Fluorescence Quantitative PCR Research Articles

Functional Study on the Key Gene LaLBD37 Related to the Lily Bulblets Formation.

Oriental hybrid lilies, known for their vibrant colors, diverse flower shapes, and long blooming seasons, require annual bulb propagation in horticultural production. This necessity can lead to higher production costs and limit their use in landscaping. The LA hybrid lily 'Aladdin' has shown strong self-reproduction capabilities in optimal cultivation environments, producing numerous high-quality underground stem bulblets. This makes it a valuable model for studying bulblet formation in lilies under natural conditions. Through transcriptome data analysis of different developmental stages of 'Aladdin' bulblets, the LaLBD37 gene, linked to bulblet formation, was identified. Bioinformatics analysis, subcellular localization studies, and transcriptional activation activity tests were conducted to understand the characteristics of LaLBD37. By introducing the LaLBD37 gene into 'Sorbonne' aseptic seedlings via Agrobacterium-mediated transformation, resistant plants were obtained. Positive plants were identified through various methods such as GUS activity detection, PCR, and fluorescence quantitative PCR. Phenotypic changes in positive plants were observed, and various physiological indicators were measured to confirm the role of LaLBD37 in bulblet formation, including soluble sugar content, starch content, sucrose synthase activity, and endogenous hormone levels. The findings suggest that the LaLBD37 gene plays a significant role in promoting the development of lily bulblets, offering insights for enhancing the reproductive capacity of Oriental hybrid lilies and exploring the molecular mechanisms involved in lily bulb regeneration.

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.

Effect of Sakuranetin on Microglia-Mediated Neuroinflammation After Spinal Cord Injury

Objective To investigate the effects of sakuranetin (SK) on motor functions in the mouse model of spinal cord injury (SCI) and decipher the mechanism.Methods Fifty-four C57BL/6J mice were randomized into sham,SCI,and SK groups.The mice in the sham group underwent only laminectomy at T9,while those in the SCI and SK groups were subjected to spinal cord contusion injury at T9.Behavioral tests were conducted at different time points after surgery to evaluate the motor functions of mice in each group.The pathological changes in the tissue were observed to assess the extent of SCI in each group.The role and mechanism of SK in SCI were predicted by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses.Reverse transcription real-time fluorescence quantitative PCR,ELISA,and immunofluorescence were employed to evaluate the inflammation and activation of microglia in SCI mice.BV2 cells in vitro were classified into control (Con),lipopolysaccharide (LPS),and LPS+SK groups.The effects of SK intervention on the release of inflammatory cytokines and the activation of BV2 cells were evaluated.Furthermore,the phosphatidylinositol-3-kinase(PI3K)/protein kinase B (AKT) signaling pathway activator insulin-like growth factor-1 (IGF-1) was used to treat the SK-induced BV2 cells in vitro (SK+IGF-1 group),and SK was used to treat the IGF-1-induced BV2 cells in vitro (IGF-1+SK group).Western blotting was conducted for molecular mechanism validation.Results Behavioral tests and histological staining results showed that compared with the SCI group,the SK group exhibited improved motor abilities and reduced area of damage in the spinal cord tissue (all P<0.001).The GO enrichment analysis predicted that SK may be involved in the inflammation following SCI.The KEGG enrichment analysis predicted that SK regulated the PI3K/Akt pathway to exert the neuroprotective effect.The results from in vitro and in vivo experiments showed that SK lowered the levels of tumor necrosis factor-α,interleukin-6,and interleukin-1β and inhibited the activation of microglia (all P<0.05).The results of Western blotting showed that SK down-regulated the phosphorylation levels of PI3K and Akt (all P<0.001) and inhibited the IGF-1-induced elevation of PI3K and Akt phosphorylation levels (all P<0.001).Conversely,IGF-1 had the opposite effects (P=0.001,P<0.001).The results of reverse transcription real-time fluorescence quantitative PCR,ELISA,and immunofluorescence showed that the SK+IGF-1 group had higher levels of inflammatory cytokines and more activated microglia than the SK group(all P<0.05).Conclusion SK may suppress the activation of the PI3K/Akt pathway to inhibit the inflammation mediated by SCI-induced activation of microglia,ameliorate the pathological damage of the spinal cord tissue,and promote the recovery of motor functions in SCI mice.

