Quantitative Real-time PCR Analysis Research Articles

HYQTD Drug-containing Serum Alleviates H2O2-induced Endothelial Oxidative Damage by Increasing Mitochondrial ATP Synthesis and Inhibiting ROS.

Atherosclerosis (AS) is caused by the endothelium injury associated with oxidative stress. Previous studies have shown that the Phlegm-Eliminating and Stasis- Transforming Decoction (Huayu Qutan Decoction, HYQTD) has mitochondrial protective function. The objective of this research was to explore how HYQTD drug-containing serum (HYQTD-DS) could potentially protect mitochondrial energy production in endothelial cells (ECs) from injury caused by hydrogen peroxide (H2O2)-induced oxidative damage in AS through SIRT1/PGC-1α/ Nrf2 pathway. After preparation of containing serum, the cells were divided into various categories, such as control group, H2O2 group (an oxidative damage model), HYQTD group, Selisistat (EX527, a SIRT1 inhibitor) combined with H2O2 group, and EX527 combined with HYQTD group. The evaluation of oxidative stress involved measuring reactive oxygen species (ROS) and malondialdehyde (MDA) generation, as well as Superoxide Dismutase (SOD) activity. Mitochondrial function and ultrastructure were measured by Transmission electron microscopy (TEM), mitochondrial membrane potential (MMP), rate of oxygen consumption (OCR), respiratory chain complex activities, and ATP production. The key proteins and gene levels in the SIRT1/PGC-1α/Nrf2 pathway was quantified by quantitative real-time PCR (RT-PCR) and Western blotting analysis. We found oxidative stress, mitochondrial damage, and mitochondrial energy disorder in H2O2-induced ECs. However it indicated a marked reversal after pretreated with HYQTD-DS. Mechanistically, EX527 induced increased oxidative stress, worse mitochondrial dysfunction, and less ATP synthesis. We demonstrated that HYQTD-DS attenuated oxidative stress, improved mitochondrial function, and up-regulated mitochondrial ATP synthesis by activating SIRT1/PGC- 1α/Nrf2 pathway-induced mitochondrial biogenesis and its downstream NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDV2).

Effects of Environmentally Relevant Concentrations of Roundup on Oxidative-Nitrative Stress, Cellular Apoptosis, Prooxidant-Antioxidant Homeostasis, Renin andCYP1A Expressions in Goldfish: Molecular Mechanisms Underlying Kidney Damage During Roundup Exposure.

Roundup is one of the most widely used glyphosate-based harmful herbicides in the United States as well as globally, which poses a severe risk for terrestrial and aquatic organisms. In order to identify the detrimental effects of Roundup exposure in aquatic organisms, we investigated the environmentally relevant concentrations of Roundup exposure (low dose: 0.5 μg/L and high dose: 5.0 μg/L for 2 weeks) on renin expression, oxidative-nitrative stress biomarkers (e.g., 2,4-dinitrophenol, DNP; and 3-nitrotyrosine protein, NTP), prooxidant-antioxidant enzymes expressions (e.g., superoxide dismutase, SOD; and catalase, CAT), cellular apoptosis, and cytochrome P450 1A (CYP1A) mRNA levels in the kidneys of goldfish (Carassius auratus). Histopathological and in situ TUNEL analyses showed widespread tissue disruption (e.g., bowman's capsule shrinkage, melanin pigment formation, etc.) and induced apoptotic nuclei in the kidneys of goldfish. Immunohistochemical and quantitative real-time PCR (qRT-PCR) analyses showed a significant (p < 0.05) increase in the expression of renin, DNP, NTP, SOD, and CAT, as well as CYP1A mRNA levels in the kidneys of fish exposed to Roundup. These results suggest that environmentally relevant concentrations of Roundup disrupt kidney architecture by inducing oxidative-nitrative stress, cellular apoptosis, and change in osmoregulatory enzymes (i.e., renin) and prooxidant-antioxidant systems in the kidneys of teleost fishes.

