged RNA binding proteins driven by I-KCKT-GAL4. When applied to the general mRNA-binder, poly(A)-binding protein, the RNA profile obtained by this method identified 98.8% of transcripts found after progenitor cell sorting, and had low background noise despite being derived from whole animal lysate. We also mapped the targets of the more selective RNA binder, Fragile Mental Retardation Protein, using enhanced CLIP, and report for the first time its binding motif in Drosophila cells. This method will therefore enable the RNA profiling of wildtype and mutant intestinal progenitor cells from intact flies exposed to normal and altered environments, as well as the identification of RNA-protein interactions critical for stem cell function. Summary StatementWe report a dissection-free method to identify proximity-based RNA-protein interactions in an in vivo stem cell population, enabling molecular analysis of these cells at unprecedented speed and resolution.

genetics↗

bioRxiv2020

Bi-compartmentalized stem cell organization of the corneal limbal niche

Stem cells exist in precise locations within tissues, yet how their organization supports tissue architecture and function is poorly understood. The limbus is the presumptive stem cell niche of the corneal epithelium. Here, we visualize the live limbus and track the activity of single stem cells in their native environment by 2-photon microscopy. We identify previously unknown niche compartments and show that long implicated slow-cycling cells form separate lineages in the outer limbus, with only local clonal dynamics. Instead, we find distinct stem cells in the pericorneal limbus to be required for corneal regeneration. Unbiased photolabeling captures their progeny exiting the niche, then moving centripetally in unison before undergoing terminal differentiation. This study demonstrates how a compartmentalized stem cell organization coordinates tissue regeneration.One Sentence Summary In vivo live imaging of the regenerating cornea reveals distinct stem cell activities in the limbal nicheCompeting Interest StatementThe authors have declared no competing interest.View Full Text

developmental biology↗

bioRxiv2020

Biodegradation of polyester polyurethane by Aspergillus flavus G10

Polyurethanes (PU) are integral to many aspects of our daily lives. Due to the extensive use of and difficulties in recycling or reusing PU, it mostly accumulates as waste. Various bacteria and fungi have been reported to degrade PU. We examined the fungus Aspergillus flavus G10 in that regard, after isolating it from the guts of Gryllus bimaculatus, a common cricket species. We observed surficial and chemical changes of PU with atomic force microscopy, scanning electron microscopy, and attenuated total reflectance Fourier-transform infrared spectroscopy. We measured physical changes as loss in tensile stress, stretching force, and weight of PU after incubations. Fungal hydrolysis of urethane bonds in the polymer backbone was demonstrated by detecting the formation of methylene di-aniline during incubations. Trapped CO2 during incubations equaled 52.6% of PU carbon. Biodegradation of PU was maximal by fungi cultured on a malt extract medium at 25 °C, pH 12, and 14:10 hrs light to dark ratio. Pretreating PU films with UV light or 1% FeSO4 or NaCl solutions further enhanced the rate of biodegradation. A range of techniques are needed to fully characterize the degradation of PU or other plastic polymers and to optimize conditions for their microbial degradation.View Full Text

bioengineering↗

bioRxiv2020

Reading frame restoration at the EYS locus, and allele-specific chromosome removal after Cas9 cleavage in human embryos

The correction of disease-causing mutations in human embryos could reduce the burden of inherited genetic disorders in the fetus and newborn, and improve the efficiency of fertility treatments for couples with disease-causing mutations in lieu of embryo selection. Here we evaluate the repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frame-shift mutation causing blindness. We show that the most common repair outcome is microhomology-mediated end joining, which occurs during the first cell cycle in the zygote, leading to embryos with non-mosaic restoration of the reading frame. However, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, upon entry into mitosis, loss of one or both chromosomal arms. Thus, Cas9 allows for the modification of chromosomal content in human embryos in a targeted manner, which may be useful for the prevention of trisomies. HighlightsO_LICas9-mediated DSB induction and repair by end joining occurs within hours C_LIO_LIEnd joining provides an efficient way to restore reading frames without mosaicism C_LIO_LIUnrepaired DSBs persist through mitosis and result in frequent chromosome loss C_LI

genetics↗

bioRxiv2020

Combinatorial Algorithms for Strain Level Metagenomic Microbial Detection and Quantification

