Regulation Of Relations Research Articles

Transcriptome Reveals Molecular Mechanisms of Neuroendocrine Regulation of Allometric Growth in the Red Swamp Crayfish Procambarus clarkii.

Allometric growth is a typical characteristic of crustaceans, which mainly occurs among individuals, life stages, tissues, and between sexes. The red swamp crayfish Procambarus clarkii is an economically important crustacean species in the world. To date, the molecular regulatory mechanisms of neuroendocrine system in the allometric growth of P. clarkii remain unclear. In this study, P. clarkii exhibiting significant allometric growth among individuals were sampled from three full-sibling families. The brain, eyestalk, nerve cord, and Y-organ were dissected for transcriptome analysis. Key functional genes were identified by random forest and DESeq2 methods. The gene pathways were enriched utilizing Kyoto Encyclopedia Genes and Genomes (KEGG) analysis. Gene topological analysis was established through weighted gene co-expression network analysis (WGCNA), and hub genes were screened by protein-protein interaction (PPI) networks. Transcriptomic analysis results were validated via qRT-PCR. RNA-Seq identified 31 differentially expressed genes (DEGs) (7 up- and 24 downregulated); 301 DEGs (23 up- and 278 downregulated); 1308 DEGs (474 up- and 834 downregulated); and 64 DEGs (52 up- and 12 downregulated) in the brain, eyestalk, Y-organ, and nerve cord, respectively. Crucial functional genes such as CHIA in the brain and perlucin-like in the eyestalk were notably identified. WGCNA revealed two hub modules, while PPI networks identified neuroendocrine regulators module which hub genes mainly including CP1876-like and cuticle protein AM1199-like, and structural components module which hub genes mainly including CUB& CCP Domain-Containing Protein, ARRDC, and E3 Ubiquitin protein ligase MCYCBP2-like. Correspondingly, the significant gene pathways such as amino sugar and nucleotide sugar metabolism (pcla00520) and insect hormone biosynthesis (pcla00981) were enriched. The results revealed the complex interactions and regulatory relationships of hub genes within hub modules to coordinate molting and growth. The results of RNA-Seq analysis were validated by the consistency of gene expression in qRT-PCR. In present study, key functional genes in the neuroendocrine system regulating allometric growth among individuals were identified, and significant pathways mainly include hormone synthesis were screened, thus constructing a neuroendocrine molecular regulatory network for the allometric growth of P. clarkii. Building on these investigations, a comprehensive mechanism whereby neuroendocrine regulators interact with structural components to coordinate molting and growth was proposed. The result would provide valuable insights into the molecular regulatory mechanisms of allometric growth, highlighting the interplay between the neuroendocrine system and relevant tissues.

RGIE: A Gene Selection Method Related to Radiotherapy Resistance in Head and Neck Squamous Cell Carcinoma.

Head and Neck Squamous Cell Carcinoma (HNSCC) is a malignant tumor with a high degree of malignancy, invasiveness, and metastasis rate. Radiotherapy, as an important adjuvant therapy for HNSCC, can reduce the postoperative recurrence rate and improve the survival rate. Identifying the genes related to HNSCC radiotherapy resistance (HNSCC-RR) is helpful in the search for potential therapeutic targets. However, identifying radiotherapy resistance-related genes from tens of thousands of genes is a challenging task. While interactions between genes are important for elucidating complex biological processes, the large number of genes makes the computation of gene interactions infeasible. We propose a gene selection algorithm, RGIE, which is based on ReliefF, Gene Network Inference with Ensemble of Trees (GENIE3) and Feature Elimination. ReliefF was used to select a feature subset that is discriminative for HNSCC-RR, GENIE3 constructed a gene regulatory network based on this subset to analyze the regulatory relationship among genes, and feature elimination was used to remove redundant and noisy features. Nine genes (SPAG1, FIGN, NUBPL, CHMP5, TCF7L2, COQ10B, BSDC1, ZFPM1, GRPEL1) were identified and used to identify HNSCC-RR, which achieved performances of 0.9730, 0.9679, 0.9767, and 0.9885 in terms of accuracy, precision, recall, and AUC, respectively. Finally, qRT-PCR validated the differential expression of the nine signature genes in cell lines (SCC9, SCC9-RR). RGIE is effective in screening genes related to HNSCC-RR. This approach may help guide clinical treatment modalities for patients and develop potential treatments.

N6-methyladenosine (m6A)-circHECA from secondary hair follicle of cashmere goats: identification, regulatory network and expression regulated potentially by methylation of its host gene promoter.

