Quantitative Reverse Transcription-polymerase Chain Reaction Research Articles

Neutrophil Biomarkers Can Predict Cardiotoxicity of Anthracyclines in Breast Cancer.

Doxorubicin (DOX), a commonly used anticancer agent, causes cardiotoxicity that begins with the first dose and may progress to heart failure years after treatment. An inflammatory response associated with neutrophil recruitment has been recognized as a mechanism of DOX-induced cardiotoxicity. This study aimed to validate mRNA expression of the previously identified biomarkers of DOX-induced cardiotoxicity, PGLYRP1, CAMP, MMP9, and CEACAM8, and to assay their protein expression in the peripheral blood of breast cancer patients. Blood samples from 40 breast cancer patients treated with DOX-based chemotherapy were collected before and after the first chemotherapy cycle and > 2 years after treatment. The protein and gene expression of PGLYRP1/Tag7, CAMP/LL37, MMP9/gelatinase B, and CEACAM8/CD66b were determined using ELISA and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Receiver operating characteristic (ROC) curve analysis was used to determine the diagnostic value of each candidate biomarker. Patients with cardiotoxicity (n = 20) had significantly elevated levels of PGLYRP1, CAMP, MMP9, and CEACAM8 at baseline, after the first dose of DOX-based chemotherapy, and at > 2 years after treatment relative to patients without cardiotoxicity (n = 20). The first dose of DOX induced significantly higher levels of all examined biomarkers in both groups of patients. At > 2 years post treatment, the levels of all but MMP9 dropped below the baseline. There was a good correlation between the expression of mRNA and the target proteins. We demonstrate that circulating levels of PGLYRP1, CAMP, MMP9, and CEACAM8 can predict the cardiotoxicity of DOX. This novel finding may be of value in the early identification of patients at risk for cardiotoxicity.

NAT10-mediated ac4C acetylation of TFRC promotes sepsis-induced pulmonary injury through regulating ferroptosis

BackgroundSepsis-induced pulmonary injury (SPI) is a common complication of sepsis with a high rate of mortality. N4-acetylcytidine (ac4C) is mediated by the ac4C “writer”, N-acetyltransferase (NAT)10, to regulate the stabilization of mRNA. This study aimed to investigate the role of NAT10 in SPI and the underlying mechanism.MethodsTwenty-three acute respiratory distress syndrome (ARDS) patients and 27 non-ARDS volunteers were recruited. A sepsis rat model was established. Reverse transcription-quantitative polymerase chain reaction was used to detect the expression of NAT10 and transferrin receptor (TFRC). Cell viability was detected by cell counting kit-8. The levels of Fe2+, glutathione, and malondialdehyde were assessed by commercial kits. Lipid reactive oxygen species production was measured by flow cytometric analysis. Western blot was used to detect ferroptosis-related protein levels. Haematoxylin & eosin staining was performed to observe the pulmonary pathological symptoms.ResultsThe results showed that NAT10 was increased in ARDS patients and lipopolysaccharide-treated human lung microvascular endothelial cell line-5a (HULEC-5a) cells. NAT10 inhibition increased cell viability and decreased ferroptosis in HULEC-5a cells. TFRC was a downstream regulatory target of NAT10-mediated ac4C acetylation. Overexpression of TFRC decreased cell viability and promoted ferroptosis. In in vivo study, NAT10 inhibition alleviated SPI.ConclusionNAT10-mediated ac4C acetylation of TFRC aggravated SPI through promoting ferroptosis.Graphical

Real-time quantitative reverse transcription PCR assay for the detection of Nuomin virus – An emerging tick-borne virus

Nuomin virus (NOMV), an emerging tick-borne virus (TBVs) identified in 2020, has been associated with fever, headache, and potential liver dysfunction in infected individuals. This study presents a novel TaqMan real-time quantitative PCR method designed for the rapid, sensitive, and specific detection of NOMV, facilitating early diagnosis. Utilizing Beacon Designer software 8.0, we optimized the PCR assay including the development of primers and probes to precisely target the conserved region of the NOMV genome, followed by optimization of primer and probe concentrations and annealing temperature. The resulting assay demonstrated robust performance, with standard curve represented by the equation y=−3.29x+39.42, a high correlation coefficient (R2 = 0.995) and an efficiency 99.53 %. Importantly, the method exhibited exceptional specificity, which did not yield cross-reacting signals from other TBVs, including Songling virus (SGLV), Beiji virus (BJNV), tick-borne encephalitis virus (TBEV), Yezo virus (YEZV), Alongshan virus (ALSV), and severe fever with thrombocytopenia syndrome bunyavirus (SFTSV). The assay’s detection limit was remarkably low, reaching 10 copies/μL, representing a 100-fold increase compared to semi-nested RT-PCR. Additionally, it demonstrated excellent repeatability, with coefficients of variation for intra- and inter-group tests consistently below 3 %. Clinical evaluations confirmed the assay’s superior performance, highlighting its high specificity, sensitivity, and reproducibility for NOMV detection. In conclusion, the method developed in this study provides a valuable tool to support timely management of NOMV infections, with significant implications for clinical practice.

