Biology subjects

Zhang, H.

Publications and source records attributed to Zhang, H..

At least 829 records · Page 46Linked to original sources

bioRxiv2023

CytoSIP: An annotated structural atlas for interactions involving cytokine or cytokine receptor

Cytokines primarily interact with specific cytokine receptors on the cell surface as essential signal transduction pathways in many physiological and pathological processes. Therapeutic agents targeting cytokine-cytokine receptor (CK-CKR) interactions lead to the disruption in cellular signaling function and have been demonstrated effective in the treatment of many diseases including tumors. However, a lack of universal and quick access to annotated structural surface regions on CK/CKR has limited the progress of a structure-driven approach to the development of targeted macromolecular drugs and precision medicine therapeutics. Herein we develop CytoSIP (Single nucleotide polymorphisms (SNPs), Interface, and Phenotype), a rich internet application based on a database of atomic interactions around hotspots in experimentally determined CK/CKR structural complexes. The content of the CytoSIP database includes the following key features: (1) SNPs on CK/CKR; (2) interactions involving CK/CKR at the domain level, including CK/CKR interfaces, oligomeric interfaces, epitopes, or other drug targeting surfaces; and (3) diseases and phenotypes associated with CK/CKR or SNPs. The database introduces a unique tri-level SIP data model to link genetic variants (atomic level) to disease phenotypes (organism level) using protein structure (complexes) as an underlying framework (molecule level). Moreover, CytoSIP implements screening criteria and tools to allow customized selection of relevant subset of CK/CKR for the study of interest. This reduces the time and resources needed to interrogate large datasets and allows rapid screening of cytokines and cytokine receptor proteins interfaces for hotspots targeted drug design and any other specific cellular signaling/function mechanisms and their correlation to pathologies. The CytoSIP framework crafted herein bridges CK/CKR genotype with phenotype, facilitating not only the panoramic investigation of the context-dependent crosstalk between CK/CKR but also the development of targeted therapeutic agents. CytoSIP portal website is publicly accessible at https://CytoSIP.biocloud.top.

immunology↗

bioRxiv2023

Haplotype-resolve genome assembly and resequencing provide insights into the origin and domestication of modern rose

Modern rose (Rosa hybrida) is a recently formed interspecific hybrid and has become one of the most important and widely cultivated ornamentals. Here, we report the haplotype-resolved chromosome-scale genome assembly of the tetraploid R. hybrida Samantha and a genome variation map of 233 Rosa accessions involving various wild species, and old and modern cultivars. The two subgenomes of Samantha show no significant global bias in gene loss but substantial expression bias, and homoeologous exchanges are frequently observed between subgenomes. Population genomic and genomic composition analyses reveal the contributions of wild Rosa species to modern roses, and highlight that R. odorata and its derived cultivars contribute more to modern roses than R. chinensis Old Blush. Furthermore, selective sweeps during modern rose breeding associated with major agronomic traits, including continuous flowering, floral organ identity, flower senescence, and disease resistance, are identified. This study provides insights into the genetic basis of modern rose origin and breeding history, and offers unprecedented genomic resources for rose improvement.

genomics↗

bioRxiv2023

Microstructural dynamics of motor learning and sleep-dependent consolidation: a diffusion imaging study

Diffusion-weighted magnetic resonance imaging (DWI) allows the observation of (micro)structural brain remodelling in cortical and subcortical regions after time-limited motor learning. Post-learning sleep consolidation should lead to long-term microstructural brain changes, but concrete evidence remains limited. Here, we used both conventional diffusion tensor imaging (DTI) and Neurite Orientation Dispersion & Density Imaging (NODDI), that estimates dendritic and axonal complexity in white and grey matter, to investigate the microstructural brain mechanisms underlying time- and sleep-dependent motor memory consolidation. Sixty-one young healthy adults underwent 2 DWI sessions, with sequential motor training in between, on day 1 followed by the experimental night of total sleep deprivation or regular sleep. After 3 recovery nights at home, they underwent 2 further DWI sessions separated by a motor retraining session. Sequential motor learning resulted in immediate modifications in structural parameters in occipitoparietal and temporal regions, as well as in subcortical regions of interest. Similar changes were observed following relearning but at a smaller magnitude. Regarding delayed consolidation effects, learning-related changes only partially persisted 3 days after initial learning, and no post-learning sleep effect was detected. Our results show rapid motor learning-related remodelling, reflecting temporary processes in learning-related neuronal brain plasticity. Post-learning sleep-related cellular changes remain to be evidenced, possibly using more sophisticated brain imaging measures and spanning more extended timescales to allow the expression of structural changes.

