Biology subjects

Yao, Y.

Publications and source records attributed to Yao, Y..

At least 109 records · Page 6Linked to original sources

bioRxiv2024

An intracranial dissection of human escape circuits

Predators attack at different spatiotemporal scales, spurring prey to elicit escape responses that range from simple motor reactions and strategic planning that involve more complex cognitive processes. Recent work in humans suggests that escape relies on two distinct circuits: the reactive and cognitive fear circuits. However, the specific involvement of these two circuits in different stages of human escaping remains poorly characterized. In this study, we recorded intracranial electroencephalography (iEEG) from epilepsy patients while they performed a modified flight initiation distance (FID) task. We found brain regions in the cognitive fear circuit, including the ventromedial prefrontal cortex and hippocampus, encoded the threat level during the information processing stage. The actual escaping stage, especially under rapid attack, prominently activated areas within the reactive fear circuit, including the midcingulate cortex and amygdala. Furthermore, we observed a negative correlation between the high gamma activity (HGA) of the amygdala and the HGA of the vmPFC and HPC under rapid attacks. This indicates that the amygdala may suppress the activity of the cognitive fear circuit under rapid attacks, enabling the organism to react quickly to ensure survival under the imminent threat. These findings highlight the distinct roles of the reactive and cognitive fear circuits in human escaping and provide accounts for the importance of fear in human survival decisions.

neuroscience↗

bioRxiv2024

Unraveling wheat endosperm development: epigenetic regulation and novel regulators for enhanced yield and quality

Starch content and seed storage protein (SSP) composition are critical factors influencing wheat grain yield and quality. To uncover the molecular mechanisms governing their biosynthesis, we conducted transcriptome and epigenome profiling across key endosperm developmental stages, revealing that chromatin accessibility, H3K27ac, and H3K27me3 collectively regulate SSP and starch genes with varying impact. Population transcriptome and phenotype analyses highlighted the crucial role of accessible promoter regions as a genetic variation resource, influencing grain yield and quality in a core collection of wheat accessions. By integrating time-serial RNA-seq and ATAC-seq data, we constructed a hierarchical transcriptional regulatory network (TRN) governing starch and SSP biosynthesis, identifying 42 high-confidence novel candidates. These candidates exhibited overlap with genetic regions associated with grain size and quality traits, and their functional significance was validated through expression-phenotype association analysis among wheat accessions and TILLING mutants. In-depth functional analysis of wheat abscisic acid insensitive 3-A1 (TaABI3-A1) with genome editing knock-out lines demonstrated its role in promoting SSP accumulation while repressing starch biosynthesis through transcriptional regulation. An elite haplotype of TaABI3-A1 with higher grain weight was identified during the breeding process in China, and its superior trait was associated with altered TaABI3-A1 expression levels. Additionally, we identified the potential upstream regulator, wheat GAGA-binding transcription factor 1 (TaGBP1), influencing TaABI3-A1 expression. Our study provides novel and high-confidence regulators, presenting an effective strategy for understanding the regulation of SSP and starch biosynthesis and contributing to breeding enhancement.

plant biology↗

bioRxiv2024

HortGenome Search Engine, a universal genomic search engine for horticultural crops

Horticultural crops comprising fruit, vegetable, ornamental, beverage, medicinal and aromatic plants play essential roles in food security and human health, as well as landscaping. With the advances of sequencing technologies, genomes for hundreds of horticultural crops have been deciphered in recent years, providing a basis for understanding gene functions and regulatory networks and for the improvement of horticultural crops. However, these valuable genomic data are scattered in warehouses with various complex searching and displaying strategies, which increases learning and usage costs and makes comparative and functional genomic analyses across different horticultural crops very challenging. To this end, we have developed a lightweight universal search engine, HortGenome Search Engine (HSE; http://hort.moilab.net), which allows querying genes, functional annotations, protein domains, homologs, and other gene-related functional information of more than 400 horticultural crops. In addition, four commonly used tools, including BLAST, Batch Query, Enrichment analysis, and Synteny Viewer, have been developed for efficient mining and analysis of these genomic data.

bioinformatics↗

bioRxiv2023

TaNF-Y-PRC2 orchestrates temporal control of starch and protein synthesis in wheat

