Quantitative Real-time PCR Analysis Research Articles

Selection of reference genes for quantitative real-time PCR analysis in exogenous hormone-treated Lycoris aurea.

Selection of reference genes for quantitative real-time PCR analysis in exogenous hormone-treated Lycoris aurea.

ZF-HD gene family in rapeseed (Brassica napus L.): genome-wide identification, phylogeny, evolutionary expansion and expression analyses

BackgroundThe zinc finger-homeodomain (ZF-HD) transcription factor family is widely involved in regulating plant growth and fruit filling, as well as responding to various abiotic stress. Rapeseed (Brassica napus L.), the second largest oil-producing crop in the world, is an annual or biennial herb of Brassica in Cruciferae. However, there is currently no systematic study on the evolutionary relationship and stress response of ZF-HD transcription factors in rapeseed.ResultsIn this study, 60 ZF-HD genes in B. napus (BnZHDs) were identified and named based on the chromosomal location. The evolutionary relationships, classifications, gene structures, motif compositions, chromosome localization, and gene replication events in these BnZHD genes were systematically analyzed. These 60 BnZHD members were divided into seven groups. According to the phylogenetic tree and repetitive events, subfamilies MIF, and V may have undergone stronger expansions during the evolutionary process. Interestingly, segmental duplications may have a more important contribution, which distinguishes them from other dicotyledon plants. To further investigate the evolutionary relationship of the ZF-HD family, we constructed eleven comparative genomic maps of homologous genes between rapeseed and different representative monocotyledonous and dicotyledonous plants. Finally, the gene expression pattern of 15 BnZHD genes from different subfamilies under different tissues, fruit developmental stages, and different abiotic stress were analyzed. The expression profile from real-time quantitative PCR analysis showed different expression patterns of BnZHD gene in B. napus. We found that certain BnZHD genes are preferentially expressed in specific tissues of B. napus, while most genes are expressed in multiple tissues. For example, BnZHD37, BnZHD53, and BnZHD55 may be sensitive to different hormones. Under different stresses, the expression of BnZHD3, BnZHD4, BnZHD7, BnZHD38, BnZD45, and BnZHD53 significantly increased in roots, stems, and leaves within 24 h. These genes may play important roles in the growth, development, and environmental adaptation of rapeseed.ConclusionsThese findings provide a basis for a comprehensive understanding of the ZF-HD family in rapeseed, which will provide information for further research on the functional characteristics of the BnZHD genes.

Genome-wide identification of HIPP and mechanism of SlHIPP4/7/9/21/26/32 mediated phytohormones response to Cd, osmotic, and salt stresses in tomato

Heavy-metal-associated isoprenylated plant proteins (HIPPs) contributed to abiotic tolerance in vascular plants. Up to now, the HIPP gene family of tomato (Solanum lycopersicum L.) had not been thoroughly understood. In the present study, 34 SlHIPP genes were identified from the tomato genome using the Hidden Markov Model (HMM). The phylogenetic analysis revealed that the evolution of SlHIPPs was highly conserved. The cis-acting element analysis indicated that SlHIPP genes might be involved in phytohormones and abiotic stresses. We constructed venn diagram with 17 genes containing stress-related motifs as well as 15 genes and 19 genes expressing in leaves and roots in RNA-seq data, suggesting that SlHIPP4/7/9/21/26/32 were selected as candidate genes for study. The quantitative real-time PCR (qRT-PCR) analysis showed that 6 candidate genes were indicated to be involved in osmotic and salt stress tolerance and SlHIPP7/21/26/32 responded to cadmium (Cd) tolerance. The virus-induced silencing of 6 candidate genes caused growth inhibition in stress conditions, further illustrating that 6 candidate genes played a positive role in abiotic conditions. Importantly, the phytohormone analysis implied that 6 candidate genes mediated abscisic acid (ABA), salicylic acid (SA), gibberellin (GA3), auxin (IAA), or methyl jasmonate (MeJA) responses to Cd, osmotic, or salt stress tolerance. These findings indicated that SlHIPP4/7/9/21/26/32 were key regulators of abiotic stress responses in tomato seedlings, functioning through multiple phytohormone pathways.

Long Non-Coding RNA LINC01116 Promotes the Proliferation of Lung Adenocarcinoma by Targeting miR-9-5p/CCNE1 Axis.

Long non-coding RNA (lncRNA) LINC01116 is crucial in promoting cell proliferation, invasion and migration in solid tumours, including lung adenocarcinoma (LUAD). LINC01116 acts as a competing endogenous RNAs (ceRNA) that binds competitively to microRNAs and plays a critical role in tumour migration and invasion. However, other mechanisms of action besides the ceRNA theory have been rarely reported and remain to be elucidated further. The differences in RNA and protein levels in cells and tissues were assessed through real-time quantitative PCR and Western blot analysis. Invitro functional assays and invivo xenograft models were used to analyse the function of LINC01116 in LUAD. Thus, the molecular correlation between miR-9-5p and CCNE1 was investigated through direct and indirect mechanism experiments. LINC01116, miR-9-5p and CCNE1 were upregulated in LUAD cell lines and tissues and were associated with a poor prognosis in patients. LINC01116 depletion inhibited proliferation but facilitated cell apoptosis. AGO2-RNA binding protein immunoprecipitation (AGO2-RIP) experiments confirmed that AGO2 binds to LINC01116 and miR-9-5p, indicating that LINC01116 interacts with miR-9-5p. The overexpression of miR-9-5p and CCNE1 effectively counteracts the biological effects of LINC01116 knockdown on reduced proliferation and cell cycle arrest in LUAD cells. The downregulation of miR-9-5p significantly reduces the CCNE1 level in A549 cells, and the upregulation of LINC01116 counteracts the downregulation of miR-9-5p effect, restoring the expression level of CCNE1. Our data demonstrated that LINC01116 regulates the expression of CCNE1 by positively regulating miR-9-5p, thereby affecting cell cycle, proliferation and participating in the development of LUAD.

