Biology subjects

Yang, W.

Publications and source records attributed to Yang, W..

At least 127 records · Page 7Linked to original sources

bioRxiv2024

Revolutionizing large-scale DNA synthesis with microchip-based massive in parallel synthesis system

DNA synthesis serves as the fundamental enabling technology of engineering biology, aiming to provide DNA molecules of designed composition, length, and complexity at scale and low cost. Current high-throughput DNA synthesis technologies rely on intricate chip manufacturing and microfluidic systems to provide large-scale synthetic oligonucleotides, at the expense of low concentration and limited compatibility in the processing of longer DNA constructs assembly. Here, we report a microchip-based massive in parallel synthesis (mMPS), pioneering an "identification-sorting-synthesis-recycling" iteration mechanism to microchips for high throughput DNA synthesis. In comparison to microarray-based methods, we demonstrate that our method can increase the DNA product concentration by 4 magnitudes (to picomole-scale per sequence) and greatly simplifies the downstream processes for large-scale gene synthesis construction. By the construction of 1.97 million-diversity variant libraries that cover 1,254 human protein domains, we demonstrated the uniformity of the constructed variant libraries using mMPS-derived oligos is greatly improved, with amino acid distribution highly consistent as designed. In addition, by synthesizing 285 1kb-to-3kb genes with varying degrees of sequence complexity from previously reported strains A501 and 3DAC, potential ancestor of early archaea and bacteria, our result shows that the overall gene assembly success rate using mMPS-derived oligos is increased by 10-fold in comparison to other methods. Our mMPS technology holds the potential to close the gap between the quality and cost of writing DNA in increasing demand across many sectors of research and industrial activities.

synthetic biology↗

bioRxiv2024

Universal function of WRI1 involved in integrated regulation of multiple latex metabolism pathways in Natural Rubber

Hevea brasiliensis and Taraxacum kok-saghyz are sources of nature rubber (NR) and recent research has focused on finding a method to increase NR quality and production. NR consists mainly of cis-1,4-polyisoprene, which is synthesized from sucrose by a series of reactions during carbon metabolism. WRINKLED1 (WRI1) is a transcription factor (TF) that coordinates many genes involved in carbon metabolism and lipid biosynthesis. Here, we isolated and characterized two orthologues of WRI in rubber-producing plants H. brasiliensisa and T. kok-saghyz, which are highly expressed in their latex. Subcellular localization and ectopic expression in Arabidopsis thaliana indicated that the TFs, HbWRI1 and TkWRI1, are involved in lipid accumulation. Overexpression of HbWRI1 and TkWRI1 in T. kok-saghyz substantially enhanced NR quality and production, including dry rubber content, molecular weight, and the diameter of rubber particles in the latex. Conversely, they were decreased in TkWRI1 repression. Furthermore, based on the activated expression in transgenic T. kok-saghyz latex and the existence of AW-box element in the promoter regions, 16 direct downstream genes of HbWRI1 and TkWRI1 were identified by a dual-luciferase reporter assay and yeast one-hybrid assay, and their products were responsible for both NR biosynthesis and lipid metabolism. These results reveal a regulatory module that HbWRI1 and TkWRI1 TFs can positively regulate NR quality and production in T. kok-saghyz latex by synergistically regulating the entire NR biosynthesis. This module is expected to be utilized to develop superior varieties to enhance NR quality and production.

biochemistry↗

bioRxiv2024

Accurate Cell Abundance Quantification using Multi-positive and Unlabeled Self-learning

Quantifying the abundance of different cell types in pathological samples can help to uncover the correlations between cell composition and pathological conditions, offering deeper insights into the roles of different cell types in complex diseases. Conventional methods for cell abundance estimation often employ unsupervised clustering or supervised learning to identify cell types and estimate their proportions. However, these methods face challenges in accurately quantifying cell abundances, as clustering results could be unreliable and supervised methods may misclassify cell types not presented in the training data. We introduce CleverXMBD1 (denoted as Clever) for quantifying cell abundance from complex samples using a multi-class classifier trained with a confidence-based multi-positive and unlabeled loss function. Our evaluations show that Clever consistently and substantially outperforms existing methods in quantifying cell type abundance across multiple single cell datasets derived from different modalities, including CyTOF and image mass cytometry.

