Quantitative Real-time PCR Analysis Research Articles

Inhibition of the rapamycin-insensitive mTORC1 /4E-BP1 axis attenuates TGF-β1-induced fibrotic response in human Tenon's fibroblasts

Subconjunctival fibrosis is the major cause of failure in both conventional and modern minimally invasive glaucoma surgeries (MIGSs) with subconjunctival filtration. The search for safe and effective anti-fibrotic agents is critical for improving long-term surgical outcomes. In this study, we investigated the effect of inhibiting the rapamycin-insensitive mTORC1/4E-BP1 axis on the transforming growth factor-beta 1(TGF-β1)-induced fibrotic responses in human Tenon's fibroblasts (HTFs), as well as in a rat model of glaucoma filtration surgery (GFS). Primary cultured HTFs were treated with 3 ng/mL TGF-β1 for 24 h, followed by treatment with 10 μM CZ415 for additional 24 h. Rapamycin (10 μM) was utilized as a control for mTORC1/4E-BP1 signaling insensitivity. The expression levels of fibrosis-associated molecules were measured using quantitative real-time PCR, Western blotting, and immunofluorescence analysis. Cell migration was assessed through the scratch wound assay. Additionally, a rat model of GFS was employed to evaluate the anti-fibrotic effect of CZ415 in vivo. Our findings indicated that both rapamycin and CZ415 treatment significantly reduced the TGF-β1-induced cell proliferation, migration, and the expression of pro-fibrotic factors in HTFs. CZ415 also more effectively inhibited TGF-β1-mediated collagen synthesis in HTFs compared to rapamycin. Activation of mTORC1/4E-BP signaling following TGF-β1 exposure was highly suppressed by CZ415 but was only modestly inhibited by rapamycin. Furthermore, CZ415 was found to decrease subconjunctival collagen deposition in rats post GFS. Our results suggest that rapamycin-insensitive mTORC1/4E-BP1 signaling plays a critical role in TGF-β1-driven collagen synthesis in HTFs. This study demonstrated that inhibition of the mTORC1/4E-BP1 axis offers superior anti-fibrotic efficacy compared to rapamycin and represents a promising target for improving the success rate of both traditional and modern GFSs.

Evaluation and validation of suitable reference genes for quantitative real-time PCR analysis in lotus (Nelumbo nucifera Gaertn.)

The qRT-PCR technique has been regarded as an important tool for assessing gene expression diversity. Selection of appropriate reference genes is essential for validating deviation and obtaining reliable and accurate results. Lotus (Nelumbo nucifera Gaertn) is a common aquatic plant with important aesthetic, commercial, and cultural values. Twelve candidate genes, which are typically used as reference genes for qRT-PCR in other plants, were selected for this study. These candidate reference genes were cloned with, specific primers designed based on published sequences. In particular, the expression level of each gene was examined in different tissues and growth stages of Lotus. Notably, the expression stability of these candidate genes was assessed using the software programs geNorm and NormFinder. As a result, the most efficient reference genes for rootstock expansion were TBP and UBQ. In addition, TBP and EF-1α were the most efficient reference genes in various floral tissues, while ACT and GAPDH were the most stable genes at all developmental stages of the seed. CYP and GAPDH were the best reference genes at different stages of leaf development, but TUA was the least stable. Meanwhile, the gene expression profile of NnEXPA was analyzed to confirm the validity of the findings. It was concluded that, TBP and GAPDH were identified as the best reference genes. The results of this study may help researchers to select appropriate reference genes and thus obtain credible results for further quantitative RT-qPCR gene expression analyses in Lotus.

Evaluation of the cytotoxic activities of the essential oil from Pistacia atlantica Desf. oleoresin on human gastric cancer cell lines.

Numerous herbal products have been the subject of research regarding their potential role in cancer prevention or adjuvant therapy. Pistacia atlantica and its main phytochemicals have garnered significant attention for their potential anti-cancer effects. The study aimed to assess the growth inhibitory effects of P. atlantica essential oil (PAEO) on MKN-45 and AGS cells. This study quantified the volatile compounds in PAEO using Gas Chromatography-Mass Spectrometry (GC-MS). Subsequently, MKN-45 and AGS cells were treated with varying concentrations of PAEO (5%, 2.5%, 1.25%, 0.625%, 0.3125%, 0.156%, 0.0781%, 0.0391%, 0.0195%) for 24h. Cell viability was evaluated through the MTT assay. The impact of PAEO on gene expression was investigated by quantifying the mRNA levels of Bax and Bcl2 in the various experimental groups using quantitative Real-Time PCR (qRT-PCR) analysis. Additionally, flow cytometry was utilized to evaluate apoptosis in the treated cells. The analysis of PAEO revealed that α-pinene was the predominant monoterpene, constituting 87.9% of the oil composition. The cytotoxic effects of PAEO were evaluated, and it was found that the oil significantly reduced the viability of MKN-45 and AGS cells. The IC50 for MKN-45 cells was determined to be 1.94 × 10-3% after 24h of treatment, while for AGS cells the IC50 was 2.8 × 10-3% after 24h. Additionally, the research revealed that PAEO triggered a notable rise in apoptotic cells in both AGS and MKN-45 cell lines. Moreover, at the molecular level, the findings indicated an increase in Bax expression and a decrease in Bcl2 mRNA expression, providing further evidence of the induction of apoptosis in both MKN-45 and AGS cell lines following PAEO treatment. The findings of this study offer evidence supporting the cytotoxic effects of PAEO on gastric cancer cell lines by promoting apoptosis. The findings suggest that PAEO may offer potential as a therapeutic candidate in managing and treating gastric cancer.

