qRT-PCR Detection Research Articles

B-211 Quantum dot-DNA nanocomposite enhanced single-molecule flow (SiM-Flow) for ultrasensitive microRNA detection in metastatic castrate-resistant prostate cancer

Abstract Background MicroRNAs (miRs) like miR-375 and miR-1290 are associated with cancer progression and chemoresistance in metastatic castrate-resistant prostate cancer (mCRPC). However, these prognostic biomarkers are typically present in low abundance (0.1∼1 fM) in patient plasma, which is close to the detection limit of the current gold standard techniques, quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Therefore, we tested a nanomaterial that incorporates quantum dots (QDs) and DNA amplicons for enhanced in-solution counting applications in plasma from a cohort of mCRPC patients. The QD-DNA nanocomposite increases the fluorescent intensity of an individual amplicon for ultrasensitive flow-counting and provides higher multiplex capacity due to the narrow emission band of QD for simple instrumentation. Methods QDs were synthesized with spectrally distinct emission bands by tuning the size or composition of CdSe core. QDs were then coated with multidentate polymer allowing further bioconjugations and better solubility in aqueous solution. The oligonucleotides complementary to amplicons generated from selected miR targets (prognostic biomarkers: miR-1290 and miR-375; normalization biomarkers: miR-16, miR-30a-5p, and cel-39) were then conjugated to QDs with optimized ratio. Total RNA was extracted from a screening cohort of 14 mCRPC patients from Huntsman Cancer Institute where platelet-poor plasma was uniformly processed with exogenous spike-in, cel-39, added in. qRT-PCR was performed using TaqMan miRNA assays, and SiM-Flow was conducted using a commercial flow cytometry to count QD barcoded enzymatic-extended miRs nanoparticles. The DNA template used for extending miRs was designed by our lab and purchased from IDT-DNA. Pearson correlation and interclass agreement analysis were applied to evaluate the concordance between the two assays. The mCRPC cohort was followed for clinical outcomes, and survival analysis for the time of progression from mCRPC to death/last follow-up was performed to determine the prognostic value of the QD-DNA nanoparticle counts. The Cox proportional hazards model was applied to linearly incorporate multiple hazard factors of patients for multivariate survival analysis. Results We demonstrated the multiplex ability of QDs with less than 5% false positive rate. The machine learning-guided optimized assay shows a 1000-time improved limit of detection of assay compared to the published literature. Interclass agreement > 0.9 and Pearson correlation of 0.94 between SiM-Flow and RT-qPCR on human plasma extracts suggest high specificity of SiM-Flow on miR detection. Also, miR-375 was identified among all the samples through SiM-Flow, although it was unidentified by qRT-PCR in 14 samples. Higher levels of miR-1290, miR-375, and prostate-specific antigen were significantly associated with poor overall survival (p=0.019) in the initial survival analysis. Survival analysis replication is ongoing in an independent mCRPC cohort. Conclusions SiM-Flow was able to detect target miRs in mCRPC patients’ plasma with a high agreement with qRT-PCR and lower detection limit and demonstrated multiplex ability with a simple optical set-up. This shows technological advancement in the detection of cancer-associated biomarkers and will aid in precision medicine therapeutic and point-of-care applications with both prognostic and predictive potential.

Ultra-early stage lower-grade gliomas: How can we define and differentiate these easily misdiagnosed gliomas through intraoperative molecular diagnosis.

Some lower-grade gliomas (LGG) are difficult to distinguish morphologically from glial cell proliferation or inflammatory changes during surgery, leading to a high risk of incorrect diagnosis. It is crucial to differentiate between the two for making surgical decisions. We define these critical cases as "ultra early stage lower-grade gliomas (UES-LGG)". We analyzed 11 out of 13 cases diagnosed with "gliosis" or "inflammatory changes" during surgery who tested positive for isocitrate dehydrogenase (IDH). Additionally, we conducted qRT-PCR detection on 35 samples diagnosed with LGG during surgery and analyzed their DNA content within an effective circulating threshold range to infer the critical value between UES-LGG and LGG. We conducted experiments using five standardized samples to infer the limited range of accurate detection of UES-LGG during surgery. In the comparative analysis of 11 samples and 35 samples, it was found that while there was no significant difference in the average DNA detection concentration between the two groups (159.36 ± 83.3 ng/μL and 146.83 ± 122.43 ng/μL), there was a notable statistical variance in the detection threshold for positive mutations (31.78 ± 1.14 and 26.14 ± 2.69, respectively). This suggests that the IDH mutation rate may serve as an indicator for differentiation between the two groups. Subsequently, DNA was extracted from standardized IDH mutant samples and subjected to gradient dilution for detection purposes. The results indicated a consistent increase in detection threshold as detection concentration decreased. When the detection concentration fell below <0.1 ng/μL, it became impossible to carry out effective threshold range detections. To further identify the precise detection interval, we conducted gradient division once again and sought to simulate the functional relationship between DNA copy number and cycle threshold within this interval. The research revealed that when the minimum detection concentration exceeded 250 copies/μL, a 100% detection rate could be achieved. This article defines UES-LGG as a tumor type easily misdiagnosed in clinical practice due to its extremely low positivity rate during surgery. The popularization of qRT-PCR based intraoperative molecular diagnosis greatly reduces errors caused by manual detection and improves disease detection rates during surgery. It provides a theoretical basis for more accurate surgical plans for surgeons.

