qRT-PCR Analysis Research Articles

Genome-Wide Identification of Superoxide Dismutase (SOD) Gene Family in Cymbidium Species and Functional Analysis of CsSODs Under Salt Stress in Cymbidium sinense

Superoxide dismutase (SOD) enzymes are essential for reducing oxidative damage resulting from overabundant reactive oxygen species under abiotic stress. While the SOD gene family has been extensively studied in many species, research focusing on Cymbidium species remains limited. In this study, a comprehensive analysis of the SOD gene family in three Cymbidium genomes was conducted. A total of 23 SOD genes were identified, with nine SODs in C. sinense, eight in C. ensifolium, and six in C. goeringii. These SOD genes were categorized into three clades: Cu/Zn-SOD, Fe-SOD, and Mn-SOD, with the Cu/Zn-SOD being the most abundant in these three types. This classification was supported by analyses of conserved domains, motifs, and phylogenetic relationships. Cis-element prediction showed that stress-responsive elements were identified in most SODs. Transcriptomic data revealed that seven CsSODs exhibited a border expression in all sequenced tissues, while two exhibited undetectable expression levels. Further qRT-PCR analysis showed that all CsSODs were upregulated under salt stress, with some exhibiting significant changes in expression. These findings all highlight the crucial role of CsSODs in the salt stress response and provide valuable insights for further breeding salt-tolerance varieties of C. sinense.

Lipid-encapsulated gold nanoparticles: an advanced strategy for attenuating the inflammatory response in SARS-CoV-2 infection

BackgroundNanodrugs play a crucial role in biomedical applications by enhancing drug delivery. To address safety and toxicity concerns associated with nanoparticles, lipid-nanocarrier-based drug delivery systems have emerged as a promising approach for developing next-generation smart nanomedicines. Ginseng has traditionally been used for various therapeutic purposes, including antiviral activity. This study aimed to prepare a biocompatible and therapeutically potent Korean ginseng nanoemulsion (KGS-NE) using ginseng seed oil (GSO), optimize its encapsulation and drug delivery efficiency, and evaluate its antiviral activity.ResultsVarious techniques were utilized to confirm the KGS-NE formation. Energy-dispersive X-ray spectroscopy identified gold nanoparticles with the highest Au peak at 2.1 keV. Selected area diffraction patterns revealed crystallographic structures. FT-IR spectrometry detected functional groups, with peaks at 2922.09 cm−1 (alkene C–H stretching), 1740.24 cm−1 (aldehyde C=O stretching), and 1098.07 cm−1 (C–O stretching in secondary alcohol). Storage stability studies showed that KGS-NE maintained its size and stability for 6 months at 4 °C. The KGS-NE exhibited a dose-dependent suppression of HCoV-OC43 viral replication in Vero E6 cells. RNA sequencing analysis unveiled differentially expressed genes (DEGs) specifically involved in the ABC transporters signaling pathway. KGS-NE oral administration facilitated the recovery of mice induced with the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, as confirmed by inflammatory markers expression in lung tissue. In the Syrian hamster infected with the SARS-CoV-2 model, the lungs dissected showed enlarged morphology and induced inflammatory cytokines. This effect was mitigated with KGS-NE oral administration, as observed through H&E and qRT-PCR analysis. Biochemical analysis at various oral administration concentrations demonstrated that KGS-NE had no adverse effects on the kidney and liver.ConclusionsOur findings strongly suggest that oral administering KGS-NE in mice and Syrian hamster models has the potential to effectively mitigate lung inflammation against coronavirus. This indicates a promising new strategy for developing the antiviral nano-agent against SARS-CoV-2.Graphical

Investigation into the influence of mild hypothermia on regulating ferroptosis through the P53-SLC7A11/GPX4 signaling pathway in sepsis-induced acute lung injury