GDSL Lipase Gene HTA1 Negatively Regulates Heat Tolerance in Rice Seedlings by Regulating Reactive Oxygen Species Accumulation.

High temperature is a significant environmental stress that limits plant growth and agricultural productivity. GDSL lipase is a hydrolytic enzyme with a conserved GDSL sequence at the N-terminus, which has various biological functions, such as participating in plant growth, development, lipid metabolism, and stress resistance. However, little is known about the function of the GDSL lipase gene in the heat tolerance of rice. Here, we characterized a lipase family protein coding gene HTA1, which was significantly induced by high temperature in rice. Rice seedlings in which the mutant hta1 was knocked out showed enhanced heat tolerance, whereas the overexpressing HTA1 showed more sensitivity to heat stress. Under heat stress, hta1 could reduce plant membrane damage and reactive oxygen species (ROS) levels and elevate the activity of antioxidant enzymes. Moreover, real-time quantitative PCR (RT-qPCR) analysis showed that mutant hta1 significantly activated gene expression in antioxidant enzymes, heat response, and defense. In conclusion, our results suggest that HTA1 negatively regulates heat stress tolerance by modulating the ROS accumulation and the expression of heat-responsive and defense-related genes in rice seedlings. This research will provide a valuable resource for utilizing HTA1 to improve crop heat tolerance.

Study of proanthocyanidin promotes osteogenic differentiation of human periodontal ligament stem cells through the transcription factor EB-induced autophagy-lysosome pathway

Objective: To investigate the mechanism of proanthocyanidin (PA) in regulating the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs), and to explore the effects of PA on the expression and nuclear translocation of transcription factor EB (TFEB) and on the autophagy-lysosome pathway. Methods: PDLSCs were divided into control group and PA group, which were subjected to RNA sequencing analysis (RNA Seq) to detect differentially expressed genes. The osteogenic differentiation ability and autophagy level were observed by real-time fluorescence quantitative PCR (RT-qPCR) analysis, alkaline phosphatase (ALP) staining and transmission electron microscope (TEM), respectively. Scratch assay and Transwell assay were used to detect the migration ability of PDLSCs. Lysotracker and immunofluorescence staining were used to detect the biogenesis of lysosomes. The total protein expression of transcription factor EB (TFEB) as well as that in cytoplasm and nucleus were detected by Western blotting. Confocal laser scanning microscope (CLSM) was used to observe the nuclear translocation of TFEB. The PDLSCs were treated with small interfering RNA (siRNA) technology to knock down the expression levels of TFEB gene with or without PA treatment. Western blotting was used to analyze the expressions of autophagy-related proteins Beclin1 and microtubule-associated protein 1 light chain 3 (LC3B), as well as osteogenic-related proteins runt-related transcription factor 2 (RUNX2), ALP, and osteocalcin in PDLSCs. Results: Compared with the control group, the osteogenic-related and autophagy-related genes showed differential expression in PDLSCs after PA treatment (P<0.05). The mRNA expression levels of osteogenic-related genes RUNX2 (2.32±0.15) and collagen type Ⅰ alpha 1 (COL1α1) (1.80±0.18), as well as the autophagy related genes LC3B (1.87±0.08) and Beclin1 (1.63±0.08) were significantly increased in the PA group, compared with the control group (1.01±0.16, 1.00±0.10, 1.00±0.07, 1.00±0.06, respectively, all P<0.01). Compared with the control group, the PA group had higher ALP activity, and more autophagosomes and autophagolysosomes observed by TEM. PA promoted the migration of PDLSCs (P<0.05) and the increased number of lysosomes and the expression of lysosomal associated membrane protein 1 (LAMP1). In the PA group, the relative expression level of total TFEB protein (1.49±0.07) and the nuclear/cytoplasmic expression of TFEB protein (1.52±0.12) were significantly higher than the control group (1.00±0.11, 1.00±0.13, respectively) (t=6.43, P<0.01; t=5.07, P<0.01). The relative nuclear/cytoplasmic fluorescence intensity of TFEB in the PA group (0.79±0.09) was increased compared with the control group (0.11±0.08) (t=8.32, P<0.01). Knocking down TFEB significantly reduced the expression of TFEB (1.00±0.15 vs 0.64±0.04), LAMP1 (1.00±0.10 vs 0.69±0.09), Beclin1 (1.00±0.05 vs 0.60±0.05), and LC3B Ⅱ/Ⅰ (1.00±0.06 vs 0.73±0.07) in PDLSCs (P<0.05, P<0.05, P<0.01, P<0.01). When TFEB gene was knocked down, the expression levels of Beclin1 (1.05±0.11), LC3B Ⅱ/Ⅰ (1.02±0.09), RUNX2 (1.04±0.10), ALP (1.04±0.16), and osteocalcin (1.03±0.15) proteins were significantly decreased in the PA group compared with the pre-knockdown period (1.28±0.03, 1.44±0.11, 1.38±0.11, 1.62±0.11, 1.65±0.17, respectively) (P<0.05, P<0.01, P<0.05, P<0.01, and P<0.01, respectively). Conclusions: PA promotes the osteogenic differentiation of PDLSCs through inducing the expression and nuclear translocation of TFEB and activating the autophagy-lysosome pathway.