Resistant starch confers protection of dietary against diabetic cardiomyopathy

Long-term dysfunction of glucose metabolism causes cardiac dysfunction called diabetic cardiomyopathy (DCM). To investigate the effect and underlying mechanism of RS on the process of DCM, mouse models induced by a high-fat diet (HFD) and streptozotocin (STZ) were fed RS (2 g/kg/day) and vehicle treatment (by oral gavage) for 14 weeks. Various analyses, including qRT-PCR, western blot, immunofluorescence staining, histology staining, cardiac function, and diversity detection of intestinal microbiota were performed. RS intervention could directly improve myocardial fibrosis, hypertrophy, apoptosis, and cardiac insufficiency in DCM. These beneficial effects may be achieved by elevating the expression of IGF-1, activating the ERK phosphorylation. Furthermore, by carrying out nano LC-MS/MS analyses and 16S rDNA sequencing, we found RS might primarily affect proteins in the cytoplasm involved in post-translational modification, protein conversion, and signal transduction mechanisms. RS altered intestinal microbiota and improved intestinal mucosal permeability towards a favorable direction in DCM. This multidimensional assessment of RS suggests that might be a promising approach towards the treatment of DCM.

Identification of autophagy gene family in potato and the role of StATG8a in salt and drought stress.

Autophagy is a highly conserved method of recycling cytoplasm components in eukaryotes. It plays an important role in plant growth and development, as well as in response to biotic and abiotic stresses. Although autophagy-related genes (ATGs) have been identified in several crop species, their particular role in potato (Solanum tuberosum L.) remains unclear. Several transcription factors and signaling genes in the transgenic lines of the model plant Arabidopsis thaliana, such as AtTSPO, AtBES1, AtPIP2;7, AtCOST1 as well as AtATI1/2, ATG8f, GFP-ATG8F-HA, AtDSK2, AtNBR1, AtHKT1 play crucial functions under drought and salt stresses, respectively. In this study, a total of 29 putative StATGs from 15 different ATG subfamilies in the potato genome were identified. Their physicochemical properties, evolutionary connections, chromosomal distribution, gene duplication, protein-protein interaction network, conserved motifs, gene structure, interspecific collinearity relationship, and cis-regulatory elements were analyzed. The results of qRT-PCR detection of StATG expression showed that 29 StATGs were differentially expressed in potato's leaves, flowers, petiole, stem, stolon, tuber, and root. StATGs were dynamically modulated by salt and drought stresses and up-regulated under salt and drought conditions. Our results showed that the StATG8a localized in the cytoplasm and the nucleus. Potato cultivar "Atlantic" overexpressing or downregulating StATG8a were constructed. Based on physiological, biochemical, and photosynthesis parameters, potato lines overexpressing StATG8a exhibited 9 times higher drought and salt tolerance compared to non-transgenic plants. In contrast, the potato plants with knockdown expression showed a downtrend in drought and salt tolerance compared to non-transgenic potato lines. These results could provide new insights into the function of StATG8a in salt and drought response and its possible mechanisms.

Transplantation of miR-145a-5p modified M2 type microglia promotes the tissue repair of spinal cord injury in mice

BackgroundThe traumatic spinal cord injury (SCI) can cause immediate multi-faceted function loss or paralysis. Microglia, as one of tissue resident macrophages, has been reported to play a critical role in regulating inflammation response during SCI processes. And transplantation with M2 microglia into SCI mice promotes recovery of motor function. However, the M2 microglia can be easily re-educated and changed their phenotype due to the stimuli of tissue microenvironment. This study aimed to find a way to maintain the function of M2 microglia, which could exert an anti-inflammatory and pro-repair role, and further promote the repair of spinal cord injury.MethodsTo establish a standard murine spinal cord clip compression model using Dumont tying forceps. Using FACS, to sort microglia from C57BL/6 mice or CX3CR1GFP mice, and further culture them in vitro with different macrophage polarized medium. Also, to isolate primary microglia using density gradient centrifugation with the neonatal mice. To transfect miR-145a-5p into M2 microglia by Lipofectamine2000, and inject miR-145a-5p modified M2 microglia into the lesion sites of spinal cord for cell transplanted therapy. To evaluate the recovery of motor function in SCI mice through behavior analysis, immunofluorescence or histochemistry staining, Western blot and qRT-PCR detection. Application of reporter assay and molecular biology experiments to reveal the mechanism of miR-145a-5p modified M2 microglia therapy on SCI mice.ResultsWith in vitro experiments, we found that miR-145a-5p was highly expressed in M2 microglia, and miR-145a-5p overexpression could suppress M1 while promote M2 microglia polarization. And then delivery of miR-145a-5p overexpressed M2 microglia into the injured spinal cord area significantly accelerated locomotive recovery as well as prevented glia scar formation and neuron damage in mice, which was even better than M2 microglia transplantation. Further mechanisms showed that overexpressed miR-145a-5p in microglia inhibited the inflammatory response and maintained M2 macrophage phenotype by targeting TLR4/NF-κB signaling.ConclusionsThese findings indicate that transplantation of miR-145a-5p modified M2 microglia has more therapeutic potential for SCI than M2 microglia transplantation from epigenetic perspective.Graphical

