Real-time Fluorescence Quantitative PCR Research Articles

Ameliorative Effect of Hedysarum polybotrys Polysaccharide on Neural Tissue Fibrosis in Diabetic Peripheral Neuropathy Mice and its Mechanisms

Objective: This study aimed to investigate the role of Hedysarum polybotrys polysaccharide (HPS) in ameliorating neural tissue fibrosis in diabetic peripheral neuropathy (DPN) mice. Methods: Fifty DPN mice were selected and randomly divided into five groups (n = 10), which were the model group, positive control group (receiving only 4 mg/(kg-d) of α-lipoic acid), high-dose HPS group (200 mg/(kg-d) of HPS was given per day), medium-dose HPS group (100 mg/(kg-d) of HPS was given per day), and low-dose HPS group (50 mg/(kg-d) of HPS given daily). In addition, non-diabetic C57BL/6 wild-type mice were selected as the normal group (n = 10). The expression levels of Keap1 and Nrf2 proteins and their mRNAs in the sciatic nerve tissues of mice in each group were analyzed by Western blot technique and real-time fluorescence quantitative PCR. Results: Compared with the normal group, the expression of Keap1 protein and mRNA was increased, while the expression of Nrf2 protein and mRNA was decreased in the sciatic nerve of mice in the model group (P < 0.05). Compared with the model group, Keap1 protein and mRNA expression decreased, while Nrf2 protein and mRNA expression increased in the control and high and medium dose HPS groups of mice (P < 0.05). Conclusion: HPS may inhibit fibrosis of neural tissue and ameliorate nerve injury in DPN mice by regulating the Keap1/Nrf2 signaling pathway. This effect was associated with enhanced antioxidant capacity, promotion of Nrf2 activation, and increased antioxidant gene expression by HPS. Therefore, HPS has a potential therapeutic value to ameliorate DPN-associated nerve injury.

Functional Verification of Transcription Factor comp54181_c0 in Monascus purpureus.

Monacolin K is a valuable secondary metabolite produced after a period of fermentation by Monascus purpureus; however, our current understanding of the regulatory mechanisms of its synthesis remains incomplete. This study conducted functional analysis on the key transcription factor, comp54181_c0, that is involved in the synthesis of monacolin K in Monascus. Mutant strains with either knockout or overexpression of comp54181_c0 were constructed using CRISPR/Cas9. A comparison between the knockout and overexpression strains revealed changes in fungal morphology and growth, with a significant increase in the production of Monascus pigments and monacolin K when comp54181_c0 was absent. Real-time fluorescence quantitative PCR analysis revealed that comp54181_c0 significantly influenced the transcription of key genes related to monacolin K biosynthesis in Monascus. In conclusion, our study elucidates the crucial role of comp54181_c0 in Monascus, enriches our understanding of fungal secondary metabolite development and regulation, and provides a foundation for the development and regulation of Monascus and monacolin K production.

A droplet digital PCR method for the detection of scale drop disease virus in yellowfin seabream (Acanthopagrus latus).

In this study, a ddPCR method for the detection of scale drop disease virus (SDDV) in yellowfin seabream (Acanthopagrus latus) was established based on Real-time fluorescence quantitative PCR detection methods and principles. The reaction conditions were optimized, and the sensitivity, specificity, accuracy, and reproducibility were assessed. The results showed that threshold line position was determined to be 1900 by the ddPCR method; the optimum annealing temperature for SDDV detection by the ddPCR method was 60°C; the limit of detection was 1.4-1.7 copies/μL; the results of specific detection of other common viruses, except for SDDV specific amplification, were all negative; and the relative standard deviation (RSD) for the reproducibility validation was 0.77%. The samples of yellowfin seabream (Acanthopagrus latus) liver, spleen, kidney, heart, intestine, brain, blood, muscle, skin and ascites with three replicates, respectively, were tested using the ddPCR method, and the results were consistent with clinical findings. The ddPCR method established in this study has the advantages of high sensitivity, high specificity, good reproducibility and simple steps for the quantitative detection of SDDV, which could be used for the nucleic acid detection of clinical SDDV samples, and provided a new quantitative method for the diagnosis of yellowfin seabream SDDV in the early stage of pathogenesis.