The objective of this study was to identify the N6-methyladenosine (m6A)- circHECA molecule in secondary hair follicles (SHFs) of cashmere goats, and generate its potential regulatory network, as well as explore the potential relationship between transcriptional pattern of m6A-circHECA and promoter methylation of its host gene (HECA). The validation of circHECA m6A sites was performed using methylation immunoprecipitation (Me-RIP) along with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) technique. The nucleus and cytoplasm localizations of m6AcircHECA were performed using SHF stem cells of cashmere goats with RT-qPCR analysis. Based on in-silico analysis, the regulatory networks of m6A-circHECA were generated with related signal pathway enrichment. The methylation level of promoter region of m6A-circHECA host gene (HECA) was assessed by the bisulfite sequencing PCR (BSPPCR) technique. The m6A-circHECA was confirmed to contain four m6A modification sites including m6A-213, m6A-297, m6A-780, and m6A-927, and it was detected mainly in cytoplasm of the SHF stem cells of cashmere goats. The integrated regulatory network analysis showed directly or indirectly complex regulatory relationships between m6A-circHECA of cashmere goats and its potential target molecules: miRNAs, mRNAs, and proteins. The regulatory network and pathway enrichment indicated that m6A-circHECA might play multiple roles in the SHF physiology process of cashmere goats through directly or indirectly interacting or regulating its potential target molecules. A higher methylation level of promoter region of HECA gene in SHFs of cashmere goats might cause the lower expression of m6A-circHECA. The m6A-circHECA might play multiple roles in SHF physiology process of cashmere goats through miRNA mediated pathways along with directly or indirectly interaction with its target proteins. The promoter methylation of m6A-circHECA host gene (HECA) most likely was implicated in its expression inhibition in SHFs of cashmere goats.

Microtubule-Rab5 mutual-influential system screening based on gene-regulatory networks map in Rab5 RNAi eye-degeneration model

Rabs are involved in neuronal development and protrusion formation. Existing studies support the notion that manipulation or mutation of Rab genes could lead to functional changes in neurons. However, whether Rabs gene-manipulation induced Drosophila eye-degeneration remains unknown. By down-regulating Rab5, but not Rab7, we first constructed a compound eye injury model in Drosophila. As the distribution, content, and even maturation of Rab5-positive endosomes are influenced by cytoskeletal proteins, like actin or tubulin-related proteins, the existence of a bidirectional regulatory relationship between Rab5 and the cytoskeleton remains unclear and worth researching. Through complete transcriptome sequencing combined immunofluorescence testing, we confirmed that down-regulation of Rab5 affected the increase of α-Tub84B (alternatively named TubA84B) but not γ-tubulin. Based on Weighted Gene Co-Expression Network Analysis (WGCNA) and multi-tissue screening verification, this study proposes that the apoptosis-related factors–Rab5–TubA84B have conserved regulatory functions with cooperative expression. Gene manipulation confirmed that apoptotic factors, especially rpr, strongly regulate Rab5, and may ultimately influence microtubule structure through complex routes, including the Rab5 variance and the intracellular configuration ratio of α-Tubulin to Glyceraldehyde-3-phosphate dehydrogenase.

The Role of Panax Notoginseng Saponins on Neuronal Oxidative Stress and Autophagy Response in Craniocerebral Injury Model Rats

This study assessed how IL-6 affects the body’s autophagy status by regulating JAK-STAT3 and explored the mechanism by which IL-6 inhibits JAK-STAT3 signaling pathway-mediated autophagy, and how Panax notoginseng total saponins promote neuronal cell regeneration in rats. A model of neuronal oxidative stress in craniocerebral injury model rats was established, and IL-6 levels were detected by ELISA. Rat neuronal cells were isolated and cultured, while dual-luciferase gene reporter experiments analyzed the targeting relationship between IL-6 and JAK. qRT-PCR detected expression of neuronal autophagy-related genes (JAK, STAT3, ULK1, OsATG7, FAM176A, and Beclin 1). The level of IL-6 in the craniocerebral injury model was significantly higher than that in the control group. IL-6 binds to 3′-UTR of JAK, and transfection with IL-6 inhibitor increases the relative luciferase activity. pMIR-JAK-mut group has no significant effect. There is a targeted regulatory relationship between IL-6 and JAK. Adding total saponins of Panax notoginseng combined with nursing intervention can promote reduction genetic expression related to neuronal autophagy damage. IL-6 can target and regulate JAK gene. IL-6 can promote neuronal development by regulating JAK-STAT3. Autophagy repairs damage and inhibits the autophagy state of cells. Panax notoginseng total saponins are effective medicinal components extracted from high quality panax notoginseng according to extraction and separation technology. The results from this study provide a better understanding of pathogenesis of neuronal oxidative stress in craniocerebral injury model rats and find potential intervention targets, which will provide more treatment methods for neuronal oxidative stress damage in craniocerebral injury model rats.

