Quantitative Real-time PCR Experiments Research Articles

Comprehensive Expression Analysis of the WRKY Gene Family in Phoebe bournei under Drought and Waterlogging Stresses.

In response to biotic and abiotic stresses, the WRKY gene family plays a crucial role in plant growth and development. This study focused on Phoebe bournei and involved genome-wide identification of WRKY gene family members, clarification of their molecular evolutionary characteristics, and comprehensive mapping of their expression profiles under diverse abiotic stress conditions. A total of 60 WRKY gene family members were identified, and their phylogenetic classification revealed three distinct groups. A conserved motif analysis underscored the significant conservation of motif 1 and motif 2 among the majority of PbWRKY proteins, with proteins within the same class sharing analogous gene structures. Furthermore, an examination of cis-acting elements and protein interaction networks revealed several genes implicated in abiotic stress responses in P. bournei. Transcriptomic data were utilized to analyze the expression patterns of WRKY family members under drought and waterlogged conditions, with subsequent validation by quantitative real-time PCR (RT-qPCR) experiments. Notably, PbWRKY55 exhibited significant expression modulation under drought stress; PbWRKY36 responded prominently to waterlogging stress; and PbWRKY18, PbWRKY38, and PbWRKY57 demonstrated altered expression under both drought and waterlogging stresses. This study revealed the PbWRKY candidate genes that potentially play a pivotal role in enhancing abiotic stress resilience in P. bournei. The findings have provided valuable insights and knowledge that can guide further research aimed at understanding and addressing the impacts of abiotic stress within this species.

RPLP0/TBP are the most stable reference genes for human dental pulp stem cells under osteogenic differentiation.

Validation of the reference gene (RG) stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction (RT-qPCR) data normalisation. Commonly, in an unreliable way, several studies use genes involved in essential cellular functions [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA, and β-actin] without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes. Furthermore, such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recommend two or more genes. It impacts the credibility of these studies and causes distortions in the gene expression findings. For tissue engineering, the accuracy of gene expression drives the best experimental or therapeutical approaches. To verify the most stable RG during osteogenic differentiation of human dental pulp stem cells (DPSCs) by RT-qPCR. We cultivated DPSCs under two conditions: Undifferentiated and osteogenic differentiation, both for 35 d. We evaluated the gene expression of 10 candidates for RGs [ribosomal protein, large, P0 (RPLP0), TATA-binding protein (TBP), GAPDH, actin beta (ACTB), tubulin (TUB), aminolevulinic acid synthase 1 (ALAS1), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (YWHAZ), eukaryotic translational elongation factor 1 alpha (EF1a), succinate dehydrogenase complex, subunit A, flavoprotein (SDHA), and beta-2-microglobulin (B2M)] every 7 d (1, 7, 14, 21, 28, and 35 d) by RT-qPCR. The data were analysed by the four main algorithms, ΔCt method, geNorm, NormFinder, and BestKeeper and ranked by the RefFinder method. We subdivided the samples into eight subgroups. All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm. The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs. Either the ΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes. However, geNorm analysis showed RPLP0/EF1α in the first place. These algorithms' two least stable RGs were B2M/GAPDH. For BestKeeper, ALAS1 was ranked as the most stable RG, and SDHA as the least stable RG. The pair RPLP0/TBP was detected in most subgroups as the most stable RGs, following the RefFinfer ranking. For the first time, we show that RPLP0/TBP are the most stable RGs, whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.

RPLP0/TBP are the most stable reference genes for human dental pulp stem cells under osteogenic differentiation

BACKGROUND Validation of the reference gene (RG) stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction (RT-qPCR) data normalisation. Commonly, in an unreliable way, several studies use genes involved in essential cellular functions [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA, and β-actin] without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes. Furthermore, such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recommend two or more genes. It impacts the credibility of these studies and causes distortions in the gene expression findings. For tissue engineering, the accuracy of gene expression drives the best experimental or therapeutical approaches. AIM To verify the most stable RG during osteogenic differentiation of human dental pulp stem cells (DPSCs) by RT-qPCR. METHODS We cultivated DPSCs under two conditions: Undifferentiated and osteogenic differentiation, both for 35 d. We evaluated the gene expression of 10 candidates for RGs [ribosomal protein, large, P0 (RPLP0 ), TATA-binding protein (TBP ), GAPDH , actin beta (ACTB ), tubulin (TUB ), aminolevulinic acid synthase 1 (ALAS1 ), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (YWHAZ ), eukaryotic translational elongation factor 1 alpha (EF1a ), succinate dehydrogenase complex, subunit A, flavoprotein (SDHA ), and beta-2-microglobulin (B2M )] every 7 d (1, 7, 14, 21, 28, and 35 d) by RT-qPCR. The data were analysed by the four main algorithms, ΔCt method, geNorm, NormFinder, and BestKeeper and ranked by the RefFinder method. We subdivided the samples into eight subgroups. RESULTS All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm. The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs. Either the ΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes. However, geNorm analysis showed RPLP0/EF1α in the first place. These algorithms’ two least stable RGs were B2M/GAPDH. For BestKeeper, ALAS1 was ranked as the most stable RG, and SDHA as the least stable RG. The pair RPLP0/TBP was detected in most subgroups as the most stable RGs, following the RefFinfer ranking. CONCLUSION For the first time, we show that RPLP0/TBP are the most stable RGs, whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.

