Biology subjects

Li, R.

Publications and source records attributed to Li, R..

At least 325 records · Page 18Linked to original sources

bioRxiv2023

Magnetogenetic cell activation using endogenous ferritin

The ability to precisely control the activity of defined cell populations enables studies of their physiological roles and may provide therapeutic applications. While prior studies have shown that magnetic activation of ferritin-tagged ion channels allows cell-specific modulation of cellular activity, the large size of the constructs made the use of adeno-associated virus, AAV, the vector of choice for gene therapy, impractical. In addition, simple means for generating magnetic fields of sufficient strength have been lacking. Toward these ends, we first generated a novel anti-ferritin nanobody that when fused to transient receptor potential cation channel subfamily V member 1, TRPV1, enables direct binding of the channel to endogenous ferritin in mouse and human cells. This smaller construct can be delivered in a single AAV and we validated that it robustly enables magnetically induced cell activation in vitro. In parallel, we developed a simple benchtop electromagnet capable of gating the nanobody-tagged channel in vivo. Finally, we showed that delivering these new constructs by AAV to pancreatic beta cells in combination with the benchtop magnetic field delivery stimulates glucose-stimulated insulin release to improve glucose tolerance in mice in vivo. Together, the novel anti-ferritin nanobody, nanobody-TRPV1 construct and new hardware advance the utility of magnetogenetics in animals and potentially humans.

bioengineering↗

bioRxiv2023

Evolution of chromosome arm aberrations in breast cancer through genetic network rewiring

The basal breast cancer subtype is enriched for triple-negative breast cancer (TNBC) and displays consistent large chromosomal deletions. Here, we characterize the evolution and maintenance of chromosome 4p (chr4p) loss in basal breast cancer. TCGA data analysis showed recurrent deletion of chr4p in basal breast cancer. Phylogenetic analysis of a unique panel of 23 primary tumor/patient-derived xenograft basal breast cancers revealed early evolution of chr4p deletion. Mechanistically we show that Chr4p loss is associated with enhanced proliferation. Gene function studies identified an unknown gene, C4orf19, within chr4p, which suppressed proliferation when overexpressed and is a novel member of a PDCD10-GCKIII kinase module, we name as PGCA1. Genome-wide pooled overexpression screens using a barcoded library of human open reading frames, identified chromosomal regions, including chr4p, that suppress proliferation when overexpressed in a context-dependent manner implicating network interactions. Together this sheds light on the early emergence of complex aneuploid karyotypes involving chr4p and adaptive landscapes shaping breast cancer genomes.

genomics↗

bioRxiv2023

Concerted epithelial and stromal changes during progression of Barrett's Esophagus to invasive adenocarcinoma exposed by multi-scale, multi-omics analysis

Esophageal adenocarcinoma arises from Barretts esophagus, a precancerous metaplastic replacement of squamous by columnar epithelium in response to chronic inflammation. Multi-omics profiling, integrating single-cell transcriptomics, extracellular matrix proteomics, tissue-mechanics and spatial proteomics of 64 samples from 12 patients paths of progression from squamous epithelium through metaplasia, dysplasia to adenocarcinoma, revealed shared and patient-specific progression characteristics. The classic metaplastic replacement of epithelial cells was paralleled by metaplastic changes in stromal cells, ECM and tissue stiffness. Strikingly, this change in tissue state at metaplasia was already accompanied by appearance of fibroblasts with characteristics of carcinoma-associated fibroblasts and of an NK cell-associated immunosuppressive microenvironment. Thus, Barretts esophagus progresses as a coordinated multi-component system, supporting treatment paradigms that go beyond targeting cancerous cells to incorporating stromal reprogramming. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/544265v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@64c375org.highwire.dtl.DTLVardef@15c6ef2org.highwire.dtl.DTLVardef@177dee0org.highwire.dtl.DTLVardef@c945e7_HPS_FORMAT_FIGEXP M_FIG Graphical AbstractTo obtain a comprehensive picture of the coordinated changes in epithelial, stromal and immune compartments during development of Barretts-associated esophageal adenocarcinoma, patient-matched samples corresponding to various phases of disease progression were collected from 12 patients, each of which had at a given time point lesions at multiple stages progression (matched-normal, metaplasia, dysplasia, and carcinoma). Matched "normal" gastric tissues were also collected. These sample were analyzed by single cell RNA-sequencing (scRNAseq) for single-cell resolution transcriptomics and Copy Number Variant (CNV), by proteomics for extracellular matrix (ECM) proteins, by Atomic Force Microscopy (AFM for tissue stiffness and by CODEX spatial proteomics imaging The integrative multi-omics analysis exposed drastic alterations in cell type composition and shifts in cell states in all three compartments. A large subpopulation of fibroblasts absent in the normal esophagus and characteristic of dysplasia and adenocarcinoma sample, that based on markers would indeed be considered cancer associated fibroblasts (CAF), appeared already in the metaplastic phase. This fibroblast subpopulation had transcriptomes virtually indistinguishable with fibroblasts of the cancer free gastric epithelium in these patients C_FIG