OsOZ1, a zinc finger motif-containing protein, is essential for chloroplast development through RNA editing and intron splicing in rice

Abstract The chloroplast is the primary organelle for photosynthesis in plants. A clade of proteins containing RanBP2-type zinc-finger domain, known as the Organelle Zinc (OZ) finger family, are known or predicted to be targeted to mitochondria or chloroplast. While some OZ proteins have been studied in Arabidopsis, there are few reports of their roles in rice. In this study, we characterized the Organelle Zinc finger family gene OsOZ1 and generated the oz1 mutants using the CRISPR-Cas9 genome editing system in rice. The oz1 mutants exhibited a seedling-lethal chlorosis phenotype, with a significantly reduced chlorophyll content at the third-leaf stage compared to the wild type. Transmission electron microscopy and quantitative real-time PCR analysis revealed that the oz1 mutants displayed abnormal chloroplast development relative to the wild type, along with significant alterations in gene expression levels related to chloroplast development and photosynthesis. The oz1 mutants demonstrated defects in chloroplast RNA editing, particularly in atpA-1148, ndhB-467, ndhB-586, ndhB-737 and ndhB-830, while the splicing of ndhA, trnG and ycf3-1 was significantly decreased in the oz1 mutants compared to the wild type. The yeast two-hybrid assay demonstrated that OsOZ1 interacts with the PPR protein OsSLC1, the ORRM protein OsORRM1 and the MORF/RIP protein OsMORF9. This suggests that OsOZ1 may form complexes with OsSLC1, OsORRM1 and OsMORF9, thereby participating in chloroplast RNA editing and splicing activities. Overall, these findings indicate that OsOZ1 plays a crucial role in chloroplast development by regulating the editing and splicing of chloroplast RNA in rice.

Phytoalexin deficient 4 is associated with the lesion mimic trait in watermelon clalm mutant (Citrullus lanatus)

In watermelon (Citrullus lanatus), lesion mimic is a rare, valuable trait that can be used by breeders for selection at early growth stages. In this study, we tested a seven-generation family to determine the inheritance and genetic basis of this trait. As revealed by analysis of the lesion mimic mutant clalm, this trait is controlled by a single dominant gene. Whole genome resequencing–bulked segregant analysis demonstrated that this gene is located on chromosome 4 from 3,760,000 bp to 7,440,000 bp, a region corresponding to a physical distance of 3.68 Mb encompassing approximately 72 annotated genes. There are 6 genes with non-synonymous mutation SNP sites. The predicted target gene, ClCG04G001930, encodes a Phytoalexin deficient 4 (PAD4), a protein that plays an important regulatory role in leaf senescence in many plant species. According to quantitative real-time PCR analysis, the expression level of ClCG04G001930 was significantly higher in the clalm mutant than in normal watermelon. Twenty-five SNPs were identified in the ClCG04G001930 gene of F2 individuals of the clalm mutant. Overexpression the ClCG04G001930 gene, designated as ClPAD4, yielded transgenic lines whose leaves gradually developed chlorotic lesions over 3 weeks. RNA interference of the ClPAD4 yielded transgenic lines whose cotyledon prone to diseased over 2 weeks. Our results suggest that ClPAD4 might be the candidate gene responsible for lesion mimic in the clalm mutant. Our findings may serve as a foundation for elucidating the mechanism underlying the molecular metabolism of programmed cell death and should be useful for marker-assisted selection breeding in watermelon.

Melatonin inhibits ferroptosis through the ATF3/GPX4 signaling pathway to relieve myocardial ischemia-reperfusion injury in rats.

Melatonin (MEL), functioning as a circulating hormone, is important for the regulation of ferroptosis in different health scenarios and acts as a crucial antioxidant in cardiovascular diseases. However, its specific function in ferroptosis related to myocardial ischemia-reperfusion injury (MIRI) remains to be fully elucidated. In our research, we utilized a rat model of MIRI induced by coronary artery ligation, along with a cell model subjected to hypoxia/reoxygenation (H/R). We evaluated relevant genes and proteins by real-time fluorescent quantitative PCR and Western blot analysis. To evaluate myocardial tissue damage and cell injury, we employed cell counting kit-8 assays, flow cytometry, hematoxylin-eosin staining, and 2,3,5-triphenyltetrazolium chloride staining techniques. Our results show that administering MEL notably reduces the concentrations of cTnT, CK-MB, and lactate dehydrogenase in the serum of MIRI rats, mitigates the extent of myocardial infarction, improves the recovery of pathological conditions in myocardial tissues, and reduces the concentrations of Fe2+, malondialdehyde (MDA), and reactive oxygen species (ROS) in the myocardial tissue, while also promoting increased glutathione levels. Moreover, MEL can also restore the reduced viability of H9C2 cells caused by H/R or ferroptosis inducers (RSL3), reduce the cellular content of Fe2+, MDA, and ROS, and inhibit ferroptosis. Mechanistically, MEL promotes the expression of GPX4 by downregulating the expression of ATF3, thereby inhibiting ferroptosis in cardiomyocytes and ultimately alleviating the process of MIRI. Our study demonstrates that MEL ameliorates MIRI by inhibiting ferroptosis.