Reference gene evaluation for normalization of gene expression studies with lymph tissue and node‑derived stromal cells of patients with oral squamous cell carcinoma.

Profiling studies using reverse transcription quantitative PCR (RT-qPCR) require reliable normalization to reference genes to accurately interpret the results. A stable reference gene panel was established to profile metastatic and non-metastatic lymph nodes in patients with oral squamous cell carcinoma. The stability of 18S ribosomal RNA (18SrRNA), ribosomal Protein Lateral Stalk Subunit P0 (RPLP0), ribosomal Protein L27 (RPL27), TATA-box binding protein (TBP), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), beta-actin (ACTB), glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and vimentin (VIM) was evaluated, as reference genes for profiling patient-derived lymph node stromal cells (LNSCs; N=8; N0:6, N+:2) and lymph node tissues (Patients:14, Nodes=20; N0:7; N+:13). The genes were initially assessed based on their expression levels, specificity, and stability rankings to identify the best combination of reference genes. VIM was excluded from the final analysis because of its low expression (high quantification cycle >32) and multiple peaks in the melting curve. The stability analysis was performed using Reffinder, which utilizes four tools; geNorm, NormFinder, BestKeeper and Comparative ∆Ct methods, thereby enabling the computing of a comprehensive ranking. Evaluation of the gene profiles indicated that while RPLP0 and 18SrRNA were stable in both lymph node tissues and LNSCs, HPRT1, RPL27 were uniquely stable in these tissues whereas ACTB and TBP were most stable in LNSCs. The present study identified the most stable reference gene panel for the RT-qPCR profiling of lymph node tissues and patient-derived LNSCs. The observation that the gene panel differed between the two model systems further emphasized the need to evaluate the reference gene subset based on the disease and cellular context.

The Role of αvβ3 Integrin in Lamina Cribrosa Cell Mechanotransduction in Glaucoma.

Purpose: Glaucoma, one of the leading causes of irreversible blindness, is a common progressive optic neuropathy characterised by visual field defects and structural changes to the optic nerve head (ONH). There is extracellular matrix (ECM) accumulation and fibrosis of the lamina cribrosa (LC) in the ONH, and consequently increased tissue stiffness of the LC connective tissue. Integrins are cell surface proteins that provide the key molecular link connecting cells to the ECM and serve as bidirectional sensors transmitting signals between cells and their environment to promote cell adhesion, proliferation, and remodelling of the ECM. Here, we investigated the expression of αVβ3 integrin in glaucoma LC cell, and its effect on stiffness-induced ECM gene transcription and cellular proliferation rate in normal (NLC) and glaucoma (GLC) LC cells, by down-regulating αVβ3 integrin expression using cilengitide (a known potent αVβ3 and αVβ5 inhibitor) and β3 integrin siRNA knockdown. Methods: GLC cells were compared to age-matched controls NLC to determine differential expression levels of αVβ3 integrin, ECM genes (Col1A1, α-SMA, fibronectin, vitronectin), and proliferation rates. The effects of αVβ3 integrin blockade (with cilengitide) and silencing (with a pool of four predesigned αVβ3 integrin siRNAs) on ECM gene expression and proliferation rates were evaluated using both reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting in the human NLC cells cultured on soft (4 kPa) and stiff (100 kPa) substrate and in GLC cells grown on standard plastic plates. Results: αVβ3 integrin gene and protein expression were enhanced (p < 0.05) in GLC cells as compared to NLC. Both cilengitide and siRNA significantly reduced αVβ3 expression in GLC. When NLC were grown in the stiff substrate, cilengitide and siRNA also significantly reduced the increased expression in αVβ3, ECM components, and proliferation rate. Conclusions: Here, we provide evidence of cilengitide- and siRNA-mediated silencing of αVβ3 integrin expression, and inhibition of ECM synthesis in LC cells. Therefore, αVβ3 integrin may be a promising target for the development of novel anti-fibrotic therapies for treating the LC cupping of the ONH in glaucoma.

