Biology subjects

Wu, Y.

Publications and source records attributed to Wu, Y..

At least 793 records · Page 44Linked to original sources

bioRxiv2021

AN ECONOMIC, SQUARE-SHAPED FLAT-FIELD ILLUMINATION MODULE FOR TIRF-BASED SUPER-RESOLUTION MICROSCOPY

Super-resolution (SR) microscopy allows complex biological assemblies to be observed with remarkable resolution. However, the presence of uneven Gaussian-shaped illumination hinders its use in quantitative imaging or high-throughput assays. Methods developed to circumvent this problem are often expensive, hard-to-implement, or not applicable to total internal reflection fluorescence (TIRF) imaging. We herein demonstrate a cost-effective method to overcome these challenges using a small square-core multimodal optical fibre as the coupler. We characterise our method with synthetic, recombinant and cellular systems imaged under TIRF and highly inclined and laminated optical sheet (HILO) illuminations to demonstrate its ability to produce highly uniform images under all conditions.

biophysics↗

bioRxiv2021

Circuit-specific sonogenetic stimulation of the deep brain elicits distinct signaling and behaviors in freely moving mice

Sonogenetics uses heterologously-expressed proteins to sensitize neurons to ultrasound, enabling selective, non-invasive, and deep brain stimulation. However, its ability to modulate specific circuits or induce behavioral changes remains to be studied and characterized. Here, we demonstrate that sonogenetics enables efficient activation of well-defined neural circuits by transcranial low-intensity, low-frequency ultrasonic stimulation with high spatiotemporal resolution. Targeted neurons in subcortical regions were made to express a mechanosensitive ion channel (MscL-G22S). Ultrasound could trigger activity in MscL-expressing neurons in the dorsal striatum without increased activation in neighboring regions, and increase locomotion in freely-moving mice. Ultrasound stimulation of MscL-expressing neurons in the ventral tegmental area could activate the mesolimbic pathway to trigger dopamine release in the nucleus accumbens and modulate appetitive conditioning. In MscL-expressing cells, neuronal responses to ultrasound pulses were rapid, reversible and repeatable. Altogether, we show that sonogenetics can selectively manipulate targeted cells to activate defined neural pathways and affect behaviors.

neuroscience↗

bioRxiv2021

SHIP164 is a Chorein Motif Containing Lipid Transport Protein that Controls Membrane Dynamics and Traffic at the Endosome-Golgi Interface.

Cellular membranes differ in protein and lipid composition as well as in the protein-lipid ratio. Thus, progression of membranous organelles along traffic routes requires mechanisms to control bilayer lipid chemistry and their abundance relative to proteins. The recent structural and functional characterization of VPS13-family proteins has suggested a mechanism through which lipids can be transferred in bulk from one membrane to another at membrane contact sites, and thus independently of vesicular traffic. Here we show that SHIP164 (UHRF1BP1L) shares structural and lipid transfer properties with these proteins and is localized on a subpopulation of vesicle clusters in the early endocytic pathway whose membrane cargo includes the cation-independent mannose-6-phosphate receptor (MPR) and ATG9. Loss of SHIP164 disrupts retrograde traffic of these organelles to the Golgi complex. Our findings raise the possibility that bulk transfer of lipids to endocytic membranes may play a role in their traffic.

cell biology↗

bioRxiv2021

Mapping neurotransmitter systems to the structural and functional organization of the human neocortex

Neurotransmitter receptors support the propagation of signals in the human brain. How receptor systems are situated within macroscale neuroanatomy and how they shape emergent function remains poorly understood, and there exists no comprehensive atlas of receptors. Here we collate positron emission tomography data from >1 200 healthy individuals to construct a whole-brain 3-D normative atlas of 19 receptors and transporters across 9 different neurotransmitter systems. We find that receptor profiles align with structural connectivity and mediate function, including neurophysiological oscillatory dynamics and resting state hemodynamic functional connectivity. Using the Neurosynth cognitive atlas, we uncover a topographic gradient of overlapping receptor distributions that separates extrinsic and intrinsic psychological processes. Finally, we find both expected and novel associations between receptor distributions and cortical thinning patterns across 13 disorders. We replicate all findings in an independently collected autoradiography dataset. This work demonstrates how chemoarchitecture shapes brain structure and function, providing a new direction for studying multi-scale brain organization.

neuroscience↗

bioRxiv2021

Cytonemes coordinate asymmetric signaling and organization in the Drosophila muscle progenitor niche