cancer biology↗

bioRxiv2023

scATAnno: Automated Cell Type Annotation for single-cell ATAC-seq Data

Recent advances in single-cell epigenomic techniques have created a growing demand for scATAC-seq analysis. One key analysis task is to determine cell type identity based on the epigenetic data. We introduce scATAnno, a python package designed to automatically annotate scATAC-seq data using large-scale scATAC-seq reference atlases. This workflow generates the reference atlases from publicly available datasets enabling accurate cell type annotation by integrating query data with reference atlases, without the use of scRNA-seq data. To enhance annotation accuracy, we have incorporated KNN-based and weighted distance-based uncertainty scores to effectively detect cell populations within the query data that are distinct from all cell types in the reference data. We compare and benchmark scATAnno against 7 other published approaches for cell annotation and show superior performance in multiple data sets and metrics. We showcase the utility of scATAnno across multiple datasets, including peripheral blood mononuclear cell (PBMC), Triple Negative Breast Cancer (TNBC), and basal cell carcinoma (BCC), and demonstrate that scATAnno accurately annotates cell types across conditions. Overall, scATAnno is a useful tool for scATAC-seq reference building and cell type annotation in scATAC-seq data and can aid in the interpretation of new scATAC-seq datasets in complex biological systems.

bioinformatics↗

bioRxiv2023

Syntenin and CD63 Promote Exosome Biogenesis from the Plasma Membrane by Blocking Cargo Endocytosis

Exosomes are small extracellular vesicles important in health and disease. Syntenin is thought to drive the biogenesis of CD63 exosomes by recruiting Alix and the ESCRT machinery to endosomes, initiating an endosome-mediated pathway of exosome biogenesis. Contrary to this model, we show here that syntenin drives the biogenesis of CD63 exosomes by blocking CD63 endocytosis, thereby allowing CD63 to accumulate at the plasma membrane, the primary site of exosome biogenesis. Consistent with these results, we find that inhibitors of endocytosis induce the exosomal secretion of CD63, that endocytosis inhibits the vesicular secretion of exosome cargo proteins, and that high-level expression of CD63 itself also inhibits endocytosis. These and other results indicate that exosomes bud primarily from the plasma membrane, that endocytosis inhibits their loading into exosomes, that syntenin and CD63 are expression-dependent regulators of exosome biogenesis, and that syntenin drives the biogenesis of CD63 exosomes even in Alix knockout cells.

cell biology↗

bioRxiv2023

GPI-anchored Gas1 protein regulates cytosolic proteostasis in yeast

Decline in protein homeostasis (proteostasis) is a hallmark of cellular aging and aging-related diseases. Maintaining a balanced proteostasis requires a complex network of molecular machineries that govern protein synthesis, folding, localization, and degradation. Under proteotoxic stress, misfolded proteins that accumulate in cytosol can be imported into mitochondria for degradation via mitochondrial as guardian in cytosol (MAGIC) pathway. Here we report an unexpected role of yeast Gas1, a cell wall-bound glycosylphosphatidylinositol (GPI)-anchored {beta}-1,3-glucanosyltransferase, in differentially regulating MAGIC and ubiquitin-proteasome system (UPS). Deletion of Gas1 inhibits MAGIC but elevates polyubiquitination and UPS-mediated protein degradation. Interestingly, we found that Gas1 exhibits mitochondrial localization attributed to its C-terminal GPI anchor signal. But this mitochondria-associated GPI anchor signal is not required for mitochondrial import and degradation of misfolded proteins via MAGIC. By contrast, catalytic inactivation of Gas1 via the gas1E161Qmutation inhibits MAGIC but not its mitochondrial localization. These data suggest that the glucanosyltransferase activity of Gas1 is important for regulating cytosolic proteostasis.