BackgroundSepsis-induced acute lung injury (S-ALI) significantly contributes to unfavorable clinical outcomes. Emerging evidence suggests a novel role for ferroptosis in the pathophysiology of ALI, though the precise mechanisms remain unclear. Mild hypothermia (32–34 °C) has been shown to inhibit inflammatory responses, reduce oxidative stress, and regulate metabolic processes. P53 has been reported to downregulate the transcriptional activity of solute carrier family 7 member 11 (SLC7A11), thereby limiting cystine uptake. This reduction in cystine availability compromises the activity of Glutathione peroxidase 4 (GPX4), a cystine-dependent enzyme, ultimately increasing cellular susceptibility to ferroptosis. However, it remains unclear whether mild hypothermia exerts protective effects through the P53-SLC7A11/GPX4 signaling pathway. This study investigates the influence of mild hypothermia on ferroptosis mediated by the P53-SLC7A11/GPX4 pathway in S-ALI.MethodsThis study utilized both in vivo and in vitro models. In the vivo model, 64 Sprague–Dawley rats were employed, with 40 analyzed for survival outcomes. Sepsis was induced using the cecum ligation and puncture (CLP) method, after which rats were subjected to either normothermic (36–38 °C) or mild hypothermic (32–34 °C) conditions for a duration of 10 h. Twelve hours post-surgery, blood samples, bronchoalveolar lavage fluid, and lung tissue samples were harvested for histological analysis, measurement of inflammatory markers, wet/dry ratios, blood gas analysis, assessment of oxidative stress and ferroptosis, Western blotting, and RT-qPCR analysis. In the in vitro model, RLE-6TN cells were exposed to lipopolysaccharide (LPS) for 24 h under normothermic and mild hypothermic conditions. These cells were then evaluated for cell viability, inflammatory markers, oxidative stress levels, ferroptosis markers, as well as Western blot and RT-qPCR analyses.ResultsCLP-induced sepsis led to elevated levels of inflammatory markers, increased lung injury scores, and heightened oxidative stress markers. These detrimental effects were significantly ameliorated by mild hypothermia. Furthermore, mild hypothermia reversed the modified expression of P53, SLC7A11, and GPX4 signaling molecules. Notably, mild hypothermia also improved the 5-day survival rate of CLP rats.ConclusionMild hypothermia attenuates S-ALI and modulates ferroptosis through the P53-SLC7A11/GPX4 signaling pathway.

Undernutrition affects metabolism and immune response in subcutaneous adipose tissue of pregnant ewes.

Pregnant ewes mobilize body fat to increase energy supply for fetal growth and development upon undernutrition, which disrupts the metabolic homeostasis of the body. However, the comprehensive metabolic changes in subcutaneous adipose tissue upon undernutrition are poorly understood. In this study, an undernutrition sheep model was established to investigate the effects of undernutrition on metabolic changes, immune response, and inflammation in subcutaneous fat through transcriptome, RT-qPCR, and metabolome analysis. Results showed that undernutrition changed the total transcriptional and metabolic profiles of adipose tissue. Compared to the controls, differentially expressed genes (DEGs) involved in fatty acid synthesis, triglyceride genesis, and lipid transport were downregulated in undernourished ewes, while DEGs related to fatty acid and triglyceride degradation were upregulated. Almost all lipid-related differential metabolites (DMs) were downregulated. DEGs and DMs involved in glucose metabolism and glycogen degradation were downregulated, while glycogen synthesis and carbohydrate transport were upregulated. DEGs linked to amino acid degradation were upregulated and some amino acids and derivatives were downregulated. KEGG pathway analysis showed complement and coagulation cascades were enriched significantly by DEGs, and DEGs related to coagulation, macrophage, and inflammation were upregulated while DEGs associated with the complement system were downregulated. Undernutrition during late gestation disrupted the metabolism of lipids, carbohydrates, and amino acids in adipose tissue, which weakened the complement system and immune response and may have ultimately led to inflammation.