SIGRN: Inferring Gene Regulatory Network with Soft Introspective Variational Autoencoders

Gene regulatory networks (GRNs) exhibit the complex regulatory relationships among genes, which are essential for understanding developmental biology and uncovering the fundamental aspects of various biological phenomena. It is an effective and economical way to infer GRNs from single-cell RNA sequencing (scRNA-seq) with computational methods. Recent researches have been done on the problem by using variational autoencoder (VAE) and structural equation model (SEM). Due to the shortcoming of VAE generating poor-quality data, in this paper, a soft introspective adversarial gene regulatory network unsupervised inference model, called SIGRN, is proposed by introducing adversarial mechanism in building a variational autoencoder model. SIGRN applies “soft” introspective adversarial mode to avoid training additional neural networks and adding additional training parameters. It demonstrates superior inference accuracy across most benchmark datasets when compared to nine leading-edge methods. In addition, method SIGRN also achieves better performance on representing cells and generating scRNA-seq data in most datasets. All of which have been verified via substantial experiments. The SIGRN method shows promise for generating scRNA-seq data and inferring GRNs.

GATv2EPI: Predicting Enhancer–Promoter Interactions with a Dynamic Graph Attention Network

Background: The enhancer–promoter interaction (EPI) is a critical component of gene regulatory networks, playing a significant role in understanding the complexity of gene expression. Traditional EPI prediction methods focus on one-to-one interactions, neglecting more complex one-to-many and many-to-many patterns. To address this gap, we utilize graph neural networks to comprehensively explore all interaction patterns between enhancers and promoters, capturing complex regulatory relationships for more accurate predictions. Methods: In this study, we introduce a novel EPI prediction framework, GATv2EPI, based on dynamic graph attention neural networks. GATv2EPI leverages epigenetic information from enhancers, promoters, and their surrounding regions and organizes interactions into a network to comprehensively explore complex EPI regulatory patterns, including one-to-one, one-to-many, and many-to-many relationships. To avoid overfitting and ensure diverse data representation, we implemented a connectivity-based sampling method for dataset partitioning, which constructs graphs for each chromosome and assigns entire connected subgraphs to training or test sets, thereby preventing information leakage and ensuring comprehensive chromosomal representation. Results: In experiments conducted on four cell lines—NHEK, IMR90, HMEC, and K562—GATv2EPI demonstrated superior EPI recognition accuracy compared to existing similar methods, with a training time improvement of 95.29% over TransEPI. Conclusions: GATv2EPI enhances EPI prediction accuracy by capturing complex topological structure information from gene regulatory networks through graph neural networks. Additionally, our results emphasize the importance of epigenetic features surrounding enhancers and promoters in EPI prediction.

Machine Learning Inference of Gene Regulatory Networks in Developing Mimulus Seeds

The angiosperm seed represents a critical evolutionary breakthrough that has been shown to propel the reproductive success and radiation of flowering plants. Seeds promote the rapid diversification of angiosperms by establishing postzygotic reproductive barriers, such as hybrid seed inviability. While prezygotic barriers to reproduction tend to be transient, postzygotic barriers are often permanent and therefore can play a pivotal role in facilitating speciation. This property of the angiosperm seed is exemplified in the Mimulus genus. In order to further the understanding of the gene regulatory mechanisms important in the Mimulus seed, we performed gene regulatory network (GRN) inference analysis by using time-series RNA-seq data from developing hybrid seeds from a viable cross between Mimulus guttatus and Mimulus pardalis. GRN inference has the capacity to identify active regulatory mechanisms in a sample and highlight genes of potential biological importance. In our case, GRN inference also provided the opportunity to uncover active regulatory relationships and generate a reference set of putative gene regulations. We deployed two GRN inference algorithms—RTP-STAR and KBoost—on three different subsets of our transcriptomic dataset. While the two algorithms yielded GRNs with different regulations and topologies when working with the same data subset, there was still significant overlap in the specific gene regulations they inferred, and they both identified potential novel regulatory mechanisms that warrant further investigation.

Polyphyllin VII enhances the sensitivity of endometrial carcinoma cells to medroxyprogesterone acetate through upregulating miR‑33a‑5p expression.