The endosperm in cereal grains is instrumental in determining grain yield and seed quality, as it controls the production of starch and protein. In this study, we identified a specific TaNF-Y trimeric complex, consisting of TaNF-YA3-D, TaNF-YB7-B, and TaNF-YC6-B, exhibiting robust expression within endosperm during grain filling stage in wheat. Knock-down of either TaNF-YA3 or TaNF-YC6 led to less starch but more gluten proteins. Detailed analyses have unveiled that the TaNF-Y indirectly boosts starch biosynthesis genes by reducing TaNAC019, a repressor of TaAGPS1a, TaSuS2, thereby regulating starch biosynthesis. Conversely, the TaNF-Y directly inhibits the expression of gliadin and low molecular weight (LMW)-GS coding genes, including TaGli-{gamma}-700 and TaLMW-400. Furthermore, the TaNF-Y components interact with TaSWN, the histone methyltransferase subunit of Polycomb repressive complex 2 (PRC2), to repress the expression of TaNAC019, TaGli-{gamma}-700, and TaLMW-400 through H3K27me3 modification. Notably, weak mutation of TaFIE, core subunit of PRC2, has reduced starch but elevated gliadin and LMW-GS levels. Intriguingly, DNA variations of TaNF-Y components are widely associated with seed developmental traits. In particular, variation within the coding region of TaNF-YB7-B is linked to differences in starch and protein content. Distinct haplotypes of TaNF-YB7-B affect its interaction with TaSWN, influencing the repression of targets like TaNAC019 and TaGli-{gamma}-700. Our findings illuminate the intricate molecular mechanisms governing epigenetic regulation by the TaNF-Y-PRC2 for wheat endosperm development. Manipulating the TaNF-Y complex holds potential for optimizing grain yield and enhancing quality.

plant biology↗

bioRxiv2023

SntB triggers the antioxidant pathways to regulate development and aflatoxin biosynthesis in Aspergillus flavus

The epigenetic reader SntB was identified as an important transcriptional regulator of growth, development, and secondary metabolite synthesis in Aspergillus flavus. However, the underlying molecular mechanism is still unclear. In this study, sntB gene deletion ({Delta}sntB), complementary (Com-sntB), and HA tag fused to sntB (sntB-HA) strains were constructed by using the homologous recombination method, respectively. Our results revealed that deletion of sntB inhibited the processes of mycelia growth, conidial production, sclerotia formation, aflatoxin synthesis, and ability to colonize host, and the defective phenotype of knockout strain {Delta}sntB could be restored in its complementary strain Com-sntB. Chromatin immunoprecipitation sequencing (ChIP-seq) of sntB-HA and WT, and RNA sequencing (RNA-seq) of {Delta}sntB and WT strains revealed that SntB played key roles in oxidative stress response of A. flavus. The function of catC gene (encode a catalase) was further analyzed based on the integration results of ChIP-seq and RNA-seq. In {Delta}sntB strain, the relative expression level of catC was significantly higher than in WT strain, while a secretory lipase encoding gene (G4B84_008359) was down-regulated. Under the stress of oxidant menadione sodium bisulfite (MSB), the deletion of sntB obvious down-regulated the expression level of catC. After deletion of catC gene, the mycelia growth, conidial production, and sclerotia formation were inhibited, while ROS level and aflatoxin production were increased compared to the WT strain. Results also showed that the inhibition rate of MSB to {Delta}catC strain was significantly lower than that of WT group and AFB1 yield of the {Delta}catC strain was significantly decreased than that of WT strain under the stress of MSB. Our study revealed the potential machinery that SntB regulated fungal morphogenesis, mycotoxin anabolism, and fungal virulence through the axle of from SntB to fungal virulence and mycotoxin bio-synthesis, i.e. H3K36me3 modification-SntB-Peroxisomes-Lipid hydrolysis-fungal virulence and mycotoxin bio-synthesis. The results of this study shed light into the SntB mediated transcript regulation pathways of fungal mycotoxin anabolism and virulence, which provided potential strategy for control the contamination of A. flavus and its aflatoxins.

pharmacology and toxicology↗

bioRxiv2023

High order expression dependencies finely resolve cryptic states and subtypes in single cell data