Nrf2 Regulates Basal Glutathione Production in Astrocytes

Astrocytes produce and export glutathione (GSH), an important thiol antioxidant essential for protecting neural cells from oxidative stress and maintaining optimal brain health. While it has been established that oxidative stress increases GSH production in astrocytes, with Nrf2 acting as a critical transcription factor regulating key components of the GSH synthetic pathway, the role of Nrf2 in controlling constitutive GSH synthetic and release mechanisms remains incompletely investigated. Our data show that naïve primary mouse astrocytes cultured from the cerebral cortices of Nrf2 knockout (Nrf2−/−) pups have significantly less intracellular and extracellular GSH levels when compared to astrocytes cultured from Nrf2 wild-type (Nrf2+/+) pups. Key components of the GSH synthetic pathway, including xCT (the substrate-specific light chain of the substrate-importing transporter, system xc−), glutamate-cysteine ligase [catalytic (GCLc) and modifying (GCLm) subunits], were affected. To wit: qRT-PCR analysis demonstrates that naïve Nrf2−/− astrocytes have significantly lower basal mRNA levels of xCT and both GCL subunits compared to naïve Nrf2+/+ astrocytes. No change in mRNA levels of glutathione synthetase (GS) or the GSH exporting transporter, Mrp1, was found. Western blot analysis reveals reduced protein levels of both subunits of GCL, while (seleno)cystine uptake into Nrf2−/− astrocytes was reduced compared to Nrf2+/+ astrocytes, confirming decreased system xc− activity. These findings suggest that Nrf2 regulates the basal production of GSH in astrocytes through constitutive transcriptional regulation of GCL and xCT.

Venlafaxine induces Teratogenesis and alters SHH Gene Expression and Protein Biochemistry of Developing Gallus sp. Embryos

The use of antidepressant drugs during pregnancy is increasing globally. SNRIs and SSRIs are most widely used for treating panic disorders. Venlafaxine is an antidepressant that inhibits serotonin and norepinephrine reuptake. Given the increasing prevalence of antidepressant use during pregnancy, Venlafaxine exposure during the early stages of development could lead to changes in protein dynamics and disrupt essential gene pathways, raising concerns about the potential teratogenic effects of antidepressant exposure during pregnancy. This study explores the developmental impact of Venlafaxine, a commonly prescribed serotonin-norepinephrine reuptake inhibitor (SNRI), on protein biochemistry and gene expression in the developing Gallus gallus domesticus embryos. Significant changes in protein profiles were observed among control and Venlafaxine-treated groups. Sonic Hedgehog (SHH) gene, a key regulator of embryonic patterning and organogenesis, specifically neural tube formation, and limb development, is known for its crucial role in embryonic development. qRT-PCR analysis of Venlafaxine-treated embryos showed changes in SHH gene expression suggesting that Venlafaxine may target SHH gene expression, and potentially alter signalling pathways crucial for normal embryogenesis. However, detailed studies are needed to understand the long-term consequences of prenatal and foetal antidepressant exposure on embryonic growth and development. These changes may lead to abnormal growth patterns or congenital defects, emphasizing the need for caution when prescribing Venlafaxine during pregnancy. Keywords: Antidepressants, Venlafaxine, SNRI, Gallus gallus domesticus, Sonic Hedgehog (SHH), Protein Biochemistry, qRT-PCR

The members of zinc finger-homeodomain (ZF-HD) transcription factors are associated with abiotic stresses in soybean: insights from genomics and expression analysis