cell biology↗

bioRxiv2023

Mitochondrial DNA Variations Modulate Alveolar Epithelial Mitochondrial Function and Oxidative Stress in Newborn Mice Exposed to Hyperoxia

Oxidative stress is an important contributor to bronchopulmonary dysplasia (BPD), a form of chronic lung disease that is the most common morbidity in very preterm infants. Mitochondrial functional differences due to inherited and acquired mutations influence the pathogenesis of disorders in which oxidative stress plays a critical role. We previously showed using mitochondrial-nuclear exchange (MNX) mice that mitochondrial DNA (mtDNA) variations modulate hyperoxia-induced lung injury severity in a model of BPD. In this study, we studied the effects of mtDNA variations on mitochondrial function including mitophagy in alveolar epithelial cells (AT2) from MNX mice. We also investigated oxidant and inflammatory stress as well as transcriptomic profiles in lung tissue in mice and expression of proteins such as PINK1, Parkin and SIRT3 in infants with BPD. Our results indicate that AT2 from mice with C57 mtDNA had decreased mitochondrial bioenergetic function and inner membrane potential, increased mitochondrial membrane permeability and were exposed to higher levels of oxidant stress during hyperoxia compared to AT2 from mice with C3H mtDNA. Lungs from hyperoxia-exposed mice with C57 mtDNA also had higher levels of pro-inflammatory cytokines compared to lungs from mice with C3H mtDNA. We also noted changes in KEGG pathways related to inflammation, PPAR and glutamatergic signaling, and mitophagy in mice with certain mito-nuclear combinations but not others. Mitophagy was decreased by hyperoxia in all mice strains, but to a greater degree in AT2 and neonatal mice lung fibroblasts from hyperoxia-exposed mice with C57 mtDNA compared to C3H mtDNA. Finally, mtDNA haplogroups vary with ethnicity, and Black infants with BPD had lower levels of PINK1, Parkin and SIRT3 expression in HUVEC at birth and tracheal aspirates at 28 days of life when compared to White infants with BPD. These results indicate that predisposition to neonatal lung injury may be modulated by variations in mtDNA and mito-nuclear interactions need to be investigated to discover novel pathogenic mechanisms for BPD.

developmental biology↗

bioRxiv2023

Quantitative Microbiology with Microscopy: Effects of Projection and Diffraction

Time-resolved live-cell imaging using widefield microscopy is instrumental in quantitative microbiology research. It allows us to track and measure the size, shape, and content of individual microbial cells over time. However, the small size of microbial cells poses a significant challenge in interpreting image data, as it approaches the dimensions of the microscopes depth of field and experiences diffraction effects. As a result, 2D widefield images of microbial cells contain projected 3D information, blurred by the 3D point spread function. In this study, we employ computer simulations and targeted experiments to investigate the impact of diffraction and projection on our ability to quantify the size and content of microbial cells from 2D microscopic images. This study points to some new and often unconsidered artefacts resulting from the interplay of projection and diffraction effects, within the context of quantitative microbiology. These artefacts introduce substantial errors and biases in size and fluorescence quantification, and even single-molecule counting, making the elimination of these errors a complex task. Awareness of these artefacts is crucial for designing strategies to accurately interpret micrographs of microbes. To address this, we present new experimental designs and machine learning based analysis methods that account for these effects, resulting in accurate quantification of microbiological processes.

microbiology↗

bioRxiv2023

Exercise Mitigates Flow Recirculation and Activates Mechanosensitive Transcriptome to Uncover Endothelial SCD1-Catalyzed Anti-Inflammatory Metabolites

