Biology subjects

Wei, R.

Publications and source records attributed to Wei, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Degradation of PET plastic with engineered environmental bacteria

Polyethylene terephthalate (PET) is one of the most widely used plastic materials in the food and textile industry. Consequently, post-consumer PET waste is a common environmental pollutant that leaks into the environment in the form of macro and microplastics with concerning health impacts. There is a pressing need to identify novel and sustainable solutions to process the abundance of PET waste contributing to this pollution. While there is extensive research into enzymes able to hydrolyse PET in vitro, a similar solution for discarded or difficult-to-collect PET based on whole-cell microbial catalysts is missing. In this work we report the engineering of environmental bacteria to use PET as a growth substrate. This was achieved by isolating a strain of Pseudomonas umsongensis able to use the PET monomer terephthalate as carbon source, engineering the strain to effectively secrete the high-activity PET hydrolase PHL7 through the addition of a recombinant TAT secretion leader sequence, and enhancing the bioavailability of PET by transforming it into an amorphous and macroporous structure by pre-treatment with an organic solvent. Our findings demonstrate the direct microbial consumption of PET, which could lead to improved and more sustainable upcycling strategies for this plastic.

synthetic biology↗

bioRxiv2024

Finding the E-Channel Proton Loading Sites by Calculating the Ensemble of Protonation Microstates

The aerobic electron transfer chain builds a proton gradient by proton coupled electron transfer reactions through multiple proteins. Complex I is the first enzyme in this chain. On transfer of two electrons from NADH to quinone four protons are pumped from the N- (negative, higher pH) to the P- (positive, lower) side. Protons move pthrough three linear antiporter paths, with a few amino acids and waters providing the route and the E-channel, which is a complex of competing paths, with clusters of interconnected protonatable residues. Proton loading sites (PLS) are residues that transiently bind protons as they are transported from N- to P-compartments. PLS can be individual residues or extended clusters. The program MCCE uses Monte Carlos sampling to analyze the E-channel proton binding in equilibrium with individual Molecular Dynamics snapshots from trajectories of Thrmus thermuphillus Complex I in the apo, quinone or quinol bound states. At pH 7, the five E-channel subunits (Nqo4, Nqo7, Nqo8, Nqo10, and Nqo11) have accepted >25,000 protonation microstates, each with different residues protonated. The microstate explosion is tamed by analyzing interconnected clusters of residues along the proton transfer paths. A proton is bound and released to five coupled residues in a cluster on the protein N-side and to six coupled residues in the protein center. Loaded microstates bind protons to sites closer to the P-side in the forward pumping direction. MCCE microstate analysis shows the strong coupling of proton binding amongst individual residues in the two PLS clusters.

biophysics↗

bioRxiv2024

From Bulk to Binding: Decoding the Entry of PET into Hydrolase Binding Pockets

Plastic-degrading enzymes hold promise for biocatalytic recycling of poly(ethylene terephthalate) (PET), a key synthetic polymer. Despite their potential, the current activity of PET hydrolases is not sufficient for industrial use. To unlock their full potential, a deep mechanistic understanding followed by protein engineering is required. Using cuttingedge molecular dynamics simulations and free energy analysis methods, we uncover the entire pathway from the initial binding of two PET hydrolases - the thermophilic leaf-branch compost cutinase (LCC) and polyester hydrolase 1 (PES-H1) - to an amorphous PET material to a PET chain entering the active site and adopting a hydrolyzable geometry. Our results reveal the initial PET binding and elucidate its non-specific nature driven by electrostatic and hydrophobic forces. Upon PET entry into the active site, we uncover that this process can occur via one of three key pathways and detect barriers to it arising from both PET-PET and PET-enzyme interactions, with specific residues identified by in silico and in vitro mutagenesis. These insights not only advance our understanding of PET degradation mechanisms and pave the way for targeted enzyme enhancement strategies, but also offer an innovative approach applicable to enzyme studies across disciplines.

bioengineering↗

bioRxiv2024

Mitochondrial fission surveillance is coupled to Caenorhabditis elegans DNA and chromosome segregation integrity