Effect of selenium on the dysfunction of rat salivary glands induced by 131I and expression of insulin-like growth factors and aquaporins.

To investigate the effects of selenium on functional and histopathological changes and mRNA expression levels of insulin-like growth factors 1 and 2 (IGF-1 and -2) and aquaporins 4 and 5 (AQP-4 and -5) in 131I-induced damaged rat parotid glands. Rats were divided into three groups: iodotherapy-with-selenium, iodotherapy-only, and control. Rats in the iodotherapy-with-selenium group were intragastrically administered 131I on the first day and selenomethionine through drinking water. Rats in the iodotherapy-only group were only administered 131I. Changes in parotid gland function were evaluated using the functional parameters of salivary gland dynamics imaging pre-experiment and on days 7, 30, and 90 post-treatment. Immunofluorescence and quantitative real-time PCR analyses detected IGF-1, IGF-2, AQP-4, and AQP-5 expression levels in tissues. The gland-to background ratio at a maximum count (G/BGmax), Tmax/Tmin, and Smax values were significantly impacted over time in the iodotherapy-with-selenium group; on day 30, the G/BGmax value was significantly higher than that in the iodotherapy-only group. Histopathological analysis revealed that on days 30 and 90, the iodotherapy-with-selenium group displayed greater parotid gland repair than the iodotherapy-only group. In the iodotherapy-with-selenium group, fluorescence intensity and mRNA levels of AQP-5 increased with the selenium supplementation period, reaching significantly higher levels on days 30 and 90 than in the iodotherapy-only group. Whereas the fluorescence intensity and mRNA levels of IGF-1 in the iodotherapy-with-selenium group were significantly higher on day 7 than on day 30 in the iodotherapy-only group. Selenium may repair 131I-induced tissue and functional damage in rat salivary glands by upregulating AQP-5 and IGF-1 expression.

Genome-wide characterization of the MADS-box gene family in Paeonia ostii and expression analysis of genes related to floral organ development

BackgroundPaeonia section Moutan DC. is a significant perennial subshrub, the ornamental value of which heavily depends on the type of flower it possesses. MADS-box transcription factors have a particular impact on the intricate process of floral organ development and differentiation. The release of the whole-genome data from Paeonia ostii now allows us to conduct a thorough investigation of the tree peony MADS-box gene family.ResultsIn this study, we identified 110 MADS-box genes in Paeonia ostii that were classified into 5 subgroups. Gene structure, domain and motif analyses revealed the conservation of the structure of these subgroups. Analysis of the cis-acting elements revealed that the 110 PoMADS genes contained different kinds of hormones and stress-related cis-acting elements in their promoter regions. Quantitative real-time PCR analysis was employed to validate the expression patterns of some PoMADS genes related to floral organ development. Genome collinearity analysis with Arabidopsis and grape revealed the conservation of PoMADS genes during evolution. A total of 857 SSRs were identified by analysing the genome sequences of identified genes. We additionally created protein‒protein interaction networks for PoMADS proteins and analysed proteins that could interact among PoMADSs in Arabidopsis thaliana and grape.ConclusionThese findings offer fundamental insights for understanding the function of the MADS-box gene family, which can aid in the selection and breeding of tree peony varieties with high ornamental value in addition to supporting the understanding of the process of tree peony floral organogenesis.

NKX6.3 modulation of mitotic dynamics and genomic stability in gastric carcinogenesis