Cloning and functional verification of the CmHSP17.9 gene from chrysanthemum.

Small molecular heat shock proteins (sHSPs) belong to the HSP family of molecular chaperones. Under high-temperature stress, they can prevent the aggregation of irreversible proteins and maintain the folding of denatured proteins to enhance heat resistance. In this study, the CmHSP17.9-1 and CmHSP17.9-2 genes, which were cloned from chrysanthemum (Chrysanthemum×morifolium 'Jinba') by homologous cloning, had a complete open reading frame of 480 bp each, encoding 159 amino acids. The protein subcellular localization analysis showed that CmHSP17.9-1 and CmHSP17.9-2 were located in the cytoplasm and mostly aggregated in granules, especially around the nucleus. Real-time quantitative PCR (qRT-PCR) analysis showed that the relative expression level of the CmHSP17.9-1 and CmHSP17.9-2 genes was highest in the terminal buds of the chrysanthemum, followed by the leaves. CmHSP17.9-1 and CmHSP17.9-2 overex-pression vectors were constructed and used to transform the chrysanthemum; overexpression of these genes led to the chrysanthemum phenotypes being less affected by high-temperature, and the antioxidant capacity was enhanced. The results showed that chrysanthemum with overex-pression of the CmHSP17.9-1 and CmHSP17.9-2 genes had stronger tolerance than the wild type chrysanthemum after high-temperature treatment or some degree of heat exercise, and overex-pression of the CmHSP17.9-1 gene led to stronger heat resistance than that of the CmHSP17.9-2 gene, providing an important theoretical basis for the subsequent molecular breeding and pro-duction applications of chrysanthemum.

Gefitinib metabolism-related lncRNAs for the prediction of prognosis, tumor microenvironment and drug sensitivity in lung adenocarcinoma

The complete compound of gefitinib is effective in the treatment of lung adenocarcinoma. However, the effect on lung adenocarcinoma (LUAD) during its catabolism has not yet been elucidated. We carried out this study to examine the predictive value of gefitinib metabolism-related long noncoding RNAs (GMLncs) in LUAD patients. To filter GMLncs and create a prognostic model, we employed Pearson correlation, Lasso, univariate Cox, and multivariate Cox analysis. We combined risk scores and clinical features to create nomograms for better application in clinical settings. According to the constructed prognostic model, we performed GO/KEGG and GSEA enrichment analysis, tumor immune microenvironment analysis, immune evasion and immunotherapy analysis, somatic cell mutation analysis, drug sensitivity analysis, IMvigor210 immunotherapy validation, stem cell index analysis and real-time quantitative PCR (RT-qPCR) analysis. We built a predictive model with 9 GMLncs, which showed good predictive performance in validation and training sets. The calibration curve demonstrated excellent agreement between the expected and observed survival rates, for which the predictive performance was better than that of the nomogram without a risk score. The metabolism of gefitinib is related to the cytochrome P450 pathway and lipid metabolism pathway, and may be one of the causes of gefitinib resistance, according to analyses from the Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Immunological evasion and immunotherapy analysis revealed that the likelihood of immune evasion increased with risk score. Tumor microenvironment analysis found most immune cells at higher concentrations in the low-risk group. Drug sensitivity analysis found 23 sensitive drugs. Twenty-one of these drugs exhibited heightened sensitivity in the high-risk group. RT-qPCR analysis validated the characteristics of 9 GMlncs. The predictive model and nomogram that we constructed have good application value in evaluating the prognosis of patients and guiding clinical treatment.