The Effects of Mixed Robinia pseudoacacia and Quercus variabilis Plantation on Soil Bacterial Community Structure and Nitrogen-Cycling Gene Abundance in the Southern Taihang Mountain Foothills.

Mixed forests often increase their stability and species richness in comparison to pure stands. However, a comprehensive understanding of the effects of mixed forests on soil properties, bacterial community diversity, and soil nitrogen cycling remains elusive. This study investigated soil samples from pure Robinia pseudoacacia stands, pure Quercus variabilis stands, and mixed stands of both species in the southern foothills of the Taihang Mountains. Utilizing high-throughput sequencing and real-time fluorescence quantitative PCR, this study analyzed the bacterial community structure and the abundance of nitrogen-cycling functional genes within soils from different stands. The results demonstrated that Proteobacteria, Acidobacteria, and Actinobacteria were the dominant bacterial groups across all three forest soil types. The mixed-forest soil exhibited a higher relative abundance of Firmicutes and Bacteroidetes, while Nitrospirae and Crenarchaeota were most abundant in the pure R. pseudoacacia stand soils. Employing FAPROTAX for predictive bacterial function analysis in various soil layers, this study found that nitrogen-cycling processes such as nitrification and denitrification were most prominent in pure R. pseudoacacia soils. Whether in surface or deeper soil layers, the abundance of AOB amoA, nirS, and nirK genes was typically highest in pure R. pseudoacacia stand soils. In conclusion, the mixed forest of R. pseudoacacia and Q. variabilis can moderate the intensity of nitrification and denitrification processes, consequently reducing soil nitrogen loss.

Mechanisms of levan in ameliorating hyperuricemia: Insight into levan on serum metabolites, gut microbiota, and function in hyperuricemia rats

This study aims to investigate the effects of levan on the progression of hyperuricemia (HUA) rats and elucidate its underlying mechanisms. After levan intervention, both low and high-dose groups exhibited a significant decrease in serum uric acid (UA) levels, reaching 71.0 % and 77.5 %, respectively, compared to the model group. Furthermore, levan could alleviate renal pathological damage caused by glomerular cell vacuolation, inflammatory infiltration and collagen deposition. The results of enzyme activity assay and real-time fluorescence quantitative PCR showed that levan decreased UA production by inhibiting adenosine deaminase (ADA) activity and gene expression in liver; it upregulated ATP-binding cassette subfamily G member 2 protein (ABCG2) and organic anion transporter 1 (OAT1) transporter gene expression in the kidney, promoting UA excretion. Gut microbiome analysis indicated that levan regulated gut flora dysbiosis induced by HUA, resulting in up-regulated the abundance of beneficial bacteria (Muribaculaceae, Faecalibaculum, Bifidobacterium, and Lactobacillus) and decreased conditioned pathogenic bacteria (Escherichia_Shigella and Proteus). Non-targeted metabolomics showed changes in various serum metabolites associated with glycerophospholipid metabolism, lipid metabolism, and inflammation following oral administration of levan. Therefore, levan may be a promising functional dietary supplement for regulating the gut flora and remodeling of metabolic disorders in individuals with HUA.

Chemosensory protein 22 in Riptortus pedestris is involved in the recognition of three soybean volatiles

Riptortus pedestris (Hemiptera: Alydidae), a common agricultural pest, is the major causative agent of “soybean staygreen.” However, the interactions between chemosensory proteins (CSPs) in R. pedestris and host plant volatiles have yet to be comprehensively studied. In this study, we performed real-time fluorescence quantitative polymerase chain reaction (PCR) to analyze the antennal expression of RpedCSP22 and subsequently analyzed the interactions between 21 soybean volatiles, five aggregation pheromones, and RpedCSP22 protein in vitro using a protein expression system, molecular docking, site-directed mutagenesis, and fluorescence competitive binding experiments. The RpedCSP22 protein showed binding affinity to three soybean volatiles (benzaldehyde, 4-ethylbenzaldehyde, and 1-octene-3-ol), with optimal binding observed under neutral pH conditions, and lost binding ability after site-directed mutagenesis. In subsequent RNA interference (RNAi) studies, gene silencing was more than 90 %, and in silenced insects, electroantennographic responses were reduced by more than 75 % compared to non-silenced insects. Moreover, Y-tube olfactory behavioral assessments revealed that the attraction of R. pedestris to the three soybean volatiles was significantly attenuated. These findings suggest that RpedCSP22 plays an important role in the recognition of host plant volatiles by R. pedestris andprovides a theoretical basis for the development of novel inhibitors targeting pest behavior.

