Potential Regulatory Pathways Research Articles

RBPWorld for exploring functions and disease associations of RNA-binding proteins across species.

RNA-binding proteins (RBPs) play key roles in a wide range of physiological and pathological processes. To facilitate the investigation of RBP functions and disease associations, we updated the EuRBPDB and renamed it as RBPWorld (http://research.gzsys.org.cn/rbpworld/#/home). Leveraging 998 RNA-binding domains (RBDs) and 87 RNA-binding Proteome (RBPome) datasets, we successfully identified 1 393 413 RBPs from 445 species, including 3030 human RBPs (hRBPs). RBPWorld includes primary RNA targets of diverse hRBPs, as well as potential downstream regulatory pathways and alternative splicing patterns governed by various hRBPs. These insights were derived from analyses of 1515 crosslinking immunoprecipitation-seq datasets and 616 RNA-seq datasets from cells with hRBP gene knockdown or knockout. Furthermore, we systematically identified 929 RBPs with multi-functions, including acting as metabolic enzymes and transcription factors. RBPWorld includes 838 disease-associated hRBPs and 970 hRBPs that interact with 12 disease-causing RNA viruses. This provision allows users to explore the regulatory roles of hRBPs within the context of diseases. Finally, we developed an intuitive interface for RBPWorld, facilitating users easily access all the included data. We believe that RBPWorld will be a valuable resource in advancing our understanding of the biological roles of RBPs across different species.

Identifying the HIV-Resistance-Related Factors and Regulatory Network via Multi-Omics Analyses.

For research on HIV/AIDS, it is important to elucidate the complex viral-host interaction, host dependency factors (HDFs), and restriction factors. However, the regulatory network of HIV-resistance-related factors remains not well understood. Therefore, we integrated four publicly available HIV-related transcriptome datasets, along with three datasets on HIV-infection-related DNA methylation, miRNA, and ChIP-seq, to predict the factors influencing HIV resistance and infection. Our approach involved differential analysis, functional annotation, and protein-protein interaction network analysis. Through comprehensive analyses, we identified 25 potential HIV-resistance-related genes (including shared EGF) and 24 HIV-infection-related hub genes (including shared JUN). Additionally, we pinpointed five key differentially methylated genes, five crucial differentially expressed microRNAs, and five significant pathways associated with HIV resistance. We mapped the potential regulatory pathways involving these HIV-resistance-related factors. Among the predicted factors, RHOA, RAD51, GATA1, IRF4, and CXCL8 have been validated as HDFs or restriction factors. The identified factors, such as JUN, EGF, and PLEK, are potential HDFs or restriction factors. This study uncovers the gene signatures and regulatory networks associated with HIV-1 resistance, suggesting potential targets for the development of new therapies against HIV/AIDS.

Investigating the mechanism of METTL16-dependent m6A modification regulating the SAMD11 protein signaling pathway to inhibit thyroid cancer phenotypes

Despite substantial progress in the research and treatment of thyroid cancer, many areas in the molecular mechanisms remain to be explored. This study aims to comprehensively and deeply investigate the key role and potential molecular mechanisms of RNA methyltransferase METTL16 in the development and progression of thyroid cancer. Firstly, through bioinformatics analysis of tumor databases, we examined the correlation between METTL16 expression levels and patient prognosis. Subsequently, immunofluorescence experiments on clinical patient tissue microarrays were conducted to validate these findings. We also compared the nucleic acid and protein expression levels of METTL16 in different cell lines. By integrating bioinformatics analysis of public databases, laboratory molecular biology experiments, and comprehensive data analysis, we revealed the high expression of METTL16 in clinical tissues and thyroid cancer cells, and confirmed its role in regulating the biological characteristics of cell proliferation, migration, and invasion in thyroid cancer through in vitro and in vivo experiments. Additionally, we identified SAMD11 as a target gene of METTL16 and further validated its importance and potential regulatory pathways in thyroid cancer.

The Potential Related Genes and Mechanisms Involved in Improving the Treadmill Exercise Ability of APP/PS1 Mice.