Scutellaria baicalensis water decoction ameliorates lower respiratory tract infection by modulating respiratory microbiota

BackgroundThe pathogenesis of lower respiratory tract infections (LRTIs) has been demonstrated to be strongly associated with dysbiosis of respiratory microbiota. Scutellaria baicalensis, a traditional Chinese medicine, is widely used to treat respiratory infections. However, whether the therapeutic effect of S. baicalensis on LRTIs depends upon respiratory microbiota regulation is largely unclear. PurposeTo investigate the potential effect and mechanism of S. baicalensis on the respiratory microbiota of LRTI mice. MethodsA mouse model of LRTI was established using Klebsiella pneumoniae or Streptococcus pneumoniae. Antibiotic treatment was administered, and transplantation of respiratory microbiota was performed to deplete the respiratory microbiota of mice and recover the destroyed microbial community, respectively. High-performance liquid chromatography (HPLC) was used to determine and quantify the chemical components of S. baicalensis water decoction (SBWD). Pathological changes in lung tissues and the expressions of serum inflammatory cytokines, including interleukin-17A (IL-17A), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), were determined by hematoxylin and eosin (H&E) staining and enzyme-linked immunosorbent assay (ELISA), respectively. Quantitative real-time PCR (qRT-PCR) analysis was performed to detect the mRNA expression of GM-CSF. Metagenomic sequencing was performed to evaluate the effect of SBWD on the composition and function of the respiratory microbiota in LRTI mice. ResultsSeven main components, including scutellarin, baicalin, oroxylin A-7-O-β-d-glucuronide, wogonoside, baicalein, wogonin, and oroxylin A, were identified and their levels in SBWD were quantified. SBWD ameliorated pulmonary pathological injury and inflammatory responses in K. pneumoniae and S. pneumoniae-induced LRTI mice, as evidenced by the dose-dependent reductions in the levels of serum inflammatory cytokines, IL-6 and TNF-α. SBWD may exert a bidirectional regulatory effect on the host innate immune responses in LRTI mice and regulate the expressions of IL-17A and GM-CSF in a microbiota-dependent manner. K. pneumoniae infection but not S. pneumoniae infection led to dysbiosis in the respiratory microbiota, evident through disturbances in the taxonomic composition characterized by bacterial enrichment, including Proteobacteria, Enterobacteriaceae, and Klebsiella. K. pneumoniae and S. pneumoniae infection altered the bacterial functional profile of the respiratory microbiota, as indicated by increases in lipopolysaccharide biosynthesis, metabolic pathways, and carbohydrate metabolism. SBWD had a certain trend on the regulation of compositional disorders in the respiratory flora and modulated partial microbial functions embracing carbohydrate metabolism in K. pneumoniae-induced LRTI mice. ConclusionSBWD may exert an anti-infection effect on LRTI by targeting IL-17A and GM-CSF through respiratory microbiota regulation. The mechanism of S. baicalensis action on respiratory microbiota in LRTI treatment merits further investigation.

RNA-Seq analysis reveals the gene expression changes of Bemisia tabaci in response to destruxin A treatment

ABSTRACT Bemisia tabaci is an important agricultural pest that has been causing significant economic losses to crops across the globe. Destruxins are secondary metabolites of entomopathogenic fungi which can be used as a potential biopesticide against B. tabaci. However, little is known about the molecular mechanism involved in the regulation of the immune response of B. tabaci post-destruxin application. In this study, we explored the molecular responses of B. tabaci exposed to destruxin A (DA) using RNA-Seq. A total of 1702, 616, and 555 differentially expressed genes (DEGs) were identified in B. tabaci after 4, 8, and 12 h of DA treatment. In addition, 39 putative detoxification-related DEGs including 29 cytochrome P450s (P450s), 5 glutathione S-transferases (GSTs), and 5 carboxylesterases (CarEs) were also identified. Quantitative real-time PCR analysis indicated that the expression profiles of 19 random DEGs were consistent with the RNA-Seq results. These findings serve as valuable information for a better understanding of the interaction and molecular mechanisms involved in the defense response of B. tabaci against DA.

Lncrna CASC15 Activated By TCF12 Promote Colorectal Cancer Progression via EMT.

Colorectal cancer (CRC) is one of the most common gastrointestinal malignancies. LncRNA CASC15 has also been found to play a vital role in malignant tumors. Our objective is to explore the role of CASC15 in colorectal cancer and its regulation of EMT and to clarify the reasons for its up-regulated expression in CRC. Quantitative real-time PCR was performed to evaluate the expression of CASC15 in CRC. The biology function of CASC15 on CRC was assessed by in vitro experiments, including CCK8, colony formation, transwell assays and flow cytometry. Luciferase reporter assays were used to confirm the regulation between TCF12 and CASC15. Quantitative real-time PCR and western blot analysis were used to evaluate the biomarkers associated with epithelial-mesenchymal transition (EMT). We found that CASC15 was remarkably upregulated in CRC and positively correlated with poorer relapse-free survival. CASC15 knockdown significantly suppressed the proliferation and migration of CRC. Furthermore, CASC15 downregulation mediated apoptosis of CRC. Mechanistically, TCF12 activates CASC15 transcription to mediate its up-regulation, which activates EMT and promotes CRC progression. Our study identified TCF12/CASC15/EMT as a new regulatory signal axis of CRC. CASC15 may be a new molecular marker and target for CRC.

