Quantitative Real-time PCR Research Articles

The Clinical Diagnostic Value of the Super-enhancer-associated Long Noncoding RNA RP11-803D5.4 and AC005592.2 in Colorectal Cancer.

Super-enhancer-associated long noncoding RNAs (SE-lncRNAs) play crucial roles in CRC pathogenesis. RP11-803D5.4 and AC005592.2 were identified as SE-lncRNAs of interest via microarray analysis, and our study aimed to evaluate their clinical value in CRC diagnosis and prognosis assessment. Fluorescence quantitative real-time PCR (qRT-PCR) was used to measure the expression of RP11-803D5.4 and AC005592.2 in the tissues and serum of CRC patients. Receiver operating characteristic (ROC) curves were generated to determine the predictive value of the two SE-lncRNAs. Functional assays were applied to assess the ability of RP11-803D5.4 to promote the proliferation, migration, and invasion of CRC cells. The two SE-lncRNAs were significantly upregulated in CRC tissue and serum samples vs. corresponding controls. ROC curve analysis indicated that RP11-803D5.4 (AUC=0.842) and AC005592.2 (AUC=0.811) had a high diagnostic performance for CRC. The combination of RP11-803D5.4, AC005592.2, and CEA had an AUC of 0.946 and distinguished CRC patients and healthy controls better than SE-lncRNA alone. The serum levels of RP11-803D5.4 and AC005592.2 were strongly correlated with their tissue expression levels. The expression levels of the two SE-lncRNAs were significantly lower in postoperative samples than in preoperative samples. Furthermore, similar to the findings of previous studies on AC005592.2, high RP11-803D5.4 expression promoted the proliferation, invasion, and migration of CRC cells. The findings suggested that RP11-803D5.4 and AC005592.2 are upregulated in CRCact and are crucial promoters of CRC progression. They also suggested that they might serve as noninvasive biomarkers for diagnosing CRC.

Development of a SYBR Green-based RT-qPCR assay for the rapid detection of peste-des-petits-ruminants virus.

Peste-des-petits-ruminants (PPR) is primarily a disease of small ruminants caused by peste-des-petits-ruminants virus (PPRV; Paramyxoviridae, Morbillivirus caprinae), formerly the small ruminant morbillivirus. PPRV can cause significant morbidity and mortality in small ruminants and a significant economic impact. Conventional reverse-transcription PCR (RT-PCR), and probe-based and SYBR Green-based RT quantitative real-time PCR (RT-qPCR), are employed for the molecular detection of PPRV. Here we describe a SYBR Green-based RT-qPCR for rapid and sensitive detection of PPRV. We designed the specific primers from the conserved region of the fusion gene (F) of PPRV. The standard curve of the established RT-qPCR assay had a good linear relationship. The developed assay was also 3 log units more sensitive than the conventional RT-PCR, with a detection limit of 13.6 copies and an efficiency of 98.2%. There was no cross-reactivity with other caprine respiratory viruses, namely bluetongue virus, goatpox virus, and orf virus. The positive detection rate of clinical samples was 11 of 64 (17.2%) versus 10 of 64 (15.6%) by conventional RT-PCR. We confirmed our results by sequencing the full F and N genes of the isolates. Our SYBR Green RT-qPCR can be used as a fast, economical, and sensitive alternative to RT-PCR for the detection of PPRV.

Altered Sertoli Cell Function Contributes to Spermatogenic Arrest in Dogs with Chronic Asymptomatic Orchitis

Acquired infertility due to chronic asymptomatic orchitis (CAO) is a common finding in male dogs. It is characterized by spermatogenic arrest, a significant reduction in spermatogonia, immune cell infiltration and a disruption of the blood–testis barrier. Sertoli cells are a key factor for spermatogenesis and the testicular micromilieu. We hypothesize altered Sertoli cell function to be involved in the pathogenesis of canine CAO. Consequently, the aim was to gain further insights into the spermatogonial stem cell niche and Sertoli cell function in CAO-affected dogs. Therefore, the testicular expression of the Sertoli cell-derived factors bFGF, GDNF, WNT5A, BMP4, CXCL12 and LDHC were evaluated in 15 CAO testis tissues and 10 normospermic controls by relative quantitative real-time PCR (qPCR). Additionally, the protein expression patterns of bFGF, GDNF and WNT5A were visualized immunohistochemically (IHC). This study revealed an overexpression of bFGF (IHC, p < 0.0001), GDNF (qPCR, p = 0.0036), WNT5A (IHC, p = 0.0066) and CXCL12 (qPCR, p = 0.0003) and a reduction in BMP4 (qPCR, p = 0.0041) and LDHC (qPCR, p = 0.0003) in CAO-affected testis in dogs, clearly confirming impaired Sertoli cell function in canine CAO. Sertoli cell function is essential for spermatogenesis and must be considered for potential therapeutic approaches.