BackgroundZinc finger homeodomain (ZF-HD) belongs to the plant-specific transcription factor (TF) family and is widely involved in plant growth, development and stress responses. Despite their importance, a comprehensive identification and analysis of ZF-HD genes in the soybean (Glycine max) genome and their possible roles under abiotic stress remain unexplored.ResultsIn this study, 51 ZF-HD genes were identified in the soybean genome that were unevenly distributed on 17 chromosomes. All GmZF-HD genes contained a conserved ZF-HD_dimer domain and had diverse physicochemical features. Furthermore, the GmZF-HD gene structures exhibited 3 to 10 conserved motifs, and most of them showed intronless gene structures. Phylogenetic analysis categorized them into eight major groups with the highest closeness to dicots including Brassica rapa and Malus domestica. The cis-element analysis recognized plant growth and development (10%), phytohormones (31%) and stress-responsive (59%) elements. Synteny analysis identified 73 segmental and 1 tandem duplicated genes that underwent purifying selection. The collinearity analysis revealed that GmZF-HD genes showed higher homology with dicot species, indicating common ancestors with close evolutionary relationships. A total of 94 gma-miRNAs from 41 diverse miRNA families were identified, targeting 40 GmZF-HD genes, with GmZF-HD6 being most targeted by 7 miRNAs, and gma-miR4993 emerging as the dominant miRNA family. Different TFs including ERF, LBD, BBR-BPC and MYB, etc., were predicted in all 51 GmZF-HD genes upstream regions and visualized in the network. Expression profiling through RNA-Seq showed diverse expressions of GmZF-HD genes in different tissues including seeds, roots, shoots and leaves under diverse conditions. Further, the qRT-PCR analysis demonstrated that all tested GmZF-HD genes were significantly induced in soybean leaves, mainly the GmZF-HD5/6/13/39 and GmZF-HD45 genes were significantly upregulated (2.5 to 8.8 folds) under the tested stress treatments compared to control, highlighting their potential roles in response to stresses in soybean.ConclusionOverall, this study reveals comprehensive insights into the ZF-HD genes in soybeans and provides a valuable contribution towards functional studies for soybean improvement under stress conditions.

The 14-3-3 protein CaTFT7 interacts with transcription factor CaHDZ27 to positively regulate pepper immunity against Ralstonia solanacearum

Abstract Bacterial wilt, caused by Ralstonia solanacearum, is a devastating disease affecting plants in the Solanaceae family. In our previous study, CaHDZ27 was shown to act crucially in the pepper defense response to R. solanacearum. However, the molecular basis underlying CaHDZ27 function remains unexplored. In this study, we demonstrate that CaHDZ27 is post-translationally regulated by the 14-3-3 protein CaTFT7, which functions as a positive regulator in pepper immunity against R. solanacearum. RT-qPCR analysis revealed that CaTFT7 is transcriptionally induced by R. solanacearum infection. The data from virus-induced gene silencing revealed that CaTFT7 positively affects pepper immunity, which was further confirmed by the data of CaTFT7-overexpressing Nicotiana benthamiana. CaTFT7 interacted with CaHDZ27, thereby promoting the stability of CaHDZ27 and enhancing CaHDZ27 binding to the promoter of cysteine-rich receptor-like protein kinase 5 (CaCRK5), a gene that positively affects pepper defense against R. solanacearum. The above data indicated that CaTFT7 enhanced CaHDZ27 stability and promoted its ability to activate pepper immunity, shedding light on the mechanisms underlying pepper resistance to bacterial wilt.

Integration of Metabolomics and Transcriptomics to Reveal the Antitumor Mechanism of Dendrobium officinale Polysaccharide-Based Nanocarriers in Enhancing Photodynamic Immunotherapy in Colorectal Cancer

Background: The mechanism of Dendrobium officinale polysaccharide-based nanocarriers in enhancing photodynamic immunotherapy in colorectal cancer (CRC) remains poorly understood. Methods: The effects of TPA-3BCP-loaded cholesteryl hemisuccinate–Dendrobium officinale polysaccharide nanoparticles (DOP@3BCP NPs) and their potential molecular mechanism of action in a tumor-bearing mouse model of CRC were investigated using non-targeted metabolomics and transcriptomics. Meanwhile, a histopathological analysis (H&E staining, Ki67 staining, and TUNEL assay) and a qRT-PCR analysis revealed the antitumor effects of DOP@3BCP NPs with and without light activation. Results: Through metabolomics and transcriptomics analysis, we found an alteration in the metabolome and functional pathways in the examined tumor tissues. The metabolic analysis showed 69 and 60 differentially expressed metabolites (DEMs) in positive- and negative-ion modes, respectively, in the treated samples compared to the Control samples. The transcriptomics analysis showed that 1352 genes were differentially expressed among the three groups. The differentially regulated functional pathways were primally related to the antitumor immune response. The results of the pathological histology assay and qRT-PCR analysis verified the findings of the integrated metabolomics and transcriptomics analysis. Conclusions: Overall, our findings elucidate the potential antitumor mechanisms of the D. officinale polysaccharide-based nanocarrier in enhancing photodynamic immunotherapy in CRC.