Alzheimer's disease (AD) causes a decline in skeletal muscle function, which can further exacerbate the cognitive dysfunction of patients with AD. It has been widely established that exercise improves AD brain pathology, but the role of skeletal muscle in AD is still poorly understood. In this study, we investigated the effects of treadmill exercise on the exercise ability of APP/PS1 transgenic AD mice and explored potential gene expression changes in their skeletal muscle. The APP/PS1 mice were subjected to a treadmill exercise for 12 weeks, followed by the Morris water maze and the open field test. After behavioral experiments, the changes in morphology, area, collagen fiber deposition, and ultrastructure of the skeletal muscle were determined; the balance of skeletal muscle protein synthesis and decomposition was analyzed; and changes in gene expression were investigated using RNA-Seq. We found that this exercise strategy can promote the learning and memory abilities of AD mice, reduce their anxiety-like behavior, improve their exercise ability, alleviate skeletal muscle atrophy, and optimize the microstructure. It can also enhance skeletal muscle protein synthesis and decomposition and improve several signaling pathways, such as the JAK-STAT, Wnt, and NOD-like receptors while decreasing calcium, cAMP, cGMP-PKG, and other signaling pathways. Six KEGG enrichment signaling pathways were downregulated and five signaling pathways were upregulated in the AD mice compared with wild-type mice, and these pathways were precisely reversed after the treadmill exercise. The expression of transcription factors such as Fosb and Egr1 in the skeletal muscle of AD mice decreased, followed by a decrease in the regulated target genes Socs1, Srrm4, and Il1b, a trend that was reversed following the exercise intervention. After exercise, AD mice exhibited a similar gene expression to that of wild-type mice, indicating enhanced exercise ability. The potential regulatory pathways and related genes identified in this study provide valuable insights for the clinical management and treatment of AD.

Roles of naïve CD4+ T cells and their differentiated subtypes in lung adenocarcinoma and underlying potential regulatory pathways

BackgroundNaïve CD4+ T cells and their differentiated counterparts play a significant regulatory role in the tumor immune microenvironment, yet their effects on lung adenocarcinoma (LUAD) are not fully understood.MethodsWe utilized Mendelian randomization to assess the causal association between naïve CD4+ T cells and LUAD. Employing a modified single-sample Gene Set Enrichment Analysis (ssGSEA) algorithm with The Cancer Genome Atlas (TCGA) database, we determined the infiltration levels of naïve CD4+ T cells and their differentiation subtypes and investigated their correlation with clinical characteristics. Potential regulatory pathways of T helper cells were identified through Mantel tests and Kyoto Encyclopedia of Genes and Genomes (KEGG) database enrichment analysis.ResultsMendelian randomization analysis revealed an inhibitory effect of naïve CD4+ T cells on LUAD (false discovery rate < 0.05), which was corroborated by observational experiments using TCGA database. Specifically, T helper cell type 2 demonstrated a promotive effect on LUAD in terms of overall, disease-free, and progression-free survival (p < 0.05). Moreover, regulatory T cells exhibited a protective effect on LUAD in terms of disease-specific survival (p < 0.01). Concurrently, we explored the overall impact of naïve CD4+ T cell differentiation subtypes on LUAD, revealing upregulation in pathways such as neutrophil degranulation, MAPK family signaling pathways, and platelet activation, signaling, and aggregation.ConclusionNaïve CD4+ T cells and their differentiated counterparts play essential regulatory roles in the tumor immune microenvironment, demonstrating bidirectionality in their effects.Thus, elucidating the mechanisms and developing novel cell differentiation-inducing agents will benefit anti-cancer therapy.

Vitamin D Relieves Epilepsy Symptoms and Neuroinflammation in Juvenile Mice by Activating the mTOR Signaling Pathway via RAF1: Insights from Network Pharmacology and Molecular Docking Studies.

Epilepsy is a common neurological disorder, and the exploration of potential therapeutic drugs for its treatment is still ongoing. Vitamin D has emerged as a promising treatment due to its potential neuroprotective effects and anti-epileptic properties. This study aimed to investigate the effects of vitamin D on epilepsy and neuroinflammation in juvenile mice using network pharmacology and molecular docking, with a focus on the mammalian target of rapamycin (mTOR) signaling pathway. Experimental mouse models of epilepsy were established through intraperitoneal injection of pilocarpine, and in vitro injury models of hippocampal neurons were induced by glutamate (Glu) stimulation. The anti-epileptic effects of vitamin D were evaluated both in vivo and in vitro. Network pharmacology and molecular docking analysis were used to identify potential targets and regulatory pathways of vitamin D in epilepsy. The involvement of the mTOR signaling pathway in the regulation of mouse epilepsy by vitamin D was validated using rapamycin (RAPA). The levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) were assessed by enzyme-linked immunosorbent assay (ELISA). Gene and protein expressions were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining was used to analyze the apoptosis of hippocampal neurons. In in vivo experiments, vitamin D reduced the Racine scores of epileptic mice, prolonged the latency of epilepsy, and inhibited the production of TNF-α, IL-1β, and IL-6 in the hippocampus. Furthermore, network pharmacology analysis identified RAF1 as a potential target of vitamin D in epilepsy, which was further confirmed by molecular docking analysis. Additionally, the mTOR signaling pathway was found to be involved in the regulation of mouse epilepsy by vitamin D. In in vitro experiments, Glu stimulation upregulated the expressions of RAF1 and LC3II/LC3I, inhibited mTOR phosphorylation, and induced neuronal apoptosis. Mechanistically, vitamin D activated the mTOR signaling pathway and alleviated mouse epilepsy via RAF1, while the use of the pathway inhibitor RAPA reversed this effect. Vitamin D alleviated epilepsy symptoms and neuroinflammation in juvenile mice by activating the mTOR signaling pathway via RAF1. These findings provided new insights into the molecular mechanisms underlying the anti-epileptic effects of vitamin D and further supported its use as an adjunctive therapy for existing anti-epileptic drugs.