CASC8 activates the pentose phosphate pathway to inhibit disulfidptosis in pancreatic ductal adenocarcinoma though the c-Myc-GLUT1 axis

PurposeGlucose starvation induces the accumulation of disulfides and F-actin collapse in cells with high expression of SLC7A11, a phenomenon termed disulfidptosis. This study aimed to confirm the existence of disulfidptosis in pancreatic ductal adenocarcinoma (PDAC) and elucidate the role of Cancer Susceptibility 8 (CASC8) in this process.MethodsThe existence of disulfidptosis in PDAC was assessed using flow cytometry and F-actin staining. CASC8 expression and its clinical correlations were analyzed using data from The Cancer Genome Atlas (TCGA) and further verified by chromogenic in situ hybridization assay in PDAC tissues. Cells with CASC8 knockdown and overexpression were subjected to cell viability, EdU, transwell assays, and used to establish subcutaneous and orthotopic tumor models. Disulfidptosis was detected by flow cytometry and immunofluorescence assays. RNA sequencing and metabolomics analysis were performed to determine the metabolic pathways which were significantly affected after CASC8 knockdown. We detected the glucose consumption and the NADP+/NADPH ratio to investigate alterations in metabolic profiles. RNA immunoprecipitation combined with fluorescence in situ hybridization assay was used to identify protein-RNA interactions. Protein stability, western blotting and quantitative real-time PCR assays were performed to reveal potential molecular mechanism.ResultsDisulfidptosis was observed in PDAC and could be significantly rescued by disulfidptosis inhibitors. CASC8 expression was higher in PDAC samples compared to normal pancreatic tissue. High CASC8 expression correlated with a poor prognosis for patients with PDAC and contributed to cancer progression in vitro and in vivo. Furthermore, CASC8 was associated with disulfidptosis resistance under glucose starvation conditions in PDAC. Mechanistically, CASC8 interacted with c-Myc to enhance the stability of c-Myc protein, leading to the activation of the pentose phosphate pathway, a reduction of the NADP+/NADPH ratio and ultimately inhibiting disulfidptosis under glucose starvation conditions.ConclusionsThis study provides evidence for the existence of disulfidptosis in PDAC and reveals the upregulation of CASC8 in this malignancy. Furthermore, we demonstrate that CASC8 acts as a crucial regulator of the pentose phosphate pathway and disulfidptosis, thereby promoting PDAC progression.

Oral Cancer-Derived miR-762 Suppresses T-Cell Infiltration and Activation by Horizontal Inhibition of CXCR3 Expression

Oral squamous cell carcinoma (OSCC) is an immune-cold tumor characterized by an immunosuppressive microenvironment with low cytotoxic activity to eliminate tumor cells. Tumor escape is one of the initial steps in cancer development. Understanding the underlying mechanisms of cancer escape can help researchers develop new treatment strategies. In this study, we prove the oral oncogenic miR-762 can suppress T-cell recruitment and cytotoxic activation in the tumor microenvironment (TME) through horizontal transmission from OSCC cells to adaptive immune T cells. Public database analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine the prognosis and expression of miR-762 in OSCC. T-cell activation by flow cytometry, qRT-PCR, IL-12 secretion, and T-cell recruitment and cytotoxicity abilities were conducted in the miR-762 manipulation T-cell and OSCC-T-cell co-culture system. A luciferase reporter and CXCR3 protein expression were also carried out to validate the direct interaction between CXCR3 and microRNA (miR)-762. This horizontal transmission of miR-762 directly suppresses CXCR3 expression in T cells, inhibiting CXCR3-induced T-cell migration and downstream T-cell cytotoxic activity by disrupting AKT activation. Additionally, miR-762 transmission suppressed T-cell activation marker expression, T-cell proliferation, IL-12 secretion, and T-cell cytotoxicity. In conclusion, our findings reveal a novel miR-762/CXCR3 axis that regulates the immunosuppressive microenvironment in OSCC and may be a potential RNA-targeted therapeutic approach to restore the anti-tumor immune response in OSCC treatment.

Suppression of METTL3 expression attenuated matrix stiffness-induced vaginal fibroblast-to-myofibroblast differentiation and abnormal modulation of the extracellular matrix in pelvic organ prolapse.