Alterations in anterior lens capsule structure and LTBP-2 expression in primary angle-closure glaucoma

ObjectiveThis study investigated the role of latent-transforming growth factor β-binding protein 2 (LTBP-2) in primary angle-closure glaucoma (PACG) by analysing its expression and the ultrastructure of the anterior lens capsule...

RNAi-mediated silencing of transferrin promotes entomopathogens lethality in Odontotermes formosanus (Shiraki)

Transferrin (Tsf) is a highly conserved multifunctional protein involved in insect physiology, defense and development that has been developed as a novel RNA interference (RNAi)-based target for pest control. The function study of the Tsf gene in Odontotermes formosanu (Shiraki) was evaluated for synergistic control of this agroforestry pest with Serratia marcescens (SM1), Bacillus thuringiensis (Bt) or Beauveria bassiana (Bb). The Tsf gene of O. formosanus was identified and characterized. Real-time fluorescent quantitative PCR (qPCR) analysis demonstrated that OfTsf was most highly expressed in the male dealate of O. formosanus, and OfTsf was highly expressed in the hemolymph. OfTsf expression was considerably elevated after SM1, Bt or Bb infection. Furthermore, dsOfTsf treatment was effective in increasing the virulence of entomopathogens to O. formosanus. In addition, OfTsf expression was markedly upregulated in O. formosanus fed with oxidative stress inducers; reactive oxygen species (ROS) levels were significantly increased after dsOfTsf treatment. Therefore, OfTsf gene played an important role in defending against entomopathogen infection and antioxidant stress. Most importantly, our work suggested OfTsf as a potential RNAi target for the control of O. formosanus.

Effects of pleiotrophin (PTN) on the FAK inhibitor Y15 in breast cancer cells

Overexpression of PTN (Pleiotrophin) in breast cancer cells stimulates breast cell proliferation and anti-apoptosis via the FAK/Src signaling pathway.1,2,4,5-Benzenetetramine tetrahydrochloride (C6H14Cl4N4) known as Y15 is an inhibitor that specifically blocks phosphorylation of Y397-FAK and total FAK phosphorylation. The exogenous PTN protein was induced to be expressed by a prokaryotic expression strain. The addition of PTN protein decreased the sensitivity of breast cancer cells to Y15, as shown by both MTT and flow cytometry assays. Breast cancer cells transfected with sh-RNA knockdown PTN were analyzed by real-time fluorescence quantitative PCR reaction (RT-PCR) and protein immunoblotting 24 h after transfection. MTT assay data showed a significant increase in the IC50 value of Y15 against breast cancer cells in the presence of PTN protein. Flow cytometry data confirmed the dose-dependent anti-apoptotic effect of PTN protein in Y15-treated breast cancer cells. After transfection with sh-RNA, Y15 further inhibited the FAK pathway, greatly reducing cell survival and promoting apoptosis. Western blotting analysis showed that knockdown of PTN protein with sh-RNA resulted in increased apoptosis and elevated levels of apoptotic proteins, such as cleaved PARP and cleaved caspase-3 in Y15-induced breast cancer cells. Finally, it was hypothesized that PTN and Y15 were associated with a significant increase in the IC50 value of PTN and Y15 in breast cancer cells by western blotting analysis. It was hypothesized that PTN and Y15 regulate the proliferation and apoptosis of breast cancer cells through the AKT/PI3K pathway. The results demonstrated that PTN affects the sensitivity of the kinase inhibitor Y15, and PTN silencing and Y15 may inhibit the survival of breast cancer cells through the AKT/PI3K pathway, which lays the foundation for subsequent in-depth research on the clinical treatment of breast cancer.

The diagnostic value of serum exosomal miRNA-587 combined with hypersensitive C-reactive protein as noninvasive biomarker in early-stage non-small cell lung cancer

