Biology subjects

Dou, Y.

Publications and source records attributed to Dou, Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Loop-mediated Isothermal Amplification (LAMP) assay for reliable detection of Xanthomonas axonopodis pv. vasculorum

Xanthomonas axonopodis pv. vasculorum (Xav), the causative agent of sugarcane gumming disease, represents a significant threat to global sugarcane production due to its systemic and destructive nature. Despite the economic implications, a field-deployable, Xav-specific diagnostic tool has not been developed. This resulted in a loop-mediated isothermal amplification (LAMP) assay targeting the pelL gene, unique to Xav strains, as a rapid and precise diagnostic assay. The selection of the pelL gene was informed by comprehensive in silico analyses of Xav genomes and related Xanthomonas species and other close relatives. Validation against the NCBI GenBank database and internally sequenced genomes confirmed the genes exclusivity to Xav. Subsequent primers for both endpoint PCR and LAMP assays were designed using the pelL gene region. The LAMP assay underwent extensive testing against inclusivity and exclusivity panels. Use of exclusivity panel, comprising 81 strains from related species, other bacterial genera, and host genomes, demonstrated the assays specificity with no false positives. The assay exhibited a detection limit of 1 pg, and its effectiveness was unimpeded by crude host lysate (sugarcane). Further validation through multi-device and multi-operator testing underscored the assays 100% reproducibility and robustness. Application to infected plant samples resulted in the detection of all infected specimens without any false positives or negatives. This novel LAMP assay is accurate and reliable tool for Xav detection, with promising applications in routine diagnostics, biosecurity measures, microbial forensics, and epidemiological research.

plant biology↗

bioRxiv2024

Loss of ZNRF3/RNF43 Unleashes EGFR in Cancer

ZNRF3 and RNF43 are closely related transmembrane E3 ubiquitin ligases with significant roles in development and cancer. Conventionally, their biological functions have been associated with regulating WNT signaling receptor ubiquitination and degradation. However, our proteogenomic studies have revealed EGFR as the protein most negatively correlated with ZNRF3/RNF43 mRNA levels in multiple human cancers. Through biochemical investigations, we demonstrate that ZNRF3/RNF43 interact with EGFR via their extracellular domains, leading to EGFR ubiquitination and subsequent degradation facilitated by the E3 ligase RING domain. Overexpression of ZNRF3 reduces EGFR levels and suppresses cancer cell growth in vitro and in vivo, whereas knockout of ZNRF3/RNF43 stimulates cell growth and tumorigenesis through upregulated EGFR signaling. Together, these data highlight ZNRF3 and RNF43 as novel E3 ubiquitin ligases of EGFR and establish the inactivation of ZNRF3/RNF43 as a driver of increased EGFR signaling, ultimately promoting cancer progression. This discovery establishes a connection between two fundamental signaling pathways, EGFR and WNT, at the level of cytoplasmic membrane receptors, uncovering a novel mechanism underlying the frequent co-activation of EGFR and WNT signaling in development and cancer.

cancer biology↗

bioRxiv2023

DNA sensor ABCF1 phase separates with cccDNA to inhibit HBV replication

Objectives: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) contributes to viral persistence and recurrence. However, it is still less known how the host immune system responds to cccDNA and suppresses HBV replication. Therefore, it is essential to explore host proteins that interact with cccDNA and efficiently suppress HBV replication. Design: The interaction of cccDNA and ABCF1 was assessed by ChIP and pull-down assay. HBV replication was assessed in different cell models, including cccDNA surrogate-transfected and HBV-infected hepatocytes models. Phase separation of recombinant ABCF1 fusion proteins with cccDNA was observed by fluorescence microscopy. Results: Our data found that ABCF1 interacts with cccDNA to form phase-separated condensates via the poly-glutamine (PolyQ) of N-terminal intrinsically disordered low-complexity domain (LCD). Subsequently, ABCF1-cccDNA phase separation not only activates the type I/III interferon (IFN-I/III) pathway but also prevents Pol II accumulation on cccDNA to inhibit HBV transcription. Conclusions: Taken together, our study showed that ABCF1 acts as an antiviral restriction factor of HBV cccDNA by phase-separation-driven innate immune signaling and transcription inhibition. These findings shed new light on the understanding of host defense against cccDNA and provide a novel promising therapeutic strategy for HBV infection.