bioinformatics↗

bioRxiv2024

Gene encoding CC-NBS-LRR protein on rye chromosome 1RS confers wheat stripe rust resistance

Stripe rust, a globally widespread disease, stands as one of the most significant threats to wheat cultivation. The 1BL/1RS translocation, renowned for its robust resistance to both rust and powdery mildew, has historically played an important role in wheat breeding and production. The gene for resistance to stripe rust on the 1RS is known as Yr9 and plays an important role in the production of wheat, but over the course of long-term breeding had lost its resistance due to the evolution of stripe rust towards greater and greater virulence. In this paper, we cloned the stripe rust resistance gene, Yr9, from triticale by genetic mapping approach. The Yr9 encodes a typical nucleotide-binding leucine-rich repeat (NLR) protein. Both transgenic and overexpression of Yr9 in highly stripe rust susceptible wheat varieties conferred complete resistance to the stripe rust races CYR17 and partial resistance to the stripe rust races CYR32, CYR33, and CYR34. In addition, the Yr9 allele in the 1BL/1RS translocation line also showed the same level of resistance to stripe rust. Both two alleles loses resistance when deployed in the field or inoculated with mixed physiological races collected from the field. Our findings provide valuable insights for breeders to strategically incorporate disease resistance genes and provides a foundation for further understanding how pathogenic bacteria might evolve to evade recognition via NLR type proteins. SignificanceThe 1BL/1RS translocation between wheat and rye is the most successful case of exogenous gene application in plant genetic improvement and has been used in wheat breeding for over 50 years. Here we report the cloning of a stripe rust resistance gene Yr9 located on rye chromosome 1RS using a triticale population. The Yr9 encodes a coiled-coil nucleotide-binding site leucine-rich repeat (CC-NBS-LRR) protein that show complete resistance to the stripe rust races CYR17 and partial resistance to the stripe rust races CYR32, CYR33, and CYR34, albeit demonstrating susceptibility under field conditions. Our findings position Yr9 as an ideal candidate gene to study the mechanism of inactivation of disease resistance genes as a result of pathogen evolution.

genetics↗

bioRxiv2024

Endogenous Huntingtin aggregates are a huge organized scaffold and mold for Golgi apparatus impaired by mutant Huntingtin protein

Abstract Huntingtin (HTT) is a naturally aggregating protein that causes Huntington's disease (HD) when its polyglutamine (polyQ) tract exceeds 38 repeats. Despite its importance, the biology of HTT aggregates remains poorly defined. Here, using high resolution imaging of cells derived from HD families, we redefine polyQ assemblies, traditionally regarded as pathogenic aggregates, as dynamic and structurally organized intracellular compartments resembling knitted-fabric patches. These assemblies closely associate with and encircle the Golgi apparatus, integrating Golgi ribbons and stacks to form a previously unrecognized polyQ assembly-Golgi complex. Mechanistically, fragmentation of polyQ assemblies is dynamically coupled with mitotic Golgi fragmentation, whereas inhibition of ADP-ribosylation factor (ARF) function by Brefeldin A disrupts and fragments the complex, revealing an intimate structural and functional coupling between polyQ assemblies and Golgi architecture. The presence of mutant HTT (mHTT) destabilizes these assemblies and complexes, altering their response to nutrient deprivation and autophagy enhancers but not to antisense oligonucleotide (ASO) therapy. Functionally, polyQ assemblies in HD cells compromise the scaffolding capacity of the Golgi apparatus, clathrin-coated vesicles, and ARF1, impair Golgi glycosylation, and promote aberrant neuronal firing activity. Collectively, these findings establish polyQ assemblies as dynamic structural regulators of Golgi organization and function and demonstrate that mHTT disrupts the homeostatic dynamics of the polyQ assembly-Golgi complex, leading to Golgipathy.