Abstract Objectives Lung cancer is a highly prevalent and life-threatening disease worldwide, with non-small cell lung cancer (NSLC) accounting for around 80 % of all cases. Exosomes contain important genetic information for humans that could be employed, especially for early screening of tumors. Accordingly, we aimed to use exosomal miRNA (ex-miRNA) in early NSCLC diagnosis. Methods The extracted ex-miRNAs were validated through transmission electron microscopy, particle size potentiometer, and western blot analyses. Microarray was used to verify ex-miRNAs, and 20 miRNAs were selected. Herein, we obtained 240 blood samples from NSCLC patients (101 in the early stage) and 234 from healthy donors. Our study deployed real-time fluorescence quantitative PCR (qRT-PCR) for detecting significantly down-regulated miR-587 expression. In addition, the hypersensitive C-reactive protein (Hs-CRP) levels were measured in patient samples. Results The results of calculating the area under the curve (AUC) revealed that the diagnostic efficiency of miR-587 and Hs-CRP were 0.771 and 0.863, respectively. Meanwhile, the combined diagnostic efficiency of both increased to 0.901. In patients with early NSCLC, the diagnostic efficiency of miR-587, Hs-CRP, and combined AUC were 0.726, 0.873, and 0.899, respectively. This indicates that the accuracy of early NSCLC diagnosis is very high. Finally, we combined miR-587 and Hs-CRP with CEA and NSE for NSCLC (AUC=0.956) and early-stage patients (AUC=0.921). Conclusions In this study, miR-587 and Hs-CRP have significant diagnostic efficiency for NSCLC, especially the combination of CEA and NSE that could indicate early NSCLC diagnosis.

Ge-Zhi-Jie-Jiu decoction alleviates alcoholic liver disease through multiple signaling pathways

Ethnopharmacological relevanceAlcoholic liver disease (ALD) is a growing public health concern caused by excessive alcohol consumption, but effective treatments are limited. Ge-Zhi-Jie-Jiu decoction (JJY) is a modified traditional Chinese herbal remedy that aims to alleviate ALD. This formula contains various components such as Ge Hua, Ge Gen, Zhi Ju Zi, and other medicinal-food herbs. However, the specific pharmacotherapeutic compounds of JJY and its pharmacological mechanisms remain unclear. Aim of the studyThis study aimed to elucidate the molecular mechanism and pharmacodynamic basis of JJY in treating ALD. Materials and methodsUPLC-Q-Orbitrap HRMS, HPLC fingerprinting, and LC-MS techniques were used for the composition identification and quality control of JJY. The pharmacological components and molecular mechanisms of JJY in anti-ALD were then predicted using network pharmacology and molecular docking approaches. Ultimately, an acute alcoholic liver injury mouse model was developed, and the potential mechanisms were verified by hematoxylin-eosin (H&E), Oil Red O, and TUNEL staining, real-time fluorescence quantitative PCR (RT-qPCR), Western blot (WB) and molecular docking analysis. ResultsThe results showed that the main components of JJY are organic acids, flavonoids, and isoflavonoids, in which puerarin, daidzein, glycitein, ononin, quercetin, and tectorigenin can be used as the indicator components of JJY. In addition, JJY might ameliorate ALD through several pathways, including potentially promoting alcohol metabolism via alcohol-metabolizing enzymes, and possibly inhibiting oxidative stress, inflammation and apoptosis via the Nrf2/Keap1/HO-1 and MAPK signaling pathways. Furthermore, JJY may also alleviated hepatic lipid accumulation through the PPARα signaling pathway. ConclusionsJJY has significant anti-ALD efficacy with multiple mechanisms. This study offers a solid experimental foundation for JJY's development as a medicine with anti-ALD characteristics and elucidates its probable active components.

Anti-neoplastic effect of heterophyllin B on ovarian cancer via the regulation of NRF2/HO-1 in vitro and in vivo

BackgroundHeterophyllin B (HB) is a cyclic peptide with anti-neoplastic effect on many cancers. However, its effect and mechanism of action in ovarian cancer cells are still unknown. PurposeThe primary objective of this study was to assess the impact of HB on the proliferation of ovarian cancer (OC) cells and delve into the underlying mechanisms involved. MethodsWe performed CCK-8 assays, HE staining, KI67 staining, clonogenic formation assays, Annexin V-FITC/PI staining, tumor invasion assays, and migration assays to detect the effects of HB on cell viability, proliferation, apoptosis, migration, and invasion in ovarian cancer cells. Additionally, real-time fluorescent quantitative PCR (qPCR) and Western blotting were utilized for verification. The expression of NF-E2-related factor 2 (NRF2) and heme oxygenase 1 (HMOX1/HO-1) signaling molecules was detected using qPCR and Western blotting. A specific inducer, Hemin, was used to activate HO-1 and Nrf2 overexpression, in order to verify the pharmacological mechanism of HB on ovarian cancer cells. The binding relationship between HB and NRF2 was investigated through molecular docking. ResultsHB treatment inhibited the viability of OC cells, meanwhile it showed suppressive effect on the proliferation, migration, and invasion of OC cells, Meanwhile, HB could promote the apoptosis of tumor cells. For the mechanisms, we found that HB treatment could significantly down-regulate the levels of NRF2/HO-1. Consistent with the results of in vitro experiments, administration of HB significantly delayed tumor growth in OVCAR8 xenografted nude mice, and inhibited the expression of Ki67, Nrf2 and HO-1. ConclusionThis study demonstrated that HB had anti-neoplastic effect on OC by inhibiting Nrf2/HO-1 signaling pathway and may be a potential drug for the treatment of OC.