Correlation between TEX14 and ADAM17 expressions in colorectal cancer tissues of elderly patients and neoplasm staging, invasion, and metastasis

BACKGROUND Colorectal cancer (CRC) is one of the most frequently encountered malignant tumors in clinical settings. Proteins encoded by the testis-expressed gene 14 (TEX14) are imperative for spermatogenesis, necessitating intercellular bridges between germ cells. Anomalous expression of TEX14 has also been associated with the proliferation and differentiation of certain tumor cells. Recombinant A disintegrin and metalloprotease 17 (ADAM17) is known as a membrane-bound protease that regulates cellular activities and signal transduction by hydrolyzing various substrate proteins on the cell membrane. We hypothesize that TEX14 and ADAM17 may serve as potential biomarkers influencing the staging, invasion, and metastasis of CRC. AIM To probe the correlation between TEX17 and ADAM17 profiles in the CRC tissues of elderly patients and their association with CRC staging, invasion, and metastasis. METHODS We gathered data from 86 elderly patients diagnosed pathologically with CRC between April 2020 and December 2023. For each patient, one sample of cancer tissue and one sample of adjacent normal tissue were harvested. Real-time fluorescence quantitative PCR measured the mRNA profiles of TEX14 and ADAM17. Immunohistochemistry ascertained the positivity rates of TEX14 and ADAM17 expressions. Clinical pathological features of neoplasm staging, invasion, and metastasis were collected, and the association between TEX14 and ADAM17 expressions and clinical pathology was evaluated. RESULTS The mRNA and expression profiles of TEX14 and ADAM17 were significantly elevated in CRC tissues. The positivity rates of TEX14 and ADAM17 proteins in CRC tissues were 70.93% and 77.91%, respectively. There were no significant differences in age, sex, pathological type, and tumor diameter between TEX14 and ADAM17-positive and -negative patients. Patients with higher tumor differentiation degree, deeper infiltration and TNM stages ranging from III to IV exhibited higher positivity rates of TEX14 and ADAM17. Patients with lymph node metastasis and distant metastasis showed higher positivity rates of TEX14 and ADAM17 than those without. Positive expressions of TEX14 and ADAM17 were highly correlated with tumor staging, invasion, and metastasis. CONCLUSION TEX14 and ADAM17 profiles were significantly elevated in the CRC tissues of elderly patients, and their high expressions were associated with tumor staging, invasion, and metastasis.

Moxibustion with seed-size moxa cones alleviates colonic injury in mice with ulcerative colitis by regulating TLR4/MyD88/NF-κB signaling pathway.

To observe the effect of moxibustion with seed-size moxa cones on the Toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear transcription factor-κB(NF-κB) signaling pathway in mice with ulcerative colitis(UC), so as to explore the therapeutic mechanism of moxibustion with seed-size moxa cones on colonic injury in UC. Forty male C57BL/6 mice were randomly divided into blank group, model group, moxibustion group, and western medicine group, with 10 mice in each group. The UC mouse model was established by 3% DSS solution by free drinking for 7 consecutive days. Mice in the moxibustion group were treated with seed-size moxa cones at "Zhongwan"(CV12), "Tianshu"(ST25) and "Shangjuxu"(ST37), 3 moxa cones per point, with each cone applied for approximately 30 s, while mice in the western medicine group were orally administered with 300 mg/kg mesalazine solution, which were both conducted once a day for 7 consecutive days. The general condition of mice was observed every 2 days, and the disease activity index (DAI) score was calculated. HE staining was used to observe the morphology of colonic tissue in mice. ELISA was used to detect the serum interleukin(IL)-1β, tumor necrosis factor(TNF)-α, IL-6, and IL-8 contents. Immunohistochemistry was used to detect the positive expression of TLR4 and MyD88 in colonic tissue of mice. Real-time fluorescence quantitative PCR was used to detect the expression levels of TLR4, MyD88, and NF-κB p65 mRNAs in colonic tissue. Compared with the blank group, varying degrees of soft or watery stools were observed, colon length and body weight were decreased(P<0.01) in mice of the model group, while DAI score, colon weight index, mucosal damage score, colonic pathological score, serum IL-1β, TNF-α, IL-6, and IL-8 contents, positive expressions of TLR4 and MyD88, and TLR4, MyD88, and NF-κB p65 mRNA expressions in colonic tissue were increased(P<0.01). Compared with the model group, improved fecal characteristics were observed, colon length and body weight were increased(P<0.01) in mice of the moxibustion group and western medicine group, while DAI scores, colon weight indexes, mucosal damage scores, colonic pathological score, serum contents of IL-1β, TNF-α, IL-6, and IL-8, positive expressions of TLR4 and MyD88, and TLR4, MyD88, and NF-κB p65 mRNA expressions in colonic tissue were decreased(P<0.01, P<0.05). There was no significant difference in the above indicators between the moxibustion group and the western medicine group. Moxibustion with seed-size moxa cones may alleviate colonic injury in UC mice by regulating the TLR4/MyD88/NF-κB signaling pathway and reducing the release of inflammatory factors.