The identification and quantification of microbial abundance at the species or strain level from sequencing data is crucial for our understanding of human health and disease. Existing approaches for microbial abundance estimation either use accurate but computationally expensive alignment-based approaches for species-level estimation or less accurate but computationally fast alignment-free approaches that fail to classify many reads accurately at the species or strain-level. Here we introduce CAMMiQ, a novel combinatorial solution to the microbial identification and abundance estimation problem, which performs better than the best used tools on simulated and real datasets with respect to the number of correctly classified reads (i.e., specificity) by an order of magnitude and resolves possible mixtures of similar genomes. As we demonstrate, CAMMiQ can better distinguish between single cells deliberately infected with distinct Salmonella strains and sequenced using scRNA-seq reads than alternative approaches. We also demonstrate that CAMMiQ is also more accurate than the best used approaches on a variety of synthetic genomic read data involving some of the most challenging bacterial genomes derived from NCBI RefSeq database; it can distinguish not only distinct species but also closely related strains of bacteria. The key methodological innovation of CAMMiQ is its use of arbitrary length, doubly-unique substrings, i.e. substrings that appear in (exactly) two genomes in the input database, instead of fixed-length, unique substrings. To resolve the ambiguity in the genomic origin of doubly-unique substrings, CAMMiQ employs a combinatorial optimization formulation, which can be solved surprisingly quickly. CAMMiQs index consists of a sparsified subset of the shortest unique and doubly-unique substrings of each genome in the database, within a user specified length range and as such it is fairly compact. In short, CAMMiQ offers more accurate genomic identification and abundance estimation than the best used alternatives while using similar computational resources. Availabilityhttps://github.com/algo-cancer/CAMMiQ

bioinformatics↗

bioRxiv2020

Parabrachial neuron types categorically encode thermoregulation variables during heat defense

Heat defense is crucial for survival and fitness, and its dysregulation may result in deaths due to poor management. Transmission of thermosensory signals into hypothalamic thermoregulation centers represent a key layer of regulation in heat defense. However, the mechanism by which these signals are transmitted into the hypothalamus remains poorly understood. Here, we reveal that glutamatergic prodynorphin and cholecystokinin neuron populations in the lateral parabrachial (LPB) are progressively recruited to defend elevated body temperature. These two nonoverlapping neuron types form circuitries with downstream preoptic hypothalamic neurons to inhibit BAT thermogenesis and activate tail vasodilation, respectively. Both circuitries are selectively activated by warm temperatures and are required for fever limiting. The prodynorphin circuitry is further required for regulation of energy expenditure and weight homeostasis. Thus, these findings establish that the genetic and functional specificity of heat defense neurons occurs as early as in the LPB and uncover categorical neuron types for encoding two heat defense variables, which may provide targets for treating thermoregulation disorders.

neuroscience↗

bioRxiv2020

One-Step Rapid Quantification of SARS-CoV-2 Virus Particles via Low-Cost Nanoplasmonic Sensors in Generic Microplate Reader and Point-of-Care Device

The spread of SARS-CoV-2 virus in the ongoing global pandemics has led to infections of millions of people and losses of many lives. The rapid, accurate and convenient SARS-CoV-2 virus detection is crucial for controlling and stopping the pandemics. Diagnosis of patients in the early stage infection are so far limited to viral nucleic acid or antigen detection in human nasopharyngeal swab or saliva samples. Here we developed a method for rapid and direct optical measurement of SARS-CoV-2 virus particles in one step nearly without any sample preparation using a spike protein specific nanoplasmonic resonance sensor. We demonstrate that we can detect as few as 30 virus particles in one step within 15 minutes and can quantify the virus concentration linearly in the range of 103 vp/ml to 106 vp/ml. Measurements shown on both generic microplate reader and a handheld smartphone connected device suggest that our low-cost and rapid detection method may be adopted quickly under both regular clinical environment and resource-limited settings.

bioengineering↗

bioRxiv2020

An emerging role of Prevotella histicola on estrogen-deficiency induced bone loss through the gut microbiota-bone axis

