Biology subjects

Wang, B.

Publications and source records attributed to Wang, B..

At least 541 records · Page 30Linked to original sources

bioRxiv2021

A high-accuracy and high-efficiency digital volume correlation method to characterize in-vivo optic nerve head biomechanics from optical coherence tomography

In-vivo optic nerve head (ONH) biomechanics characterization is emerging as a promising way to study eye physiology and pathology. We propose a high-accuracy and high-efficiency digital volume correlation (DVC) method for the purpose of characterizing the in-vivo ONH deformation from volumes acquired by optical coherence tomography (OCT). Using a combination of synthetic tests and analysis of OCTs from monkey ONHs subjected to acute and chronically elevated intraocular pressure, we demonstrate that our proposed methodology overcomes several challenges for conventional DVC methods. First, it accounts for large ONH rigid body motion in the OCT volumes which could otherwise lead to analysis failure; second, sub-voxel-accuracy displacement can be guaranteed despite high noise and low image contrast of some OCT volumes; third, computational efficiency is greatly improved, such that the memory consumption of our method is substantially lower than with conventional methods; fourth, we introduce a parameter measuring displacements confidence. Test of image noise effects showed that the proposed DVC method had displacement errors smaller than 0.028 voxels with speckle noise and smaller than 0.037 voxels with Gaussian noise; The absolute (relative) strain errors in the three directions were lower than 0.0018 (4%) with speckle noise and than 0.0045 (8%) with Gaussian noise. Compared with conventional DVC methods, the proposed DVC method had substantially improved overall displacement and strain errors under large body motions (lower by up to 70%), with 75% lower computation times, while saving about 30% memory. The study thus demonstrates the potential of the proposed technique to investigate ONH biomechanics.

bioengineering↗

bioRxiv2021

Sift-PULs: A public repository for specific functional polysaccharide utilization loci

BackgroundPolysaccharide utilization loci (PULs) were bacterial gene clusters encoding genes responsible for polysaccharide utilization process. PUL studies are blooming in recent years but the biochemical characterization speed is relative slow. There is a growing demand for PUL database with function annotations. ResultsUsing signature genes corresponding for specific polysaccharide, 10422 PULs specific for 6 polysaccharides (agar, alginate, pectin, carrageenan, chitin and {beta}-manan) from various bacterial phyla were predicted. Then online website of specific functional polysaccharide utilization loci (Sift-PULs) was constructed. Sift-PULs provides a repository where users could browse, search and download interested PULs without registration. ConclusionsThe key advantage of Sift-PULs is to assign a function annotation of each PUL, which is not available in existing PUL databases. PULs functional annotation lays a foundation for studying novel enzymes, new pathways, PUL evolution or bioengineering. The website is available on https://sift-puls.org

bioinformatics↗

bioRxiv2021

A rear-engine drives adherent tissue migration in vivo

During animal embryogenesis, homeostasis and disease, tissues push and pull on their surroundings to move forward. Although the force-generating machinery is known, it is unknown how tissues exert physical stresses on their substrate to generate motion in vivo. Here, we identify the force transmission machinery, the substrate, and the stresses that a tissue, the zebrafish posterior lateral line primordium, generates during its migration. We find that the primordium couples actin flow through integrins to the basement membrane for forward movement. Talin/integrin-mediated coupling is required for efficient migration and its loss is partly compensated for by increased actin flow. Using Embryogram, an approach to measure stresses in vivo, we show that the primordiums rear exerts high stresses, indicating that this tissue pushes itself forward with its back. This unexpected strategy likely also underlies the motion of other tissues in animals

developmental biology↗

bioRxiv2021

Asymmetric Arp2/3-mediated actin assembly facilitates clathrin-mediated endocytosis at stalled sites in genome-edited human stem cells.