Electroacupuncture promotes angiogenesis by regulating miR-142-5p and activating ADAMTS1/PI3K/AKT pathway in ischemic stroke rats.

To observe the effect of electroacupuncture on miR-142-5p and ADAMTS1/PI3K/AKT pathway in rats with ischemic stroke, so as to explore the regulatory mechanism of electroacupuncture on angiogenesis after ischemic stroke. This study was divided into two parts. The first part of the experiment：SD rats were randomly divided into sham operation group, model group and electroacupuncture group. There were 20 rats in each group. The middle cerebral artery occlusion (MCAO) rat model was prepared using a modified Longa's method. In the electroacupuncture group, "Shuigou" (GV26) was selected for electroacupuncture intervention (4 Hz/20 Hz) for 30 min each time. The rats in the electroacupuncture group were given electroacupuncture immediately after successful modeling, once a day for 4 times. Hunter score and TTC staining were used to observe the neurological deficits and infarct volumes respectively；HE staining was used to observe the cortical pathological changes；immunohistochemistry was used to determine the changes of cerebral microvascular density. Real-time quantitative PCR and Western blot were used to observe the miR-142-5p expression, mRNA and protein expression levels of ADAMTS1, VEGF, PI3K, AKT, eNOS in ischemic cortex. The second part of the experiment：The rats were randomly divided into electroacupuncture+control group and electroacupuncture+miR-142-5p Antagomir group with 8 rats in each group. MCAO model was established after injection. Electroacupuncture+control group was given 0.9% sodium chloride solution injected into the right ventricle.The rats in the electroacupuncture+miR-142-5p Antagomir group were injected with miR-142-5p inhibitor into the right ventricle 30 min before modeling. Rats in electroacupuncture+control group and electroacupuncture+miR-142-5p Antagomir group were all given the same electroacupuncture treatment. Real-time fluorescence quantitative PCR was used to observe the effect of miR-142-5p Antagomir on the expression of miR-142-5p and ADAMTS1 mRNA. The effect of miR-142-5p Antagomir on ADAMTS1 protein was observed by Western blot. In the first part of the experiment, compared with the sham operation group, the Hunter score in the model group was significantly increased (P<0.01)；the volume of cerebral infarction in the model group was significantly increased (P<0.01)；the degree of brain edema and neuronal necrosis and the density of cerebral microvessels was increased；the cerebral microvascular density was significantly increased (P<0.01)；the expression levels of miR-142-5p and the mRNA expression levels of VEGF, AKT and eNOS were significantly decreased (P<0.01, P<0.05), and the protein expression levels of VEGF, p-AKT and eNOS were significantly down-regulated (P<0.01), while the mRNA expression levels of ADAMTS1 and PI3K, and the protein expression levels of ADAMTS1 and p-PI3K were all up-regulated (P<0.01, P<0.05) in the model group. Compared with the model group, after intervention, the Hunter score in the electroacupuncture group was decreased (P<0.01), the volume of cerebral infarction was significantly decreased (P<0.01)；the degree of brain edema and neuronal necrosis were alleviated；the cerebral microvascular density was significantly increased (P<0.01)；the expression of miR-142-5p and the mRNA expression of VEGF, PI3K, AKT and eNOS were increased (P<0.01), the protein expressions of VEGF, p-PI3K, p-AKT and eNOS were increased (P<0.01, P<0.05), while the mRNA and protein expression of ADAMTS1 were decreased (P<0.05, P<0.01). After injection of miR-142-5p inhibitor, compared with electroacupuncture+control group, the expression of miR-142-5p in electroacupuncture+miR-142-5p Antagomir group was decreased(P<0.05), while the mRNA and protein expression of ADAMTS1 were increased (P<0.01, P<0.05). Electroacupuncture at GV26 can improve the neurological damage of ischemic stroke rats, reduce the volume of cerebral infarction and promote angiogenesis. The mechanism may be associated with the function of electroacupuncture in promoting the expression of miR-142-5p, so as to inhibit the expression of its target gene ADAMTS1, mediate the up-regulation of VEGF expression, activate PI3K/AKT pathway, promote the release of eNOS, and participate in promoting angiogenesis in ischemic stroke rats.