microbiology↗

bioRxiv2023

Spatially resolved molecular and cellular atlas of the mouse brain

A comprehensive atlas of genes, cell types, and their spatial distribution across a whole mammalian brain is fundamental for understanding function of the brain. Here, using snRNA-seq and Stereo-seq techniques, we generated a mouse brain atlas with spatial information for 308 cell clusters with single-cell resolution involving over 6 million cells as well as for 29,655 genes. We have identified new astrocyte clusters, and demonstrated that distinct cell clusters exhibit preference for cortical subregions. In addition, we identified 155 genes exhibiting regional specificity in the brainstem, and 513 long non-coding RNA exhibited regional specificity in the adult brain. Parcellation of brain regions based on spatial transcriptomic information showed large overlap with that by traditional method. Furthermore, we have uncovered 411 transcription factor regulons with spatiotemporal specificity during development. Thus, our study has discovered genes and regulon with spatiotemporal specificity, and provided a high-resolution spatial transcriptomic atlas of the mouse brain.

neuroscience↗

bioRxiv2023

Cell lineage analysis with somatic mutations reveals late divergence of neuronal cell types and cortical areas in human cerebral cortex

The mammalian cerebral cortex shows functional specialization into regions with distinct neuronal compositions, most strikingly in the human brain, but little is known in about how cellular lineages shape cortical regional variation and neuronal cell types during development. Here, we use somatic single nucleotide variants (sSNVs) to map lineages of neuronal sub-types and cortical regions. Early-occurring sSNVs rarely respect Brodmann area (BA) borders, while late-occurring sSNVs mark neuron-generating clones with modest regional restriction, though descendants often dispersed into neighboring BAs. Nevertheless, in visual cortex, BA17 contains 30-70% more sSNVs compared to the neighboring BA18, with clones across the BA17/18 border distributed asymmetrically and thus displaying different cortex-wide dispersion patterns. Moreover, we find that excitatory neuron-generating clones with modest regional restriction consistently share low-mosaic sSNVs with some inhibitory neurons, suggesting significant co-generation of excitatory and some inhibitory neurons in the dorsal cortex. Our analysis reveals human-specific cortical cell lineage patterns, with both regional inhomogeneities in progenitor proliferation and late divergence of excitatory/inhibitory lineages.

neuroscience↗

bioRxiv2023

Division of labor among H3K4 Methyltransferases Defines Distinct Facets of Homeostatic Plasticity

Heterozygous mutations in any of the six H3K4 methyltransferases (KMT2s) result in monogenic neurodevelopmental disorders, indicating nonredundant yet poorly understood roles of this enzyme family in neurodevelopment. Recent evidence suggests that histone methyltransferase activity may not be central to KMT2 functions; however, the enzymatic activity is evolutionarily conserved, implicating the presence of selective pressure to maintain the catalytic activity. Here, we show that H3K4 methylation is dynamically regulated during prolonged alteration of neuronal activity. The perturbation of H3K4me by the H3.3K4M mutant blocks synaptic scaling, a form of homeostatic plasticity that buffers the impact of prolonged reductions or increases in network activity. Unexpectedly, we found that the six individual enzymes are all necessary for synaptic scaling and that the roles of KMT2 enzymes segregate into evolutionary-defined subfamilies: KMT2A and KMT2B (fly-Trx homologs) for synaptic downscaling, KMT2C and KMT2D (Trr homologs) for upscaling, and KMT2F and KMT2G (dSet homologs) for both directions. Selective blocking of KMT2A enzymatic activity by a small molecule and targeted disruption of the enzymatic domain both blocked the synaptic downscaling and interfered with the activity-dependent transcriptional program. Furthermore, our study revealed specific phases of synaptic downscaling, i.e., induction and maintenance, in which KMT2A and KMT2B play distinct roles. These results suggest that mammalian brains have co-opted intricate H3K4me installation to achieve stability of the expanding neuronal circuits.

