Biology subjects

Zhao, Y.

Publications and source records attributed to Zhao, Y..

At least 415 records · Page 23Linked to original sources

bioRxiv2024

Perinatal Reduction of Genetically Aberrant Neurons from Human Cerebral Cortex

Human neurons are postmitotic and long-lived, requiring precise genomic regulation to maintain function over a lifetime. Normal neuronal function is highly dependent on gene dosage, with copy number variants (CNVs) and heterozygous point mutations associated with a host of neurodevelopmental and neuropsychiatric conditions [1-5]. Here, we investigated the landscape of somatic CNVs arising in fetal human brains, and how they change over development, to understand the processes that generate normal neuronal genomes. We sequenced 2,158 single neurons from human postmortem brain using two distinct single cell whole genome sequencing (scWGS) approaches. Tn5-transposase based (TbA) scWGS of 1,327 neurons from 16 individuals ranging in age from gestational week 14 to 90 years old resulted in 8,765 CNVs. Primary template amplification (PTA) was used to assess for CNV from 831 neurons from 12 individuals. Up to 46% of neurons in prenatal cortex showed aberrant genomes, characterized by widespread CNVs of multiple chromosomes, but this proportion reduces by 4-5 fold after birth across the two scWGS approaches (p=7.5x10-6, Fisher meta-analysis). We identified micronuclei in the developing cortex in situ, reflecting chromosomal material missegregated during neurodevelopment [6-8]. Neurons with widespread CNVs were eliminated in the perinatal period, while neurons with smaller CNV burden slowly declined during postnatal aging. CNVs in surviving neurons were depleted for genes that are dosage-sensitive or involved in neurodevelopmental disorders (p<0.05), suggesting selective elimination of neurons with CNVs involving these critical genes. We surveyed 44,861 nuclei with 10X Genomics scATAC/RNAseq and determined that neurons with high CNV burdens also showed abnormal expression of synaptic gene sets, suggesting that abnormal synaptic gene regulation contributes to neuronal elimination. Elimination of defective neuronal genomes during synaptogenesis may represent a critical process of genome quality control and a vulnerable target of factors that contribute to neurodevelopmental disease.

neuroscience↗

bioRxiv2024

Disruption of the Novel Nested Gene Aff3ir Mediates Disturbed Flow-Induced Atherosclerosis in Mice

Disturbed shear stress-induced endothelial atherogenic responses are pivotal in the initiation and progression of atherosclerosis, contributing to the uneven distribution of atherosclerotic lesions. This study investigates the role of Aff3ir-ORF2, a novel nested gene variant, in disturbed flow-induced endothelial cell activation and atherosclerosis. We demonstrate that disturbed shear stress significantly reduces Aff3ir-ORF2 expression in athero-prone regions. Using three distinct mouse models with manipulated AFF3ir-ORF2 expression, we demonstrate that AFF3ir-ORF2 exerts potent anti-inflammatory and anti-atherosclerotic effects in ApoE-/- mice. RNA sequencing revealed that interferon regulatory factor 5 (IRF5), a key regulator of inflammatory processes, mediates inflammatory responses associated with AFF3ir-ORF2 deficiency. AFF3ir-ORF2 interacts with IRF5, promoting its retention in the cytoplasm, thereby inhibiting the IRF5-dependent inflammatory pathways. Notably, IRF5 knockdown in AFF3ir-ORF2 deficient mice almost completely rescues the aggravated atherosclerotic phenotype. Moreover, endothelial-specific AFF3ir-ORF2 supplementation using the CRISPR/Cas9 system significantly ameliorated endothelial activation and atherosclerosis. These findings elucidate a novel role for AFF3ir-ORF2 in mitigating endothelial inflammation and atherosclerosis by acting as an inhibitor of IRF5, highlighting its potential as a valuable therapeutic approach for treating atherosclerosis.