Exercise modulates vascular plasticity in multiple organ systems; however, the metabolomic transducers underlying exercise and vascular protection in the disturbed flow-prone vasculature remain under-investigated. We simulated exercise-augmented pulsatile shear stress (PSS) to mitigate flow recirculation in the lesser curvature of the aortic arch. When human aortic endothelial cells (HAECs) were subjected to PSS ({tau}ave = 50 dyne{middle dot}cm-2, {partial}{tau}/{partial}t = 71 dyne{middle dot}cm-2{middle dot}s-1, 1 Hz), untargeted metabolomic analysis revealed that Stearoyl-CoA Desaturase (SCD1) in the endoplasmic reticulum (ER) catalyzed the fatty acid metabolite, oleic acid (OA), to mitigate inflammatory mediators. Following 24 hours of exercise, wild-type C57BL/6J mice developed elevated SCD1-catalyzed lipid metabolites in the plasma, including OA and palmitoleic acid (PA). Exercise over a 2-week period increased endothelial SCD1 in the ER. Exercise further modulated the time-averaged wall shear stress (TAWSS or{tau} ave) and oscillatory shear index (OSIave), upregulated Scd1 and attenuated VCAM1 expression in the disturbed flow-prone aortic arch in Ldlr-/- mice on high-fat diet but not in Ldlr-/-Scd1EC-/- mice. Scd1 overexpression via recombinant adenovirus also mitigated ER stress. Single cell transcriptomic analysis of the mouse aorta revealed interconnection of Scd1 with mechanosensitive genes, namely Irs2, Acox1 and Adipor2 that modulate lipid metabolism pathways. Taken together, exercise modulates PSS ({tau}ave and OSIave) to activate SCD1 as a metabolomic transducer to ameliorate inflammation in the disturbed flow-prone vasculature.

physiology↗

bioRxiv2023

Aging-associated Decline in Vascular Smooth Muscle Cell Mechanosensation is Mediated by Piezo1 Channel

Aging of the vasculature is associated with detrimental changes in vascular smooth muscle cell (VSMC) mechanosensitivity to extrinsic forces in their surrounding microenvironment. However, how chronological aging alters VSMCs ability to sense and adapt to mechanical perturbations remains unexplored. Here, we show defective VSMC mechanosensation in aging measured with ultrasound tweezers-based micromechanical system, force instantaneous frequency spectrum and transcriptome analyses. The mechanobiological study reveals that aged VSMCs adapt a relatively inert solid-like state with altered actin cytoskeletal integrity, resulting in an impairment in their mechanosensitivity and dynamic mechanoresponse to mechanical perturbations. The aging-associated decline in mechanosensation behaviors is mediated by hyperactivity of Piezo1-dependent calcium signaling. Inhibition of Piezo1 alleviates vascular aging and partially restores the loss in dynamic contractile properties in aged cells. Altogether, our study reveals the novel signaling pathway underlying aging-associated aberrant mechanosensation in VSMC and identifies Piezo1 as a potential therapeutic mechanobiological target to alleviate vascular aging.

bioengineering↗

bioRxiv2023

Single-cell RNA-sequencing identifies a novel subset of CD9+ reparative macrophages modulated by CCN2 derived from Nlrx1 deficient cardiomyocytes in the infarcted heart

RationaleNlrx1, a pattern recognition receptor belonging to the NLR family, is highly expressed in the heart and is significantly increased after myocardial infarction in vivo, however, the role of Nlrx1 in post-infarction inflammation and healing is poorly understood. Although the effects of infiltrating macrophages on survival of cardiomyocytes and healing of injured hearts have been intensely studied, whether or not and how surviving cardiomyocytes might influence the phenotype and function of macrophages in the infarcted hearts was largely unexplored. ObjectiveTo investigate the role of Nlrx1 in post-infarction inflammation and healing and to dissect the underlying mechanism. Methods and resultsSingle cell RNA-sequencing of 23,805 cells isolated from infarcted hearts revealed 7 clusters of infarct macrophages including CCR2+, Isg+, Mki67+ proliferative, and LYVE1+ cardiac resident macrophages, and identified a novel subset of CD9+Il1rn+ macrophages, which were characterized by high expression of Cd9, Il1rn, Gpnmb and Spp1, and downregulation of proinflammatory cytokines and chemokines. CD9+Il1rn+ macrophages were largely found in infarcted hearts of Nlrx1KO mice, and were transcriptionally unique with reparative phenotype. Loss of Nlrx1 attenuated ventricular dysfunction and adverse remodeling, and decreased post-infarction mortality. Flow cytometry analysis showed markedly increased number of CD9+ macrophages and higher mean fluorescence intensity of CD9 in Nlrx1KO mice. RNA-seq analysis revealed that CD9+ macrophages of Nlrx1KO infarcted hearts exhibited a reparative and anti-inflammatory phenotype as featured by upregulation of wound healing-associated genes including CD9, Spp1 and Fn1, and downregulation of proinflammatory genes. Conditioned medium from Nlrx1 deficient cardiomyocytes upon hypoxia stimulation skewed macrophage polarization towards a reparative and inflammation-suppressed phenotype. Gain-and loss-of function of Nlrx1 combined with RNA-seq analysis identified CCN2, whose expression and secretion were significantly increased in Nlrx1 deficient cardiomyocytes upon hypoxia, as a cardiomyocytes-derived mediator responsible, at least partly, for reprograming macrophage towards a reparative phenotype. In vivo, CCN2 expression was markedly elevated in Nlrx1KO infarcted hearts. Moreover, exogenous CCN2 partially mediated macrophage transition towards a reparative phenotype. ConclusionsCD9+ IL1rn+ macrophages modulated by Nlrx1 deficient cardiomyocytes-derived CCN2, is a novel subset of reparative macrophages contributing to infarct healing. Inhibition of myocardial Nlrx1 and/or activation of CCN2 to boost CD9+ IL1rn+ reparative macrophages may represent new therapeutics for infarct healing following ischemic injury.