neuroscience↗

bioRxiv2023

Soil intake modifies the gut microbiota and alleviates ovalbumin-induced mice asthma inflammation

BackgroundLow cleanliness living environment (LCLE) can increase gut microbial diversity and prevent allergic diseases, whereas gut microbial dysbiosis is closely related to the pathogenesis of asthma. Our previous studies suggested that soil in the LCLE is a key factor in shaping intestinal microbiota. ObjectiveWe aimed to explore if sterilized soil intake as prebiotics while being incubated with microbes in the air can attenuate mice asthma symptoms by modifying gut microbiota. Methods16S rRNA gene sequencing was used to analyze the gut microbial composition, in combination with immune parameters measured in the lung and serum samples. Results16S rRNA gene sequencing results showed significant differences in the fecal microbiota composition between the test and control mice, with a higher abundance of Allobaculum, Alistipes, and Lachnospiraceae_UCG-001, which produce short-chain fatty acids and are beneficial for health in the test mice. Soil intake downregulated the concentrations of IL-6, IL-4, IL-17F, TNF-, and IL-22 in serum and increased the expression of IFN-{gamma}, which regulated the Th1/Th2 balance in lung by polarizing the immune system toward Th1, strongly alleviating ovalbumin-induced asthma inflammation. The effect of sensitization on gut microbiota was greater than that of air microbes and age together, but weaker than that of soil. ConclusionSoil intake had a significant therapeutic effect on mouse asthma, possibly by promoting the growth of multiple beneficial bacteria. The results indicated that the development of soil-based prebiotic products might be used for allergic asthma management and our study provides further evidence for the hygiene hypothesis. ImportanceExposure to a low cleanliness living environment (LCLE), of which soil is an important component, can shape the gut microbiota and support immune tolerance, preventing allergic diseases such as eczema and asthma. However, with the rapid progress of urbanization, it is impossible to return to farm-like living and we are becoming disconnected from the soil. Here, our study found that ingesting sterilized soil and living in an LCLE have the same protective effects on asthma inflammation. Ingestion of sterilized soil significantly altered the gut microbial composition and exerted significant therapeutic effects on asthmatic mice. However, edible sterilized soil possesses more advantages than LCLE exposure, such as the absence of pathogenic bacteria, safer, and convenience. The results indicate that the development of soil-based prebiotic products might be used for allergic asthma management and our study further supports the hygiene hypothesis. NotificationThe article is currently undergoing peer review in the World Allergy Organization Journal.

microbiology↗

bioRxiv2023

Human-specific lncRNAs contributed critically to human evolution by distinctly regulating gene expression

What genes and regulatory sequences critically differentiate modern humans from apes and archaic humans, which share highly similar genomes but show distinct phenotypes, has puzzled researchers for decades. Previous studies examined species-specific protein-coding genes and related regulatory sequences, revealing that birth, loss, and changes in these genes and sequences can drive speciation and evolution. However, investigations of species-specific lncRNA genes and related regulatory sequences, which regulate substantial genes, remain limited. We identified human-specific (HS) lncRNAs from GENCODE-annotated human lncRNAs, predicted their DNA binding domains (DBDs) and binding sites (DBSs), analyzed DBS sequences in modern humans (CEU, CHB, and YRI), archaic humans (Altai Neanderthals, Denisovans, and Vindija Neanderthals), and chimpanzees, and investigated how HS lncRNAs and their DBSs have influenced gene expression in archaic and modern humans. Our results suggest that these lncRNAs and DBSs have substantially reshaped gene expression. This reshaping has evolved continuously from archaic to modern humans, enabling humans to adapt to new environments and lifestyles, promoting brain evolution, and resulting in cross-population differences. The parallel analysis of gene expression in GTEx tissues by HS TFs and their DBSs indicates that HS lncRNAs have reshaped gene expression in the brain more significantly than HS TFs.