cell biology↗

bioRxiv2024

Neural population variance explains adaptation differences during learning

Variability, a ubiquitous feature of neural activity, plays an integral role in behavior. However, establishing a causal relationship between neural signals and behavior is difficult. By defining a mathematical mapping between neural spiking activity and behavior, we investigate the role of spiking variability in adaptation during a brain-computer interface (BCI) behavior in male rhesus macaques (Macaca mulatta, n=2). Recent BCI evidence demonstrates that creating novel neural patterns is harder than repurposing existing patterns to respond to changes in external input. However, what limits the ability to repurpose, or adapt, patterns under different magnitudes of change is less well-characterized. Here, we present evidence that variance in neural spiking activity reveals differences in learnability between easy and hard adaptation conditions and across sessions. Furthermore, our study illuminates the limitations in neural changes underlying behavior within a neurofeedback paradigm. Significance StatementVariability in neural activity is a major driver of behavioral variability, though it is unclear how variability is balanced with stable neural activity as new behaviors become more practiced. By using a brain-computer interface methodology, we define a mathematical mapping between neural spiking activity and a behavioral control signal. Through thoughtful manipulation of this mapping, we incite the subjects (rhesus macaques) to learn and adapt neural activity to regain behavioral proficiency. We find that metrics of neural population variability are differentially modulated depending on difficulty of the imposed manipulation. Our exciting results provide important implications for brain-computer interface applications as well as our understanding of learning and adaptation more broadly. Our work represents an important step forwards towards understanding population neural dynamics in this critical component of behavior.

bioengineering↗

bioRxiv2024

scMODAL: A general deep learning framework for comprehensive single-cell multi-omics data alignment with feature links

Recent advancements in single-cell technologies have enabled comprehensive characterization of cellular states through transcriptomic, epigenomic, and proteomic profiling at single-cell resolution. These technologies have significantly deepened our understanding of cell functions and disease mechanisms from various omics perspectives. As these technologies evolve rapidly and data resources expand, there is a growing need for computational methods that can integrate information from different modalities to facilitate joint analysis of single-cell multi-omics data. However, integrating single-cell omics datasets presents unique challenges due to varied feature correlations and technology-specific limitations. To address these challenges, we introduce scMODAL, a deep learning framework tailored for single-cell multi-omics data alignment using feature links. scMODAL integrates datasets with limited known positively correlated features, leveraging neural networks and generative adversarial networks to align cell embeddings and preserve feature topology. Our experiments demonstrate scMODALs effectiveness in removing unwanted variation, preserving biological information, and accurately identifying cell subpopulations across diverse datasets. scMODAL not only advances integration tasks but also supports downstream analyses such as feature imputation and feature relationship inference, offering a robust solution for advancing single-cell multi-omics research.

bioinformatics↗

bioRxiv2024

SC-MAMBA2: Leveraging State-Space Models for Efficient Single-Cell Ultra-Long Transcriptome Modeling

AO_SCPLOWBSTRACTC_SCPLOWThe rapid advancement of single-cell sequencing technology has significantly deepened our understanding of cellular heterogeneity, yet it concurrently presents substantial challenges for the unified modeling of single-cell data. Simultaneously, pre-trained foundation models have achieved notable success in domains such as natural language processing and image analysis. However, extending these models to accommodate ultra-long single-cell transcriptome sequences, characterized by an extensive number of genes, remains a formidable task. In this study, we introduce SC-MAMBA2, based on the MAMBA2 architecture, meticulously designed with a bidirectional modeling approach tailored for single-cell transcriptomics data. As the first single-cell foundation model to integrate state-space models (SSMs) underlying MAMBA2 architecture, SC-MAMBA2 features over 625 million parameters, covers more than 60,000 genes, and was pre-trained on a dataset of over 57 million cells, making it the most comprehensive solution for processing ultra-long transcriptome sequences. Extensive bench-marking across a diverse array of downstream tasks consistently demonstrates that SC-MAMBA2 surpasses state-of-the-art models, delivering superior accuracy and enhanced computational efficiency.