AO_SCPLOWBSTRACTC_SCPLOWSingle cells are typically typed by clustering in reduced dimensional transcriptome space. Here we introduce Stator, a novel method, workflow and app that reveals cell types, subtypes and states without relying on local proximity of cells in gene expression space. Rather, Stator derives higher-order gene expression dependencies from a sparse gene-by-cell expression matrix. From these dependencies the method multiply labels the same single cell according to type, sub-type and state (activation, differentiation or cell cycle sub-phase). By applying the method to data from mouse embryonic brain, and human healthy or diseased liver, we show how Stator first recapitulates other methods cell type labels, and then reveals combinatorial gene expression markers of cell type, state, and disease at higher resolution. By allowing multiple state labels for single cells we reveal cell type fates of embryonic progenitor cells and liver cancer states associated with patient survival.

molecular biology↗

bioRxiv2023

Extracellular Matrix Protein 1 Attenuates Hepatic Fibrosis by Inhibiting TSP-, ADAMTS-, and MMP-Mediated Latent TGF-β1 Activation

ObjectiveExtracellular Matrix Protein 1 (ECM1) serves as a gatekeeper of hepatic fibrosis by maintaining transforming growth factor-{beta}1 (TGF-{beta}1) in its latent form. ECM1 knockout (KO) causes latent (L) TGF-{beta}1 activation, resulting in hepatic fibrosis with rapid mortality. In chronic liver disease (CLD), ECM1 decreases with increasing CLD severity. We investigate the regulatory role of ECM1 in TGF-{beta}1 bioavailability and its impact on CLD progression. DesignRNAseq was performed to analyze hepatic gene expression. Functional assays were performed using hepatic stellate cells (HSCs), Ecm1-KO and Fxr-KO mice, patient liver tissue, and computer simulations. ResultsExpression of LTGF-{beta}1 activators, including thrombospondins (TSPs), ADAMTS proteases, and matrix metalloproteinases (MMPs) increased along with pro-fibrotic gene expression in liver tissue of Ecm1-KO mice. In HSCs, overexpression of ECM1 prevented TSP-1-, ADAMTS1-, and MMP-2/9-mediated LTGF-{beta}1 activation. In vitro interaction assays demonstrated that ECM1 inhibited LTGF-{beta}1 activation by interacting with TSP-1 and ADAMTS1 via their respective, intrinsic KRFK or KTFR amino acid sequences, and by suppressing MMP-2/9 proteolytic activity. In mice, ECM1 overexpression attenuated KRFK-induced LTGF-{beta}1 activation, while KTFR treatment reversed Ecm1-KO- and Fxr-KO-mediated liver injury. In patients with CLD, ECM1 expression was inversely correlated with TSP-1, ADAMTS1, MMP-2/9 expression and LTGF-{beta}1 activation. And these results were complemented by a computational compartment model representing the key network of cellular phenotypes and predicted interactions in liver fibrogenesis. ConclusionOur findings underscore the hepatoprotective effect of ECM1, which interferes with mediators of LTGF-{beta}1 activation, suggesting ECM1 or its representative peptide as potential anti-fibrotic therapies in CLD. What is already known on this topic?[tpltrtarr] ECM1 expression is negatively correlated with CLD progression. [tpltrtarr]ECM1 maintains liver homeostasis by keeping TGF-{beta}1 latency. What this study adds?[tpltrtarr] ECM1 inhibits LTGF-{beta}1 activation through interfering with key activators, including TSP-1, ADAMTS1, MMP-2, and MMP-9. [tpltrtarr]ECM1 interacts with TSP-1 and ADAMTS1 via their respective, intrinsic KRFK or KTFR amino acid motifs, and suppresses MMP-2/9 proteolytic activity. [tpltrtarr]In vivo, ECM1 overexpression mitigates KRFK peptide-induced LTGF-{beta}1 activation, while KTFR peptide rescues Ecm1-KO- and Fxr-KO-induced liver injury. [tpltrtarr]ECM1 expression inversely correlates with TSP-1, ADAMTS1, MMP-2/9 expression and LTGF-{beta}1 activation in CLD patients. How might this study affect research, practice or policy?[tpltrtarr] Considering severe adverse effects associated with anti-fibrotic treatments utilizing TGF-{beta}1 receptor inhibitors, our findings indicate that restoration of ECM1 expression or phenocopying peptides might represent a novel and safe route to urgently needed anti-fibrotic therapies in CLD.

cell biology↗

bioRxiv2023

Elevation-dependent patterns of snow-gum dieback are moderated by trait differences between montane and subalpine forests.