pathology↗

bioRxiv2024

Interaction between histone residue H3K56 and mismatch repair protein MutSβ drives CAG repeat expansion

DNA repair protein MutS{beta} promotes CAG*CTG repeat expansions, which cause [~]20 untreatable neurodegenerative disorders including Huntingtons disease (HD). However, how MutS{beta} is recruited onto chromatin to enable repeat expansion is unknown. Here, we show that H3K56 interacts with MutS{beta} and brings it to chromatin. The H3K56-MutS{beta} interaction is regulated by H3K56 acetylation (H3K56ac) conditions, with acetylation inhibiting but deacetylation facilitating MutS{beta} chromatin recruitment. Blocking the H3K56-MutS{beta} interaction by either disrupting the MutS{beta} aromatic packet or increasing the H3K56ac level by removing histone deacetylases stabilizes CAG repeats, while depleting histone acetyltransferase p300/CBP promotes the H3K56-MutS{beta} interaction and CAG*CTG repeat instability, including expansion of CAG repeats of the huntingtin gene. Therefore, our study suggests a novel mechanism, by which MutS{beta} induces triplet repeat expansion, and new strategies of blocking the H3K56-MutS{beta} interaction to prevent HD and other triplet repeat expansion-caused disorders.

molecular biology↗

bioRxiv2024

Brainwaves Monitoring via Human Midbrain Organoids Microphysiological Analysis Platform: MAP

Understanding the development and pathogenesis of the human midbrain is critical for developing diagnostics and therapeutics for incurable neurological disorders including Parkinsons disease (PD)1-3. While organoid models are introduced to delineate midbrain-related pathogenesis based on experimental flexibility4-6, there is currently a lack of tools with high fidelity for tracing the long-term dynamics of intact brain networks-- an essential portrait of physiological states7,8. Here, we report a brain organoid microphysiological analysis platform (MAP) designed for long-term physiological development and in-situ real-time monitoring, akin to electroencephalogram (EEG), of midbrain organoids. We successfully achieved the on-chip homogeneous organogenesis of midbrain organoids and in-situ, non-disturbing electrophysiological tracking of the midbrain network activities. Throughout our long-term EEG monitoring via MAP, we captured the early-stage electrophysiological evolution of midbrain development, transitioning from discontinuous brief brainwave bursts to complex broadband activities. Furthermore, our midbrain organoid MAP facilitated the modeling and monitoring of neurotoxin-induced Parkinsonism, replicating the pathological dynamics of midbrain circuitry and exhibiting PD-like alterations in beta oscillation. We envision that the modeling and monitoring of brain organoid MAP will significantly enhance our understanding of human neurophysiology, neuropathogenesis, and drug discovery of neurodegenerative diseases.

bioengineering↗

bioRxiv2024

Epigenomic analysis of hepatocellular carcinoma reveals aberrant cis-regulatory changes and dysregulated retrotransposons with prognostic potentials

Hepatocellular carcinoma (HCC) exhibits widespread epigenetic alterations, yet their impact on cis-regulatory elements (CREs) and retrotransposons remains poorly understood. Here, we present an integrated epigenomic and transcriptomic analysis of HCC tumors and matched tumor-adjacent normal tissues. We identified extensive DNA hypomethylation coupled with changes in histone modifications at partially methylated domains, CREs, and retrotransposons. These epigenetic aberrations were associated with dysregulated expression of genes involved in cell cycle regulation, immune response, and extracellular matrix organization. Notably, our findings revealed a novel mechanism for the transcriptional dysregulation of GPC3, a key HCC biomarker and immunotherapeutic target. We observed that GPC3 upregulation is driven by both the reactivation of a fetal liver super enhancer and hypomethylation of GPC3-associated CpG islands. Moreover, we found that DNA hypomethylation-driven aberrant expression of retrotransposons carries prognostic significance in HCC. Patients with high expression of a long non-coding RNA driven by a HERVE-int element exhibited more aggressive tumors, poorer clinical outcomes, and molecular features associated with favorable immunotherapy response. Together, our study provides a comprehensive resource for understanding the role of epigenetic dysregulation in HCC and identifies retrotransposon-associated transcripts as potential biomarkers.