Comprehensive evaluation of genes related to basement membrane in hepatocellular carcinoma.

In all mammals, the basement membrane serves as a pivotal extracellular matrix. Hepatocellular carcinoma (HCC) is a challenge among numerous cancer types shaped by basement membrane-related genes (BMGs). Our research established an innovative prognostic model that is highly accurate in its prediction of HCC prognoses and immunotherapy efficacy to summarize the crucial role of BMGs in HCC. We obtained HCC transcriptome analysis data and corresponding clinical data from The Cancer Genome Atlas (TCGA). To augment our dataset, we incorporated 222 differentially expressed BMGs identified from relevant literature. A weighted gene coexpression network analysis (WGCNA) of 10158 genes demonstrated four modules that were connected to HCC. Additionally, 66 genes that are found at the intersection of BMGs and HCC-related genes were designated as hub HCC-related BMGs. MMP1, ITGA2, P3H1, and CTSA comprise the novel model that was engineered using univariate and multivariate Cox regression analysis. Furthermore, the International Cancer Genome Consortium (ICGC) and Gene Expression Omnibus (GEO) datasets encouraged the BMs model's validity. The overall survival (OS) of individuals with HCC may be precisely predicted in the TCGA and ICGC databases utilizing the BMs model. A nomogram based on the model was created in the TCGA database at similar time, and displayed a favorable discriminating ability for HCC. Particularly, when compared to the patients at an elevated risk, the patients with a low-risk profile presented different tumor microenvironment (TME) and hallmark pathways. Moreover, we discovered that a lower risk score of HCC patients would display a greater response to immunotherapy. Finally, quantitative real-time PCR (qRT-PCR) experiments were used to verify the expression patterns of BMs model. In summary, BMs model demonstrated efficacy in prognosticating the survival probability of HCC patients and their immunotherapeutic responsiveness.

Transcriptome Study of rd1Mouse Brain and Association with Parkinson's Disease.

Degeneration of the retina is intrinsically associated with the pathogenesis and progression of neurodegenerative diseases. However, the cellular and molecular mechanisms underlying the association between neurodegeneration and retinal degeneration are still under exploration due to the complexity of the connectivity network of the nervous system. In this study, RNA-seq data from the brains of model retinitis pigmentosa (RP) mice and previously studied Parkinson's disease (PD) mice were analyzed to explore the commonalities between retinal degenerative and neurodegenerative diseases. Differentially expressed genes in RP were compared with neurodegenerative disease-related genes and intersecting genes were identified, including Cnr1 and Septin14. These genes were verified by quantitative real-time reverse transcription PCR and Western blotting experiments. The key proteins CNR1 and SEPTIN14 were found to be potential cotherapeutic targets for retinal degeneration and neurodegenerative disease. In conclusion, understanding the commonalities between retinal degenerative diseases and neurodegenerative processes in the brain will not only facilitate the interpretation of the underlying pathomechanisms but also contribute to early diagnosis and the development of new therapeutic strategies.

Cannabis Sativa Oil Promotes Social Interaction and Ultrasonic Communication by Acting on Oxytocin Pathway.

Objective: Cannabis sativa is the most used recreational drug worldwide. In recent years, there has been a growing interest in the potential therapeutic benefits of medicinal cannabis to treat a variety of psychiatric and neurological conditions. In particular, cannabidiol (CBD), a nonpsychoactive cannabis constituent, has been investigated for its potential prosocial effects on behavior, although the molecular mechanisms underlying this effect are still largely unknown. The aim of this study was to investigate the effect of a C. sativa oil CBD rich (CS oil) on social interaction and ultrasonic communication in mice. Study Design: Twenty-seven adult male mice (B6; 129P F2) were treated daily with vehicle or CS oil for 2 weeks. At Day 14, mice were tested for behavior (social interaction test and ultrasonic communication). Forty minutes before the behavioral tests, mice were exposed to intranasal treatment with vehicle or the oxytocin receptor antagonist, L-371,257. After behavioral tests, VH- and CS oil-treated mice were sacrificed, RNA was extracted from the hypothalamus and used for quantitative Real Time-PCR experiments. Results: We found that a 2-week treatment with the CS oil on mice exerted a prosocial effect associated with an increase in ultrasonic vocalizations. These effects were inhibited by pretreating mice with an oxytocin receptor antagonist. In addition, at the molecular level, we found that CS oil treatment caused a significant increase in oxytocin and a decrease in oxytocin receptor expression levels in the brain hypothalamus. Conclusion: Our results suggest that CS oil promotes social behavior by acting on oxytocin pathway.