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.

Effective Mixed-Type Tissue Crusher and Simultaneous Isolation of RNA, DNA, and Protein from Solid Tissues Using a TRIzol-Based Method

One of the major challenges of studying biomarkers in tumor samples is the low quantity and quality of isolated RNA, DNA, and proteins. Additionally, the extraction methods ideally should obtain macromolecules from the same tumor biopsy, allowing better-integrated data interpretation. In this work, an in-house, low-cost, mixed-type tissue crusher combining blade and beating principles was made and the simultaneous isolation of macromolecules from human cells and tissues was achieved using TRIzol. RT-qPCR, genotyping, SDS-PAGE, and Western blot analysis were used to validate the approach. For tissue samples, RNA, DNA, and proteins resulted in an average yield of 677 ng/mg, 225 ng/mg, and 1.4 µg/mg, respectively. The same approach was validated using cell lines. The isolated macromolecule validation included the detection of mRNA levels of ATP-binding cassette (ABC) transporters through RT-qPCR, genotyping of TNFR1 (rs767455), and protein visualization through SDS-PAGE following Coomassie blue staining and Western blot. This work contributed to filling a gap in knowledge about TRIzol efficiency for the simultaneous extraction of RNA, DNA, and proteins from a single human tissue sample. A low-cost, high yield, and quality method was validated using target biomarkers of multidrug resistance mechanisms. This approach might be advantageous for future biomarker studies using different tissue specimens.

Elevated MRPS23 expression facilitates aggressive phenotypes in breast cancer cells.

Mitochondrial ribosomal protein S23 (MRPS23), encoded by a nuclear gene, is a well-known driver of proliferation in cancer. It participates in mitochondrial protein translation, and its expression association has been explored in many types of cancer. However, MRPS23 expression associations are rarely reported in breast cancer (BC). In this study, we explored the MRPS23 expression in BC cells compared with the non-tumoral breast cells. Overexpression and knockdown analysis of MRPS23 were performed in BC cells. Transfection efficiency was evaluated by western blot and qRT-PCR analysis. The role of MRPS23 in the malignant biological behaviors of BC cells was investigated using in-vitro experiments. Our results demonstrate that MRPS23 was aberrantly overexpressed at both the transcript and protein levels in BC cells. Additional findings reveal that deficiency of MRPS23 is associated with a decrease in cell proliferation/viability and compromised cell migration/invasion in BC cells. Relative to the sh-Ctrl group, the expression levels of cadherin, SNAI 1, and TWIST 1 decreased in the MRPS23 knockdown BC cells. We further found a significant decrease in the expression levels of Cyclin D1, Axin 2, LEF1, NKD1, and Survivin in MRPS23 knockdown cells. In conclusion, we found an association between MRPS23 knockdown and the metastasis ability of BC cells. These findings reveal that MRPS23 significantly decreases the migration and invasion of BC, thus inhibiting BC progression. We confirmed for the first time that MRPS23 expression determines the metastasis features of BC. Hence, the findings justify the key role of this protein in BC progression; therefore, it may be a potential therapeutic target for BC therapy.

Characterization of Quality Marker for Mume Fructus Pulp by Ultra-Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry-Based Untargeted Metabolomics Combined With Biological Activities.