genomics↗

bioRxiv2023

Deciphering deep-sea chemosynthetic symbiosis by single-nucleus RNA-sequencing

Bathymodioline mussels dominate deep-sea methane seep and hydrothermal vent habitats and obtain nutrients and energy primarily through chemosynthetic endosymbiotic bacteria in the bacteriocytes of their gill. However, the molecular mechanisms that orchestrate mussel host-symbiont interactions remain unclear. Here, we constructed a comprehensive cell atlas of the gill in the mussel Gigantidas platifrons from the South China Sea methane seeps (1100m depth) using single-nucleus RNA sequencing (snRNA-seq) and whole-mount in situ hybridisation. We identified 13 types of cells, including three previously unknown ones, and uncovered unknown tissue heterogeneity. Every cell type has a designated function in supporting the gills structure and function, creating an optimal environment for chemosynthesis, and effectively acquiring nutrients from the endosymbiotic bacteria. Analysis of snRNA-seq of in situ transplanted mussels clearly showed the shifts in cell state in response to environmental oscillations. Our findings provide insight into principles of host-symbiont interaction and the bivalves environmental adaption mechanisms.

ecology↗

bioRxiv2023

Large differences in collateral blood vessel abundance among individuals arise from multiple genetic variants

Collateral blood flow varies greatly among humans for reasons that remain unclear, resulting in significant differences in ischemic tissue damage. A similarly large variation has also been found in mice that is caused by genetic background-dependent differences in the extent of collateral formation, termed collaterogenesis--a unique angiogenic process that occurs during development and determines collateral number and diameter in the adult. Previous studies have identified several quantitative trait loci (QTL) linked to this variation. However, understanding has been hampered by the use of closely related inbred strains that do not model the wide genetic variation present in the "outbred" human population. The Collaborative Cross (CC) multiparent mouse genetic reference panel was developed to address this limitation. Herein we measured the number and average diameter of cerebral collaterals in 60 CC strains, their 8 founder strains, 8 F1 crosses of CC strains selected for abundant versus sparse collaterals, and 2 intercross populations created from the latter. Collateral number evidenced 47-fold variation among the 60 CC strains, with 14% having poor, 25% poor-to-intermediate, 47% intermediate-to-good, and 13% good collateral abundance, that was associated with large differences in post-stroke infarct volume. Genome-wide mapping demonstrated that collateral abundance is a highly polymorphic trait. Subsequent analysis identified: 6 novel QTL circumscribing 28 high-priority candidate genes harboring putative loss-of-function polymorphisms (SNPs) associated with low collateral number; 335 predicted-deleterious SNPs present in their human orthologs; and 32 genes associated with vascular development but lacking protein coding variants. This study provides a comprehensive set of candidate genes for future investigations aimed at identifying signaling proteins within the collaterogenesis pathway whose variants potentially underlie genetic-dependent collateral insufficiency in brain and other tissues.

genetics↗

bioRxiv2023

FOXK2 amplification and overexpression promotes breast cancer development and chemoresistance

Activation of oncogenes through DNA amplification/overexpression plays an important role in cancer initiation and progression. Chromosome 17 has many cancer-associated genetic anomalies. This cytogenetic anomaly is strongly associated with poor prognosis of breast cancer. FOXK2 gene is located on 17q25 and encodes a transcriptional factor with a forkhead DNA binding domain. By integrative analysis of public genomic datasets of breast cancers, we found that FOXK2 is frequently amplified and overexpressed in breast cancers. FOXK2 overexpression in breast cancer patients is associated with poor overall survival. FOXK2 knockdown significantly inhibits cell proliferation, invasion and metastasis, and anchorage-independent growth, as well as causes G0/G1 cell cycle arrest in breast cancer cells. Moreover, inhibition of FOXK2 expression sensitizes breast cancer cells to frontline anti-tumor chemotherapies. More importantly, co-overexpression of FOXK2 and PI3KCA with oncogenic mutations (E545K or H1047R) induces cellular transformation in non-tumorigenic MCF10A cells, suggesting that FOXK2 is an oncogene in breast cancer and is involved in PI3KCA-driven tumorigenesis. Our study identified CCNE2, PDK1, and Estrogen receptor alpha (ESR1) as direct transcriptional targets of FOXK2 in MCF-7 cells. Blocking CCNE2- and PDK1-mediated signaling by using small molecule inhibitors has synergistic anti-tumor effects in breast cancer cells. Furthermore, FOXK2 inhibition by gene knockdown or inhibitors for its transcriptional targets (CCNE2 and PDK1) in combination with PI3KCA inhibitor, Alpelisib, showed synergistic anti-tumor effects on breast cancer cells with PI3KCA oncogenic mutations. In summary, we provide compelling evidence that FOXK2 plays an oncogenic role in breast tumorigenesis and targeting FOXK2-mediated pathways may be a potential therapeutic strategy in breast cancer.