genomics↗

bioRxiv2024

Target-conditioned diffusion generates potent TNFR superfamily antagonists and agonists

Despite progress in designing protein binding proteins, the shape matching of designs to targets is lower than in many native protein complexes, and design efforts have failed for TNF receptor (TNFR1) and other protein targets with relatively flat and polar surfaces. We hypothesized that free diffusion from random noise could generate shape-matched binders for challenging targets, and tested this on TNFR1. We obtain designs with low picomolar affinity whose specificity can be completely switched to other family members using partial diffusion. Designs function as antagonists or as superagonists when presented at higher valency for OX40 and 4-1BB. The ability to design high-affinity and specificity antagonists and agonists for pharmacologically important targets in silico presages a new era in which binders are made by computation rather than immunization or random screening approaches.

biochemistry↗

bioRxiv2024

Small mammals in biodiversity hotspot harbor viruses of epidemic potential

Metagenomic sequencing has transformed our understanding of viral diversity in wildlife and its threat to human health. Despite this progress, many studies have lacked systematic and ecologically informed sampling, which has left numerous potentially emergent viruses undiscovered, and the drivers of their ecology and evolution poorly understood. We conducted an extensive analysis of viruses in the lung, spleen, and gut of 1,688 animals from 38 mammalian species across 428 sites in Yunnan, China--a hotspot for zoonotic diseases. We identified 162 mammalian viral species, including 102 novel species and 24 posing potential risks to humans due to their relationships with known pathogens associated with serious diseases and their ability to cross major host species barriers. Our findings offer an in-depth view of virus organotropism, cross-species transmission, host sharing patterns, and the ecological factors influencing viral evolution, all of which are critical for anticipating and mitigating future zoonotic outbreaks.

microbiology↗

bioRxiv2024

Sub-3 A resolution protein structure determination by single-particle cryo-EM at 100 keV

Cryo-electron microscopy (cryo-EM) has revolutionized structural biology by providing high-resolution insights into biological macromolecules. Here, we present sub-3 [A] resolution structures determined using the 100 keV Tundra cryogenic transmission electron microscope (cryo-TEM), equipped with the newly developed Falcon C direct electron detector (DED). Our results demonstrate that this lower voltage microscope, when combined with advanced electron optics and detectors, can achieve high-resolution reconstructions that were previously only attainable with higher voltage systems. The implementation of an extreme-brightness field emission gun (XFEG) and an SP-TWIN objective lens significantly enhanced the spatial and temporal coherence of the system. Furthermore, the semi-automated sample loader minimized contamination and drift, allowing extended data collection sessions without manual intervention. The high detective quantum efficiency (DQE) of Falcon C further improved the signal-to-noise ratio, which is critical for achieving high-resolution structures. We validated the performance of this microscope by determining the structures of various biological samples, including apoferritin, T20S proteasome, GABAA receptor, haemoglobin, and human transthyretin ranging in size from 440 kDa to 50 kDa. The highest resolutions achieved were 2.1 [A] for apoferritin, 2.7 [A] for the 20S proteasome, 2.8 [A] for the GABAA receptor, 5.0 [A] for haemoglobin, and 3.5 [A] for transthyretin. We also explored a larger specimen, a 3.9 MDa Adeno-associated virus (AAV9) capsid and resolved it a 2.8 [A]. This work highlights the potential of 100 keV TEMs to make high-resolution cryo-EM more accessible to the structural biology community. Furthermore, it sets a precedent for the use of lower voltage TEMs in routine cryo-EM studies, not only for screening grids for single particle analysis but also for achieving high-resolution structures of protein samples.

