Biology subjects

Wu, Y.

Publications and source records attributed to Wu, Y..

At least 703 records · Page 39Linked to original sources

bioRxiv2022

Efficient repair of human homozygous genetic mutation by CRISPR/Cas9 mediated interlocus gene conversion

DNA double-strand breaks (DSBs) induced by gene editing tools are primarily resolved either by non-homologous end joining (NHEJ) or homology-directed repair (HDR) using exogenous synthetic DNA templates. Repaired by error-prone NHEJ may lead to unexpected indels at the targeted site. In the case of most genetic disorders, HDR-mediated precise correction using an exogenous homologous sequence is ideal. However, the therapeutic application of HDR might be especially challenging given the requirement for the codelivery of exogenous DNA templates with toxicity into cells, and the low efficiency of HDR could also limit its clinical application. Here, we used hematopoietic stem cells (HSCs) with genetic mutations to cause {beta}-thalassemia in HBB coding regions and discovered that many cells are actually repaired by CRISPR/Cas9-mediated gene conversion (GC) independent of exogenous synthetic DNA templates. We show that pathogenic mutations in the HBB coding regions of HSCs can be repaired efficiently through CRISPR/GC using the paralog gene HBD as the internal template. Electroporations of Cas9 for ribonucleoprotein with sgRNA into haematopoietic stem and progenitor cells (HSPCs) with a variety of pathogenic gene mutations also resulted in effective conversion of mutations to normal wild-type sequences without exogenous DNA template. Moreover, the edited HSCs can repopulate the haematopoietic system and generate erythroid cells with a greatly reduced propensity for thalassemia after transplantations. Thus, CRISPR/GC, independent of exogenous DNA templates, holds great promise for gene therapy of genetic diseases.

genetics↗

bioRxiv2022

Re-expression of CA1 and entorhinal activity patterns preserves temporal context memory at long timescales

Converging, cross-species evidence indicates that memory for time is supported by hippocampal area CA1 and entorhinal cortex. However, limited evidence characterizes how these regions preserve temporal memories over long timescales (e.g., months). At long timescales, memoranda may be encountered in multiple temporal contexts, potentially creating interference. Here, using 7T fMRI, we measured CA1 and entorhinal activity patterns as human participants viewed thousands of natural scene images distributed, and repeated, across many months. We show that memory for an images original temporal context was predicted by the degree to which CA1/entorhinal activity patterns from the first encounter with an image were re-expressed during re-encounters occurring minutes to months later. Critically, temporal memory signals were dissociable from predictors of recognition confidence, which were carried by distinct medial temporal lobe expressions. These findings suggest that CA1 and entorhinal cortex preserve temporal memories across long timescales by coding for and reinstating temporal context information.

neuroscience↗

bioRxiv2022

eIF3 mRNA selectivity profiling reveals eIF3k as a cancer-relevant regulator of ribosome content

eIF3, whose subunits are frequently overexpressed in cancer, regulates mRNA translation from initiation to termination, but mRNA-selective functions of individual subunits remain poorly defined. Using multi-omic profiling upon acute depletion of eIF3 subunits, we observed that while eIF3a, b, e, and f markedly differed in their impact on eIF3 holo-complex formation and translation, they were each required for cancer cell proliferation and tumor growth. Remarkably, eIF3k showed the opposite pattern with depletion promoting global translation, cell proliferation, tumor growth, and stress resistance through repressing the synthesis of ribosomal proteins, especially RPS15A. Whereas ectopic expression of RPS15A mimicked the anabolic effects of eIF3k depletion, disruption of eIF3 binding to the 5-UTR of RSP15A mRNA negated them. Supported by mathematical modeling, our data uncovers eIF3k-l as a mRNA-specific module which, through controlling RPS15A translation, serves as a rheostat of ribosome content to secure spare translational capacity that can be mobilized during stress.

cell biology↗

bioRxiv2022

Active bulging promotes biofilm formation in a bacterial swarm

Microbial communities such as biofilms are commonly found at interfaces. However, it is unclear how the physical environment of interfaces may contribute to the development and behavior of surface-associated microbial communities. Combining multi-mode imaging, single-cell tracking and numerical simulations, here we discovered that an interfacial process denoted as "active bulging" promotes biofilm formation. During this process, an initially two-dimensional layer of swarming bacteria spontaneously develops scattered liquid bulges; the bulges have a higher propensity to transit from motile to sessile biofilm state, presumably due to the enrichment of pre-existing immotile cells in the colony. We further demonstrate that the formation of liquid bulges can be controlled reversibly by manipulating the speed and local density of cells with light. Our findings reveal a unique physical mechanism of biofilm formation and provide a new strategy for biofilm patterning in engineered living materials as well as for directed self-assembly in active fluids.

