No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-△△Ct Method
Abstract
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-DeltaDeltaCr) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-DeltaDeltaCr) method. In addition, we present the derivation and applications of two variations of the 2(-DeltaDeltaCr) method that may be useful in the analysis of real-time, quantitative PCR data. (C) 2001 Elsevier science.
... 1.94 means 94% amplification efficiency). Relative expression levels were determined with efficiency correction, which considers differences in primer pair amplification efficiencies between target and reference gene and results in a more reliable estimation of the "relative expression ratio" through the 2ΔCt method [7][8]. Expression data and associated technical errors on duplicates were calculated using the gene expression module of the BIORAD iQ5 software, which follows models and error propagation rules outlined in the geNorm manual. ...
... In this respect, the genes coding for hydrophilins, proteins linked to the stress due to the dehydration of the microorganisms, have been of particular interest. In recent years, they have been the subject of some studies to determine the levels of expression of these genes [1][2][3][4][5][6][7][8][9]. The ability of micro-organisms to adapt rapidly to changing environmental conditions is essential for their survival. ...
... In strain F12-7, the TIF11 and YJL144W genes were the least expressed. The TIF11 and YJL144W genes have been reported to be important in strengthening the ability of yeasts to resist water stress [1][2][3][4][5][6][7][8][9]. Our result was in line with those of Cordero-Otero et al. [9]. ...
... The reaction conditions involved enzyme activation at 95˚Cx30 sec, followed by 50 cycles of denaturation at 95˚Cx5 sec, 53˚Cx20 sec for annealing, and 60˚Cx30 sec for extension (15). To analyze the relative changes in the quantitative RT-PCR, we employed the 2(-ΔΔCt) method (16). Following these reactions, high-resolution melting curve analysis was conducted by heating the mixture from 60-99˚C using a ramping degree of 4.8˚C/sec. ...
... Real time PCR was performed and analyzed following the previously described protocol (15,16). cDNA was generated as described above. ...
... For the analysis of the relative quanti cation of gene expression, Microsoft Excel was used using the 2 -ΔΔCT method proposed by Livak and Schmittgen, (2001). Heatmaps with corresponding dendrograms were based on a complete binding cluster by the Euclidean distance method using the heatmap function. ...
Black Sigatoka (SN) is a disease that causes leaf lesions that impede the photosynthesis process, culminating in fruits of poor quality banana. Hence, environmentally correct measures, which can present sustainable control of the disease in the field, are extremely important. Therefore, the objective of this study was to verify glycerol-mediated resistance responses in banana plants of the Grande Naine cultivar susceptible to BS. The experiment was conducted in randomized blocks with 5 treatments, namely: control, 0, 3, 5 and 8% glycerol and three replicates. The severity of the disease after the first symptoms was assessed every seven days. For gene expression analysis, the signal transduction genes (STRANS) and the recognition and signaling genes (KINLRR) and the reference genes, L2MU and 25S, were used. Histochemistry was performed to detect calosis with the aid of relative dyes of lugol and aniline blue. By means of leaf tissue clearing, it was possible to visualize pathogen structures from 72 HAI. In the analysis of calosis, the production of the compound was identified in the first evaluations in all glycerol treatments. The target resistance genes, STRANS and KINLRR, had higher expression in the treatments with 5-8% glycerol. From the results presented, glycerol appears to be a good resistance inducer for banana plants when used at a dose of 5%, but further studies are needed to verify the best time to apply treatments, inoculation, and doses to be used.
... GADPH expression was used as control. Samples were tested in triplicate and data were analyzed using the 2 -∆∆Ct method (11). ...
DNA methylation data for identification of epigenetic targets of resveratrol in triple negative breast cancer cells, Data in Brief, http://dx.
... Gene expression of SmNPP5 or SmAP was measured by quantitative real time PCR (RT-qPCR), with custom TaqMan gene expression systems from Applied Biosystems, CA using primers and probes, as previously [19,23]. Alpha tubulin was used as the endogenous control gene for relative quantification, as described [32], employing the ΔΔCt method [33]. Results obtained from parasites treated with the irrelevant, control siRNA were used for calibration. ...
Background
Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. The intravascular worms acquire the nutrients necessary for their survival from host blood. Since all animals are auxotrophic for riboflavin (vitamin B2), schistosomes too must import it to survive. Riboflavin is an essential component of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD); these support key functions of dozens of flavoenzymes.
Methods
Here, using a combination of metabolomics, enzyme kinetics and in silico molecular analysis, we focus on the biochemistry of riboflavin and its metabolites in Schistosoma mansoni (Sm).
Results
We show that when schistosomes are incubated in murine plasma, levels of FAD decrease over time while levels of FMN increase. We show that live schistosomes cleave exogenous FAD to generate FMN and this ability is significantly blocked when expression of the surface nucleotide pyrophosphatase/phosphodiesterase ectoenzyme SmNPP5 is suppressed using RNAi. Recombinant SmNPP5 cleaves FAD with a Km of 178 ± 5.9 µM and Kcat/Km of 324,734 ± 36,347 M− 1.S− 1. The FAD-dependent enzyme IL-4I1 drives the oxidative deamination of phenylalanine to produce phenylpyruvate and H2O2. Since schistosomes are damaged by H2O2, we determined if SmNPP5 could impede H2O2 production by blocking IL-4I1 action in vitro. We found that this was not the case; covalently bound FAD on IL-4I1 appears inaccessible to SmNPP5. We also report that live schistosomes can cleave exogenous FMN to generate riboflavin and this ability is significantly impeded when expression of a second surface ectoenzyme (alkaline phosphatase, SmAP) is suppressed. Recombinant SmAP cleaves FMN with a Km of 3.82 ± 0.58 mM and Kcat/Km of 1393 ± 347 M− 1.S− 1.
Conclusions
The sequential hydrolysis of FAD by tegumental ecto-enzymes SmNPP5 and SmAP can generate free vitamin B2 around the worms from where it can be conveniently imported by the recently described schistosome riboflavin transporter SmaRT. Finally, we identified in silico schistosome homologs of enzymes that are involved in intracellular vitamin B2 metabolism. These are riboflavin kinase (SmRFK) as well as FAD synthase (SmFADS); cDNAs encoding these two enzymes were cloned and sequenced. SmRFK is predicted to convert riboflavin to FMN while SmFADS could further act on FMN to regenerate FAD in order to facilitate robust vitamin B2-dependent metabolism in schistosomes.
... Gene expression was normalized to GAPDH, which served as an internal reference. The 2 -ΔΔCq method was used for data analysis (17). ...
... The primers used were as follows: miR-372 forward (F), 5'-ACA CTC CAG CTG GGA AAG TGC TGC GAC ATTT-3' and reverse (R), 5'-GTG CAG GGT CCG AGGT-3'; HOXA-AS2 F, 5'-CCC GTA GGA AGA ACC GAT GA-3' and R, 5'-TTT AGG CCT TCG CAG ACA GC-3'; U6 F, 5'-CTC GCT TCG GCA GCA CAT ATA CT-3' and R, 5'-ACG CTT CAC GAA TTT GCG TGTC-3'; and GAPDH F, 5'-GGG AAA CTG TGG CGT GAT-3' and R, 5'-GAG TGG GTG TCG CTG TTGA-3'. Relative gene expression levels were calculated from the data of three independent experiments using the 2 -∆∆Cq method (19). U6 was used as the internal reference for miR-372 and GAPDH was used as the internal reference for HOXA-AS2. ...