Subalpine forests worldwide face the synergistic threats of global warming and increased biotic attack, and the collapse or transition of subalpine forests is predicted in south-eastern Australia under future climates. The recent widespread dieback of subalpine snow gum forests due to increased activity of a native wood-boring longicorn beetle suggests this process may already be underway. We investigated how variation in tree tissue traits and environmental conditions correlated with elevation-dependent spatial patterns of forest mortality. We hypothesised that increased vulnerability of subalpine snow gums to wood-borer-mediated dieback at intermediate elevations was associated with poorly-resolved differences in traits between montane (Eucalyptus pauciflora subsp. pauciflora) and subalpine (E. pauciflora subsp. niphophila) snow-gum subspecies. We first sought to characterise variation and elevation-dependent transitions in 20 structural and drought-related functional traits among 120 healthy trees distributed along a 1000m elevation transect which spanned the subspecies transition zone. Secondly, we surveyed 774 trees across 53 sites between 1280-1980m asl. to explore associations between borer-damage severity, elevation, subspecies, and a subset of traits that differed between subspecies. These snow-gum subspecies exhibited mean differences and/or divergent elevation responses in 25% of traits surveyed, indicating contrasting suites of traits between montane and subalpine subspecies. Increased borer-damage severity across the montane-to-subalpine subspecies transition was correlated with lower bark thickness, whereas reduced borer damage at the highest elevations was associated with greater precipitation and lower temperatures. Our results suggest that due to possessing distinct traits associated with increased borer susceptibility, subalpine snow-gum forests will be subject to increased risk of severe borer-mediated forest dieback under warmer and drier future climates. Identifying traits contributing to species distribution limits and biotic-agent vulnerability remains critical for predicting, monitoring, and possibly mitigating forest and vegetation declines under future climates.

ecology↗

bioRxiv2023

Immediate impact of the 2021 harmful algal bloom in southeast Hokkaido on the rocky intertidal community and its spatial variation

There has been a limited number of studies on the effects of harmful algal blooms (HABs) on natural rocky intertidal ecosystems. From mid-September to early November 2021, an unprecedented HAB caused by Karenia selliformis hit the Pacific coast of southeast Hokkaido, Japan, for the first time, causing massive mortalities among marine organisms. To clarify the immediate impacts of the HAB on abundance of 10 rocky intertidal species in four functional groups (macroalgae, sessile invertebrates, molluscan grazers, and molluscan carnivores), we focused on two questions. 1) How did the HAB affect the abundance of each species differently at the regional scale? 2) How did the impacts of the HAB on the abundance of each functional groups vary spatially, and was the spatial variation of the HAB impacts related to the spatial distribution of cell density of HAB species? To study these issues, we compared census data for 17 years before the HAB and within one month after it for five shores on the southeast coast of Hokkaido. Results showed that two macroalgae species and all three molluscan grazer species declined significantly after the HAB. Moreover, the decrease of molluscan grazers was significantly correlated with cell density. These results suggest that the impacts of the HAB in southeast Hokkaido on abundance of rocky intertidal organisms are highly variable depending on species and locality, presumably because of differences in species-specific tolerances to HAB toxins and spatial variation in the density of the HAB organisms.

ecology↗

bioRxiv2023

Tropism of AAV.CPP.16 in the respiratory tract and its application for a CRISPR-based gene therapy against SARS-CoV-2

Efficient gene delivery vectors are essential for developing gene therapies for respiratory diseases. Here, we report that AAV.CPP.16, a novel AAV9-derived adeno-associated virus vector, can efficiently transduce airway epithelium systems and lung parenchyma cells in both mice and non-human primates after intranasal administration. AAV.CPP.16 outperforms AAV6 and AAV9, two wild-type AAVs with demonstrated tropism to respiratory tract tissues, and can target major cell types in the respiratory tract and the lung. We also report an "all-in-one", CRISPR-Cas13d-based AAV gene therapy vector that targets the highly conserved RNA-dependent RNA polymerase (Rdrp) gene in SARS-CoV-2, and show the potential of such gene therapy against a broad range of circulating and emergent SARS-CoV-2 variants. Thus, AAV.CPP.16 could be a useful gene delivery vector for treating genetic respiratory diseases and airborne infections including for developing a potential prophilaxis to SARS-CoV-2.