biophysics↗

bioRxiv2022

Defence systems provide synergistic anti-phage activity in E. coli

Bacterial defence against phage predation involves diverse defence systems acting both individually and concurrently, yet their interactions remain poorly understood. We investigated >100 defence systems in 42,925 bacterial genomes and identified numerous instances of their non-random co-occurrence and mutual exclusion. For several pairs of defence systems significantly co-occurring in Escherichia coli strains, we demonstrate synergistic anti-phage activity, including tmn synergising with defence systems containing sensory switch ATPase domains. Some of the defence system pairs that are mutually exclusive in E. coli significantly co-occur in other taxa, and one tested mutually exclusive pair showed synergy. These findings demonstrate compatibility and synergy between defence systems, allowing bacteria to adopt flexible strategies for phage defence, driven by specific environmental pressures. The evolution of bacterial immune repertoires seems to be shaped largely by selection for resistance to host-specific phages rather than by negative epistasis.

microbiology↗

bioRxiv2022

Targeted protein degradation reveals BET bromodomains as the cellular target of Hedgehog Pathway Inhibitor-1

Target deconvolution of small molecule hits from phenotypic screens presents a major challenge. Illustrative of these are the many screens that have been conducted to find inhibitors for the Hedgehog (Hh) signaling pathway - a major developmental pathway with many implications in health and disease - with many hits but very few identified cellular targets. We here present a strategy for target identification based on Proteolysis-Targeting Chimeras (PROTACs), combined with label-free quantitative proteomics. We developed a PROTAC based on the downstream Hedgehog Pathway Inhibitor-1 (HPI-1), a phenotypic screen hit with unknown cellular target. Using our Hedgehog Pathway PROTAC (HPP) we identified and validated BET bromodomains to be the cellular targets of HPI-1. Furthermore, we found that HPP-9 has a unique mechanism of action as a long-acting Hh pathway inhibitor through prolonged BET bromodomain degradation. Collectively, we provide a powerful PROTAC-based approach for target deconvolution, that has answered the longstanding question of the cellular target of HPI-1 and yielded the first PROTAC that acts on the Hh pathway.

cell biology↗

bioRxiv2022

ATG9 vesicles comprise the seed membrane of mammalian autophagosomes

During autophagosome biogenesis, the incorporation of transmembrane proteins into the expanding phagophore is not readily observed. In addition, the membrane surface area of the organelle expands rapidly, while the volume of the autophagosome is kept low. Several recent studies have suggested a model of membrane expansion that explains how these attributes are maintained. The autophagosome expands predominantly through the direct protein-mediated transfer of lipids through the lipid transfer protein ATG2. As these lipids are only introduced into the cytoplasmic-facing leaflet of the expanding phagophore, full membrane growth also requires lipid scramblase activity. ATG9 has been demonstrated to harbor scramblase activity and is essential to autophagosome formation, however if and when it is integrated into mammalian autophagosomes remains unclear. Here we show that in the absence of lipid transport, ATG9 vesicles are already fully competent to collect proteins normally found on mature autophagosomes, including LC3-II. Further, through the novel use of styrene-maleic acid lipid particles as a nanoscale interrogation of protein organization on intact membranes, we show that ATG9 is fully integrated in the same membranes as LC3-II, even on maturing autophagosomes. The ratios of these two proteins at different stages of maturation demonstrate that ATG9 proteins are not continuously integrated, but rather are present on the seed vesicles only and become diluted in the rapidly expanding autophagosome membrane. Thus, ATG9 vesicles are the seed membrane from which mammalian autophagosomes form.