... Therefore, we will place more emphasis on investigating this subject. In addition, acquiring a deeper understanding of the complex genetic pathways that control the fibrogenic process might aid in the identification of epigenetic signatures as diagnostic or prognostic markers and in the development of innovative therapeutic strategies (Garofalo et al., 2009;Livak et al., 2001). A primary goal of this research is to determine how miRNA 122 and 221 contribute to liver fibrosis, in addition be looking at lipid levels and they relate to those genes. ...
... Non-specific amplification was controlled through control reactions lacking template, and with no-RT controls. Log2 fold changes were calculated using the 2 -ΔΔCt method (63), and statistical significance between groups was determined using a two-tailed unpaired t-test. ...
Background
Myocarditis leads to dilated cardiomyopathy (DCM) with one-third failing to recover normal ejection fraction (EF50%), and there is a critical need for prognostic biomarkers to assess risk of nonrecovery. Cardiac myosin (CM) autoantibodies (AAbs) cross-reactive with the β−adrenergic receptor (βAR) are associated with myocarditis/DCM, but their potential for prognosis and functional relevance is not fully understood.
Methods
CM AAbs and myocarditis-derived human monoclonal antibodies (mAbs) were investigated to define pathogenic mechanisms and CM epitopes of nonrecovery. Myocarditis patients who do not recover ejection fraction (EF<50%) by one year were studied in a longitudinal (n=41) cohort. Sera IgG and human mAbs were investigated for autoreactivity with CM and CM peptides by ELISA, protein kinase A (PKA) activation, and transcriptomic analysis in H9c2 heart cell line.
Results
CM AAbs were significantly elevated in nonrecovered compared to recovered patients and correlated with reduced EF (<50%). CM epitopes specific to nonrecovery were identified. Transcriptomic analysis revealed serum IgG and mAb 2C.4 induced fibrosis/apoptosis pathways in vitro similar to isoproterenol treated cells. Sera IgG and 2C.4 activated PKA in an IgG and βAR-dependent manner. Endomyocardial biopsies from myocarditis/DCM revealed IgG+ trichrome+ tissues.
Conclusions
CM AAbs were significantly elevated in nonrecovered patients, suggesting novel prognostic relevance. CM AAbs correlated with lower EF, and Ab-induced fibrosis/apoptosis pathways suggested a role for CM AAbs in patients who do not recover and develop irreversible heart failure. Homology between CM and βARs supports mechanisms related to cross-reactivity of CM AAbs with the βAR, a potential AAb target in nonrecovery.
... Each qRT-PCR consisted of three technical replicates for each of three bio replicates. The 2-ΔΔCt method (Livak and Schmittgen, 2001) was used to calculate the relative expression levels of each gene and Wilcoxon rank sum exact test was performed to examine the relative expression difference. ...
Root-knot nematodes (RKNs) are a major pest of Solanum and other economically important crops worldwide. Two species of RKNs ( Meloidogyne chitwoodi and Meloidogyne hapla ) are persistent threats to potato growers of the United States. These RKNs infect potato roots and tubers, causing tuber blemishes that decrease potato market value and significantly impact the profitability of the infected potato crop. Due to environmental, health, and economic concerns, the longstanding control methods of using soil fumigants and post-plant nematicides are not favored by producers and consumers. Therefore, deploying RKN resistant cultivars is an alternative method to control RKN damage. However, there is no genetic resistance to RKN in commercially-available, cultivated potatoes. Therefore, the critical first step to breed a RKN resistant plant is to identify a genetic source of RKN resistance. A wild Solanum species, Solanum sisymbriifolium, also known as litchi tomato, can effectively control several agronomically important species of plant parasitic nematodes. Solanum sisymbriifolium is completely resistant to RKNs; only a few nematodes enter the plant roots and those that do, cannot establish a feeding site. To understand its ability to prevent RKNs from forming feeding sites, we performed transcriptomic analysis of S. sisymbriifolium roots inoculated with the Northern root knot nematode, M. hapla . Combined with the annotation of the recently published S. sisymbriifolium genome assembly, we discovered 13 differentially expressed resistance-related genes upon nematode inoculation. By transforming potatoes with candidate resistance genes from S. sisymbriifolium , we aim to understand the strong genetic resistance in S. sisymbriifolium and whether those genes are necessary and sufficient to drive resistance to RKN in potatoes. This information will help us understand gene functions and help us generate RKN resistance in relevant Solanum crops.
... Gene-specific primers (Table S1) were designed using the NCBI Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), and validation was done by qRT-PCR (CFX96 Touch Real-Time System; Bio-Rad) using the PowerUp SYBR Green Master Mix (Applied Biosystems). The relative gene expression was determined through the DDCT method (Livak and Schmittgen, 2001) using the lentil actin gene (Lcu.2RBY.L011470.1) as an internal control (Sen Gupta et al., 2017;Yuan et al., 2021). ...
Developing early maturing lentil has the potential to minimize yield losses, mainly during terminal drought. Whole‐genome resequencing (WGRS) based QTL‐seq identified the loci governing earliness in lentil. The genetic analysis for maturity duration provided a good fit to 3:1 segregation (F2), indicating earliness as a recessive trait. WGRS of Globe Mutant (late parent), late‐flowering, and early‐flowering bulks (from RILs) has generated 1124.57, 1052.24 million raw and clean reads, respectively. The QTL‐Seq identified three QTLs (LcqDTF3.1, LcqDTF3.2, and LcqDTF3.3) on chromosome 3 having 246244 SNPs and 15577 insertions/deletions (InDels) and 13 flowering pathway genes. Of these, 11 exhibited sequence variations between bulks and validation (qPCR) revealed a significant difference in the expression of nine candidate genes (LcGA20oxG, LcFRI, LcLFY, LcSPL13a, Lcu.2RBY.3g060720, Lcu.2RBY.3g062540, Lcu.2RBY.3g062760, LcELF3a, and LcEMF1). Interestingly, the LcELF3a gene showed significantly higher expression in late‐flowering genotype and exhibited substantial involvement in promoting lateness. Subsequently, an InDel marker (I‐SP‐383.9; LcELF3a gene) developed from LcqDTF3.2 QTL region showed 82.35% PVE (phenotypic variation explained) for earliness. The cloning, sequencing, and comparative analysis of the LcELF3a gene from both parents revealed 23 SNPs and InDels. Interestingly, a 52 bp deletion was recorded in the LcELF3a gene of L4775, predicted to cause premature termination of protein synthesis after 4 missense amino acids beyond the 351st amino acid due to the frameshift during translation. The identified InDel marker holds significant potential for breeding early maturing lentil varieties.
... The primer sequences used are listed in Table 1. Relative expression levels were calculated using the 2 −ΔΔCt method (Livak and Schmittgen 2001). ...
Cervical cancer (CC) is a malignant tumor primarily caused by the persistent infection with high-risk strains of human papillomavirus. This study investigates the aberrant expression of Tripartite Motif Containing 8 (TRIM8) in CC and its impact on cell proliferation, invasion, and migration. Expression levels of TRIM8, Proliferating Cell Nuclear Antigen, and Suppressor of Cytokine Signaling 1 (SOCS1) were assessed in CC cell lines. CC cells were transfected with si-TRIM8, followed by cell counting kit-8 (CCK-8) assay, colony formation assay, and Transwell assay. Protein immunoprecipitation assay was employed to examine TRIM8’s binding with SOCS1, and the ubiquitination level of SOCS1 was determined after MG132 treatment. Rescue experiments were conducted using si-SOCS1 and si-TRIM8 in combination. Results indicate upregulation of TRIM8 in CC cells. Inhibition of TRIM8 suppressed cell viability, proliferation, invasion, and migration. TRIM8 promoted CC cell proliferation, invasion, and migration of CC cells through ubiquitination-mediated degradation of SOCS1. Inhibition of SOCS1 partially reversed the inhibitory effects of si-TRIM8 on the proliferation, invasion, and migration of CC cells. In conclusion, TRIM8 enhances CC cell proliferation, invasion, and migration via ubiquitination-mediated degradation of SOCS1.
