Biology subjects

Zhao, Y.

Publications and source records attributed to Zhao, Y..

At least 649 records · Page 36Linked to original sources

bioRxiv2023

Docking Domain Engineering in a Modular Polyketide Synthase and its Impact on Structure and Function

Modular polyketide synthases (PKSs) are attractive targets for the directed, biosynthetic production of platform chemicals and pharmaceuticals by protein engineering. In this study, we analyze docking domains from the 6-deoxyerythronolide B synthase, SYNZIP domains, and the SpyCatcher:SpyTag complex as engineering tools to couple the polypeptides VemG and VemH to functional venemycin synthases. Our data show that the high-affinity interaction or covalent connection of modules, enabled by SYNZIP domains and the SpyCatcher:SpyTag complex, can be advantageous, e.g., in synthesis at low protein concentrations, but their rigidity and steric demand decrease synthesis rates. However, we also show that efficiency can be recovered when inserting a hinge region distant from the rigid interface. This study demonstrates that engineering approaches should take the conformational properties of modular PKSs into account and establishes a three-polypeptide split-venemycin synthase as an exquisite in vitro platform for the analysis and engineering of modular PKSs.

biochemistry↗

bioRxiv2023

HIV viral protein R induces loss of DCT1-type renal tubules

Hyponatremia and salt wasting is a common occurance in patients with HIV/AIDS, however, the understanding of its contributing factors is limited. HIV viral protein R (Vpr) contributes to HIV-associated nephropathy. To investigate the effects of Vpr on the expression level of the Slc12a3 gene, encoding the Na-Cl cotransporter, which is responsible for sodium reabsorption in distal nephron segments, we performed single-nucleus RNA sequencing of kidney cortices from three wild-type (WT) and three Vpr-transgenic (Vpr Tg) mice. The results showed that the percentage of distal convoluted tubule (DCT) cells was significantly lower in Vpr Tg mice compared with WT mice (P < 0.05), and that in Vpr Tg mice, Slc12a3 expression was not different in DCT cell cluster. The Pvalb+ DCT1 subcluster had fewer cells in Vpr Tg mice compared with WT (P < 0.01). Immunohistochemistry demonstrated fewer Slc12a3+ Pvalb+ DCT1 segments in Vpr Tg mice. Differential gene expression analysis comparing Vpr Tg and WT in the DCT cluster showed Ier3, an inhibitor of apoptosis, to be the most downregulated gene. These observations demonstrate that the salt-wasting effect of Vpr in Vpr Tg mice is mediated by loss of Slc12a3+ Pvalb+ DCT1 segments via apoptosis dysregulation.

pathology↗

bioRxiv2023

Endothelial cell derived microvesicles modulate cerebrovascular and brain aging via miR-17-5p and serve as potential biomarkers for vascular aging

Vascular aging, characterized by brain endothelial cells (ECs) senescence and dysfunction, has been known to lead to various age-related cerebrovascular and neurodegenerative diseases. However, its underlying mechanisms remain elusive. ECs derived microvesicles (EMVs) and exosomes (EEXs) carry the characteristics of parent cells and transfer their contents to modulate the functions of recipient cells, holding the potential to evaluate or regulate vascular aging. Here, we found that young or aged ECs released EMVs were more effective than their released EEXs on alleviating or aggravating mice cerebrovascular and brain aging as indicated by SA-{beta}-gal staining, cerebral blood flow, blood brain barrier function, aging related markers and cognitive ability test. We further identified that these EMVs regulated cerebrovascular and brain aging by transferring miR-17-5p and could modulate ECs senescence and functions via miR-17-5p/PI3K/Akt pathway. Plasma EMVs and their contained miR-17-5p (EMV-miR-17-5p) were significantly increased or decreased in the elderly, and were closely correlated with vascular aging. Receiver Operating Characteristic (ROC) analysis showed that the area under the curve was 0.724 for EMVs, 0.77 for EMV-miR-17-5p and 0.815 for their combination for distinguishing vascular aging. Our results revealed the novel roles for EMVs that could more effectively modulate vascular and brain aging than EEXs by regulating ECs functions through miR-17-5p/PI3K/Akt pathway, and also suggested that EMVs and EMV-miR-17-5p represent promising biomarkers and therapeutic targets for vascular aging.