cell biology↗

bioRxiv2022

Membrane remodeling properties of the Parkinson's disease protein LRRK2

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are responsible for late-onset autosomal dominant Parkinsons disease (PD). LRRK2 has been implicated in a wide range of physiological processes including membrane repair in the endolysosomal system. Here, using cell free systems, we report that purified LRRK2 directly binds acidic lipid bilayers with a preference for highly curved bilayers. While this binding is nucleotide independent, LRRK2 can also deform low curvature liposomes into narrow tubules in a guanylnucleotide-dependent but ATP-independent way. Moreover, assembly of LRRK2 into scaffolds at the surface of lipid tubules can constrict them. We suggest that an interplay between the membrane remodeling and signaling properties of LRRK2 may be key to its physiological function. LRRK2, via its kinase activity, may achieve its signaling role at sites where membrane remodeling occurs. Significance StatementLRRK2 is one of the most frequently mutated gene in familial Parkinsons disease. While much has been learned about its molecular properties, upstream regulators and protein substrates of its kinase activity, its precise function remains unclear. Recent evidence has pointed to a role of LRRK2 in membrane repair in the endo/lysosomal system. Here we show that purified LRRK2 has membrane remodeling properties. We suggest that its ability to sense and induce membrane curvature may be key to its function in membrane dynamics. These properties may help coordinate a direct role of LRRK2 at the membrane interface with its the signaling role of its kinase domain.

cell biology↗

bioRxiv2022

Acrylamide Induces Protein Aggregation in CNS through Suppression of FoxO1-mediated Autophagy.

Proteotoxic stress is a major stimulus and risk factor for the neuropathogenesis in central nervous system (CNS), which is tightly associated with neurodegenerative diseases. Here, we identify acrylamide (ACR), a type-2 alkene that is commonly detected in deep-fried starch food and used in water industry and biomedical laboratories, as a potent and universal inducer of proteotoxic stress in mouse brain, as well as in cultured mouse neural stem cells, neurons and astrocytes, three major cell types in CNS. Aggregations of ubiquitin-labeled misfolded proteins and neurodegeneration-related proteins including amyloid precursor protein and presenilin 1 were drastically induced by ACR, leading to cell-defensive aggresome formation that was able to temporarily counteract with ACR toxicity. However, a defected clearance of aggresomes eventually led to apoptotic cell death, which was largely attributed to the breakdown of cytoskeleton and impairment of macroautophagy/autophagy, as evidenced by an aggregation of filament actin and an almost complete loss of LC3-positive autophagosomes. A series of core autophagy-related genes responsible for autophagosome formation were down-regulated, indicating an ACR-induced transcriptional suppression of autophagy. In addition, FoxO1, the master transactivator of these genes, were both transcriptionally repressed and nuclear excluded by ACR. Overexpression of FoxO1 rescued ACR-induced autophagy defects and attenuated the proteotoxicity. In summary, we spotlight proteotoxic stress as a novel feature of ACR neurotoxicity in CNS, and implicate FoxO1 as a critical therapeutic target.

pathology↗

bioRxiv2022

PhaeoEpiView: An epigenome browser of the newly assembled genome of the model diatom Phaeodactylum tricornutum

MotivationRecent advances in DNA sequencing technologies in particular of long reads type greatly improved genomes assembly leading to discrepancies between both published annotations and epigenome tracks which did not keep pace with new assemblies. This comprises the availability of accurate resources which penalizes the progress in research. ResultsHere, we used the latest improved telomere to telomere assembly of the model pennate diatom Phaeodactylum tricornutum to lift over the gene models from Phatr3, a previously annotated reference genome. We used the lifted genome annotation including genes and transposable elements to map the epigenome landscape, namely DNA methylation and post translational modifications of histones providing the community with PhaeoEpiView, a browser that allows the visualization of epigenome data as well as transcripts on an updated reference genome to better understand the biological significance of the mapped data on contiguous genome rather than a fragmented one. We updated previously published histone marks with a more accurate mapping using monoclonal antibodies instead of polyclonal and deeper sequencing. PhaeoEpiView will be continuously updated with the newly published epigenomic data making it the largest and richest epigenome browser of any stramenopile. We expect that PhaeoEpiView will be a standard tool for the coming era of molecular environmental studies where epigenetics holds a place of choice. AvailabilityPhaeoEpiView is available at: https://PhaeoEpiView.univ-nantes.fr

bioinformatics↗

bioRxiv2022

Genome-wide investigation of multiplexed CRISPR-Cas12a-mediated editing in rice

We previously reported highly specific genome editing in rice by CRISPR-Cas9 and Cas12a with single DNA double strand break (DSB) (Tang et al., 2018). Two concurrent DSBs by CRISPR-Cas9 could generate defined deletions (Zhou et al., 2014), inversions (Schmidt et al., 2020), and translocations (Beying et al., 2020). Off-target effects of many simultaneous DSBs are unknown in plants. Here, we used whole-genome sequencing (WGS) to investigate off-target effects in rice plants edited by highly multiplexable CRISPR-Cas12a systems (Zhang et al., 2021). Our WGS study revealed highly specific multiplexed genome editing by CRISPR-Cas12a. We found low-frequency large chromosomal deletion and duplication events in plants that endured many (e.g., >50) simultaneous DSBs, but not in plants that endured a lower order DSBs (e.g., <10). While our short reads sequencing may not capture all chromosomal rearrangements, our results nevertheless shed important light on the analysis and regulation of engineered crops derived from multiplexed genome editing.