... The data were processed using Stratagene MX3005P Mxpro Mx3005p V4 10 software (Stratagene, Amsterdam, The Netherlands). The normalization of gene expression was performed using the ACT1 housekeeping gene, following the 2 −ΔΔCT method [61]. The fold changes were determined as the mean normalized expression of treated strains relative to the mean normalized expression of the untreated one. ...
Invasive candidiasis poses a worldwide threat because of the rising prevalence of antifungal resistance, resulting in higher rates of morbidity and mortality. Additionally, Candida species, which are opportunistic infections, have significant medical and economic consequences for immunocompromised individuals. This study explores the antifungal potential of chitosan to mitigate caspofungin resistance in caspofungin-resistant Candida albicans, C. krusei, and C. tropicalis isolates originating from human and animal sources using agar well diffusion, broth microdilution tests, and transmission electron microscope (TEM) analysis of treated Candida cells. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was performed to assess the expression of SAGA complex genes (GCN5 and ADA2) and the caspofungin resistance gene (FKS) in Candida species isolates after chitosan treatment. The highest resistance rate was observed to ketoconazole (80%) followed by clotrimazole (62.7%), fluconazole (60%), terbinafine (58%), itraconazole (57%), miconazole (54.2%), amphotericin B (51.4%), voriconazole (34.28%), and caspofungin (25.7%). Nine unique FKS mutations were detected, including S645P (n = 3 isolates), S645F, L644F, S645Y, L688M, E663G, and F641S (one isolate in each). The caspofungin minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values before chitosan treatment ranged from 2 to 8 µg/mL and 4 to 16 µg/mL, respectively. However, the MIC and MFC values were decreased after chitosan treatment (0.0625–1 µg/mL) and (0.125–2 µg/mL), respectively. Caspofungin MIC was significantly decreased (p = 0.0007) threefold following chitosan treatment compared with the MIC values before treatment. TEM analysis revealed that 0.5% chitosan disrupted the integrity of the cell surface, causing irregular morphologies and obvious aberrant changes in cell wall thickness in caspofungin-resistant and sensitive Candida isolates. The cell wall thickness of untreated isolates was 0.145 μm in caspofungin-resistant isolate and 0.125 μm in sensitive isolate, while it was significantly lower in chitosan-treated isolates, ranging from 0.05 to 0.08 μm when compared with the cell wall thickness of sensitive isolate (0.03 to 0.06 μm). Moreover, RT-qPCR demonstrated a significant (p < 0.05) decrease in the expression levels of histone acetyltransferase genes (GCN5 and ADA2) and FKS gene of caspofungin-resistant Candida species isolates treated with 0.5% chitosan when compared with before treatment (fold change values ranged from 0.001 to 0.0473 for GCN5, 1.028 to 4.856 for ADA2, and 2.713 to 12.38 for FKS gene). A comparison of the expression levels of cell wall-related genes (ADA2 and GCN5) between caspofungin-resistant and -sensitive isolates demonstrated a significant decrease following chitosan treatment (p < 0.001). The antifungal potential of chitosan enhances the efficacy of caspofungin against various caspofungin-resistant Candida species isolates and prevents the development of further antifungal resistance. The results of this study contribute to the progress in repurposing caspofungin and inform a development strategy to enhance its efficacy, appropriate antifungal activity against Candida species, and mitigate resistance. Consequently, chitosan could be used in combination with caspofungin for the treatment of candidiasis.
... Real-time quantitative PCR was performed using the StepOnePlus real-time PCR system. Expression analysis was done using the double delta ct method as previously described 14 . ...
... The housekeeping gene ribosomal protein 49 (BmRP49) (Gene ID: 778453) was used as an internal reference to normalize the data. Relative expression level of genes was analyzed using the 2 −△△Ct method [31]. All the data were the means of three independent biological replicates. ...
Background
Transcription factor lark has been demonstrated to play multiple functions in Drosophila, but the function of this gene in embryo development remains to be elucidated.
Results
In this study, CRISPR/Cas9 gene-editing method was used to construct a Bmlark mutant strain of Bombyx mori to investigate the roles of this gene. The results showed that the homozygous mutant Bmlark−/− was lethal. The Bmlark−/− embryos showed obvious developmental defects, such as defective sclerotization and melanization of exoskeleton. Transcriptomic comparison of Bmlark−/− and wild-type embryos showed that the differentially expressed genes (DEGs) were mainly enriched in the structure and metabolism processes of chitin and cuticles. While the expression levels of chitin metabolism-related enzyme genes did not significantly change, the expression levels of 63 putative cuticle protein genes showed significant difference in the mutant embryos as compared to the wild-type embryos. The expression levels of transcription factor POUM2 and eight wing disc cuticle protein genes (WCPs) were also changed. While the expression level of TH in the tyrosine-mediated pigmentation pathway was up-regulated in the mutant embryos, the expression levels of the four key pigment synthesis genes DDC, aaNAT, Laccase2A, and yellow-f2 were significantly down-regulated.
Conclusions
The expression levels of 63 putative cuticle protein genes, eight WCPs and five pigment synthesis genes were significantly changed in Bmlark mutants. These results suggest that Bmlark is essential for normal development of cuticle and tyrosine-mediated melanization in silkworm embryo.
... Quantitative detection was performed using a Quant-StudioTM 5 real-time quantitative PCR instrument (Thermo Fisher, USA). Using β-actin as the internal reference gene, the relative expression of each gene was calculated by the 2 −∆∆CT method [33]. All RT-qPCR primers used in this study were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., and the details are given in Table 2. ...
The aim of this study was to investigate the in vitro antioxidant activity of zinc ascorbate (AsA-Zn), its effects on the growth performance of and liver function in Magang geese under heat stress, and its potential mechanism. At AsA-Zn concentrations of 7.5, 15, 30, and 60 µmol/L, the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS·⁺) radical scavenging rate increased significantly by 120.85%, 53.43%, 36.12%, and 0.99%, respectively, compared with that of ascorbic acid (AsA), indicating that AsA-Zn had better antioxidant performance in vitro. In this study, Magang geese were divided into a control group (basal diet, CON) and experimental groups, who received the basal diet supplemented with 400 mg/kg AsA or 30 (AsA-Zn30), 60 (AsA-Zn60), or 90 (AsA-Zn90) mg/kg AsA-Zn. AsA-Zn supplementation considerably reduced the feed-to-gain ratio, whereas both AsA and AsA-Zn significantly increased the thymus index. Moreover, AsA-Zn supplementation improved serum protein levels, lipid metabolism, liver function, and antioxidant capacity while reducing hepatocyte vacuolar degeneration. Furthermore, supplementation with AsA-Zn60 significantly increased the total antioxidant capacity, glutathione peroxidase activity, and superoxide dismutase activity and decreased the malondialdehyde content in the serum, liver, and hepatic mitochondria (P < 0.05), with more pronounced effects in the AsA-Zn60 group. Moreover, supplementation with ASA-Zn regulated the Nrf 2 signaling pathway and significantly increased the expression of genes encoding antioxidant-related factors in the liver. In conclusion, AsA-Zn has good antioxidant activity, and AsA-Zn supplementation may improve the antioxidant capacity of heat-stressed geese and promote their growth. Supplementation with 30 mg/kg AsA-Zn is recommended.