Hovenia dulcis (Guaizao) polysaccharide ameliorates hyperglycemia through multiple signaling pathways in rats with type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) poses a significant threat to human health, with its incidence and mortality rates increasing annually. This study investigated the hypoglycemic effects and underlying mechanisms of pure Hovenia dulcis (Guaizao) polysaccharide (HDPs-2A) in rats subjected to a high-fat and high-sugar diet combined with streptozotocin-induced T2DM. Oral administration of HDPs-2A resulted in significant increases in body weight and liver glycogen levels compared to untreated controls. Moreover, a reduction in fasting blood glucose levels, alleviation of hyperinsulinemia, enhanced glucose tolerance, and improved insulin resistance were observed in the HDPs-2A-treated group. HDPs-2A also effectively reversed diabetes-induced dyslipidemia, as evidenced by decreased total cholesterol and triglyceride levels, alongside increased high-density lipoprotein cholesterol levels. Histopathological analyses confirmed that HDPs-2A partially repaired liver tissue damage by mitigating oxidative stress responses in the liver. Additionally, treatment with HDPs-2A significantly elevated short-chain fatty acid levels in T2DM rats. Real-time quantitative PCR and Western blot analyses indicated that HDPs-2A significantly enhanced the expression of InsR, IRS2, PI3K, Akt, and GLUT4, suggesting that HDPs-2A regulates insulin resistance and glycometabolism through the activation of the PI3K/Akt signaling pathway. Furthermore, HDPs-2A appeared to modulate the expression of GS, GSK-3β, and FoxO1 to improve glucose metabolism and reduce insulin resistance. It also improved glucose metabolism by activating the AMPK pathway and modulating G6Pase and PEPCK expression. This study provides novel insights into the antidiabetic effects of HDPs, positioning them as promising nutritional agents for the management of T2DM.

Profiling mRNA encoding glucocorticoid receptor α in saliva: Relationship to hair cortisol levels in individuals aged 15-25 years

ObjectiveWe assessed levels of mRNA encoding two glucocorticoid receptor (GR) isoforms (GRα and GRβ) in saliva and examined their relationship with hair cortisol levels and dental caries experience. DesignAdolescents and young adults were assessed for dental caries experience, and hair cortisol was measured by ELISA. RNA was extracted from whole saliva using TRIzol, followed by quantitative real-time PCR analysis of GRα, GRβ, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ResultsGRβ mRNA was not detectable in most samples, whereas GRα mRNA was observed in all samples. There were significantly lower levels of GRα mRNA in individuals with elevated hair cortisol levels than in those with normal cortisol levels. Levels of GRα mRNA did not differ significantly in individuals with dental caries experience compared to individuals with no caries experience. ConclusionsWe identified and quantified mRNA encoding GRα in saliva. Its levels were inversely associated with hair cortisol (a marker of chronic stress). Although caries experience was associated with hair cortisol levels, there was no significant association between GRα levels and caries experience. Chronic stress has been proposed to be associated with reduced expression of GRα and this association appears to hold for GRα mRNA levels in saliva.

Fluoxetine treatment reverses chronic stress-induced promotion on Fk506-binding protein 5 expression and multiple effects on glucocorticoid receptor phosphorylation in the paraventricular nucleus of mice