cell biology↗

bioRxiv2024

Potent and Selective SETDB1 Covalent Negative Allosteric Modulator Reduces Methyltransferase Activity in Cells

A promising drug target, SETDB1, is a dual Kme reader and methyltransferase, which has been implicated in cancer and neurodegenerative disease progression. To help understand the role of the triple Tudor domain (3TD) of SETDB1, its Kme reader, we first identified a low micromolar small molecule ligand, UNC6535, which occupies simultaneously both the TD2 and TD3 reader binding sites. Further optimization led to the discovery of UNC10013, the first covalent 3TD ligand targeting Cys385 of SETDB1. UNC10013 is potent with a kinact/KI of 1.0 x 106 M-1s-1 and demonstrated proteome-wide selectivity. In cells, negative allosteric modulation of SETDB1-mediated Akt methylation was observed after treatment with UNC10013. Therefore, UNC10013 is a potent, selective and cell-active covalent ligand for the 3TD of SETDB1, demonstrating negative allosteric modulator properties and making it a promising tool to study the biological role of SETDB1 in disease progression. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/615363v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@6db8e2org.highwire.dtl.DTLVardef@1852ee3org.highwire.dtl.DTLVardef@c95bb6org.highwire.dtl.DTLVardef@1a43951_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

LRRC8 complexes are adenosine nucleotide release channels regulating platelet activation and arterial thrombosis

Platelet shape and volume changes are early mechanical events contributing to platelet activation and thrombosis. Here, we identify single-nucleotide polymorphisms in Leucine-Rich Repeat Containing 8 (LRRC8) protein subunits that form the Volume-Regulated Anion Channel (VRAC) which are independently associated with altered mean platelet volume. LRRC8A is required for functional VRAC in megakaryocytes (MKs) and regulates platelet volume, adhesion, and agonist-stimulated activation, aggregation, ATP secretion and calcium mobilization. MK-specific LRRC8A cKO mice have reduced arteriolar thrombus formation and prolonged arterial thrombosis without affecting bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced ATP/ADP release to amplify agonist-stimulated calcium and PI3K-AKT signaling via P2X1, P2Y1 and P2Y12 receptors. Small-molecule LRRC8 channel inhibitors recapitulate defects observed in LRRC8A-null platelets in vitro and in vivo. These studies identify the mechanoresponsive LRRC8 channel complex as an ATP/ADP release channel in platelets which regulates platelet function and thrombosis, providing a proof-of-concept for a novel anti-thrombotic drug target.

cell biology↗

bioRxiv2024

Long-read whole-genome sequencing-based concurrent haplotyping and aneuploidy profiling of single cells

Long-read whole-genome sequencing (lrWGS) enhances haplotyping by providing more phasing information per read compared to short-read sequencing. However, its use for single-cell haplotype phasing remains underexplored. This proof-of-concept study examines lrWGS data from single cells for small variant (SNV and indel) calling and haplotyping using the Genome in a Bottle (GIAB) Ashkenazi trio. lrWGS was performed on single-cell (1 cell) and multi-cell (10 cells) samples from the offspring. Chromosome-length haplotypes were obtained by leveraging both long reads and pedigree information. These haplotypes were further refined by replacing them with matched parental haplotypes. In single-cell and multi-cell samples, 92% and 98% of heterozygous SNVs, and 74% and 78% of heterozygous indels were accurately haplotyped. Applied to human embryos for preimplantation genetic testing (PGT), lrWGS demonstrated 100% consistency with array-based methods for detecting monogenic disorders, without requiring phasing references. Aneuploidies were accurately detected, with insights into the mechanistic origins of chromosomal abnormalities inferred from the parental unique allele fractions. We show that lrWGS-based concurrent haplotyping and aneuploidy profiling of single cells provides an alternative to current PGT methods, with applications potential in areas such as cell-based prenatal diagnosis and animal and plant breeding.