Molecular mechanisms underlying the modulation of T-cell proliferation and cytotoxicity by immobilized CCL21 and ICAM1.

Adoptive cancer immunotherapy, using engineered T-cells, expressing chimeric antigen receptor or autologous tumor infiltrating lymphocytes became, in recent years, a major therapeutic approach for diverse types of cancer. However, despite the transformative potential of adoptive cancer immunotherapy, this field still faces major challenges, manifested by the apparent decline of the cytotoxic capacity of effector CD8+ T cells upon their expansion. To address these challenges, we have developed an ex vivo "synthetic immune niche" (SIN), composed of immobilized CCL21 and ICAM1, which synergistically induce an efficient expansion of antigen-specific CD8+ T cells while retaining, and even enhancing their cytotoxic potency. To explore the molecular mechanisms through which a CCL21+ICAM1-based SIN modulates the interplay between the proliferation and cytotoxic potency of antigen-activated and CD3/CD28-activated effector CD8+ T cells, we performed integrated analysis of specific differentiation markers via flow cytometry, together with gene expression profiling. On day 3, the transcriptomic effect induced by the SIN was largely similar for both dendritic cell (DC)/ovalbumin (OVA)-activated and anti-CD3/CD28-activated cells. Cell proliferation increased and the cells exhibited high killing capacity. On day 4 and on, the proliferation/cytotoxicity phenotypes became radically "activation-specific"; The DC/OVA-activated cells lost their cytotoxic activity, which, in turn, was rescued by the SIN treatment. On longer incubation, the cytotoxic activity further declined, and on day7, could not be rescued by the SIN. SIN stimulation following activation with anti-CD3/CD28 beads induced a major increase in the proliferative phenotype while transiently suppressing their cytotoxicity for 2-3 days and fully regaining their killing activity on day 7. Potential molecular regulatory pathways of the SIN effects were identified, based on transcriptomic and multispectral imaging profiling. These data indicate that cell proliferation and cytotoxicity are negatively correlated, and the interplay between them is differentially regulated by the mode of initial activation. The SIN stimulation greatly enhances the cell expansion, following both activation modes, while displaying high survival and cytotoxic potency at specific time points following stimulation, suggesting that it could effectively reinforce adoptive cancer immunotherapy.

GGTLC1 knockdown inhibits the progression of endometrial cancer by regulating the TGF-β/Smad signaling pathway

PurposeEndometrial cancer (EC) poses a serious risk to females worldwide; thus, a deep understanding of EC is urgently required. The role and mechanisms of gamma-glutamyltransferase light chain 1 (GGTLC1) in EC remain obscure. This study aims to elucidate the function and mechanisms underlying GGTLC1's involvement in EC. MethodsBioinformatic tools and databases were used to analyze GGTLC1 and its associated gene expression in EC tissues. Functional enrichment explorations and immune infiltration analyses were conducted, together with investigation into the methylation status of GGTLC1. Western blotting and Quantitative real-time PCR quantified expression levels. Additional experimental methodologies elucidated the role of GGTLC1 in EC progression. Transcriptome sequencing identified potential regulatory pathways for GGTLC1, and tumor growth was evaluated in vivo using HEC-1A cells in nude mice. ResultsGGTLC1 was upregulated and negatively correlated with immune cell infiltration and DNA methylation in EC. Cell migration and proliferation were reduced following GGTLC1 knockdown, together with arrest at the G0/G1 phase and an upsurge in apoptosis. Compared to the knockdown group, TGF-β/Smad signaling pathway was up-regulated in the negative control group of EC cells by transcriptome analysis. The levels of TGF-β, pSmad2, and pSmad3 followed the same decreasing trend, whereas Smad3 and Smad2 protein levels remained unchanged. ConclusionKnockdown of GGTLC1 attenuates EC development through the TGF-β/Smad pathway, positioning GGTLC1 as a promising target for EC treatment.