BackgroundFluoxetine is widely used as a first-line antidepressant. However, the molecular mechanisms for its antidepressant effects are still not fully understood. Hyperactivation of the hypothalamic-pituitary-adrenal (HPA) axis is a core pathogenic mechanism contributing to depression, and fluoxetine treatment prevents this dysfunction. The glucocorticoid receptor (GR) is a major negative feedback regulator of the HPA axis, while Fk506-binding protein 5 (Fkbp5) is a negative regulator of the GR signaling. Therefore, we examined the effects of fluoxetine on Fkbp5 and the GR signaling in the hypothalamic paraventricular nucleus (PVN) of depressed mice. MethodsMice were exposed to chronic social defeat stress (CSDS), chronic unpredictable mild stress (CUMS), or chronic restraint stress (CRS) with or without fluoxetine treatment (intraperitoneally injected, 20 mg/kg) and examined for changes in depression-like behaviors and HPA axis activity as well as Fkbp5 expression and GR phosphorylation in the PVN. We then examined if adeno-associated virus (AAV)-mediated Fkbp5 overexpression in the PVN affected the antidepressant actions of fluoxetine in mice. ResultsFluoxetine treatment significantly mitigated CSDS-, CUMS-, and CRS-induced depression-like behaviors and HPA axis hyperactivity in mice. Subsequent western blotting analyses showed that fluoxetine treatment fully reversed not only chronic stress-induced upregulation of Fkbp5 and CRH but also chronic stress-induced increase in Ser203 phosphorylation and decrease in Ser211 and Ser234 phosphorylation in GR in the PVN. Moreover, quantitative real-time reverse transcription PCR (qRT-PCR) analyses revealed that the enhanced mRNA levels of Fkbp5 and CRH in PVN neurons of mice subjected to CSDS/CUMS/CRS were also notably reversed by fluoxetine administration. Conversely, Fkbp5 overexpression in the PVN significantly eliminated the antidepressant effects of fluoxetine in mice without affecting their locomotor activity. ConclusionThese results together suggest that fluoxetine treatment reverses chronic stress-induced promotion on Fkbp5 expression and multiple effects on GR phosphorylation in the PVN of mice. Significance statementThe selective serotonin reuptake inhibitor fluoxetine (sold as Prozac) is a widely used treatment for depression, but the full spectrum of therapeutic mechanisms is still debated. Recent evidence suggests that these therapeutic mechanisms include suppression of chronic stress-activated hypothalamus–pituitary–adrenal (HPA) axis. The current study presents the first in vivo evidence showing that suppression of HPA axis hyperactivity by fluoxetine treatment involves reversal of glucocorticoid receptor (GR) phosphorylation via modulation of the GR negative regulator Fk506-binding protein 5 (Fkbp5) in the hypothalamic paraventricular nucleus (PVN). Fluoxetine treatment not only inhibited chronic stress-induced depression-like behaviors and HPA axis hyperactivity but also reversed Fkbp5 upregulation and GR phosphorylation changes in the PVN, while adeno-associated virus (AAV)-based Fkbp5 overexpression in the PVN eliminated the antidepressant effects of fluoxetine. These findings may expand our understanding of the pharmacological effects of fluoxetine, and further identify Fkbp5 as a possible target for novel antidepressants.

Effect of Cow Bone Addition on the Humification, Heavy Metals Passivation and Fate of Resistance Genes During Swine Manure Composting

Bone meal has been used as economic and effective additive for heavy metals (HMs) pollution remediation due to the distinct components and structures that enable their favorable properties, such as its low cost, high adsorption capacity, acid-base adjustability, and ion-exchange capability. However, no attempt has been made to establish whether cow bone could promote the passivation of HMs and the removal of metal resistance genes (MRGs) and antibiotics resistance genes (ARGs) during the composting process. Two sizes of cow bone (meal (T2) and granule (T3)) were added to investigate their effects on humification, HMs passivation and the abundance of ARGs and MRGs during swine manure composting. Excitation-emission matrix (EEM)-parallel factor analysis showed that the percentage of maximum fluorescence intensity of humic-like substances were higher in T2 (91.82%) than in T3 (88.46%), implying that T2 could promote the humification process compared to T3. In comparison with control (T1), the addition of T2 and T3 could promote the change of exchangeable Cu and reducible Cu into oxidizable Cu, thus reducing the mobility factors (MF) of Cu in T2 and T3 treatments by 10.48% and 6.98%, respectively. In addition, T2 and T3 could increase exchangeable Zn into reducible Zn and oxidizable Zn, thereby reducing the MF of Zn in T2 and T3 treatments by 18.80% and 2.0%, respectively. Quantitative Real-time PCR (qPCR) analysis revealed that the total abundances of MRGs were decreased by 100% in T2 and T3 treatments, and T2 decreased the total relative abundance of ARGs. Furthermore, the relative abundance of ARGs and MRGs had significantly correlated with intI1 and bio-available of Cu and Zn, which was triggered by selective pressure of HMs and horizontal gene transfer. The present study suggested that cow bone meal as additives can be a feasible approach to promote the passivation of HMs and enhance the removal of MGRs and ARGs by decreasing horizontal gene transfer and selective pressure by bioavailable HMs.

Identification and functional analysis of the transcriptional factor GeERF4B-1 in Gelsemium elegans