immunology↗

bioRxiv2023

Doxorubicin inhibits phosphatidylserine decarboxylase and confers broad-spectrum antifungal activity

O_LIAs phospholipids of cell membranes, phosphatidylethanolamine (PE) and phosphatidylserine (PS) play crucial roles in glycerophospholipid metabolism. Broadly, some phospholipid biosynthesis enzymes serve as potential fungicide targets. Therefore, revealing the functions and mechanism of PE biosynthesis in plant pathogens would provide potential targets for crop disease control. C_LIO_LIWe performed analyses including phenotypic characterizations, lipidomics, enzyme activity, site-directed mutagenesis, and chemical inhibition assays to study the function of PS decarboxylase-encoding gene MoPSD2 in rice blast fungus Magnaporthe oryzae. C_LIO_LIThe Mopsd2 mutant was defective in development, lipid metabolism and plant infection. The PS level increased while PE decreased in Mopsd2, consistent with the enzyme activity. Furthermore, chemical doxorubicin inhibited the enzyme activity of MoPsd2 and showed antifungal activity against ten phytopathogenic fungi including M. oryzae and reduced disease severity of two crop diseases in the field. Three predicted doxorubicin-interacting residues are important for MoPsd2 functions. C_LIO_LIOur study demonstrates that MoPsd2 is involved in de novo PE biosynthesis and contributes to the development and plant infection of M. oryzae and that doxorubicin shows broad-spectrum antifungal activity as a fungicide candidate. The study also implicates that bacterium Streptomyces peucetius, which biosynthesizes doxorubicin, could be potentially used as an eco-friendly biocontrol agent. C_LI

pathology↗

bioRxiv2023

Metabolic regulation of misfolded protein import into mitochondria

Mitochondria are the cellular energy hub and central target of metabolic regulation. Mitochondria also facilitate proteostasis through pathways such as the mitochondria as guardian in cytosol (MAGIC) whereby cytosolic misfolded proteins (MPs) are imported into and degraded inside mitochondria. In this study, a genome-wide screen in yeast uncovered that Snf1, the yeast AMP-activated protein kinase (AMPK), inhibits the import of MPs into mitochondria while promoting mitochondrial biogenesis under glucose starvation. We show that this inhibition requires a downstream transcription factor regulating mitochondrial gene expression and is likely to be conferred through substrate competition and mitochondrial import channel selectivity. We further show that Snf1/AMPK activation protects mitochondrial fitness in yeast and human cells under stress induced by MPs such as those associated with neurodegenerative diseases.

cell biology↗

bioRxiv2023

Distinct Myeloid Derived Suppressor Cell Populations Promote Tumor Aggression in Glioblastoma

The diversity of genetic programs and cellular plasticity of glioma-associated myeloid cells, and thus their contribution to tumor growth and immune evasion, is poorly understood. We performed single cell RNA-sequencing of immune and tumor cells from 33 glioma patients of varying tumor grades. We identified two populations characteristic of myeloid derived suppressor cells (MDSC), unique to glioblastoma (GBM) and absent in grades II and III tumors: i) an early progenitor population (E-MDSC) characterized by strong upregulation of multiple catabolic, anabolic, oxidative stress, and hypoxia pathways typically observed within tumor cells themselves, and ii) a monocytic MDSC (M-MDSC) population. The E-MDSCs geographically co-localize with a subset of highly metabolic glioma stem-like tumor cells with a mesenchymal program in the pseudopalisading region, a pathognomonic feature of GBMs associated with poor prognosis. Ligand-receptor interaction analysis revealed symbiotic cross-talk between the stemlike tumor cells and E-MDSCs in GBM, whereby glioma stem cells produce chemokines attracting E-MDSCs, which in turn produce growth and survival factors for the tumor cells. Our large-scale single-cell analysis elucidated unique MDSC populations as key facilitators of GBM progression and mediators of tumor immunosuppression, suggesting that targeting these specific myeloid compartments, including their metabolic programs, may be a promising therapeutic intervention in this deadly cancer. One-Sentence SummaryAggressive glioblastoma harbors two unique myeloid populations capable of promoting stem-like properties of tumor cells and suppressing T cell function in the tumor microenvironment.