genomics↗

bioRxiv2024

Germinated brown rice research: bibliometric analysis of progress, hotspots and trends

To gain a deeper understanding of global research trends and focal points in germinated brown rice, this article takes the relevant literature on germinated brown rice in the core database of Web of Science as the research object. By using bibliometric analysis, the literature on germinated brown rice published from 2003 to 2023 is deeply analyzed, and the global research progress, hotspots and future development trend of germinated brown rice are summarized. Since 2018, research on germinated brown rice has been rapidly developing, with a significant surge in interest since 2020. China leads in the number of publications, institutions, and core authors in this research area. The primary research topics include the nutritional value, physiological active components, and industrial applications of germinated brown rice. Current research frontiers involve identifying, evaluating, and enhancing bioactive substances in germinated brown rice for food applications. Research in this field remains active, and application scenarios are becoming increasingly diverse. Future studies may explore new directions in related equipment and ingredients.

bioinformatics↗

bioRxiv2024

Association analysis of PAEP, KRT10 and BMP7 Genes SNPs with reproductive traits in Kele pigs

The aims of this study were to investigate the effects of single nucleotide polymorphisms (SNPs) in progestogen-associated endometrial protein (PAEP), keratin 10 (KRT10), and bone morphogenetic protein 7 (BMP7) genes on reproductive traits (total number of piglets born, number of piglets born alive, litter birth weight, number of piglets weaned and litter weight weaned) in Kele pigs. We used a total of 255 multiparous Kele sow (2-4 parities) as research materials. SNPs were identified by PCR amplification instrument and sequence alignment software DANMAN. The population genetic characteristics of SNPs were analyzed using the SHEsis online software. Bioinformatics analysis of SNPs were conducted using RNAfold, SOPMA, SWISS-MODEL, and Swiss-PdbViewer programs. The associations between the SNPs and reproductive traits in Kele pigs were analyzed through SPSS 22.0 software. In this study, nine SNPs were identified in the three genes: g.1884992 T>C (exon 4), g.1885152 G>C (intron 4) and g.1887834 G>A (intron 4) in PAEP, g.21643703 C>T (intron 4), g.21643714 G>A (intron 4) and g.21643741 G>A (exon 5) in KRT10, and g.57647887 G>A (intron 3), g.57647990 C>T (intron 3) and g.57648145 C>G (intron 3) in BMP7. In SNPs g.1884992 T>C of PAEP, missense mutation eventuated structural changes in mRNA and proteins secondary structure. In SNPs g.21643741 G>A of KRT10, synonymous mutation led to alteration in mRNA secondary structure. For PAEP, the CC genotype in SNPs g.1885152 G>C and the AA genotype in SNPs g.1887834 G>A showed significantly higher values than other genotypes in all reproduction traits except for litter birth weight, preliminarily identified as favorable genotypes. For KRT10, the GG genotype in SNPs g.21648641 G>A showed significant superiority than AA genotype (P<0.05) in all aspects except for litter birth weight, and notably surpassed the GA genotype in total number of piglets born (P<0.05), preliminarily recognized as a favorable genotype. Regarding BMP7, the GA genotype in SNPs g.57647887 G>A and the CT genotype in SNPs g.57647990 C>T exhibited significantly higher number of piglets born alive and number of piglets born alive compared to other genotypes (P<0.05). And the GG genotype in SNPs g.57648145 C>G was significantly associated with higher litter birth weight (P<0.05). The result of diplotype analyses indicated that the H3H3 (CCGGGG) of PAEP and H3H3 (CCGGAA) of KRT10 had a significant effect on the five traits. For BMP7, the H4H4 (AATTGG) diplotype showed a significant influence on all genotypes except litter birth weight.