As a widely used Chinese herbal medicine, Mume Fructus pulp (MFP) has rich nutritional value and biological activity, but its quality control research is relatively scarce. The objective of the study was to evaluate the quality difference between MFPs from different origins and its adulterant apricot pulp (APP), and to identify potential quality markers. The chemical compositions were identified by untargeted metabolomics analysis based on ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry combined with feature-based molecular networking. The effect of the test samples on the gene expression of the pro-inflammatory cytokines was investigated by RT-qPCR and ELISA analyses to determine their anti-inflammatory potential. Antioxidant activity was assessed by a free radical scavenging assay. Subsequently, the relationship between biological activity and chemical composition was analyzed by chemometrics, and the quality index of MFP was preliminarily determined. Based on network pharmacology and molecular docking, quality markers of MFP and their possible molecular mechanisms were further determined. There were significant differences in the contents of flavonoids and organic acids, as well as the anti-inflammatory and antioxidant activities of MFPs from different habitats. Its adulterant APP was not only different from MFP in composition, but also had significantly weaker biological activity. Sixteen compounds were positively correlated with the antioxidant or anti-inflammatory effects of MFP. Combined with network pharmacology and molecular docking, citric acid, maleic acid, rutin, quercetin, isoquercetin, spiraeoside, and hesperidin were identified as potential quality markers of MFP. This study showed the potential of untargeted metabolomic analysis combined with bioactivity assays in the identification of herbal quality markers.

HDC1 Promotes Primary Root Elongation by Regulating Auxin and K+ Homeostasis in Response to Low-K+ Stress

Plants frequently encounter relatively low and fluctuating potassium (K+) concentrations in soil, with roots serving as primary responders to this stress. Histone modifications, such as de-/acetylation, can function as epigenetic markers of stress-inducible genes. However, the signaling network between histone modifications and low-K+ (LK) response pathways remains unclear. This study investigated the regulatory role of Histone Deacetylase Complex 1 (HDC1) in primary root growth of Arabidopsis thaliana under K+ deficiency stress. Using a hdc1-2 mutant line, we observed that HDC1 positively regulated root growth under LK conditions. Compared to wild-type (WT) plants, the hdc1-2 mutant exhibited significantly inhibited primary root growth under LK conditions, whereas HDC1-overexpression lines displayed opposite phenotypes. No significant differences were observed under HK conditions. Further analysis revealed that the inhibition of hdc1-2 on root growth was due to reduced apical meristem cell proliferation rather than cell elongation. Notably, the root growth of hdc1-2 showed reduced sensitivity compared to WT after auxin treatment under LK conditions. HDC1 may regulate root growth by affecting auxin polar transport and subsequent auxin signaling, as evidenced by the altered expression of auxin transport genes. Moreover, the organ-specific RT-qPCR analyses unraveled that HDC1 negatively regulates the expression of CBL-CIPK-K+ channel-related genes such as CBL1, CBL2, CBL3, AKT1, and TPK1, thereby establishing a molecular link between histone deacetylation, auxin signaling, and CBLs-CIPKs pathway in response to K+ deficiency.

Influence of Enterococcus faecium, a probiotic component, on ion channels in colon cancer

Aims: This study aimed to investigate the effect of probiotic bacterium Enterococcus faecium (E. faecium) on the gene expression of ion channels in colon cancer cells. Methods: The cytotoxic effect on SW480 colon cancer cell line was analyzed by MTT cell proliferation test using E. faecium bacterial cell culture free supernatants. The effect of E. faecium on ion channel genes in SW480 cells was determined by qRT PCR analysis. STRING analysis was used to reveal protein-protein interactions of ion channel proteins. Bioinformatic analysis of healthy and colon cancer patient data on ion channels was revealed with GEPIA platform. Results: The SW480 cell line's viability was enhanced by E. fecieum bacterial cell culture free supernatants dosages of 0.5 and 1.09 mg/ml, but it dramatically declined between 2.187 and 17.5 mg/ml doses, according the MTT study. Analysis of gene expression revealed that the TRPV2 and TRPM8 genes had significantly increased. The examined ion channel proteins were discovered to be substantially linked to one another based on STRING analysis. The TRPM2 gene we looked at revealed a notable rise in colon cancer patients based on data from both healthy and colon cancer patients on the GEPIA platform. Conclusion: E. faecium has been shown to have many beneficial effects on health. Our study has shown that it also has an effect on ion channels in cancer cells and that ion channels are of great importance for cell survival and death.

Molecular Mechanisms of circKIF4A in Breast Cancer Progression.