Gelsemium elegans, a vine plant of Loganiaceae, has both medicinal and forage values. However, it is susceptible to low temperatures during growth. Exploring low temperature response genes is of great significance for cold resistance breeding of G. elegans. Ethylene response factors (ERFs) are the transcription factors of the AP2/ERF superfamily and play a crucial role in plant stress response. In this study, based on the unigene GeERF involved in the response to low temperature stress in the transcriptome of G. elegans, a full-length cDNA sequence of the transcription factor GeERF4B-1 was cloned from the leaves of G. elegans by reverse transcription-polymerase chain reaction (RT-PCR). Bioinformatics analysis showed that GeERF4B-1 had an open reading frame of 759 bp, encoding a protein composed of 252 amino acid residues and with a relative molecular weight of 27 kDa. The deduced protein was predicted to be an unstable, alkaline, and hydrophilic protein. The phylogenetic tree showed that GeERF4B-1 was in the same clade as the B-4 subfamily of the ERF family. The results of the subcellular localization experiment revealed that GeERF4B-1 was located in the nucleus. Real time quantitative PCR (RT-qPCR) analysis indicated that GeERF4B-1 was expressed in the root, stem, and leaf of G. elegans, with the highest expression level in the root. Compared with the control, the treatments with a low temperature (4 ℃), methyl jasmonate (MeJA), and abscisic acid (ABA) up-regulated the expression level of GeERF4B-1, which reached the peak at 24-48 h. This result revealed that GeERF4B-1 actively responded to low temperature, MeJA, and ABA stresses. However, both sodium chloride (NaCl) and drought treatments down-regulated the expression of GeERF4B-1. In addition, a prokaryotic expression vector of GeERF4B-1 was constructed, and a fusion protein of approximately 52 kDa was yielded after induced expression. The results of the plate stress assay showed that compared with the control, the prokaryotic strain expressing GeERF4B-1 demonstrated enhanced tolerance to low temperatures and sensitivity to salt and mannitol stresses. This study provides theoretical references and potential genetic resources for breeding G. elegans varieties with stress resistance.

Ershen Zhenwu Decoction Suppresses Myocardial Fibrosis of Chronic Heart Failure with Heart–Kidney Yang Deficiency by Down-Regulating the Ras Homolog Gene Family Member A/Rho-Associated Coiled-Coil Kinases Signaling Pathway

Ethnopharmacological significanceThe therapeutic efficacy of Ershen Zhenwu Decoction (ESZWD)—a famous formulation from Xin’an for patients with chronic heart failure heart–kidney Yang deficiency (CHF-HKYD)—is well established. Still, the underlying molecular mechanism is not clear. Aim of the studyThis study investigated mechanisms by which ESZWD suppresses cardiac pathology, including myocardial fibrosis, in CHF-HKYD model rats and Ang II-stimulated cardiac fibroblasts (CFs). Materials and methodsThe components in ESZWD were analyzed by ultra-high-performance liquid chromatography coupled with Quadrupole Time-Of-Flight mass spectrometry (UHPLC-Q-TOF-MS). CHF-HKYD model was established in the male Sprague–Dawley rats through bilateral thyroidectomy and intraperitoneal administration of 0.02% doxorubicin (DOX), twice weekly for 3 weeks. Subsequently, the CHF-HKYD model rats were randomly categorized into the Model, ESZWD-L (3.96 g/kg/d ESZWD), ESZWD-M (7.92 g/kg/d ESZWD), ESZWD-H (15.84 g/kg/d ESZWD), and Sac/Val (68 mg/kg/d sacubitril/valsartan) groups and treated daily for 4 weeks. As a control, the sham surgery group (Sham) was used. Primary cardiac fibroblasts (CFs) were categorized into Control, Model, ESZWD, and Sac/Val groups. Then, the CFs were stimulated with Ang-II. The ESZWD and Sac/Val groups were incubated with different concentrations of drug-containing sera and their effects on CF viability were assessed via the CCK-8 assay. The ESZWD and Sac/Val groups received drug-containing serum concentrations determined by CCK-8 assay results. The cardioprotective effects of ESZWD were determined using echocardiography, Hematoxylin & Eosin (H&E) staining, Masson staining, and Sirius red staining, and the Enzyme Linked Immunosorbent Assay (ELISA). ESZWD's effects on the Ras Homolog Gene Family Member A (RhoA) / Rho-Associated Coiled-Coil Kinases (ROCKs) signaling pathway and myocardial fibrosis were assessed by Western blotting and Quantitative Real-Time PCR (qRT-PCR) analyses. Immunofluorescence was used to observe fibrotic markers in CFs. ResultsESZWD treatment improved cardiac function in the CHF-HKYD rats by significantly reducing myocardial fibrosis and ventricular remodeling. ESZWD treatment increased the rats' body temperature (Tb) and 24-hour urine volume, left ventricular ejection fraction (LVEF) and LV fractional shortening (LVFS), and decreased LV internal systolic diameter (LVIDs), LV internal diastolic diameter (LVIDd), and heart weight/ body weight (HW/BW) compared to the Model group. In comparison to the model rats, ESZWD treatment decreased serum levels of B-type natriuretic peptide precursor (NT-proBNP), tumor necrosis factor-alpha (TNF-α), interleukin-11 (IL-11), and IL-17A. ESZWD treatment significantly down-regulated the protein and mRNA expression levels of collagen I A1, α-SMA, RhoA, ROCK1, and ROCK2 in the heart tissues of the CHF-HKYD rats and the Ang II-stimulated CFs. ConclusionESZWD significantly improved cardiac function and attenuated myocardial fibrosis and inflammation in the CHF-HKYD rats by inhibiting the RhoA/ROCKs signaling pathway.