Fibrosis of the connective tissue in the vaginal wall predominates in pelvic organ prolapse (POP), which is characterized by excessive fibroblast-to-myofibroblast differentiation and abnormal deposition of the extracellular matrix (ECM). Our study aimed to investigate the effect of ECM stiffness on vaginal fibroblasts and to explore the role of methyltransferase 3 (METTL3) in the development of POP. Polyacrylamide hydrogels were applied to create an ECM microenvironment with variable stiffness to evaluate the effects of ECM stiffness on the proliferation, differentiation, and expression of ECM components in vaginal fibroblasts. METTL3small interfering RNA and an overexpression vector were transfected into vaginal fibroblasts to evaluate the effects of METTL3silencing and overexpression on matrix stiffness-induced vaginal fibroblast-to-myofibroblast differentiation and abnormal modulation of the ECM. Both procedures were detected by 5-ethynyl-2'-deoxyuridine (EdU) staining, Western blotting (WB), quantitative real-time polymerase chain reaction (RT-qPCR), and immunofluorescence (IF). Vaginal fibroblasts from POP patients exhibited increased proliferation ability, increased expression of α-smooth muscle actin (α-SMA), decreased expression of collagen I/III, and significantly decreased expression of tissue inhibitors of matrix metalloproteinases (TIMPs) in the stiff matrix (P<0.05). Compared with those from non-POP patients, vaginal wall tissues from POP patients demonstrated a significant increase in METTL3 content (P<0.05). However, silencing METTL3 expression in vaginal fibroblasts with high ECM stiffness resulted in decreased proliferation ability, decreased α-SMA expression, an increased ratio of collagen I/III, and increased TIMP1 and TIMP2 expression. Conversely, METTL3 overexpression significantly promoted the process of increased proliferation ability, increased α-SMA expression, decreased ratio of collagen I/III and decreased TIMP1 and TIMP2 expression in the soft matrix (P<0.05). Elevated ECM stiffness can promote excessive proliferation, differentiation, and abnormal ECM modulation, and the expression of METTL3 plays an important role in alleviating or aggravating matrix stiffness-induced vaginal fibroblast-to-myofibroblast differentiation and abnormal ECM modulation.

Phospholipase C Beta 2 as a Key Regulator of Tumor Progression and Epithelial-Mesenchymal Transition via PI3K/AKT Signaling in Renal Cell Carcinoma

Background: Renal cell carcinoma (RCC) represents the most common form of invasive kidney cancer in adults. Among the components critical to cellular regulation is Phospholipase C Beta 2 (PLCB2), a member of the phospholipase C enzyme family. This enzyme plays a vital role in managing key cellular functions such as growth, differentiation, migration, and survival. Despite its significant importance, the specific expression patterns and molecular mechanisms of PLCB2 in the progression of RCC are not well understood. Methods: This investigation employed a combination of bioinformatics analyses, scRNA-seq, functional assays, transcriptome sequencing, real-time quantitative PCR (RT-PCR), immunofluorescence, rescue experiments, and Western blotting to explore the regulatory function of PLCB2 in driving the epithelial-mesenchymal transition (EMT) in RCC through the PI3K/AKT signaling pathway. Results: PLCB2 expression is significantly elevated in RCC samples, and this increase is inversely correlated with patient prognosis. The knockdown of PLCB2 in RCC cell lines leads to a marked reduction in cell proliferation, invasion, migration, and EMT. Transcriptome sequencing further revealed that PLCB2 is significantly associated with the PI3K/AKT pathway. Notably, the PI3K activator 740Y-P was able to reverse the reductions in migration, invasion, and EMT caused by the PLCB2 knockdown. Conclusions: Our findings underscore the pivotal role of PLCB2 in regulating RCC invasion and metastasis by modulating the EMT via the PI3K/AKT signaling pathway. This highlights PLCB2 not only as a key prognostic biomarker, but also as a promising therapeutic target in the treatment of advanced-stage RCC, offering new avenues for more effective interventions.

Comparative Transcriptomic Analysis Provides Insight into Spatiotemporal Expression Patterns of Pivotal Genes During Critical Growth Stages in Min Pig Breed

The growth and development of animals are dynamic processes characterized by fluctuations. Min pigs, a local breed renowned for their superior meat quality, present an intriguing yet poorly understood relationship between this quality and their growth and development patterns. To elucidate this relationship, we employed a multi-faceted approach that included comparative transcriptomics, quantitative real-time PCR (qRT-PCR), selection pressure analysis of key genes, and three-dimensional protein structure simulations. Our findings revealed that 150 days (150 d) of age represented a pivotal turning point in the growth and development of Min pigs. Thirteen key genes exhibiting significant differential expression between early and late growth stages were identified. Notably, the CDK2 gene demonstrated specific high expression in the hind limb muscles and adipose tissues during the later growth stages. Comparative analysis with the African warthog revealed that while the CDK2 protein structure remained conserved, base mutations in upstream and downstream non-coding regions resulted in strong positive selection pressure on the CDK2 gene. These results suggest that CDK2 plays a crucial role in defining the spatiotemporal characteristics of meat development during the domestication of Min pigs. This study provides critical insights into the growth and development patterns of domestic pigs and offers a robust scientific foundation for improving meat quality traits through domestication.