BackgroundGastric cancer remains a significant global health challenge, characterized by poor prognosis and high mortality rates. Mitotic integrity and genomic stability are crucial in maintaining cellular homeostasis and preventing tumorigenesis. The transcription factor NKX6.3 has emerged as a potential regulator of these processes in gastric epithelial cells, prompting an investigation into its role in gastric cancer development.MethodsWe employed a combination of in vitro and in vivo techniques to elucidate the impact of NKX6.3 depletion on mitotic dynamics and genomic stability in gastric epithelial cells. Quantitative real-time PCR and Western blot analyses were conducted to assess the expression of mitosis-related genes and proteins. Flow cytometry was utilized to evaluate cell cycle distribution, while immunofluorescence microscopy enabled the visualization of mitotic abnormalities. Statistical analyses, including Student’s t-test and ANOVA, were performed to determine the significance of our findings.ResultsOur results demonstrate that NKX6.3 depletion leads to significant mitotic defects, characterized by increased chromosome misalignment and lagging chromosomes during anaphase. These abnormalities corresponded with elevated levels of genomic instability markers, indicating compromised genomic integrity. Furthermore, the loss of NKX6.3 resulted in altered expression of key regulatory proteins involved in mitosis and DNA repair pathways, suggesting a mechanistic link between NKX6.3 and the maintenance of genomic stability in gastric epithelial cells. Depletion of NKX6.3 resulted in accelerated cell cycle progression and the formation of abnormal mitotic figures, leading to genomic instability characterized by increased DNA content and structural abnormalities. In both in vitro and xenograft models, the depletion of NKX6.3 significantly upregulated AurkA and TPX2, which correlated with gains in DNA copy number. An inverse relationship was observed between NKX6.3 expression and the levels of AurkA and TPX2 in human gastric cancer tissues.ConclusionsThis study highlights the essential role of NKX6.3 in regulating mitotic integrity and genomic stability in gastric carcinogenesis. The findings suggest that targeting NKX6.3 may offer a novel therapeutic strategy for improving treatment outcomes in gastric cancer by restoring mitotic fidelity and genomic stability.Trial registrationThis study was not registered.

Identification of HXK Gene Family and Expression Analysis of Salt Tolerance in Buchloe dactyloides.

Buchloe dactyloides is one of the typical ecological grass species, characterized by its strong salt tolerance. Hexokinase (HXK) plays a crucial role in plant growth, development, and resistance to abiotic stresses. To understand the function of HXKs in the salt tolerance of B. dactyloides, this study identified and analyzed the HXK gene family members using the whole-genome data of B. dactyloides. Additionally, transcriptomic methods were employed to investigate the expression levels and stress response patterns of the HXK family genes under salt stress. The results showed that 25 HXK genes were identified in the B. dactyloides HXK gene family, which were classified into three subfamilies based on the phylogenetic tree. Members within the same subfamily exhibited similar gene structures and conserved motifs. The promoter regions of BdHXKs contained numerous cis-regulatory elements associated with plant hormone responses, plant growth and development, and resistance to abiotic stresses. Quantitative real-time PCR analysis provided preliminary evidence that the BdHXK5, BdHXK7, and BdHXK23 genes might play important roles in the salt tolerance regulation of B. dactyloides. These findings offer a theoretical foundation for further elucidating the functions and molecular regulatory mechanisms of BdHXKs under salt stress. This study has provided a theoretical basis for the breeding of new varieties of ecological restoration grasses with stronger salt tolerance and better growth and development. This is of great significance for the improvement and ecological restoration of saline-alkali land.

Prioritization of candidate genes regulating the dwarfness in rice by integration of whole-genome and transcriptome analyses.

Dwarfism is a major trait for developing lodging-resistant rice cultivars. Gamma irradiation-induced mutagenesis has proven to be an effective method for generating dwarf rice mutants. In this research, we isolated a dwarf mutant from Anna R (4) in the M2 generation and subsequently stabilized the trait through successive selfing of progeny across the M3-M7 generations. We then employed whole-genome re-sequencing (WGRS) and RNA sequencing (RNA-seq) analyses of Anna R (4) and the mutant (designated as ACM-20001) to elucidate the underlying mechanisms and identify candidate genes associated with dwarfness. Numerous genetic variations were identified between Anna (R) 4 and ACM-20001 through WGRS. In total, 2049 genetic variants, including 343 InDels and 1706 nonsynonymous SNPs, were identified across 697 genes. Additionally, RNA-seq analysis revealed 2,881 differentially expressed genes between the wild-type Anna (R) 4 and the mutant ACM-20001, with 1,451 genes up-regulated and 1,430 genes down-regulated in ACM-20001 compared to Anna (R) 4. By integrating WGRS and RNA-seq data with functional annotation analysis, we identified the most likely candidate genes (i.e., Os02g0506400, Os05g0515200, Os06g0154200 and Os08g0250900) related to dwarfness. Quantitative real-time PCR analysis verified the expression of these genes. Collectively, our study provides valuable insights in to the genes and mechanisms underlying dwarfness in rice. Further studies are required to elucidate the roles of these candidate genes in dwarfness, which contribute to advancements rice breeding programs.