Age-dependent decline of B3AR agonist-mediated activation of FAP UCP-1 expression in murine models of chronic rotator cuff repair.

Amibegron, a β3-adrenergic receptor (B3AR) agonist, has recently been shown to provide therapeutic effects for chronic rotator cuff (RC) tears by inducing the expression of uncoupling protein 1 (UCP-1), a marker of brown fat, in fibroadipogenic progenitors (FAPs). However, it remains to be seen if these beneficial effects hold true with age and in older, more clinically relevant populations. This study seeks to understand the impacts of aging on the efficacy of amibegron to treat chronic RC tears. Young (4-month-old) and aged (33-month-old) C57BL/6 mice underwent a RC injury procedure with delayed repair (DR). Mice were equally randomized to receive amibegron or dimethyl sulfoxide (DMSO) treatments after repair. Functional ability was measured at baseline and 6-weeks after DR. Wet muscle weight and histology of injured and contralateral supraspinatus were also analyzed 6-weeks post-DR. For in vitro histology and real-time quantitative PCR experiments, FAPs were isolated from young and aged mice via fluorescence-activated cell sorting. Young and aged FAPs were treated with amibegron or DMSO either immediately after seeding (early exposure) or 8-days after seeding (late exposure). In vitro results showed that amibegron-mediated FAP UCP-1 expression decreases with age. In vivo data demonstrated that aged mice have a decreased responsiveness to amibegron and decreased propensity for intramuscular FAP UCP-1 expression. Further, delayed amibegron treatment with RC repair did not lead to improvements in muscle atrophy and functional outcomes. Our findings demonstrate that age and the timing of interventions play a critical role in FAP-targeted therapeutics for chronic injuries.

The Downregulation of MMP23B Facilitates the Suppression of Vitality and Induction of Apoptosis in Endometrial Cancer Cells.

Endometrial cancer is a malignant tumor that commonly occurs in the female reproductive system and its incidence is still increasing. The mechanism of the development of endometrial cancer has not yet been fully clarified, so we need to continuously study the relevant mechanisms of endometrial cancer and continue to explore its biomarkers in order to discover more precise and effective treatment methods for endometrial cancer. RT-qPCR (Real-Time quantitative Polymerase Chain Reaction) experiments were used to detect the expression level of MMP23B (Matrix Metalloproteinase 23B) in endometrial cancer cells; the clinical data of the TCGA (The Cancer Genome Atlas) database were downloaded, and gene expression profiles were analyzed to investigate the correlation between MMP23B (Matrix Metalloproteinase 23B) and the survival prognosis of endometrial cancer, and functional enrichment analysis was performed on MMP23B (Matrix Metalloproteinase 23B) related genes. After silencing MMP23B (Matrix Metalloproteinase 23B), CCK8 (Cell Counting Kit-8), RT-qPCR (Real-Time quantitative Polymerase Chain Reaction), scratch assay, and transwell assay were used to detect cell viability, levels of apoptotic factors, migration rate, and invasion number of endometrial cancer, respectively. MMP23B (Matrix Metalloproteinase 23B) was highly expressed in endometrial cancer, which is closely related to a poor survival prognosis for endometrial cancer, and may act on endometrial cancer through apoptosis-related functions. The downregulation of MMP23B (Matrix Metalloproteinase 23B) reduced the cell viability of endometrial cancer cells, upregulated the expression levels of CASP3 (Caspase-3), CASP8 (Caspase-8) and CASP9 (Caspase-9) in cells, and inhibited cell migration and invasion.

Genome-wide identification and expression-pattern analysis of sulfate transporter (SULTR) gene family in cotton under multiple abiotic stresses and fiber development.