The GA2ox Gene Family in Solanum pennellii: Genome-Wide Identification and Expression Analysis Under Salinity Stresses

-GA 2-oxidases (GA2oxs), a class of enzymes, inhibit the biosynthesis of bioactive gibberellins (GAs) in plants. The GA2 oxidase gene is crucial for regulating the passivation process of active GA and is widely involved in hormone signaling and abiotic stress processes. -To examine the potential effects of the GA2 oxidase gene on Solanum pennellii, one of the important stress-tolerance wild species of tomato, a systematic analysis was performed to study the structure, phylogenetic tree, genomic locus, and upstream cis-regulatory elements of SpGA2ox genes. The expression patterns of the SpGA2ox family in various tissues were analyzed on the basis of published RNA-seq data, and the changes in SpGA2ox expression in the leaves of seedlings were detected under salinity stress and GA treatment by real-time fluorescence quantitative PCR. -We identified nine SpGA2ox genes in S. pennellii. They were located on chromosomes 1, 2, 4, 7, 8, and 10. The SpGA2ox family was clearly divided into three groups through phylogenetic relationship analysis, namely, five in C19-GA2ox class I, one in C19-GA2ox class II, and three in C20-GA2ox class. And cis-element analysis provided the basis for understanding the function of growth, development, hormones, and abiotic stress of GA2ox genes in S. pennellii. The expression patterns of the SpGA2ox family were different in three classes, and SpGA2ox1 exhibited higher expression levels in the stem compared to other tissues. The expression levels of all SpGA2ox genes increased significantly under salt stress and decreased by treatment with GA3. With the largest changes in relative expression levels, SpGA2ox3 and SpGA2ox8 might exert key effects on the regulation of GA synthesis and the response to salt stress. -The present study may be instrumental for further investigation into the impact of SpGA2oxs on responses to abiotic stress and provide potential targets for the genetic improvement of S. pennellii.

SLAMF8 Disrupts Epithelial Barrier in Chronic Rhinosinusitis with Nasal Polyps via M1 Macrophage Polarization.

Recent studies show that M1 macrophages accumulate predominantly in non-eosinophilic chronic rhinosinusitis with nasal polyps (neCRSwNP). However, the precise mechanisms regulating M1 macrophages and their impact on the epithelial barrier remain unclear. We aim to investigate the expression and regulatory role of SLAMF8, a molecule exclusively expressed in myeloid cells, in M1 macrophage polarization and its potential contribution to neCRSwNP development. We evaluated SLAMF8 expression and its correlation with clinical variables using real-time quantitative polymerase chain reaction and Western blot in sinonasal mucosa samples from CRSwNP and control subjects. Immunofluorescence staining confirmed the co-expression of SLAMF8 with macrophages. After SLAMF8 knockdown, we explored the influence on macrophage M1 polarization and the effect on epithelial-mesenchymal transition (EMT) process and tight junction integrity in epithelial cells through an indirect co-culture system of M1 macrophages with human nasal epithelial cells. SLAMF8 was highly expressed on M1 macrophages in polyp tissues, notably in neCRSwNP, and correlated with disease severity indices only in neCRSwNP. SLAMF8 knockdown in THP-1 cells reduced M1 macrophage markers (CD86, iNOS, and NLRP3) and decreased secretion of inflammatory cytokines (IL-1β, IL-6, and TNF-α). Co-culture with M1 macrophage supernatant after SLAMF8 knockdown enhanced epithelial viability, reduced EMT and apoptosis, and upregulated tight junction markers Occludin and Claudin-4 in nasal epithelial cells. SLAMF8 elevation correlates with the EMT, epithelial tight junction and disease severity in neCRSwNP. The SLAMF8 upregulation promotes M1 macrophage polarization, by which facilitates EMT and impairs nasal epithelial barrier function. SLAMF8 may represent a novel therapeutic target for neCRSwNP.