neuroscience↗

bioRxiv2023

Multi-species polyploidization, chromosome shuffling, and genome extraction in Zea/Tripsacum hybrids

By hybridization and special sexual reproduction, we sequentially aggregated Zea mays, Zea perennis, and Tripsacum dactyloides in an allohexaploid, backcrossed it with maize, derived self-fertile allotetraploids of maize and Z. perennis by natural genome extraction, extended their first six selfed generations, and finally constructed amphitetraploid maize using nascent allotetraploids as a genetic bridge. Transgenerational chromosome inheritance, subgenomes stability, chromosome pairings and rearrangements, and their impacts on an organisms fitness were investigated by fertility phenotyping and molecular cytogenetics techniques GISH and FISH. Results showed that diversified sexual reproductive methods produced highly differentiated progenies (2n=35-84) with varying proportions of subgenomic chromosomes, of which one individual (2n=54, MMMPT) overcame self-incompatibility barriers and produced self-fertile nascent near-allotetraploid by preferentially eliminating Tripsacum chromosomes. Nascent near-allotetraploid progenies showed persistent chromosome changes, intergenomic translocations, and rDNA variations for at least up to the first six selfed generations; however, ploidy tended to stabilize at near-tetraploid level (2n=40) with full integrity of 45SrDNA pairs, and a trend of decreasing variations by advancing generations with an average of 25.53, 14.14, and 0.37 maize, Z. perennis, and T. dactyloides chromosomes, respectively. The mechanisms for three genome stabilities and karyotype evolution for formatting new polyploid species were discussed.

genetics↗

bioRxiv2023

Structure of human NaV1.6 channel reveals Na+ selectivity and pore blockade by 4,9-anhydro-tetrodotoxin

The sodium channel NaV1.6 is widely expressed in neurons of the central and peripheral nervous systems, which plays a critical role in regulating neuronal excitability. Dysfunction of NaV1.6 has been linked to epileptic encephalopathy, intellectual disability and movement disorders. Here we present cryo-EM structures of human NaV1.6/{beta}1/{beta}2 alone and complexed with a guanidinium neurotoxin 4,9-anhydro-tetrodotoxin (4,9-ah-TTX), revealing molecular mechanism of NaV1.6 inhibition by the blocker. In the apo-form structure, two potential Na+ binding sites were revealed in the selectivity filter, suggesting a possible mechanism for Na+ selectivity and conductance. In the 4,9-ah-TTX-bound structure, 4,9-ah-TTX binds to a pocket similar to the tetrodotoxin (TTX) binding site, which occupies the Na+ binding sites and completely blocks the channel. Molecular dynamics simulation results show that subtle conformational differences in the selectivity filter affect the affinity of TTX analogues. Taken together, our results provide important insights into NaV1.6 structure, ion conductance, and inhibition.

biophysics↗

bioRxiv2023

Antithetic effects of agonists and antagonists on the structural fluctuations of TRPV1 channel

Transient receptor potential vanilloid member 1 (TRPV1) is a heat and capsaicin receptor that allows cations to permeate and cause pain. As the molecular basis for temperature sensing, the heat capacity ({Delta}Cp) model (D. E. Clapham, C. Miller, Proc. Natl. Acad. Sci. U. S. A. 108, 19492-19497 (2011).) has been proposed and experimentally supported. Theoretically, heat capacity is proportional to a variance in enthalpy, presumably related to structural fluctuation; however, the fluctuation of TRPV1 has not been directly visualized. In this study, we directly visualized single-molecule structural fluctuations of the TRPV1 channels in a lipid bilayer with the ligands resiniferatoxin (RTX: agonist, 1000 times hotter than capsaicin) and capsazepine (CPZ: antagonist) by high-speed atomic force microscopy (HS-AFM). We observed the structural fluctuations of TRPV1 in an apo state and found that RTX binding enhances structural fluctuations, while CPZ binding suppresses fluctuations. These ligand-dependent differences in structural fluctuation would play a key role in the gating of TRPV1.