KLF7 enhances the invasion and migration of colorectal cancer cells via the miR-139-5p/TPD52 axis

ABSTRACT In this study, we aimed to investigate the molecular mechanism of Krüppel-like factor 7 (KLF7) in colorectal cancer (CRC) cell invasion and migration. The expression pattern of KLF7 in CRC tissues and the correlation between KLF7 expression and clinical symptoms of CRC were analyzed. CRC cell lines were transfected with si-KLF7, followed by qRT-PCR or western blot detection of KLF7, miR-139-5p, and tumor protein D52 (TPD52) expression, cell counting kit-8 (CCK-8) assay to detect cell viability, and transwell detection of invasion and migration. Chromatin immunoprecipitation (ChIP) analyzed the enrichment KLF7 in the miR-139-5p promoter. The dual-luciferase reporter assay verified the binding relationship between KLF7 and miR-139-5p, and between miR-139-5p and TPD52. In the subcutaneous tumorigenesis experiment, tumor growth was observed and ki67-positive expression was detected. KLF7 is abundantly expressed in CRC cells KLF7 silencing inhibits CRC cell viability, invasion, and migration. KLF7 represses miR-139-5p expression by binding to the miR-139-5p promoter. miR-139-5p targets TPD52 expression. miR-13-5p inhibition or TPD52 overexpression partially counteracted the effect of KLF7 silencing in CRC cells. KLF7 silencing suppresses tumor growth in vivo. In conclusion, KLF7 suppresses miR-139-5p expression by binding to the miR-139-5p promoter, thereby upregulating TPD52 expression and enhancing CRC cell invasion and migration.

Selection of Stable Reference Genes for QRT-PCR in Tree Peony 'Doulv' and Functional Analysis of PsCUC3.

(1) Background: Tree peonies display extensive cultivar diversity due to widespread hybridization, resulting in a complex genetic architecture. This complexity complicates the selection of universal reference genes across different cultivars for qRT-PCR analyses. Paeonia suffruticosa 'Doulv', notable for its unique green blooms in China, exhibits chlorosis post-flowering and features petaloid stamens and pistils. (2) Methods: Based on published literature and RNA-seq data from 'Doulv', nine candidate reference genes-ACT (Actin), TUB (β-Tubulin), UBC (Ubiquitin Conjugating Enzyme), UBQ (Ubiquitin), UPL (Ubiquitin Protein Ligase), PP2A (Protein Phosphatase 2A), PP2C (Protein Phosphatase 2C), MBF1A (Multiprotein Bridging Factor 1A), and GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase)-were selected. Their expression stability was assessed across various tissues and developmental stages of 'Doulv' flowers using qRT-PCR, with evaluations conducted via GeNorm_v3.5, NormFinder_v20, and BestKeeper_v1.0. Gene cloning and expression analyses of PsCUC3, including its subcellular localization, were performed. (3) Results: GAPDH and ACT were identified as the most stable reference genes in petaloid stamens across various developmental stages of 'Doulv', whereas UBC and MBF1A were optimal across different tissues. Notably, specific conserved amino acids in PsCUC3 from 'Doulv' diverged from those in NAM/CUC3 proteins of other species, impacting its protein structure. PsCUC3 expression analysis revealed no correlation with chlorophyll content in petaloid stamens but an association with petaloid organ development. Furthermore, PsCUC3 was predominantly localized in the nucleus. (4) Conclusions: This study comprehensively evaluated suitable reference genes using GeNorm_v3.5, NormFinder_v20, and BestKeeper_v1.0 software, establishing a robust qRT-PCR detection system for 'Doulv' peony. These results provide a solid experimental foundation for further research on 'Doulv' peony. Building on this experimental foundation, the functional analysis of the PsCUC3 gene was conducted. The findings suggest a potential association between the PsCUC3 gene and floral morphology alterations in 'Doulv', identifying PsCUC3 as crucial for understanding the molecular mechanisms influencing floral structure in tree peonies.