Asymmetric signaling and organization in the stem-cell niche determine stem-cell fates. Here, we investigate the basis of asymmetric signaling and stem-cell organization using the Drosophila wing-disc that creates an adult muscle progenitor (AMP) niche. We show that AMPs extend polarized cytonemes to contact the disc epithelial junctions and adhere themselves to the disc/niche. Niche-adhering cytonemes localize FGF-receptor to selectively adhere to the FGF-producing disc and receive FGFs in a contact-dependent manner. Activation of FGF signaling in AMPs, in turn, reinforces disc-specific cytoneme polarity/adhesion, which maintains their disc-proximal positions. Loss of cytoneme-mediated adhesion promotes AMPs to lose niche occupancy and FGF signaling, occupy a disc-distal position, and acquire morphological hallmarks of differentiation. Discrete AMP organization and diversification patterns are determined by localized expression and presentation patterns of two different FGFs in the wing-disc and their polarized target-specific distribution through niche-adhering cytonemes. Thus, cytonemes are essential for asymmetric signaling and niche-specific AMP organization.

developmental biology↗

bioRxiv2021

The Ribb-osome: Ribbon boosts ribosomal protein gene expression to coordinate organ form and function

Cell growth is well defined for the late (post-embryonic) stages of development, but evidence for early (embryonic) cell growth during post-mitotic morphogenesis is quite limited. Here, we identify early cell growth as a key characteristic of tubulogenesis in the Drosophila embryonic salivary gland (SG). A BTB/POZ domain nuclear factor, Ribbon (Rib), mediates this early cell growth. Rib binds the transcription start site of nearly every SG-expressed ribosomal protein gene (RPG) and is required for full expression of all RPGs tested. Rib binding to RPG promoters in vitro is weak and not sequence-specific, suggesting that specificity is achieved through co-factor interactions. Consistent with this hypothesis, we demonstrate Ribs ability to physically interact with each of the three known contributors to RPG transcription. Surprisingly, Rib-dependent early cell growth in another tubular organ--the embryonic trachea--is not mediated by direct RPG transcription. These findings support a model of early cell growth sustained by transcriptional regulatory networks customized for organ form and function.

developmental biology↗

bioRxiv2021

Yn-situ: a robust single RNA molecule in situ detection method

We describe a cost-effective, highly sensitive, and quantitative method for in situ detection of single RNA molecules in tissue sections. This method, dubbed Yn situ, standing for Y-branched probe in situ hybridization, uses a single-strand DNA preamplifier with multiple initiation sites that trigger hybridization chain reaction (HCR) to detect polynucleotide. We characterized the performance of this method and compared it to other approaches in the postnatal mouse olfactory epithelia. We find that the Yn situ method, in conjunction with an improved fixation step, is sensitive enough to allow detection of single molecules using a single pair of probes targeting a short nucleotide sequence. A set of 5-probes can produce quantitative results with smaller puncta and higher signal-to-noise ratio than the 20-probe sets commonly required for HCR and RNA-Scope. We show that the high sensitivity and wide dynamic range allow quantification of genes expressed at different levels in the olfactory sensory neurons. We describe key steps of this method to enable broad utility by individual laboratories.

bioengineering↗

bioRxiv2021

Machine Learning Prediction of Non-Coding Variant Impact in Human Retinal Cis-Regulatory Elements

PurposePrior studies demonstrate the significance of specific cis-regulatory variants in retinal disease, however determining the functional impact of regulatory variants remains a major challenge. In this study, we utilize a machine learning approach, trained on epigenomic data from the adult human retina, to systematically quantify the predicted impact of cis-regulatory variants. MethodsWe used human retinal DNA accessibility data (ATAC-seq) to determine a set of 18.9k high-confidence putative cis-regulatory elements. 80% of these elements were used to train a machine learning model utilizing a gapped k-mer support vector machine-based approach. In silico saturation mutagenesis and variant scoring was applied to predict the functional impact of all potential single nucleotide variants within cis-regulatory elements. Impact scores were tested in a 20% hold-out dataset and compared to allele population frequency, phylogenetic conservation, transcription factor (TF) binding motifs, and existing massively parallel reporter assay (MPRA) data. ResultsWe generated a model that distinguishes between human retinal regulatory elements and negative test sequences with 95% accuracy. Among a hold-out test set of 3.7k human retinal CREs, all possible single nucleotide variants (SNVs) were scored. Variants with negative impact scores correlated with reduced population allele frequency, higher phylogenetic conservation of the reference allele, disruption of predicted TF binding motifs, and massively-parallel reporter expression. ConclusionsWe demonstrated the utility of human retinal epigenomic data to train a machine learning model for the purpose of predicting the impact of non-coding regulatory sequence variants. Our model accurately scored sequences and predicted putative transcription factor binding motifs. This approach has the potential to expedite the characterization of pathogenic non-coding sequence variants in the context of unexplained retinal disease.