biophysics↗

bioRxiv2024

Hippocampal neuronal activity is aligned with action plans

Neurons in the hippocampus are correlated with different variables, including space, time, sensory cues, rewards, and actions, where the extent of tuning depends on ongoing task demands. However, it remains uncertain whether such diverse tuning corresponds to distinct functions within the hippocampal network or if a more generic computation can account for these observations. To disentangle the contribution of externally driven cues versus internal computation, we developed a task in mice where space, auditory tones, rewards, and context were juxtaposed with changing relevance. High-density electrophysiological recordings revealed that neurons were tuned to each of these modalities. By comparing movement paths and action sequences, we observed that external variables had limited direct influence on hippocampal firing. Instead, spiking was influenced by online action plans modulated by goal uncertainty. Our results suggest that internally generated cell assembly sequences are selected and updated by action plans toward deliberate goals. The apparent tuning of hippocampal neuronal spiking to different sensory modalities might emerge due to alignment to the afforded action progression within a task rather than representation of external cues.

neuroscience↗

bioRxiv2024

Architecture of the ATP-driven motor for protein import into chloroplasts

Thousands of nuclear-encoded proteins are transported into chloroplasts through the TOC-TIC translocon spanning the chloroplast envelope membranes. A motor complex pulls the translocated proteins out of the TOC-TIC complex into the chloroplast stroma by hydrolyzing ATP. The Orf2971-FtsHi complex was suggested to serve as the ATP-hydrolyzing motor in Chlamydomonas reinhardtii, but little is known about its architecture and assembly. Here, we report the 3.2-[A] resolution structure of the Chlamydomonas Orf2971-FtsHi complex. The 20-subunit complex spans the chloroplast inner envelope with two bulky modules protruding into the intermembrane space and stromal matrix. Six subunits form a hetero-hexamer potentially providing the pulling force through ATP hydrolysis. The remaining subunits, including potential enzymes/chaperones, likely facilitate the complex assembly and regulate its proper function. Our results provide the structural foundation for mechanistic understanding of chloroplast protein translocation.

biochemistry↗

bioRxiv2024

Extracellular Vesicles heterogeneity through the lens of multiomics

Extracellular vesicles (EVs) are heterogenous in size, biogenesis, cargo and function. Beside small EVs, aggressive tumor cells release a population of particularly large EVs, namely large oncosomes (LO). This study provides the first resource of label-free quantitative proteomics of LO and small EVs obtained from distinct cancer cell types (prostate, breast, and glioma). This dataset was integrated with a SWATH Proteomic assay on LO, rigorously isolated from the plasma of patients with metastatic prostate cancer (PC). Proteins enriched in LO, which were identified also at the RNA level, and found in plasma LO significantly correlated with PC progression. Single EV RNA-Seq of the PC cell-derived LO confirmed some of the main findings from the bulk RNA-Seq, providing first evidence that single cell technologies can be successfully applied to EVs. This multiomics resource of cancer EVs can be leveraged for developing a multi-analyte approach for liquid biopsy.

cancer biology↗

bioRxiv2024

Bootstrap Evaluation of Association Matrices (BEAM) for Integrating Multiple Omics Profiles with Multiple Outcomes

MotivationLarge datasets containing multiple clinical and omics measurements for each subject motivate the development of new statistical methods to integrate these data to advance scientific discovery. ModelWe propose bootstrap evaluation of association matrices (BEAM), which integrates multiple omics profiles with multiple clinical endpoints. BEAM associates a set omic features with clinical endpoints via regression models and then uses bootstrap resampling to determine statistical significance of the set. Unlike existing methods, BEAM uniquely accommodates an arbitrary number of omic profiles and endpoints. ResultsIn simulations, BEAM performed similarly to the theoretically best simple test and outperformed other integrated analysis methods. In an example pediatric leukemia application, BEAM identified several genes with biological relevance established by a CRISPR assay that had been missed by univariate screens and other integrated analysis methods. Thus, BEAM is a powerful, flexible, and robust tool to identify genes for further laboratory and/or clinical research evaluation. AvailabilitySource code, documentation, and a vignette for BEAM are available on GitHub at: https://github.com/annaSeffernick/BEAMR. The R package is available from CRAN at: https://cran.r-project.org/package=BEAMR. ContactStanley.Pounds@stjude.org Supplementary InformationSupplementary data are available at the journals website.