Identifying the key motif essential for enhancing plant defense against Botrytis cinerea by β-glucanase from Bacillus velezensis LJ02

The glycosyl hydrolase β-glucanase elicits plant immune responses against pathogens and enhances plant immunity by activating signaling pathways. The specific functional domains responsible for disease prevention remain unclear. In this study, transient expression of β-glucanase significantly increased leaves resistance to Botrytis cinerea in Nicotiana benthamiana systemic leaves. Through sequence alignment and similarity analysis, five conserved motifs in the amino acid sequence of β-glucanase were identified, and five deletion mutants were generated to investigate its essential regions further. Notably, the N-terminal amino acid sites 5-54 deletion mutation of β-glucanase decreased resistance to B. cinerea infection. These results indicate that N-terminal amino acids 5-54 (N54) are crucial for β-glucanase induced N. benthamiana defense response and for enhancing resistance to B. cinerea. Further analysis using real-time quantitative fluorescent PCR (qRT-PCR) revealed a significant reduction in gene expression within the N54 region compared to that of unmutated β-glucanase. Additionally, there was a notable reduction in the relative expression levels of FRK, CYP71D20, WRKY7, WRKY8, ACRE31 and PTI genes. Therefore, the first 50 amino acids at positions 5-54 within the N-terminal domain were essential for triggering plant defense responses and enhancing resistance against B. cinerea infection. This study provides an important theoretical foundation for systematic investigation into key functional domains within β-glucanase that trigger defensive responses in plants against B. cinerea.

Melatonin alleviates LPS-induced depression-like behavior in mice by inhibiting ferroptosis by regulating RNA methylation-mediated SIRT6/Nrf2/HO-1 pathway.

The objective of this study is to investigate the impact of ferroptosis on depression and elucidate the molecular mechanism underlying melatonin's inhibitory effect on ferroptosis in the treatment of depression. In this study, a depression-like behavior model was induced in mice using LPS, and the effect of melatonin on depression-like behavior was evaluated through behavioral experiments (such as forced swimming test (FST) and sucrose preference test (SPT)). Additionally, molecular biological techniques (including real-time fluorescence quantitative PCR, Western blotting, immunoprecipitation) were employed to detect the expression levels and interactions of METTL3, SIRT6 and ferroptosis-related genes in mouse brain tissue. Furthermore, both in vitro and in vivo experiments were conducted to verify the regulatory effect of melatonin on Nrf2/HO-1 pathway and explore its potential molecular mechanism for regulating ferroptosis. Melatonin was found to significantly ameliorate depression-like behavior in mice, as evidenced by reduced immobility time in the forced swimming test and increased sucrose intake in the sucrose preference test. Subsequent investigations revealed that melatonin modulated SIRT6 stability through METTL3-mediated ubiquitination of SIRT6, leading to its degradation. As a deacetylase, SIRT6 plays a pivotal role in cellular metabolism regulation and antioxidative stress response. This study elucidated potential signaling pathways involving Nrf2/HO-1 through which SIRT6 may exert its effects. The findings suggest that melatonin can improve depressive behavior by suppressing ferroptosis and protecting neurons through its antioxidant properties. Additionally, targeting the Nrf2/HO-1 pathway via METTL3 and NEDD4 regulation may be a potential therapeutic approach for depression.

Influences and mechanisms of iron input for methane productions in peatlands.