Regulatory roles of long non-coding RNAs in short-term heat stress in adult worker bees

Long non-coding RNAs (lncRNAs) are crucial modulators of post-transcriptional gene expression regulation, cell fate determination, and disease development. However, lncRNA functions during short-term heat stress in adult worker bees are poorly understood. Here, we performed deep sequencing and bioinformatic analyses of honeybee lncRNAs. RNA interference was performed by using siRNA targeting the most highly expressed lncRNA. The silencing effect on lncRNA and the relative expression levels of seven heat shock protein (HSP) genes, were subsequently examined. Overall, 7,842 lncRNAs and 115 differentially expressed lncRNAs (DELs) were identified in adult worker bees following heat stress exposure. Structural analysis revealed that the overall expression abundance, length of transcripts, exon number, and open reading frames of lncRNAs were lower than those of mRNAs. GO analysis revealed that the target genes were mainly involved in “metabolism,” “protein folding,” “response to stress,” and “signal transduction” pathways. KEGG analysis indicated that the “protein processing in endoplasmic reticulum” and “longevity regulating pathway-multiple species” pathways were most enriched. Quantitative real-time polymerase chain reaction (qRT-PCR) detection of the selected DELs confirmed the reliability of the sequencing data. Moreover, the siRNA experiment indicated that feeding siRNA yielded a silencing efficiency of 77.51% for lncRNA MSTRG.9645.5. Upon silencing this lncRNA, the expression levels of three HSP genes were significantly downregulated (p < 0.05), whereas those of three other HSP genes were significantly upregulated (p < 0.05). Our results provide a new perspective for understanding the regulatory mechanisms of lncRNAs in adult worker bees under short-term heat stress.

Identification of osteoporosis ferroptosis-related markers and potential therapeutic compounds based on bioinformatics methods and molecular docking technology

Research background and purposeOsteoporosis (OP) is one of the most common bone diseases worldwide, characterized by low bone mineral density and susceptibility to pathological fractures, especially in postmenopausal women and elderly men. Ferroptosis is one of the newly discovered forms of cell death regulated by genes in recent years. Many studies have shown that ferroptosis is closely related to many diseases. However, there are few studies on ferroptosis in osteoporosis, and the mechanism of ferroptosis in osteoporosis is still unclear. This study aims to identify biomarkers related to osteoporosis ferroptosis from the GEO (Gene Expression Omnibus) database through bioinformatics technology, and to mine potential therapeutic small molecule compounds through molecular docking technology, trying to provide a basis for the diagnosis and treatment of osteoporosis in the future.Materials and methodsWe downloaded the ferroptosis-related gene set from the FerrDb database (http://www.zhounan.org/ferrdb/index.html), downloaded the data sets GSE56815 and GSE7429 from the GEO database, and used the R software “limma” package to screen differentially expressed genes (DEGs) from GSE56815, and intersected with the ferroptosis gene set to obtain ferroptosis-related DEGs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed by the R software “clusterProfiler” package. The random forest model was further screened to obtain essential ferroptosis genes. R software “corrplot” package was used for correlation analysis of essential ferroptosis genes, and the Wilcox test was used for significance analysis. The lncRNA-miRNA-mRNA-TF regulatory network was constructed using Cytoscape software. The least absolute shrinkage and selection operator (LASSO) was used to construct a disease diagnosis model, and a Receiver operating characteristic (ROC) curve was drawn to evaluate the diagnostic performance, and then GSE7429 was used to verify the reliability of the diagnosis model. Molecular docking technology was used to screen potential small molecule compounds from the Drugbank database. Finally, a rat osteoporosis model was constructed, and peripheral blood mononuclear cells were extracted for qRT-PCR detection to verify the mRNA expression levels of crucial ferroptosis genes.ResultSix DEGs related to ferroptosis were initially screened out. GO function and KEGG pathway enrichment analysis showed that ferroptosis-related DEGs were mainly enriched in signaling pathways such as maintenance of iron ion homeostasis, copper ion binding function, and ferroptosis. The random forest model identified five key ferroptosis genes, including CP, FLT3, HAMP, HMOX1, and SLC2A3. Gene correlation analysis found a relatively low correlation between these five key ferroptosis genes. The lncRNA-miRNA-mRNA-TF regulatory network shows that BAZ1B and STAT3 may also be potential molecules. The ROC curve of the disease diagnosis model shows that the model has a good diagnostic performance. Molecular docking technology screened out three small molecule compounds, including NADH, Midostaurin, and Nintedanib small molecule compounds. qRT-PCR detection confirmed the differential expression of CP, FLT3, HAMP, HMOX1 and SLC2A3 between OP and normal control group.ConclusionThis study identified five key ferroptosis genes (CP, FLT3, HAMP, HMOX1, and SLC2A3), they were most likely related to OP ferroptosis. In addition, we found that the small molecule compounds of NADH, Midostaurin, and Nintedanib had good docking scores with these five key ferroptosis genes. These findings may provide new clues for the early diagnosis and treatment of osteoporosis in the future.