Breast cancer (BC) is the most frequently diagnosed malignancy worldwide, necessitating continued research into its molecular mechanisms. Circular RNAs (circRNAs) are increasingly recognized for their role in various cancers, including BC. This study explores the role of circRNA kinesin family member 4A (circKIF4A) in BC progression and its underlying molecular mechanisms. BC cell lines were cultured, and circKIF4A expression was knocked down. Cell viability, proliferation, migration, and invasion were assessed using the Cell Counting Kit-8, colony formation assay, and Transwell assays. The expression of circKIF4A, miR-874-3p, and glycerophosphodiester phosphodiesterase domain-containing 5 (GDPD5) was quantified using qRT-PCR and Western blot analysis. Subcellular fractionation was performed to localize circKIF4A within the cell. The interactions between circKIF4A and miR-874-3p, as well as between miR-874-3p and GDPD5, were evaluated using RNA pull-down and dual-luciferase assays. Rescue experiments were conducted with miR-874-3p inhibition or GDPD5 overexpression to confirm the mechanistic pathway. circKIF4A was found to be upregulated in BC cells. Its knockdown significantly inhibited cell proliferation, migration, and invasion. circKIF4A acts as a sponge for miR-874-3p, reducing its expression. miR-874-3p targets and suppresses GDPD5, a key regulator in BC cell growth. Silencing miR-874-3p or overexpressing GDPD5 reversed the tumor-suppressive effects of circKIF4A knockdown. circKIF4A promotes BC cell proliferation and invasiveness by regulating the miR-874-3p/GDPD5 axis. These findings highlight a potential therapeutic target in BC and contribute to the understanding of circRNA involvement in cancer progression.

Expression of Agrobacterium Isopentenyl transferase (IPT) gene in wheat improves drought tolerance.

Drought, as an abiotic stressor, globally limits cereal productivity, leading to early aging of leaves and lower yields. The expression of the isopentenyl transferase (IPT) gene, which is involved in cytokinin (CK) biosynthesis, can delay drought-induced leaf senescence. In this study, the Agrobacterium Isopentenyl transferase (IPT) gene was introduced into two local hexaploid wheat cultivars, NR-421 and FSD-2008. The expression cassette was developed containingthe IPT gene under transcriptional regulation of the stress-inducible promoter 'Dehydrin,' sourced from Hordeum vulgare. The gene expression cassette was assembled in pSB219M, a modified transformation vector for monocots, equipped with both an antibiotic (spectinomycin) and an herbicide selection marker (BASTA). Initial screening of transgenic plants involved BASTA selection (2 and 3mg/L) and was subsequently confirmed through PCR analysis. The transformation efficiencies of NR-421 and FSD-2008 were 0.4% and 0.3%, respectively. The qRT-PCR analysis under stress conditions showed a 13.5-fold higher expression of the IPT gene in T2 transgenic plants of NR-421 and a 5.8-fold higher expression in those of FSD-2008 than in non-transgenic controls. Under stress conditions, the wheat transgenic plants exhibited increased chlorophyll and relative water content. Additionally, for total soluble proteins, two transgenic lines from the NR-421 variety showed a significant increase, whereas no notable change was observed in the FSD-2008 transgenics. Moreover, the transgenic lines displayed increased plant height, higher fresh and dry biomass, and increased seed weight compared to the non-transgenic controls. These findings highlight that stress-inducible expression of the IPT gene in wheat leads to enhanced grain yield and subsequently improved drought tolerance.

Brassica rapa receptor-like cytoplasmic kinase BrRLCK1 negatively regulates freezing tolerance in transgenic Arabidopsis via the CBF pathway.