Gut microbiota (GM)-bone axis has emerged as a crucial mediator in the maintenance of bone homeostasis. Estrogen-deficiency induced bone loss is closely associated with an altered GM. However, the underlying mechanisms remain not fully understood. To this end, feces samples collected from the postmenopausal patients with osteoporosis (PMO) and with normal bone mass (PMN) were examined by 16s rRNA gene sequencing. We found that GM in PMO group was featured by a significant decreased proportion of genus Prevotella in comparison with that in the PMN group. Next, Prevotella histicola (P.histicola), a typical specie of Prevotella, was orally given to the mice following the ovariectomy (OVX) procedure and it significantly prevented OVX induced bone loss. Mechanistically, the protective effects of P.histicola on bone mass were found to be associated with the inhibition of osteoclastic resorption by attenuating osteoclastogenic cytokines expression and ameliorating gut permeability. Thus, P.histicola may prevent estrogen-deficiency induced bone loss through the GM-bone axis.

microbiology↗

bioRxiv2020

stLearn: integrating spatial location, tissue morphology and gene expression to find cell types, cell-cell interactions and spatial trajectories within undissociated tissues

Spatial Transcriptomics is an emerging technology that adds spatial dimensionality and tissue morphology to the genome-wide transcriptional profile of cells in an undissociated tissue. Integrating these three types of data creates a vast potential for deciphering novel biology of cell types in their native morphological context. Here we developed innovative integrative analysis approaches to utilise all three data types to first find cell types, then reconstruct cell type evolution within a tissue, and search for tissue regions with high cell-to-cell interactions. First, for normalisation of gene expression, we compute a distance measure using morphological similarity and neighbourhood smoothing. The normalised data is then used to find clusters that represent transcriptional profiles of specific cell types and cellular phenotypes. Clusters are further sub-clustered if cells are spatially separated. Analysing anatomical regions in three mouse brain sections and 12 human brain datasets, we found the spatial clustering method more accurate and sensitive than other methods. Second, we introduce a method to calculate transcriptional states by pseudo-space-time (PST) distance. PST distance is a function of physical distance (spatial distance) and gene expression distance (pseudotime distance) to estimate the pairwise similarity between transcriptional profiles among cells within a tissue. We reconstruct spatial transition gradients within and between cell types that are connected locally within a cluster, or globally between clusters, by a directed minimum spanning tree optimisation approach for PST distance. The PST algorithm could model spatial transition from non-invasive to invasive cells within a breast cancer dataset. Third, we utilise spatial information and gene expression profiles to identify locations in the tissue where there is both high ligand-receptor interaction activity and diverse cell type co-localisation. These tissue locations are predicted to be hotspots where cell-cell interactions are more likely to occur. We detected tissue regions and ligand-receptor pairs significantly enriched compared to background distribution across a breast cancer tissue. Together, these three algorithms, implemented in a comprehensive Python software stLearn, allow for the elucidation of biological processes within healthy and diseased tissues.

bioinformatics↗

bioRxiv2020

Repurposing screen identifies Amlodipine as an inducer of PD-L1 degradation and antitumor immunity

Cancer cell expression of PD-L1 leads to T cells exhaustion by transducing co-inhibitory signal, and further understanding the regulation of PD-L1 in cancer cells may provide additional therapeutic strategies. Here by drug repurposing screen we identified amlodipine as a potent inhibitor of PD-L1 expression in cancer cells. Further survey of calcium-associated pathways revealed calpains-dependent stabilization of the PD-L1 protein. Intracellular calcium delivered an operational signal to calpain-dependent Beclin-1 cleavage, blocking autophagic degradation of PD-L1 accumulated on recycling endosome (RE). Blocking calcium flux by amlodipine depleted PD-L1 expression and increased CD8+ T cell infiltration in tumor tissues but not in myocardium, causing dose-dependent tumor suppression in vivo. Rescuing PD-L1 expression eliminated the effects of amlodipine, suggesting the PD-L1 dependent effect of amlodipine. These results reveal a calcium-dependent mechanism controlling PD-L1 degradation, and highlight calcium flux blockade as a potential strategy for combinatorial immunotherapy.

cancer biology↗

bioRxiv2020

The effect of autophagy and mitochondrial fission on Harderian gland is greater than apoptosis in male hamsters during different photoperiods