bioengineering↗

bioRxiv2023

Ultrasonic reporters of calcium for deep tissue imaging of cellular signals

Calcium imaging has enabled major biological discoveries. However, the scattering of light by tissue limits the use of standard fluorescent calcium indicators in living animals. To address this limitation, we introduce the first genetically encoded ultrasonic reporter of calcium (URoC). Based on a unique class of air-filled protein nanostructures called gas vesicles, we engineered URoC to produce elevated nonlinear ultrasound signal upon binding to calcium ions. With URoC expressed in mammalian cells, we demonstrate noninvasive ultrasound imaging of calcium signaling in vivo during drug-induced receptor activation. URoC brings the depth and resolution advantages of ultrasound to the in vivo imaging of dynamic cellular function and paves the way for acoustic biosensing of a broader variety of biological signals.

bioengineering↗

bioRxiv2023

A blood-brain-barrier penetrant AAV gene therapy rescues neurological deficits in mucolipidosis IV mice.

Mucolipidosis IV (MLIV) is a rare, autosomal recessive, lysosomal disease characterized by intellectual disability, motor deficits and progressive vision loss. Using AAV9 and AAV-PHP.B as delivery vectors, we previously demonstrated the feasibility of modifying disease course in a mouse model of MLIV by the human MCOLN1 gene transfer. Here, using a primate-enabling capsid AAV.CPP.16 (CPP16), we constructed a new, clinic-oriented MCOLN1 gene expression vector and demonstrated its efficacy in the preclinical model of MLIV. Systemic administration of CPP16-MCOLN1 in adult symptomatic Mcoln1-/- mice at a dose of 1e12 vg per mouse resulted in MCOLN1 expression in the brain and peripheral tissues, alleviated brain pathology, rescued neuromotor function, and completely prevented paralysis. Notable expression of MCOLN1 transcripts was also detected in the retina of the mouse that had exhibited significant degeneration at the time of the treatment. However, no increase of retinal thickness was observed after the gene therapy treatment. Our results suggest a new AAV-based systemic gene replacement therapy for the treatment of MLIV that could be translated into clinical studies.

genetics↗

bioRxiv2023

G protein subunit Gγ13-mediated signaling pathway is critical to the inflammation resolution and functional recovery of severely injured lungs

Tuft cells are a group of rare epithelial cells that can detect pathogenic microbes and parasites. Many of these cells express signaling proteins initially found in taste buds. It is, however, not well understood how these taste signaling proteins contribute to the response to the invading pathogens or to the recovery of injured tissues. In this study, we conditionally nullified the signaling G protein subunit G{gamma}13 and found that the number of ectopic tuft cells in the injured lung was reduced following the infection of the influenza virus H1N1. Furthermore, the infected mutant mice exhibited significantly larger areas of lung injury, increased macrophage infiltration, severer pulmonary epithelial leakage, augmented pyroptosis and cell death, greater bodyweight loss, slower recovery, worsened fibrosis and increased fatality. Our data demonstrate that the G{gamma}13-mediated signal transduction pathway is critical to tuft cells-mediated inflammation resolution and functional repair of the damaged lungs.To our best knowledge, it is the first report indicating subtype-specific contributions of tuft cells to the resolution and recovery.

immunology↗

bioRxiv2023

Identifying Temporal Correlations Between Natural Single-shot Videos and EEG Signals

AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSObjectiveC_ST_ABSElectroencephalography (EEG) is a widely used technology for recording brain activity in brain-computer interface (BCI) research, where understanding the encoding-decoding relationship between stimuli and neural responses is a fundamental challenge. Recently, there is a growing interest in encoding-decoding natural stimuli in a single-trial setting, as opposed to traditional BCI literature where multi-trial presentations of synthetic stimuli are commonplace. While EEG responses to natural speech have been extensively studied, such stimulus-following EEG responses to natural video footage remain underexplored. ApproachWe collect a new EEG dataset with subjects passively viewing a film clip and extract a few video features that have been found to be temporally correlated with EEG signals. However, our analysis reveals that these correlations are mainly driven by shot cuts in the video. To avoid the confounds related to shot cuts, we construct another EEG dataset with natural single-shot videos as stimuli and propose a new set of object-based features. Main ResultsWe demonstrate that previous video features lack robustness in capturing the coupling with EEG signals in the absence of shot cuts, and that the proposed object-based features exhibit significantly higher correlations. Furthermore, we show that the correlations obtained with these proposed features are not dominantly driven by eye movements. Additionally, we quantitatively verify the superiority of the proposed features in a match-mismatch (MM) task. Finally, we evaluate to what extent these proposed features explain the variance in coherent stimulus responses across subjects. SignificanceThis work provides valuable insights into feature design for video-EEG analysis and paves the way for applications such as visual attention decoding.

bioengineering↗

bioRxiv2023

Multi-omics analysis reveals signatures of selection and loci associated with complex traits in pigs

Selection signatures that contribute to phenotypic diversity, especially morphogenesis in pigs, remain to be further elucidated. To reveal the regulatory role of genetic variations in phenotypic differences between Eastern and Western pig breeds, we performed a systematic analysis based on seven high-quality de novo assembled genomes, 1,081 resequencing data representing 78 domestic breeds, 162 methylomes, and 162 transcriptomes of skeletal muscle from Tongcheng (Eastern) and Landrace (Western) pigs at 27 developmental stages. Selective sweep uncovers different genetic architectures behind divergent selection directions for the Eastern and Western breeds. Notably, two loci showed functional alterations by almost fixed missense mutations. By integrating time-course transcriptome and methylome, we revealed differences in developmental timing during myogenesis between Eastern and Western breeds. Genetic variants under artificial selection have critical regulatory effects on progression patterns of heterochronic genes like GHSR and BDH1, by the interaction of local DNA methylation status, particularly during embryonic development. Altogether, our work not only provides valuable resources for understanding pig complex traits, but also contributes to human biomedical research.

genomics↗

bioRxiv2023

Deep Whole-Genome Resequencing of Marek's Disease Virus Strains Reveals Intrastrain Variation

Current strategies to understand the molecular basis of Mareks disease virus (MDV) virulence primarily consist of cataloguing divergent nucleotides between strains with different phenotypes. However, each MDV strain is typically represented by a single consensus genome despite the confirmed existence of mixed viral populations. To assess the reliability of single-consensus interstrain genomic comparisons, we obtained two additional consensus genomes of vaccine strain CVI988 (Rispens) and two additional consensus genomes of the very virulent strain Md5 by sequencing viral stocks and cultured field isolates. In conjunction with the published genomes of CVI988 and Md5, this allowed us to perform 3-way comparisons between consensus genomes of the same strain. We found that consensus genomes of CVI988 can vary in as many as 236 positions involving 13 open reading frames (ORFs). In contrast, we found that Md5 genomes varied only in 11 positions involving a single ORF. Phylogenomic analyses showed all three Md5 consensus genomes clustered closely together, while also showing that CVI988GenBank.BAC diverged from CVI988Pirbright.lab and CVI988USDA.PA.field. Comparison of CVI988 consensus genomes revealed 19 SNPs in the unique regions of CVI988GenBank.BAC that were not present in either CVI988Pirbright.lab or CVI988USDA.PA.field. Finally, we evaluated the genomic heterogeneity of CVI988 and Md5 populations by identifying positions with >2% read support for alternative alleles in two ultra-deeply sequenced samples. We were able to confirm that both populations of CVI988 and Md5 were mixed, exhibiting a total of 29 and 27 high-confidence minor variant positions, respectively. We did not find any evidence of minor variants in the positions corresponding to the 19 SNPs in the unique regions of CVI988GenBank.BAC. Taken together, our findings confirm that consensus genomes of the same strain of MDV can vary and suggest that multiple consensus genomes per strain are needed in order to maximize the accuracy of interstrain genomic comparisons.

microbiology↗

bioRxiv2023

CLOCK Mediates Delay in Cardiac Myofibroblast Formation and Attenuates Progression of Myocardial Fibrosis through Inhibition of Smad 3 Transcriptional Activity