cancer biology↗

bioRxiv2023

BIG participates in the Arg/N-degron pathways and the hypoxia response in Arabidopsis thaliana.

BIG (also known as DOC1 and TIR3) is an 0.5 MDa protein that has been associated with multiple important functions in signalling and development through forward genetic screens in Arabidopsis thaliana. However, the biochemical function(s) of BIG are unknown. Here, we investigated whether BIG plays a role in the Arg/N-degron pathways, protein regulatory mechanisms in which substrate protein fate is influenced by the N-terminal (Nt) residue. In Arabidopsis, PROTEOLYSIS1 (PRT1) is an E3 ligase with specificity for aromatic amino acids, whereas PROTEOLYSIS6 (PRT6) targets basic N-terminal residues. We crossed a big loss-of-function allele to prt6 and prt1 mutants and examined the stability of protein substrates. Stability of model N-degron pathway substrates was enhanced in prt6-1 big-2 and prt1-1 big-2 relative to the respective single mutants. Abundance of the PRT6 physiological substrates, HYPOXIA RESPONSIVE ERF (HRE)2 and VERNALIZATION (VRN)2 was similarly increased in prt6 big double mutants, without increase in transcripts. Accordingly, hypoxia marker expression was enhanced in prt6 big double mutants, in a manner requiring arginyltransferase activity and RAP-type ERFVII transcription factors. Transcriptomic analysis of roots not only demonstrated synergistically increased expression of a plethora of hypoxia responsive genes in the double mutant relative to prt6 but also revealed other roles for PRT6 and BIG, including regulation of suberin deposition through both ERFVII-dependent and independent mechanisms, respectively. Our results show that BIG acts together with PRT6 to regulate the hypoxia response and wider processes. Significance StatementThe N-degron pathways are a group of protein regulatory mechanisms that play important roles in plant growth, development, and response to biotic and abiotic stresses. Despite rapid progress in the last decade, key enzymatic components of the pathways remain to be identified. BIG (also known as DOC1 and TIR3) is a protein of approximately 0.5 MDa, associated with multiple, distinct roles in plants but the precise biochemical functions of this protein have remained enigmatic until now. Here we identify BIG as a new component of plant N-degron pathways that acts together with the N-recognin E3 ligase PROTEOLYSIS6 (PRT6) to control the hypoxia response and other functions in Arabidopsis thaliana.

plant biology↗

bioRxiv2023

ATR kinase supports normal proliferation in the early S phase by preventing replication resource exhaustion.

The ATR kinase, which coordinates cellular responses to DNA replication stress, is also essential for the proliferation of normal unstressed cells. Although its role in the replication stress response is well defined, the mechanisms by which ATR supports normal cell proliferation remain elusive. Here, we show that ATR is dispensable for the viability of G0-arrested naive B cells. However, upon cytokine-induced proliferation, Atr-deficient B cells initiate DNA replication efficiently in early S phase, but by mid-S phase they display dNTP depletion, fork stalling, and replication failure. Nonetheless, productive DNA replication can be restored in Atr-deficient cells by pathways that suppress origin firing, such as downregulation of CDC7 and CDK1 kinase activities. Together, these findings indicate that ATR supports the proliferation of normal unstressed cells by tempering the pace of origin firing during the early S phase to avoid exhaustion of dNTPs and other replication factors.

genetics↗

bioRxiv2023

Type-2 diabetes alters hippocampal neural oscillations and disrupts synchrony between hippocampus and cortex