Liraglutide mitigates dexamethasone-induced fatty acid synthase (FASN) and the cluster of differentiation36 (CD36) expression: a potential treatment for glucocorticoid-induced non-alcoholic fatty liver disease (NAFLD).

The clinical use of dexamethasone (DXM) is associated with the development of non-alcoholic fatty liver disease (NAFLD). However, the mechanisms by which DXM-induced NAFLD is still incompletely known. Therefore, the current study aims to test the hypothesis that DXM-induced NAFLD is mediated by dysregulation of key genes involved in lipid metabolism and liraglutide (LG) can ameliorate these effects. The histopathological and biochemical analysis assessed the effects of DXM and/or LG in liver tissue. The computational analysis was performed to detect the glucocorticoid response elements (GRE) in the promotor regions of FASN and CD36 genes. The effects of DXM and LG on the expression of FASN and CD36 were determined by real-time quantitative reverse transcription PCR (RT-qPCR), western blot (WB) and immunohistochemical (IHC) analyses. The NAFLD induced by high-fat diet (HFD) and DXM was manifested by increased levels of liver enzymes, deterioration of histological architecture of the liver tissue and accumulation of fat droplets. Computational analysis revealed that the promotor regions of FASN and CD36 harper several GRE. Most importantly, treatment with DXM decreased phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) levels, while LG upregulate it. In addition, treatment with DXM increased expression of FASN and CD36, whereas LG ameliorated these effects in a dose-dependent manner. DXM-induced NAFLD is mediated by upregulation of FASN and CD36 expression which may be attributed to GRE. LG coupling with DXM mitigates this induction by downregulating FASN and CD36 levels and therefore mitigates the development of NAFLD.

Copper Tolerance of Trichoderma koningii Tk10

Copper (Cu) is an essential micronutrient for all living organisms, serving as a cofactor for numerous enzymes. However, excessive copper concentrations can be harmful. To investigate copper tolerance in fungi, a copper-tolerant strain was isolated from soil and identified as Trichoderma koningii Tk10, with optimized culture conditions being established. Additionally, copper-related genes were analyzed through whole-genome sequencing. The results indicated that Tk10 exhibits a maximum copper tolerance of 5.4 mmol/L and a maximum adsorption rate of 51.5% under optimal cultivation conditions. Whole-genome sequencing revealed six genes associated with copper tolerance, including one superoxide dismutase gene, one peroxidase gene, and three catalase genes linked to copper stress. Furthermore, the enzyme activities of the catalase, superoxide dismutase, and peroxidase significantly increased, reaching levels that were 8.02, 4.12, and 3.88 times higher than those observed in the control group, respectively. A real-time quantitative PCR analysis indicated that three of these genes were significantly upregulated in response to copper stress. The findings of this study provide valuable insights into copper tolerance in filamentous fungi.

Genome-Wide Analysis and Expression Profiling of Watermelon VQ Motif-Containing Genes Under Abiotic and Biotic Stresses

Valine-glutamine (VQ) motif-containing proteins play important roles in diverse plant developmental processes and signal transduction in response to biotic and abiotic stresses. However, no systematic investigation has been conducted on VQ genes in watermelon. In this study, we identified 31 watermelon VQ genes, which were classified into six subfamilies (I–VI). All of the deduced proteins contained a conserved FxxxVQxL/F/VTG motif. Eleven ClVQs were involved in segment duplication, which was the main factor in the expansion of the VQ family in watermelon. Numerous stress- and hormone-responsive cis-elements were detected in the putative promoter region of the ClVQ genes. Green fluorescent protein fusion proteins for ten selected ClVQs were localized in the nucleus, but three ClVQs also showed signals in cell membranes and the cell wall, thus confirming their predicted divergent functionality. Quantitative real-time PCR (qRT-PCR) analysis indicated that the majority of ClVQ genes were specifically or preferentially expressed in certain tissues or organs, especially in the male flower. Analyses of RNA-sequencing data under osmotic, cold, and drought stresses and Cucumber green mottle mosaic virus (CGMMV) infection revealed that the majority of ClVQ genes, especially those from subfamily IV, were responsive to these stresses. The results provide useful information for the functional characterization of watermelon ClVQ genes to unravel their biological roles.

Anti-atherosclerotic effects of LncRNA NEXN-AS1 by regulation of the canonical inflammasome pathway of pyroptosis via NEXN.