Sulfate transporter (SULTR) proteins are in charge of the transport and absorption on sulfate substances, and have been reported to play vital roles in the biological processes of plant growth and stress response. However, there were few reports of genome-wide identification and expression-pattern analysis of SULTRs in Hibiscus mutabilis. Gossypium genus is a ideal model for studying the allopolyploidy, therefore two diploid species (G. raimondii and G. arboreum) and two tetraploid species (G. hirsutum and G. barbadense) were chosen in this study to perform bioinformatic analyses, identifying 18, 18, 35, and 35 SULTR members, respectively. All the 106 cotton SULTR genes were utilized to construct the phylogenetic tree together with 11 Arabidopsis thaliana, 13 Oryza sativa, and 8 Zea mays ones, which was divided into Group1-Group4. The clustering analyses of gene structures and 10 conserved motifs among the cotton SULTR genes showed the consistent evolutionary relationship with the phylogenetic tree, and the results of gene-duplication identification among the four representative Gossypium species indicated that genome-wide or segment duplication might make main contributions to the expansion of SULTR gene family in cotton. Having conducted the cis-regulatory element analysis in promoter region, we noticed that the existing salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA) elements could have influences with expression levels of cotton SULTR genes. The expression patterns of GhSULTR genes were also investigated on the 7 different tissues or organs and the developing ovules and fibers, most of which were highly expressed in root, stem, sepal, receptacel, ovule at 10 DPA, and fiber at 20 and 25 DPA. In addition, more active regulatory were observed in GhSULTR genes responding to multiple abiotic stresses, and 12 highly expressed genes showed the similar expression patterns in the quantitative Real-time PCR experiments under cold, heat, salt, and drought treatments. These findings broaden our insight into the evolutionary relationships and expression patterns of the SULTR gene family in cotton, and provide the valuable information for further screening the vital candidate genes on trait improvement.

Research on the mechanism of Guanyu Zhixie Granule in intervening gastric ulcers in rats based on network pharmacology and multi-omics.

Guanyu Zhixie Granule (GYZXG) is a traditional Chinese medicine compound with definite efficacy in intervening in gastric ulcers (GUs). However, the effect mechanisms on GU are still unclear. This study aimed to explore its mechanism against GU based on amalgamated strategies. The comprehensive chemical characterization of the active compounds of GYZXG was conducted using UHPLC-Q/TOF-MS. Based on these results, key targets and action mechanisms were predicted through network pharmacology. GU was then induced in rats using anhydrous ethanol (1 mL/200 g). The intervention effects of GYZXG on GU were evaluated by measuring the inhibition rate of GU, conducting HE staining, and assessing the levels of IL-6, TNF-α, IL-10, IL-4, Pepsin (PP), and epidermal growth factor (EGF). Real-time quantitative PCR (RT-qPCR) was used to verify the mRNA levels of key targets and pathways. Metabolomics, combined with 16S rRNA sequencing, was used to investigate and confirm the action mechanism of GYZXG on GU. The correlation analysis between differential gut microbiota and differential metabolites was conducted using the spearman method. For the first time, the results showed that nine active ingredients and sixteen targets were confirmed to intervene in GU when using GYZXG. Compared with the model group, GYZXG was found to increase the ulcer inhibition rate in the GYZXG-M group (p < 0.05), reduce the levels of IL-6, TNF-α, PP in gastric tissue, and increase the levels of IL-10, IL-4, and EGF. GYZXG could intervene in GU by regulating serum metabolites such as Glycocholic acid, Epinephrine, Ascorbic acid, and Linoleic acid, and by influencing bile secretion, the HIF-1 signaling pathway, and adipocyte catabolism. Additionally, GYZXG could intervene in GU by altering the gut microbiota diversity and modulating the relative abundance of Bacteroidetes, Bacteroides, Verrucomicrobia, Akkermansia, and Ruminococcus. The differential gut microbiota was strongly associated with serum differential metabolites. KEGG enrichment analysis indicated a significant role of the HIF-1 signaling pathway in GYZXG's intervention on GU. The changes in metabolites within metabolic pathways and the alterations in RELA, HIF1A, and EGF mRNA levels in RT-qPCR experiments provide further confirmation of this result. GYZXG can intervene in GU induced by anhydrous ethanol in rats by regulating gut microbiota and metabolic disorders, providing a theoretical basis for its use in GU intervention.