Experimental Investigation into the Design, Optimization, Toxicity, and Anti-Viral Efficacy of Proliposomes Loaded with Ivermectin Against Infectious Bronchitis Virus Using an Embryonated Chicken Egg Model

Background: Infectious bronchitis virus (IBV) causes a significant illness in birds, making it a leading source of financial loss in the poultry business. The objective of this study was to assess the effectiveness of proliposomes (PLs) containing ivermectin (IVM) against IBV using embryonated chicken eggs (ECEs). Methods: A three-factor, two-level (23) full factorial design was employed; carrier/lipid phase ratio (A), stearyl glycyrrhetinate amount (B), and phospholipid type (C) were studied as independent variables, while product yield (PY), entrapment efficiency (EE), particle size (PS), polydispersity index (PDI), zeta potential (ZP), and cumulative percentage of drug released after 6 h (Q6h) were characterized. The selected formulations (PL2 and PL6) were subjected to further characterizations, including IVM toxicity and anti-viral activity. Results: The PY% ranged from 88.6 ± 2.19% to 98.8 ± 0.45%, EE% was from 71.8 ± 2.01% to 96.1 ± 0.51%, PS was from 330.1 ± 55.65 nm to 1801.6 ± 45.61 nm, PDI was from 0.205 ± 0.06 to 0.603 ± 0.03, ZP was from −18.2 ± 0.60 mV to −50.1 ± 1.80 mV, and Q6h was from 80.95 ± 1.36% to 88.79 ± 2.03%. IVM-loaded PLs had lower toxicity in ECEs than pure IVM; the mortality rate was substantially reduced in IBV-infected ECEs injected with PL2 rather than pure IVM. As further evidence of IVM’s anti-viral action against IBV, quantitative real-time polymerase chain reaction (qRT-PCR) revealed that the PL2-treated group exhibited further reduction in IBV’s copies in comparison with the pure IVM-treated group. Conclusions: PLs loaded with IVM are an innovative and potentially effective way to inhibit IBV.

Anti-angiogenic therapy as potential treatment for adenomyosis

Adenomyosis is characterized by abnormal uterine bleeding, dysmenorrhea and subfertility. Increased expression of angiogenesis markers in adenomyosis presents a treatment opportunity and was studied in an adenomyosis mouse model. Mice were administered tamoxifen (1 mg/kg) on neonatal days 2–5. At six weeks of age, mice received oral treatment with axitinib 3 mg/kg (‘dose I/AX3’, n = 34), axitinib 25 mg/kg (‘dose II/AX25’ n = 34), or with vehicle-only (‘placebo’, n = 34). The prevalence and severity of adenomyosis were assessed. An adenomyosis severity index was calculated by multiplying mean grade/mouse by the percentage affected surface area. Angiogenesis-related gene expression was evaluated using real-time quantitative PCR. 101 mice completed adenomyosis induction and could be analyzed. The prevalence of adenomyosis was 30/33 (90.0%) in dose I, 29/34 (85.3%) in dose II, and 30/34 (88.2%) in placebo treated mice (p = 0.78). High grade (2/3) adenomyosis was significantly less prevalent in mice treated with axitinib dose II (n = 19, 55.9%) than in the placebo group (n = 27, 79.4%, p < 0.05). The adenomyosis severity index was reduced by 48% in the axitinib-treated groups (dose I, p < 0.05). The expression of angiogenic growth factors was reduced in the dose I and II axitinib-treated groups compared to the placebo-treated group. Following these promising first results, further research should focus on commonality among different angiostatic drugs, potential side effects, as well as the method and timing of application.

Clinical significance analysis of microRNA-199a-3p in gingival crevicular fluid for patients with chronic periodontitis.

The aim was to investigate the clinical performance of microRNA-199a-3p (miR-199a-3p) in patients with chronic periodontitis. 91 patients with chronic periodontitis and 78 healthy individuals were enrolled for the research subjects. MiR-199a-3p expression was detected using real-time quantitative PCR (RT-qPCR) assay. Pearson correlation analysis was used for the relevance of miR-199a-3p with inflammatory mediators. Receiver operating characteristic (ROC) and logistic regression were conducted for the evaluation of the diagnostic performance and risk factors of chronic periodontitis. Bioinformatics analysis was utilized for miR-199a-3p-related genes. MiR-199a-3p was distinctly decreased in gingival crevicular fluid from patients with chronic periodontitis. The area under the curve (AUC) was 0.978 to discriminate chronic periodontitis patients from healthy individuals. The negative correlation was observed between miR-199a-3p and inflammatory factors. Logistic regression showed that miR-199a-3p was an independently protective factor for the occurrence of chronic periodontitis. Bioinformatics analysis revealed that the predictive regulated genes of miR-199a-3p mainly concentrated in inflammatory-associated signaling pathways. MiR-199a-3p was attenuated in patients with chronic periodontitis and an underlying diagnostic biomarker for the disease.