Down-regulation of ESRP2 inhibits breast cancer cell proliferation via inhibiting cyclinD1

Epithelial splicing regulatory protein 2 (ESRP2),an important alternative splicing protein of mRNA, is reported to have a dual role in tumors, which can promote or inhibit the occurrence and development of tumors. However, the function and mechanism of ESRP2 in breast cancer (BC) remain unclear. The distribution of ESRP2 expression in breast cancer and the correlation between ESRP2 expression and the overall survival rate were detected by The Cancer Genome Atlas (TCGA) database. Gene Ontology(GO)analysis, containing biological process, cellular components, and molecular function, was utilized to evaluate the potential mechanism of ESRP2 in breast cancer. The ESRP2 expression in breast cancer cell lines was detected by real-time quantitative PCR analysis (RT-qPCR) and western blotting. Cell clone was performed to examine the proliferation of ESRP2 knockdown in MCF-7 cells. The cell cycle was measured by flow cytometry assays. The role of ESRP2 knockdown in synergistic effect with chemotherapeutic agents was also determined by MTT assay. Bioinformatics analysis demonstrated that the ESRP2 gene was elevated in breast cancer cells and its overexpression was strongly correlated with shorter overall survival. GO analysis revealed that ESRP2 expression was related to cell proliferation. ESRP2 mRNA and protein expression were elevated in breast cancer cell lines, compared to the normal human breast cell line MCF-10 A. Dwon-regulation of ESRP2 inhibited cell proliferation and promoted the sensitivity of chemotherapy drug, Cisplatin(DDP) and Paclitaxel (TAXOL), in MCF-7 cells.Additionally, ESRP2 knockdown obstructed the cell cycle at the G1 phase and caused a decrease in cyclinD1 protein expression. These findings reveal that ESRP2 is highly expressed in breast cancer and is correlated with poor prognosis in breast cancer patients. ESRP2 knockdown can inhibit MCF-7 cell proliferation by arresting the cell cycle at the G1 phase and promoting the sensitivity of chemotherapy drugs (DDP and TAXOL)in MCF-7 cells. ESRP2 may be required for the regulation of breast cancer progression, as well as a critical target for the clinical treatment of breast cancer.

XRCC1 is linked to poor prognosis in adenocarcinoma of the esophagogastric junction after radiotherapy: transcriptome and alternative splicing events analysis.

This study aimed to (i) investigate the relationship between X-ray repair cross-complementing protein 1 gene (XRCC1) and prognosis in patients with adenocarcinoma of the esophagogastric junction (AEG), and (ii) analyze the roles of XRCC1 in human gastric adenocarcinoma (AGS) cells following X-ray radiation. A total of 46 AEG patients were enrolled and examined for XRCC1 protein by immunohistochemistry. XRCC1 was knocked down in AGS cells by transfection, and AGS cells were subsequently exposed to 6Gy of X-ray radiation. XRCC1 mRNA and protein expression was examined via quantitative real-time PCR (qRT-PCR) and Western blot analysis. The apoptosis of AGS cells was examined by flow cytometer. RNA-sequencing technology was used to identified differentially expressed genes and alternative splicing events following XRCC1 knockdown and radiation exposure. XRCC1 positivity was strongly associated with distant metastasis, pathological tumor-node-metastasis (pTNM) classification, and radiotherapy resistance in AEG patients. A significant difference in progression-free survival was observed between AEG patients with low and high XRCC1 protein expression. The knockdown of XRCC1 notably exacerbated the effects of X-ray radiation on apoptosis in AGS cells. Additionally, X-ray radiation modified the expression of genes related to apoptosis and immune response in XRCC1-knockdown AGS cells. Furthermore, the generation of splice variants was influenced by XRCC1 knockdown in AGS cells. XRCC1 may serve as a key oncogene that elucidates the role of alternative splicing events in the progression of AEG following X-ray treatment.

Evaluation and Validation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis of the Gene Expression in Macadamia integrifolia

Macadamia is an economically significant crop, with its kernel oil being abundant in monounsaturated fatty acids (MUFA). Analyzing the expression of genes related to MUFA biosynthesis is essential for understanding the complex regulatory networks in Macadamia. However, there are few reports on the identification of suitable reference genes for use as internal controls in this species. Consequently, selecting a reliable reference gene for gene expression studies under various conditions is critical. In this study, we evaluated the expression stability of 11 traditional housekeeping genes: α-tubulin (TUBa), β-tubulin (TUBb), malate dehydrogenase (MDH), 18S ribosomal RNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), α-elongation factor 1 (EF1a), β-elongation factor 1 (EF1b), ubiquitin (UBQ), ubiquitin-conjugating enzyme (UBC), cyclophilin (CYP), and actin (ACT) under abiotic stresses, hormonal treatments and in variety of plant tissues using the online tool RefFinder, which integrates four commonly used software programs: ΔCt, geNorm (version 3.4), NormFinder (version 0953), and BestKeeper (version 1.0). A comprehensive expression stability ranking was established by integrating results from these four methods based on the geometric mean. The findings indicated that ACT was the most stable gene across all samples, including those subjected to cold stress, NaCl stress, PEG stress, ABA treatment, MeJA treatment, and both stem and leaf tissues. EF1b was identified as the most stable gene in GA treatment and heat stress samples, while UBC and CYP were ranked highest in ethrel treatment and root tissue samples, respectively. Finally, the reliability of these findings was further validated using the target gene SAD through qRT-PCR. In summary, this study evaluated and validated appropriate reference genes for qRT-PCR, which will facilitate future investigations into the molecular mechanisms in Macadamia.