Transcriptomic and Metabolomic Research on the Germination Process of Panax ginseng Overwintering Buds.

Ginseng (Panax ginseng C. A. Meyer) is a perennial plant with a long dormancy period. While some researchers employ gibberellin and other substances to stimulate premature germination, this method is limited to laboratory settings and cannot be applied to the field cultivation of ginseng. The mechanism underlying the germination of ginseng overwintering buds remains largely unexplored. Understanding the internal changes during the dormancy release process in the overwintering buds would facilitate the discovery of potential genes, metabolites, or regulatory pathways associated with it. In this study, we approximately determined the onset of dormancy release through morphological observations and investigated the process of dormancy release in ginseng overwintering buds using transcriptomic and metabolomic approaches. Our analyses revealed that the germination process of ginseng overwintering buds is regulated by multiple plant hormones, each acting at different times. Among these, abscisic acid (ABA) and gibberellic acid (GA) serve as classical signaling molecules regulating the dormancy process, while other hormones may promote the subsequent growth of overwintering buds. Additionally, metabolic pathways associated with arginine may be involved in the dormancy release process. Polyamines synthesized downstream may promote the growth of overwintering buds after dormancy release and participate in subsequent reproductive growth. This study provides insights into the germination process of ginseng overwintering buds at the molecular level and serves as a reference for further exploration of the detailed mechanism underlying ginseng overwintering germination in the future.

Physiological and molecular responses in phosphorus-hyperaccumulating Polygonum species to high phosphorus exposure.

Phosphorus (P)-hyperaccumulators for phytoextraction from P-polluted areas generally show rapid growth and accumulate large amounts of P without any toxicity symptom, which depends on a range of physiological processes and gene expression patterns that have never been explored. We investigated growth, leaf element concentrations, P fractions, photosynthetic traits, and leaf metabolome and transcriptome response in amphibious P-hyperaccumulators, Polygonum hydropiper and P. lapathifolium, to high-P exposure (5 mmol L-1), with 0.05 mmol L-1 as the control. Under high-P exposure, both species demonstrated good growth, allocating more P to metabolite P and inorganic P (Pi) accompanied by high potassium and calcium. The expression of a cluster of unigenes associated with photosynthesis was maintained or increased in P. lapathifolium, explaining the increase in net photosynthetic rate and the rapid growth under high-P exposure. Metabolites of trehalose metabolism, including trehalose 6-phosphate and trehalose, were sharply increased in both species by the high-P exposure, in line with the enhanced expression of associated unigenes, indicating that trehalose metabolic pathway was closely related to high-P tolerance. These findings elucidated the physiological and molecular responses involved in the photosynthesis and trehalose metabolism in P-hyperaccumulators to high-P exposure, and provides potential regulatory pathways to improve the P-phytoextraction capability.

Abstract 5413: In vitro modeling of the migration ability of lung adenocarcinoma cell lines under hypoxic conditions

Abstract Hypoxia is a common condition in the tumor microenvironment that contributes to tumor cell migration, invasion, and metastasis, partly through inducing epithelial-mesenchymal transition (EMT). One of the key proteins in this process is hypoxia-inducible factor 1 (HIF-1), which regulates the expression of various genes as transcription factor and is vital for the cellular response in tissue hypoxia. Several studies have investigated the causation between hypoxic conditions and increased in vitro tumor cell motility and the role of potential regulatory signaling pathways. Understanding the precise molecular mechanisms underlying hypoxia-induced responses is crucial for better targeting tumor progression. However, establishing sound and predictive models is urgently needed to achieve this.In this study, we investigated the migration ability of four non-small cell lung cancer cell lines carrying different mutations of the growth factor pathway (H1975: EGFR T790M, PF901: BRAF V600E, PF139: KRAS G12C mutant, and H838: triple WT) under normoxic, hypoxic, and hypoxia-mimicking CoCl2-treated conditions. The motility of the cells was investigated by 48 h video microscopy (single cell random migration) and 72 h scratch assay. We analyzed the expression of crucial genes and proteins, including HIF-1α, as well as molecules associated with epithelial-mesenchymal transition (vimentin, n-cadherin, and e-cadherin), proliferation (PCNA), and apoptosis (PARP), using Western blot and qPCR techniques. Our work showed no change in cellular motility between any of the cell lines following treatment with CoCl2 compared to normoxic conditions. However, PF139 and PF901 cell lines showed a significant decrease in single-cell motility upon hypoxia. Interestingly, immunoblot analysis revealed stabilization of HIF-1α in response to both CoCl2 and hypoxia in the meantime. Additionally, we observed cell line-dependent changes in EMT marker expression upon CoCl2 treatment and hypoxia. Our work complements previous results published by our group and others on the tumor-promoting role of hypoxia, demonstrating that short-term exposure to hypoxia may act as an acute stressor for tumor cell lines. Our work suggests that the tumor progression-promoting effects are tissue-specific and should be investigated using long-term hypoxia models. Acknowledgments: This work was supported by the Hungarian Thematic Excellence Program (TKP2021-EGA-44) and the National Laboratories Program - National Laboratory for Tumor Biology (NLP-17). Sára Eszter Surguta is a grantee of the ITM-NKFIA funded Cooperative Doctoral Program (1018567). Citation Format: Sára Eszter Surguta, Marcell Baranyi, Laura Svajda, Ivan Ranđelović, Mihály Cserepes, József Tóvári. In vitro modeling of the migration ability of lung adenocarcinoma cell lines under hypoxic conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5413.