Genome-wide identification, molecular evolution, and functional characterization of fructokinase gene family in apple reveal its role in improving salinity tolerance

Fructokinase (FRK) is a regulator of fructose signaling in plants and gateway proteins that catalyze the initial step in fructose metabolism through phosphorylation. Our previous study demonstrated that MdFRK2 protein exhibit not only high affinity for fructose, but also high enzymatic activity due to sorbitol. However, genome-wide identification of the MdFRK gene family and their evolutionary dynamics in apple are yet to be reported. A systematic genome-wide analysis in this study identified a total of nine MdFRK gene members, which could phylogenetically be clustered into seven groups. Chromosomal location and synteny analysis of MdFRKs revealed that their expansion in the apple genome is primarily driven by tandem and segmental duplication events. Divergent expression patterns of MdFRKs were observed in four source-sink tissues and at five different apple fruit developmental stages, which suggested their potential crucial roles in the apple fruit development and sugar accumulation. Reverse transcription-quantitative PCR (RT-qPCR) identified candidate NaCl or drought stress responsive MdFRKs, and transgenic apple plants overexpressing MdFRK2 exhibited considerably enhanced salinity tolerance. Our results will be useful for understanding the functions of MdFRKs in the regulation of apple fruit development and salt stress response.

PPAR gamma and PGC-1alpha activators protect against diabetic nephropathy by suppressing the inflammation and NF-kappaB activation.

Inflammation plays a critical role in the progression of diabetic nephropathy. Peroxisome proliferator-activated receptor gamma (PPARγ) and its coactivator PPARγ coactivator-1 alpha (PGC-1α) enhance mitochondrial biogenesis and cellular energy metabolism but inhibit inflammation. However, the molecular mechanism through which these two proteins cooperate in the kidney remains unclear. The aim of the present study was to investigate this mechanism. HK-2 human proximal tubular cells were stimulated by inflammatory factors, the expression of PPARγ and PGC-1α were determined via reverse transcription-quantitative polymerase chain reaction (PCR) and western blotting (WB), and DNA binding capacity was measured by an EMSA. Furthermore, db/db mice were used to establish a diabetic nephropathy model and were administered PPARγ and PGC-1α activators. Kidney injury was evaluated microscopically, and the inflammatory response was assessed via WB, immunohistochemistry and immunofluorescence staining. Besides, HK-2 cells were stimulated by high glucose and inflammatory factors with and without ZLN005 treatment, the expression of PPARγ, PGC-1α, p-p65 and p65 were determined via qPCR and WB. Our results revealed that both TNF-α and IL-1β significantly decreased PPARγ and PGC-1 expression in vitro. Cytokines obviously decreased PPARγ DNA binding capacity. Moreover, we detected rapid activation of the NF-κB pathway in the presence of TNF-α or IL-1β. PPARγ and PGC-1α activators effectively protected against diabetic nephropathy and suppressed NF-κB expression both in db/db mice and HK-2 cells. PPARγ and its coactivator PGC-1α actively participate in protecting against renal inflammation by regulating the NF-κB pathway, which highlights their potential as therapeutic targets for renal diseases.

New molecular mechanisms of quercetin in improving recurrent spontaneous abortion based on in-depth network pharmacology and molecular docking.

The increasing prevalence of recurrent spontaneous abortion (RSA) poses significant physical and psychological challenges for affected individuals. Quercetin, a natural plant flavonoid, shows promise in reducing miscarriage rates, yet its precise mechanism remains elusive. This study uses network pharmacology, molecular docking, and experimental validation to explore the molecular pathways through which quercetin mitigates RSA. Quercetin-related target genes were sourced from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), and RSA target genes were retrieved from the Comparative Toxicogenomics Database (CTD), with overlapping targets identified using Venn diagrams. All genes were visualized using the STRING database, and core targets were selected with Cytoscape 3.7.3. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using the DAVID and Reactome online resources. Subsequently, HTR-8/SVneo cells were stimulated with lipopolysaccharide (LPS) and treated with varying concentrations of quercetin (1, 5, and 10μM), then subjected to CCK-8, wound healing, transwell, and annexin V-FITC/PI apoptosis assays. Reverse-transcription quantitative PCR was used to determine the mRNA expression levels of IL-1β, TNF-α, and IL-6 in LPS-induced cells post-quercetin intervention, and western blotting was used to measure AKT1, MMP9, and caspase-3 protein levels. A total of 139 quercetin-associated target genes were identified from the TCMSP database, and 98 disease-associated target genes were obtained from the CTD, resulting in 25 shared target genes. Gene ontology enrichment highlighted the involvement of these targets in positive regulation of apoptosis, response to hypoxia, and intrinsic apoptotic signaling pathway in response to DNA damage. KEGG pathway analysis indicated enrichment in pathways related to interleukin-4 and interleukin-13 signaling, cytokine signaling in the immune system, and apoptosis. Molecular docking studies revealed robust binding of quercetin with MMP9, AKT1, IL-1β, TNF, and caspase-3. In vitro experiments demonstrated that quercetin enhanced LPS-induced cell activity, fostering proliferation, migration, and invasion, and reducing apoptosis. Moreover, quercetin reduced IL-1β, TNF-α, and IL-6 mRNA expression, increased AKT1 and MMP9 protein levels, and reduced caspase-3 expression. Quercetin could mitigate the incidence of RSA by modulating inflammatory responses and apoptotic processes, through upregulation of AKT1 and MMP9, and downregulation of caspase-3, IL-1β, TNF-α, and IL-6. Quercetin opens up a new way of thinking about treating RSA.