Transcriptomic Profiling Reveals 17β-Estradiol Treatment Represses Ubiquitin-Proteasomal Mediators in Skeletal Muscle of Ovariectomized Mice.

With a decline of 17β-estradiol (E2) at menopause, E2 has been implicated in the accompanied loss of skeletal muscle mass and strength. We aimed at characterizing transcriptomic responses of skeletal muscle to E2 in female mice, testing the hypothesis that genes and pathways related to contraction and maintenance of mass are differentially expressed in ovariectomized mice with and without E2 treatment. Soleus and tibialis anterior (TA) muscles from C57BL/6 ovariectomized mice treated with placebo (OVX) or E2 (OVX + E2) for 60 days, or from skeletal muscle-specific ERα knockout (skmERαKO) mice and wild-type littermates (skmERαWT), were used for genome-wide expression profiling, quantitative real-time PCR and immunoblotting. Computational detection of estrogen response elements (EREs) was performed with EREFINDER. We found 155 significantly regulated probe sets in response to E2 (p ≤ 0.001). Pathway analyses identified proteasome and ubiquitin-mediated proteolysis as two downregulated pathways in the E2 group. We confirmed downregulation (p ≤ 0.05) in levels of Fbxw7, Psmb6, Ube2h and Ubxn1, as well as pro-apoptotic Bnip3 and inflammatory factor Nfkbia. Computational analysis identified ERE in the promoter regions of Psmb6, Ube2h, Bnip3 and Nfkbia. The overall content of ubiquitinated proteins was modestly but significantly lower in TA muscles from OVX + E2 vs. OVX mice (p = 0.039). There were no differences between skmERαKO and skmERαWT mice or between skmERαKO/OVX and skmERαKO/OVX + E2 mice for any genes assessed, indicating that ERα is required for E2 regulation of those genes. These results suggest that a mechanism whereby E2 protects against losses of skeletal muscle mass and strength is regulation of ubiquitin-proteasomal mediators.

Mechanistic insights of methylcinnamate in improving oxidative stress and inflammation in acetaminophen-induced hepatotoxic mice by upregulating Nrf2 pathway.

Methylcinnamate (MC), a safe flavoring agent naturally found in Occimum basilicum L. is reported to have an anti-inflammatory responses in various disease models. Acetaminophen (APAP) toxicity is a significant contributor to acute liver injury, which leads to oxidative stress and inflammation. The transcriptional factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulated the cellular defense mechanisms aid to antioxidant response facilitation and reduction in inflammation against various disorders. This study evaluated the protective effects of MC in APAP-induced hepatotoxicity in mice and its anti-oxidant, anti-inflammatory, and Nrf2 mechanisms were studied. In-vitro 2,2-diphenyl-1-picrylhydrazyl assay showed the antioxidant capacity of MC. Mice were pretreated with MC (25, 50, 75, and 100mg/kg) orally for 7 days. After a fasting period of 16h, hepatotoxicity was induced by injecting APAP 300mg/kg intraperitoneal on day 7. Liver profile, oxidative test, and histopathological changes were studied. Gene expression of interlukin-1β (IL-1β), interlukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), cytochrome P450 2E1 (CYP2E1), Nrf2, and NAD(P)H dehydrogenase (quinone) 1 (NQO-1) were estimated by real time quantitative polymerase chain reaction (RT-qPCR). IL-1β, IL-6, and TNF-α concentrations were also analyzed by enzyme-linked immunosorbent assay (ELISA). The MC treatment showed a notable reduction in alanine transaminase, aspartate aminotransferase and alkaline phosphatase activities, and total bilirubin level of serum. Moreover, MC significantly attenuated oxidative stress by rising the antioxidant enzymes catalase, glutathione, and superoxide dismutase and reducing the malondialdehyde and nitric oxide levels in the liver. Furthermore, MC successfully mitigated the levels of IL-1β, IL-6, and TNF-α, which were estimated through RT-qPCR and ELISA. The RT-qPCR revealed a CYP2E1 enzyme inhibition and significant upregulation of hepatic Nrf2 and NQO-1 levels after MC therapy. Histopathological analysis showed improvement in liver injury within the MC treatment groups. It was concluded from this study that pretreatment of MC had successfully protected the liver through anti-inflammatory, anti-oxidant activity upon subsequent activation of Nrf2.