Exploring the impact of insufficient thermal ablation on hepatocellular carcinoma: NDST2 overexpression mechanism and its role in facilitating growth and invasion of residual cancer cells

Objective Incomplete thermal ablation (ITA) fosters the malignancy of residual cells in Hepatocellular carcinoma (HCC) with unclear mechanisms now. This study aims to investigate the expression changes of NDST2 following ITA of HCC and its impact on residual cancer cells. Methods An in vitro model of heat stress-induced liver cancer was constructed to measure the expression of NDST2 using Quantitative Real-Time PCR and Western blotting experiments. The sequencing data from nude mice were used for validation. The clinical significance of NDST2 in HCC was evaluated by integrating datasets. Gene ontology and pathway analysis were conducted to explore the potential signaling pathways regulated by NDST2. Additionally, NDST2 was knocked down in heat stress-induced HCC cells, and the effects of NDST2 on these cells were verified using Cell Counting Kit-8 assays, scratch assays, and Transwell assays. Results NDST2 expression levels are elevated in HCC, leading to a decrease in overall survival rates of HCC patients. Upregulation of immune checkpoint levels in high NDST2-expressing HCC may contribute to immune evasion by liver cancer cells. Additionally, the low mutation rate of NDST2 in HCC suggests a relatively stable expression of NDST2 in this disease. Importantly, animal and cell models treated with ITA demonstrate upregulated expression of NDST2. Knockdown of NDST2 in heat stress-induced liver cancer cells results in growth inhibition associated with gene downregulation. Conclusion The upregulation of NDST2 can accelerate the progression of residual HCC after ITA, suggesting a potential role for NDST2 in the therapeutic efficacy and prognosis of residual HCC.

Screening and Analysis of Core Genes for Osteoporosis Based on Bioinformatics Analysis and Machine Learning Algorithms.

This study aimed to identify osteoporosis-related core genes using bioinformatics analysis and machine learning algorithms. mRNA expression profiles of osteoporosis patients were obtained from the Gene Expression Profiles (GEO) database, with GEO35958 and GEO84500 used as training sets, and GEO35957 and GSE56116 as validation sets. Differential gene expression analysis was performed using the R software "limma" package. A weighted gene co-expression network analysis (WGCNA) was conducted to identify key modules and modular genes of osteoporosis. Kyoto Gene and Genome Encyclopedia (KEGG), Gene Ontology (GO), and gene set enrichment analysis (GSEA) were performed on the differentially expressed genes. LASSO, SVM-RFE, and RF machine learning algorithms were used to screen for core genes, which were subsequently validated in the validation set. Predicted microRNAs (miRNAs) from the core genes were also analyzed, and differential miRNAs were validated using quantitative real-time PCR (qPCR) experiments. A total of 1280 differentially expressed genes were identified. A disease key module and 215 module key genes were identified by WGCNA. Three core genes (ADAMTS5, COL10A1, KIAA0040) were screened by machine learning algorithms, and COL10A1 had high diagnostic value for osteoporosis. Four core miRNAs (has-miR-148a-3p, has-miR-195-3p, has-miR-148b-3p, has-miR-4531) were found by intersecting predicted miRNAs with differential miRNAs from the dataset (GSE64433, GSE74209). The qPCR experiments validated that the expression of has-miR-195-3p, has-miR-148b-3p, and has-miR-4531 was significantly increased in osteoporosis patients. This study demonstrated the utility of bioinformatics analysis and machine learning algorithms in identifying core genes associated with osteoporosis.

Poplar glutathione S-transferase PtrGSTF8 contributes to reactive oxygen species scavenging and salt tolerance

Glutathione S-transferases (GSTs) constitute a protein superfamily encoded by a large gene family and play a crucial role in plant growth and development. However, their precise functions in wood plant responses to abiotic stress are not fully understood. In this study, we isolated a Phi class glutathione S-transferase-encoding gene, PtrGSTF8, from poplar (Populus alba × P. glandulosa), which is significantly up-regulated under salt stress. Moreover, compared with wild-type (WT) plants, transgenic tobacco plants exhibited significant salt stress tolerance. Under salt stress, PtrGSTF8-overexpressing tobacco plants showed a significant increase in plant height and root length, and less accumulation of reactive oxygen species. In addition, these transgenic tobacco plants exhibited higher superoxide dismutase, peroxidase, and catalase activities and reduced malondialdehyde content compared with WT plants. Quantitative real-time PCR experiments showed that the overexpression of PtrGSTF8 increased the expression of numerous genes related to salt stress. Furthermore, PtrMYB108, a MYB transcription factor involved in salt resistance in poplar, was found to directly activate the promoter of PtrGSTF8, as demonstrated by yeast one-hybrid assays and luciferase complementation assays. Taken together, these findings suggest that poplar PtrGSTF8 contributes to enhanced salt tolerance and confers multiple growth advantages when overexpressed in tobacco.