Construction of circRNA-mediated ceRNA network and immunoassay for investigating pathogenesis of COPD.

The chronic respiratory condition known as chronic obstructive pulmonary disease (COPD) was one of the main causes of death and disability worldwide. This study aimed to explore and elucidate new targets and molecular mechanisms of COPD by constructing competitive endogenous RNA (ceRNA) networks. GSE38974 and GSE106986 were used to select DEGs in COPD samples and normal samples. Cytoscape software was used to construct and present protein-protein interaction (PPI) network, mRNA-miRNA co-expression network and ceRNA network. The CIBERSORT algorithm and the Lasso model were used to screen the immune infiltrating cells and hub genes associated with COPD, and the correlation between them was analyzed. COPD cell models were constructed in vitro and the expression level of ceRNA network factors mediated by hub gene was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In this study, 852 differentially expressed genes were screened in the GSE38974 dataset, including 439 upregulated genes and 413 downregulated genes. Gene clustering analysis of PPI network results was performed using the Minimum Common Tumor Data Element (MCODE) in Cytoscape, and seven hub genes were screened using five algorithms in cytoHubba. CCL20 was verified as an important hub gene based on mRNA-miRNA co-expression network, GSE106986 database validation and the analysis of ROC curve results. Finally, we successfully constructed the circDTL-hsa-miR-330-3p-CCL20 network by Cytoscape. Immune infiltration analysis suggested that CCL20 can co-regulate immune cell migration and infiltration through chemokines CCL7 and CXCL3. In vitro experiments, the expression of circDTL and CCL20 was increased, while the expression of hsa-miR-330-3p was decreased in the COPD cell model. By constructing the circDTL-hsa-miR-330-3p-CCL20 network, this study contributes to a better understanding of the molecular mechanism of COPD development, which also provides important clues for the development of new therapeutic strategies and drug targets.

A novel system with robust compatibility and stability for detecting Sugarcane yellow leaf virus based on CRISPR-Cas12a.

Sugarcane yellow leaf virus (SCYLV) can reduce sugarcane productivity. A novel detection system based on reverse transcription-multienzyme isothermal rapid amplification (RT-MIRA) combined with CRISPR-Cas12a, named RT-MIRA-CRISPR-Cas12a, was developed. This innovative approach employs crude leaf extract directly as the reaction template, streamlining the extraction process for simplicity and speed. Combining RT-MIRA and CRISPR-Cas12a in one reaction tube increases the ease of operation while reducing the risk of aerosol contamination. In addition, it exhibits sensitivity equivalent to qPCR, boasting a lower detection limit of 25 copies. Remarkably, the entire process, from sample extraction to reaction completion, requires only 52-57 minutes, just a thermostat water bath. The result can be observed and judged by the naked eye.IMPORTANCESugarcane yellow leaf disease (SCYLD) is an important viral disease that affects sugarcane yield. There is an urgent need for rapid, sensitive, and stable detection methods. The reverse transcription-multienzyme isothermal rapid amplification combined with CRISPR-Cas12a (RT-MIRA-CRISPR-Cas12a) method established in this study has good specificity and high sensitivity. In addition, the system showed good compatibility and stability with the crude leaf extract, as shown by the fact that the crude extract of the positive sample could still be stably detected after 1 week when placed at 4°C. RT-MIRA-CRISPR-Cas12a, reverse transcription polymerase chain reaction (RT-PCR), and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to detect SCYLV on 33 sugarcane leaf samples collected from the field, and it was found that the three methods reached consistent conclusions. This Cas12a-based detection method proves highly suitable for the rapid on-site detection of the SCYLV.