Type 2 diabetes mellitus (T2DM) increases the risk of neurological diseases, yet how brain oscillations change as age and T2DM interact is not well characterized. To delineate the age and diabetic effect on neurophysiology, we recorded local field potentials with multichannel electrodes spanning the somatosensory cortex and hippocampus (HPC) under urethane anesthesia in diabetic and normoglycemic control mice, at 200 and 400 days of age. We analyzed the signal power of brain oscillations, brain state, sharp wave associate ripples (SPW-Rs), and functional connectivity between the cortex and HPC. We found that while both age and T2DM were correlated with a breakdown in long-range functional connectivity and reduced neurogenesis in the dentate gyrus and subventricular zone, T2DM further slowed brain oscillations and reduced theta-gamma coupling. Age and T2DM also prolonged the duration of SPW-Rs and increased gamma power during SPW-R phase. Our results have identified potential electrophysiological substrates of hippocampal changes associated with T2DM and age. The perturbed brain oscillation features and diminished neurogenesis may underlie T2DM-accelerated cognitive impairment.

neuroscience↗

bioRxiv2023

Sex-biased regulatory changes in the placenta of native highlanders contribute to adaptive fetal development

Compared with lowlander migrants, native Tibetans have a higher reproductive success at high altitude though the underlying mechanism remains unclear. Here, we compared the transcriptome and histology of full-term placentas between native Tibetans and Han migrants. We found that the placental trophoblast shows the largest expression divergence between Tibetans and Han, and Tibetans show decreased immune response and endoplasmic reticulum stress. Remarkably, we detected a sex-biased expression divergence, where the male-infant placentas show a greater between-population difference than the female-infant placentas. The umbilical cord plays a key role in the sex-biased expression divergence, which is associated with the higher birth weight of the male newborns of Tibetans. We also identified adaptive histological changes in the male-infant placentas of Tibetans, including larger umbilical artery wall and umbilical artery intima and media, and fewer syncytial knots. These findings provide valuable insights into the sex-biased adaptation of human populations, with significant implications for medical and genetic studies of human reproduction.

evolutionary biology↗

bioRxiv2023

Inactive PARP1 causes embryonic lethality and genome instability in a dominant-negative manner.

PARP1 is recruited and activated by DNA strand breaks, catalyzing the generation of poly-ADP-ribose (PAR) chains from NAD+. PAR relaxes chromatin and recruits other DNA repair factors, including XRCC1 and DNA Ligase 3, to maintain genomic stability. Here we show that, in contrast to the normal development of Parp1-null mice, heterozygous expression of catalytically inactive Parp1 (E988A, Parp1+/A) acts in a dominant-negative manner to disrupt murine embryogenesis. As such, all the surviving F1 Parp1+/A mice are chimeras with mixed Parp1+/AN (neoR retention) cells that act similarly to Parp1+/-. Pure F2 Parp1+/A embryos were found at Mendelian ratios at the E3.5 blastocyst stage but died before E9.5. Compared to Parp1-/- cells, genotype and expression-validated pure Parp1+/A cells retain significant ADP-ribosylation and PARylation activities but accumulate markedly higher levels of sister chromatin exchange and mitotic bridges. Despite proficiency for homologous recombination and non-homologous end-joining measured by reporter assays and supported by normal lymphocyte and germ cell development, Parp1+/A cells are hypersensitive to base damages, radiation, and Topoisomerase I and II inhibition. The sensitivity of Parp1+/A cells to base damages and Topo inhibitors in particular exceed Parp1-/- controls. The findings show that the enzymatically inactive PARP1 protein has a dominant negative role and establishes a clear physiological difference between PARP1 inactivation vs. deletion. As a result, the enzymatically inactive PARP1 has a much more deteriorating impact on normal tissues than previously estimated, providing a mechanism for the on-target side effect of PARP inhibitors used for cancer therapy. Significance StatementPARP1 is the primary target of PARP enzymatic inhibitors. The use of PARP inhibitors for cancer therapy is based not only on the extreme sensitivity of BRCA1/2-deficient cancer cells to PARP1 inhibition but also on the nonessential role of PARP1 in normal tissues. Here we show that in contrast to the normal development of Parp1-null mice, the mouse model expressing the catalytically inactive Parp1 on only one allele (E988A, Parp1+/A) dies embryonically with high levels of genomic instability. The results reveal the severe dominant-negative impact of catalytically inactive PARP1, indicating the presence of enzymatically inactive PARP1 is much more damaging to normal tissues than previously anticipated. These findings provide a mechanism for clinical PARP inhibitors unexpected normal tissue toxicity.