Heat shock protein 90 and prolyl hydroxylase 2 co-regulate hypoxia-inducible factor-1α expression in porcine small intestinal epithelial cells under heat stress

Heat stress (HS) poses a substantial threat to animal growth and development, resulting in declining performance and economic losses. The intestinal system is susceptible to HS and undergoes intestinal hyperthermia and pathological hypoxia. Hypoxia-inducible factor-1α (HIF-1α), a key player in cellular hypoxic adaptation, is influenced by prolyl-4-hydroxylase 2 (PHD2) and heat shock protein 90 (HSP90). However, the comprehensive regulation of HIF-1α in the HS intestine remains unclear. This study aims to explore the impact of HS on pig intestinal mucosa and the regulatory mechanism of HIF-1α. Twenty-four Congjiang Xiang pigs were divided into the control and five HS-treated groups (6, 12, 24, 48, and 72 h). Ambient temperature and humidity were maintained in a thermally-neutral state (temperature–humidity index (THI) < 74) in the control group, whereas the HS group experienced moderate HS (78 < THI <84). Histological examination revealed villus exfoliation after 12 h of HS in the duodenum, jejunum, and ileum, with increasing damage as HS duration extended. The villus height to crypt depth ratio (V/C) decreased and goblet cell number increased with prolonged HS. Quantitative real-time PCR, Western blot, and immunohistochemistry analysis indicated increased expression of HIF-1α and HSP90 in the small intestine with prolonged HS, whereas PHD2 expression decreased. Further investigation in IPEC-J2 cells subjected to HS revealed that overexpressing PHD2 increased PHD2 mRNA and protein expression, while it decreases HIF-1α. Conversely, interfering with HSP90 expression substantially decreased both HSP90 and HIF-1α mRNA and protein levels. These results suggest that HS induces intestinal hypoxia with concomitant small intestinal mucosal damage. The expression of HIF-1α in HS-treated intestinal epithelial cells may be co-regulated by HSP90 and PHD2 and is possibly linked to intestinal hyperthermia and hypoxia.

Interleukin-10 supplementation protects against oxalate-mediated injury in mouse kidneys and bone-marrow derived macrophages

Approximately 10% of the United States population will experience a kidney stone (KS). Dietary oxalate promotes calcium oxalate (CaOx) crystals, which are precursors to CaOx KS. Macrophages rely on Interleukin-10 (IL-10) anti-inflammatory cytokine signaling to phagocytose and remove CaOx crystals. We recently reported that oxalate causes macrophages to become M1-like (pro-inflammatory) as well as have increased Interleukin-6 (IL-6; pro-inflammatory) and reduced IL-10 cytokine secretion. The purpose of this study was to assess the effects of IL-10 supplementation on mouse kidneys and bone-marrow derived macrophages exposed to oxalate. We hypothesized that exogenous IL-10 treatment prevents oxalate-mediated injury in mouse kidneys and bone-marrow derived macrophages (BMDMs). Mice with a C57BL/6 background were fed Hydroxy-L-proline (HLP; an oxalate precursor) for 7 or 14 days ± IL-10 intraperitoneal injections (25 μg/kg for 2 days) before collecting kidneys and BMDMs. Real-Time Quantitative Reverse Transcription PCR, fluorescence assays, and histological analyses were performed. Mice fed HLP had increased IL-6 and M1 macrophage expression in their kidneys over time. These mice also had reduced IL-10 and M2 macrophage gene expression. In contrast, mice fed HLP with IL-10 supplementation had increased M2 macrophage levels as well as reduced inflammation in their kidneys. Consistent with these findings, BMDM isolated from mice fed HLP displayed similar results. These cells also had reduced mitochondrial membrane polarization and lysosomal staining and increased cellular ROS; whereas, exogenous IL-10 treatment protected BMDMs from oxalate mediated injury. These findings suggest that IL-10 signaling may be important for preventing oxalate-induced injury and inflammation in mouse kidneys and BMDMs. NIH R01 DK129885 (TM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The wheat pathogenesis-related protein (TdPR1.2) enhanced tolerance to abiotic and biotic stresses in transgenic Arabidopsis plants.

In plants, the pathogenesis-related (PR) proteins have been identified as important regulators of biotic and abiotic stresses. PR proteins branch out into 19 different classes (PR1-PR19). Basically, all PR proteins display a well-established method of action, with the notable exception of PR1, which is a member of a large superfamily of proteins with a common CAP domain. We have previously isolated and characterized the first PR1 from durum wheat, called TdPR-1.2. In the current research work, TdPR1.2 gene was used to highlight its functional activities under various abiotic (sodium chloride (100mM NaCl) and oxidative stresses (3mM H2O2), hormonal salicylic acid (SA), abscisic acid (ABA) and jasmonic acid (JA), and abiotic stresses (Botrytis cinerea and Alternaria solani). Enhancement survival index was detected in Arabidopsis transgenic plants expressing TdPR1.2 gene. Moreover, quantitative real-time reverse transcription PCR (qRT-PCR) analysis demonstrated induction of antioxidant enzymes such as catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD). It equally revealed a decrease of malondialdehyde (MDA) as well as hydrogen peroxide (H2O2) levels in transgenic Arabidopsis plants compared to control lines, confirming the role of TdPR1.2 in terms of alleviating biotic and abiotic stresses in transgenic Arabidopsis plants. Eventually, RT-qPCR results showed a higher expression of biotic stress-related genes (PR1 and PDF1.2) in addition to a downregulation of the wound-related gene (LOX3 and VSP2) in transgenic lines treated with jasmonic acid (JA). Notably, these findings provide evidence for the outstanding functions of PR1.2 from durum wheat which can be further invested to boost tolerance in crop plants to abiotic and biotic stresses.