Activation of Piezo1 channels enhances spontaneous contractions of isolated human bladder strips via acetylcholine release from the mucosa

Enhanced spontaneous bladder contractions (SBCs) have been thought one of the important underlying mechanisms for detrusor overactivity (DO). Piezo1 channel has been demonstrated involved in bladder function and dysfunction in rodents. We aimed to investigate the modulating role of Piezo1 in SBCs activity of human bladder. Human bladder tissues were obtained from 24 organ donors. SBCs of isolated bladder strips were recorded in organ bath. Piezo1 expression was examined with reverse transcription-quantitative polymerase chain reaction and immunofluorescence staining. ATP and acetylcholine release in cultured human urothelial cells was measured. Piezo1 is abundantly expressed in the bladder mucosa. Activation of Piezo1 with its specific agonist Yoda1 (100 nM–100 μM) enhanced the SBCs activity in isolated human bladder strips in a concentration-dependent manner. The effect of Yoda1 mimicked the effect of a low concentration (30 nM) of carbachol, which can be attenuated by removing the mucosa, blocking muscarinic receptors with atropine (1 μM), and blocking purinergic receptors with pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate (PPADS, 30 μM), but not by tetrodotoxin (1 μM). Activation of urothelial Piezo1 with Yoda1 (30 μM) or hypotonic solution induced the release of ATP and acetylcholine in cultured human urothelial cells. In patients with benign prostatic hyperplasia, greater Piezo1 expression was observed in bladder mucosa from patients with DO than patients without DO. We conclude that upregulation and activation of Piezo1 may contribute to DO generation in patients with bladder outlet obstruction by promoting the urothelial release of ATP and acetylcholine. Inhibition of Piezo1 may be a novel therapeutic approach in the treatment of overactive bladder.

Construction of a cuproptosis‑related lncRNA signature to predict biochemical recurrence of prostate cancer.

Biochemical recurrence (BCR) is common in prostate cancer (PCa), and patients with BCR usually have a poor prognosis. Cuproptosis is a unique type of cell death, and copper homeostasis is crucial to the occurrence and development of malignancies. The present study aimed to explore the prognostic value of cuproptosis-related long non-coding RNAs (lncRNAs; CRLs) in PCa and to develop a predictive signature for forecasting BCR in patients with PCa. Using The Cancer Genome Atlas database, transcriptomic, mutation and clinical data were collected from patients with PCa. A total of 121 CRLs were identified using Pearson's correlation coefficient. Subsequently, a 6-CRL signature consisting of AC087276.2, CNNM3-DT, AC090198.1, AC138207.5, METTL14-DT and LINC01515 was created to predict the BCR of patients with PCa through Cox and least absolute shrinkage and selection operator regression analyses. Kaplan-Meier curve analysis demonstrated that high-risk patients had a low BCR-free survival rate. In addition, there was a substantial difference between the high- and low-risk groups in the immune microenvironment, immune therapy, drug sensitivity and tumor mutational burden. A nomogram integrating the Gleason score, 6-CRL signature and clinical T-stage was established and evaluated. Finally, the expression of signature lncRNAs in PCa cells was verified through reverse transcription-quantitative PCR. In conclusion, the 6-CRL signature may be a potential tool for making predictions regarding BCR in patients with PCa, and the prognostic nomogram may be considered a practical tool for clinical decision-making.

Interplay of toxic metal levels and endoplasmic reticulum stress gene profile in type 2 diabetes mellitus

The increasing global prevalence of type 2 diabetes mellitus (T2DM) necessitates investigating its complex etiology. This study aimed to explore the relationship between exposure to toxic metals, expression of endoplasmic reticulum stress response (ERSR) genes, and various biochemical parameters, including glycated hemoglobin (HbA1c), insulin resistance (HOMA-IR)/sensitivity (QUICKI), lipid profile, and estimated glomerular filtration rate (eGFR) in T2DM patients. T2DM patients and control subjects were matched for age, gender, and lifestyle factors. Biochemical parameters, toxic metal levels, and ERSR gene expression were analyzed using inductively coupled plasma mass spectrometry (ICPMS) and quantitative reverse transcription PCR (qRT-PCR), respectively. T2DM patients exhibit dysregulated lipid profiles and significantly higher fasting blood sugar (FBS), HbA1c, and insulin levels (all p < 0.0001). The insulin sensitivity was lower in T2DM patients (0.32 ± 0.09) than in the control group (0.35 ± 0.02, p = 0.02). Insulin resistance was significantly higher in the T2DM group (5.38 ± 3.15) than in the control group (1.98 ± 0.86, p = 0.0001). Nickel (4.75 ± 2.45 ppb, p < 0.0001) and arsenic (1.85 ± 1.78 ppb, p < 0.0001) levels were significantly elevated in T2DM patients. There was significant upregulation of ER stress genes: GRP78, CHOP, IRE1, ATF4, ATF6, and XBP1 (all p < 0.0001), while PERK was significantly down regulated (0.68-fold, p < 0.0001). Nickel levels were positively correlated with HOMA-IR (r = 0.49, p < 0.0001) and HbA1c (r = 0.35, p = 0.002). Arsenic levels were correlated with insulin (r = 0.34, p < 0.0001), insulin resistance (r = 0.51,p < 0.0001), HbA1c (r = 0.53, p < 0.0001), Arsenic levels (β = 0.37, p < 0.001), XBP1 (β = 0.36, p < 0.0001) independently associated with HbA1c.This study has revealed a significant association between arsenic exposure and the upregulation of XBP1 at the onset of T2DM. The overexpression of XBP1 and high levels of arsenic were independently associated with HbA1c and insulin resistance.