Mitochondrial fission and fusion are tightly regulated to specify mitochondrial abundance, localization, and arrangement during cell division as well as in the diverse differentiated cell types and physiological states. However, the regulatory pathways for such mitochondrial dynamics are less explored than the mitochondrial fission and fusion components. Here we report a large-scale screen for genes that regulate mitochondrial fission. Mitochondrial fission defects cause a characteristic asymmetric fluorescent pattern in embryos carrying mitochondrial stress reporter genes. Using this asymmetric activation, we performed RNAi screens that identified 3 kinase genes from a [~]500-kinase library and another 11 genes from 3,300 random genes that function in mitochondrial fission. Many of these identified genes play roles in chromosome segregation. We find that chromosome missegregation and genome instability lead to dysregulation of mitochondrial fission in a manner independent of Drp-1. ATL-1, the C. elegans ATR orthologue, plays a protective role in alleviating the mitochondrial fission defect caused by chromosome missegregation. This establishes a screening paradigm for identifying mitochondrial fission regulators which reveals the role of ATR in surveilling mitochondrial fission to mitigate dysregulation caused by improper chromosome segregation.

cell biology↗

bioRxiv2023

Highly multiplexed, image-based pooled screens in primary cells and tissues with PerturbView

Optical pooled screening (OPS) is a highly scalable method for linking image-based phenotypes with cellular perturbations. However, it has thus far been restricted to relatively low-plex phenotypic readouts in cancer cell lines in culture, due to limitations associated with in situ sequencing (ISS) of perturbation barcodes. Here, we developed PerturbView, an OPS technology that leverages in vitro transcription (IVT) to amplify barcodes prior to ISS, enabling screens with highly multiplexed phenotypic readouts across diverse systems, including primary cells and tissues. We demonstrate PerturbView in iPSC-derived neurons, primary immune cells, and tumor tissue sections from animal models. In a screen of immune signaling pathways in primary bone marrow-derived macrophages, PerturbView uncovered both known and novel regulators of NF{kappa}B signaling. Furthermore, we combined PerturbView with spatial transcriptomics in tissue sections from a mouse xenograft model, paving the way to in vivo screens with rich optical and transcriptomic phenotypes. PerturbView broadens the scope of OPS to a wide range of models and applications.

systems biology↗

bioRxiv2023

CpG Island Definition and Methylation Mapping of the T2T-YAO Genome

Precisely defining and mapping all cytosine positions and their clusters, known as CpG islands (CGIs), as well as their methylation status are pivotal for genome-wide epigenetic studies, especially when population-centric reference genomes are ready for timely application. Here we first align the two high-quality reference genomes, T2T-YAO and T2T-CHM13, from different ethnic backgrounds in a base-by-base fashion and compute their genome-wide density-defined and position-defined CGIs. Second, mapping some representative genome-wide methylation data from selected organs onto the two genomes, we find that there are about 4.7-5.8% sequence divergency of variable categories depending on quality cutoffs. Genes among the divergent sequences are mostly associated with neurological functions. Moreover, CGIs associated with the divergent sequences are significantly different with respect to CpG density and observed CpG/expected CpG (O/E) ratio between the two genomes. Finally, we find that the T2T-YAO genome not only has a greater CpG site coverage than that of the T2T-CHM13 genome when whole-genome bisulfite sequencing (WGBS) data from the European and American populations are mapped to each reference, but also show more hyper-methylated CpG sites as compared to the T2T-CHM13 genome. Our study suggests that future genome-wide epigenetic studies of the Chinese populations rely on both acquisition of high-quality methylation data and subsequent precision CGI mapping based on the Chinese T2T reference.

bioinformatics↗

bioRxiv2023

Histone lactylation antagonizes senescence and skeletal muscle aging via facilitating gene expression reprogramming

One of the prominent drivers of cellular senescence and/or aging is epigenetic alteration, through which orchestrated regulation of gene expression is achieved during the processes. Accumulating endeavors have been devoted to identifying histone modifications-related mechanisms underlying senescence and aging. Here, we show that histone lactylation, a recently identified histone modification bridging metabolism, epigenetic regulation of gene expression and cellular activities in response to internal and external cues, plays a crucial role in counteracting senescence as well as mitigating dysfunctions of skeletal muscle in aged mice. Mechanistically, the abundance of histone lactylation is markedly decreased during senescence and aging but restored following manipulation of the metabolic environment. Genome-wide distribution profiling and gene expression network analysis uncover that the maintenance of histone lactylation level is critical for suppressing senescence and aging programs via targeting of proliferation- and homeostasis-related pathways. We also confirmed that the level of histone lactylation is not only controlled by glycolysis but also regulated by NAD+ content in vivo. More intriguingly, running exercise enhances the level of histone lactylation and reconstructs the cell landscape and communications of mouse skeletal muscle, leading to rejuvenation and functional improvement. Our study highlights the role of histone lactylation in regulating senescence as well as aging-related tissue function, implying that this modification could be used as a novel marker of senescence, and provides a potential target for aging intervention via metabolic manipulation.