. Pyroptosis is closely related to many chronic diseases including atherosclerosis, but the potential pathomechanisms are still unclear. This research aimed to explore how lncRNAs may contribute to pyroptosis and the potential mechanisms. . We performed in vitro assays to investigate the effects of a relatively newly discovered lncRNA, NEXN-AS1, on pyroptosis. Two types of vascular wall cells, namely, human vascular smooth muscle cells (VSMCs) and endothelial cells (ECs), were used as cell models in this study. A previously constructed lentivirus vector was used to overexpress lncRNA NEXN-AS1 and a small interfering RNA (siRNA) mimic against NEXN to knockdown NEXN. The mRNA and protein levels of molecules of pyroptosis in the canonical inflammasome pathway, including nucleotide-binding oligomerization segment-like receptor family 3 (NLRP3), caspase-1, gasdermin D (GSDMD), interleukin-1β (IL-1β), and interleukin-18 (IL-18) were detected using quantitative real-time PCR and western blot analysis, respectively. . We observed that lentivirus-mediated overexpression of lncRNA NEXN-AS1 increased the expression levels of NEXN and markedly reduced the expression of critical molecules involved in pyroptosis, including NLRP3, caspase-1, GSDMD, IL-1β, and IL-18, in both VSMCs and ECs. Furthermore, NEXN knockdown could reverse the effects of lncRNA NEXN-AS1 overexpression on pyroptosis. lncRNA NEXN-AS1 could act as an target for maintaining endothelium homeostasis, plaque stability, and delaying the progression of atherosclerosis.

Identification and characterisation of temporal abundance of microRNAs in synovial fluid from an experimental equine model of osteoarthritis.

MicroRNAs, a class of small noncoding RNAs, serve as post-transcriptional regulators of gene expression and are present in a stable and quantifiable form in biological fluids. MicroRNAs may influence intra-articular responses and the course of disease, but very little is known about their temporal changes in osteoarthritis. To identify miRNAs and characterise the temporal changes in their abundance in SF from horses with experimentally induced osteoarthritis. We hypothesised that the abundance of miRNA would change during disease progression. In vivo experiments. RNA extracted from synovial fluid obtained sequentially (Day 0, 28 and 70) from nine horses with experimentally induced osteoarthritis was subjected to small RNA sequencing using the Illumina Hiseq 4000 sequencing platform. Differentially abundant miRNAs underwent further validation and mapping of temporal abundance (Day 0, 14, 17, 21, 28, 35, 42, 49, 56, 63 and 70 days after osteoarthritis induction) by microfluidic reverse transcription quantitative real-time PCR. Bioinformatic analyses were performed to predict potential biological associations and target genes of the differentially abundant microRNAs. Small RNA sequencing revealed 61 differentially abundant microRNAs at an early osteoarthritis stage (Day 28), and subsequent reverse transcription quantitative real-time PCR analysis validated 20 of these. Significant biological functions of the differentially abundant microRNAs were apoptosis, necrosis, cell proliferation and cell invasion. Following validation, four microRNAs (miRNA-199b-3p, miRNA-139-5p, miRNA-1839 and miRNA-151-5p) were detected in more than 50% of the synovial fluid samples and had higher abundance in osteoarthritic than in control joints. Limited sample size. This is the first study to determine longitudinal changes in synovial fluid microRNA abundance in an equine model of osteoarthritis. Larger studies are needed in naturally occurring osteoarthritis to interrogate putative changes identified by this study.

Irisin reshapes bone metabolic homeostasis to delay age-related osteoporosis by regulating the multipotent differentiation of BMSCs via Wnt pathway.