Calcitriol alleviates noise-induced hearing loss by regulating the ATF3/DUSP1 signalling pathway

BackgroundCalcitriol (Cal) is the most active metabolite of vitamin D and has antioxidant and anti-inflammatory properties. The aim of this study was to investigate the role of Cal in noise-induced hearing loss (NIHL) to further elucidate the mechanism of noise-induced oxidative stress in the mouse cochlea. MethodsC57BL/6 J mice were given six intraperitoneal injections of Cal (500 ng/kg/d). After 14 days of noise exposure, auditory brainstem response (ABR) thresholds, and the cochlear outer hair cell loss rate were analysed to evaluate auditory function. Real-time fluorescence quantitative PCR, immunofluorescence and western blotting were performed in vitro after the treatment of cochlear explants with 100 µM tert-butyl hydroperoxide (TBHP) for 2.5 h and HEI-OC1 cells with 250 µM TBHP for 1.5 h. ResultsIn vivo experiments confirmed that Cal pretreatment mitigated NIHL and outer hair cell death. The in vitro results demonstrated that Cal significantly reduced TBHP-induced cochlear auditory nerve fibre degradation and spiral ganglion neuron damage. Moreover, treatment with Cal inhibited the expression of oxidative stress-related factors (3-NT and 4-HNE) and DNA damage-related factors (γ-H2A.X) and attenuated TBHP-induced apoptosis in cochlear explants and HEI-OC1 cells. A total of 1479 upregulated genes and 1443 downregulated genes were screened in cochlear tissue 1 h after noise exposure. The level of transcription factor 3 (ATF3) was significantly elevated in HEI-OC1 cells after TBHP stimulation. Gene Transcription Regulation Database （GTRD）and Cistrome database analyses revealed that the downstream target gene of ATF3 is dual specificity phosphatase 1 (DUSP1). Cistrome DB Toolkit database results showed that the transcription factor of DUSP1 was ATF3. In addition, the ChIP-PCR results indicated that ATF3 might be a direct transcription factor of DUSP1. ConclusionThe results of our study suggest that Cal attenuates NIHL and inhibits noise-induced apoptosis by regulating the ATF3/DUSP1 signalling pathway.

A nomogram including admission serum glycated albumin/albumin ratio to predict mortality in patients with severe fever with thrombocytopenia syndrome

BackgroundSevere fever with thrombocytopenia syndrome (SFTS) is a novel tick-borne infectious disease with a high fatality rate. Although several nomograms based on demographic and laboratory data have been reported to predict the prognosis of SFTS in recent studies, baseline serum glycated albumin (GA)/albumin (ALB) ratio included risk model has not been evaluated for the prediction of clinical outcome.MethodsA total of 214 SFTS patients with integral data admitted to our hospital from May, 2020 to November, 2022 were included in this study. SFTS infection was confirmed by real time fluorescent quantitative PCR (qRT-PCR). The demographic characteristics, clinical and laboratory data were collected with in 24 h of admission and 1 to 2 days before discharge and were analysed retrospectively.ResultsFiffty-seven patients (26.6%) died. Multivariate logistic regression analysis showed that age, aspartate aminotransferase (AST), blood glucose (GLU), GA/ALB ratio, neutrophil counts (NEU) and lymphocyte percentage (LYM%) were the independent risk factors for mortality. A nomogram by these factors was created using RMS package in the R program. Area under the receiver operating characteristic (ROC) curve (AUC) of this nomogram was 0.88 (95% CI: 0.83, 0.93). This model showed the excellent net benefit, as revealed by the decision curve analysis. GA/ALB ratios were also independent risk factors for poor out clinical come in subgroups of patients with hyperglycemia on admission and with diabetes history. Nomograms were constructed by the independent risk factors in the subgroups. AUCs of the nomograms in the subgroups showed high predictive values for adverse prognosis.ConclusionsGA/ALB ratios were independent risk factors for mortality in all SFTS patients and subgroups of with hyperglycemia on admission and diabetes history. The nomograms including GA/ALB ratio had high predictive value for adverse clinical outcome.The nomograms provide a basis for clinical decision-making for the treatment of SFTS patients in different clinical settings.