Expression and prognostic significance of KAP1 gene in malignant pleural mesothelioma

Objective: To explore the expression of KAP1 (KRAB-associated protein 1, KAP1) in Malignant pleural mesothelioma (MPM) based on the cancer genome atlas (TCGA) and clinical trials. And elucidate the correlation between the expression of KAP1 and the clinical pathological parameters of patients with MPM and its prognosis. Methods: In April 2022, Based on the second generation KAP1mRNA sequencing data and clinicopathological data of MPM patients downloaded from TCGA database, the correlation between KAP1mRNA expression and clinical parameters was analyzed, and the correlation between KAP1 protein expression and clinicopathological parameters and its prognostic value were analyzed based on Chuxiong data set cohort clinical samples. The expression of KAP1 mRNA in MPM samples and matched normal tumor adjacent tissues was detected by qRT-PCR, and the expression of KAP1 protein in MPM and normal pleural tissues was detected by immunohistochemistry and Westernblotting. To construct a Kaplan-Meier model to explore the effect of KAP1 expression on the prognosis of MPM patients, and to analyze the prognostic factors of MPM patients by Cox regression. Results: qRT-PCR and Western blotting detection showed that the expression levels of KAP1 gene in four different MPM cells (NCI-H28, NCI-H2052, NCI-H2452, and MTSO-211H) were significantly higher than those in normal pleural mesothelial cells Met-5A. qRT-PCR, Western blotting and IHC results demonstrated that the mRNA and protein expression levels of KAP1 in MPM tissues was significantly higher than that in matching normal mesothelial tissues, and the expression level of KAP1 protein was correlated with TP 53 protein expression levels and serum CEA levels (P<0.05) . The mRNA expression level was significantly correlated with the prognosis, The overall survival time of mesothelioma patients with high KAP1mRNA expression was significantly shorter (HR=3.7, Logrank P<0.001) . Tumor type, age and the mRNA expression were related to the prognosis of MPM patients (P<0.05) . Multivariate analysis showed that tumor type and KAP1 mRNA expression level were independent prognostic factors of MPM patients (P<0.05) . Conclusion: In this study, TCGA database and Chuxiong cohort experiment samples were used to collect the relevant information of KAP1 expression in malignant melanoma tissues. It was confirmed that KAP1 is highly expressed in MPM tissues. The mRNA expression level and pathological type are correlated with the prognosis of patients.

Stress response membrane protein OsSMP2 negatively regulates rice tolerance to drought.

In a gene chip analysis, rice (Oryza sativa) OsSMP2 gene expression was induced under various abiotic stresses, prompting an investigation into its role in drought resistance and abscisic acid signaling. Subsequent experiments, including qRT-PCR and β-glucuronidase activity detection, affirmed the OsSMP2 gene's predominant induction by drought stress. Subcellular localization experiments indicated the OsSMP2 protein primarily localizes to the cell membrane system. Overexpressing OsSMP2 increased sensitivity to exogenous abscisic acid, reducing drought resistance and leading to reactive oxygen species accumulation under drought stress. Conversely, in simulated drought experiments, OsSMP2-silenced transgenic plants showed significantly longer roots compared with the wild-type Nipponbare. These results suggest that OsSMP2 overexpression negatively affects rice drought resistance, offering valuable insights into molecular mechanisms, and highlight OsSMP2 as a potential target for enhancing crop resilience to drought stress.

Biomimetic fabrication of sr-silk fibroin co-assembly hydroxyapatite based microspheres with angiogenic and osteogenic properties for bone tissue engineering

Bone defects caused by trauma, tumor resection, or developmental abnormalities are important issues in clinical practice. The vigorous development of tissue engineering technology provides new ideas and directions for regenerating bone defects. Hydroxyapatite (HAp), a bioactive ceramic, is extensively used in bone tissue engineering because of its excellent osteoinductive performance. However, its application is challenged by its single function and conventional environment-unfriendly synthesis methods. In this study, we successfully "green" synthesized sr-silk fibroin co-assembly hydroxyapatite nanoparticles (Sr-SF-HA) using silk fibroin (SF) as a biomineralized template, thus enabling it to have angiogenic activity and achieving the combination of organic and inorganic substances. Then, the rough composite microspheres loaded with Sr-SF-HA (CS/Sr-SF-HA) through electrostatic spraying technology and freeze-drying method were prepared. The CCK-8 test and live/dead cell staining showed excellent biocompatibility of CS/Sr-SF-HA. Alkaline phosphatase (ALP) staining, alizarin red staining (ARS), immunofluorescence, western blotting, and qRT-PCR test showed that CS/Sr-SF-HA activated the expression of related genes and proteins, thus inducing the osteogenic differentiation of rBMSCs. Moreover, tube formation experiments, scratch experiments, immunofluorescence, and qRT-PCR detection indicated that CS/Sr-SF-HA have good angiogenic activity. Furthermore, in vivo studies showed that the CS/Sr-SF-HA possesses excellent biocompatibility, vascular activity, as well as ectopic osteogenic ability in the subcutaneous pocket of rats. This study indicates that the construction of CS/Sr-SF-HA with angiogenic and osteogenic properties has great potential for bone tissue engineering.

Comprehensive bioinformatics analysis reveals the role of cuproptosis-related gene Ube2d3 in myocardial infarction.