physiology↗

bioRxiv2023

Size-Dependent Expression of the Fission Yeast Cdc13 Cyclin is Conferred by Translational Regulation

The major fission yeast cyclin, Cdc13, has been shown to increase in concentration in correlation with cell size, and has been proposed to thereby regulate cell size at division. However, the mechanism of its cell-size regulation has been unknown. Here, we show that Cdc13 is regulated post-transcriptionally. Its transcript is not expressed in a size-dependent manner, rather a size-dependent concentration of protein is expressed from a size-independent concentration of mRNA. Moreover, we show that the expression of Cdc13 is, in fact, size dependent, as opposed to simply size-correlated due to time-dependent expression. We identify a 20-amino-acid motif, which includes the APC D-box degron, as necessary and sufficient for size-dependent expression, which allowed us to construct a size-independent allele of cdc13. Using this allele, we show that size-dependent expression of Cdc13 is not required for size control in fission yeast cells.

cell biology↗

bioRxiv2023

DPHL v2: An updated and comprehensive DIA pan-human assay library for quantifying more than 14,000 proteins.

A comprehensive pan-human spectral library is critical for biomarker discovery using mass spectrometry (MS)-based proteomics. DPHL v1, a previous pan-human library built from 1096 data-dependent acquisition (DDA) MS data of 16 human tissue types, allows quantifying 10,943 proteins. However, a major limitation of DPHL v1 is the lack of semi-tryptic peptides and protein isoforms, which are abundant in clinical specimens. Here, we generated DPHL v2 from 1608 DDA-MS data acquired using Orbitrap mass spectrometers. The data included 586 DDA-MS newly acquired from 17 tissue types, while 1022 files were derived from DPHL v1. DPHL v2 thus comprises data from 24 sample types, including several cancer types (lung, breast, kidney, and prostate cancer, among others). We generated four variants of DPHL v2 to include semi-tryptic peptides and protein isoforms. DPHL v2 was then applied to a publicly available colorectal cancer dataset with 286 DIA-MS files. The numbers of identified and significantly dysregulated proteins increased by at least 21.7% and 14.2%, respectively, compared with DPHL v1. Our findings show that the increased human proteome coverage of DPHL v2 provides larger pools of potential protein biomarkers.

bioinformatics↗

bioRxiv2022

MYSM1 co-activates ERα action via histone and non-histone deubiquitination to confer antiestrogen resistance in breast cancer

Endocrine resistance is a crucial challenge in estrogen receptor alpha (ER)-positive breast cancer (BCa) therapy. Aberrant alteration in modulation of E2/ER signaling pathway has emerged as the putative contributor for endocrine resistance in BCa. Thus, identification the efficient ER cofactor remains necessary for finding a potential therapeutic target for endocrine resistance. Herein, we have demonstrated that Myb like, SWIRM and MPN domains 1 (MYSM1) as a histone deubiquitinase is a novel ER co-activator with established Drosophila experimental model. Our results showed that MYSM1 participated in up-regulation of ER action via histone and non-histone deubiquitination. We provided the evidence to show that MYSM1 was involved in maintenance of ER stability via ER deubiquitination. Furthermore, silencing MYSM1 induced enhancement of histone H2A ubiquitination as well as reduction of histone H3K4me3 and H3Ac levels at cis regulatory elements on promoter of ER-regulated gene. In addition, MYSM1 depletion attenuated cell proliferation/growth in BCa-derived cell lines and xenograft models. Knockdown of MYSM1 increased the sensitivity of antiestrogen agents in BCa cells. MYSM1 was highly expressed in clinical BCa samples, especially in aromatase inhibitor (AI) non-responsive tissues. These findings clarify the molecular mechanism of MYSM1 as an epigenetic modifier in regulation of ER action and provide a potential therapeutic target for endocrine resistance in BCa.