... In the 20 µL reaction, the programmed sequence consisted of an initial stage of 95°C for 30 s, followed by 40 cycles each with steps at 95°C for 5 s, 60°C for 30 s, and 72°C for 20 s. The 5´-3´ primer sequences for qRT-PCR analysis are given in Table-2 [21][22][23][24][25]. Transformed gene expression levels were calculated by the 2 -∆∆Ct formula using normalization to β-actin, as reported by Livak and Schmittgen [26]. ...
Background and Aim: Artemisia annua (AA), used as a growth promoter in poultry, lowers feed costs and enhances economic efficiency. This study aimed to assess the impact of varying AA concentrations on broiler chicken growth, gene expression, and profitability.
... Primer efficiency and specificity were determined using a dilution series of the cDNA before use. The F. fulva actin gene was used as a reference for normalisation of gene expression as per Mesarich et al. (2014), with results analysed according to the 2 ÀDCt method (Livak & Schmittgen, 2001). Results were the average of three biological replicates. ...
Leaf mould, caused by Fulvia fulva, is a devastating disease of tomato plants. In many commercial tomato cultivars, resistance to this disease is governed by the Cf‐9 locus, which encodes five paralogous receptor‐like proteins. Two of these proteins confer resistance: Cf‐9C recognises the previously identified F. fulva effector Avr9 and provides resistance during all plant growth stages, while Cf‐9B recognises the yet‐unidentified F. fulva effector Avr9B and provides mature plant resistance only. In recent years, F. fulva strains have emerged that can overcome the Cf‐9 locus, with Cf‐9C circumvented through Avr9 deletion. To understand how Cf‐9B is circumvented, we set out to identify Avr9B.
Comparative genomics, transient expression assays and gene complementation experiments were used to identify Avr9B, while gene sequencing was used to assess Avr9B allelic variation across a world‐wide strain collection.
A strict correlation between Avr9 deletion and resistance‐breaking mutations in Avr9B was observed in strains recently collected from Cf‐9 cultivars, whereas Avr9 deletion but no mutations in Avr9B were observed in older strains.
This research showcases how F. fulva has evolved to sequentially break down the Cf‐9 locus and stresses the urgent need for commercial tomato cultivars that carry novel, stacked resistance genes active against this pathogen.
... Quantification of car1 transcripts in As(III) and As(V) The relative changes of gene expression were calculated using the method described by Livak and Schmittgen (2001). ...
An arsenate reductase (Car1) from the Bacteroidetes species Rufibacter tibetensis 1351T was isolated from the Tibetan Plateau. The strain exhibits resistance to arsenite [As(III)] and arsenate [As(V)] and reduces As(V) to As(III). Here we shed light on the mechanism of enzymatic reduction by Car1. AlphaFold2 structure prediction, active site energy minimization, and steady‐state kinetics of wild‐type and mutant enzymes give insight into the catalytic mechanism. Car1 is structurally related to calcineurin‐like metallophosphoesterases (MPPs). It functions as a binuclear metal hydrolase with limited phosphatase activity, particularly relying on the divalent metal Ni²⁺. As an As(V) reductase, it displays metal promiscuity and is coupled to the thioredoxin redox cycle, requiring the participation of two cysteine residues, Cys74 and Cys76. These findings suggest that Car1 evolved from a common ancestor of extant phosphatases by incorporating a redox function into an existing MPP catalytic site. Its proposed mechanism of arsenate reduction involves Cys74 initiating a nucleophilic attack on arsenate, leading to the formation of a covalent intermediate. Next, a nucleophilic attack of Cys76 leads to the release of As(III) and the formation of a surface‐exposed Cys74‐Cys76 disulfide, ready for reduction by thioredoxin.
... Reactions were carried out with CFX PCR System (Bio-rad Laboratories, Hercules, CA). The expression of each gene was compared between groups using the 2 −ΔCT method 33 . For all qPCR reactions, every sample was ampli ed in duplicate. ...
The microbiome-gut-brain axis plays a role in anxiety and social development and is of growing interest in neuropsychiatic conditions, including autism spectrum disorder (ASD). The present study investigated the behavioral phenotype and the molecular profile of neuropeptide Y (NPY), an anxiolytic peptide, in microbiome-gut-brain communication of Nf1 +/− mice, a well-established animal model of ASD. Sex differences, up to date poorly investigated in animal models, were specifically addressed. Our data revealed that females Nf1 +/− exhibited more prominent anxious-like behavior. In addition, molecular analyses indicated sex-related differences in expression of NPY and NPY receptors’ transcripts in transgenic animals, with a more prominent effect in females. In addition, the analysis of microbiota revealed sex-specific changes in the Lactobacillus content which correlated with NPY and Y2 receptor changes in transgenic females. Remarkably, the Y2 receptor exhibited sex-dependent expression in both gut and brain of Nf1 +/− mice, suggesting its potential as a molecular biomarker for ASD symptoms, namely social anxiety and gastrointestinal issues. For the first time, our findings suggest NPY-mediated regulation of gut-brain communication to be altered in autism and hold potential for the development of new interventions addressing sex-specific aspects of ASD.
... All reactions were performed in triplicate, and contamination was monitored by including a no-template control in each RT-qPCR run, consisting of a reaction mixture without template DNA. Quantitative RT-qPCR cycle threshold (CT) values were normalized using the housekeeping gene GAPDH, and fold changes were determined using the 2 -ΔΔCT method (8). The findings of the data analysis were presented as mean ± standard deviation (SD) using SPSS Data Editor version 23.0 (IBM, USA). ...
Background and Objectives: Enterococcus faecalis is known as common pathogen for endodontic infections and cause secondary and refractory pulp periapical periodontitis. The bacteria can opportunistically colonize periodontal pockets and presents a possibility of infection developing in other organs. This research will investigate the dissemination of E. faecalis from the gingival tissue to the heart and kidney. Materials and Methods: Three groups were formed, consisting of twelve male Sprague Dawley rats: a control group designated as 0-day, and experimental groups labeled as 7-days and 14-days. Periodontitis induced by concurrent infection with sterile wire 0.2 mm insertion and E. faecalis inoculation is performed into the gingival sulcus located between the maxillary right 1 st and 2 nd molar teeth area. After euthanasia, tissue samples around the maxillary gingiva, maxillary jaw samples, kidney and heart tissues were obtained for quantitative Real-Time PCR assay and histopathological analysis. Results: Results showed at 7-days, there was an upregulation of E. faecalis gene expression in the gingiva, heart, and kidney samples as well as infiltration of the inflammatory cells at 7-days post induction, which consequently decreased at 14-days. Conclusion: Thus, the study suggests dissemination of E. faecalis from gingival tissue to the heart, kidney which could be probable link between periodontal disease, heart, and kidney disease.
... Therefore, we will place more emphasis on investigating this subject. In addition, acquiring a deeper understanding of the complex genetic pathways that control the fibrogenic process might aid in the identification of epigenetic signatures as diagnostic or prognostic markers and in the development of innovative therapeutic strategies (Garofalo et al., 2009;Livak et al., 2001). A primary goal of this research is to determine how miRNA 122 and 221 contribute to liver fibrosis, in addition be looking at lipid levels and they relate to those genes. ...
Many biological processes depend on microRNAs (miRNAs), and disorders
caused by a defect in the functioning in these genes may manifest in a
variety of ways, including metabolic disorders. Therefore, the current
research aims to investigate the gene expression of miRNA 122 and 221
and their impact on the lipid profile of individuals suffering from liver
fibrosis. Present research is a case – control study, included blood samples
collection from 44 fibrotic patients and 44 health individuals as control.