neuroscience↗

bioRxiv2023

Antibody Humanization via Protein Language Model and Neighbor Retrieval

Antibody (Ab), also known as immunoglobulin (Ig), is an essential macromolecule involved in human immune response and plays an increasingly vital role in drug discovery. However, the development of antibody drugs heavily relies on humanization of murine antibodies, which often necessitates multiple rounds of sequence optimizations through laborious experimental processes. In recent years, the remarkable capabilities of machine learning have revolutionized the field of natural sciences and have also demonstrated promising applications in the field of antibody humanization. Here, we present Protein-LAnguage-model-knN (PLAN), a machine learning model leveraging protein language model and information retrieval for improving humanization of antibodies. Further, we propose DE, a computed value shows a positive correlation with antigen-binding affinity. Our in silico experimental results demonstrate that 1) the PLAN-humanized sequences average humanness score reaches 0.592, improving over the best existing method by 44.7%; 2) a 63% overlap between the PLAN-proposed mutations and the mutations validated through wet lab experiments, which is 16.7% higher than the best existing result; 3) comparable antigen-binding affinity after DE guided back mutation.

bioinformatics↗

bioRxiv2023

Modeling kidney development, disease, and plasticity with clonal expandable nephron progenitor cells and nephron organoids

Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here we report manipulation of p38 and YAP activity creates a synthetic niche that allows the long-term clonal expansion of primary mouse and human NPCs, and induced NPCs (iNPCs) from human pluripotent stem cells. Cultured iNPCs resemble closely primary human NPCs, generating nephron organoids with abundant distal convoluted tubule cells, which are not observed in published kidney organoids. The synthetic niche reprograms differentiated nephron cells into NPC state, recapitulating the plasticity of developing nephron in vivo. Scalability and ease of genome-editing in the cultured NPCs allow for genome-wide CRISPR screening, identi-fying novel genes associated with kidney development and disease. A rapid, efficient, and scala-ble organoid model for polycystic kidney disease was derived directly from genome-edited NPCs, and validated in drug screen. These technological platforms have broad applications to kidney development, disease, plasticity, and regeneration.

developmental biology↗

bioRxiv2023

Using macromolecular electron densities to improve the enrichment of active compounds in virtual screening

The quest for more effective virtual screening algorithms is hindered by the scarcity of training data, calling for innovative approaches. This study presents the first use of experimental electron density (ED) data for improving active compound enrichment in virtual screening, supported by EDs ability to reflect the time-averaged behavior of ligands and solvents in the binding pocket. Experimental ED-based grid matching score (ExptGMS) was developed to score compounds by measuring the degree of matching between their binding conformations and a series of multi-resolution experimental ED grids. The efficiency of ExptGMS was validated using both in-silico tests with the Directory of Useful Decoys-Enhanced dataset and wet-lab tests on Covid-19 3CLpro-inhibitors. ExptGMS improved the active compound enrichment in top-ranked molecules by approximately 20%. Furthermore, ExptGMS helped identify four new and active inhibitors of 3CLpro, with the top showing an IC50 value of 1.9 {micro}M. To facilitate the use of ExptGMS, we developed an online database containing experimental ED grids for over 17,000 proteins.

bioinformatics↗

bioRxiv2023

A generic binding pocket for small molecule IKs activators at the extracellular inter-subunit interface of KCNQ1 and KCNE1 channel complexes