Genome-Wide Identification of the bHLH Gene Family in Callerya speciosa Reveals Its Potential Role in the Regulation of Isoflavonoid Biosynthesis.

Callerya speciosa (Champ. ex Benth.) Schot is a significant leguminous plant valued for its edible tuberous roots, which are a plentiful source of isoflavonoids. Basic helix-loop-helix (bHLH) transcription factors (TFs) have been reported to regulate secondary metabolism in plants, especially flavonoid biosynthesis. However, the bHLH genes in C. speciosa have not yet been reported, and their regulatory role in isoflavonoid biosynthesis remains unexplored. Here, 146 CsbHLH genes were identified in the C. speciosa genome, classifying them into 23 subfamilies based on the gene structures and phylogenetic relationships. All the CsbHLH proteins contained both motifs 1 and 2, whereas motif 8 was only distributed in subgroup III (d + e). Collinearity analysis demonstrated that fragmental replications are the primary driver of CsbHLH evolution, with the majority of duplicated CsbHLH gene pairs experiencing selective pressure. Nine candidate CsbHLH genes were found to play a potential role in regulating isoflavonoid biosynthesis through a combination of gene-to-metabolite correlation analysis and weighted gene co-expression network analysis (WGCNA). Additionally, the cis-regulatory elements and response to MeJA of these nine genes were characterized and confirmed through quantitative real-time PCR (qRT-PCR) analysis. Among them, three CsbHLHs (CsbHLH9, CsbHLH89, and CsbHLH95) were selected for further investigation. Yeast two-hybrid (Y2H), dual-luciferase (LUC) assays, bimolecular fluorescence complementation (BiFC) assays, and transient transformation demonstrated that CsbHLH9 acted as a transcriptional activator through its interaction with CsMYB36 and binding to the promoters of isoflavonoid biosynthesis genes in a MeJA-induced manner, such as CsIFR2, CsI3'H2, and CsCHS4, to promote isoflavonoid (calycosin, calycosin-7-o-glucoside, and formononetin) accumulation. Our results establish a basis for the functional analysis of bHLH genes and investigations into the molecular mechanisms underlying isoflavonoid biosynthesis in C. speciosa.

PlFabG improves high-temperature resistance in herbaceous peony by increasing saturated fatty acids

The herbaceous peony (Paeonia lactiflora Pall.) is renowned for its striking flowers, which are highly valued in the cut flower industry. However, in the middle and lower reaches of the Yangtze River, the elevated summer temperatures negatively affect the plant's flowering capacity in the subsequent year. 3-oxoacyl-ACP reductase (FabG) is a component of the type II fatty acid synthesis (FAS II) system, where it plays a role in facilitating the production of saturated fatty acids. However, its role in conferring resistance to high-temperature stress remains unclear. In order to investigate the function of PlFabG in dealing with high-temperature stress, we isolated PlFabG from P. lactiflora. The gene contains an open reading frame (ORF) of 780 bp, which encodes 259 amino acids. Quantitative real-time PCR (qRT-PCR) analysis showed that the expression levels of PlFabG increased with prolonged exposure to high temperature. Additionally, plants overexpressing PlFabG maintained a relatively healthy phenotype under high-temperature stress, whereas plants with silencing PlFabG exhibited more severe leaf scorching and wilting under the same conditions. Various physiological indices indicated that PlFabG reduced reactive oxygen species (ROS) accumulation and enhanced the saturation of photosystem II. Most importantly, PlFabG helped P. lactiflora withstand high-temperature stress by increasing the proportion of saturated fatty acids, thereby maintaining cell membrane integrity. These findings elucidate the beneficial role of PlFabG in enhancing plant tolerance to high temperature and provide a strong theoretical support for the development of high-temperature tolerance in transgenic P. lactiflora.

Genome-wide characterization of PEBP genes in Mung bean (Vigna radiata L.) with functional analysis of VrFT1 in relation to photoperiod

Mung bean (Vigna radiata L.), a widely cultivated legume, belongs to the Fabaceae family’s Papilionoideae subfamily. Although Phosphatidylethanolamine-binding protein (PEBP) genes have been identified in several plant species, their presence and function in mung bean remain largely unexplored. In this study, we identified seven VrPEBP genes from mung bean and classified them into four clades: FT, MFT, TFL and FT-like. Cis-regulatory element analysis revealed that VrPEBP genes may play a role in light, hormone, and stress responses. Quantitative real-time PCR (qRT-PCR) analysis indicated that VrPEBPs were constitutively expressed in various tissues. However, tissue-specific expression patterns were observed among VrPEBP genes. Under short-day (SD) conditions, VrFT1 and VrMFT1 exhibited significantly higher expression levels than under long-day (LD) conditions at 8 and 4 h, respectively. Conversely, VrTFL2 and VrTFL3 showed significantly higher expression levels under LD conditions compared to SD conditions at 8 and 12 h, respectively. The varied expression patterns of these genes under different photoperiod suggest their potential involvement in the photoperiodic regulation of flowering in mung bean. Additionally, phenotypic analysis of transgenic Arabidopsis plants overexpressing VrFT1 revealed higher expression levels under SD conditions and predicted its role in promoting flowering. These results provide valuable insights into the evolution and function of PEBP genes in mung bean and lay the foundation for further research on their regulatory mechanisms and potential applications in mung bean improvement.