bioinformatics↗

bioRxiv2024

Dual modes of DNA N6-methyladenine maintenance by distinct methyltransferase complexes

Stable inheritance of DNA N6-methyladenine (6mA) is crucial for its biological functions in eukaryotes. Here, we identify two distinct methyltransferase (MTase) complexes, both sharing the catalytic subunit AMT1, but featuring AMT6 and AMT7 as their unique components, respectively. While the two complexes are jointly responsible for 6mA maintenance methylation, they exhibit distinct enzymology, DNA/chromatin affinity, genomic distribution, and knockout phenotypes. AMT7 complex, featuring high MTase activity and processivity, is connected to transcription-associated epigenetic marks, including H2A.Z and H3K4me3, and is required for the bulk of maintenance methylation. In contrast, AMT6 complex, with reduced activity and processivity, is recruited to initiate maintenance methylation immediately after DNA replication. These two complexes coordinate in maintenance methylation. By integrating signals from both replication and transcription, this mechanism ensures the faithful and efficient transmission of 6mA as an epigenetic mark in eukaryotes. Significance statementDNA N6-methyladenine (6mA) has recently been recognized as an epigenetic mark in eukaryotes. The stable inheritance of 6mA is essential for its biological functions. However, the precise mechanisms by which 6mA patterns are faithfully and efficiently transmitted remain largely unknown. Here, we have identified two distinct 6mA methyltransferase (MTase) complexes and elucidated their coordinated role in maintenance methylation. This dual- complex mechanism ensures rapid and accurate methylation at newly replicated loci with proper transcription-associated epigenetic marks.

molecular biology↗

bioRxiv2024

Systematic Targeting of Protein Complexes with Molecular COUPLrs

Molecular glues that engage protein complexes have transformed the study of cell biology and have had a direct impact on clinical oncology. However, the identification of new glue classes and their corresponding protein complexes has remained largely serendipitous. To overcome this challenge, we report the development of molecular COUPLrs, elaborated small molecules flanked by two cysteine-reactive warheads, as well as CONNECT, an integrated chemical proteomic platform for target deconvolution. By profiling a library of molecular COUPLrs across 13 cancer cell lines, we uncovered hundreds of proteins that can be coupled together, including in some cases in mutant selective fashions. We develop an advanced COUPLr for the oncogene EML4-ALK, which engages the fusion outside of its kinase domain, restricts protein dynamics, and disrupts EML4-ALK signaling. Collectively, molecular COUPLrs substantially expand the scope of proteins that can be chemically connected, providing an unbiased approach to identify small molecules that target protein complexes.

systems biology↗

bioRxiv2024

Proxi-RIMS-seq2 applied to native microbiomes uncovers hundreds of known and novel m5C methyltransferase specificities.

Methylation patterns in bacteria can be used to study Restriction-Modification (RM) or other defense systems with novel properties. While m4C and m6A methylation is well characterized mainly through PacBio sequencing, the landscape of m5C methylation is under-characterized. To bridge this gap, we performed RIMS-seq2 on microbiomes composed of resolved assemblies of distinct genomes through proximity ligation. This high-throughput approach enables the identification of m5C methylated motifs and links them to cognate methyltransferases directly on native microbiomes without the need to isolate bacterial strains. Methylation patterns can also be identified on viral DNA and compared to host DNA, strengthening evidence for virus-host interaction. Applied to three different microbiomes, the method unveils over 1900 motifs that were deposited in REBASE. The motifs include a novel 8-base recognition site (CATm5CGATG) that was experimentally validated by characterizing its cognate methyltransferase. Our findings suggest that microbiomes harbor arrays of untapped m5C methyltransferase specificities, providing insights to bacterial biology and biotechnological applications.

genomics↗