Atmospheric deposition provides a stable iron source for peatlands. The influences of Fe input on methane (CH4) productions and the underlying mechanisms remain unclear. We conducted a microcosm experiment with peat sediments collected from the Qinghai-Tibet Plateau of China to explore the effects of ferrihydrite reductionfor CH4 productions in peatlands by using geochemical analyses including 57Fe Mössbauer spectroscopy and three-dimensional fluorescence spectroscopy (3D-EEM) in combination with high-throughput sequencing of 16S rRNA and real-time fluorescence quantitative PCR (qPCR). Results showed that ferrihydrite reduction significantly increased CH4 production, being 30 times of that under the control. Selective extractions for iron oxides and 57Fe Mössbauer spectroscopy measurements revealed that no crystalline secondary iron minerals were formed during the ferrihydrite reduction process. The addition of ferrihydrite enhanced the degradation of dissolved organic matter (DOM) in peat soil, resulting in a reduction in the concentration of dissolved organic carbon (DOC). Furthermore, the relative abundance of typical fermentative microorganisms in peat sediments, including Acidobacteriota and Bacteroidota, significantly increased. Such a result indicated that reduction of ferrihydrite accelerated organic matter decomposition and increased substrate concentration required for methanogenesis. Furthermore, a co-increase in relative abundance of Geobacter, Geothrix, and Methanobacterium in the ferrihydrite-amended group suggested a potential synergistic interaction that may promote the CH4 production. Our results demonstrated that ferrihydrite reduction could significantly enhance CH4 production and play a vital role in regulating CH4 emissions in peatlands.

Distribution Characteristics of Nitrogen-Cycling Microorganisms in Deep-Sea Surface Sediments of Western South China Sea.

Nitrogen-cycling processes in the deep sea remain understudied. This study investigates the distribution of nitrogen-cycling microbial communities in the deep-sea surface sediments of the western South China Sea, using metagenomic sequencing and real-time fluorescent quantitative PCR techniques to analyze their composition and abundance, and the effects of 11 environmental parameters, including NH4+-N, NO3--N, NO2--N, PO43--P, total nitrogen (TN), total organic carbon (TOC), C/N ratio, pH, electrical conductivity (EC), SO42-, and Cl-. The phylum- and species-level microbial community compositions show that five sites can be grouped as a major cluster, with sites S1 and S9 forming a sub-cluster, and sites S13, S19, and S26 forming the other; whereas sites S3 and S5 constitute a separate cluster. This is also evident for nitrogen-cycling functional genes, where their abundance is influenced by distinct environmental conditions, including water depths (shallower at sites S1 and S9 against deeper at sites S13, S19, and S26) and unique geological features (sites S3 and S5), whereas the vertical distribution of nitrogen-cycling gene abundance generally shows a decreasing trend against sediment depth. Redundancy analysis (RDA) exploring the correlation between the 11 environmental parameters and microbial communities revealed that the NO2--N, C/N ratio, and TN significantly affect microbial community composition (p < 0.05). This study assesses the survival strategies of microorganisms within deep-sea surface sediments and their role in the marine nitrogen cycle.

Abamectin at environmentally relevant concentrations impairs bone development in zebrafish larvae

Abamectin (ABM) is a widely used pesticide in agriculture and veterinary medicine, which primarily acts by disrupting the neurological physiology of pests, leading to their paralysis and death. Its extensive application has resulted in contamination of many natural water bodies. While the adverse effects of ABM on the growth and development of non-target organisms are well documented, its impact on bone development remains inadequately studied. The present study aimed to investigate the effects of environmentally relevant concentrations of ABM (1, 5, 25 μg/L) on early bone development in zebrafish. Our results indicated that ABM significantly affected both cartilage and bone development of zebrafish larvae, accompanied by dose-dependent increase in deformity and mortality rates, as well as exacerbated apoptosis. ABM exposure led to deformities in the ceratobranchial (cb) and hyosymplectic (hs), accompanied by significant increases in the length of the palatoquadrate (pq). Furthermore, significant decreases in the CH-CH angle, Meckel's-Meckel's angle, and Meckel's-PQ angle were noted. Even at the safe concentration of 5 μg/L (1/10 of the 96 h LC50), ABM delayed the process of bone mineralization in zebrafish larvae. Real-time fluorescent quantitative PCR results demonstrated that ABM induced differential gene expression associated with cartilage and bone development in zebrafish. Thus, this study provides preliminary insights into the effects and molecular mechanisms underlying ABM's impact on the bone development of zebrafish larvae and offers new evidence for a better understanding of its toxicity.