cell biology↗

bioRxiv2023

A spatially resolved single cell genomic atlas of the adult human breast

The adult human breast comprises an intricate network of epithelial ducts and lobules that are embedded in connective and adipose tissue. While previous studies have mainly focused on the breast epithelial system, many of the non-epithelial cell types remain understudied. Here, we constructed a comprehensive Human Breast Cell Atlas (HBCA) at single-cell and spatial resolution. Our single-cell transcriptomics data profiled 535,941 cells from 62 women, and 120,024 nuclei from 20 women, identifying 11 major cell types and 53 cell states. These data revealed abundant pericyte, endothelial and immune cell populations, and highly diverse luminal epithelial cell states. Our spatial mapping using three technologies revealed an unexpectedly rich ecosystem of tissue-resident immune cells in the ducts and lobules, as well as distinct molecular differences between ductal and lobular regions. Collectively, these data provide an unprecedented reference of adult normal breast tissue for studying mammary biology and disease states such as breast cancer.

genomics↗

bioRxiv2023

Zero-shot learning enables instant denoising and super-resolution in optical fluorescence microscopy

Computational super-resolution (SR) methods, including conventional analytical algorithms and deep learning models, have substantially improved optical microscopy. Among them, supervised deep neural networks have demonstrated outstanding SR performance, however, demanding abundant high-quality training data, which are laborious and even impractical to acquire due to the high dynamics of living cells. Here, we develop zero-shot deconvolution networks (ZS-DeconvNet) that instantly enhance the resolution of microscope images by more than 1.5-fold over the diffraction limit with 10-fold lower fluorescence than ordinary SR imaging conditions in an unsupervised manner without the need for either ground truths or additional data acquisition. We demonstrate the versatile applicability of ZS-DeconvNet on multiple imaging modalities, including total internal reflection fluorescence microscopy, three-dimensional (3D) wide-field microscopy, confocal microscopy, lattice light-sheet microscopy, and multimodal structured illumination microscopy (SIM), which enables multi-color, long-term, super-resolution 2D/3D imaging of subcellular bioprocesses from mitotic single cells to multicellular embryos of mouse and C. elegans.

bioengineering↗

bioRxiv2023

Single-cell transcriptomic landscape deciphers novel olfactory neuroblastoma subtypes and intratumoral heterogeneity

Olfactory neuroblastoma (ONB) is an uncommon malignant tumor known to originate from the olfactory epithelial. The complex tumor ecosystem of this pathology remained unclear. Here, we explored the cellular components with ONB tumors based on scRNA profiles of 96,325 single-cells derived from 10 tumors and 1 olfactory mucosa sample. We discovered 11 major cell types, including 6 immune cell, 3 stromal cell subtypes and epithelial cluster in the tumor microenvironment (TME), and identified 5 common expression programs from malignant epithelial cells. We analyzed subclusters of TME and the interactions among different cell types in the TME. An innovative three-classification of ONB was established via scRNA analysis. Markers for categorizing tumor samples into new subtypes were elucidated. Different responses towards certain chemotherapy regimens could be inferred according to the molecular features of three tumor types. Relative abundance of immunosuppressive TAMs indicated the benefits of immunotherapies targeting myeloid cells.

cancer biology↗

bioRxiv2022

Comparison of proton transfer paths to the QA and QB sites of the Rb. sphaeroides photosynthetic reaction centers

The photosynthetic bacterial reaction centers from purple non-sulfur bacteria use light energy to drive the transfer of electrons from cytochrome c to ubiquinone. Ubiquinone bound in the QA site cycles between quinone, QA, and anionic semiquinone, QA*-, being reduced once and never binding protons. In the QB site, ubiquinone is reduced twice by QA*-, binds two protons and is released into the membrane as the quinol, QH2. The network of hydrogen bonds formed in a molecular dynamics trajectory was drawn to investigate proton transfer pathways from the cytoplasm to each quinone binding site. QA is isolated with no path for protons to enter from the surface. In contrast, there is a complex and tangled network requiring residues and waters that can bring protons to QB. There are three entries from clusters of surface residues centered around HisH126, GluH224, and HisH68. The network is in good agreement with earlier studies, Mutation of key nodes in the network, such as SerL223, were previously shown to slow proton delivery. Mutational studies had also shown that double mutations of residues such as Asp M17 and AspL210 along multiple paths in the network presented here slow the reaction, while single mutations do not. Likewise, mutation of both HisH126 and HisH128, which are at the entry to two paths reduce the rate of proton uptake.