Bone aging is linked to changes in the lineage differentiation of bone marrow stem cells (BMSCs), which show a heightened tendency to differentiate into adipocytes instead of osteoblasts. The therapeutic potential of irisin in addressing age-related diseases has garnered significant attention. More significantly, irisin has the capacity to enhance bone mass recovery and sustain overall bone health. Its mechanism of action in preventing osteoporosis has generated considerable interest within the research community. Nonetheless, the targeting effect of irisin on age-related osteoporosis and its underlying molecular biological mechanisms remain unclear. The specific role of irisin in osteogenic-adipogenic differentiation in young or aging BMSCs was evaluated by multiple cells staining and quantitative real-time PCR (RT-qPCR) analysis. RNA-seq and protein Western blotting excavated and validated the key pathway by which irisin influences the fate determination of aging BMSCs. The macroscopic and microscopic changes of bone tissue in aging mice were examined using Micro-computed tomography (Micro-CT) and morphological staining. It was noted that irisin affected the multilineage differentiation of BMSCs in a manner dependent on the dosage. Simultaneously, the Wnt signaling pathway might be a crucial mechanism through which irisin sustains the bone-fat balance in aging BMSCs and mitigates the decline in pluripotency. In vivo, irisin reduced bone marrow fat deposition in aging mice and effectively alleviating the occurrence of bone loss. Irisin mediates the Wnt signaling pathway, thereby influencing the fate determination of BMSCs. In addition, it is essential for preserving metabolic equilibrium in the bone marrow microenvironment and significantly contributes to overall bone health. The findings provide new evidence for the use of iris extract in the treatment of age-related osteoporosis.

Characterization of the ligand-binding properties of odorant-binding protein 38 from Riptortus pedestris when interacting with soybean volatiles.

Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) is a major soybean pest throughout East Asia that relies on its advanced olfactory system for the perception of plant-derived volatile compounds and aggregation pheromones for conspecific and host plant localization. Odorant binding proteins (OBPs) facilitate the transport of odorant compounds across the sensillum lymph within the insect olfactory system, enabling their interaction with odorant receptors (ORs). Real-time quantitative PCR (qRT-PCR) analyses, fluorescence-based competitive binding assays, and molecular docking analyses were applied to assess the expression and ligand-binding properties of OBP38 from R. peddestris. The qRT-PCR analyses revealed high levels of RpedOBP38 expression in the antennae without any apparent sex bias, and it was also highly expressed in the adult stage. Recombinant RpedOBP38 was prepared by expressing it in E. coli BL21 (DE3) followed by its purification with a Ni-chelating affinity column. RpedOBP38 was found to bind most strongly to trans-2-decenal (Ki = 7.440) and trans-2-nonenal (Ki = 10.973), followed by β-pinene, (+) -4-terpineol, carvacrol, methyl salicylate, and (-)-carvone. The 3D structure of RpedOBP38 contains six α-helices and three interlocked disulfide bridges comprising a stable hydrophobic binding pocket. In a final series of molecular docking analyses, several polar (e.g., His 94, Glu97) and nonpolar (e.g., Leu29, Ile59) residues were found to be involved in RpedOBP38-ligand binding. These data support a role for RpedOBP38 in the perception of volatiles derived from host plants, providing important insight into the mechanisms that govern olfactory recognition in R. pedestris, thereby informing the development of ecologically friendly approaches to managing R. pedestris infestations.

Identifying Pyroptosis-Hub Genes and Inflammation Cell Type-Related Genes in Ischemic Stroke.

Stroke is the second-leading global cause of death. The damage attributed to the immune storm triggered by ischemia-reperfusion injury (IRI) post-stroke is substantial. However, data on the transcriptomic dynamics of pyroptosis in IRI are limited. This study aimed to analyze the expression of key pyroptosis genes in stroke and their correlation with immune infiltration. Pyroptosis-related genes were identified from the obtained middle cerebral artery occlusion (MCAO) datasets. Differential expression and functional analyses of pyroptosis-related genes were performed, and differences in functional enrichment between high-risk and low-risk groups were determined. An MCAO diagnostic model was constructed and validated using selected pyroptosis-related genes with differential expression. High- and low-risk MCAO groups were constructed for expression and immune cell correlation analysis with pyroptosis-related hub genes. A regulatory network between pyroptosis-related hub genes and miRNA was also constructed, and protein domains were predicted. The expression of key pyroptosis genes was validated using an MCAO rat model. Twenty-five pyroptosis genes showed differential expression, including four hub genes, namely WISP2, MELK, SDF2L1, and AURKB. Characteristic genes were verified using real-time quantitative PCR analyses. The high- and low-risk groups showed significant expression differences for WISP2, MELK, and SDF2L1. In immune infiltration analysis, 12 immune cells showed differences in expression in MCAO samples. Further analysis demonstrated significant positive correlations between the pyroptosis-related hub gene SDF2L1 and immune cell-activated dendritic cells in the high-risk group and immune cell natural killer cells in the low-risk group. This study identified four pyroptosis-related hub genes, with elevated WISP2, MELK, and SDF2L1 expression closely associated with the high-risk group. The analysis of inflammatory cell types in immune infiltration can predict ischemic stroke risk levels and help to facilitate treatment.