After RNA extraction, gene expression of miRNA 122 and 221 evaluated by
RT-qPCR. FUJI DRI-CHEM SLIDE are used to determine total Cholesterol,
triglyceride, HDL and LDL concentration. The results of the current study
showed higher concentration of cholesterol, triglycerides and LDL in
patients (316.12 ± 96, 256.08 ± 41 and 147.48 ± 11.69 ml/dl respectively)
compared to control (136.24 ± 10.5, 126.48 ± 6.96 and117.72 ± 3.59 ml/dl
respectively) (P< 0.05) while a lower HDL concentration detected in
patients (53.8 ± 4.89 ml/dl) compared to controls (59.28 ± 6.31 ml/dl).
The results of the gene expression analysis showed a lower mean fold
change (2-∆∆CT) for miRNA 122 in patients (3.75) compared to controls
(7.44) while we found a higher mean fold change for miRNA221 in patients
(2.70) compared with control (0.04). Increase or decrease in gene
expression for miRNA 122 was inversely related to the level of lipids in the
patients' blood, while a moderate positive relationship appeared between
the rate of gene expression for miRNA 221 and the level of lipids in
patients. In conclusion, we found that 122 and 221 are associated with
fibrosis, and that high or low gene expression of these genes moderately
affects the level of lipids, which in turn is one of the most important causes
of liver fibrosis
... The quantitative real-time PCR (qRT-PCR) experiments were performed on LightCycler 480 II PCR System (Mannheim, Germany), with GhActin selected as the internal reference gene for normalization purposes [47]. Quantification of gene transcript levels was performed using the 2 -∆∆CT method with three biological replicates and three technical replicates [48]. The expression data was visualized by GraphPad Prism software (version: 10) and the 'pheatmap' R-package to generate comprehensive and informative representations of gene expression patterns within the studied gene set [46]. ...
Background
Monoacylglycerol lipase (MAGL) genes belong to the alpha/beta hydrolase superfamily, catalyze the terminal step of triglyceride (TAG) hydrolysis, converting monoacylglycerol (MAG) into free fatty acids and glycerol.
Results
In this study, 30 MAGL genes in upland cotton have been identified, which have been classified into eight subgroups. The duplication of GhMAGL genes in upland cotton was predominantly influenced by segmental duplication events, as revealed through synteny analysis. Furthermore, all GhMAGL genes were found to contain light-responsive elements. Through comprehensive association and haplotype analyses using resequencing data from 355 cotton accessions, GhMAGL3 and GhMAGL6 were detected as key genes related to lipid hydrolysis processes, suggesting a negative regulatory effect.
Conclusions
In summary, MAGL has never been studied in upland cotton previously. This study provides the genetic mechanism foundation for the discover of new genes involved in lipid metabolism to improve cottonseed oil content, which will provide a strategic avenue for marker-assisted breeding aimed at incorporating desirable traits into cultivated cotton varieties.
... The 2 −∆∆Cq method was employed to calculate relative mRNA MCM8 expression. 22 ...
MCM8 is a helicase, which participates in DNA replication and tumorigenesis and is upregulated in many human cancers, including lung cancer (LC); however, the function of MCM8 in LC tumour progression is unclear. In this study, we found that MCM8 was expressed at high levels in LC cells and tissues. Further, MCM8 upregulation was associated with advanced tumour grade and lymph node metastasis, and indicated poor prognosis. Silencing of MCM8 suppressed cell growth and migration in vitro and in vivo, while ectopic MCM8 expression promoted cell cycle progression, as well as cell migration, proliferation, and apoptosis. Mechanistically, DNAJC10 was identified as a downstream target of MCM8, using gene array and CO‐IP assays. DNAJC10 overexpression combatted the inhibitory activity of MCM8 knockdown on LC progression, while silencing DNAJC10 alleviated the oncogenic function of MCM8 overexpression. MCM8 expression was positively correlated with that of DNAJC10 in LC samples from The Cancer Genome Atlas database, and DNAJC10 upregulation was also associated with poor overall survival of patients with LC. This study indicated that MCM8/DNAJC10 axis plays an important role in in LC development, and maybe as a new potential therapeutic target or a diagnostic biomarker for treating patients with LC.
... The collected blood of golden pompano was centrifuged at 1500 rpm for 10 minutes, and the 85 supernatant was taken with a pipette and the collected serum was placed at -80 for the determination. Table 3. Data were 99 analyzed by 2 −ΔΔCT method [29] using β-actin as reference. ...
... RT-qPCR was performed using a CFX96™ Real-Time PCR detection system (Bio-Rad, USA). All genes expression levels were standardized by using the 16S rRNA gene expression level as an internal standard and quantified as previously described (Livak and Schmittgen, 2001). All experiments were repeated three times. ...
... Data were analyzed using CFX Maestro software (Bio-Rad). Relative mRNA levels for genes were normalized to the geometric mean of the endogenous controls GAPDH, and RPL13A using the 2^-ΔΔCt method 60 . ...
During pulmonary mucormycosis, inhaled sporangiospores adhere to, germinate, and invade airway epithelial cells to establish infection. We provide evidence that HIF1α plays dual roles in airway epithelial cells during Mucorales infection. We observed an increase in HIF1α protein accumulation and increased expression of many known HIF1α-responsive genes during in vitro infection, indicating that HIF1α signaling is activated by Mucorales infection. Inhibition of HIF1α signaling led to a substantial decrease in the ability of R. delemar to invade cultured airway epithelial cells. Transcriptome analysis revealed that R. delemar infection induces the expression of many pro-inflammatory genes whose expression was significantly reduced by HIF1α inhibition. Importantly, pharmacological inhibition of HIF1α increased survival in a mouse model of pulmonary mucormycosis without reducing fungal burden. These results suggest that HIF1α plays two opposing roles during mucormycosis: one that facilitates the ability of Mucorales to invade the host cells and one that facilitates the ability of the host to mount an innate immune response.
... For qPCR, the reaction mixture comprised approximately 20ng of cDNA template, 10 µM each of forward and reverse primers and 1x SYBR Green PCR Master Mix Kit (Takara Bio Inc, Japan), along with 20 µl of nuclease-free water. The qPCR cycle threshold (Ct) values were used to calculate the relative gene expression level displayed as 2-Ct (Livak and Schmittgen, 2001). ...
Background: The objective of this study was to assess the effects of dietary levamisole supplementation feed on the growth, hemato-biochemical parameters, disease resistance and the expression of genes related to growth and immunity in Asian seabass, Lates calcarifer. Methods: Triplicate groups of fish (n=20) with an average weight of 3.29±0.4 g were fed with feed supplemented with levamisole (C, 0.0 mg/Kg; LVT1, 75 mg/Kg; LVT2, 150 mg/Kg; LVT3, 300 mg/Kg; LVT4, 600 mg/Kg) for 60 days. At the end of the experiment growth, haemato-biochemical parameters and disease resistance to Aeromonas veronii were assessed. Relative expressions of growth and immune genes were analyzed by quantitative real-time PCR (qPCR). Result: Dietary incorporation with levamisole in the range of 75-600 mg/Kg diet improved the haemato-biochemical indices, survival rates and resistance to Aeromonas veronii challenge (CAK4/SRLAAH/2022) at a concentration of 2.5 10 4 cfu/ml. Levamisole incorporation at 300 mg/Kg feed (LVT3) resulted in significant (p<0.05) improvements in various parameters such as feed efficiency, growth rate, increased disease resistance and upregulated expression of both growth and immune-related genes in comparison to the control (C). Significant improvements in haemato-biochemical indices such as hemoglobin (Hb), white blood cells (W BC), packed cell volume (PCV), erythrocytes (Ery), glucose (GLU), cholesterol (Cho) and triglyceride (TG) levels were also recorded.