genetics↗

bioRxiv2024

Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

Mechanistic understanding of the immune checkpoint receptor PD1 is largely based on mouse models, but human and mouse PD1 orthologs exhibit only 59.6% identity in amino acid sequences. Here we show that human PD1 is more inhibitory than mouse PD1 due to stronger interactions with the ligands PDL1 and PDL2 and with the effector phosphatase Shp2. A novel motif highly conserved among PD1 orthologs in vertebrates except in rodents is primarily responsible for the differential Shp2 recruitment. Evolutionary analysis suggested that rodent PD1 orthologs uniquely underwent functional relaxation, particularly during the K-Pg boundary. Humanization of the PD1 intracellular domain disrupted the anti-tumor activity of mouse T cells while increasing the magnitude of anti-PD1 response. Together, our study uncovers species-specific features of the PD1 pathway, with implications to PD1 evolution and differential anti-PD(L)1 responses in mouse models and human patients.

immunology↗

bioRxiv2024

Lysosomal LRRC8 complex regulates lysosomal pH, morphology and systemic glucose metabolism

The lysosome integrates anabolic signalling and nutrient-sensing to regulate intracellular growth pathways. The leucine-rich repeat containing 8 (LRRC8) channel complex forms a lysosomal anion channel and regulates PI3K-AKT-mTOR signalling, skeletal muscle differentiation, growth, and systemic glucose metabolism. Here, we define the endogenous LRRC8 subunits localized to a subset of lysosomes in differentiated myotubes. We show LRRC8A regulates leucine-stimulated mTOR, lysosome size, number, pH, and expression of lysosomal proteins LAMP2, P62, LC3B, suggesting impaired autophagic flux. Mutating a LRRC8A lysosomal targeting dileucine motif sequence (LRRC8A-L706A;L707A) in myotubes recapitulates the abnormal AKT signalling and altered lysosomal morphology and pH observed in LRRC8A KO cells. In vivo, LRRC8A-L706A;L707A KI mice exhibit increased adiposity, impaired glucose tolerance and insulin resistance characterized by reduced skeletal muscle glucose-uptake, and impaired incorporation of glucose into glycogen. These data reveal a lysosomal LRRC8 mediated metabolic signalling function that regulates lysosomal activity, systemic glucose homeostasis and insulin-sensitivity.

cell biology↗

bioRxiv2024

Unveil the Molecular Interplay between Aminoglycosides and Pseudouridine in IRES Translation

Eukaryotic ribosomes are enriched with pseudouridine, particularly at the functional centers targeted by antibiotics. Here we investigated the roles of pseudouridine in aminoglycoside-mediated translation inhibition by comparing the structural and functional properties of the wild-type ribosomes and those lacking pseudouridine (cbf5-D95A). We showed that the cbf5-D95A ribosomes have decreased thermostability and high sensitivity to aminoglycosides. When presented with an internal ribosome entry site (IRES) RNA, elongation factor eEF2, GTP, sordarin, hygromycin B preferentially binds to the cbf5-D95A ribosomes during initiation by blocking eEF2 binding and stalls the ribosomes in a non-rotated conformation, further hindering translocation. Hygromycin B binds to the inter-subunit bridge B2a that is known to be sensitive to pseudouridine, revealing a functional link between pseudouridine and aminoglycoside inhibition. Our results suggest that pseudouridine enhances both thermostability and conformational fitness of the ribosomes, thereby influencing their susceptibility to aminoglycosides. HighlightsO_LILoss of pseudouridine increases cell sensitivity to aminoglycosides C_LIO_LIPseudouridine enhances ribosome thermostability C_LIO_LIHygromycin B competes with eEF2 for the non-rotated ribosome C_LIO_LIHygromycin B deforms the codon-anticodon duplex C_LI

biochemistry↗

bioRxiv2024

MdLRR-RLK1-MdATG3 module enhances the resistance of apples to abiotic stress via autophagy