Repair of Mechanical Cartilage Damage Using Exosomes Derived from Deer Antler Stem Cells.

Articular cartilage has limited self-repair capacity, and current clinical treatment options for cartilage defects are inadequate. However, deer antler cartilage possesses unique regenerative properties, with the ability to rapidly repair itself. This rapid self-repair process is closely linked to the paracrine factors released by deer antler stem cells. These findings present potential for the development of cell-free therapies for cartilage defects in clinical settings. The aim of this study was to investigate a novel method for repairing cartilage. A rat model with articular cartilage defects was established through surgery. Hydrogels loaded with exosomes (Exos) derived from antler stem cells (ASC-Exos) were implanted into the rat cartilage defects. The extent of cartilage damage repair was assessed using histological methods. The effects of ASC-Exos on chondrocytes and rat bone marrow mesenchymal stem cells (BMSCs) were evaluated using cell viability assays, proliferation assays, and scratch assays. Additionally, the maintenance of the chondrocyte phenotype by ASC-Exos was assessed using real-time fluorescence quantitative PCR (qPCR) and western blot analysis. The protein components contained of the Exos were identified using data-independent acquisition (DIA) mass spectrometry. ASC-Exos significantly promoted the repair of cartilage tissue damage. The level of cartilage repair in the experimental group (ASC-Exos) was higher than that in the positive control (human adipose-derived stem cells, hADSC-Exos) and negative control (dulbecco's modified eagle medium) groups (p < 0.05). In vitro experiments demonstrated that ASC-Exos significantly enhanced the proliferation abilities of chondrocytes and the proliferation abilities and the migration abilities of BMSCs (p < 0.05). ASC-Exos up-regulated the expression levels of Aggrecan, Collagen II (COLII), and Sox9 mRNA and proteins in chondrocytes. Analysis of ASC-Exos protein components revealed the presence of active components such as Serotransferrin (TF), S100A4, and Insulin-like growth factor-binding protein 1 (IGF1). ASC-Exos have a significant effect on cartilage damage repair, which may be attributed to their promotion of chondrocyte and BMSCs proliferation and migration, as well as the maintenance of chondrocyte phenotype. This effect may be mediated by the presence of TF, S100A4, and IGF1.

Multiplex methylation detection assays using a blocking FRET probe with machine learning-assisted quantitative melting curve method targeting early-stage breast cancer

Breast cancer (BC) is the second most prevalent form of cancer, and poses a significant threat to public health. DNA methylation is an ideal marker for the early detection of BC. Fluorescence quantitative polymerase chain reaction (PCR)-based DNA methylation detection is simpler and faster but is constrained by its multiplexing capability and specificity. To address this, we developed a multiplex quantitative methylation PCR assay for the simultaneous analysis of methylation status at multiple sites specific to BC (cg11754974, cg13828440, cg18637238, and cg16652347). The machine learning model was trained using 1200 cases of multipeak data to enhance the melting curve resolution. Performance testing demonstrated the method’s ability to selectively amplify methylated genes at a DNA concentration of 1 × 105 copies μL−1, with high replicability (coefficient of variation <5 %) and mutation detection capabilities as low as 10 %. When applied to 80 clinical BC samples, the assay effectively distinguished patients with early-stage BC from normal controls, achieving an area under the curve of 0.8938, sensitivity of 83.33 %, and specificity of 84.62 %. Our essay exhibits superior clinical performance when compared to the quantitative methylation-specific PCR assays for noninvasive detection of early-stage BC, which is poised to become a favorable clinical diagnostic method for early-stage BC owing to its simplicity, speed, and capacity to improve diagnostic accuracy.