The cardiac IKs ion channel comprises KCNQ1, calmodulin, and KCNE1 in a dodecameric complex which provides a repolarizing current reserve at higher heart rates and protects from arrhythmia syndromes that cause fainting and sudden death. Pharmacological activators of IKs are therefore of interest both scientifically and therapeutically for treatment of IKs loss-of-function disorders. One group of chemical activators are only active in the presence of the accessory KCNE1 subunit and here we investigate this phenomenon using molecular modeling techniques and mutagenesis scanning in mammalian cells. A generalized activator binding pocket is formed extracellularly by KCNE1, the domain-swapped S1 helices of one KCNQ1 subunit and the pore/turret region made up of two other KCNQ1 subunits. A few residues, including K41, A44 and Y46 in KCNE1, W323 in the KCNQ1 pore, and Y148 in the KCNQ1 S1 domain, appear critical for the binding of structurally diverse molecules, but in addition, molecular modeling studies suggest that induced fit by structurally different molecules underlies the generalized nature of the binding pocket. Activation of IKs is enhanced by stabilization of the KCNQ1-S1/KCNE1/pore complex, which ultimately slows deactivation of the current, and promotes outward current summation at higher pulse rates. Our results provide a mechanistic explanation of enhanced IKscurrents by these activator compounds and provide a map for future design of more potent therapeutically useful molecules.

pharmacology and toxicology↗

bioRxiv2023

Semi-conservative transmission of DNA N6-adenine methylation in a unicellular eukaryote

While DNA N6-adenine methylation (6mA) is best known in prokaryotes, its presence in eukaryotes has generated great interest recently. Biochemical and genetic evidence supports that AMT1, a MT-A70 family methyltransferase (MTase), is crucial for 6mA deposition in unicellular eukaryotes. Nonetheless, 6mA transmission mechanism remains to be elucidated. Taking advantage of Single Molecule Real-Time Circular Consensus Sequencing (SMRT CCS), here we provide definitive evidence for semi-conservative transmission of 6mA, showcased in the unicellular eukaryote Tetrahymena thermophila. In wildtype (WT) cells, 6mA occurs at the self-complementary ApT dinucleotide, mostly in full methylation (full-6mApT); hemi-methylation (hemi-6mApT) is transiently present on the parental strand of newly replicated DNA. In {Delta}AMT1 cells, 6mA predominantly occurs as hemi-6mApT. Hemi-to-full conversion in WT cells is fast, robust, and likely processive, while de novo 6mA deposition in {Delta}AMT1 cells is slow and sporadic. In Tetrahymena, regularly spaced 6mA clusters coincide with linker DNA of the canonical nucleosome arrays in the gene body. Importantly, in vitro methylation of human chromatin by reconstituted AMT1 complex recapitulates preferential targeting of hemi-6mApT sites in linker DNA, supporting AMT1s intrinsic and autonomous role in maintenance methylation. We conclude that 6mA is transmitted by a semi-conservative mechanism: full-6mApT is split by DNA replication into hemi-6mApT, which is restored to full-6mApT by AMT1-dependent maintenance methylation. Our study dissects AMT1-dependent maintenance methylation and AMT1-independent de novo methylation, reveals a molecular pathway for 6mA transmission with striking similarity to 5-methyl cytosine (5mC) transmission at the CpG dinucleotide, and establishes 6mA as a bona fide eukaryotic epigenetic mark.

molecular biology↗

bioRxiv2022

MOF-mediated Histone H4 Lysine 16 Acetylation Governs Mitochondrial and Ciliary Functions by Controlling Gene Promoters

Histone H4 lysine 16 acetylation (H4K16ac), governed by the histone acetyltransferase (HAT) MOF, orchestrates critical functions in gene expression regulation and chromatin interaction. However, how does MOF and H4K16ac control cellular function and regulate mammalian tissue development remains unclear. Furthermore, whether the function of MOF is mediated by MSL or NSL, two distinct MOF-containing HAT complexes, have not been determined during mammalian development. Here we show that conditional deletion of Mof but not Kansl1, an essential component of the NSL complex, causes severe defects during murine skin development. In the absence of Mof and H4K16ac, basal epithelial progenitors of mammalian skin fail to establish the basement membrane and cell polarity, causing the failure of self-renewal. Furthermore, epidermal differentiation and hair growth are severely compromised, leading to barrier defects and perinatal lethality. Single-cell and bulk RNA-seq, in combination with MOF ChIP-seq, reveal that MOF regulated genes are highly enriched in mitochondria and cilia. Mechanistically, MOF coordinates with RFX2 transcription factor, which preferentially binds to gene promoters, to regulate ciliary and mitochondrial genes. Importantly, genetic deletion of Uqcrq, a nuclear-encoded, essential subunit for electron transport chain (ETC) Complex III, recapitulates the defects of epidermal differentiation and hair follicle growth observed in MOF cKO. Together, this study reveals the requirement of MOF-mediated epigenetic mechanism for mitochondria and cilia, and demonstrates the important function of the MOF/ETC axis for mammalian skin development.