Genome-wide characterization, transcriptome profiling, and functional analysis of the ALMT gene family in Medicago for aluminum resistance

Aluminum (Al) is the major limiting factor affecting plant productivity in acidic soils. Al3+ ions exhibit increased solubility at a pH below 5, leading to plant root tip toxicity. Alternatively, plants can perceive very low concentrations of Al3+, and Al triggers downstream signaling even at pH 5.7 without causing Al toxicity. The ALUMINUM-ACTIVATED-MALATE-TRANSPORTER (ALMT) family members act as anion channels, with some regulating the secretion of malate from root apices to chelate Al, which is a crucial mechanism for plant Al resistance. To date, the role of the ALMT gene family within the legume Medicago species has not been fully characterized. In this study, we investigated the ALMT gene family in M. sativa and M. truncatula and identified 68 MsALMTs and 18 MtALMTs, respectively. Phylogenetic analysis classified these genes into five clades, and synteny analysis uncovered genuine paralogs and orthologs. The real-time quantitative reverse transcription PCR (qRT-PCR) analysis revealed that MtALMT8, MtALMT9, and MtALMT15 in clade 2-2b are expressed in both roots and root nodules, and MtALMT8 and MtALMT9 are significantly upregulated by Al in root tips. We also observed that MtALMT8 and MtALMT9 can partially restore the Al sensitivity of Atalmt1 in Arabidopsis. Moreover, transcriptome analysis examined the expression patterns of these genes in M. sativa in response to Al at both pH 5.7 and pH 4.6, as well as to protons, and found that Al and protons can independently induce some Al-resistance genes. Overall, our findings indicate that MtALMT8 and MtALMT9 may play a role in Al resistance, and highlight the resemblance between the ALMT genes in Medicago species and those in Arabidopsis.

Longevity and Potential Mechanisms of Fenpropathrin Resistance in Asian Citrus Psyllid, Diaphorina citri Kuwayama

The stability of resistance to fenpropathrin was assessed using five populations of Diaphorina citri with varying initial resistances ranging from fully susceptible (SS) to fully resistant (RR). Furthermore, we quantified the relative expression of voltage-gated sodium channel (VGSC) genes in crosses of field-selected and laboratory-susceptible D. citri lines after eight months without insecticide selection. We found that resistance to fenpropathrin remained elevated up to eight months after exposure to fenpropathrin. A real-time quantitative PCR analysis using the susceptible baseline population revealed that levels of VGSC gene expression were significantly higher in the RS75 cross and the RR100 fully resistant line eight months after their last fenpropathrin exposure. Our results suggest that while fenpropathrin resistance is likely unstable under field conditions when interbreeding with susceptible individuals is possible, resistance can remain stable for at least 8 months if those populations are isolated. Further, insecticide rotation and the maintenance of susceptible reservoirs of individuals should mitigate fenpropathrin resistance in D. citri over time. The development of a VGSC gene biomarker may be a useful tool for monitoring pyrethroid resistance in D. citri going forward.

Genome‑wide identification and expression analysis of the UBC gene family in wheat (Triticum aestivum L.)

BackgroundUbiquitination is an important regulatory step of selective protein degradation in the plant UPS (ubiquitin–proteasome system), which is involved in various biological processes in eukaryotes. Ubiquitin-conjugating enzymes play an intermediate role in the process of protein ubiquitination reactions and thus play an essential role in regulating plant growth and response to adverse environmental conditions. However, a genome-wide analysis of the UBC gene family in wheat (Triticum aestivum L.) has not yet been performed.ResultsIn this study, the number, physiochemical properties, gene structure, collinearity, and phylogenetic relationships of TaUBC family members in wheat were analyzed using bioinformatics methods. The expression pattern of TaUBC genes in different tissues/organs and developmental periods, as well as the transcript levels under abiotic stress treatment, were analyzed using RNA-Seq data and qRT-PCR. Meanwhile, favorable haplotypes of TaUBC25 were investigated based on wheat resequencing data of 681 wheat cultivars from the Wheat Union Database. The analyses identified a total of 93 TaUBC family members containing a UBC domain in wheat genome. These genes were unevenly distributed across 21 chromosomes, and numerous duplication events were observed between gene members. Based on phylogenetic analysis, the TaUBC family was divided into 13 E2 groups and a separate UEV group. We investigated the expression of TaUBC family genes under different tissue/organ and stress conditions by quantitative real-time PCR (qRT-PCR) analysis. The results showed that some TaUBC genes were specifically expressed in certain tissues/organs and that most TaUBC genes responded to NaCl, PEG6000, and ABA treatment with different levels of expression. In addition, we performed association analysis for the two haplotypes based on key agronomic traits such as thousand-kernel weight (TKW), kernel length (KL), kernel weight (KW), and kernel thickness (KT), examining 122 wheat accessions at three environmental sites. The results showed that TaUBC25-Hap II had significantly higher TKW, KL, KW, and KT than TaUBC25-Hap I. The distribution analysis of haplotypes showed that TaUBC25-Hap II was preferred in the natural population of wheat.ConclusionOur results identified 93 members of the TaUBC family in wheat, and several genes involved in grain development and abiotic stress response. Based on the SNPs detected in the TaUBC sequence, two haplotypes, TaUBC25-Hap I and TaUBC25-Hap II, were identified among wheat cultivars, and their potential value for wheat breeding was validated by association analysis. The above results provide a theoretical basis for elucidating the evolutionary relationships of the TaUBC gene family and lay the foundation for studying the functions of family members in the future.