Myocardial infarction (MI) caused by severe coronary artery disease has high incidence and mortality rates, making its prevention and treatment a central and challenging aspect of clinical work for cardiovascular practitioners. Recently, researchers have turned their attention to a novel mechanism of cell death caused by Cu2+, cuproptosis. This study integrated data from three MI-related bulk datasets downloaded from the Gene Expression Omnibus (GEO) database, and identified 16 differentially expressed genes (DEGs) related to cuproptosis by taking intersection of the 6378 DEGs obtained by differential analysis with 49 cuproptosis-related genes. Four hub genes, Dbt, Dlat, Ube2d1 and Ube2d3, were screened out through random forest analysis and Lasso analysis. In the disease group, Dbt, Dlat, and Ube2d1 showed low expression, while Ube2d3 exhibited high expression. Focusing on Ube2d3 for subsequent functional studies, we confirmed its high expression in the MI group through qRT-PCR and Western Blot detection after successful construction of a MI mouse model by left anterior descending (LAD) coronary artery ligation, and further clarified the correlation of cuproptosis with MI development by detecting the levels of cuproptosis-related proteins. Moreover, through in vitro experiments, Ube2d3 was confirmed to be highly expressed in oxygen-glucose deprivation (OGD)-treated cardiomyocytes AC16. In order to further clarify the role of Ube2d3, we knocked down Ube2d3 expression in OGD-treated AC16 cells, and confirmed Ube2d3's promoting role in the hypoxia damage of AC16 cells by inducing cuproptosis, as evidenced by the detection of MTT, TUNEL, LDH release and cuproptosis-related proteins. In summary, our findings indicate that Ube2d3 regulates cuproptosis to affect the progression of MI.

Multi-omics analysis of miRNA-mediated intestinal microflora changes in crucian carp Carassius auratus infected with Rahnella aquatilis.

Infection by an emerging bacterial pathogen Rahnella aquatilis caused enteritis and septicemia in fish. However, the molecular pathogenesis of enteritis induced by R. aquatilis infection and its interacting mechanism of the intestinal microflora associated with microRNA (miRNA) immune regulation in crucian carp Carassius auratus are still unclear. In this study, C. auratus intraperitoneally injected with R. aquatilis KCL-5 was used as an experimental animal model, and the intestinal pathological changes, microflora, and differentially expressed miRNAs (DEMs) were investigated by multi-omics analysis. The significant changes in histopathological features, apoptotic cells, and enzyme activities (e.g., lysozyme (LYS), alkaline phosphatase (AKP), alanine aminotransferase (ALT), aspartate transaminase (AST), and glutathione peroxidase (GSH-Px)) in the intestine were examined after infection. Diversity and composition analysis of the intestinal microflora clearly demonstrated four dominant bacteria: Proteobacteria, Fusobacteria, Bacteroidetes, and Firmicutes. A total of 87 DEMs were significantly screened, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the potential target genes were mainly involved in the regulation of lipid, glutathione, cytosine, and purine metabolism, which participated in the local immune response through the intestinal immune network for IgA production, lysosome, and Toll-like receptor (TLR) pathways. Moreover, the expression levels of 11 target genes (e.g., TLR3, MyD88, NF-κB, TGF-β, TNF-α, MHC II, IL-22, LysC, F2, F5, and C3) related to inflammation and immunity were verified by qRT-PCR detection. The correlation analysis indicated that the abundance of intestinal Firmicutes and Proteobacteria was significantly associated with the high local expression of miR-203/NF-κB, miR-129/TNF-α, and miR-205/TGF-β. These findings will help to elucidate the molecular regulation mechanism of the intestinal microflora, inflammation, and immune response-mediated miRNA-target gene axis in cyprinid fish.

PpAmy1 Plays a Role in Fruit-Cracking by Regulating Mesocarp Starch Hydrolysis of Nectarines.

Nectarine [Prunus persica (L.) Batsch var.] fruits are highly susceptible to cracking during the ripening process, which significantly decreases their commercial value. In this study, we investigated the underlying mechanism of nectarine fruit-cracking using two nectarine varieties, namely, "Qiannianhong" (cracking-susceptible) and "CR1012" (cracking-resistant). Our findings indicate that nectarine fruit-cracking occurs during the second stage of fruit expansion. Despite no differences in epicarp cell size between "Qiannianhong" and "CR1012", the mesocarp cells of "Qiannianhong" were larger than those of "CR1012". Moreover, a comparison of starch hydrolysis between the two varieties revealed that "CR1012" had higher starch content in the mesocarp but lower soluble sugar content compared to "Qiannianhong". Additionally, by testing the α-amylase and β-amylase activity of the mesocarp, our results showed a difference only in α-amylase activity between the two varieties. Furthermore, qRT-PCR detection indicated a higher expression level of the PpAmy1 (α-amylase synthesis gene) in "Qiannianhong" compared to "CR1012". To further investigate the role of PpAmy1, we employed RNAi technology to suppress its expression in "Qiannianhong" fruits. The results showed a significant reduction in α-amylase activity, starch hydrolysis, soluble sugar content, cell size of the mesocarp, and fruit-cracking. These findings underscore the pivotal role of PpAmy1 in the occurrence of nectarine fruit cracking.