Wenjing decoction: Mechanism in the treatment of dysmenorrhea with blood stasis syndrome through network pharmacology and experimental verification

Ethnopharmacological relevanceTraditional Chinese Medicine (TCM) formula Wenjing Decoction (WJD) longstanding efficacy in enhancing blood circulation, resolving blood stasis, and mitigating dysmenorrhea symptoms. Despite its prevalent application, the specific mechanism underlying effect of WJD remains elusive. ObjectiveThe purpose of this study is to examine the material basis of Wenjing Decoction and explore the effect of WJD on rat models of dysmenorrhea with blood stasis syndrome and elucidate its mechanism. MethodsIn this study, we initially identified the chemical constituents of WJD using liquid chromatography-mass spectrometry (LC-MS). Subsequently, we employed network pharmacology to predict the mechanism of WJD in treating acute blood stasis dysmenorrhea. To further investigate the role of WJD, we established a rat model of acute blood stasis. We monitored changes in blood coagulation indexes, IL-6, TNF-α, NO, and COX-2 in rats before and after administration to confirm the successful establishment of the rat model and evaluate the therapeutic effect of WJD on dysmenorrhea and acute blood stasis. Finally, real-time fluorescence quantitative PCR (qPCR) and Western blot (WB) were utilized to investigate its mechanism. ResultsThrough LC-MS analysis, 69 chemical substances were identified in WJD. Network pharmacology study revealed that the mechanism of WJD in treating BSS may be associated with the PI3K/AKT/NF-κB pathway. Following administration, the WJD group showed gradual recovery of physical signs and coagulation index to a healthy level. Additionally, the levels of IL-6, TNF-α, and COX-2 decreased in a dose-dependent manner, whereas NO levels increased. Results from QPCR and WB detection indicated increased expression levels of p-PI3K, p-AKT, Bcl-2, and eNOS, and decreased expression levels of Bax, NFκBp65, ICAM1, and VCAM1. ConclusionThe results show that WJD significantly improves the characterization, dysmenorrhea index, and coagulation-related factors in BSS rats. Through network pharmacological prediction, real-time fluorescence quantitative PCR, and Western blot analysis, it is postulated that the beneficial effects of WJD on dysmenorrhea may be linked to the PI3K/AKT/NF-κB signaling pathway. These findings offer a theoretical foundation for the advancement and utilization of WJD.

Genome-Wide Identification of MsICE Gene Family in Medicago sativa and Expression Analysis of the Response to Abiotic Stress

To predict the role of the MsICE gene family in the response to abiotic stress, in this study, bioinformatics analysis and real-time fluorescence quantitative PCR were performed. Alfalfa (Medicago sativa) is one of the most economically valuable crops globally. Inducer of CBF expression (ICE), which is part of the basic helix–loop–helix (bHLH) transcription factor (TF) family, acts as a key regulator of cold tolerance. Despite this, there is little information available about ICE genes in alfalfa. Therefore, we studied the function of ICE TFs in alfalfa. We identified 11 MsICE genes from the alfalfa genome and classified them into two groups. Analysis of the protein motif and gene structure revealed relatively high conservation among subgroups of the tightly clustered MsICE genes. Through synteny analysis, we detected duplication events in the MsICE gene family, suggesting that the ICE gene family was formed through fragment duplications. All the MsICE proteins were located in the nucleus according to subcellular localization predictions. The promoter cis-regulatory elements of MsICE genes are largely involved in light (Box 4), hormone (ABRE), and stress (MYB) responses. The MsICE01/MsICE07/MsICE09/MsICE10/MsICE11 genes contained MYB- and MYC-binding motifs, indicating an association with abiotic stress. The specific expression patterns of MsICE genes in leaves were revealed by examining their expression patterns in different tissues. These findings suggest that these genes may sense external environmental changes through leaves. Abiotic stress can cause striking upregulation of MsICE07 (PCA score: −4.03) and MsICE10 (PCA score: −4.05) expression. In this study, candidate genes associated with cold stress were identified, and subsequent molecular biological analyses allowed elucidation of the biological functions of these genes in alfalfa. This research provides a theoretical foundation for enhancing alfalfa yield and quality under cold conditions.