Upregulation of phosphatase and tensin homolog deleted on chromosome ten inhibits lung cancer cell proliferation by suppressing the oncogene polo-like kinase 1 and inducing autophagy

Objective: Lung cancer is one of the main causes of cancer-related mortality globally, and it poses considerable therapeutic challenges. Polo-like kinase 1 (PLK1) exhibits upregulation in lung cancer, and PLK1 silencing promotes autophagy in lung cancer cells, which inhibits tumor progression. The phosphatase and tensin homolog deleted on chromosome ten (PTEN) acts as a tumor suppressor gene. This study aimed to investigate whether PTEN regulates autophagy and inhibits lung cancer-cell proliferation by suppressing PLK1. Material and Methods: In this study, we evaluated cell proliferation by silencing or overexpressing PLK1 and PTEN in A549 cells through 5-ethynyl-2'-deoxyuridine labeling and cloning experiments. The autophagy levels were detected through transmission electron microscopy, real-time quantitative polymerase chain reaction, and Western blot. Finally, the results of in vitro experiment were further verified using an in vivo xenograft tumor animal model. Results: The upregulation of PTEN suppressed PLK1 expression in lung cancer cells and reduced their proliferation rate. In addition, the overexpression of PTEN has been associated with the growth of lung cancer tumors. In parallel, the levels of autophagy of lung cancer cells rose in response to PTEN upregulation in vivo and in vitro. Conclusion: This study revealed that PTEN promotes the autophagy of lung cancer cells and inhibits cell proliferation and tumor growth by suppressing PLK1 expression. This finding provides a new strategy for lung cancer treatment by utilizing the autophagy-regulating effect of PTEN to inhibit lung cancer growth by targeting PLK1.

Phenotypic, physiological and molecular changes of some wheat varieties under drought stress

Water stress poses a significant challenge to wheat production, adversely affecting both field productivity and grain quality in the face of climate change and diminishing water resources. It reduces vegetative growth and disrupts physiological processes, which negatively impact yield components like grain size and protein content. Consequently, selecting drought-tolerant varieties is critical for enhancing resilience in arid regions. This study examined ten wheat varieties belonging to the genus Triticum aestivum L. (Abba 99, Adna 99, Baraka, Bohooth 10, Bohooth 22, Jihan 99, Bora, Dijla, Sham 6 and Wafia) under three water stress levels: 0 MPa (S0), -1.48 MPa (S1), and -2.95 MPa (S2) using polyethylene glycol (PEG6000). Phenotypic traits measured included plant height, leaf area, stem diameter, wet and dry weight, along with chlorophyll content. Real-time quantitative Polymerase Chain Reaction (RT-qPCR) was used to assess the expression of SOD and BADH-1 genes. Results indicated that Adana 99 exhibited significant drought resistance, recording the highest measurements in plant height (35 cm), leaf area (15.50 cm²), stem diameter (1.80 mm), wet weight (0.80 g), dry weight (0.55 g) and total chlorophyll content (45.85 and 48.14) at 15 and 30 days, respectively, under S2. The SOD gene expression peaked at 8.57 in S2, an eightfold increase from S0. Similarly, BADH-1 gene expression was recorded at 8 in S2, also an eightfold increase. In contrast, Baraka and Wafia showed the lowest expressions for the SOD (0.001) and BADH-1 (0.002) genes under S2, negatively affecting their phenotypic and physiological traits. These findings underscore the importance of selecting drought-resistant varieties for sustainable productivity under harsh environmental conditions.

Effect of glyphosate on renal function: A study integrating epidemiological and experimental evidence.

Glyphosate, a widely used herbicide globally, has prompted concerns regarding its potential health impacts. This study aimed to explore the link between glyphosate exposure and renal function by combining NHANES, a zebrafish model, and metabolomics. A cross-sectional analysis of 2013-2014 NHANES data investigated the relationship between glyphosate exposure and renal function [albumin-to-creatinine ratio (ACR) and estimated glomerular filtration rate (eGFR)]. A subsequent zebrafish experiment was conducted to verify this association. Embryos (0.75 hpf-96 hpf) were exposed to different glyphosate concentrations dissolved in water (0, 30, 60, 90, 120 μg/mL). The underlying mechanism of the association between glyphosate and renal function was explored by the real-time quantitative polymerase chain reaction (RT-qPCR) and non-targeted metabolomics analysis [embryos (0.75 hpf-96 hpf) were exposed to 90 μg/mL glyphosate]. 1170 participants were enrolled in the NHANES study. The NHANES-based study found a positive association between glyphosate and ACR [0.07 (0.01, 0.13)]. Higher urinary glyphosate levels, particularly in the third quartile group, were negatively linked to eGFR [-3.72 (-5.98, -1.46)]. Further zebrafish experiments indicated that zebrafish exposed to 90 μg/mL glyphosate exhibited increased mortality rates, higher fluorescence intensity, up-regulated the havcr1 expression level, and cystic dilatation of the kidney. Non-targeted metabolomics analysis identified differential metabolites (e.g., 5-Hydroxyindole acetic acid) and pathways (e.g., ABC transporters) influenced by glyphosate. Glyphosate exposure is negatively associated with renal function in community adults. The damage to the kidneys caused by glyphosate may be mediated through the regulation of metabolic pathways, and the specific mechanisms require further experimental investigation.