biophysics↗

bioRxiv2022

Characterizing protein protonation microstates using Monte Carlo sampling

Proteins are polyelectrolytes with acidic or basic amino acids making up {approx}25% of the residues. The protonation state of all Asp, Glu, Arg, Lys, His and other protonatable residues, cofactors and ligands define each protonation microstate. As all of these residues will not be fully ionized or neutral, proteins exist in a mixture of microstates. The microstate distribution changes with pH. As the protein environment modifies the proton affinity of each site the distribution may also change in different reaction intermediates or as ligands are bound. Particular protonation microstates may be required for function, while others exist simply because there are many states with similar energy. Here, the protonation microstates generated in Monte Carlo sampling in MCCE are characterized in HEW lysozyme as a function of pH and bacterial photosynthetic reaction centers (RCs) in different reaction intermediates. The lowest energy and highest probability microstates are compared. The {Delta}G, {Delta}H and {Delta}S between the four protonation states of Glu35 and Asp52 in lysozyme are shown to be calculated with reasonable precision. A weighted Pearson correlation analysis identifies coupling between residue protonation states in RCs and how they change when the quinone in the QB site is reduced.

biophysics↗

bioRxiv2021

Spatial charting of single cell transcriptomes in tissues

Single cell RNA sequencing (scRNA-seq) methods can profile the transcriptomes of single cells but cannot preserve spatial information. Conversely, spatial transcriptomics (ST) assays can profile spatial regions in tissue sections, but do not have single cell genomic resolution. Here, we developed a computational approach called CellTrek that combines these two datasets to achieve single cell spatial mapping. We benchmarked CellTrek using a simulation study and two in situ datasets. We then applied CellTrek to reconstruct cellular spatial structures in existing datasets from normal mouse brain and kidney tissues. We also performed scRNA-seq and ST experiments on two ductal carcinoma in situ (DCIS) tissues and applied CellTrek to identify tumor subclones that were restricted to different ducts, and specific T cell states adjacent to the tumor areas. Our data shows that CellTrek can accurately map single cells in diverse tissue types to resolve their spatial organization.

genomics↗

bioRxiv2021

Salicylaldehyde ester-mediated protein semi-synthesis enables studies on the tetra-acetylation of HMGB1

HMGB1 (high-mobility group box 1) protein is a nonhistone chromatin-associated protein that has been widely reported to be a representative damage-associated molecular pattern (DAMP) and to play a pivotal role in proinflammatory process once it is in an extracellular location. Accumulating evidence has shown HMGB1 undergoes extensive PTMs that remarkably regulated its conformation, localization, and intermolecular interaction. However, the PTMrelated study has been dramatically hindered by the difficulty to access to homogenous proteins with site-specific PTMs of interest. Here, we introduce a protein semi-synthesis strategy via salicylaldehyde ester-mediated chemical ligations (Ser/Thr ligation and Cys/Pen ligation, STL/CPL). This methodology has enabled us to generate N-terminal acetylated HMGB1 proteins in high purity. Further studies revealed that the acetylation on N-terminus regulates its interaction with heparin and modulates its stability, representing a regulatory switch to control the HMGB1s activity.

biochemistry↗

bioRxiv2021

Selective cleavage of ncRNA and antiviral activity by human RNase2/EDN in a macrophage infection model