MsSPL12 is a positive regulator in alfalfa (Medicago sativa L.) salt tolerance.

Over expression of MsSPL12 improved alfalfa salt tolerance by reducing Na+ accumulation and increasing antioxidant enzyme activity and regulating down-stream gene expression. Improvement of salt tolerance is one of the major goals in alfalfa breeding. Here, we demonstrated that MsSPL12, an alfalfa transcription factor gene highly expressed in the stem cells, plays a positive role in alfalfa salt tolerance. MsSPL12 is localized in the nucleus and shows transcriptional activity in the presence of its C-terminus. To investigate MsSPL12 function in plant response to salt stress, we generated transgenic plants overexpressing either MsSPL12 or a chimeric MsSPL12-SRDX gene that represses the function of MsSPL12 by using the Chimeric REpressor gene-Silencing Technology (CRES-T), and observed that overexpression of MsSPL12 increased the salt tolerance of alfalfa transgenic plants associated with an increase in K+/Na+ ratio and relative water content (RWC) under salt stress treatment, but a reduction in electrolyte leakage (EL), reactive oxygen species (ROS), malondialdehyde (MDA), and proline (Pro) compared to wild type (WT) plants. However, transgenic plants overexpressing MsSPL12-SRDX showed an inhibited plant growth and a reduced salt tolerance. RNA-sequencing and quantitative real-time PCR analyses revealed that MsSPL12 affected the expression of plant abiotic resistance-related genes in multiple physiological pathways. The potential MsSPL12-mediated regulatory pathways based on the differentially expressed genes between the MsSPL12 overexpression transgenics and WT controls were predicted. In summary, our study proves that MsSPL12 is a positive regulator in alfalfa salt tolerance and can be used as a new candidate for manipulation to develop forage crops with enhanced salt tolerance.

Identification of a novel inflammatory-related gene signature to evaluate the prognosis of gastric cancer patients.

Gastric cancer (GC) is a highly aggressive malignancy with a heterogeneous nature, which makes prognosis prediction and treatment determination difficult. Inflammation is now recognized as one of the hallmarks of cancer and plays an important role in the aetiology and continued growth of tumours. Inflammation also affects the prognosis of GC patients. Recent reports suggest that a number of inflammatory-related biomarkers are useful for predicting tumour prognosis. However, the importance of inflammatory-related biomarkers in predicting the prognosis of GC patients is still unclear. To investigate inflammatory-related biomarkers in predicting the prognosis of GC patients. In this study, the mRNA expression profiles and corresponding clinical information of GC patients were obtained from the Gene Expression Omnibus (GEO) database (GSE66229). An inflammatory-related gene prognostic signature model was constructed using the least absolute shrinkage and selection operator Cox regression model based on the GEO database. GC patients from the GSE26253 cohort were used for validation. Univariate and multivariate Cox analyses were used to determine the independent prognostic factors, and a prognostic nomogram was established. The calibration curve and the area under the curve based on receiver operating characteristic analysis were utilized to evaluate the predictive value of the nomogram. The decision curve analysis results were plotted to quantify and assess the clinical value of the nomogram. Gene set enrichment analysis was performed to explore the potential regulatory pathways involved. The relationship between tumour immune infiltration status and risk score was analysed via Tumour Immune Estimation Resource and CIBERSORT. Finally, we analysed the association between risk score and patient sensitivity to commonly used chemotherapy and targeted therapy agents. A prognostic model consisting of three inflammatory-related genes (MRPS17, GUF1, and PDK4) was constructed. Independent prognostic analysis revealed that the risk score was a separate prognostic factor in GC patients. According to the risk score, GC patients were stratified into high- and low-risk groups, and patients in the high-risk group had significantly worse prognoses according to age, sex, TNM stage and Lauren type. Consensus clustering identified three subtypes of inflammation that could predict GC prognosis more accurately than traditional grading and staging. Finally, the study revealed that patients in the low-risk group were more sensitive to certain drugs than were those in the high-risk group, indicating a link between inflammation-related genes and drug sensitivity. In conclusion, we established a novel three-gene prognostic signature that may be useful for predicting the prognosis and personalizing treatment decisions of GC patients.