Some winter rapeseed (Brassica rapa) varieties can endure extremely low temperatures (-20°C to -32°C). However, because of a lack of mutant resources, the molecular mechanisms underlying cold tolerance in B. rapa remain unclear. In this study, we identified a low-temperature-sensitive mutant receptor-like cytoplasmic kinase (RLCK), BrRLCK1, using the B. rapa--Arabidopsis (Arabidopsis thaliana) full-length cDNA-overexpressing gene hunting system mutant library. BrRLCK1, localized to the plasma membrane and retained its localization under low temperatures. Phylogenetic analysis showed that BrRLCK1 is highly conserved across six widely cultivated Brassica species, but exhibits complexity due to genome hybridization and polyploidization. Notably, β-glucuronidase activity and qRT-PCR analysis showed that B. rapa BrRLCK1 and its homologous gene BrRLCK2 were mainly expressed in the main root, shoot, and leaves, with their expression being activated by low temperatures. Transgenic Arabodipsis expressing BrRLCK1 and BrRLCK2 reduced freezing tolerance and promoted root elongation. These combined results indicated that low temperatures can activate the expression of BrRLCK1 and BrRLCK2, negatively regulating freezing tolerance via the C-repeat-binding factor (CBF) pathway.

RNA binding protein ALYREF regulates ferroptosis to facilitate LUAD growth and metastasis via promoting SLC7A11 mRNA stability

Ferroptosis is of great significance in carcinogenesis as it interconnects with a multiplicity of biological processes. Meanwhile, its function and regulatory role in lung cancer remains ambiguous. In this study, we discovered by WB and IHC that ALYREF has a higher expression in lung adenocarcinoma (LUAD) tissues compared with normal ones. By the direct observation of mitochondria with electron microscopy, we also identified that the knockdown of ALYREF limits the proliferation of LUAD in vitro as well as in vivo and prompts LUAD cells to go through ferroptosis. Mechanistically, our RNA-immunoprecipitation experiment combined with qRT-PCR analysis confirmed that ALYREF could interact with SLC7A11. ALYREF mainly binds to the 3’UTR region of SLC7A11 mRNA, increasing the mRNA stability of SLC7A11 and reducing intracellular Reactive Oxygen Species (ROS) and Methylene diphenyl diamine (MDA) thereby inhibiting ferroptosis and eventually promoting the proliferation and metastasis of LUAD cells. Importantly, our study reveals for the first time that ALYREF can regulate LUAD by inhibiting ferroptosis. Based on the evidences mentioned above, we have good reason to believe that the role of ALYREF as a new oncological factor could be proved and, acting along the ALYREF-SLC7A11-ferroptosis axis, utilized as a promising therapeutic target for LUAD.

Agathisflavone Inhibits Viability and Modulates the Expression of miR-125b, miR-155, IL-6, and Arginase in Glioblastoma Cells and Microglia/Macrophage Activation.

Glioblastomas (GBM) are malignant tumours with poor prognosis. Treatment involves chemotherapy and/or radiotherapy; however, there is currently no standard treatment for recurrence, and prognosis remains unfavourable. Inflammatory mediators and microRNAs (miRNAs) influence the aggressiveness of GBM, being involved in the communication with the cells of the tumour parenchyma, including microglia/macrophages, and maintaining an immunosuppressive microenvironment. Hence, the modulation of miRNAs and inflammatory factors may improve GBM treatments. In this study, we investigated the effects of agathisflavone, a biflavonoid purified from Cenostigma pyramidale (Tul.), on the growth and migration of GBM cells, on the expression of inflammatory cytokines and microRNAs, as well on the response of microglia. Agathisflavone (5-30 μM) induced a dose- and time-dependent reduction in the viability of both human GL-15 and rat C6 cells, as determined by the MTT test, and reduced cell migration, as determined by cell scratch assay. RT-qPCR analysis revealed that agathisflavone (5 μM) down-regulated the expression of miR-125b and miR-155 in the secretome derived from GL-15 cells, which was associated with upregulation of the mRNA expression of IL-6 and arginase-1 immunoregulatory factors. Exposure of human microglia/macrophage to the secretome from GL-15 GMB cells modulated proliferation and morphology, effects that were modulated by agathisflavone treatment. These results demonstrate the effect of flavonoids on the growth of GBM cells, which impacts cells in the microenvironment and can be considered for preclinical studies for adjuvant treatments.