genomics↗

bioRxiv2022

TIST: Transcriptome and Histopathological Image Integrative Analysis for Spatial Transcriptomics

Sequencing-based spatial transcriptomics (ST) is an emerging technique to study in situ gene expression patterns at the whole-genome scale. In addition to transcriptomic data, the technique usually generates matched histopathological images for the same tissue sample. ST data analysis is complicated by severe technical noise; matched histopathological images with high spatial continuity and resolution introduce complementary cellular phenotypical information and provide a chance to mitigate the noise in ST data. Hence, we propose a novel ST data analysis method called transcriptome and histopathological image integrative analysis for spatial transcriptomics (TIST), which integrates the information from sequencing-based ST data and histopathological images. TIST uses a Markov random field (MRF) model to learn the macroscopic cellular features from histopathological images and devises a random-walk-based strategy to integrate the extracted image features, the transcriptomic features and the location information for spatial cluster (SC) identification and gene expression enhancement. We tested TIST both on simulated datasets and on 33 real datasets; we found that TIST achieved superior performance on multiple tasks, which illustrates the utility of this method in facilitating the discovery of biological insights from sequencing-based ST data.

bioinformatics↗

bioRxiv2022

Novel methods to identify TCM constitution of hyperlipidemic patients and predict atherosclerotic diseases

Hyperlipidemia can accelerate the progression of atherosclerosis, ultimately leading to cardiovascular disease. TCM constitution has been widely used as an indicator of health status and susceptibility to diseases. However, there still lack of objective, credible ways to identify TCM constitution of hyperlipidemic patients, and the connection between TCM constitution and atherosclerotic diseases in individuals with hyperlipidemia is unknown. This study aims to identify differentially expressed microRNAs (DEMs) as biomarkers of the TCM constitution of hyperlipidemic patients and explore the potential connection between TCM constitution and atherosclerotic diseases. In the study, we randomly recruited 10 hyperlipidemic patients with asthenic constitution (HAC), 10 hyperlipidemic patients with strong constitution (HSC), and 10 hyperlipidemic patients with normal constitution (HNC) and collected serum miRNA. After analyzing the miRNA expression profiles, we found that hsa-miR-338-3p may be a potential blood biomarker for the identification of the TCM constitution of hyperlipidemic patients. Moreover, the HSC classification is likely a cardiocerebrovascular disease predisposition and is closely related to the inflammatory process and glucose and lipid metabolism, which contribute to the development of atherosclerotic diseases.

bioinformatics↗

bioRxiv2022

Three-dimensional structured illumination microscopy with enhanced axial resolution

We present two distinct, complementary methods for improving axial resolution in three-dimensional structured illumination microscopy (3D SIM) with minimal or no modification to the optical system. First, we show that placing a mirror directly opposite the sample enables 4-beam interference with higher spatial frequency content than 3D SIM illumination, offering near-isotropic imaging with [~]120 nm lateral and 160 nm axial resolution. Second, we develop an improved deep learning method that can be directly applied to 3D SIM data, obviating the need for additional hardware. This procedure results in [~]120 nm isotropic resolution and can be combined with denoising to facilitate volumetric imaging spanning dozens of time points. We demonstrate the potential of these advances by imaging a variety of cellular samples, delineating the nanoscale distribution of vimentin and microtubule filaments, observing the relative positions of caveolar coat proteins and lysosomal markers, and visualizing rich cytoskeletal dynamics within T-cells in the early stages of immune synapse formation.

cell biology↗

bioRxiv2022

Genomic mapping of the modifiers of Teosinte crossing barrier 1 (Tcb1)