genomics↗

bioRxiv2021

MiR-339-3p aggravates rat vascular inflammation induced by AT1R autoantibodies by down-regulating BKα protein expression

The abnormality of large-conductance calcium-activated potassium channels (BK channels) is an important factor in inducing vascular inflammation. BK channel agonists can readily recover BK channel function and improve vascular inflammation. However, it is not clear how to improve BK dysfunction caused by downregulation of BK channel protein expression. This study found that angiotensin II-1 receptor autoantibodies (AT1-AA), which are widely present in the body of various types of cardiovascular diseases, can down-regulate the expression of BK channel protein and induce vascular inflammation. Further research found that the elevated neural precursor cells expressed developmentally downregulated 4-like (NEDD4L) protein level is involved in the down-regulation of BK channel subunit (BK) protein level by AT1-AA. Bioinformatics analysis and experiments have confirmed that miR-339-3p plays an irreplaceable role in the high expression of NEDD4L and the low expression of BK, and aggravates the vascular inflammation induced by AT1-AA. Overall, AT1-AA increased miR-339-3p expression (targeting BK via the miR-339-3p/NEDD4L axis or miR-339-3p alone), reduced BK protein expression in VSMCs, and induced vascular inflammation. The results of the study indicate that miR-339-3p may become a new target for reversing vascular inflammation in AT1-AA-positive patients.

molecular biology↗

bioRxiv2021

CsMYB4a from Camellia sinensis Regulates the Auxin Signaling Pathway by Interacting with CsIAA4

Members of the R2R3-MYB4 subgroup are well-known negative regulatory transcription factors of phenylpropane and lignin pathways. In this study, we found that transgenic tobacco plants overexpressing a R2R3-MYB4 subgroup gene from Camellia sinensis (CsMYB4a) showed inhibited growth that was not regulated by phenylpropane and lignin pathways, and these plants exhibited altered sensitivity to synthetic auxin 1-naphthaleneacetic acid (-NAA) treatment. An auxin/indole-3-acetic acid 4 (AUX/IAA4) gene from Camellia sinensis (CsIAA4) participating in the regulation of the auxin signal transduction pathway was screened from the yeast two-hybrid library with CsMYB4a as the bait protein, and tobacco plants overexpressing this gene showed a series of auxin-deficiency phenotypes, such as dwarfism, small leaves, reduced lateral roots, and a shorter primary root. CsIAA4 transgenic tobacco plants were less sensitive to exogenous -NAA than control plants, which was consistent with the findings for CsMYB4a transgenic tobacco plants. The knockout of the endogenous NtIAA4 gene (a CsIAA4 homologous gene) in tobacco plants alleviated growth inhibition in CsMYB4a transgenic tobacco plants. Furthermore, protein-protein interaction experiments proved that domain II of CsIAA4 is the key motif for the interaction between CsIAA4 and CsMYB4a and that the degradation of CsIAA4 is prevented when CsMYB4a interacts with CsIAA4. In summary, our results suggest that CsMYB4a is a multifunctional transcription factor that regulates the auxin signaling pathway, phenylpropane and lignin pathways. This study provides new insights into the multiple functions of R2R3-MYB4 subgroup members as a group of well-known negative regulatory transcription factors. One-sentence summaryCsMYB4a act as multifunctional transcription factor that regulates the auxin signaling pathway, phenylpropane and lignin pathways.

physiology↗

bioRxiv2021

CD47 cross-dressing by extracellular vesicles expressing CD47 inhibits phagocytosis without transmitting cell death signals

Transgenic CD47 overexpression is an encouraging approach to ameliorating xenograft rejection and alloresponses to pluripotent stem cells, and the efficacy correlates with the level of CD47 expression. However, CD47, upon ligation, also transmits signals leading to cell dysfunction or death, raising a concern that overexpressing CD47 could be harmful. Here, we unveiled an alternative source of cell surface CD47. We showed that extracellular vesicles (EVs), including exosomes (Exos), released from normal or tumor cells overexpressing CD47 (transgenic or native) can induce efficient CD47 cross-dressing on pig or human cells. Like the autogenous CD47, CD47 cross-dressed on cell surfaces is capable of interacting with SIRP to inhibit phagocytosis. However, ligation of the autogenous, but not cross-dressed, CD47 induced cell death. Thus, CD47 cross-dressing provides an alternative source of cell surface CD47 that may elicit its anti-phagocytic function without transmitting harmful signals to the cells. CD47 cross-dressing also suggests a previously unidentified mechanism for tumor-induced immunosuppression. Our findings should help to further optimize the CD47 transgenic approach that may improve outcomes by minimizing the harmful effects of CD47 overexpression.