Establishment of a Real-Time Fluorescence Isothermal Recombinase-Aided Amplification Method for the Detection of H9 Avian Influenza Virus.

The H9 subtype of avian influenza virus (AIV) has been characterized by its rapid spread, wide range of prevalence, and continuous evolution in recent years, leading to an increasing ability for cross-species transmission. This not only severely impacts the economic benefits of the aquaculture industry, but also poses a significant threat to human health. Therefore, developing a rapid and sensitive detection method is crucial for the timely diagnosis and prevention of H9 AIVs. In this study, a real-time fluorescent reverse transcription recombinase-aided isothermal amplification (RT-RAA) technique targeting the hemagglutinin (HA) of H9 AIVs was established. This technique can be used for detection in just 30 min at a constant temperature of 42 °C, and it exhibits good specificity without cross-reactivity with other viruses. Sensitivity tests revealed that the detection limit of RT-RAA was 163 copies per reaction, and the visual detection limit was 1759 copies per reaction at a 95% confidence interval, both of which are capable of detecting low concentrations of standards. Furthermore, RT-RAA was applied to detect 155 clinical samples, and compared to real-time fluorescent quantitative PCR (RT-qPCR), RT-RAA demonstrated high accuracy, with a specificity of 100% and a kappa value of 0.96, indicating good correlation. Additionally, with the assistance of a portable blue imaging device, we can visually observe the amplification products, greatly facilitating rapid detection in resource-limited environments. The RT-RAA detection method developed in this study does not require expensive equipment or highly skilled staff, making it beneficial for the accurate and low-cost detection of H9 AIVs.

Establishment of a Rapid Detection Method for Highly Virulent Klebsiella Pneumoniae Based on Multienzyme Isothermal Rapid Amplification Technology.

This study aimed to establish a method for the rapid detection of highly virulent Klebsiella pneumoniae (hvKP) by using multienzyme isothermal rapid amplification (MIRA) technology. The laboratory can quickly, accurately, and conveniently diagnose highly virulent Klebsiella pneumoniae infection. For this study, 7 laboratory standard strains and 184 clinical isolates (including 70 strains of Klebsiella pneumoniae) were collected and screened for highly virulent Klebsiella pneumoniae based on its colony morphology, wire drawing test, and next-generation sequencing (NGS) results. Based on the nucleic acid sequence of the peg344 gene of highly virulent Klebsiella pneumoniae on GenBank (no. AP006726.1), specific conserved regions were selected to design MIRA and real-time fluorescence quantitative PCR (qPCR) specific primers and probes. The MIRA and qPCR methods were used to detect the tested strain, and the specificity, sensitivity, and clinical performance of the MIRA method for detecting hvKP were evaluated. In total, 21 cases of hvKP were screened from clinical isolates. The MIRA detection method utilizes specific primers and probes to transmit significant fluorescence signals at 39°C, and the detection process takes 30 minutes. The specificity test results showed that only hvKP had a specific amplification curve, while the rest of non-highly virulent Klebsiella pneumoniae (non-hvKP) had no specific amplification curve. The sensitivity test results showed that the sensitivity of MIRA for detecting hvKP is 7 × 102 CFU/mL, which is consistent with the sensitivity of the real-time fluorescence qPCR method. A simultaneous detection of 184 clinical isolates was accomplished by using MIRA and qPCR methods. Twenty-one strains of hvKP have specific amplification curves, while the remaining 163 strains of non-hvKP have no specific amplification curves. The accuracy of both methods for detecting hvKP is 100%. The established multienzyme isothermal rapid amplification (MIRA) has the following characteristics: a short detection time, high sensitivity, and a strong specificity, and it can be used as a powerful tool for an early diagnosis and epidemiological monitoring of hvKp.

Transcriptome-wide identification of the Hsp70 gene family in Pugionium cornutum and functional analysis of PcHsp70-5 under drought stress.