LRR-RLKs play a key role in plant responses to stress, although their physiological functions under abiotic stress are not yet fully understood. MdLRR-RLK1 was found to regulate plant growth and development and to increase tolerance to salt and drought stress. MdLRR-RLK1 interacts with MdATG3, which ubiquitinates and degrades MdLRR-RLK1. MdATG3 enhances salt and drought tolerance through autophagy, interacting with MdATG8F/I-like in apple. These findings reveal the interaction between MdLRR-RLK1 and MdATG3, suggesting mechanisms that regulate apple growth and resistance to abiotic stress.

molecular biology↗

bioRxiv2024

A T6SS DNase effector induces nuclear DNA Damage to trigger apoptosis via activation of the cGAS-STING-TNF axis

In the relentless battle for survival, bacteria have evolved sophisticated weaponry like the Type VI secretion system (T6SS) to not only outcompete rivals but also manipulate host cell processes to their advantage. Apoptosis, a controlled form of programmed cell death without triggering inflammatory responses, is often exploited by pathogens. While many bacterial pathogens are known to induce apoptosis, the role of T6SS effectors in this process remains unexplored. In this study, we discovered that Yersinia pseudotuberculosis (Yptb) utilizes its T6SS to secrete a DNase effector, TkeA, which induces apoptosis in host cells. Mechanistically, the translocation of TkeA into host cells causes nuclear DNA damage, leading to the release of fragmented DNA into the cytoplasm. This, in turn, activates the DNA-sensing cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway. The activation of the cGAS-STING pathway by TkeA subsequently triggers apoptosis in host cells via extrinsic pathways, with tumor necrosis factor (TNF) signaling playing a critical role. Additionally, TkeA also mediates interbacterial competition, promoting the colonization of Yptb in mices gut. Our findings reveal that the T6SS-secreted DNase effector TkeA acts as a trans-kingdom effector, enhancing interbacterial competition and inducing apoptosis in host cells by activating the cGAS-STING-TNF axis. This discovery adds a new dimension to the mechanisms employed by bacterial pathogens to manipulate host cell death pathways by using their secretion systems. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/613802v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@1bfd70org.highwire.dtl.DTLVardef@19bfc89org.highwire.dtl.DTLVardef@1e0bf9dorg.highwire.dtl.DTLVardef@4412b9_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTS* Yptb T6SS secretes a trans-kingdom DNase effector TkeA * TkeA degrades bacterial DNA to mediate interbacterial competition * TkeA causes nuclear DNA damage in host cells to induce apoptosis * TkeA induces apoptosis via the cGAS-STING-TNF axis

microbiology↗

bioRxiv2024

Age-related decline of PKA-RIIβ level in SNc dopaminergic neurons underlies PD pathogenesis

The cyclic-AMP dependent protein kinase A (Protein kinase A, PKA) regulates dopaminergic function in the substantia nigra pars compacta (SNc). However, whether PKA is involved in the pathogenesis of Parkinsons disease (PD) is unknown. Here, by collecting and analyzing the current worldwide SNc transcriptomic datasets of PD patients, we found a decline of PKA-RII{beta} subunit level in the SNc of PD patients. The decreased PKA-RII{beta} subunit level was positively correlated with decreased dopamine synthesis and increased oxidative stress in the SNc of PD patients. PKA-RII{beta} subunit is expressed in the striatum and the SNc. PKA-RII{beta} gene knockout mice (RII{beta}-KO) showed a age-related parkinsonism at 12 months of age. Using Cre-LoxP system, we observed that RII{beta} re-expression in the SNc dopaminergic neurons rescued parkinsonism of RII{beta}-KO mice. RII{beta} re-expression in striatal neurons did not affect parkinsonism of RII{beta}-KO mice. The spontaneous parkinsonism could be developed in 12-month-old SNc dopaminergic neuron-specific RII{beta}-deficient mice. Single-nucleus RNA sequencing revealed decreased PKA activity, reduced dopamine synthesis and raised oxidative stress in the SNc dopaminergic neurons of RII{beta}-KO mice. Adeno-associated virus (AAV)-mediated gene therapy targeting PKA-RII{beta} in the SNc dopaminergic neurons rescued parkinsonism in PD mouse model. Taken together, these findings indicate that PKA-RII{beta} may be a key factor of human genetic etiologies of PD. The therapy targeting PKA-RII{beta} in the SNc dopaminergic neurons may be promising for PD treatment.