Molecular and functional characterization of short peptidoglycan recognition proteins in Vesicomyidae clam

Within cold seep environments, the Vesicomyidae clam emerges as a prevalent species, distinguished by its symbiotic relationship with microorganisms housed within its organ gill. Given the extreme conditions and the symbiotic nature of this association, investigating the host's immune genes, particularly immune recognition receptors, is essential for understanding their role in facilitating host-symbiotic interactions. Three short peptidoglycan recognition proteins (PGRPs) were identified in the clam. AmPGRP-S1, -S2, and -S3 were found to possess conserved amidase binding sites and Zn2+ binding sites. Quantitative Real-time PCR (qRT-PCR) analysis revealed differential expression patterns among the PGRPs. AmPGRP-S1 and AmPGRP-S2 exhibited elevated expression levels in the gill, while AmPGRP-S3 displayed the highest expression in the adductor muscle. Functional experiments demonstrated that recombinant AmPGRP-S1, -S2, and -S3 (rAmPGRPs) exhibited binding capabilities to both L-PGN and D-PGN (peptidoglycan). Notably, rAmPGRP-S1 and -S2 possessed Zn2+-independent amidase activity, while rAmPGRP-S3 lacked this enzymatic function. rAmPGRPs were shown to bind to five different bacterial species. Among these, rAmPGRP-S1 inhibited Escherichia coli and Bacillus subtilis, while rAmPGRP-S2 and -S3 inhibited Bacillus subtilis in the absence of Zn2+. In the presence of Zn2+, rAmPGRP-S1 and -S2 exhibited enhanced inhibitory activity against Staphylococcus aureus or Bacillus subtilis. These findings suggest that AmPGRPs may play a pivotal role in mediating the interaction between the host and endosymbiotic bacteria, functioning as PGN and microbe receptors, antibacterial effectors, and regulators of host-microbe symbiosis. These results contribute to our understanding of the adaptive mechanisms of deep-sea organisms to the challenging cold seep environments.

Integrative Lipid Pseudotargeted Metabolomics and Amino Acids Targeted Metabolomics Unravel the Therapeutic Mechanism of Rhizoma Paridis Saponins on Experimental Colitis of Damp-Heat Type.

Inflammatory bowel disease (IBD) is a serious disease that affects the metabolism and inflammatory responses of human beings. From the perspective of traditional Chinese medicine, damp-heat syndrome is one of the main syndromes of IBD. Rhizoma Paridis, also known as the root of Paris polyphylla, a well-known herbal medicine used in China, is used to treat IBD with damp-heat syndrome (IBD-DH). However, uncertainty still exists regarding the underlying mechanisms and the impact of Rhizoma Paridis on IBD-DH. The rats in the model (DAT) and medication administration (Rhizoma Paridis total saponins (RPTS) and Pennogenin (PN)) groups were given a high temperature and high humidity environment, high fat and high sugar diet combined with 2,4,6-trinitrobenzene sulfonic acid (TNBS) to establish the model of experimental colitis of damp-heat type, and the normal control group (RNC) rats were given a normal diet at normal temperature and humidity. Damp-heat control group (DNC) was set with the same condition as DAT without TNBS. Hematoxylin-Eosin (HE) staining was used to observe the histopathological morphology of the rat colorectum. The expression of the metabolism-related genes (Phospholipase A2 (sPLA2, cPLA2), and phosphatidylethanolamine N-methyltransferase (PEMT)) was assessed by using real-time quantitative PCR analysis (RT-qPCR). And the levels of the metabolism-related proteins (sPLA2, cPLA2), S100A8/9, Arg-1, and cytokines were detected by enzyme-linked immunosorbent assay (ELISA) kit. To investigate lipids and amino acids which closely associated with the IBD and IBD-DH, lipid pseudotargeted metabolomics with UHPLC-TQ/MS analysis method, as well as targeted quantitative amino acid analysis were performed. Our data showed that RPTS (50 mg/kg) and PN (20 mg/kg) significantly ameliorated the severity of TNBS-induced colitis and downregulated the levels of circulating proinflammatory cytokines. Compared with RNC group, lipid pseudotargeted metabolomics demonstrated that glycerophospholipids, sphingolipids, carnitine, and glycerolipids were the four most perturbed lipid classes, and amino acids targeted metabolomics demonstrated that serine, N-acetylneuraminic acid, histidine, proline, taurine, and kynurenine changed significantly in DAT group . Correlation analyses showed tight associations between most of differential metabolites and proinflammatory cytokines. RPTS and PN both regulated glycerophospholipid metabolism and sphingolipid metabolism. However, both of them did not have a significant effect on amino acid modulation. RPTS and PN potentially regulated sPLA2, cPLA2, and PEMT. These results showed that RPTS (50 mg/kg) and PN (20 mg/kg) effectively alleviated rats' colitis of damp-heat type, affected cytokines, and altered lipid metabolism without significant modulation on amino acid metabolism.