Genome-wide analysis of the MATE gene family and expression analysis of subfamily III genes in response to aluminium toxicity in pineapple

ABSTRACT Pineapple has strong adaptability to acidic soil and has become a significant economic fruit crop in the tropics and subtropics. The multidrug and toxin compound extrusion (MATE) gene family plays crucial roles in plant growth, development and responses to adverse stresses, especially in acidic soils. In this study, forty-two AcMATE genes were identified and classified into six subfamilies based on phylogenetic analysis. The chromosomal distribution, physical characteristics, phylogenetic relationships, and gene and protein structures of pineapple MATE gene family members were systematically analysed. Our results showed that six AcMATE genes can be further clustered into subfamily III with similar conserved motifs and exon-intron patterns, and are unevenly distributed on four pineapple chromosomes. Citrate exuding motif (CEM) was present in all six AcMATE proteins of subfamily III. Finally, quantitative real-time PCR experiments indicated that subfamily III AcMATE genes were highly expressed in roots and distinctly upregulated under Al stresses. In conclusion, our study illustrated that the AcMATE genes of subfamily III may function as an Al-induced citrate transporter in alleviating Al toxicity to adapt acidic soil in pineapple.

Microglia differential genes and their functions in paraquat-induced Parkinson's disease-like in mice's brains based on single-cell RNA sequencing

Objective: To analyze the differential genes and related signaling pathways of microglia subpopulations in Parkinson's disease (PD) -like mouse brains induced by paraquat (PQ) based on single-cell RNA sequencing, and provide clues to elucidate the mechanism of PQ-induced PD-like changes in the brain of animals. Methods: In September 2021, six male 6-week-old C57BL/6 mice were randomly divided into control group and experimental group (three mice in each group) . The mice were injected with saline, 10.0 mg/kg PQ intraperitoneally, once every three days, and 10 consecutive injections were used for modeling. After infection, the brains of mice were taken and 10×Genomics single-cell RNA sequencing was performed. Microglia subpopulations were screened based on gene expression characteristics, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. The differential genes of microglia subpopulations between the experimental group and control group were further screened, and functional enrichment analysis was performed using bioinformatics tools. Mouse microglia (BV2 cells) were treated with 0, 60, 90 μmol/L PQ solution, respectively. And real-time fluorescence quantitative PCR experiments were conducted to validate the expressions of differential genes hexokinase 2 (Hk2) , ATPase H+ Transporting V0 Subunit B (Atp6v0b) and Neuregulin 1 (Nrg1) . Results: Cluster 7 and Cluster 20 were identified as microglia subpopulations based on the signature genes inositol polyphosphate-5-phosphatase d, Inpp5d (Inpp5d) and transforming growth factor beta receptor 1 (Tgfbr1) , and they reflected the microglia-activated M2 phenotype. The bioinformatics analysis showed that the characteristic genes of identified microglia subpopulations were enriched in endocytosis. In terms of molecular function, it mainly enriched in transmembrane receptor protein kinase activity and cytokine binding. The up-regulated genes of Cluster 7 were mainly enriched in lysosomal pathway, endocytosis pathway, and down-regulated genes were mainly enriched in neurodegenerative disease and other signaling pathways. The up-regulated genes of Cluster 20 were mainly enriched in signaling pathways related to PD, and down-regulated genes were mainly enriched in cyclic adenosine 3', 5'-monophosphate (cAMP) signaling pathways, neurological development, synaptic function and other signaling pathways. The results of real-time fluorescence quantitative PCR showed that the expressions of Hk2 mRNA and Atp6v0b mRNA increased and the expression of Nrg1 mRNA decreased in the 90 μmol/L PQ-treated BV2 cells compared with the 0 μmol/L, and the differences were statistically significant (P<0.05) . Conclusion: Microglia are activated in the PQ-induced PD-like mouse model and polarized toward the M2 phenotype. And their functions are associated with lysosomal (endocytosis) , synaptic functions and the regulation of PD-related pathways.

Medicinal cannabis oil improves anxiety-like and depressive-like behaviors in CCS mice via the BDNF/TRPC6 signaling pathway