Endometrial carcinoma (EC) often exhibits resistance to hormone therapies, such as medroxyprogesterone acetate (MPA), highlighting the need for novel strategies to enhance therapeutic efficacy. The present study aimed to investigate the effects of polyphyllin VII (PPVII) on the efficacy of MPA in EC, focusing on the regulatory role of microRNA (miR)-33a-5p. Briefly, an MPA-resistant Ishikawa cell line (Ishikawa/MPA-R), maintained with 10 µM MPA, was established and transfected with negative control (NC) and miR-33a-5p inhibitors. Following treatment with PPVII and MPA, the proliferation capacity and apoptosis levels of the Ishikawa and Ishikawa/MPA-R cells were evaluated using reverse transcription-quantitative polymerase chain reaction, MTT assay, clonogenic assay, flow cytometry, western blotting and dual-luciferase assay. Next, the expression levels of miR-33a-5p and F-box and leucine rich repeat protein 16 (FBXL16) were measured, and the regulatory relationship between miR-33a-5p and FBXL16 was analyzed. Significant reductions in cell viability were observed in all groups following treatment with increased concentrations of MPA and PPVII, with the greatest effect observed in the combined MPA + PPVII group (P<0.01). The apoptosis levels of the Ishikawa/MPA-R cells were significantly increased in all drug treatment groups, particularly in the MPA + PPVII group (P<0.05). PPVII treatment significantly increased the expression level of miR-33a-5p in Ishikawa/MPA-R cells (P<0.01). In the PPVII + miR-33a-5p inhibitor group, the Ishikawa/MPA-R cells exhibited an upregulation in the viability (P<0.01), colony formation ability (P<0.01), proportion in the G1 phase (P<0.05) and the protein expression levels of cyclin D1 (P<0.01) and cyclin-dependent kinase 4 (P<0.01), and a reduction in the miR-33a-5p expression (P<0.01), apoptosis levels (P<0.05), proportion in the S (P<0.05) and G2 phases and the levels of Bcl-2-associated X protein (P<0.001). The FBXL16 protein expression in Ishikawa/MPA-R cells was significantly higher compared with Ishikawa cells, and the mRNA and protein expression levels of FBXL16 were markedly elevated in the PPVII + miR-33a-5p inhibitor group compared with the PPVII + NC group (P<0.01). These findings suggested that PPVII upregulated the expression of miR-33a-5p, enhanced the sensitivity of EC cells to MPA and potentially exerted anticancer effects in EC through the synergistic action of the miR-33a-5p/FBXL16 axis in combination with MPA.

EfficientNet-resDDSC: A Hybrid Deep Learning Model Integrating Residual Blocks and Dilated Convolutions for Inferring Gene Causality in Single-Cell Data.

Gene Regulatory Networks (GRNs) reveal complex interactions between genes in organisms, crucial for understanding the life system's operation. The rapid development of biotechnology, especially single-cell RNA sequencing (scRNA-seq), has generated a large amount of scRNA-seq data, which can be analyzed to explore the regulatory relationships between genes at the single-cell level. Previous models used to construct GRNs mainly aim at constructing associative relationships between genes, but usually fail to accurately reveal the causality between genes. Therefore, we present a hybrid deep learning model called EfficientNet-resDDSC (the EfficientNet with Residual Blocks and Depthwise Separable Dilated Convolutions) to infer causality between genes. The model inherits the basic structure of EfficientNet-B0 and incorporates residual blocks as well as dilated convolutions. The model's ability to extract low-level features at the primary stage is enhanced by introducing residual blocks. The model combines Depthwise Separable Convolution (DSC) in the inverted linear bottleneck layers with the dilated convolutions to expand the model's receptive fields without increasing the computational effort. This design enables the model to comprehensively reveal potential relationships among different genes in high-dimensional and high-noise single-cell data. In comparison with the five existing deep learning network models, EfficientNet-resDDSC's overall performance is significantly better than others on four datasets. In this study, EfficientNet-resDDSC was further applied to construct GRNs for breast cancer patients, focusing on the related regulatory genes of the key gene BRCA1, which contributes to the advancement of breast cancer research and treatment strategies.

MiR-PC-3p-241582_34 Contributes to the Infection of Athetis lepigone by Regulating the Expression of HSWP4 in Nosema bombycis.