cell biology↗

bioRxiv2022

A pair of NLRs coordinately modulates NRC3-mediated ETI responses to facilitate age-dependent immunity

Two NLRs (Nucleotide-binding and Leucine-rich repeat Receptors) adjacent to each other on a locus, termed as paired NLRs, may act separately for effector recognition and subsequent signaling activation to mediate effector-triggered immunity (ETI) in many plants. However, it is largely unknown about their distribution and functions in Solanaceae species, in which NLR-Hs (Helpers NLR REQUIRED FOR CELL DEATHs) have been extensively studied. Here, we identified paired NLRs in Solanaceae species and found they harbor paired NLRs ranging from 6 to 100, which are significantly negatively correlated with the numbers of NLR-Hs. N. benthamiana has six paired NLRs, among which silencing of NRCX exhibits phenotypes of dwarfism and accelerated senescence. Importantly, NRCX-silencing phenotypes could be restored by simultaneously silencing its head-to-head NLR pair, thus we named it as NRCY. NRCX/Y pair is specific in Solanaceae species. NRCY contains non-canonical walker B and MHD motifs, but could not induce autoactive cell death in N. benthamiana. Instead of that, silencing NRCY impaired cell death triggered by Sw5b-Nsm and NRC3D480V, indicating NRCY is also an NLR modulator like NRCX. Furthermore, NRCX suppression of Sw5b-Nsm and NRC3-mediated cell death is dependent on NRCY. Remarkably, we found that NRCX and NRCY expressions were induced during plant senescence, while NRCY was induced more than NRCX. Accordingly, the plant resistance was stronger during maturation, indicated NRCX/Y might be involved in age-dependent resistance. Our study reveals one of the paired NLRs coordinately regulates ETI to facilitate age-dependent immunity.

plant biology↗

bioRxiv2022

CAbiNet: Joint visualization of cells and genes based on a gene-cell graph

In routine single-cell RNA-sequencing (scRNA-seq) analysis workflows, cells are commonly visualized in 2D to show the patterns in the data. However, these visualization approaches do not give any information about the genes that define the cell groups or clusters. It is therefore desirable to display cells and genes simultaneously such that by their relative position to each other information about the genes expression in a cluster can be obtained. Here we propose "Correspondence Analysis based Biclustering on Networks" (CAbiNet) as a novel approach to jointly visualize cells and genes by a non-linear embedding approach, called biMAP. The biMAP allows for easy and interactive exploration of cells and their corresponding marker genes in a single plot. CabiNet additionally offers an intuitive way to perform biclustering jointly on cells and genes, providing a simplified workflow to annotate cell types on the biMAP. CAbiNet is accessible through GitHub as an R package.

bioinformatics↗

bioRxiv2022

Molecular insights into the gating mechanisms of voltage-gated calcium channel CaV2.3

High-voltage-activated R-type CaV2.3 channel plays pivotal roles in many physiological activities and is implicated in epilepsy, convulsions, and other neurodevelopmental impairments. Here, we determine the high-resolution cryo-electron microscopy (cryo-EM) structure of human CaV2.3 in complex with the 2{delta}1 and {beta}1 subunits. The VSDII is stabilized in the resting state. Electrophysiological experiments elucidate that the conformational change of VSDII in response to variation in membrane potential is not required for channel activation, whereas the other VSDs are essential for channel opening. The intracellular gate is blocked by the W-helix. A pre-W-helix adjacent to the W-helix can significantly regulate closed-state inactivation (CSI) by modulating the association and dissociation of the W-helix with the gate. Electrostatic interactions formed between the negatively charged domain on S6II, which is exclusively conserved in the CaV2 family, and nearby regions at the alpha-interacting domain (AID) and S4-S5II helix are identified. Further functional analyses indicate that these interactions are critical for the open-state inactivation (OSI) of CaV2 channels.