BackgroundPost-traumatic stress disorder (PTSD) refers to a chronic impairing psychiatric disorder occurring after exposure to the severe traumatic event. Studies have demonstrated that medicinal cannabis oil plays an important role in neuroprotection, but the mechanism by which it exerts anti-PTSD effects remains unclear. MethodsThe chronic complex stress (CCS) simulating the conditions of long voyage stress for 4 weeks was used to establish the PTSD mice model. After that, behavioral tests were used to evaluate PTSD-like behaviors in mice. Mouse brain tissue index was detected and hematoxylin-eosin staining was used to assess pathological changes in the hippocampus. The indicators of cell apoptosis and the BDNF/TRPC6 signaling activation in the mice hippocampus were detected by western blotting or real-time quantitative reverse transcription PCR experiments. ResultsWe established the PTSD mice model induced by CCS, which exhibited significant PTSD-like phenotypes, including increased anxiety-like and depression-like behaviors. Medicinal cannabis oil treatment significantly ameliorated PTSD-like behaviors and improved brain histomorphological abnormalities in CCS mice. Mechanistically, medicinal cannabis oil reduced CCS-induced cell apoptosis and enhanced the activation of BDNF/TRPC6 signaling pathway. ConclusionsWe constructed a PTSD model with CCS and medicinal cannabis oil that significantly improved anxiety-like and depressive-like behaviors in CCS mice, which may play an anti-PTSD role by stimulating the BDNF/TRPC6 signaling pathway.

Exosome secretion related gene signature predicts chemoresistance in patients with colorectal cancer

BackgroundColorectal cancer (CRC) is a highly heterogeneous malignancy, and patients often have different responses to treatment. In this study, the genetic characteristics related to exosome formation and secretion procedure were used to predict chemoresistance and guide the individualized treatment of patients. MethodsFirstly, seven microarray datasets in Gene Expression Omnibus (GEO) and RNA-Seq dataset from the Cancer Genome Atlas (TCGA) were used to analysis the transcriptome profiles and associated characteristics of CRC patients. Then, a predictive model based on gene features linked to exosome formation and secretion was created and validated using Least Absolute Shrinkage and Selection Operator (LASSO) regression analysis and Support Vector Machine-Recursive Feature Elimination (SVM-RFE) machine learning. Finally, we evaluated the model using chemoresistant/chemosensitive cells and tissues by immunofluorescence (IF), western blot (WB), quantitative real-time PCR (qRT-PCR) and immunocytochemistry (IHC) experiments, and the predictive value of integrated model in the clinical validation cohort were performed by Receiver Operating Characteristic (ROC) and Kaplan-Meier (K-M) curves analyses. ResultsWe established a risk score signature based on three genes related to exosome secretion in CRC. Better Overall Survival (OS) and greater chemosensitivity were seen in the low-risk group, whereas the high-risk group exhibited chemoresistance and a subpar response to immune checkpoint blockade (ICB) therapy. Higher expression of the model genes EXOC2, EXOC3 and STX4 were observed in chemoresistant cells and specimens. The AUC of 5-year disease-free survival (DFS) was 0.804. Compared with that in the low-risk group, patients' DFS was found to be significantly worse in the high-risk group. ConclusionsIn summary, the gene signature related to exosome formation and secretion could reliably predict patients' chemosensitivity and ICB treatment response, which providing new independent biomarkers for the treatment of CRC.

Comparative transcriptomic analysis reveals the molecular changes of acute pancreatitis in experimental models

BACKGROUND Acute pancreatitis (AP) encompasses a spectrum of pancreatic inflammatory conditions, ranging from mild inflammation to severe pancreatic necrosis and multisystem organ failure. Given the challenges associated with obtaining human pancreatic samples, research on AP predominantly relies on animal models. In this study, we aimed to elucidate the fundamental molecular mechanisms underlying AP using various AP models. AIM To investigate the shared molecular changes underlying the development of AP across varying severity levels. METHODS AP was induced in animal models through treatment with caerulein alone or in combination with lipopolysaccharide (LPS). Additionally, using Ptf1α to drive the specific expression of the hM3 promoter in pancreatic acinar cells transgenic C57BL/6J- hM3/Ptf1α(cre) mice were administered Clozapine N-oxide to induce AP. Subsequently, we conducted RNA sequencing of pancreatic tissues and validated the expression of significantly different genes using the Gene Expression Omnibus (GEO) database. RESULTS Caerulein-induced AP showed severe inflammation and edema, which were exacerbated when combined with LPS and accompanied by partial pancreatic tissue necrosis. Compared with the control group, RNA sequencing analysis revealed 880 significantly differentially expressed genes in the caerulein model and 885 in the caerulein combined with the LPS model. Kyoto Encyclopedia of Genes and Genomes enrichment analysis and Gene Set Enrichment Analysis indicated substantial enrichment of the TLR and NOD -like receptor signaling pathway, TLR signaling pathway, and NF-κB signaling pathway, alongside elevated levels of apoptosis-related pathways, such as apoptosis, P53 pathway, and phagosome pathway. The significantly elevated genes in the TLR and NOD -like receptor signaling pathways, as well as in the apoptosis pathway, were validated through quantitative real-time PCR experiments in animal models. Validation from the GEO database revealed that only MYD88 concurred in both mouse pancreatic tissue and human AP peripheral blood, while TLR1 , TLR7 , RIPK3 , and OAS2 genes exhibited marked elevation in human AP. The genes TUBA1A and GADD45A played significant roles in apoptosis within human AP. The transgenic mouse model hM3/Ptf1α(cre) successfully validated significant differential genes in the TLR and NOD -like receptor signaling pathways as well as the apoptosis pathway, indicating that these pathways represent shared pathological processes in AP across different models. CONCLUSION The TLR and NOD receptor signaling pathways play crucial roles in the inflammatory progression of AP, notably the MYD88 gene. Apoptosis holds a central position in the necrotic processes of AP, with TUBA1A and GADD45A genes exhibiting prominence in human AP.