Athetis lepigone is a recurring pest in the maize seedling stage under the wheat-maize no-tillage direct seeding system in China's summer maize region. Our previous research identified a highly pathogenic Nosema bombycis to A. lepigone, which spore wall protein plays an important role in the infection process. However, the regulatory mechanism of this spore wall protein is still unclear. In this study, we explored the regulatory mechanism of miRNAs on spore wall proteins. Transcriptome sequencing results showed that expression of the spore wall protein, HSWP4, significantly increased in the germination group compared to dormancy group. Silencing of HSWP4 reduced the number of microsporidian spores breaking through the midgut wall cells of A. lepigone. Association analysis of small RNA and mRNA revealed that the targeting site of miR-PC-3p-241582_34 on HSWP4 was located in the CDS region, and miR-PC-3p-241582_34 had a significant negative regulatory relationship with HSWP4. The dual luciferase reporter assay demonstrated that miR-PC-3p-241582_34 significantly affected the luciferase activity of the HSWP4-3'UTR expression vector (P < 0.05). Delivery of miRNA mimics decreased the expression of HSWP4 and inhibited the behavior of microsporidian spores breaking through the midgut wall of A. lepigone. On the other hand, delivery of inhibitors produced opposite results, indicating that the miR-HSWP4 pathway plays an important role in microsporidian infection of A. lepigone. This study provides a new theoretical basis for understanding the pathogenic mechanism and gene regulation of microsporidia, as well as for the green control of A. lepigone.

Sc2GWAS: a comprehensive platform linking single cell and GWAS traits of human.

Identifying cell populations associated with risk variants is essential for uncovering cell-specific mechanisms that drive disease development and progression. Integrating genome-wide association studies (GWAS) with single-cell RNA sequencing (scRNA-seq) has become an effective strategy for detecting trait-cell relationships. The accumulation of trait-related single cell data has led to an urgent need for its comprehensively processing. To address this, we developed sc2GWAS (https://bio.liclab.net/sc2GWAS/), which aims to document large-scale GWAS trait-cell regulatory pairs at single-cell resolution and provide comprehensive annotations and enrichment analyses for these related pairs. The current version of sc2GWAS curates a total of 15078310 candidate trait-cell pairs from>6300000 individual cells, offering a valuable resource for exploring complex regulatory relationships between traits and cells. We applied strict quality control measures on both scRNA-seq data and GWAS data, ensuring the reliability and accuracy of the datasets for the identification of trait-relevant cells and genes. In addition, sc2GWAS provides ranked lists of trait-relevant genes and extensive (epi) genetic annotations, making it a valuable resource for downstream analyses. We demonstrate the utility of the platform by investigating Alzheimer's disease, where we identified significant associations between the disease and microglial cells, with the APOE gene emerging as particularly significant. This platform facilitates detailed research into complex trait-celland trait-gene interactions, we anticipate that sc2GWAS will become a comprehensive and valuable platform for exploring GWAS trait-cell regulatory mechanisms.

Qingre Huoxue decoction attenuates myocardial ischemia‒reperfusion injury by regulating the autophagy‒endoplasmic reticulum stress axis via FAM134B-mediated ER-phagy

BackgroundAutophagy‒endoplasmic reticulum (ER) stress axis dysregulation is linked to myocardial ischemia‒reperfusion injury (MIRI), which counteracts the benefits of acute myocardial infarction (AMI) reperfusion therapy. Qingre Huoxue decoction (QRHX) improves the short- and long-term prognosis of AMI after percutaneous coronary intervention and alleviates myocardial injury in AMI rats by stimulating autophagy via the PI3K/Akt pathway. We aimed to further explore the efficacy of QRHX in treating MIRI and its regulatory relationship with FAM134B-mediated ER-phagy.Materials and methodsRats were administered different concentrations of QRHX for 2 weeks, and then MIRI was induced. Ultra-performance liquid chromatography‒tandem mass spectrometry (UPLC‒MS) was used to examine the levels of the main pharmacological metabolites of the serum of rats treated with QRHX. H9c2 cells were pretreated with QRHX-mediating serum (QRHX-MS) for 24 h before being exposed to hypoxia/reoxygenation (H/R). The mechanisms underlying the effects of QRHX-MS were further studied via rescue experiments involving FAM134B knockdown. The myocardial infarct size, cardiac function, morphology and the expression of apoptosis-, autophagy-, and ER stress-related proteins and genes were assessed. The colocalization of autophagosomes with lysosomes and the localization of proteins involved in ER-phagy or autophagic flux was examined.ResultsQRHX decreased the myocardial infarct size and oxidative stress, improved cardiac function and alleviated morphological changes in a dose-dependent manner in MIRI rats by promoting autophagic flux to inhibit ER stress and ER stress-related apoptosis, which was related to FAM134B-mediated ER-phagy, as revealed by autophagy analysis. UPLC‒MS analysis of QRHX-MS revealed 20 major active metabolites of QRHX-MS, including baicalin, cryptotanshinone, 3,4-dihydroxybenzaldehyde and caffeic acid. QRHX-MS attenuated H/R-induced cardiomyocyte injury and apoptosis by increasing autophagic flux to suppress ER stress and ER stress-related apoptotic protein and gene expression. When autophagic flux was inhibited or FAM134B was knocked down in H9c2 cells followed by QRHX-MS pretreatment, the protective effect of QRHX was partially reversed.ConclusionQRHX alleviates myocardial injury, apoptosis and infarct size expansion in MIRI by regulating the autophagy‒ER stress axis via FAM134B-mediated ER-phagy.