Actin assembly facilitates vesicle formation in several trafficking pathways. Clathrin-mediated endocytosis (CME) shows elevated actin assembly dependence under high membrane tension. Why actin assembly at CME sites occurs heterogeneously even within the same cell, and how assembly forces are harnessed, are not fully understood. Here, endocytic dynamics, actin presence, and geometry of CME proteins from three different functional modules, were analyzed using three-dimensional (3D) super-resolution microscopy, live-cell imaging, and machine-learning-based computation. When hundreds of CME events were compared, sites with actin assembly showed a distinct signature, a delay between completion of coat expansion and vesicle scission, indicating that actin assembly occurs preferentially at stalled CME sites. N-WASP is recruited to one side of CME sites where it is positioned to stimulate asymmetric actin assembly. We propose that asymmetric actin assembly rescues stalled CME sites by pulling vesicles into the cell much like a bottle opener pulls off a bottle cap.

cell biology↗

bioRxiv2021

Dynamics of glioma-associated microglia and macrophages reveals their divergent roles in the immune response of brain

Glioma microenvironment contains numerous myeloid cells, including brain-resident microglia and recruited monocytes and macrophages (Mo/M{varphi}). When studied collectively, these cells presented pro-tumor effects. Yet, little is known about the differences among these myeloid populations. Using single-cell sequencing analysis, we studied the phenotypic characteristics, spatial variances, and dynamic changes of these relatively heterogeneous cell populations. Microglia populations with distinct spatial distribution presented different functional states, including tumor-associated subsets with phagocytic and lipid metabolism signature. Notably, this subset of glioma-associated microglia shared similar trait in a diverse spectrum of neuropathogenesis. In contrast, Mo/M{varphi} highly expressed genes related to angiogenesis, tumor invasion, and immune evasion. Moreover, identifying the Mo/M{varphi} subsets had prognostic and classificatory value in clinical application. These results thus eliminate the long-existing ambiguity about the role of microglia and Mo/M{varphi} in glioma pathogenesis, and reveal their prognostic and therapeutic value for glioma patients.

immunology↗

bioRxiv2021

High Bandwidth Power Electronics and Magnetic Nanoparticles for Multichannel Magnetogenetic Neurostimulation

ObjectiveWe present a power electronic system and magnetic nanoparticles for multiplexed magnetogenetic neurostimulation with three channels spanning a wide frequency range and rapid channel switching capability. This enables selective heating of magnetic nanoparticles with different coercivity using various frequency-amplitude combinations of the magnetic field. Such multiplexed operation could provide the technical means for selective magnetogenetic neurostimulation beyond its spatial focality limits. ApproachThe electronic system uses a hybrid of silicon metal-oxide-semiconductor and gallium-nitride field-effect transistors, which generate the required high-amplitude current up to 500 A in the sub-MHz range and the high-frequency current in the MHz range, respectively. Via three discrete resonance capacitor banks, the system generates an alternating magnetic field in the same liquid-cooled field coil at three distinct frequency channels spanning 50 kHz to 4 MHz. Fast switching between channels is achieved with high-voltage contactors connecting the coil to different capacitor banks. We characterized the system by the output channels frequencies, field strength, and switching time, as well as the systems overall operation stability. Three types of iron oxide nanoparticles with different coercivity are developed to form three magnetothermal channels. Specific absorption rate and infrared thermal imaging measurements are performed with the nanoparticles to characterize their heating and demonstrate selective actuation for all three channels. Main resultsThe system achieved the desired target field strengths for three frequency channels (70 kA/m at 50 kHz, 10 kA/m at 500 kHz, and 1 kA/m at [≥]2 MHz), with rapid switching speed between channels on the order of milliseconds. The system can operate continuously for at least two hours at 30% duty cycle with 125 Arms load in the coil, corresponding to a stimulation protocol of cycling the three channels at target strength with 3 s pulses and 7 s interpulse intervals. The nanoparticles were heated with selectivity between 2.3x and 9x for their respective frequency channel. The systems intended use was thus validated with three distinct channels available for magnetothermal heating. SignificanceWe describe the first combination of a power electronic system and magnetic nanoparticles that achieves three stimulation channels. Selective actuation of nanoparticles is demonstrated for each channel using the same field coil, including a novel composition responding to magnetic fields in the MHz range. This approach could improve the speed and flexibility of frequency-multiplexed magnetogenetic neural stimulation.

bioengineering↗

bioRxiv2021

Do we really understand the role of the prefrontal cortex in placebo analgesia?