Photoperiod is an important factor of mammalian seasonal rhythm. We studied the morphological differences in HG which is a vital photosensitive organ of male striped dwarf hamsters (Cricetulus barabensis), under different photoperiods (short photoperiod, SP; moderate photoperiod, MP; long photoperiod, LP), and further investigated the molecular mechanisms related to these morphological differences. Results showed that body weight, carcass weight, and HG weight were lower in SP and LP. Protein expression of bax/bcl2 and Cytochrome C showed no significant differences, indicating that the level of apoptosis remained stable. Protein aggregation of LC3 and protein expression of LC3II/LC3I were higher in SP than. Furthermore, comparison of changes in the HG ultrastructure demonstrated autolysosome formation in the LP, which suggesting the lowest autophagy level in MP. Protein expression levels of ATP synthase and mitochondrial fission factor were highest in the MP, whereas citrate synthase, dynamin-related protein1, and fission1 remained unchanged in three groups. In summary, the significant up-regulation of autophagy under short and long photoperiod may be the main factor leading to the loss of HG weight and reduced mitochondrial energy supply.

cell biology↗

bioRxiv2020

Human IgG cell neutralizing monoclonal antibodies block SARS-CoV-2 infection

The coronavirus induced disease 19 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a worldwide threat to human lives, and neutralizing antibodies present a great therapeutic potential in curing affected patients. We purified more than one thousand memory B cells specific to SARS-CoV-2 S1 or RBD (receptor binding domain) antigens from 11 convalescent COVID-19 patients, and a total of 729 naturally paired heavy and light chain fragments were obtained by single B cell cloning technology. Among these, 178 recombinant monoclonal antibodies were tested positive for antigen binding, and the top 13 binders with Kd below 0.5 nM are all RBD binders. Importantly, all these 13 antibodies could block pseudoviral entry into HEK293T cells overexpressing ACE2, with the best ones showing IC50s around 2-3 nM. We further identified 8 neutralizing antibodies against authentic virus with IC50s within 10 nM. Among these, 414-1 blocked authentic viral entry at IC50 of 1.75 nM and in combination with 105-38 could achieve IC50 as low as 0.45 nM. Meanwhile, we also found that 3 antibodies could cross-react with the SARS-CoV spike protein. Altogether, our study provided a panel of potent human neutralizing antibodies for COVID19 as therapeutics candidates for further development.

immunology↗

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

A single dose SARS-CoV-2 simulating particle vaccine induces potent neutralizing activities

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for which a vaccine is urgently needed to control its spreading. To facilitate the representation of a native-like immunogen without being infectious, here, we reported a SARS-CoV-2 vaccine candidate (designated ShaCoVacc) by incorporating spike-encoding mRNA inside and decorating spike protein on the surface of the virus simulating particles (VSPs) derived from lentiviral particles. We characterized the mRNA copy number, glycosylation status, transduction efficiency, and innate immune property of the new vaccine platform. Importantly, we showed the ShaCoVacc induced strong spike-specific humoral immune responses and potent neutralizing activities by a single injection. Additionally, we disclosed the epitopes of spike-specific antibodies using peptide microarray and revealed epitopes susceptible to specific neutralizing antibodies. These results support further development of ShaCoVacc as a candidate vaccine for COVID-19 and VSP may serve as a new vaccine platform for emerging infectious diseases.

bioengineering↗

bioRxiv2020

Generating Synthetic Signaling Networks for in Silico Modeling Studies

Predictive models of signaling pathways have proven to be difficult to develop. Traditional approaches to developing mechanistic models rely on collecting experimental data and fitting a single model to that data. This approach works for simple systems but has proven unreliable for complex systems such as biological signaling networks. Thus, there is a need to develop new approaches to create predictive mechanistic models of complex systems. To meet this need, we developed a method for generating artificial signaling networks that were reasonably realistic and thus could be treated as ground truth models. These synthetic models could then be used to generate synthetic data for developing and testing algorithms designed to recover the underlying network topology and associated parameters. We defined the reaction degree and reaction distance to measure the topology of reaction networks, especially to consider enzymes. To determine whether our generated signaling networks displayed meaningful behavior, we compared them with signaling networks from the BioModels Database. This comparison indicated that our generated signaling networks had high topological similarities with BioModels signaling networks with respect to the reaction degree and distance distributions. In addition, our synthetic signaling networks had similar behavioral dynamics with respect to both steady states and oscillations, suggesting that our method generated synthetic signaling networks comparable with BioModels and thus could be useful for building network evaluation tools. HighlightsO_LIWe provided a Julia script to generate synthetic signaling networks. C_LIO_LIWe defined reaction degree and distance to measure the reaction network topology. C_LIO_LIWe provided the Python scripts to calculate the reaction network topology. C_LIO_LIThe synthetic signaling networks had topological similarities with the BioModels. C_LIO_LIThe synthetic signaling networks had dynamic similarities with the BioModels. C_LI