Long non-coding RNA HOTAIR promotes tumorigenesis by affecting proliferation, invasion, migration and apoptosis of liver cancer cells.

Increasing evidence shows that Hox transcript antisense RNA (HOTAIR) plays a vital role in liver cancer initiation and progression by affecting the proliferation, invasion, migration and apoptosis of liver cancer cells. However, the underlying mechanism of how HOTAIR exerts its functions in liver cancer cells remains unclear. Previous studies have shown that HOTAIR affects the invasion and migration of liver cancer cells by regulating the expression of E-cadherin. Snail2, a transcription factor involved in epithelial-mesenchymal transition, directly binds to the E-boxes of theE-cadherinpromoter to repress its transcription. The aim of the study was to examine the correlation between HOTAIR and Snail2 in the HOTAIR/Snail2/E-cadherin signal pathway and explore the role of HOTAIR in the proliferation, invasion, migration and apoptosis of liver cancer cells. 50 matched normal liver tissues and 373 liver cancer tissues were analysed and evaluated. HepG2 and SNU-387 cells were cultured and transfected with plasmids knocking down HOTAIR to disrupt HOTAIR expression. Cell scratch and transwell assays were performed to examine the migration and invasion of HepG2 and SNU-387 cells; in addition, the expression of MMP2 and MMP9 was detected by immunoblotting analysis, RT-qPCR analysis, immunofluorescence analysis, and bioinformatics analysis, which elucidated the regulatory relationship between HOTAIR and Snail2. We used flow cytometry and JC-1 probe analysis assays to clarify the function of HOTAIR in liver cancer cell apoptosis. The HOTAIR mRNA was upregulated in liver cancer tissues, which was related to worse overall survival. HOTAIR induced the expression of matrix metalloproteinase-9 (MMP9) and metalloproteinase-2 (MMP2), leading to degradation of extracellular matrix. HOTAIR knockdown significantly reduced the doubling time and inhibited cell migration and invasion of liver cancer cells. Furthermore, HOTAIR depletion induced mitochondrial-related apoptosis in HepG2 and SNU-387 cell lines. In this study, we proposed a novel mechanism in which HOTAIR promotes invasion and migration of liver cancer cells by regulating the nuclear localization of Snail2.

The effects of PstR, a PadR family transcriptional regulatory factor, in Plesiomonas shigelloides are revealed by transcriptomics

BackgroundPlesiomonas shigelloides is a gram-negative opportunistic pathogen associated with gastrointestinal and extraintestinal diseases in humans. There have been reports of specific functional genes in the study of P. shigelloides, but there are also many unknown genes that may play a role in P. shigelloides pathogenesis as global regulatory proteins or virulence factors.ResultsIn this study, we found a transcriptional regulator of the PadR family in P. shigelloides and named it PstR (GenBank accession number: EON87311.1), which is present in various pathogenic bacteria but whose function has rarely been reported. RNA sequencing (RNA-Seq) was used to analyze the effects of PstR on P. shigelloides, and the results indicated that PstR regulates approximately 9.83% of the transcriptome, which includes impacts on motility, virulence, and physiological metabolism. RNA-seq results showed that PstR positively regulated the expression of the flagella gene cluster, which was also confirmed by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Luminescence screening assay. Meanwhile, the ΔpstR mutant strains lacked flagella and were non-motile, as confirmed by motility assays and transmission electron microscopy (TEM). Additionally, PstR also positively regulates T3SS expression, which aids in P. shigelloides’ capacity to infect Caco-2 cells. Meanwhile, we also revealed that PstR negatively regulates fatty acid degradation and metabolism, as well as the regulatory relationship between PsrA, a regulator of fatty acid degradation and metabolism, and its downstream genes in P. shigelloides.ConclusionsOverall, we revealed the effects of PstR on motility, virulence, and physiological metabolism in P. shigelloides, which will serve as a foundation for future research into the intricate regulatory network of PstR in bacteria.