Differentially expressed long non-coding RNAs and mRNAs of cadmium exposure on learning disability of offspring rats

BackgroundCadmium (Cd) exposure has been found to have detrimental effects on the development of the central nervous system and cognitive ability in children. However, there is ongoing debate regarding the impact of maternal Cd exposure on the cognitive ability of offspring. In this study, we aimed to investigate the mechanisms underlying the influence of maternal Cd exposure on the cognitive ability of offspring rats.MethodsHere, we constructed a model of cadmium poisoning in first-generation rats through gavage. The cognitive and memory abilities of its offspring were evaluated by water maze experiment. Then, we used the gene chip to find out the key genes, and we performed qRT-PCR detection of these genes. Subsequently, enrichment analysis was employed to identify pathways. Finally, we constructed a co-expression network consisting of LncRNAs and mRNAs to elucidate the biological functions and regulatory mechanisms of LncRNAs.ResultsThe results of the water maze trial demonstrated that the offspring of rats exposed to cadmium in the first generation had reduced cognitive and memory abilities. Through an analysis of gene expression in the hippocampus of the cadmium-treated rats' offspring and the control group, we identified a correlation between the islet secretion pathway and the cognitive impairment observed in the offspring. Utilizing various algorithms, we identified Cpa1 and Prss1 as potential key genes associated with the cognitive impairment caused by cadmium. The results of qRT-PCR demonstrated a decrease in the expression levels of these genes in the hippocampus of the cadmium-treated rats’ offspring. In addition, in the co-expression network, we observed that Cpa1 was co-expressed with 11 LncRNAs, while Prss1 was associated with 4 unexplored LncRNAs. Furthermore, we conducted an analysis to examine the relationship between Cpa1, Prss1-related transcription factors, and LncRNAs.ConclusionOverall, this study provides novel insights into the molecular effects of first generation Cd exposure on the cognitive ability of offspring. The target genes and signaling pathways investigated in this study could serve as potential targets for improving neurodevelopment and cognitive ability in children.

Research of miR-29a on TGF-β1/Smad3 pathway in pulmonary fibrosis induced by neodymium oxide

Objective: To exploring the regulatory effect of miR-29a on the transforming growth factor-β1 (TGF-β1) /Smad homolog 3 (Smad3) pathway during the process of rare earth neodymium oxide (Nd(2)O(3)) induced pulmonary fibrosis in mice. Methods: In March 2021, 72 SPF grade C57/BL6J male mice were selected and randomly divided into a control group, Nd(2)O(3) group, Nd(2)O(3)+miR-29a agomir group, and Nd(2)O(3)+NC agomir group, with 18 mice in each group. The Nd(2)O(3) group, Nd(2)O(3)+miR-29a agomir group, and Nd(2)O(3)+NC agomir group were treated with non exposed tracheal instillation, with a dust concentration of 250 mg/ml and a dust volume of 0.1 ml. The control group was given the same volume of physiological saline. After exposure to Nd(2)O(3), 0.1 ml (5 nmol) of miR-29a agomir was injected into the tail vein of mice in the Nd(2)O(3)+miR-29a agomir group every 3 days, while 0.1 ml of NC agomir was injected into the tail vein of mice in the Nd(2)O(3)+NC agomir group. On the 7 th, 14 th, and 28 th days after dust exposure, 6 mice were killed in each group, and the lung tissue of the mice was taken out. HE staining was used to observe the pathological status of the mouse lung tissue; ELISA method was used to detect the levels of TGF-β1 and connective tissue growth factor (CTGF) in lung tissue; Use qRT-PCR detection method to detect the expression level of TGF-β1 mRNA; Using immunofluorescence assay to detect the expression level of Smad3 in mouse lung tissue; Use bioinformatics websites such as TargetScan7 and miRDB to predict the target gene of miR-29a. When the metrological date were satisfied with normal distribution, Mean±SD was used for comparison between groups, t test was used for two indepent samples, and LSD method was used when the variance was homogeneity in pairwise comparison. Results: HE staining showed that the Nd(2)O(3) group of mice showed obvious infiltration of inflammatory cells and structural disorder of alveoli in the early stage of lung tissue. At 28 days, the collagen fibers in the mouse lung tissue increased and the lung tissue showed fibrotic honeycomb like changes. The degree of pulmonary fibrosis in the Nd(2)O(3)+miR-29a agomir group of mice was significantly reduced; The content of TGF-β1 and CTGF in the lung tissue of mice in the Nd(2)O(3)+miR-29a agomir group was lower than that in the Nd(2)O(3)+NC agomir group (P<0.05) ; The relative expression level of TGF-β1 in the lung tissue of mice in the Nd(2)O(3)+miR-29a agomir group was lower than that in the Nd(2)O(3)+NC agomir group (P<0.05) ; The expression level of Smad3 in the nucleus of the Nd(2)O(3)+miR-29a agomir group was lower than that of the Nd(2)O(3)+NC agomir group (P<0.05). The prediction results of bioinformatics websites have found 152 downstream target genes related to miR-29a, among which FBN1, MAP2K6, KPNB1, COL1A2, SNIP1, LAMC1, and SP1 genes may be related to the regulatory effect of miR-29a on TGF-β1/Smad3 signaling pathway. Conclusion: miR-29a may affect lung fibrosis induced by rare earth Nd(2)O(3) exposure in mice by regulating TGF-β1/Smad3 signaling pathway. Overexpression of miR-29a may inhibit TGF-β1/Smad3 signaling pathway and reduce the degree of pulmonary fibrosis in mice.