Tanshinone IIA acts as a regulator of lipogenesis to overcome osimertinib acquired resistance in lung cancer

Osimertinib is a novel epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), acting as the first-line medicine for advanced EGFR-mutated NSCLC. Recently, the acquired resistance to osimertinib brings great challenges to the advanced treatment. Therefore, it is in urgent need to find effective strategy to overcome osimertinib acquired resistance. Here, we demonstrated that SREBP pathway-driven lipogenesis was a key mediator to promote osimertinib acquired resistance, and firstly found Tanshinone IIA (Tan IIA), a natural pharmacologically active constituent isolated from Salvia miltiorrhiza, could overcome osimertinib-acquired resistance in vitro and in vivo via inhibiting SREBP pathway-mediated lipid lipogenesis by using LC-MS based cellular lipidomics analysis, quantitative real-time PCR (qRT-PCR) analysis, western blotting analysis, flow cytometry, small interfering RNAs transfection, and membrane fluidity assay et al. The results showed that SREBP1/2-driven lipogenesis was highly activated in osimertinib acquired resistant NSCLC cells, while knockdown or inhibition of SREBP1/2 could restore the sensitivity of NSCLC to osimertinib via altered the proportion of saturated phospholipids and unsaturated phospholipids in osimertinib acquired-resistant cells. Furthermore, Tanshinone IIA (Tan IIA) could reverse the acquired resistance to osimertinib in lung cancer. Mechanically, Tan IIA inhibited SREBP signaling mediated lipogenesis, changed the profiles of saturated phospholipids and unsaturated phospholipids, and thus promoted osimertinib acquired resistant cancer cells to be attacked by oxidative stress-induced damage and reduce the cell membrane fluidity. The reversal effect of Tan IIA on osimertinib acquired resistant NSCLC cells was also confirmed in vivo, which is helpful for the development of strategies to reverse osimertinib acquired resistance.

Metformin inhibits activation of NLRP3 inflammasome and inflammatory response in preeclamptic rats

Backgroud: It is widely acknowledged that Metformin (MET), an established medication for managing type 2 diabetes, possesses diverse pharmacological effects. This study aims to investigate the protective effects of MET against Nω-Nitro-L-arginine methyl ester (L-NAME)-induced preeclampsia (PE). MethodsSprague Dawley (SD) rats were exposed to 200 mg/kg L-NAME with or without prior MET treatment. Histopathological analysis was performed using Hematoxylin and Eosin staining. Serum levels of inflammatory, antiangiogenic, and angiogenic factors were quantified using ELISA kits. Immunohistochemistry (IHC) staining was employed to observe NLRP3 and IL-1β expressions in placental tissues. Western blot and Quantitative Real-Time PCR (q-PCR) analyses were conducted to assess protein and mRNA expressions of NLRP3, caspase-1, ASC, and IL-1β. ResultsWe found that MET could mitigate placental histopathological deterioration and improve pregnancy outcomes in L-NAME-induced PE rat models. MET not only suppressed L-NAME-induced elevation of antiangiogenic factors but also stimulated the production of pro-angiogenic factors. Additionally, MET treatment reversed the excessive inflammatory response induced by L-NAME. Furthermore, MET inhibited the activation of the NLRP3 inflammasome triggered by L-NAME, evidenced by the downregulation of NLRP3 expression, caspase-1, and IL-1β. ConclusionsMET demonstrates a protective effect against L-NAME-induced PE rats, potentially mediated through inhibition of the inflammatory response, downregulation of NLRP3 inflammasome expression in the placenta, and regulation of the balance between anti-angiogenic and pro-angiogenic factors.

Effect of moxibustion on the SIRT1/FOXO3a signaling pathway in ApoE-/- mice with atherosclerosis.