genetics↗

bioRxiv2023

Utilizing tumor microenvironment microbial profiles and host gene expressions for survival subtyping in diverse types of cancers

The tumor microbiome, a complex community of microbes found in tumors, has been found to be linked to cancer development, progression, and treatment outcome. However, it remains a bottleneck in distangling the relationship between the tumor microbiome and patient survival. In this study, we aimed to decode this complex relationship by developing ASD-cancer (autoencoder-based subtypes detector for cancer), a semi-supervised deep learning framework that could extract survival-related features from tumor microbiome and transcriptome data, and identify patients survival subtypes. By using samples from The Cancer Genome Atlas database, we identified two statistically distinct survival subtypes across all 20 types of cancer. Our framework provided improved risk-stratification (e.g., for Liver hepatocellular carcinoma, LIHC, log-rank test, P = 8.12E-6) compared to PCA (e.g., for LIHC, log-rank test, P = 0.87), predicted survival subtypes accurately, and identified biomarkers for survival subtypes. Additionally, we identified potential interactions between microbes and genes that may play roles in survival. For instance, in LIHC, Arcobacter, Methylocella, and Isoptericola may regulate host survival through interactions with host genes enriched in the HIF-1 signaling pathway, indicating these species as potential therapy targets. Further experiments on validation dataset have also supported these patterns. Collectively, ASD-cancer has enabled accurate survival subtyping and biomarker discovery, which could facilitate personalized treatment for a broad-spectrum types of cancers.

bioinformatics↗

bioRxiv2023

TREM2 promotes glioma progression and angiogenesis mediated by microglia/brain macrophages

TREM2, a myeloid cell-specific signaling molecule, controls essential functions of microglia and impacts on the pathogenesis of Alzheimers disease and other neurodegenerative disorders. TREM2 is also highly expressed in tumor-associated macrophages and plays detrimental roles in an experimental mouse sarcoma model. Here we studied whether TREM2 influences glioma progression. We found a sex- dependent effect of TREM2: the glioma volume is significantly attenuated in TREM2- deficient male but not female mice injected with GL261-EGFP glioma cells. The accumulation of glioma-associated microglia/macrophages (GAMs) and vascularization is reduced in male TREM2-deficient mice. A transcriptomic analysis of glioma tissue revealed that TREM2 deficiency suppresses angiogenic genes and MHC clusters. In an organotypic slice model devoid of functional vascularization, the tumor size was not affected by TREM2-deficiency. In human resection samples from glioblastoma, TREM2 is upregulated in GAMs. Based on the TCGA and CGGA databases, the TREM2 expression levels are negatively correlated with survival. Thus, the TREM2-dependent crosstalk between GAMs and the vasculature formation promotes glioma growth. Graphic abstractTREM2-dependent crosstalk between glioma-associated microglia/macrophages and the vasculature formation promotes glioma growth in male glioma mouse model. Created with BioRender.com O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/540621v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@65bec7org.highwire.dtl.DTLVardef@1d00961org.highwire.dtl.DTLVardef@117ee5eorg.highwire.dtl.DTLVardef@227994_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

UBL3 interaction with α-synuclein is downregulated by silencing MGST3

Ubiquitin-like 3 (UBL3) is a membrane-anchored protein that has been discovered to function as a protein post-translational modifier, helping to sort proteins into small extracellular vesicles (sEVs). Aggregations of alpha-synuclein (-syn) are associated with the pathology of neurodegenerative diseases such as Parkinsons disease (PD). The aggregation and toxicity of -syn can be influenced by its interactions with specific proteins. Recently, the interactions between UBL3 and -syn was uncovered. It is believed to play a role in eliminating excess -syn from neurons, or in the spread of -syn pathology in the brain associated with neurodegenerative diseases. However, the regulator that can mediate the interaction between UBL3 and -syn remains unclear. In this study, we employed the split gaussian luciferase complementation assay and RNA interference (RNAi) technology to discover that QSOX2, HTATIP2, UBE3C, MGST3, NSF, HECTD1, SAE1 and ATG3 are involved in downregulating the interaction between UBL3 and -syn. Among these proteins, silencing MGST3 had the most significant impact on the UBL3--syn interaction (with a fold change log2 of less than - 1). MGST3 is a part of the antioxidant system, and silencing MGST3 is believed to contribute to oxidative stress. We used hydrogen peroxide (H2O2) to induce oxidative stress and observed its effect on the UBL3--syn interaction. Our finding showed that an 800M concentration of H2O2 could also downregulate this interaction. However, the effect of oxidative stress caused by 800M H2O2 on the UBL3--syn interaction did not be enhanced by silencing MGST3. In conclusion, the interaction between UBL3 and -syn is downregulated by silencing MGST3.