biophysics↗

bioRxiv2022

The pan-genome and local adaptation of Arabidopsis thaliana

Arabidopsis thaliana has been used as a model species for research in a diverse collection of plant species. However, previous studies based on single reference genomes and short-read sequencing data are restricted to detecting variable genes and large structural variation (SV) underlying local adaptation. Here we de novo assemble high-quality chromosomal genomes of 38 A. thaliana ecotypes (with 6 relict ones) using PacBio-HiFi long-read sequencing. From these newly assembled genomes, we annotate several thousand new genes through pan-genomic analysis in comparison to the previous reference genome. The identified variable genes are mainly enriched in and associated with ecological adaptation and this species substantially expands its gene repertoire for local adaptation. We construct a graph-based pan-genome and identify 62,525 SVs which overlap with 14,243 genes. These genes are enriched in multiple ecological adaptation functions, including secondary metabolic processes, enzyme regulation, and biotic/abiotic stimulus. For example, a 566 bp insertion in the promoter of the light-adaptation KNAT3 gene was specific to the high-altitude relict Tibet-0 ecotype. This SV reduces the expression level of KNAT3 and promotes A. thaliana adaptation to habitats high in light radiation. In addition, compared with the SNPs, the SVs identified in this study captured the missing heritability and we detected novel SV associations with environmental variables in their native range, highlighting the value of SVs in environmental adaptation. The genome resources presented here will help pinpoint genetic changes that include both SVs and the ecotype-specific genes for local adaptation of A. thaliana and increase our understanding of the molecular mechanisms in this model species to respond to varied habitats.

genomics↗

bioRxiv2022

The compact CasPi (Cas12l) bracelet "provides" a unique structural platform for DNA manipulation

CRISPR-Cas modules serve as the adaptive nucleic-acid immune systems for prokaryotes, and provide versatile tools for nucleic-acid manipulation in various organisms. Here, we discovered a new miniature type V system, CRISPR-Cas{pi} (Cas12l) ([~]860 aa), from the environmental metagenome. Complexed with a large guide-RNA ([~]170 nt) comprising the tracrRNA and crRNA, Cas{pi} (Cas12l) recognizes a unique 5 C-rich PAM for DNA cleavage under a broad range of biochemical conditions, and generates gene editing in mammalian cells. Cryo-EM study reveals a bracelet architecture of Cas{pi} effector encircling the DNA target at 3.4-[A] resolution, substantially different from the canonical two-lobe architectures of Cas12 and Cas9 nucleases. And the large guide-RNA serves as a two-arm scaffold for effector assembly. Our study expands the knowledge of DNA targeting mechanisms by CRISPR effectors, and offers an efficient but compact platform for DNA manipulations.

biophysics↗

bioRxiv2022

Discovering Haematoma-Stimulated Circuits for Secondary Brain Injury after Intraventricular Haemorrhage by Spatial Transcriptome Analysis

Central nervous system (CNS) diseases, such as neurodegenerative disorders and brain diseases caused by acute injuries, are important yet challenging to study due to disease leisions locations and other complexities. Utilizing the powerful spatial transcriptome analysis together with novel algorithms we developed for the study, we report here for the first time a 3D trajectory map of gene expression changes in the brain following acute neural injury using a mouse model of intraventricular haemorrhage (IVH). IVH is a common and representative complication after various acute brain injuries with severe mortality and mobility implications. Our data identified three main 3D global pseudospace-time trajectory bundles which represent the main neural circuits from the lateral ventricle to the hippocampus and primary cortex induced by experimental intraventricular haematoma stimulation. Further analysis indicated a rapid response in the primary cortex, as well as a direct and integrated effect on the hippocampus after IVH. These results are informative in understanding the pathophysiological changes, including the spatial and temporal patterns of gene expression changes, in IVH patients after acute brain injuries and strategizing more effective clinical management regimens. The bioinformatics strategies will also be useful for the study of other CNS diseases.

bioinformatics↗

bioRxiv2022

M-Band Wavelet-Based Imputation of scRNA-seq Matrix and Multi-view Clustering of Cell