Pollen cross-contamination has been a major problem for maize breeders. Mechanical methods applied to avoid cross-contamination are largely ineffective and time-consuming. Cross incompatibility barriers are genetic factors involved in maize fertilization that can be used as an effective method to prevent pollen cross-contamination. Teosinte crossing barrier 1 (Tcb1) is a cross-incompatibility system in which silks possessing dominant Tcb1-s reject pollen possessing the recessive allele (tcb1). However, successful fertilization occurs when Tcb1-s pollen falls upon tcb1 silks or under self-fertilization of Tcb1-s pollen on Tcb1-s silks. Previous studies have shown that the efficacy of dominant Tcb1-s was reduced when repeatedly backcrossing with maize inbred lines suggesting the presence of modifiers to Tcb1-s. To find those modifiers, we conducted a QTL mapping experiment using the Intermated B73 x Mo17 (IBM) recombinant inbred lines (RILs) for two consecutive years. Two significant and stable QTL were identified on chromosomes 4L and 5S explained 16% and 17.6% of the total phenotypic variation (R2), and both had negative additive effects. Further investigation of these QTL regions identified twelve candidate genes that could modify Tcb1-s activity. The introgression of the Tcb1-s genetic system, and its appropriate modifying factors, could be a novel and reliable solution for cultivar isolation in maize breeding.

plant biology↗

bioRxiv2022

Cryptochrome Stabilization Ameliorates Chronic Pain

Physiological and pathological pain exhibits striking diurnal variation, but the underlying mechanisms are largely unknown. We now describe an independent molecular clock in peripheral sensory neurons and satellite glial cells of sensory ganglia. We show that it is the sensory neuron transcription-translation feedback loops (TTFLs) that are responsible for diurnal pain behaviors. This clock regulates diurnal neurophysiological responses to a range of ligands, as well as synaptic activities of primary nociceptors. Furthermore, we find that loss of Cry1 and Cry2, the repressive arm of the core TTFLs, intensifies pain responses associated with increased voltage-gated sodium channel currents. Conversely, stabilization of CRY1 and CRY2 using the small molecule KL001, reduces pain sensitivity. Our results highlight novel opportunities to address chronic pain by directly harnessing circadian mechanisms. One-Sentence SummaryA peripheral pain clock governs daily pain fluctuations, which can be harnessed for treating pain disorders.

neuroscience↗

bioRxiv2022

Structural characterization of human RPA70N association with DNA damage response proteins

The heterotrimeric Replication protein A (RPA) is the ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein and participates in nearly all aspects of DNA metabolism, especially DNA damage response. The N-terminal OB domain of the RPA70 subunit (RPA70N) is a major protein-protein interaction element for RPA. Previous crystallography studies of RPA70N with p53, DNA2 and PrimPol fragments revealed that RPA70N binds to amphipathic peptides that mimics ssDNA. NMR chemical-shift studies also provided valuable information of RPA70N residues interacting with target sequences. However, it is still not clear how RPA70N recognizes and distinguishes such a diverse group of proteins. Here we present high resolution crystal structures of RPA70N in complex with peptides from HelB, ATRIP, RMI1, WRN and BLM. The structures showed that in addition to the ssDNA mimicry mode of interaction, RPA70N employs multiple ways to bind its partners, some of which may serve to increase the avidity of RPA70N binding.

biophysics↗

bioRxiv2022

Early Life Exposure to Queen Mandibular Pheromone Mediates Persistent Transcriptional Changes in the Brain of Honey bee Foragers

How behavior in insect societies is regulated remains a fundamental question in sociobiology. In hymenopteran societies, the queen plays a crucial role in regulating group behavior by affecting individual behavior, physiology, and lifespan through worker gene expression. Honey bee (Apis mellifera) queens signal their presence via the queen mandibular pheromone (QMP). While QMP has been shown to influence the behavior of young workers, we know little about its long-term molecular impacts on workers and whether these pheromone effects depend on an early sensitive period in the life of a worker. Here we demonstrate that QMP treatment strongly impacts long-term forager gene expression in the mushroom bodies, antennal lobes, and antennae, but only if bees were treated early in life (1-2 days of age). Contrary to our expectation, foraging activity was not impacted by QMP treatment in the long-term, but genes important for division of labor, learning, chemosensory perception and aging were differentially expressed in the antennae and brain tissues, suggesting that QMP influences diverse physiological and behavioral processes in workers. Overall, our study suggests a sensitive period early in the life of workers, where the queens presence or absence has strong and potentially livelong effects on transcriptional activity in the central and peripheral nervous system. Significance statementDespite our increasing understanding of how social cues affect gene expression and behavior in social animals, we still know little about the importance of sensitive periods in cue perception for the long-term regulation of gene expression. Honey bees live in complex societies and queen pheromones play a central role in the regulation of worker behavior and division of labor. We tested the exposure to queen pheromone presence and found that there is a sensitive period in the early adult life of workers. Understanding and identifying sensitive periods and their effect on long-term gene transcription in workers in response to changes in the environment will lay an important foundation for a better understanding of how queens shape colony life.

animal behavior and cognition↗