Study on molecular mechanism of intervertebral disc degeneration by single cell hdWGCNA combined with transcriptome sequencing

BackgroundIntervertebral disc degeneration (IVDD) is one of the important causes of lower back pain, seriously affecting people's health and quality of life. This research employs single-cell analysis to identify the specific cellular subtypes and key regulatory genes associated with IVDD. MethodsWe analyzed the single-cell data and screened cells that closely associated with the development of IVDD. The differential expression of feature genes between IVDD and control groups was analyzed. Additionally, drugs and regulatory transcription factors that interact with feature genes were predicted and clinically validated by reverse transcription quantitative real-time PCR (RT-qPCR), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA). ResultsOur study identified the Chond2 cell subtype associated with IVDD and selected four feature genes influencing the development of IVDD, namely IGFBP3, ACAN, VAPA and TMEM45A, through the high-dimensional weighted gene co-expression network analysis (hdWGCNA) analysis, least absolute shrinkage and selection operator (LASSO), and random forest (RF). Besides, compared to the MDD group, IGFBP3 and TMEM45A were significantly upregulated in the SDD group, while ACAN and VAPA showed no significant difference between the two groups. ELISA testing revealed a positive correlation between IGFBP3 concentration and the grading of IVDD. Furthermore, Celecoxib may be used to treat IVDD by inhibiting IGFBP3. ConclusionOur study identified the Chond2 cell subtype associated with IVDD and selected four feature genes influencing the development of IVDD, namely IGFBP3, ACAN, VAPA and TMEM45A. Our findings establish a robust theoretical foundation for the clinical diagnosis and treatment of IVDD patients.

Insulin-like growth factor-2 mRNA-binding protein 2 facilitates post-ischemic angiogenesis by increasing the stability of fibroblast growth factor 2 mRNA and its protein expression

BackgroundPost-ischemic angiogenesis is crucial for reestablishing blood flow in conditions such as peripheral artery disease (PAD). The role of insulin-like growth factor-2 mRNA-binding protein 2 (IGF2BP2) in post-transcriptional RNA metabolism and its involvement in post-ischemic angiogenesis remains unclear. MethodsUsing a human GEO database and a hind-limb ischemia (HLI) mouse model, the predominant isoform IGF2BP2 in ischemic gastrocnemius tissue was identified. Adeno-associated virus with the Tie1 promoter induced IGF2BP2 overexpression in the HLI model, evaluating the expression of vascular structural proteins (CD31 and α-SMA) and blood flow recovery after HLI. In vitro experiments with human umbilical vein endothelial cells (HUVECs) demonstrated that lentivirus-mediated IGF2BP2 overexpression upregulates cell proliferation, migration, and tube formation. GeneCards, RNAct databases, and subsequent reverse transcription quantitative polymerase chain reaction (RT-qPCR) predicted IGF2BP2 interactions with fibroblast growth factor 2 (FGF2) mRNA, and actinomycin D treatment, binding site predictions and CLIP-seq data further confirmed this interaction. Furthermore, western blotting, enzyme-linked immunosorbent assay, and RNA immunoprecipitation followed by RT-qPCR were performed to validate IGF2BP2's interaction with FGF2 mRNA and to assess its role in stabilizing FGF2 mRNA, as well as its impact on FGF2 protein expression. ResultsHLI reduced IGF2BP2 expression in the gastrocnemius tissue, which gradually increased during blood flow recovery. IGF2BP2 overexpression in HLI mice accelerated blood flow recovery and increased capillary and small artery densities. The overexpression of IGF2BP2 in HUVECs stimulated proliferation, migration, and tube formation by interacting with FGF2 mRNA to increase its stability. This interaction resulted in increased levels of FGF2 protein and secretion, ultimately promoting angiogenesis. ConclusionsIGF2BP2 contributes to blood flow restoration post-ischemia in vivo and promotes angiogenesis in HUVECs by enhancing FGF2 mRNA stability and FGF2 protein expression and secretion. These findings underscore IGF2BP2's therapeutic potential in ischemic conditions, such as PAD.

Melatonin alleviates heme-induced ferroptosis via activating the Nrf2/HO-1 pathway in neurons.