Doublecortin-like kinase 1 promotes stem cell-like properties through the Hippo-YAP pathway in prostate cancer.

Background: Doublecortin-like kinase 1 (DCLK1) has been revealed to be involved in modulating cancer stemness and tumor progression, but its role in prostate cancer (PCa) remains obscure. Castration-resistant and metastatic PCa exhibit aggressive behaviors, and current therapeutic approaches have shown limited beneficial effects on the overall survival rate of patients with advanced PCa. This study aimed to investigate the biological role and potential molecular mechanism of DCLK1 in the progression of PCa. Methods: The role of DCLK1 in maintaining PCa stem cell-like properties was explored via gain- and loss-of-function studies, including colony formation assays, sphere formation assays and measurement of stemness-related marker expression. A set of transcriptomic data for patients with PCa was downloaded from The Cancer Genome Atlas to analyze the correlations between DCLK1 and Hippo pathway gene expression. The mechanism by which DCLK1 regulates Hippo signaling and cancer stemness was further investigated in vitro by methods such as Western blot analysis, quantitative real-time PCR analysis, immunofluorescence staining, and luciferase reporter assays and in vivo by animal studies. Results: The gain- and loss-of-function studies demonstrated that upregulating DCLK1 promoted but downregulating DCLK1 suppressed aspects of the PCa stem cell-like phenotype, including colony formation, sphere formation and the expression of stemness-related markers (c-Myc, OCT4, CD44, NANOG, SOX2, and KLF4). Importantly, bioinformatics analysis indicated that DCLK1 is closely correlated with the Hippo signaling pathway in PCa. Further in vitro assays revealed that DCLK1 inhibits the Hippo signaling pathway, leading to yes-associated protein (YAP) activation via large tumor suppressor homolog 1 (LATS1). Moreover, the effect of DCLK1 on abolishing stemness traits in PCa was observed after treatment with verteporfin, a small molecule inhibitor of YAP. Consistent with the in vitro findings, the in vivo findings confirmed that DCLK1 promoted the tumorigenicity and stem cell-like traits of PCa cells via Hippo-YAP signaling. Conclusion: DCLK1 promotes stem cell-like characteristics by inducing LATS1-mediated YAP signaling activation, ultimately leading to PCa tumor growth and progression. Thus, our findings identify an attractive candidate for the development of cancer stem cell-targeted therapies to improve treatment outcomes in advanced PCa.

Decoding the aroma of Rosa canina L.: Chemical composition and gene expression.

The aromatic profile of Rosa canina L. petals hold immense potential for the fragrance and pharmaceutical industries. This study aims to investigate the chemical composition and gene expression patterns across different floral development stages to uncover the biosynthetic pathways of floral scent. Essential oils (EOs) were extracted from petals at five developmental stages (S1-S5) and analyzed using Gas Chromatography-Mass Spectrometry (GC-MS), identifying 20 distinct compounds. RNA isolation and quantitative real-time PCR (qRT-PCR) analysis were performed to assess gene expression. Stage S3, notable for its enhanced aromatic profile, was dominated by terpenoid compounds such as β-Citronellol (1.18%), Caryophyllene (8.59%), β-Selinene (1.50%), and Caryophyllene oxide (0.50%), indicating significant upregulation of terpenoid biosynthesis genes. qRT-PCR analysis revealed that CCD1 had the highest expression in S4 (9.51-fold), while DXR and DXS peaked at S3 with fold changes of 29058.38 and 73.35, respectively. Other genes like AAT1, LIS, and GPS also showed peak expressions at S3 with fold changes of 1.33, 10.70, and 1.18, respectively. PAR exhibited the highest expression in S1, while GGPPS peaked in S4 (2.01-fold). Clustering analysis indicated distinct groupings of developmental stages and gene expression patterns, with strong correlations between specific genes and compounds, such as CCD1 with GGPPS (0.78) and β-Citronellol with Caryophyllene (0.92). Principal Component Analysis (PCA) highlighted significant contributions of AAT1, GPS, and nonadecane compounds to the overall variance. These findings provide a comprehensive understanding of the chemical and genetic factors shaping the aromatic profile of R. canina, with promising applications for both the fragrance and pharmaceutical sectors. The study's innovation lies in the detailed correlation between EO composition and gene expression, presenting new insights into the biosynthetic pathways of floral scent.