... The protocol included initial denaturation at 95 °C for 30 s, followed by 40 cycles of amplification, comprising denaturation at 95 °C for 5 s, annealing at 60 °C (mexA, mexD, and rpsL) or 62 °C (mexE and mexX) for 20 s, and extension at 72 °C for 30 s. rpsL was used as the housekeeping gene to calculate the relative expression levels of each gene. Relative target levels were calculated using the ∆∆Ct method [67]. Results represent the average enrichment measured by qPCR in at least three replicates. ...
... PBC, Boston, MA, USA. Fold changes were calculated with the 2 −∆∆CT method [39], as described by us previously [40,41], using the average of a set of multiple endogenous control genes (Actb, B2m, Gapdh, Gusb, Hsp90ab1), as recommended by the manufacturer and prior publications [42]. Statistical significance was assessed using the Bioconductor limma package on R and the false discovery rate (FDR) was calculated with the Benjamini-Hochberg correction for multiple comparisons. ...
The emerging concern about chemicals in electronic cigarettes, even those without nicotine, demands the development of advanced criteria for their exposure and risk assessment. This study aims to highlight the sensitivity of lung nuclear receptors (NRs) to electronic cigarette e-liquids, independent of nicotine presence, and the influence of the sex variable on these effects. Adult male and female C57BL/6J mice were exposed to electronic cigarettes with 0%, 3%, and 6% nicotine daily (70 mL, 3.3 s, 1 puff per min/30 min) for 14 days, using the inExpose full body chamber (SCIREQ). Following exposure, lung tissues were harvested, and RNA extracted. The expression of 84 NRs was determined using the RT² profiler mRNA array (Qiagen). Results exhibit a high sensitivity to e-liquid exposure irrespective of the presence of nicotine, with differential expression of NRs, including one (females) and twenty-four (males) in 0% nicotine groups compared to non-exposed control mice. However, nicotine-dependent results were also significant with seven NRs (females), fifty-three NRs (males) in 3% and twenty-three NRs (female) twenty-nine NRs (male) in 6% nicotine groups, compared to 0% nicotine mice. Sex-specific changes were significant, but sex-related differences were not observed. The study provides a strong rationale for further investigation.
... The thermocycling conditions were as follows: Polymerase activation for 1 cycle at 95˚C for 2 min; followed by 40 cycles of 95˚C for 15 sec and 60˚C for 1 min. Relative fold changes in RNA expression were calculated using the 2 -ΔΔCt method (42). mRNA levels were normalized to GAPDH gene expression, and miRNA levels were normalized to U6 small nuclear RNA levels. ...
... All samples were analyzed in duplicate. Relative gene expression was calculated using the 2 -ΔΔCq method (37). ...
... β-actin served as an internal control. The expression levels of targets were quantified using the 2 −∆∆Ct method [32]. ...
... The qPCR was performed with the following steps: 95 °C for 5 s, 60 °C for 5 s, and 40 cycles on a CFX Connect™ (Bio-Rad Laboratories, Hercules, CA, USA). The comparative threshold cycle (CT) method (2 −ΔΔCT ) (Livak and Schmittgen 2001) was used to analyze the relative expression levels of the target mRNAs, with the medaka cytoplasmic actin gene actb serving as an internal reference. ...
Intracellular bacteria such as those belonging to the genus Edwardsiella can survive and proliferate within macrophages. However, the detailed mechanisms underlying the host macrophage immune response and pathogen evasion strategies remain unknown. To advance the field of host macrophage research, we successfully established transgenic (Tg) Japanese medaka Oryzias latipes that possesses fluorescently visualized macrophages. As a macrophage marker, the macrophage-expressed gene 1.1 (mpeg1.1) was selected because of its predominant expression across various tissues in medaka. To validate the macrophage characteristics of the fluorescently labeled cells, May-Grünwald Giemsa staining and peroxidase staining were conducted. The labeled cells exhibited morphological features consistent with those of monocyte/macrophage-like cells and tested negative for peroxidase activity. Through co-localization studies, the fluorescently labeled cells co-localized with E. piscicida in the intestines and kidneys of infected medaka larvae, confirming the ingestion of bacteria through phagocytosis. In addition, the labeled cells expressed macrophage markers but lacked a neutrophil marker. These results suggested that the fluorescently labeled cells of Tg[mpeg1.1:mCherry/mAG] medaka were monocytes/macrophages, which will be useful for future studies aimed at understanding the mechanisms of macrophage-mediated bacterial infections.
... The following thermocycling conditions were used: Initial denaturation at 95˚C for 60 sec, followed by 40 cycles of 95˚C for 15 sec, 60˚C for 15 sec and 72˚C for 45 sec. The primers are listed in Table I. mRNA levels were quantified for each target gene using the 2 -ΔΔCq method (26), and normalized to GAPDH as an internal control. (FISH). ...
... The PCR program was as follows: 95˚C for 3 min; 40 cycles of 95˚C for 30 sec, 56˚C for 30 sec; and 72˚C for 50 sec. The expression levels of target genes were quantified relative to the expression level of GAPDH as an internal control for normalization using the 2 -ΔΔCq method (32). Primer sequences are listed in Table I. ...
... All primers used for the testing genes are described in Table S1 and Table S2. T. xiaojinensis ribosomal protein S3 (rps3) was used as a reference for target gene expression calibration [34], and qPCR results were analyzed using the relative quantitative method (2 −ΔΔct ) [35,36]. Three biological replicates. ...
Background Ophiocordyceps sinensis as one typical entomopathogenic fungus (EPF) has the long-term symbiosis process with its host Thitarodes xiaojinensis. O. sinensismainly exists in the hemolymph of the host. However, the mechanism of the host immune response to O. sinensis remains unclear.
Results Here, a multi‑omics approach was used to clarify the role of the interaction between O. sinensis and T.xiaojinensis. The infection of O. sinensis could lead to the increase of hormone levels (20-hydroxyecdysone and juvenile hormone), the enhancement of antioxidant capacity (total antioxidant capacity and glutathione S-transferase) and the response of humoral immunity based on the antibacterial peptides (AMPs) in the host T.xiaojinensis. Elevated 20E levels in the host when O. sinensis infection might contribute to the enhanced expression of AMPs. O. sinensis infection led to intestinal barrier damage and promoted the translocation of bacteria from the gut to hemocoel. Then, the presence of O. sinensis and other opportunistic pathogenic bacterium from gut disrupted the homeostasis of hemolymph microbiota and increased bacterial diversity of the hemolymph.
Conclusions Overall, this study demonstrated that O. sinensis infection damaged intestinal barrier and induced the translocation of gut bacteria and the disruption of microbial homeostasis in hemolymph. The host T.xiaojinensis activated and exploited humoral antibacterial immunity and to eliminate opportunistic bacteria. our findings reveal a novel strategy of interaction between O. sinensis and T.xiaojinensis.
... β-actin was used as the reference gene [12]. The relative gene expression levels were analyzed using the 2 −ΔΔCt method [13]. ...
Enterocytozoon hepatopenaei (EHP) is a parasite in shrimp farming. EHP mainly parasitizes the hepatopancreas of shrimp, causing slow growth, which severely restricts the economic income of shrimp farmers. To explore the pathogenic mechanism of EHP, the host subcellular construction, molecular biological characteristics, and mitochondrial condition of Litopenaeus vannamei were identified using transmission electron microscopy (TEM), real-time qPCR, an enzyme assay, and flow cytometry. The results showed that EHP spores, approximately 1 μm in size, were located on the cytoplasm of the hepatopancreas. The number of mitochondria increased significantly, and mitochondria morphology showed a condensed state in the high-concentration EHP-infected shrimp by TEM observation. In addition, there were some changes in mitochondrial potential, but apoptosis was not significantly different in the infected shrimp. The qPCR results showed that the gene expression levels of hexokinase and pyruvate kinase related to energy metabolism were both upregulated in the diseased L. vannamei. Enzymatic activity showed hexokinase and lactate dehydrogenase were significantly increased in the shrimp infected with EHP, indicating EHP infection can increase the glycolysis process and decrease the oxidative phosphorylation process of L. vannamei. Previous transcriptomic data analysis results also support this conclusion.