Multi-omics analysis reveals the unique landscape of DLD in the breast cancer tumor microenvironment and its implications for immune-related prognosis

BackgroundCuproptosis, i.e., copper-induced programmed cell death, has potential implications in cancer therapy. However, the impact of the cuproptosis-related gene (CRG) dihydrolipoyl dehydrogenase (DLD) on breast cancer (BC) prognosis remains underexplored. MethodsWe employed real-time quantitative PCR and multiplexed immunostaining techniques to quantify DLD expression in both BC and the adjacent non-cancerous tissues. Immunofluorescence analysis was employed to assess the influence of DLD on immune cells and immunological checkpoints in the BC microenvironment. DLD knockdown experiments were conducted in BC cell lines MDA-MB-468 and SK-BR-3, with knockdown efficiency validated via western blot. Subsequently, we performed the cell counting kit-8 (CCK-8) assay, clone formation assay, Transwell migration assay, and invasion assay. To construct a prognostic model, we employed a Lasso-Cox regression analysis of immune-related genes associated with DLD. Additionally, we established a competing endogenous RNA network based on CRGs to evaluate potential regulatory pathways. ResultsCompared to the adjacent tissues, BC tissues exhibited markedly elevated DLD expression levels. In vitro experiments demonstrated that DLD knockdown effectively inhibited BC cell migration, invasion, and proliferation. DLD exhibited positive correlations with CD68+ macrophages and PD-L1 in the tumor, as well as with macrophages and CD4+ T cells in the stroma. Tumor regions with high DLD expression were enriched in PD-L1 and macrophages, while stromal regions with high DLD expression contained CD4+ T cells and macrophages. The AUC values for 1-, 3-, and 5-year overall survival in TCGA-BRCA training set were 0.67, 0.66, and 0.66, respectively. A nomogram with a C-index of 0.715 indicated that risk score, tumor stage, and age could serve as independent prognostic factors for BC. ConclusionOur findings underscore the significant predictive significance of DLD in BC and its influence on the tumor microenvironment. DLD represents a promising diagnostic and prognostic marker for BC, offering novel avenues for the identification of therapeutic targets and the enhancement of immunotherapy in BC.

Efficacy and potential mechanism of atherosclerosis prevention by the active components of leech based on network pharmacology combined with animal experiments

IntroductionLeeches are flesh-eating and bloodsucking parasitic worms. They are being used as a traditional Chinese medicine for centuries in activating blood and dissolving statis, dreging the meridims and tick. Hirudin, an active peptide product present in leech, has blood anticoagulant property and can assist in the treatment of thrombosis and diseases related to blood circulation. The efficacy and potential mechanism of action of leeches in such diseases should be further explored. Materials and methodsFirst, network pharmacology was used to screen the predicted potential targets of the active constituents of leech and AS. The common targets of the active constituents of leech and AS were obtained using Venn diagram. Further, the drug-active-constituent–target network diagram, protein–protein interaction, and GO and KEGG pathway enrichment analyses were used to construct the active-constituent–AS target–pathway network diagram. Subsequently, the protein–drug molecule docking model was drawn. Finally, the results of network pharmacology were validated using a mouse model of AS. ResultsIn total, 34 active constituents of leech and 1172 AS-related gene targets were selected, took the drug action targets and potential disease targets to get the common targets, and took the top 10 of degree value as the main active constituents for the treatment of atherosclerosis. There were 89 common targets and 12 core targets. The main targets included MAPK, EGFR, PIK3CB, etc. Potential regulatory pathways included cancer pathways, EGFR tyrosine kinase inhibitor resistance, Rap1 signaling pathway, PPAR signaling pathway, PI3K-Akt signaling pathway, C-type lectin receptor signaling pathway, and AGE-RAGE signaling pathway in diabetic complications. Animal experiments using mouse model of AS confirmed that AS plaques were smaller after treatment with leeches. SRC level was measured using western blotting. Expression of SRC in myocardial tissue was remarkably lower in the mice treated with leech than in the mice from model group fed on high-fat chow. ConclusionsTo the best of our knowledge, this is the first study to explore the mechanism of action of the active components of leech in AS prevention. The active components of leeches play a coordinated role in preventing AS through multicomponent, multitarget, and multichannel mechanism of action related to inflammatory response, oxidative stress, and lipid metabolism. This study provided a reference for subsequent cellular and animal experiments.