Identification of Ferroptosis-related Genes for Diabetic Nephropathy by Bioinformatics and Experimental Validation.

The present study delves into the exploration of diagnostic biomarkers linked with ferroptosis in the context of diabetic nephropathy, unraveling their underlying molecular mechanisms. In this study, we retrieved datasets GSE96804 and GSE30529 as the training cohort, followed by screening for Differentially Expressed Genes (DEGs). By intersecting these DEGs with known ferroptosisrelated genes, we obtained the differentially expressed genes related to ferroptosis (DEFGs). Subsequently, Weighted Correlation Network Analysis (WGCNA) was carried out to identify key modules associated with Diabetic Nephropathy (DN), culminating in the identification of a significant gene. Enrichment analysis and Gene Set Enrichment Analysis (GSEA) were then carried out on the DEFGs and genes linked to the significant gene. To validate our findings, we employed cohorts GSE30528 and GSE43950, utilizing ROC curve analysis to assess diagnostic efficacy for DN, as measured by the area under the curve (AUC). Immune cell infiltration was analyzed and compared between groups using the CIBERSORT algorithm. Bayesian colocalization analysis was performed to examine the co-location of DEFGs and DN. Finally, to validate the hub genes identified, we conducted quantitative real-time polymerase chain reaction (qRT-PCR) experiments in vitro. FUZ, GLI1, GLI2, GLI3, and DVL2 were identified as the hub genes. Functional enrichment analysis demonstrated that ferroptosis and immune response play an important role in DN. ROC analysis showed that the identified genes had good diagnostic efficiency in DN. The results of the immune infiltration analysis showed that there may be crosstalk between ferroptosis and immune cells in DN. Bayesian co-localization analysis revealed the genetic correlation between the hub genes and DN. The outcomes of the qRT-PCR analyses corroborated the reliability of the identified hub genes as robust molecular markers for targeted therapy in DN. The interplay between immune inflammatory reactions and ferroptosis emerges as a crucial pathogenic mechanism, offering novel insights into the molecular therapy of DN. Furthermore, the identification of FUZ, GLI1, GLI2, GLI3, and DVL2 as potential targets holds promise for future therapeutic interventions aimed at treating DN.

MiR-125b-5p ameliorates ox-LDL-induced vascular endothelial cell dysfunction by negatively regulating TNFSF4/TLR4/NF-κB signaling

BackgroundOxidized low-density lipoprotein (ox-LDL)-induced endothelial cell dysfunction plays a crucial role in the progression of atherosclerosis (AS). Although miR-125b-5p is known to be involved in cardiovascular and cerebrovascular disorders, its function in ox-LDL-induced endothelial injury is still not well understood.MethodsAn in vitro AS cell model was established by exposing human umbilical vein endothelial cells (HUVECs) to 100 µg/mL ox-LDL for 24 h. A series of functional assays, including CCK-8 assay, flow cytometry, MDA and SOD kits, capillary-like network formation assay and ELISA assay were performed in vitro. TNFSF4/TLR4/NF-κB pathway-related protein expressions were measured by Western blot. Molecular mechanisms were elucidated through quantitative real-time PCR, western blot analysis, and luciferase reporter assays.ResultsOur investigation revealed that exposure to ox-LDL led to a downregulation in miR-125b-5p, while upregulating the expression of tumor necrosis factor (ligand) superfamily, member 4 (TNFSF4), TLR4, p-p65 and p-IkBa in HUVECs in a dose-dependent manner. We confirmed TNFSF4 as a direct target of miR-125b-5p. Ox-LDL exposure led to decreased cell viability and angiogenic capacity, along with increased apoptosis, inflammation, and oxidative stress in HUVECs. These effects were reversed by overexpressing miR-125b-5p or knocking down TNFSF4. Overexpression of TNFSF4 significantly reversed the effects brought about by miR-125b-5p in HUVECs exposed to ox-LDL. Moreover, miR-125b-5p inactivated the TLR4/NF-κB signaling pathway by negatively regulating TNFSF4.ConclusionsIn summary, our findings demonstrate that miR-125b-5p possessed an anti-inflammatory and anti-apoptosis against ox-LDL-induced HUVEC injury by regulating the TNFSF4/TLR4/NF-κB signaling, indicating that miR-125b-5p may have an important therapeutic function for AS.