Guar is a legume of industrial importance due to the presence of gum (galactomannan) in its seed endosperm. The mannose (M)/galactose (G) ratio in galactomannan molecule is critical in deciding the physico-chemical properties of guar gum. Gum with reduced galactose content has better commercial value due to better viscosity. We report down regulation of galactomannan galactosyltransferase (GMGT) which links G residues to the M backbone in galactomannan molecule. The target GMGT sequence for RNAi was directionally cloned into pENTR TM /D-TOPO® vector, followed by a LR recombination reaction with pANDA vector. The constructed gmgt RNAi cassette was delivered into guar tissue using Agrobacterium tumefaciens-mediated gene transfer. The cotyledonary node explants were pre-cultured for 72 h and co-cultivated with A. tumefaciens strain harbouring the gmgt RNAi cassette for 24 h. Maximum number of putative transformed shoots regenerated from the cotyledonary node explants cultured in vitro on Murashige and Skoog (MS) medium containing 1.5 mg/L indole-3-acetic acid (IAA), 4.0 mg/L butyric acid (BA) and 1.0 mg/L gibberellic acid (GA 3). In vitro regenerated shoots were rooted on half-strength MS medium containing 4% sucrose and 2 mg/L indole-3-butyric acid (IBA). The quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) analysis showed reduced levels of GMGT expression in the seed endosperm of 35 days after owering (DAF) stage pods. The present ndings are expected to be bene cial in taking up of gene characterization and improvement of agronomic traits in guar.
Background
The comprehensive understanding of microRNAs (miRNAs) in sheep milk during various lactation periods and their impact on milk yield and composition remains limited.
Objectives
This study aimed to investigate the expression patterns of four highly expressed miRNAs in sheep milk and their association with milk composition and yield parameters during peak and late lactation stages.
Methods
A total of 40 healthy 4‐year‐old Akkaraman (n = 20) and Awassi (n = 20) ewes registered with the Ministry of Agriculture and Forestry of the Republic of Türkiye were used in the present study. For miRNA isolation from milk, the Qiagen miRNeasy Serum/Plasma Advanced Kit was utilised following the manufacturer's instructions. The expression levels of miRNAs were assessed using Qiagen miRNA PCR Assays.
Results
The significant fold changes in the expression levels of oar‐miR‐30a‐5p, oar‐miR‐148a and oar‐miR‐181a were observed between peak and late lactation periods in the Awassi sheep breed. Conversely, only oar‐miR‐30a‐5p and oar‐miR‐148a exhibited statistically significant changes in the Akkaraman sheep breed during the same lactation periods. Furthermore, oar‐miR‐21‐5p demonstrated a significant fold change exclusively in peak lactation compared to Akkaraman and Awassi ewes.
Conclusions
The findings suggest that the expression of the analysed miRNAs is influenced by both the lactation stage and different sheep breeds. This study offers valuable insights into the relationship between key miRNA expressions in sheep milk and milk composition and yield parameters during peak and late lactation, contributing to the existing knowledge in this field.
Rice stripe mosaic virus (RSMV) is an emerging pathogen which significantly reduces rice yields in the southern region of China. It is transmitted by the leafhopper Recilia dorsalis, which overwinters in rice fields. Our field investigations revealed that RSMV infection causes delayed rice heading, resulting in a large number of green diseased plants remaining in winter rice fields. This creates a favorable environment for leafhoppers and viruses to overwinter, potentially contributing to the rapid spread and epidemic of the disease. Next, we explored the mechanism by which RSMV manipulates the developmental processes of the rice plant. A rice heading‐related E3 ubiquitin ligase, Heading date Associated Factor 1 (HAF1), was found to be hijacked by the RSMV‐encoded P6. The impairment of HAF1 function affects the ubiquitination and degradation of downstream proteins, HEADING DATE 1 and EARLY FLOWERING3, leading to a delay in rice heading. Our results provide new insights into the development regulation‐based molecular interactions between virus and plant, and highlights the importance of understanding virus‐vector‐plant tripartite interactions for effective disease management strategies.
Osteoporosis (OP) is a systemic metabolic bone disease that is characterized by decreased bone mineral density and microstructural damage to bone tissue. Recent studies have demonstrated significant advances in the research of programmed cell death (PCD) in OP. However, there is no bibliometric analysis in this research field. This study searched the Web of Science Core Collection (WoSCC) database for literature related to OP and PCD from 2000 to 2023. This study used VOSviewers 1.6.20, the “bibliometrix” R package, and CiteSpace (6.2.R3) for bibliometric and visualization analysis. A total of 2905 articles from 80 countries were included, with China and the United States leading the way. The number of publications related to PCD in OP is increasing year by year. The main research institutions are Shanghai Jiao Tong University, Chinese Medical University, Southern Medical University, Zhejiang University, and Soochow University. Bone is the most popular journal in the field of PCD in OP, and the Journal of Bone and Mineral Research is the most co‐cited journal. These publications come from 14,801 authors, with Liu Zong‐Ping, Yang Lei, Manolagas Stavros C, Zhang Wei, and Zhao Hong‐Yan having published the most papers. Ronald S. Weinstein was co‐cited most often. Oxidative stress and autophagy are the current research hot spots for PCD in OP. This bibliometric study provides the first comprehensive summary of trends and developments in PCD research in OP. This information identifies the most recent research frontiers and hot directions, which will provide a definitive reference for scholars studying PCD in OP.
One of the lignans isolated from plants within the genus Podophyllum is podophyllotoxin (PPT). PPT and its derivatives are pharmacologically active compounds with potential antiproliferative properties in several kinds of tumors. Although these compounds have been used to treat other malignancies, no PPT derivative-based chemotherapeutic agent has been used to cure tamoxifen (TAM)-resistant breast cancer in clinical trials, to the best of our knowledge. Thus, using TAM-resistant breast cancer as a disease model, the present study assessed the effects of a recently synthesized PPT derivative, bromosulfonamidine amino-PPT (BSAPPT), on TAM-resistant breast cancer. Using the tamoxifen-resistant breast cancer cell model (MCF-7/TAMR) in vitro, Cell Counting Kit-8 and colony formation assays were adopted to evaluate the effect of BSAPPT on cell proliferation. Cell apoptosis and cell cycle assays were used to assess the influence of BSAPPT on cell apoptosis and the cell cycle in MCF-7/TAMR. The targets of the potential mechanism of action were analyzed by RT-qPCR and western blotting. The present study demonstrated that BSAPPT suppressed MCF-7/TAMR cell proliferation in a dose-dependent manner. By modulating the level of expression of genes linked to both apoptosis and the cell cycle, BSAPPT triggered MCF-7/TAMR cells to undergo apoptosis and prevented them from entering the cell cycle. Consequently, BSAPPT blocked these cells from proliferating, thereby halting the malignant advancement of TAM-resistant breast cancer. Therefore, these findings indicate that new therapeutic agents involving BSAPPT may be developed to facilitate the treatment of TAM-resistant breast cancer.
Background
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, occurring mostly in individuals with chronic liver disease, but biomarkers for therapeutic diagnosis and prognosis are lacking. This study aimed to investigate the possible effect of the common food additive monosodium glutamate (MSG) and tannic acid (TA), a phenolic compound, on the key molecular actors responsible for HCC development.