Integrated transcriptomic and metabolomic analyses reveal potential regulatory pathways regulating bone metabolism pre- and postsexual maturity in hens

At the onset of sexual maturity, the increasing circulating estrogen stimulates the formation of medullary bone, which provides available calcium for eggshell formation. The bone loss of laying hens is caused by the continuous dynamic changes of structure bone leading to bone fragility and susceptibility. The degree of medullary bone mineralization in sexual maturity is positively correlated with bone quality in the late laying stage. This study aimed to explore the molecular regulation mechanism of bone metabolism pre- and postsexual maturity in hens based on the joint analysis of transcriptome and metabolome. A total of 50 Hy-line Sonia pullets with comparable body weight at 13 wk were selected. Eight pullets were killed at 15 wk (juvenile hens, JH) and 19 wk (laying hens, LH), and LHs were killed within 3 h after oviposition. Differentially expressed genes and metabolites in tibia were analyzed based on transcriptome and metabolome, and then combined to construct the relevant metabolisms and hub genes. In the LH hens, plasma levels of estrogen and tartrate-resistant acid phosphatase were significantly elevated by 1.7 and 1.3 times. In addition, the midpoint diameter, bone mineral density and bone mineral content of the tibia and femur were higher at 19 wk of age. A total of 580 differentially expressed genes were found between the JH and LH group in the tibia, including 280 up-regulated, and 300 down-regulated genes in the LH group. Gene set enrichment analysis (GSEA) showed that the intracellular biosynthesis and secretion of matrix vesicles were significantly enrichment in the LH hens. A total of 21 differential metabolites were identified between JH and LH group. Estradiol valerate positively correlated with L-theanine, tryptophan betaine, dopamine, and perindopril. Joint analysis showed that the top 20 hub genes were enriched in cholesterol biosynthesis and phospholipid metabolism, which played a key regulatory role in bone metabolism during pre- and postsexual maturity. These results provide a theoretical foundation for maintaining efficient egg production and reducing bone health problems in laying hens.

Evaluation of the roles of brassinosteroid, gibberellin and auxin for tomato internode elongation in response to low red:far-red light.

In this study, we explore the interplay between the plant hormones gibberellins (GA), brassinosteroids (BR), and Indole-3-Acetic Acid (IAA) in their collective impact on plant shade avoidance elongation under varying light conditions. We focus particularly on low Red:Far-red (R:FR) light conditions achieved by supplementing the background light with FR. We characterized the tomato internode response to low R:FR and, with RNA-seq analysis, we were able to identify some of the potential regulatory hormonal pathways. Through a series of exogenous pharmacological modulations of GA, IAA, and BR, we demonstrate that GA and BR are sufficient but also necessary for inducing stem elongation under low R:FR light conditions. Intriguingly, while IAA alone shows limited effects, its combination with GA yields significant elongation, suggesting a nuanced hormonal balance. Furthermore, we unveil the complex interplay of these hormones under light with low R:FR, where the suppression of one hormone's effect can be compensated by the others. This study provides insights into the hormonal mechanisms governing plant adaptation to light, highlighting the intricate and adaptable nature of plant growth responses. Our findings have far-reaching implications for agricultural practices, offering potential strategies for optimizing plant growth and productivity in various lighting environments.

Comparison of the transcriptome and metabolome of wheat (Triticum aestivum L.) proteins content during grain formation provides insight.

Wheat is a food crop with a large global cultivation area, and the content and quality of wheat glutenin accumulation are important indicators of the quality of wheat flour. To elucidate the gene expression regulation and metabolic characteristics related to the gluten content during wheat grain formation, transcriptomic and metabolomic analyses were performed for the high gluten content of the Xinchun 26 cultivar and the low proteins content of the Xinchun 34 cultivar at three periods (7 d, 14 d and 21 d) after flowering. Transcriptomic analysis revealed that 5573 unique differentially expressed genes (DEGs) were divided into two categories according to their expression patterns during the three periods. The metabolites detected were mainly divided into 12 classes. Lipid and lipid-like molecule levels and phenylpropanoid and polyketide levels were the highest, and the difference analysis revealed a total of 10 differentially regulated metabolites (DRMs) over the three periods. Joint analysis revealed that the DEGs and DRMs were significantly enriched in starch and sucrose metabolism; the citrate cycle; carbon fixation in photosynthetic organisms; and alanine, aspartate and glutamate metabolism pathways. The genes and contents of the sucrose and gluten synthesis pathways were analysed, and the correlation between gluten content and its related genes was calculated. Based on weighted correlation network analysis (WGCNA), by constructing a coexpression network, a total of 5 specific modules and 8 candidate genes that were strongly correlated with the three developmental stages of wheat grain were identified. This study provides new insights into the role of glutenin content in wheat grain formation and reveals potential regulatory pathways and candidate genes involved in this developmental process.