systems biology↗

bioRxiv2020

A potent neutralizing human antibody reveals the N-terminal domain of the Spike protein of SARS-CoV-2 as a site of vulnerability

The pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a global public health threat. Most research on therapeutics against SARS-CoV-2 focused on the receptor binding domain (RBD) of the Spike (S) protein, whereas the vulnerable epitopes and functional mechanism of non-RBD regions are poorly understood. Here we isolated and characterized monoclonal antibodies (mAbs) derived from convalescent COVID-19 patients. An mAb targeting the N-terminal domain (NTD) of the SARS-CoV-2 S protein, named 4A8, exhibits high neutralization potency against both authentic and pseudotyped SARS-CoV-2, although it does not block the interaction between angiotensin-converting enzyme 2 (ACE2) receptor and S protein. The cryo-EM structure of the SARS-CoV-2 S protein in complex with 4A8 has been determined to an overall resolution of 3.1 Angstrom and local resolution of 3.4 Angstrom for the 4A8-NTD interface, revealing detailed interactions between the NTD and 4A8. Our functional and structural characterizations discover a new vulnerable epitope of the S protein and identify promising neutralizing mAbs as potential clinical therapy for COVID-19.

biochemistry↗

bioRxiv2020

Integrative Single-cell RNA-Seq and ATAC-Seq Analysis of Human Foetal Liver and Bone Marrow Haematopoiesis

Regulation of haematopoiesis during human development remains poorly defined. Here, we applied single-cell (sc)RNA-Seq and scATAC-Seq analysis to over 8,000 human immunophenotypic blood cells from foetal liver and bone marrow. We inferred their differentiation trajectory and identified three highly proliferative oligopotent progenitor populations downstream from haematopoietic stem cell/multipotent progenitors (HSC/MPPs). Along this trajectory, we observed opposing patterns of chromatin accessibility and differentiation that coincided with dynamic changes in the activity of distinct lineage-specific transcription factors. Integrative analysis of chromatin accessibility and gene expression revealed extensive epigenetic but not transcriptional priming of HSC/MPPs prior to their lineage commitment. Finally, we refined and functionally validated the sorting strategy for the HSC/MPPs and achieved around 90% enrichment. Our study provides a useful framework for future investigation of human developmental haematopoiesis in the context of blood pathologies and regenerative medicine.

cell biology↗

bioRxiv2020

SARS-CoV-2 Spike Glycoprotein Receptor Binding Domain is Subject to Negative Selection with Predicted Positive Selection Mutations

COVID-19 is a highly contagious disease caused by a novel coronavirus SARS-CoV-2. The interaction between SARS-CoV-2 spike protein and the host cell surface receptor ACE2 is responsible for mediating SARS-CoV-2 infection. By analyzing the spike-hACE2 interacting surface, we predicted many hot spot residues that make major contributions to the binding affinity. Mutations on most of these residues are likely to be deleterious, leading to less infectious virus strains that may suffer from negative selection. Meanwhile, several residues with mostly advantageous mutations have been predicted. It is more probable that mutations on these residues increase the transmission ability of the virus by enhancing spike-hACE2 interaction. So far, only a limited number of mutations has been reported in this region. However, the list of hot spot residues with predicted downstream effects from this study can still serve as a tracking list for SARS-CoV-2 evolution studies. Coincidentally, one advantageous mutation, p.476G>S, started to surge in the last couple of weeks based on the data submitted to the public domain, indicating that virus strains with increased transmission ability may have already spread.

bioinformatics↗