IntroductionMyocardial fibrosis, characterized by excessive extracellular matrix deposition, leading to adverse cardiac remodeling and impaired function. The differentiation of cardiac fibroblasts into myofibroblasts plays a pivotal role in this process. The involvement of the core clock protein CLOCK in the formation of cardiac myofibroblasts and the advancement of myocardial fibrosis is not well understood. This study aims to investigate how CLOCK modulates cardiac myofibroblast differentiation and attenuates myocardial fibrosis by inhibiting Smad3 transcriptional activity. MethodsIn vivo, adeno-associated virus 9 was utilized for overexpression or silencing of the CLOCK gene via mice intravenous injection. In vitro experiments were conducted using primary cultures of cardiac fibroblasts isolated from mouse hearts to examine the expression levels of molecular markers associated with myofibroblast differentiation, such as -smooth muscle actin (-SMA) and collagen I. Additionally, a Smad3 luciferase reporter gene experiment was employed to assess the transcriptional activity of Smad3 under CLOCK regulation. ResultsThe results demonstrate that CLOCK overexpression significantly reduces the expression of -SMA and collagen I in cardiac fibroblasts, indicating a suppression of myofibroblast formation. Conversely, CLOCK knockdown enhances the expression of these fibrotic markers. Interestingly, CLOCK was found to negatively regulate the transcriptional activity of Smad3, suggesting a potential mechanism by which CLOCK exerts its inhibitory effect on cardiac fibrosis. Specifically, CLOCK interacts with Smad3, inhibiting its nuclear translocation and subsequent activation of fibro genic gene expression. ConclusionThese findings highlight the crucial role of CLOCK in the regulation of cardiac myofibroblast formation and the progression of myocardial fibrosis. CLOCK appears to exert an inhibitory effect on fibrotic signaling pathways, partially through the inhibition of Smad3 transcriptional activity. This study provides novel insights into the molecular mechanisms underlying cardiac fibrosis and emphasizes the therapeutic potential of targeting CLOCK-mediated pathways for treating myocardial fibrosis.

molecular biology↗

bioRxiv2023

TGF-β1 inhibits cholesterol metabolism in hepatocytes to facilitate cell death, EMT and signals for HSC activation.

Background and Aims: Transforming growth factor-{beta}1 (TGF-{beta}1) plays important roles in chronic liver diseases, including metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD involves various biological processes including dysfunctional cholesterol metabolism and contributes to progression to metabolic dysfunction-associated steatohepatitis (MASH) and hepatocellular carcinoma (HCC). However, the reciprocal regulation of TGF-{beta}1 signaling and cholesterol metabolism in MASLD is yet unknown. Methods: Changes in transcription of genes associated with cholesterol metabolism were assessed by RNA-Seq of murine hepatocyte cell line (AML12) and mouse primary hepatocytes (MPH) treated with TGF-{beta}1. Functional assays were performed on AML12 cells (untreated, TGF-{beta}1 treated, or subjected to cholesterol enrichment (CE) or depletion (CD)), and on mice injected with adeno-associated virus 8 (AAV8)-Control/TGF-{beta}1. Results: TGF-{beta}1 inhibited mRNA expression of several cholesterol metabolism regulatory genes, including rate-limiting enzymes of cholesterol biosynthesis in AML12 cells, MPHs, and AAV8-TGF-{beta}1-treated mice. Total cholesterol levels and lipid droplet accumulation in AML12 cells and liver tissue were also reduced upon TGF-{beta}1 treatment. Smad2/3 phosphorylation following 2 h TGF-{beta}1 treatment persisted after CE or CD and was mildly increased following CD, while TGF-{beta}1-mediated AKT phosphorylation (30 min) was inhibited by CE. Furthermore, CE protected AML12 cells from several effects mediated by 72 h incubation with TGF-{beta}1, including EMT, actin polymerization, and apoptosis. CD mimicked the outcome of long term TGF- {beta}1 administration, an effect that was blocked by an inhibitor of the type I TGF-{beta} receptor. Additionally, the supernatant of CE- or CD-treated AML12 cells inhibited or promoted, respectively, the activation of LX-2 hepatic stellate cells. Conclusions: TGF-{beta}1 inhibits cholesterol metabolism while cholesterol attenuates TGF-{beta}1 downstream effects in hepatocytes.

cell biology↗