LCN2 aggravates diabetic cataracts by promoting ferroptosis in lens epithelial cells

Abstract Background Cataracts are the leading cause of reversible blindness worldwide. Diabetic cataract (DC), a prevalent complication of diabetes mellitus, is characterized by its high occurrence, rapid progression, and severe impact. The prevalence of diabetes varies greatly between the northern and southern regions, with higher rates observed among northern residents. DC-induced lens opacity is mainly attributed to oxidative stress. However, it remains unclear whether ferroptosis, a form of regulated cell death, occurs in crystalline epithelial cells during the pathogenesis, which may represent a novel mechanism contributing to DC. Methods Transmission electron microscopy, quantitative assays for iron levels and reactive oxygen species (ROS), real-time quantitative polymerase chain reaction (RT-qPCR), western blotting, immunofluorescence, and immunohistochemistry were used to detect ferroptosis. Gene editing techniques were utilized to study the regulatory relationships among lipocalin 2 (LCN2), glutathione peroxidase 4 (GPX4), and ferritin heavy chain (FTH). Local knockdown of the LCN2 gene in B-3 cells and the eyes of Sprague Dawley (SD) rats was performed to verify and further explore the role and regulatory mechanisms of LCN2 in DC-associated ferroptosis. Results An in vitro model using high glucose levels and an in vivo model with streptozotocin-induced diabetes in SD rats were successfully established. Ferroptosis was observed in both in vitro and in vivo experiments. LCN2 protein was normally expressed in human and rat lens epithelial cells, but its expression significantly increased during ferroptosis. The ferroptosis inhibitor, ferrostatin-1 (Fer-1) effectively inhibited ferroptosis and reduced LCN2 protein expression. Notably, local knockdown of LCN2 via gene editing protected lens epithelial cells from ferroptosis in vitro and slowed the progression of DC in SD rats in vivo. Conclusion Our findings underscore the significant role of ferroptosis in the pathogenesis of DC, suggesting that selectively targeting LCN2 activation and enhancing ferroptosis resistance may offer a novel therapeutic approach for treating DC.

Safety assessment and exploration of the mechanism of toxic effects of diallyl trisulfide: Based on acute and subacute toxicity and proteomics experiments

Ethnopharmacological relevanceGarlic (Allium sativum L.) is a widely consumed spice and condiment around the world, applied both as a food and as a traditional medicine, and is a natural strengthening agent for the body's circulatory and nervous systems. Diallyl trisulfide (DATS) is the major volatile organosulfur phytochemical found in garlic, with antithrombotic, anticoagulant, and antiplatelet activities as well as antioxidant, anti-infective, and other pharmacological effects. However, the safe dose and the underlying mechanisms of its toxic effects remain elusive. Aim of the studyDATS, an important pharmacologically active compound found in garlic, has garnered attention for its ability to fight cancer, antioxidant, anti-infective, and cardioprotective. The aim of this study was to evaluate the safety of DATS and to elucidate the potential mechanisms of its toxicity. Materials and methodsIn this study, ICR mice were selected for acute and subacute toxicity experiments according to OECD guidelines. The toxicity profile of DATS was analyzed by computer prediction software. Also, key differential proteins in spleen and serum were analyzed by proteomics. The binding stability of DAST to differential proteins was analyzed by molecular docking. Additionally, the regulatory relationship between DATS and differential proteins was verified by Western blot and ELISA experiments. ResultsThe results showed that the LD50 value of DATS in acute toxicity was 188.67 mg/kg. In subacute toxicity, water consumption and food intake were reduced in both male and female mice. In addition, the spleen and small intestinal organ coefficients were significantly elevated in male mice at the high dose of DATS; the blood biochemical indices ALB and TP were also significantly elevated. HE staining results showed significant damage to the spleen, liver, small intestine, and kidney of mice at high doses of DATS. Spleen and serum proteomics analyses showed that DATS significantly inhibited ZBP1 expression and upregulated TEC. ADMETlab 2.0 software predictions identified DATS as having potential genotoxicity, dermal sensitization, carcinogenicity, and respiratory and ocular toxicity. Docking results showed that the binding energies between DATS and TEC protein (PDB: 6F3F) and ZBP1 protein (PDB: 4KA4) were −3.7 kcal/mol, −2.4 kcal/mol, respectively. Western blot results showed that DATS-H significantly inhibited the expression of ZBP1 (only in male mice) and Bcl-2 proteins. ELISA results showed that DATS-H significantly increased the level of TEC protein both in male and female mice. ConclusionsLong-term administration of high-dose DATS may carry some risk of toxicity. Based on the amount of DATS in garlic, it is recommended that adults should not take more than 359 mg of DATS and 84.5 g of garlic per day. The mechanism of toxicity may be related to the fact that DATS significantly inhibits ZBP1 expression, upregulates TEC, and promotes apoptosis. This study provides valuable toxicological data for the effective evaluation of the long-term toxicity of DATS and offers an additional experimental basis for developing DATS as a healthy food or drug.