Several reviews have strongly implicated prefrontal cortical engagement in expectation-based placebo analgesia. We recently found a robust placebo analgesic response and associated decreases in pain-related cortical activations, without observable prefrontal engagement. We hypothesized our substantial conditioning and weak verbal instructions diminished expectation-related prefrontal activation. To test this, we examined the same subjects during a conditioning procedure, in which expectancy of pain relief was high. In two conditioning sessions, noxious heat was applied to a leg region treated with an "analgesic" cream and another treated with a "moisturizing" cream. In reality, both creams were inert, but the temperature applied to the moisturizing-cream area was 2{degrees}C higher than that applied to the analgesic-cream area. Functional MRI was acquired during the second conditioning session. Pain ratings were lower for the low heat than the high heat, with corresponding reduced activations in pain-related regions. Similar to previous studies with strong expectation for pain relief, we observed more prefrontal activations during the "analgesic" than the control condition. Nevertheless, contrary to the idea of active prefrontal engagement, the relative activation was based on differences in negative BOLD signals. A literature review revealed that only a few studies conclusively showed active engagement of prefrontal cortex, i.e. increased positive BOLD signal during high expectation compared to a control, with variable timing and spatial-specificity. We suggest that this variability is due to the heterogeneous influence of cognitive, emotional and motivational factors. Future studies should attempt to unravel the multiple contributions to placebo responsiveness in the prefrontal cortex.

neuroscience↗

bioRxiv2021

NMPylation and de-NMPylation of SARS-CoV-2 Nsp9 by the NiRAN domain

Nsp12, the catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), contains two active sites that catalyze nucleotidyl-monophosphate (NMP) transfer (NMPylation). RNA synthesis is mediated by the RdRp active site that is conserved among all RNA viruses and has been a focus of mechanistic studies and drug discovery. The second active site resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. Both catalytic reactions are essential for viral replication, but the mechanism and targets of NiRAN are poorly characterized. One recent study showed that NiRAN transfers NMP to the first residue of RNA-binding protein Nsp9. Another study reported a structure of SARS-CoV-2 replicase with an extended Nsp9 in the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 Nsp12 efficiently and reversibly NMPylates the native but not the extended Nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas a substitution of a catalytic RdRp Asp residue does not. NMPylation is inhibited by nucleotide analogs, pyrophosphate, and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We hypothesize that Nsp9 remodels both active sites of Nsp12 to support initiation of RNA synthesis by RdRp and subsequent capping of the product RNA by the NiRAN domain.

biochemistry↗

bioRxiv2021

High-quality Arabidopsis thaliana genome assembly with Nanopore and HiFi long reads

Arabidopsis thaliana is an important and long-established model species for plant molecular biology, genetics, epigenetics, and genomics. However, the latest version of reference genome still contains significant number of missing segments. Here, we report a high-quality and almost complete Col-0 genome assembly with two gaps (Col-XJTU) using combination of Oxford Nanopore Technology ultra-long reads, PacBio high-fidelity long reads, and Hi-C data. The total genome assembly size is 133,725,193 bp, introducing 14.6 Mb of novel sequences compared to the TAIR10.1 reference genome. All five chromosomes of Col-XJTU assembly are highly accurate with consensus quality (QV) scores > 60 (ranging from 62 to 68), which are higher than those of TAIR10.1 reference (QV scores ranging from 45 to 52). We have completely resolved chromosome (Chr) 3 and Chr5 in a telomere-to-telomere manner. Chr4 has been completely resolved except the nucleolar organizing regions, which comprise long repetitive DNA fragments. The Chr1 centromere (CEN1), reportedly around 9 Mb in length, is particularly challenging to assemble due to the presence of tens of thousands of CEN180 satellite repeats. Using the cutting-edge sequencing data and novel computational approaches, we assembled about 4 Mb of sequence for CEN1 and a 3.5-Mb-long CEN2. We investigated the structure and epigenetics of centromeres. We detected four clusters of CEN180 monomers, and found that the centromere-specific histone H3-like protein (CENH3) exhibits a strong preference for CEN180 cluster 3. Moreover, we observed hypomethylation patterns in CENH3-enriched regions. We believe that this high-quality genome assembly, Col-XJTU, would serve as a valuable reference to better understand the global pattern of centromeric polymorphisms, as well as genetic and epigenetic features in plants.