immunology↗

bioRxiv2023

Rice OsHAK18 mediates shoot-to-root circulation of potassium and sodium and source-to-sink transport of soluble sugar

High-affinity potassium (K+) transporters HAK/KUP/KT have been identified in all genome-sequenced terrestrial plants. They play important role in K+ acquisition, translocation and enhancing salt tolerance. Here we report that the plasma-membrane-located OsHAK18 functions in K+ and sodium (Na+) circulation and sugar translocation in rice. OsHAK18 is expressed mainly, though not exclusively, in vascular tissues and particularly in the phloem. Knockout (KO) of OsHAK18 reduced K+ concentration in phloem sap and in the root but increased K+ accumulation in the shoot of both Nipponbare and Zhonghua11 cultivars, while overexpression (OX) of OsHAK18 driven by its endogenous promoter increased K+ concentration in phloem sap and in roots, and promoted Na+ retrieval from shoot to root under salt stress. Split-root experimental analysis of rubidium (Rb+) uptake and circulation indicated that OsHAK18-OX promoted Rb+ translocation from shoot to root. In addition, OsHAK18-KO increased while OsHAK18-OX reduced soluble sugar content in the shoot and affected oppositely the sugar concentration in the phloem and its content in the root. Moreover, OsHAK18-OX increased dramatically grain yield and physiological K+ utilization efficiency. Taken together, our results suggest that - unlike other OsHAKs analyzed heretofore - OsHAK18 is critical to K+ and Na+ re-circulation from shoot to root and enhances the source-to-sink translocation of a photo-assimilate. One sentence summaryRice potassium transporter OsHAK18 mediates shoot-to-root circulation of potassium and sodium and source-to-sink translocation of soluble sugar which improves potassium use efficiency and grain yield.

plant biology↗

bioRxiv2023

Intronic TNR-retained ISOPROPYLMALATE ISOMERASE LARGE SUBUNIT1 transcripts impair leaf development in Arabidopsis

Intronic trinucleotide repeat (TNR) is widely distributed in plant genomes. In Arabidopsis accession Bur-0, abnormally expanded TTC repeat in intron-3 of the ISOPROPYLMALATE ISOMERASE LARGE SUBUNIT1 (IIL1) gene causes growth defects called the irregularly impaired leaves (iil) phenotype, triggered by DNA methylation-mediated IIL1 gene silencing at elevated temperature. However, little is known about how the reduced expression of IIL1 causes the iil phenotype. We demonstrated that the iil phenotype was resulted from the relative increase of intron-3-retained IIL1 transcripts through the experiments where the iil phenotype was reproduced by introducing the IIL1 gene harboring 100 copies of TTC repeat into Col-0. The iil phenotype appeared when the total amount of the IIL1 transcripts was decreased by co-suppression and the percentage of intron-3-retained IIL1 transcripts was increased. The IIL1 gene encodes an isopropylmalate isomerase large subunit, forming heterodimers with small subunits (AtLeuD1, AtLeuD2, or AtLeuD3). In the myb28 myb29 mutant lacking AtLeuD1 and AtLeuD2, the iil phenotype was almost completely suppressed regardless of higher percentage of intron-3-retained IIL1 transcripts. The results indicated that the iil phenotype was associated with interaction with AtLeuDs, suggesting that intronic TNR-containing transcripts were translated into abnormal proteins and perturbed the metabolic pathway supporting the leaf development.

plant biology↗

bioRxiv2023

Inferring gene regulatory networks using transcriptional profiles as dynamical attractors