Genome wide association study reveals new genes for resistance to striped stem borer in rice (Oryza sativa L.).

Rice is one of the most important food crops in the world and is important for global food security. However, damage caused by striped stem borer (SSB) seriously threatens rice production and can cause significant yield losses. The development and use of resistant rice varieties or genes is currently the most effective strategy for controlling SSB. We genotyped 201 rice samples using 2849855 high-confidence single nucleotide polymorphisms (SNPs). We conducted a genome-wide association study (GWAS) based on observed variation data of 201 rice cultivars resistant to SSB. We obtained a quantitative trait locus (QTL)-qRSSB4 that confers resistance to SSB. Through annotation and analysis of genes within the qRSSB4 locus, as well as qRT-PCR detection in resistant rice cultivars, we ultimately selected the candidate gene LOC_Os04g34140 (named OsRSSB4) for further analysis. Next, we overexpressed the candidate gene OsRSSB4 in Nipponbare through transgenic methods, resulting in OsRSSB4 overexpressing lines (OsRSSB4OE). In addition, we evaluated the insect resistance of OsRSSB4OE lines using wild type (Nipponbare) as a control. The bioassay experiment results of live plants showed that after 20 days of inoculation with SSB, the withering heart rate of OsRSSB4OE-34 and OsRSSB4OE-39 lines was only 8.3% and 0%, with resistance levels of 1 and 0, respectively; however, the withering heart rate of the wild-type reached 100%, with a resistance level of 9. The results of the in vitro stem bioassay showed that, compared with the wild-type, the average corrected mortality rate of the SSB fed on the OsRSSB4OE line reached 94.3%, and the resistance reached a high level. In summary, we preliminarily confirmed that OsRSSB4 positively regulates the defense of rice against SSB. This research findings reveal new SSB resistance gene resources, providing an important genetic basis for SSB resistance breeding in rice crops.

A prognosis model for predicting immunotherapy response of esophageal cancer based on oxidative stress-related signatures.

Oxidative stress (OS) is intimately associated with tumorigenesis and has been considered a potential therapeutic strategy. However, the OS-associated therapeutic target for esophageal squamous cell carcinoma (ESCC) remains unconfirmed. In our study, gene expression data of ESCC and clinical information from public databases were downloaded. Through LASSO-Cox regression analysis, a risk score (RS) signature map of prognosis was constructed and performed external verification with the GSE53625 cohort. The ESTIMATE, xCell, CIBERSORT, TIMER, and ImmuCellAI algorithms were employed to analyze infiltrating immune cells and generate an immune microenvironment (IM). Afterward, functional enrichment analysis clarified the underlying mechanism of the model. Nomogram was utilized for forecasting the survival rate of individual ESCC cases. As a result, we successfully constructed an OS-related genes (OSRGs) model and found that the survival rate of high-risk groups was lower than that of low-risk groups. The AUC of the ROC verified the strong prediction performance of the signal in these two cohorts further. According to independent prognostic analysis, the RS was identified as an independent risk factor for ESCC. The nomogram and follow-up data revealed that the RS possesses favorable predictive value for the prognosis of ESCC patients. qRT-PCR detection demonstrated increased expression of MPC1, COX6C, CYB5R3, CASP7, and CYCS in esophageal cancer patients. In conclusion, we have constructed an OSRGs model for ESCC to predict patients' prognosis, offering a novel insight into the potential application of the OSRGs model in ESCC.

Protective effect and regulatory mechanism of salidroside on skin inflammation induced by imiquimod in psoriasis mice

Salidroside (SAL) is a glucoside of tyrosol commonly existing in the roots of Rhodiola rosea. This study unveils the protective effect of SAL on skin inflammation in imiquimod (IMQ)-induced psoriasis. The mouse model of psoriasis was established by local application of IMQ, and SAL efficacy was evaluated through PASI scoring, H&E staining, and skin tissue pathology observation. The HaCaT cell model was established by interferon (IFN)-γ induction, followed by MTT assay detection of cell viability, detection of ROS, SOD, MDA, and CAT levels in skin tissues and cells using reagent kits, ELISA detection of inflammatory factors (TNF-α, IL-6, IL-1β), and qRT-PCR detection of psoriasis-related genes (S100a9, Cxcl1, Cxcl2) as well as miR-369-3p and SMAD2 expressions. The binding relationship between miR-369-3p and SMAD2 was validated using dual-luciferase reporter assay. SAL treatment reduced PASI scores and alleviated psoriasis symptoms of IMQ-induced mice, and also augmented the viability and subsided the oxidative stress and inflammation of IFN-γ-treated HaCaT cells. SAL treatment restrained miR-369-3p expression but elevated SMAD2 expression. Mechanistically, miR-369-3p targeted SMAD2 expression. miR-369-3p overexpression or SMAD2 inhibition partially offset the alleviating effect of SAL on psoriasis skin inflammation. In conclusion, SAL alleviates skin inflammation in IMQ-induced psoriasis mice via the miR-369-3p/SMAD2 axis.