developmental biology↗

bioRxiv2022

SEPepQuant enables comprehensive protein isoform characterization in shotgun proteomics

Shotgun proteomics is essential for protein identification and quantification in biomedical research, but protein isoform characterization is inhibited by the extensive number of peptides shared across proteins, hindering our understanding of protein isoform regulation and their roles in normal and disease biology. We systematically assess the challenge and opportunities of shotgun proteomics-based protein isoform characterization using in silico and experimental data, and then present SEPepQuant, a graph theory-based approach to enable isoform characterization. Using published data from one induced pluripotent stem cell study and two human hepatocellular carcinoma studies, we demonstrate the ability of SEPepQuant in addressing the key limitations of existing methods, providing more comprehensive proteome characterization, identifying hundreds of isoform-level regulation events, and facilitating streamlined cross-study comparisons. Our analysis provides solid evidence to support a widespread role of protein isoform regulation in normal and disease processes, and SEPepQuant has broad applications to biological and translational research.

bioinformatics↗

bioRxiv2022

Rnf20 shapes the endothelial control of heart morphogenesis and function

During embryogenesis, distinct cardiac cell types form, which shape the structural and functional properties of the heart. How their activity is coordinated is largely unknown. Here we show that Rnf20 is a multifaceted regulator of cardiac morphogenesis and function. On the one hand, Rnf20 controls extracellular matrix dynamics and endothelial-cardiomyocyte crosstalk essential for second heart field development. On the other hand, it safeguards endothelial cell identity and function by maintaining physiological angiocrine signaling and preventing endothelial-to-mesenchymal transition. Endothelial-specific deletion of Rnf20 led to ventricular septal defects, myocardial thinning and cardiac dysfunction as a result of aberrant signaling and excessive extracellular matrix deposition that induced precocious cardiomyocyte binucleation and irregular contractility. Furthermore, we uncovered upstream factors (e.g. Sox9) and multiple angiocrine and extracellular matrix molecules that alter cardiomyocyte functionality upon endothelial Rnf20 loss. In summary, our work identifies a novel, endothelial-specific role of Rnf20 in regulating cardiac morphogenesis and function.

developmental biology↗

bioRxiv2022

WDR5 promotes breast cancer growth and metastasis via KMT2-independent translation regulation

Metastatic breast cancer remains a major cause of cancer related deaths in women and there are few effective therapies against this advanced disease. Emerging evidence suggests that key steps of tumor progression and metastasis are controlled by reversible epigenetic mechanisms. Using an in vivo genetic screen, we identified WDR5 as an actionable epigenetic regulator that is required for metastatic progression in models of triple-negative breast cancer. We found that knockdown of WDR5 in breast cancer cells independently impaired their tumorigenic as well as metastatic capabilities. Mechanistically, WDR5 promotes cell growth by increasing ribosomal gene expression and translation efficiency in a KMT2-independent manner. Consistently, pharmacological inhibition or degradation of WDR5 impedes cellular translation rate and the clonogenic ability of breast cancer cells. Furthermore, combination of WDR5-targeting with mTOR inhibitors leads to potent suppression of translation and proliferation of breast cancer cells. These results reveal novel therapeutic strategies to treat metastatic breast cancer.

cancer biology↗

bioRxiv2021

MLL1 Methyltransferase Activity is Regulated by Distinct Nucleosome Binding Modes