genomics↗

bioRxiv2021

Evolutionary tracking of cancer haplotypes at single-cell resolution

Cancer genomes exhibit extensive chromosomal copy number changes and structural variation, yet how allele specific alterations drive cancer genome evolution remains unclear. Here, through application of a new computational approach we report allele specific copy number alterations in 11,097 single cell whole genomes from genetically engineered mammary epithelial cells and 21,852 cells from high grade serous ovarian and triple negative breast cancers. Resolving single cell copy number profiles to individual alleles uncovered genomic background distributions of gains, losses and loss of heterozygosity, yielding evidence of positive selection of specific chromosomal alterations. In addition specific genomic loci in maternal and paternal alleles were commonly found to be altered in parallel with convergent phenotypic transcriptional effects. Finally we show that haplotype specific alterations trace the cyclical etiology of high level amplifications and reveal clonal haplotype decomposition of complex structures. Together, our results illuminate how allele and haplotype specific alterations, here determined across thousands of single cell cancer genomes, impact the etiology and evolution of structural variations in human tumours.

cancer biology↗

bioRxiv2021

The impact of mutational processes on structural genomic plasticity in cancer cells

Structural genome alterations are determinants of cancer ontogeny and therapeutic response. While bulk genome sequencing has enabled delineation of structural variation (SV) mutational processes which generate patterns of DNA damage, we have little understanding of how these processes lead to cell-to-cell variations which underlie selection and rates of accrual of different genomic lesions. We analysed 309 high grade serous ovarian and triple negative breast cancer genomes to determine their mutational processes, selecting 22 from which we sequenced >22,000 single cell whole genomes across a spectrum of mutational processes. We show that distinct patterns of cell-to-cell variation in aneuploidy, copy number alteration (CNA) and segment length occur in homologous recombination deficiency (HRD) and fold-back inversion (FBI) phenotypes. Widespread aneuploidy through induction of HRD through BRCA1 and BRCA2 inactivation was mirrored by continuous whole genome duplication in HRD tumours, contrasted with early ploidy fixation in FBI. FBI tumours exhibited copy number distributions skewed towards gains, widespread clone-specific variation in amplitude of high-level amplifications, often impacting oncogenes, and break-point variability consistent with progressive genomic diversification, which we termed serriform structural variation (SSV). SSVs were consistent with a CNA-based molecular clock reflecting a continual and distributed process across clones within tumours. These observations reveal previously obscured genome plasticity and evolutionary properties with implications for cancer evolution, therapeutic targeting and response.

cancer biology↗

bioRxiv2021

Cellular calcium levels influenced by NCA-2 impact circadian period determination in Neurospora

Intracellular calcium signaling has been implicated in control of a variety of circadian processes in animals and plants but its role in microbial clocks has remained largely cryptic. To examine the role of intracellular Ca2+ in the Neurospora clock we screened knockouts of calcium transporter genes and identified a gene encoding a calcium exporter, nca-2, uniquely as having significant period effects. Loss of NCA-2 results in an increase in cytosolic calcium level, and this leads to hyper-phosphorylation of core clock components, FRQ and WC-1, and a short period as measured by both the core oscillator and overt clock. Genetic analyses showed that mutations in certain frq phospho-sites, and in Ca2+-calmodulin-dependent kinase (camk-2), are epistatic to nca-2 in controlling the pace of the oscillator. These data are consistent with a model in which elevated intracellular Ca+2 leads to increased activity of CAMK-2 leading to enhanced FRQ phosphorylation, accelerated closure of the circadian feedback loop, and a shortened circadian period length. At a mechanistic level some CAMKs undergo more auto-phosphorylations in {Delta}nca-2, consistent with high calcium in the {Delta}nca-2 mutant influencing the enzymatic activity of CAMKs. NCA-2 interacts with multiple proteins including CSP-6, a protein known to be required for circadian output. Most importantly, expression of nca-2 is circadian clock-controlled at both the transcriptional and translational levels, and this in combination with the period effects seen in strains lacking NCA-2, firmly places calcium signaling within the larger circadian system where it acts as both an input to and output from the core clock. ImportanceCircadian rhythms are based on cell-autonomous, auto-regulatory, feedback loops formed by interlocked positive and negative arms, and they regulate myriad molecular and cellular processes in most eukaryotes including fungi. Intracellular calcium signaling is also a process that impacts a broad range of biological events in most eukaryotes. Clues have suggested that calcium signaling can influence circadian oscillators through multiple pathways; however, mechanistic details have been lacking in microorganisms. Building on prior work describing calcium transporters in the fungus Neurospora, one such transporter, NCA-2, was identified as a regulator of circadian period length. Increased intracellular calcium levels caused by loss of NCA-2 results in over-activation of calcium-responsive protein kinases, in turn leading to a shortened circadian period length. Importantly, expression of NCA-2 is itself controlled by the molecular clock. In this way calcium signaling can be seen as providing both input to and output from the circadian system.