immunology↗

bioRxiv2021

Progressive chromatin silencing of ABA biosynthesis gene permits seed germination in Arabidopsis

Seed germination represents a major developmental switch in plants that is vital to agricultural, but how this process is controlled at the chromatin level remains obscure. Here we demonstrate that successful germination in Arabidopsis requires a chromatin mechanism that progressively silences NCED6, which encodes a rate-limiting enzyme for abscisic acid (ABA) biosynthesis, through the cooperative action of the RNA-binding protein RZ-1 and the polycomb repressive complex 2 (PRC2). Simultaneous inactivation of RZ-1 and PRC2 blocks germination and synergistically derepresses NCEDs and hundreds of genes. At NCED6, by promoting H3 deacetylation and suppressing H3K4me3, RZ-1 facilitates transcriptional silencing and also a H3K27me3 accumulation process that occurs during seed germination and early seedling growth. Genome-wide analysis reveals RZ-1 is preferentially required for transcriptional silencing of many PRC2 targets early during seed germination when H3K27me3 is not yet established. We propose RZ-1 confers a novel silencing mechanism to compensate and coordinate with PRC2. Our work highlights the progressive chromatin silencing of ABA biosynthesis genes via synergized action of the RNA-binding protein RZ1 and PRC2, which is vital for seed germination.

plant biology↗

bioRxiv2021

SARS-CoV-2 causes human BBB injury and neuroinflammation indirectly in a linked organ chip platform

COVID-19 is a multi-system disease affecting many organs outside of the lungs, and patients generally develop varying degrees of neurological symptoms. Whereas, the pathogenesis underlying these neurological manifestations remains elusive. Although in vitro models and animal models are widely used in studies of SARS-CoV-2 infection, human organ models that can reflect the pathological alterations in a multi-organ context are still lacking. In this study, we propose a new strategy to probe the effects of SARS-CoV-2 on human brains in a linked alveolus-BBB organ chip platform. The new multi-organ platform allows to recapitulate the essential features of human alveolar-capillary barrier and blood-brain barrier in a microfluidic condition by co-culturing the organ-specific cells. The results reveal direct SARS-CoV-2 exposure has no obvious effects on BBB chip alone. While, infusion of endothelial medium from infected alveolus chips can cause BBB dysfunction and neuroinflammation on the linked chip platform, including brain endothelium disruption, glial cell activation and inflammatory cytokines release. These new findings suggest that SARS-CoV-2 could induce neuropathological alterations, which might not result from direct viral infection through hematogenous route, but rather likely from systemic inflammation following lung infection. This work provides a new strategy to study the virus-host interaction and neuropathology at an organ-organ context, which is not easily obtained by other in vitro models. This will facilitate to understand the neurological pathogenesis in SARS-CoV-2 and accelerate the development of new therapeutics. SUMMARYO_LIA linked human alveolus-BBB chip platform is established to explore the influences of SARS-CoV-2 on human brains in an organ-organ context. C_LIO_LISARS-CoV-2 infection could induce BBB injury and neuroinflammation. C_LIO_LIThe neuropathological changes are caused by SARS-CoV-2 indirectly, which might be mediated by systemic inflammation following lung infection, but probably not by direct viral neuroinvasion. C_LI

bioengineering↗

bioRxiv2021

A systematic genotype-phenotype map for missense variants in the human intellectual disability-associated gene GDI1

Next generation sequencing has become a common tool in the diagnosis of genetic diseases. However, for the vast majority of genetic variants that are discovered, a clinical interpretation is not available. Variant effect mapping allows the functional effects of many single amino acid variants to be characterized in parallel. Here, we combine multiplexed functional assays with machine learning to assess the effects of amino acid substitutions in the human intellectual disability-associated gene, GDI1. We show that the resulting variant effect map can be used to discriminate pathogenic from benign variants. Our variant effect map recovers known biochemical and structural features of GDI1 and reveals additional aspects of GDI1 function. We explore how our functional assays can aid in the interpretation of novel GDI1 variants as they are discovered, and to re-classify previously observed variants of unknown significance.

genetics↗

bioRxiv2021

Structure selected RBM immunogens prime polyclonal memory responses that neutralize SARS-CoV-2 variants of concern