TFAP2C/FLT3 axis reduces ferroptosis in breast cancer cells by inhibiting mitochondrial autophagy

BackgroundFMS-like tyrosine kinase 3 (FLT3), a key target protein for treating acute myeloid leukemia, has recently been found to be closely related to ferroptosis in breast cancer (BC). However, the mechanism by which FLT3 regulates ferroptosis in BC remains unknown. Whether this regulatory relationship can be exploited for BC treatment needs further exploration. MethodsThis study combined analysis from The Cancer Genome Atlas database with immunohistochemistry/quantitative reverse transcription-PCR/Western blot experiments to verify the expression of FLT3 in BC. FLT3 knockdown/overexpression plasmids were used in conjunction with mitochondrial autophagy inducers to treat BC cells, analyzing the effects of FLT3 on autophagy and ferroptosis. Key transcription factors for FLT3 were determined through predictions from the KnockTF database and dual luciferase/chromatin immunoprecipitation experiments, further analyzing the impact of this regulatory axis on autophagy and ferroptosis in BC cells. ResultsFLT3 was significantly overexpressed in BC tissues and cells. Overexpression of FLT3 could inhibit autophagy and ferroptosis in BC cells, a regulation that was restored upon the addition of mitochondrial autophagy inducers. Additionally, transcription factor AP-2 gamma (TFAP2C) could mediate the transcriptional activation of FLT3, further inhibiting ferroptosis induced by mitochondrial autophagy. ConclusionThe TFAP2C/FLT3 axis reduced ferroptosis in BC cells by inhibiting mitochondrial autophagy. These research findings elucidated the mechanism by which FLT3 regulated ferroptosis in BC and provided potential targets for BC treatment.

RNA/DNA Binding Protein TDP43 Regulates DNA Mismatch Repair Genes with Implications for Genome Stability.

TAR DNA-binding protein 43 (TDP43) is increasingly recognized for its involvement in neurodegenerative diseases, particularly amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP43 proteinopathy, characterized by dysregulated nuclear export and cytoplasmic aggregation, is present in most ALS/FTD cases and is associated with a loss of nuclear function and genomic instability in neurons. Building on prior evidence linking TDP43 pathology to DNA double-strand breaks (DSBs), this study identifies a novel regulatory role for TDP43 in the DNA mismatch repair (MMR) pathway. We demonstrate that depletion or overexpression of TDP43 affects the expression of key MMR genes, including MLH1, MSH6, MSH2, MSH3, and PMS2. Specifically, TDP43 modulates the expression of MLH1 and MSH6 proteins through alternative splicing and transcript stability. These findings are validated in ALS mice models, patient-derived neural progenitor cells and autopsied brain tissues from ALS patients. Furthermore, MMR depletion showed a partial rescue of TDP43-induced DNA damage in neuronal cells. Bioinformatics analysis of TCGA cancer database reveals significant correlations between TDP43 and MMR gene expressions and mutational burden across various cancer subtypes. These results collectively establish TDP43 as a critical regulator of the MMR pathway, with broad implications for understanding the genomic instability underlying neurodegenerative and neoplastic diseases.

Genome-wide identification of alternative splicing related with transcription factors and splicing regulators in breast cancer stem cells responding to fasting-mimicking diet

Fasting-mimicking diet (FMD) can effectively inhibit the viability of breast cancer stem cells (CSCs). However, the molecular mechanisms underlying the inhibitory function of FMD on breast CSCs remain largely unknown. Elucidating the mechanisms by which FMD suppresses breast CSCs is beneficial to targeting breast CSCs. Herein, we systematically analyze alternative splicing and RNA binding protein (RBP) expression in breast CSCs during FMD. The analysis results show that a large number of regulated alternative splicing (RAS) and differentially expressed genes (DEGs) appear responding to FMD. Further studies show that there are potential regulatory relationships between transcription factors (TFs) with RAS (RAS-TFs) and their differentially expressed target genes (RAS-TF-DEGs). Moreover, differentially expressed RNA binding proteins (DERBPs) exhibit potential regulatory functions on RAS-TFs. In short, DERBPs potentially control the alternative splicing of TFs (RAS-TFs), regulating their target gene (RAS-TF-DEG) expression, which leads to the regulation of biological processes in breast CSCs during FMD. In addition, the alternative splicing and DEGs are compared between breast CSCs and differentiated cancer cells during FMD, providing new interpretations for the different responses of the two types of cells. Our studies will shed light on the understanding of the molecular mechanisms underlying breast CSC inhibition induced by FMD.