molecular biology↗

bioRxiv2021

Reprogrammed tracrRNAs enable repurposing RNAs as crRNAs and detecting RNAs

In type II CRISPR systems, the guide RNA (gRNA) consists of a CRISPR RNA (crRNA) and a hybridized trans-acting CRISPR RNA (tracrRNA) which interacts directly with Cas9 and is essential to its guided DNA targeting function. Though tracrRNAs are diverse in sequences and structures across type II CRISPR systems, the programmability of crRNA-tracrRNA hybridization for particular Cas9 has not been studied adequately. Here, we revealed the high programmability of crRNA-tracrRNA hybridization for Streptococcus pyogenes Cas9. By reprogramming the crRNA-tracrRNA hybridized sequence, reprogrammed tracrRNAs can repurpose various RNAs as crRNAs to trigger CRISPR function. We showed that the engineered crRNA-tracrRNA pairs enable design of orthogonal cellular computing devices and hijacking of endogenous RNAs as crRNAs. We next designed novel RNA sensors that can monitor the transcriptional activity of specific genes on the host genome and detect SARS-CoV-2 RNA in vitro. The engineering potential of crRNA-tracrRNA interaction has therefore redefined the capabilities of CRISPR/Cas9 system.

synthetic biology↗

bioRxiv2021

ADP-ribose triggers neuronal ferroptosis via metabolic orchestrating

Hyperactivation of NAD+-consuming pathways frequently occurs in neurological diseases, however therapies replenishing NAD+ levels show limited therapeutic efficacy, indicating more complex underlying pathophysiology. Here, we delineate a pathogenic link between ADP-ribose --a product of NAD+ consumption--and a metabolic rewiring-dependent form of neuronal ferroptosis. We demonstrate that oxidative stress induces neurons to produce ADP-ribose through the PARP1-PARG axis. ADP-ribose directly binds and inhibits the equilibrative nucleoside transporter ENT2, remodeling de novo purine and pyrimidine synthesis by hyperactivating the inosine-hypoxanthine-xanthine oxidase and glutamine-dihydroorotate-dihydroorotate dehydrogenase axes. This overproduces superoxide radicals and drives lipid peroxidation and neuronal ferroptosis. Elevated ADP-ribose levels were observed in neurological disease models, and acute ADP-ribose exposure severely reduced mouse brain neurons in vivo. Critically, interventions blocking ADP-ribose signaling alleviated cognitive decline in mouse intracerebral hemorrhage models. Our findings characterize ADP-ribose signaling as linking NAD+ consumption to neuronal ferroptosis, and provide a theraputic strategy for neuropathologies involving NAD+ consumption and oxidative stress.

neuroscience↗

bioRxiv2021

One Cell At a Time: A Unified Framework to Integrate and Analyze Single-cell RNA-seq Data