Selective Dry Cow Therapy: Clinical Field Trial on Prevention and Cure of Intramammary Infections

Intramammary antibiotic dry cow therapy (DCT) at the end of lactation is one of the key measures in the management and control of bovine mastitis. Currently, livestock production is under pressure to reduce antibiotic consumption, emphasizing the need to avoid medicating all cows at dry-off, and instead to treat only infected cows. The study objective was to evaluate IMI cure risk, new IMI risk, and post-calving IMI risk between DCT-treated and untreated quarters over the dry period. The sample totaled 269 cows from 12 Finnish automatic milking system herds entering the dry period between 2019 and 2021. Cows eligible for the study had a final DHI test-day SCC ≥ 100,000 cells/mL and had no clinical signs of mastitis. Based on odd or even identification numbers, cows with an SCC between 100,000 and 250,000 cells/mL were sequentially assigned to either receive DCT at dry-off (DCT100-250) or remain untreated (NoDCT100-250). All cows with an SCC > 250,000 cells/mL received DCT (DCT > 250). No quarters received teat sealants. Farmers collected aseptic quarter-milk samples for microbiological quantitative real-time PCR (qPCR) analysis 0 to 4 d before dry-off and 0 to 4 d after calving. The outcome of the statistical model was the odds of a quarter having an IMI at freshening 0 to 4 d after calving. The statistical tool was a generalized linear mixed-effects model with logit link function and 2-level random intercepts, cows nested within herds. Quarter-level IMI prevalence at dry-off was NoDCT100-250 17.6% (45/256), DCT100-250 22.2% (83/374), and DCT > 250 32.0% (132/413). Quarter-level IMI prevalence at freshening was NoDCT100-250, 36.2% (84/232), DCT100-250, 13.6% (50/369), and DCT > 250, 11.6% (46/397). The untreated quarters in the NoDCT100-250 group had a 1.1 times higher risk of failing to cure over the dry period than did the treated quarters in the DCT100-250 group (unadjusted RR 1.10, 95% CI 0.94-1.28) and a 1.2 times higher risk than the treated quarters in the DCT > 250 group (unadjusted RR 1.15, 95% CI 1.00-1.33). The untreated quarters in the NoDCT100-250 group had a 1.5 times higher risk of having a new IMI over the dry period than did the treated quarters in the DCT100-250 group (unadjusted RR 1.48, 95% CI 1.35-1.63) and a 1.4 times higher risk than the treated quarters in the DCT > 250 group (unadjusted RR 1.35, 95% CI 1.23-1.49). The principal cause of new IMI was NAS, followed by Streptococcus dysgalactiae. Quarters in the NoDCT100-250 group had 6.6 times higher odds for having an IMI at freshening than did quarters in the DCT100-250 group (95% CI 3.52-12.21). According to our findings within the framework of this experiment, selectively DCT-treated cows with an SCC between 100,000 and 250,000 cells/mL at dry-off demonstrated that, although the group-specific cure risks over the dry period were high, untreated quarters had a higher risk of acquiring a new IMI over the dry period than did treated quarters, highlighting the effectiveness of DCT in reducing IMI risks. The prophylactic use of antibiotics, however, is not recommended, and in some countries is even prohibited.

Nrf2 protects against oxidative damage induced by hemoglobin in the liver of grass carp (Ctenopharyngodon idella)

Hemoglobin (Hb) releases during hemorrhaging and causes oxidative damage, further exacerbates the development of multiple diseases. Nuclear factor erythroid 2–related factor 2 (Nrf2) is a transcription factor that regulates cellular defenses against toxic and oxidative challenges. However, the regulation mechanism of Nrf2 in Hb-induced oxidative stress remains unclear in teleost. To accomplish this goal, a hemolysis model was established by injecting grass carp with phenylalanine (PHZ), and the immunofluorescence analysis (IFA) and hematoxylin and eosin (H&E) staining revealed that PHZ-induced hemolysis caused Hb accumulation and hepatic vacuolization, resulted in tissue damage. Prussian blue, Sirius red, and Masson staining results revealed significant iron deposition and extensive collagen fiber accumulation in the liver. IFA and immunohistochemical analyses demonstrated that PHZ-induced hemolysis markedly increased the production of reactive oxygen species (ROS), malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE). The quantitative real-time PCR (qRT-PCR) analysis data revealed that the PHZ-induced hemolysis also significantly upregulated the expression of antioxidant-related genes through activation of the adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK)/Nrf2 signaling pathway. To further explore the molecule regulation mechanism of PHZ-induced hemolysis, the RNA-seq analysis was performed, and the data revealed that the AMPK/Nrf2 and multiple programmed cell death pathways, including ferroptosis, autophagy, apoptosis, and necroptosis in PHZ injection groups were significant upregulated. In vitro, the hemin supplementation activated the expression of target genes in the AMPK/Nrf2 pathway detected by qRT-PCR. To further verify the regulation function of Nrf2, an Nrf2 activator (4OI) was supplemented, and the flow cytometer analysis results suggested that the Hb-induced cell damage was significantly attenuated. However, the supplementary of ML385 down-regulated the AMPK/Nrf2 pathway and aggravated the hemin induced cell death. In conclusion, these findings highlight the critical regulatory role of the AMPK/Nrf2 signaling pathway in protecting against Hb-induced oxidative damage in the liver of grass carp.