To observe the effects of moxibustion on blood lipid metabolism, pathological morphology of thoracic aorta, and the expression of silent information regulator 1 (SIRT1) and forkhead box transcription factor O3a (FOXO3a) in ApoE-/- atherosclerosis (AS) mice, so as to explore the potential mechanism of moxibustion in preventing and treating AS. Ten C57BL/6J mice were fed a normal diet as the control group, and 30 ApoE-/- mice were fed a high-fat diet to establish the AS model, which were randomly divided into the model group, simvastatin group, and moxibustion group, with 10 mice in each group. From the first day of modeling, mice in the moxibustion group received mild moxibustion treatment at "Shenque"(CV8), "Yinlingquan"(SP9), bilateral "Neiguan"(PC6) and "Xuehai"(SP10) for 30 min per time；the mice in the simvastatin group were given simvastatin orally (2.5 mg·kg-1·d-1), with both treatments given once daily, 5 times a week, with a total intervention period of 12 weeks. The body weight and general condition of the mice were observed and recorded during the intervention period. After the intervention, the contents of serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured using an automated biochemistry analyzer. Hematoxylin eosin (HE) staining was used to observe the pathological morphology of the thoracic aorta. ELISA was used to measure the contents of serum oxidized low-density lipoprotein (ox-LDL) and superoxide dismutase (SOD) activity. Western blot and real-time fluorescent quantitative PCR analysis were used to detect the expression levels of SIRT1 and FOXO3a protein and mRNA in the thoracic aorta. Compared with the control group, body weight at the 8th and 12th week, serum TC, TG, LDL-C, and ox-LDL contents of the model group mice were significantly increased(P<0.05, P<0.01), while the HDL-C contents, SOD activity, and the expression levels of SIRT1 protein and mRNA in the thoracic aorta were significantly decreased(P<0.05, P<0.01). HE staining showed thickening of the aortic intima, endothelial cell degeneration, swelling, and shedding. Compared with the model group, body weight at the 8th and 12th week, serum TC, TG, LDL-C, and ox-LDL contents of mice in the simvastatin group and moxibustion group were significantly decreased(P<0.01), while the serum SOD activity, expression levels of SIRT1 protein and mRNA in the thoracic aorta were significantly increased(P<0.01). The HDL-C contents were significantly increased in the simvastatin group(P<0.05). The thoracic aortic structure was more intact in both groups, with a more regular lumen and orderly arrangement of the elastic membrane in the media, and a slight amount of endothelial cell degeneration and swelling in the intima. There was no significant difference in the evaluated indexes between the moxibustion group and the simvastatin group and the pathological changes in the thoracic aorta were similar between the two groups. Moxibustion can reduce the body weight of AS model mice, regulate lipid levels, repair vascular intima, and alleviate endothelial damage. Its mechanism of action may be related to the regulation of the SIRT1/FOXO3a signaling pathway to improve oxidative damage.

Roles of AhR/CYP1s signaling pathway mediated ROS production in uremic cardiomyopathy

PurposeUremic cardiomyopathy (UCM) is the leading cause of chronic kidney disease (CKD) related mortality. Uremic toxins including indoxyl sulfate (IS) play important role during the progression of UCM. This study was to explore the underlying mechanism of IS related myocardial injury. MethodsUCM rat model was established through five-sixths nephrectomy to evaluate its effects on blood pressure, cardiac impairment, and histological changes using echocardiography and histological analysis. Additionally, IS was administered to neonatal rat cardiomyocytes (NRCMs) and the human cardiomyocyte cell line AC16. DHE staining and peroxide-sensitive dye 2′,7′-dichlorofluorescein diacetate (H2DCFDA) was conducted to assess the reactive oxygen species (ROS) production. Cardiomyocyte hypertrophy was estimated using wheat germ agglutinin (WGA) staining and immunofluorescence. Aryl hydrocarbon receptor (AhR) translocation was observed by immunofluorescence. The activation of AhR was evaluated by immunoblotting of cytochrome P450 1 s (CYP1s) and quantitative real-time PCR (RT-PCR) analysis of AHRR and PTGS2. Additionally, the pro-oxidative and pro-hypertrophic effects were evaluated using the AhR inhibitor CH-223191, the CYP1s inhibitor Alizarin and the ROS scavenger N-Acetylcysteine (NAC). ResultsUCM rat model was successfully established, and cardiac hypertrophy, accompanied by increased blood pressure, and myocardial fibrosis. Further research confirmed the activation of the AhR pathway in UCM rats including AhR translocation and downstream protein CYP1s expression, accompanied with increasing ROS production detected by DHE staining. In vitro experiment demonstrated a translocation of AhR triggered by IS, leading to significant increase of downstream gene expression. Subsequently study indicated a close relationship between the production of ROS and the activation of AhR/CYP1s, which was effectively blocked by applying AhR inhibitor, CYP1s inhibitor and siRNA against AhR. Moreover, the inhibition of AhR/CYP1s/ROS pathway collectively blocked the pro-hypertrophic effect of IS-mediated cardiomyopathy. ConclusionThis study provides evidence that the AhR/CYP1s pathway is activated in UCM rats, and this activation is correlated with the uremic toxin IS. In vitro studies indicate that IS can stimulate the AhR translocation in cardiomyocyte, triggering to the production of intracellular ROS via CYP1s. This process leads to prolonged oxidative stress stimulation and thus contributes to the progression of uremic toxin-mediated cardiomyopathy.