The PcHsp70-5 enhances drought stress tolerance in transgenic Arabidopsis thaliana by upregulating stress tolerance genes and antioxidant enzyme activities. Heat shock proteins (HSPs) constitute a class of evolutionarily conserved proteins synthesized by organisms in response to various adverse environmental stimuli such as elevated temperatures, drought, hormonal fluctuations, high salt concentrations, and mechanical stress. However, research on HSPs has predominantly focused on model plants and crops, whereas their functions in desert plants have not been well investigated. This study analyzed the transcriptome of Pugionium cornutum and identified the complete ORFs of 25 genes of the PcHsp70 family genes. Their expression levels under drought stress were investigated using existing RNA-seq data. PcHsp70-5 genes exhibited high expression levels in both roots and leaves under drought stress. Consequently, the PcHsp70-5 genes were cloned and transformed into Arabidopsis thaliana for further analysis of their roles in drought stress response. Real-time fluorescence quantitative PCR (qRT-PCR) analysis demonstrated that both, drought stress and ABA, induced PcHsp70-5 expression. Under drought conditions, transgenic Arabidopsis plants exhibited markedly enhanced growth compared to wild-type plants, as evidenced by improved survival rates, root length, fresh weight, chlorophyll content, and reduced levels of malondialdehyde (MDA) and hydrogen peroxide (H2O2) in leaves, indicating that PcHsp70-5 overexpression mitigated growth inhibition and oxidative damage induced by drought stress. Subsequent research revealed that PcHsp70-5 overexpression significantly augmented the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and increased the proline content in transgenic Arabidopsis under drought conditions, alongside a significant increase in the expression levels of genes related to stress tolerance. This suggests that PcHsp70-5 enhances drought stress tolerance in transgenic Arabidopsis by upregulating stress tolerance genes and antioxidant enzyme activities.

Prognostic value of KLFs family genes in renal clear cell carcinoma

Numerous studies have shown that the Krüppel-like factors (KLFs) family of transcription factors regulate various eukaryotic physiological processes including the proliferation, differentiation, senescence, death, and carcinogenesis of animal cells. In addition, they are involved in the regulation of key biological processes such as cell cycle, DNA repair, and immune response. Current studies focus on investigating the role of KLFs in normal physiological conditions and the incidence and development of diseases. However002C the significance of KLFs family genes in clear cell renal cell carcinoma (ccRCC) remains partly understood; therefore, an in-depth investigation of their role and clinical value in this cancer is desired. The study aimed to investigate the role of KLF family genes in the incidence, development, and prognosis of ccRCC, and to identify the related potential biomarkers and therapeutic targets. The expression of KLFs in the RNA sequencing data of 613 ccRCCs from the TCGA database was analyzed using R software, and UALCAN and GEPIA assessed the expression of KLF genes in ccRCC. Real-time fluorescence quantitative PCR analysis was performed using 10 pairs of paired ccRCC sample tissues and renal cancer cell lines from the First Affiliated Hospital of Nanchang University. Overall survival (OS), progression-free interval (PFI), and disease-specific survival (DSS) of Kidney Clear Cell Carcinoma (KIRC) samples at differential expressions of KLFs in the TCGA database were analyzed using the R software, followed by generating a nomogram prediction model. GSCALite assessed the interactions of KLF genes with miRNAs and generated network maps. Protein interaction network maps of 50 neighboring genes associated with KLF mutations were analyzed using STRING with GO and KEGG functional enrichment analyses. The cBioPortal determined the probability of KLF gene mutations and their impact on OS and disease-free survival (DFS) in patients with ccRCC. Immune cell infiltration of KLFs was analyzed using TIMER. Finally, GSCALite was used to analyze the drug sensitivity and associated pathways of action of KLFs. Correlation validation using cellular experiments. KLF3/5/9/15 were significantly downregulated in ccRCC tissues, whereas KLF16/17 were upregulated compared with the adjacent tissues. Patients with high mRNA levels of KLF16/17 showed significantly lower OS, PFI, and DSS, whereas KLF3/5/9 showed a reverse trend. In patients with ccRCC, a significant correlation was observed between KLF mutations and OS and DSS. Furthermore, the correlation of KLF3/5/9 with immune cell infiltration was stronger than that of KLF15/16, while KLF17 was significantly associated with the Epithelial-Mesenchymal Transition (EMT) pathway. Overexpression of KLF5 inhibits the proliferative and migratory capacity of renal cancer cells (786-O and OS-RC-2), as well as their sensitivity to relevant small molecule drugs. Our research revealed the expression levels and biological significance of KLF genes in ccRCC, particularly highlighting the potential of KLF5 as a promising biomarker and therapeutic target for effective prognosis and diagnosis of ccRCC.