Integrative analyses of whole-transcriptome sequencing reveals CeRNA regulatory network in pulmonary hypertension treated with FGF21

Noncoding RNAs have been shown to play essential roles in hypoxic pulmonary hypertension (HPH). Our preliminary data showed that HPH is attenuated by fibroblast growth factor 21 (FGF21) administration. Therefore, we further investigated the whole transcriptome RNA expression patterns and interactions in a mice HPH model treated with FGF21. By whole-transcriptome sequencing, differentially expressed mRNAs, miRNAs, lncRNAs, and circRNAs were successfully identified in normoxia (Nx) vs. hypoxia (Hx) and Hx vs. hypoxia + FGF21 (Hx + F21). Differentially expressed mRNAs, miRNAs, lncRNAs, and circRNAs regulated by hypoxia and FGF21 were selected through intersection analysis. Based on prediction databases and sequencing data, differentially co-expressed mRNAs, miRNAs, lncRNAs, and circRNAs were further screened, followed by functional enrichment analysis. MAPK signaling pathway and epigenetic modification were enriched and may play fundamental roles in the therapeutic effects of FGF21. The ceRNA regulatory network of lncRNA–miRNA–mRNA and circRNA–miRNA–mRNA was constructed with miR-7a-5p, miR-449c-5p, miR-676-3p and miR-674-3p as the core. In addition, quantitative real-time PCR experiments were employed to verify the whole-transcriptome sequencing data. The results of luciferase reporter assays highlighted the relationship between miR-449c-5p and XR_878320.1, miR-449c-5p and Stab2, miR-449c-5p and circ_mtcp1, which suggesting that miR-449c-5p may be a key regulator of FGF21 in the treatment of PH. Taken together, this study provides potential biomarkers, pathways, and ceRNA regulatory networks in HPH treated with FGF21 and will provide an experimental basis for the clinical application of FGF21 in PH.

Development of an immune-related diagnostic predictive model for oral lichen planus.

Oral lichen planus (OLP) was a chronic inflammatory disease of unknown etiology with a 1.4% chance of progressing to malignancy. However, it has been suggested in several studies that immune system disorders played a dominant role in the onset and progression of OLP. Therefore, this experiment aimed to develop a diagnostic prediction model for OLP based on immunopathogenesis to achieve early diagnosis and treatment and prevent cancer. In this study, 2 publicly available OLP datasets from the gene expression omnibus database were filtered. In the experimental group (GSE52130), the level of immune cell infiltration was assessed using MCPcounter and ssGSEA algorithms. Subsequently, differential expression analysis and gene set enrichment analysis were performed between the OLP and control groups. The resulting differentially expressed genes were intersected with immunologically relevant genes provided on the immunology database and analysis portal database (ImmPort) website to obtain differentially expressed immunologically relevant genes (DEIRGs). Furthermore, the gene ontology and kyoto encyclopedia of genes and genomes analyses were carried out. Finally, protein-protein interaction network and least absolute shrinkage and selection operator regression analyses constructed a model for OLP. Receiver operating characteristic curves for the experimental and validation datasets (GSE38616) were plotted separately to validate the model's credibility. In addition, real-time quantitative PCR experiment was performed to verify the expression level of the diagnostic genes. Immune cell infiltration analysis revealed a more significant degree of inflammatory infiltration in the OLP group compared to the control group. In addition, the gene set enrichment analysis results were mainly associated with keratinization, antibacterial and immune responses, etc. A total of 774 differentially expressed genes was obtained according to the screening criteria, of which 65 were differentially expressed immunologically relevant genes. Ultimately, an immune-related diagnostic prediction model for OLP, which was composed of 5 hub genes (BST2, RNASEL, PI3, DEFB4A, CX3CL1), was identified. The verification results showed that the model has good diagnostic ability. There was a significant correlation between the 5 hub diagnostic biomarkers and immune infiltrating cells. The development of this model gave a novel insight into the early diagnosis of OLP.