RNase2, also named the Eosinophil derived Neurotoxin (EDN), is one of the main proteins secreted by the eosinophil secondary granules. RNase2 is also expressed in other leukocyte cells and is the member of the human ribonuclease A family most abundant in macrophages. The protein is endowed with a high ribonucleolytic activity and participates in the host antiviral activity. Although RNase2 displays a broad antiviral activity, it is mostly associated to the targeting of single stranded RNA viruses. To explore RNase2 mechanism of action in antiviral host defence we knocked out RNase2 expression in the THP1 monocyte cell line and characterized the cell response to human Respiratory Syncytial Virus (RSV). We observed that RSV infection induced the RNase2 expression and protein secretion in THP1 macrophage-derived cells, whereas the knockout (KO) of RNase2 resulted in higher RSV burden and reduced cell viability. Next, by means of the cP-RNAseq methodology, which uniquely amplifies the RNA 23cyclic-phosphate-end products released by an endonuclease cleavage, we compared the ncRNA population in native and RNase2-KO cell lines. Among the ncRNAs accumulated in WT versus KO cells, we found mostly tRNA-derived fragments and secondly miRNAs. Analysis of the differential sequence coverage of tRNAs molecules in native and KO cells identified fragments derived from only few parental tRNAs, revealing a predominant cleavage at anticodon loops and secondarily at D-loops. Inspection of cleavage region identified U/C and A, at 5 and 3 sides of cleavage sites respectively (namely RNase B1 and B2 base binding subsites). Likewise, only few selected miRNAs were significantly more abundant in WT versus RNase2-KO cells, with cleavage sites located at the end of stem regions with predominance for pyrimidines at B1 but following an overall less defined nucleotide specificity. Complementarily, by screening of a tRF&tiRNA PCR array we identified an enriched population of tRNA-derived fragments associated to RNase2 expression and RSV infection. The present results confirm the contribution of the protein in macrophage response against virus infection and provide the first evidence of its cleavage selectivity against ncRNA population. A better understanding of the mechanism of action of RNase2 recognition of cellular RNA during the antiviral host defence should pave the basis for the design of novel antiviral drugs.

cell biology↗

bioRxiv2021

AFEchidna is a R package for genetic evaluation of plant and animal breeding datasets

Progeny tests play important roles in plant and animal breeding programs, and mixed linear models are usually performed to estimate variance components of random effects, estimate the fixed effects (Best Linear Unbiased Estimates, BLUEs) and predict the random effects (Best Linear Unbiased Predictions, BLUPs) via restricted maximum likehood (REML) methods in progeny test datasets. The current pioneer software for genetic assessment is ASReml, but it is commercial and expensive. Although there is free software such as Echidna or the R package sommer, the Echidna syntax is complex and the R package functionality is limited. Therefore, this study aims to develop an R package named AFEchidna based on Echidna software. The mixed linear models are conveniently implemented for users through the AFEchidna package to solve variance components, genetic parameters and the BLUP values of random effects, and the batch analysis of multiple traits, multiple variance structures and multiple genetic parameters can be also performed, as well as comparison between different models and genomic BLUP analysis. The AFEchidna package is free, please email us (yzhlinscau@163.com) to get a copy if reader is interested for it. The AFEchidna package is developed to expand free genetic assessment software with the expectation that its efficiency could be close to the commercial software.

plant biology↗

bioRxiv2021

Quickomics: exploring omics data in an intuitive, interactive and informative manner

SummaryWe developed Quickomics, a feature-rich R Shiny-powered tool to enable biologists to fully explore complex omics statistical analysis results and perform advanced analysis in an easy-to-use interactive interface. It covers a broad range of secondary and tertiary analytical tasks after primary analysis of omics data is completed. Each functional module is equipped with customizable options and generates both interactive and publication-ready plots to uncover biological insights from data. The modular design makes the tool extensible with ease. AvailabilityResearchers can experience the functionalities with their own data or demo RNA-Seq and proteomics datasets by using the app hosted at http://quickomics.bxgenomics.com and following the tutorial, https://bit.ly/3rXIyhL. The source code under GPLv3 license is provided at https://github.com/interactivereport/Quickomics for local installation. Supplementary informationSupplementary materials are available at https://bit.ly/37HP17g.

bioinformatics↗

bioRxiv2020

Bio-upcycling of polyethylene terephthalate

Over 359 million tons of plastics were produced worldwide in 2018, with significant growth expected in the near future, resulting in the global challenge of end-of-life management. The recent identification of enzymes that degrade plastics previously considered non-biodegradable opens up opportunities to steer the plastic recycling industry into the realm of biotechnology. Here, we present the sequential conversion of polyethylene terephthalate (PET) into two types of bioplastics: a medium chain-length polyhydroxyalkanoate (PHA) and a novel bio-based poly(amide urethane) (bio-PU). PET films were hydrolyzed by a thermostable polyester hydrolase yielding 100% terephthalate and ethylene glycol. A terephthalate-degrading Pseudomonas was evolved to also metabolize ethylene glycol and subsequently produced PHA. The strain was further modified to secrete hydroxyalkanoyloxy-alkanoates (HAAs), which were used as monomers for the chemo-catalytic synthesis of bio-PU. In short, we present a novel value-chain for PET upcycling, adding technological flexibility to the global challenge of end-of-life management of plastics. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/993592v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1433e0dorg.highwire.dtl.DTLVardef@19efc71org.highwire.dtl.DTLVardef@cf97f7org.highwire.dtl.DTLVardef@101f123_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