Methods
Eight HCC‐related public microarray datasets (GSE84005, GSE14520, GSE25097, GSE57958, GSE22058, GSE84402, GSE54238, and GSE36376) were extracted from the gene expression omnibus (GEO) database and analyzed to identify differentially expressed genes (DEGs). To make sense of the identified biological data and to identify hub genes, protein–protein interaction (PPI) network and enrichment analysis were performed. The mRNA expression profiles of the identified hub genes, expression changes in different stages of HCC, and their prognostic significances in HCC were determined using GEPIA, UALCAN, and Kaplan–Meier Plotter databases, respectively. Finally, mRNA expression changes of identified hub genes in the liver tissues of rats treated with MSG and TA were measured by the quantitative real‐time PCR (qPCR) method.
Results
Two up‐regulated (AURKA and CCNB2) and two down‐regulated (F9 and CYP2E1) genes were identified between the HCC tumor and adjacent non‐tumor liver tissue samples. qPCR results showed that the mRNA expression of up‐regulated DEGs involved in HCC development increased significantly in rat liver tissues exposed to MSG, while this increase was remarkably suppressed by TA treatment. It was observed that the mRNA expressions of down‐regulated DEGs involved in HCC development decreased markedly in the presence of MSG, while this decrease was alleviated with TA.
Conclusion
Our results provide new insights into pivotal molecular candidates that should be focused on in future in vivo and in vitro HCC research. Moreover, MSG may play a crucial role in HCC development and progression and TA may be used as a favorable restorative agent in HCC.
Tomato is a popular vegetable worldwide; its production is highly threatened by infection with the potato spindle tuber viroid (PSTVd). We obtained the full‐length genome sequence of previously conserved PSTVd and inoculated it on four genotypes of semi‐cultivated tomatoes selected from a local tomato germplasm resource. SC‐5, which is a PSTVd‐resistant genotype, and SC‐96, which is a PSTVd‐sensitive genotype, were identified by detecting the fruit yield, plant growth, biomass accumulation, physiological indices, and PSTVd genome titer after PSTVd inoculation. A non‐target metabolomics study was conducted on PSTVd‐infected and control SC‐5 to identify potential anti‐PSTVd metabolites. The platform of liquid chromatography‐mass spectrometry detected 158 or 123 differential regulated metabolites in modes of positive ion or negative ion. Principal component analysis revealed a clear separation of the global metabolite profile between PSTVd‐infected leaves and control regardless of the detection mode. The potential anti‐PSTVd compounds, xanthohumol, oxalicine B, indole‐3‐carbinol, and rosmarinic acid were significantly upregulated in positive ion mode, whereas echinocystic acid, chlorogenic acid, and 5‐acetylsalicylic acid were upregulated in negative ion mode. Xanthohumol and echinocystic acid were detected as the most upregulated metabolites and were exogenously applied on PSTVd‐diseased SC‐96 seedlings. Both xanthohumol and echinocystic acid had instant and long‐term inhibition effect on PSTVd titer. The highest reduction of disease symptom was induced by 2.6 mg/L of xanthohumol and 2.0 mg/L of echinocystic acid after 10 days of leaf spraying, respectively. A superior effect was seen on echinocystic acid than on xanthohumol. Our study provides a statistical basis for breeding anti‐viroid tomato genotypes and creating plant‐originating chemical preparations to prevent viroid disease.
Purpose:
To compare gene expression changes following branch retinal vein occlusion (BRVO) in the pig with and without bevacizumab (BEV) and triamcinolone acetonide (TA).
Methods:
Photothrombotic BRVOs were created in both eyes of four groups of nine pigs (2, 6, 10, and 20 days). In each group, six pigs received intravitreal injections of BEV in one eye and TA in the fellow eye, with three pigs serving as untreated BRVO controls. Three untreated pigs served as healthy controls. Expression of mRNA of vascular endothelial growth factor (VEGF), glial fibrillary acidic protein (GFAP), dystrophin (DMD), potassium inwardly rectifying channel subfamily J member 10 protein (Kir4.1, KCNJ10), aquaporin-4 (AQP4), stromal cell-derived factor-1α (CXCL12), interleukin-6 (IL6), interleukin-8 (IL8), monocyte chemoattractant protein-1 (CCL2), intercellular adhesion molecule 1 (ICAM1), and heat shock factor 1 (HSF1) were analyzed by quantitative reverse-transcription polymerase chain reaction. Retinal VEGF protein levels were characterized by immunohistochemistry.
Results:
In untreated eyes, BRVO significantly increased expression of GFAP, IL8, CCL2, ICAM1, HSF1, and AQP4. Expression of VEGF, KCNJ10, and CXCL12 was significantly reduced by 6 days post-BRVO, with expression recovering to healthy control levels by day 20. Treatment with BEV or TA significantly increased VEGF, DMD, and IL6 expression compared with untreated BRVO eyes and suppressed BRVO-induced CCL2 and AQP4 upregulation, as well as recovery of KCNJ10 expression, at 10 to 20 days post-BRVO.
Conclusions:
Inflammation and cellular osmohomeostasis rather than VEGF suppression appear to play important roles in BRVO-induced retinal neurodegeneration, enhanced in both BEV- and TA-treated retinas.
Translational relevance:
Inner retinal neurodegeneration seen in this acute model of BRVO appears to be mediated by inflammation and alterations in osmohomeostasis rather than VEGF inhibition, which may have implications for more specific treatment modalities in the acute phase of BRVO.
Accurate and timely diagnosis of oral squamous cell carcinoma (OSCC) is crucial in preventing its progression to advanced stages with a poor prognosis. As such, the construction of sensors capable of detecting previously established disease biomarkers for the early and non-invasive diagnosis of this and many other conditions has enormous therapeutic potential. In this work, we apply synthetic biology techniques for the development of a whole-cell biosensor (WCB) that leverages the physiology of engineered bacteria in vivo to promote the expression of an observable effector upon detection of a soluble molecule. To this end, we have constructed a bacterial strain expressing a novel chimeric transcription factor (Sphnx) for the detection of N-acetylneuraminic acid (Neu5Ac), a salivary biomolecule correlated with the onset of OSCC. This WCB serves as the proof-of-concept of a platform that can eventually be applied to clinical screening panels for a multitude of oral and systemic medical conditions whose biomarkers are present in saliva.
The temporal program of gene expression during a model physiological response of human cells, the response of fibroblasts to serum, was explored with a complementary DNA microarray representing about 8600 different human genes. Genes could be clustered into groups on the basis of their temporal patterns of expression in this program. Many features of the transcriptional program appeared to be related to the physiology of wound repair, suggesting that fibroblasts play a larger and richer role in this complex multicellular response than had previously been appreciated.
In this article we present validation of a real-time RT-PCR method to quantitate mRNA expression levels of atrial natriuretic peptide and c-fos in an in vitro model of cardiac hypertrophy. This method requires minimal sample and no postreaction manipulation. In real-time RT-PCR a dual-labeled fluorescent probe is degraded concomitant with PCR amplification. Input target mRNA levels are correlated with the time (measured in PCR cycles) at which the reporter fluorescent emission increases beyond a threshold level. The use of an oligo(dt) magnetic bead protocol to harvest poly(A) mRNA from cultured cells in 96-well plates minimized DNA contamination. We show that the GAPDH gene chosen for normalization of the RNA load is truly invariant throughout the biological treatments examined. We discuss two methods of calculating fold increase: a standard curve method and the DeltaDelta Ct method. Real-time quantitative RT-PCR was used to determine the time course of c-fos induction and the effect of varying doses of four known hypertrophy agents on atrial naturitic factor messenger RNA expression in cultured cardiac muscle cells. Our results agree with published data obtained from Northern blot analysis.