Wavelet analysis has been recognized as a cutting-edge and promising tool in the fields of signal processing and data analysis. However, application of wavelet-based method in single-cell RNA sequencing (scRNA-seq) data is little known. Here, we present M-band wavelet-based imputation of scRNA-seq matrix and multi-view clustering of cells (WIMC). We applied integration of M-band wavelet analysis and uniform manifold approximation and projection (UMAP) to a panel of single cell sequencing datasets by breaking up the data matrix into a trend (low frequency or low resolution) component and (M-1) fluctuation (high frequency or high resolution) components. We leverage a non-parametric wavelet-based imputation algorithm of sparse data that integrates M-band wavelet transform for recovering dropout events of scRNA-seq datasets. Our method is armed with multi-view clustering of cell types, identity, and functional states, enabling missing cell types visualization and new cell types discovery. Distinct to standard scRNA-seq workflow, our wavelet-based approach is a new addition to resolve the notorious chaotic sparsity of scRNA-seq matrix and to uncover rare cell types with a fine-resolution. Author summaryWe develop M-band wavelet-based imputation of scRNA-seq matrix and multi-view clustering of cells. Our new approach integrates M-band wavelet analysis and UMAP to a panel of single cell sequencing datasets via breaking up the data matrix into a trend (low frequency or low resolution) component and (M - 1) fluctuation (high frequency or high resolution) components. Our method enables us to efficiently impute sparse scRNA-seq data matrix and to examine multi-view clustering of cell types, identity, and functional states, potentializing missing cell types recovery, fine rare cell types discovery, as well as functional cell states exploration.

bioinformatics↗

bioRxiv2022

Optogenetic activation of visual thalamus generates artificial visual percepts

The lateral geniculate nucleus (LGN), a retinotopic relay center where visual inputs from the retina are processed and relayed to the visual cortex, has been proposed as a potential target for artificial vision. At present, it is unknown whether optogenetic LGN stimulation is sufficient to elicit behaviorally relevant percepts and the properties of LGN neural responses relevant for artificial vision have not been thoroughly characterized. Here, we demonstrate that tree shrews pretrained on a visual detection task can detect optogenetic LGN activation using an AAV2-CamKII-ChR2 construct and readily generalize from visual to optogenetic detection. We also characterize how amplitude and frequency of optogenetic stimulation affect behavioral performance. Given the importance of temporal stimulation parameters, we also study tree shrew behavioral ability to discriminate among pairs of visual flicker frequencies and demonstrate performance transfer among related discrimination problems as well as limitations. Neural recordings in LGN and primary visual cortex (V1) examining two flicker discriminations reveal that while LGN neurons multiplex information about temporal stimulation discriminations, V1 neurons explicitly encode information about one or the other discriminations but not both. Our findings confirm the LGN as a potential target for visual prosthetics and introduce a distinction in stimulus encoding between thalamic and cortical representations that could be of fundamental importance in artificial vision.

neuroscience↗

bioRxiv2022

Quantitatively mapping local quality of super-resolution microscopy by rolling Fourier ring correlation

In fluorescence microscopy, computational algorithms have been developed to suppress noise, enhance contrast, and even enable super-resolution (SR). However, the local quality of the images may vary on multiple scales, and these differences can lead to misconceptions, which is especially intractable in emerging deep-learning ones. Current mapping methods fail to finely estimate the local quality, challenging to associate the SR scale content. Here, we develop a rolling Fourier ring correlation (rFRC) framework to evaluate the reconstruction uncertainties down to SR scale. To visually pinpoint regions with low reliability, a filtered rFRC is combined with a modified resolution scaled error map (RSM), offering a comprehensive and concise map for further examination. We demonstrate their performances on various SR imaging modalities, and the resulting quantitative maps enable better SR images integrated from different reconstructions. Beyond that, we provide a strategy for learning-based restorations, allowing a direct detection of both data and model uncertainties, and expect the representative cases can inspire further advances in this rapidly developing field.

biophysics↗