Successful control of the COVID-19 pandemic depends on vaccines that prevent transmission. The full-length Spike protein is highly immunogenic but the majority of antibodies do not target the virus: ACE2 interface. In an effort to concentrate the antibody response to the receptor-binding motif (RBM) we generated a series of conformationally-constrained immunogens by inserting solvent-exposed RBM amino acid residues into hypervariable loops of an immunoglobulin molecule. Priming C57BL/6 mice with plasmid (p)DNA encoding these constructs yielded a rapid memory response to booster immunization with recombinant Spike protein. Immune sera antibodies bound strongly to the purified receptor-binding domain (RBD) and Spike proteins. pDNA primed for a consistent response with antibodies efficient at neutralizing authentic WA1 virus and two variants of concern (VOC), B.1.351 and B.1.617.2. These findings demonstrate that immunogens built on structure selection can focus the response to conserved sites of vulnerability shared between wildtype virus and VOCs and induce neutralizing antibodies across variants.

immunology↗

bioRxiv2021

Assessing computational variant effect predictors with a large prospective cohort

BackgroundCausal gene/trait relationships can be identified via observation of an excess (or reduced) burden of rare variation in a given gene within humans who have that trait. Although computational predictors can improve the power of such burden tests, it is unclear which are optimal for this task. MethodUsing 140 gene-trait combinations with a reported rare-variant burden association, we evaluated the ability of 20 computational predictors to predict human traits. We used the best-performing predictors to increase the power of genome-wide rare variant burden scans based on [~]450K UK Biobank participants. ResultsTwo predictors--VARITY and REVEL--outperformed all others in predicting human traits in the UK Biobank from missense variation. Genome-scale burden scans using the two best-performing predictors identified 1,038 gene-trait associations (FDR < 5%), including 567 (55%) that had not been previously reported. We explore 54 cardiovascular gene-trait associations (including 15 not reported in other burden scans) in greater depth. ConclusionsRigorous selection of computational missense variant effect predictors can improve the power of rare-variant burden scans for human gene-trait associations, yielding many new associations with potential value in informing mechanistic understanding and therapeutic development. The strategy we describe here is generalizable to future computational variant effect predictors, traits and organisms.

systems biology↗

bioRxiv2021

Activation of the CaMKII-Sarm1-ASK1 MAP kinase pathway protects against axon degeneration caused by loss of mitochondria

Mitochondrial defects are tightly linked to axon degeneration, yet the underlying cellular mechanisms remain poorly understood. In C. elegans, PVQ axons that lack mitochondria degenerate spontaneously with age. Using an unbiased genetic screen, we found that cell-specific activation of CaMKII/UNC-43 suppresses axon degeneration due to loss of mitochondria. Unexpectedly, CaMKII/UNC-43 protects against degeneration through the conserved Sarm1/TIR-1-ASK1/NSY-1 MAPK pathway. In addition, we show that disrupting a trafficking complex composed of calsyntenin/CASY-1, Mint/LIN-10, and kinesin suppresses axon degeneration. Further analysis indicates that disruption of this trafficking complex activates the CaMKII-Sarm1-MAPK pathway through L-type voltage-gated calcium channels. Our findings identify CaMKII as a pivot point between mitochondrial defects and axon degeneration, describe how it is regulated in this context, and uncover a surprising neuroprotective role for the Sarm1-ASK1 pathway.

neuroscience↗

bioRxiv2021

Dysfunction of novel energy sensor NFE2L1 leads to uncontrollable AMPK signal and glucose metabolism reprogramming

NFE2L1 (also called Nrf1) acts a core regulator of redox signaling and metabolism homeostasis, and thus its dysfunction results in multiple systemic metabolic diseases. However, the molecular mechanism(s) by which NFE2L1 regulates glycose and lipid metabolism is still elusive. Here, we found that the loss of NFE2L1 in human HepG2 cells led to a lethal phenotype upon glucose deprivation. The uptake of glucose was also affected by NFE2L1 deficiency. Further experiments unveiled that although the glycosylation of NFE2L1 was monitored through the glycolysis pathway, it enabled to sense the energy state and directly interacted with AMPK. These indicate that NFE2L1 can serve as a dual sensor and regulator of glucose homeostasis. In-depth sights into transcriptome, metabolome and seahorse data further unraveled that glucose metabolism was reprogrammed by disruption of NFE2L1, so as to aggravate the Warburg effect in NFE2L1-silenced hepatoma cells, along with the mitochondrial damage observed under the electron microscope. Collectively, these demonstrate that disfunction of NFE2L1 triggers the uncontrollable signaling by AMPK towards glucose metabolism reprogramming in the liver cancer development.

molecular biology↗