1The surge of single-cell RNA sequencing technologies gives rise to the abundance of large single-cell RNA-seq datasets at the scale of hundreds of thousands of single cells. Integrative analysis of large-scale scRNA-seq datasets has the potential of revealing de novo cell types as well as aggregating biological information. However, most existing methods fail to integrate multiple large-scale scRNA-seq datasets in a computational and memory efficient way. We hereby propose OCAT, One Cell At a Time, a graph-based method that sparsely encodes single-cell gene expressions to integrate data from multiple sources without most variable gene selection or explicit batch effect correction. We demonstrate that OCAT efficiently integrates multiple scRNA-seq datasets and achieves the state-of-the-art performance in cell type clustering, especially in challenging scenarios of non-overlapping cell types. In addition, OCAT efficaciously facilitates a variety of downstream analyses, such as differential gene analysis, trajectory inference, pseudotime inference and cell inference. OCAT is a unifying tool to simplify and expedite the analysis of large-scale scRNA-seq data from heterogeneous sources.

genomics↗

bioRxiv2021

A high-continuity and annotated tomato reference genome

Genetic and functional genomics studies require a high-quality genome assembly. Tomato (Solanum lycopersicum), an important horticultural crop, is an ideal model species for the study of fruit development. Here, we assembled an updated reference genome of S. lycopersicum cv. Heinz 1706 that was 799.09 Mb in length, containing 34,384 predicted protein-coding genes and 65.66% repetitive sequences. By comparing the genomes of S. lycopersicum and S. pimpinellifolium LA2093, we found a large number of genomic fragments probably associated with human selection, which may have had crucial roles in the domestication of tomato. Our results offer opportunities for understanding the evolution of the tomato genome and will facilitate the study of genetic mechanisms in tomato biology. Information for the assembled genome SLT1.0 was deposited both into the Genome Warehouse (GWH) database (https://bigd.big.ac.cn/gwh/) in the BIG Data Center under Accession Number GWHBAUD00000000.

genomics↗

bioRxiv2021

Transcriptome diversity is a systematic source of bias in RNA-sequencing data

BackgroundRNA sequencing has been widely used as an essential tool to probe gene expression. While standard practices have been established to analyze RNA-seq data, it is still challenging to detect and remove artifactual signals. Several factors such as sex, age, and sequencing technology have been found to bias these estimates. Probabilistic estimation of expression residuals (PEER) has been used to account for some systematic effects, but it has remained challenging to interpret these PEER factors. ResultsHere we show that transcriptome diversity - a simple metric based on Shannon entropy - explains a large portion of variability in gene expression, and is a major factor detected by PEER. We then show that transcriptome diversity has significant associations with multiple technical and biological variables across diverse organisms and datasets. This prevalent confounding factor provides a simple explanation for a major source of systematic biases in gene expression estimates. ConclusionsOur results show that transcriptome diversity is a metric that captures a systematic bias in RNA-seq and is the strongest known factor encoded in PEER covariates.

genomics↗

bioRxiv2021

Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection

Planarians have long been studied for their regenerative abilities. Moving forward, tools for ectopic expression of non-native proteins will be of substantial value. Using a luminescent reporter to overcome the strong autofluorescence background of planarian tissues, we demonstrate heterologous protein expression in planarian cells and live animals. Our approach is based on the introduction of mRNA through several nanotechnological and chemical transfection methods. We improve reporter expression by altering untranslated region (UTR) sequences and codon bias, facilitating measurement of expression kinetics both in isolated cells and in whole planarians using luminescence imaging. We also examine protein expression as a function of variations in the UTRs of delivered mRNA, demonstrating a framework to investigate gene regulation at the post-transcriptional level. Together, these advances expand the toolbox for the mechanistic analysis of planarian biology and establish a strong foundation for the development and expansion of transgenic techniques in this unique model system. MotivationThe study of planarians has contributed to advances in our understanding of regeneration, stem cell dynamics, and many other fundamental biological processes. However, the persistent challenge of expressing transgenes in planarians has led to the speculation that they may be resistant to transfection. In this work, we develop methods to express exogenous mRNAs in both isolated planarian cells and whole animals by optimizing delivery techniques, genetic constructs, and detection methods. These methods allow us to study transfection kinetics and post-transcriptional regulation of gene expression in a quantitative manner. Beyond planarian research, this work should also provide a broadly applicable strategy to develop similar tools for animals that are also challenging to modify genetically.

bioengineering↗