Genetic regulatory networks (GRNs) regulate the flow of genetic information from the genome to expressed messenger RNAs (mRNAs) and thus are critical to controlling the phenotypic characteristics of cells. Numerous methods exist for profiling mRNA transcript levels and identifying protein-DNA binding interactions at the genome-wide scale. These enable researchers to determine the structure and output of transcriptional regulatory networks, but uncovering the complete structure and regulatory logic of GRNs remains a challenge. The field of GRN inference aims to meet this challenge using computational modeling to derive the structure and logic of GRNs from experimental data and to encode this knowledge in Boolean networks, Bayesian networks, ordinary differential equation (ODE) models, or other modeling frameworks. However, most existing models do not incorporate dynamic transcriptional data since it has historically been less widely available in comparison to "static" transcriptional data. We report the development of an evolutionary algorithm-based ODE modeling approach that integrates kinetic transcription data and the theory of attractor matching to infer GRN architecture and regulatory logic. Our method outperformed six leading GRN inference methods, none of which incorporate kinetic transcriptional data, in predicting regulatory connections among TFs when applied to a small-scale engineered synthetic GRN in Saccharomyces cerevisiae. Moreover, we demonstrate the potential of our method to predict unknown transcriptional profiles that would be produced upon genetic perturbation of the GRN governing a two-state cellular phenotypic switch in Candida albicans. We established an iterative refinement strategy to facilitate candidate selection for experimentation; the experimental results in turn provide validation or improvement for the model. In this way, our GRN inference approach can expedite the development of a sophisticated mathematical model that can accurately describe the structure and dynamics of the in vivo GRN. Author SummaryThe establishment of distinct transcriptional programs, where specific sets of genes are activated or repressed, is fundamental to all forms of life. Sequence-specific DNA-binding proteins, often referred to as regulatory transcription factors, form interconnected gene regulatory networks (GRNs) which underlie the establishment and maintenance of specific transcriptional programs. Since their discovery, many modeling approaches have sought to understand the structure and regulatory behaviors of these GRNs. The field of GRN inference uses experimental measurements of transcript abundance to predict how regulatory transcription factors interact with their downstream target genes to establish specific transcriptional programs. However, most prior approaches have been limited by the exclusive use of "static" or steady-state measurements. We have developed a unique approach which incorporates dynamic transcriptional data into a sophisticated ordinary differential equation model to infer GRN structures that give rise to distinct transcriptional programs. Our model not only outperforms six other leading models, it also is capable of accurately predicting how changes in GRN structure will impact the resulting transcriptional programs. These unique attributes of our model, combined with "real world" experimental validation of our model predictions, represent a significant advance in the field of gene regulatory network inference.

systems biology↗

bioRxiv2023

E2F1 Mediates SOX17 Deficiency-Induced Pulmonary Hypertension

RationaleRare genetic variants and genetic variation at loci in an enhancer in SRY-Box Transcription Factor 17 (SOX17) are identified in patients with idiopathic pulmonary arterial hypertension (PAH) and PAH with congenital heart disease. However, the exact role of genetic variants or mutation in SOX17 in PAH pathogenesis has not been reported. ObjectivesTo investigate the role of SOX17 deficiency in pulmonary hypertension (PH) development. MethodsHuman lung tissue and endothelial cells (ECs) from IPAH patients were used to determine the expression of SOX17. Tie2Cre-mediated and EC-specific deletion of Sox17 mice were assessed for PH development. Single-cell RNA sequencing analysis, human lung ECs, and smooth muscle cell culture were performed to determine the role and mechanisms of SOX17 deficiency. A pharmacological approach was used in Sox17 deficiency mice for therapeutic implication. Measurement and Main ResultsSOX17 expression was downregulated in the lungs and pulmonary ECs of IPAH patients. Mice with Tie2Cre mediated Sox17 knockdown and EC-specific Sox17 deletion developed spontaneously mild PH. Loss of endothelial Sox17 in EC exacerbated hypoxia-induced PH in mice. Loss of SOX17 in lung ECs induced endothelial dysfunctions including upregulation of cell cycle programming, proliferative and anti-apoptotic phenotypes, augmentation of paracrine effect on pulmonary arterial smooth muscle cells, impaired cellular junction, and BMP signaling. E2F Transcription Factor 1 (E2F1) signaling was shown to mediate the SOX17 deficiency-induced EC dysfunction and PH development. ConclusionsOur study demonstrated that endothelial SOX17 deficiency induces PH through E2F1 and targeting E2F1 signaling represents a promising approach in PAH patients.

physiology↗