molecular biology↗

bioRxiv2023

UBL3 Interacts with Alpha-synuclein in Cells and the Interaction is Downregulated by the EGFR Pathway Inhibitor Osimertinib

Ubiquitin-like 3 (UBL3) acts as a post-translational modification (PTM) factor and regulates protein sorting into small extracellular vesicles (sEVs). sEVs have been reported as vectors for the pathology propagation of neurodegenerative diseases, such as -synucleinopathies. Alpha-synuclein (-syn) has been widely studied for its involvement in -synucleinopathies. However, it is still unknown whether UBL3 interacts with -syn, and is influenced by drugs or compounds. In this study, we investigated the interaction between UBL3 and -syn, and any ensuing possible functional and pathological implications. We found that UBL3 can interact with -syn by the Gaussia princeps based split luciferase complementation assay in cells and immunoprecipitation, while cysteine residues at its C-terminal, which are considered important as PTM factors for UBL3, were not essential for the interaction. The interaction was upregulated by 1-methyl-4-phenylpyridinium exposure. In drug screen results, the interaction was significantly downregulated by the treatment of osimertinib. These results suggest that UBL3 interacts with -syn in cells and be significantly downregulated by epidermal growth factor receptor (EGFR) pathway inhibitor osimertinib. Therefore, the UBL3 pathway may be a new therapeutic target for -synucleinopathies in the future.

molecular biology↗

bioRxiv2023

txci-ATAC-seq, a massive-scale single-cell technique to profile chromatin accessibility

Measuring chromatin accessibility is a powerful method to identify cell types and states. Performed at single-cell resolution, this assay has generated catalogs of genome-wide DNA regulatory sites, whole-organism cell atlases, and dynamic chromatin reorganization through development. However, the limited throughput of current single-cell approaches poses a challenge for implementing proper study designs, population-scale profiling, and/or very deep profiling of complex samples. To this end, we developed a 10X-compatible combinatorial indexing ATAC sequencing ("txci-ATAC-seq"), which is a combinatorial indexing framework that initially indexes ("pre-indexes") chromatin within nuclei with barcoded transposases followed by encapsulation and further barcoding using a commercialized droplet-based microfluidics platform (10X Genomics). Leveraging this molecular hashing strategy, we demonstrate that txci-ATAC-seq enables the indexing of up to 200,000 nuclei across multiple samples in a single emulsion reaction, representing a [~]22-fold increase in throughput compared to the standard workflow at the same collision rate. To improve the efficiency of this new technique, we further developed a faster version of the protocol ("Fast-txci-ATAC-seq") that separates sample pre-processing from library generation and has the potential to profile up to 96 samples simultaneously. We initially benchmarked our assay by generating chromatin accessibility profiles for 230,018 cells from five native tissues across three experiments, including human cortex (28,513 cells), mouse brain (48,997 cells), human lung (15,799 cells), mouse lung (73,280 cells), and mouse liver (63,429 cells). We also applied our method to a club cell secretory protein knockout (CC16-/-) mouse model to examine the biological and technical limitations of the mouse line. By characterizing DNA regulatory landscapes in 76,498 wild-type and 77,638 CC16-/- murine lung nuclei, our investigations uncovered previously unappreciated residual genetic deviations from the reference strain that resulted from the method of gene targeting, which employed embryonic stem cells from the 129 strain. We found that these genetic remnants from the 129 strain led to profound cell-type-specific changes in chromatin accessibility in regulatory elements near a host of genes. Collectively, we defined single-cell chromatin signatures in 384,154 nuclei from 13 primary samples across different species, organs, biological replicates, and genetic backgrounds, establishing txci-ATAC-seq as a robust, high-quality, and highly multiplexable single-cell assay for large-scale chromatin studies.

genomics↗