Transcriptomic profiling of long non-coding RNAs and messenger RNAs in the liver of mice during Toxoplasma gondii infection

BackgroundToxoplasma gondii is an intracellular protozoan parasite that can infect a wide range of warm-blooded animals, including humans. It poses significant health risks, particularly in immunocompromised individuals and during pregnancy, leading to severe disease manifestations. The liver, being a crucial organ involved in immune response and metabolic regulation, plays a critical role in the host's defense against T. gondii infection.MethodsIn this study, we utilized RNA sequencing to investigate the expression profiles of long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in the liver of mice infected with T. gondii. By employing this method, we obtained a comprehensive overview of the alterations in gene expression occurring in the liver during infection.ResultsBy comparing the infected groups to the control groups, we identified numerous differentially expressed lncRNAs DElncRNAs and DEmRNAs at two stages of infection. Specifically, at the acute infection stage, we found 628 DElncRNAs, and 6346 DEmRNAs. At the chronic infection stage, we identified 385 DElncRNAs and 2513 DEmRNAs. Furthermore, we identified 1959 commonly expressed DEmRNAs, including IL27, Nos2, and Cxcr2, across two infection stages. Enrichment and co-location analyses revealed pathways linked to immune and inflammatory responses during T. gondii infection. Notably, through co-location analysis, our analysis revealed several DElncRNAs, including Gm29156, Gm29157, and Gm28644, which are potentially implicated in the progression of liver inflammation induced by T. gondii. Additionally, functional enrichment analysis disclosed stage-specific characteristics of liver inflammation and immune response, alongside changes in metabolic regulation and immunosuppression pathways.ConclusionsOur findings provide valuable insights into the expression patterns of lncRNAs and mRNAs in the liver at different stages of T. gondii infection. We identified potential regulatory factors and pathways implicated in liver inflammation, thereby enhancing our understanding of the molecular mechanisms underlying liver inflammation and immune responses during T. gondii infection. These findings could contribute to the development of targeted therapeutic strategies for liver inflammation in the context of T. gondii infection.Graphical

Identification of Differentially Expressed mRNAs and miRNAs and Related Regulatory Networks in Cumulus Oophorus Complexes Associated with Fertilization.

Cumulus oophorus complexes (COCs) are the first extracellular barriers that sperm must pass through to fuse with oocytes, which have an important role in oocyte maturation and fertilization. However, little is known about the molecular mechanisms of COCs involved in fertilization. In this study, COCs were collected and then randomly divided into a test group that interacted with sperm and a control group that did not interact with sperm. Then, the total RNA was extracted; RNA transcriptome and small RNA libraries were prepared, sequenced, and analyzed. The results showed that 1283 differentially expressed genes (DEGs), including 560 upregulated and 723 downregulated genes. In addition, 57 differentially expressed miRNAs (DEMIs) with 35 upregulated and 22 downregulated were also detected. After the RNA-seq results were verified by RT-qPCR, 86 effective DEGs and 40 DEMIs were finally screened and a DEMI-DEG regulatory network was constructed. From this, the top ten hub target genes were HNF4A, SPN, WSCD1, TMEM239, SLC2A4, E2F2, SIAH3, ADORA3, PIK3R2, and GDNF, and they were all downregulated. The top ten hub DEMIs were miR-6876-5p, miR-877-3p, miR-6818-5p, miR-4690-3p, miR-6789-3p, miR-6837-5p, miR-6861-5p, miR-4421, miR-6501-5p, and miR-6875-3p, all of which were upregulated. The KEGG signaling pathway enrichment analysis showed that the effective DEGs were significantly enriched in the calcium, AMPK, and phospholipase D signaling pathways. Our study identified several DEGs and DEMIs and potential miRNA-mRNA regulatory pathways in COCs and these may contribute to fertilization. This study may provide novel insights into potential biomarkers for fertilization failure.