Ferroptosis of neurons is a significant cause of brain injury following intracerebral hemorrhage (ICH). As an iron-containing compound in hemoglobin, heme contributes to nerve injury post-ICH. Melatonin has been shown to mitigate the effects of ICH, yet its specific functions remain largely elusive. In this study, we aimed to explore the roles and mechanisms of melatonin in heme-induced ferroptosis subsequent to ICH. C57BL/6 mice were intracranially injected with heme and then treated with melatonin. Behavior tests [modified neurological severity score (mNSS), forelimb placing, and corner turn tests], H&E staining, Nissl staining, and Prussian blue staining were used to evaluate mouse brain tissue injury. In vitro, HT-22 cells were stimulated with heme and cell viability was determined by crystal violet staining. The iron contents were determined in heme-treated brains and cells, and the levels of 4-hydroxynonenal (4-HNE) and malonaldehyde (MDA) were assessed by ELISA. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to investigate the mRNA levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). Immunoblotting was used to analyze the protein expression of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), Nrf2, and HO-1. Finally, small interfering RNA (siRNA) was used to knock down Nrf2 in HT-22 cells. Melatonin treatment alleviated heme-induced injuries to neural function, as indicated by improved behavior in the mice. Moreover, melatonin decreased cell death and iron concentrations, increased MDA and 4-HNE levels, and reversed the decreases in GPX4, SLC7A11, Nrf2, and HO-1 induced by heme in vitro and in vivo. These results indicated that melatonin could improve the ferroptosis induced by heme. In addition, we found that Nrf2 knockdown attenuated the therapeutic effect of melatonin on neuronal ferroptosis induced by heme. In general, melatonin alleviates heme-induced ferroptosis by activating the Nrf2/HO-1 pathway, which implies that melatonin is a promising treatment for ferroptosis in ICH.

Comparative analysis of high-throughput RNA extraction kits in Naïve Non-Human Primate (NHP) tissues for downstream applications utilizing Xeno Internal Positive Control (IPC)

Ribonucleic acid (RNA) extraction and purification play pivotal roles in molecular biology and cell and gene therapy, where the quality and integrity of RNA are critical for downstream applications. Automated high-throughput systems have gained interest due to their potential for scalability and reduced labor requirements compared to manual methods. However, ensuring high-throughput capabilities, reproducibility, and reliability while maintaining RNA yield and purity remains challenging.This study evaluated and compared the performance of four commercially available high-throughput magnetic bead-based RNA extraction kits across six types of naïve non-human primate (NHP) tissue matrices: brain, heart, kidney, liver, lung, and spleen. The assessment focused on RNA purity, yield, and extraction efficiency (EE) using Xeno Internal Positive Control (IPC) spiking.Samples (∼50 mg) were homogenized via bead-beating and processed according to the manufacturer's protocol on the KingFisher Flex platform in eight replicates. RNA purity and yield were measured using a NanoDrop® spectrophotometer, while EE was evaluated via real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR).The findings indicate consistent high RNA purity across all tested extraction kits, yet substantial variation in RNA yield. Extraction efficiency exhibited variations across tissue types, with decreasing trends observed from brain to lung tissues. These results underscore the importance of careful kit selection and method optimization for achieving reliable downstream applications. The MagMAX™ mirVana™ Total RNA Isolation Kit stands out as the most accurate and reproducible, making it the preferred choice for applications requiring high RNA quality and consistency. Other kits, such as the Maxwell® HT simplyRNA Kit, offer a good balance between cost and performance, though with some trade-offs in precision. These findings highlight the importance of selecting the appropriate RNA isolation method based on the specific needs of the research, underscoring the critical role of accurate nucleic acid extraction in gene and cell therapy research.In conclusion, this study highlights the critical factors influencing RNA extraction performance, emphasizing the need for researchers and practitioners to consider both kit performance and tissue characteristics when designing experimental protocols. These insights contribute to the ongoing efforts to enhance the reproducibility and reliability of RNA extraction methods in molecular biology and cell/gene therapy applications.

Phenotypic and genotypic identification of class C and D β-lactamases in clinical isolates of Pseudomonas aeruginosa: a cross-sectional study.

The emergence of Pseudomonas aeruginosa (P. aeruginosa) antibiotic resistance is an important public health problem worldwide that can negatively affect infection control. Therefore, obtaining knowledge about antibiotic resistance mechanisms is necessary for infection control policies. This study aimed to determine the frequency of class C and D β-lactamases in P. aeruginosa strains isolated from patients referred to Ardabil hospitals using phenotypic and genotypic tests. Phenotypic detection of β-lactamases including AmpC cephalosporinase, oxacillinase (OXA)-type extended-spectrum β-lactamases (ESBLs), and OXA-type carbapenemases were performed using the disk diffusion-based methods. Amplification of genes encoding classes C (ampC and FOX genes) and D (OXA-1, OXA-2, OXA-10, OXA-23, and OXA-48 genes) β-lactamases was performed using the polymerase chain reaction (PCR) method. A quantitative reverse transcription PCR (qRT-PCR) method was used to determine the expression level of the ampC gene among multiple drug-resistant and imipenem-resistant P. aeruginosa strains. In phenotypic tests, the prevalence of AmpC cephalosporinase, OXA-type ESBLs, and OXA-type carbapenemases were 52.5%, 7.2%, and 95.8%, respectively. In genotypic tests, the prevalence of ampC, FOX, OXA-1, OXA-2, OXA-10, OXA-23, and OXA-48 genes were 100%, 0%, 4.3%, 60.8%, 42%, 29.7%, and 2.9%, respectively. In addition, the ampC gene overexpression was seen in 16 (33.3%) drug-resistant P. aeruginosa clinical isolates. Given the presence of class C and D β-lactamases in clinical isolates of P. aeruginosa in Ardabil hospitals, early detection of these strains can help prevent the spread of resistant strains in hospital environments and subsequent treatment failure.