neuroscience↗

bioRxiv2024

Porosome reconstitution therapy: A biologic rescue from cystic fibrosis

Cystic fibrosis (CF) is a genetic disorder resulting from mutations in the CF Transmembrane Conductance Regulator (CFTR) gene that codes for a chloride transporting channel at the cell plasma membrane. In CF, highly viscous mucus is secreted in the airways preventing its clearance, leading to lung infections and respiratory failure. A major challenge in treating CF patients has been the presence of more than 2,000 different CFTR mutations or due to the absence of CFTR expression. CFTR is among the 34 major proteins composing the 100 nm porosome secretory machinery in the human airway epithelia, involved in mucin secretion. The airways is coated with a thin film of mucus, composed primarily of mucin MUC5AC and MUC5B. Sputum from patients with CF show a >70% decrease in MUC5B and MUC5AC secretion. Our studies using differentiated 3D cultures of human airway epithelial cell line, also demonstrate loss of both chloride and mucus secretion following exposure to CFTR inhibitors thiazolidinone 172 or the hydrazide GlyH101. Our studies show that human bronchial epithelial (HBE) cells with {Delta}F508 CFTR mutation, affects nearly a dozen porosome proteins including CFTR. Therefore, we hypothesized that the introduction of normal functional porosomes carrying wild type CFTR into the cell plasma membrane of CF cells would rescue from all forms of CF. Air liquid interface (ALI) 3D differentiated HBE WT-CFTR cells and {Delta}F508-CFTR CF HBE cell cultures mimicking normal lung physiology, responding to CFTR inhibitors and CF corrector and modulator drugs Tezacaftor, Ivacaftor and TRIKAFTA, was used in the study. Introduction of functional porosome complexes obtained from WT-CFTR HBE cells into the plasma membrane (PM) of {Delta}F508-CFTR CF cells, was demonstrated by an increase in PM-associated CFTR using Magnify expansion microscopy. Mucin secretion assays demonstrate porosome reconstitution to restore mucin secretion more than twice as effectively as TRIKAFTA. These results are further supported by preliminary nasal potential different studies in {Delta}F508 mice, where treatment of the nasal passage with porosome isolates from WT-CFTR HBE cells, restore chloride secretion in the nasal passage of mice, a further validation of the highly effective porosome reconstitution therapy for CF.

cell biology↗

bioRxiv2024

A circular engineered sortase for interrogating histone H3 in chromatin

Reversible modification of the histone H3 N-terminal tail is critical in regulating chromatin structure, gene expression, and cell states, while its dysregulation contributes to disease pathogenesis. Understanding the crosstalk between H3 tail modifications in nucleosomes constitutes a central challenge in epigenetics. Here we describe an engineered sortase transpeptidase, cW11, that displays highly favorable properties for introducing scarless H3 tails onto nucleosomes. This approach significantly accelerates the production of both symmetrically and asymmetrically modified nucleosomes. We demonstrate the utility of asymmetrically modified nucleosomes produced in this way in dissecting the impact of multiple modifications on eraser enzyme processing and molecular recognition by a reader protein. Moreover, we show that cW11 sortase is very effective at cutting and tagging histone H3 tails from endogenous histones, facilitating multiplex "cut-and-paste" middle down proteomics with tandem mass tags. This cut-and-paste proteomics approach permits the quantitative analysis of histone H3 modification crosstalk after treatment with different histone deacetylase inhibitors. We propose that these chemoenzymatic tail isolation and modification strategies made possible with cW11 sortase will broadly power epigenetics discovery and therapeutic development.

biochemistry↗