Here we solve the single particle cryoEM structure for the MLL1 complex with nucleosome core particle (NCP) carrying histone H3 lysine 4 to methionine mutation. The MLL1 complex displays significant rotational dynamics on the NCP, a feature distinct from the yeast SET1 complex. We identified two major binding modes of the MLL1 complex on the NCP. Both binding modes anchor on the NCP through ASH2L, but they differ drastically with regard to where the MLL1 SET domain and RbBP5 bind. We show that one of the binding modes is catalytically inactive since disrupting interactions unique to this binding mode does not affect overall MLL1 activity in an NCP-specific manner. Interestingly, the inactive binding mode is in a configuration similar to that of the ySET1- NCP complex, which is intrinsically inactive on an unmodified NCP. The high rotational dynamics of the MLL1 complex as well as distinction between MLL and yeast SET1 complexes may reflect the necessity for loci-specific regulation of H3K4 methylation states in higher eukaryotes.

biochemistry↗

bioRxiv2021

BREVIPEDICELLUS and ERECTA mediate expression of AtPRX17 in preventing Arabidopsis callus retardation and browning

Efficient in vitro callus generation is fundamental to tissue culture propagation, a process required for plant regeneration and transgenic breeding for desired phenotypes. Identifying genes and regulatory elements that prevent callus retardation and browning is essential to facilitate the development of vitro callus systems. Here we show that BREVIPEDICELLUS (BP) and ERECTA (ER) pathways in Arabidopsis callus are converged to prevent callus browning and positively regulate an isoperoxidase gene AtPRX17 expression in the rapid growth callus. Loss of functions in both BP and ER resulted in markedly increasing callus browning. Transgenic lines with pro35S::AtPRX17 in the bp-5 er105 double mutant background fully rescued this phenotypic abnormality. Using plant in vitro DNA-binding assays, we observed that BP protein bound directly to the upstream sequence of AtPRX17 to promote its transcription during callus growth. ER is a universally presenting factor required for cell proliferation and growth, we show that ER positively regulates expression of a transcription factor WRKY6, which also directly binds to an additional site of the AtPRX17 promoter for its high expression. Our data reveals an important molecular mechanism in regulating expression of peroxidase isozyme to reduce Arabidopsis callus browning. HighlightBREVIPEDICELLUS and ERECTA are involved in regulating Arabidopsis callus browning by controlling expression of AtPRX17.

plant biology↗

bioRxiv2020

Comprehensive identification of somatic nucleotide variants in human brain tissue

Post-zygotic mutations incurred during DNA replication, DNA repair, and other cellular processes lead to somatic mosaicism. Somatic mosaicism is an established cause of various diseases, including cancers. However, detecting mosaic variants in DNA from non-cancerous somatic tissues poses significant challenges, particularly if the variants only are present in a small fraction of cells. Here, the Brain Somatic Mosaicism Network conducted a coordinated, multi-institutional study to: (i) examine the ability of existing methods to detect simulated somatic single nucleotide variants (SNVs) in DNA mixing experiments; (ii) generate multiple replicates of whole genome sequencing data from the dorsolateral prefrontal cortex, other brain regions, dura mater, and dural fibroblasts of a single neurotypical individual; (iii) devise strategies to discover somatic SNVs; and (iv) apply various approaches to validate somatic SNVs. These efforts led to the identification of 43 bona fide somatic SNVs that ranged in variant allele fractions from ~0.005 to ~0.28. Guided by these results, we devised best practices for calling mosaic SNVs from 250X whole genome sequencing data in the accessible portion of the human genome that achieve 90% specificity and sensitivity. Finally, we demonstrated that analysis of multiple bulk DNA samples from a single individual allows the reconstruction of early developmental cell lineage trees. Thus, this study provides a unified set of best practices to detect somatic SNVs in non-cancerous tissues. The data and methods are freely available to the scientific community and should serve as a guide to assess the contributions of somatic SNVs to neuropsychiatric diseases.

genomics↗