Leukocyte cell-derived chemotaxin 2 (LECT2) regulates liver ischemia–reperfusion injury

Background and aimHepatic ischemia–reperfusion injury (IRI) is a significant challenge in liver transplantation, trauma, hypovolemic shock, and hepatectomy, with limited effective interventions available. This study aimed to investigate the role of leukocyte cell-derived chemotaxin 2 (LECT2) in hepatic IRI and assess the therapeutic potential of Lect2-short hairpin RNA (shRNA) delivered through adeno-associated virus (AAV) vectors. Materials and methodsThis study analyzed human liver and serum samples from five patients undergoing the Pringle maneuver. Lect2-knockout and C57BL/6J mice were used. Hepatic IRI was induced by clamping the hepatic pedicle. Treatments included recombinant human LECT2 (rLECT2) and AAV-Lect2-shRNA. LECT2 expression levels and serum biomarkers including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, and blood urea nitrogen (BUN) were measured. Histological analysis of liver necrosis and quantitative reverse-transcription polymerase chain reaction were performed. ResultsSerum and liver LECT2 levels were elevated during hepatic IRI. Serum LECT2 protein and mRNA levels increased post reperfusion. Lect2-knockout mice had reduced weight loss; hepatic necrosis; and serum ALT, AST, creatinine, and BUN levels. rLECT2 treatment exacerbated weight loss, hepatic necrosis, and serum biomarkers (ALT, AST, creatinine, and BUN). AAV-Lect2-shRNA treatment significantly reduced weight loss, hepatic necrosis, and serum biomarkers (ALT, AST, creatinine, and BUN), indicating therapeutic potential. ConclusionsElevated LECT2 levels during hepatic IRI increased liver damage. Genetic knockout or shRNA-mediated knockdown of Lect2 reduced liver damage, indicating its therapeutic potential. AAV-mediated Lect2-shRNA delivery mitigated hepatic IRI, offering a potential new treatment strategy to enhance clinical outcomes for patients undergoing liver-related surgeries or trauma.

Increased CCT5 expression is a potential unfavourable factor promoting the growth of nasopharyngeal carcinoma.

Chaperonin containing TCP1 subunit 5 (CCT5) encodes the CCT5 protein subunit of chaperonin-containing TCP-1 (CCT/TRiC) complex, and is shown to be upregulated in tumour pathogenesis. The study aim was to investigate the differential expression of CCT5 between nasopharyngeal carcinoma (NPC) and noncancerous nasopharyngeal tissues, and the correlation between CCT5 expression and clinicopathological parameters/prognosis in patients with NPC. Microarray assay data were evaluated for differential expression between NPC and noncancerous nasopharyngeal tissues. CCT5 expression in NPC and noncancerous nasopharyngeal tissues was determined at mRNA and protein levels by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemistry. Relationships between CCT5 expression in NPC, clinical parameters, and prognosis were statistically analysed. CCT5-mediated cell proliferation was assessed using EdU and cell counting kit-8. Western blot and co-immunoprecipitation were utilized to explore E3 ubiquitin-protein ligase parkin (PARK2)-induced degradation of CCT5. Microarray data showed CCT5 levels to be significantly increased in NPC versus noncancerous nasopharyngeal tissues, which was confirmed by qRT-PCR and immunohistochemical assays. Increased CCT5 protein levels positively correlated with tumour size, tumour recurrence, and clinical stage, and inversely correlated with patient's overall survival. Multivariate Cox regression analysis showed that enhanced CCT5 protein expression is an independent prognostic factor for patients with NPC. Overexpression of CCT5 markedly induced NPC cell proliferation. Finally, PARK2, as a suppressive E3 ubiquitin-ligase enzyme, was shown to bind CCT5 and induce degradation in NPC. Increased CCT5 may be an unfavourable factor promoting NPC growth. Binding of PARK2 to CCT5 was associated with CCT5 degradation, suggesting that PARK2 is an upstream negative modulator in NPC.