Isoliquiritigenin Modulates the Effect of LINC01503 on Lung Squamous Carcinoma Cells

Isoliquiritigenin (ISL) is an important pharmacological constituent of Glycyrrhiza glabra, which possesses a range of physiological and pharmacological activities, as well as significant antitumor activity, and can be used as a potential drug for targeted cancer therapy. LINC01503 is an oncogene, which has been closely associated with the malignant biological processes of many cancers. The aim of this study was to investigate the effects of ISL on the proliferation, apoptosis, invasion and migration of lung squamous carcinoma cells by regulating LINC01503. Plasma was collected from lung squamous carcinoma patients and healthy individuals treated at Tangshan People's Hospital from January 2021 to December 2022. The expression of LINC01503 in lung squamous carcinoma plasma, tissues and cells was detected by real-time quantitative fluorescence polymerase chain reaction (qRT-PCR). Lung squamous carcinoma cells were treated with different concentrations of ISL for 24 h, and LINC01503 expression was detected by qRT-PCR. The cells were treated in groups: si-NC group, si-LINC01503 group, DMSO (0.1% dimethyl sulfone) group, ISL group, pc DNA3.1(+)-NC group, pc DNA3.1(+)-LINC01503 group, ISL+pc DNA3.1(+)-NC group and ISL+pc DNA3.1(+)- LINC01503 groups. CCK-8 assay, clone formation assay, flow cytometry, Transwell assay and scratch assay were used to explore the effect of LINC01503 on the functional phenotype of lung squamous carcinoma cells. Fluorescence in situ hybridization results showed that the average fluorescence intensity of LINC01503 in tissue microarrays of lung squamous carcinoma patients was higher than that in paracancerous tissues (P<0.05). The expression of LINC01503 in the plasma of patients with lung squamous carcinoma was higher than that in the plasma of healthy individuals (P<0.05). Knockdown of LINC01503 inhibited the proliferation, invasion and migration of lung squamous carcinoma cells and promoted apoptosis (P<0.05). ISL inhibited the proliferation, invasion, migration and promoted apoptosis of lung squamous carcinoma cells (P<0.05). Overexpression of LINC01503 followed by intervention with ISL reversed the promotional effect of overexpression of LINC01503 on the proliferation, invasion and migration of lung squamous carcinoma cells as well as the inhibitory effect on apoptosis (P<0.05). LINC01503 was highly expressed in lung squamous carcinoma, and LINC01503 could promote the proliferation, invasion and migration of lung squamous carcinoma cells and inhibit the apoptosis, ISL could inhibit the proliferation, invasion and migration of lung squamous carcinoma cells and promote apoptosis of lung squamous carcinoma cells by regulating the expression of LINC01503.

Construction of competitive endogenous RNA regulatory network and clinical verification for repeated implantation failure

Objective: To construct a repetitive implantation failure (RIF)-related competitive endogenous RNA (ceRNA) regulatory network and validate with clinical samples. Methods: RIF-related long non-coding RNA (lncRNA), microRNA (miRNA) and messenger RNA (mRNA) from the high-throughput gene expression omnibus (GEO) database Expression profile data set were obtained to construct a ceRNA regulatory network of lncRNA-miRNA-mRNA. At the same time, weighted gene co-expression network analysis (WGCNA) was used to explore hub genes in the network. This retrospective study collected RIF patients and controls (at least one pregnancy history after assisted conception) who underwent in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) for assisted pregnancy from 2020 to 2021 at the Reproductive Medicine Center of the First Affiliated Hospital of Zhengzhou University. In the endometrial tissue of patients with 1 pregnancy history, real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to verify the mRNA expression levels of RIF-related hub genes, and Western blotting and immunohistochemistry were used to verify protein expression levels of vascular cell adhesion molecule-1 (VCAM1). Results: A RIF-related ceRNA regulatory network consisting of 32 lncRNAs, 31 miRNAs and 88 mRNAs was constructed, and 7 RIF-related hub genes were identified using WGCNA. By intersecting 88 mRNAs and hub genes in the ceRNA network, two RIF-related key genes were obtained, i.e., VCAM1 and interleukin-2 receptor α (interleukin-2 receptor α, IL-2RA). In clinical verification, the ages of the control group and RIF group [M (Q1, Q3)] were 26.50 (25.00, 34.00) and 30.50 (25.75, 35.25) years old, respectively (P>0.05). Compared with the control group, the mRNA [0.30 (0.15, 0.42) vs 0.99 (0.69, 1.34), P=0.001] and protein expression [0.44 (0.16, 1.27) vs 2.39 (1.58, 2.58), P<0.001] of VCAM1 in the endometrium of the RIF group were both reduced. Conclusions: This study uses bioinformatics analysis methods to construct a RIF-related ceRNA regulatory network, and it is confirmed through clinical samples that the expression level of VCAM1 in the endometrial tissue of RIF patients is significantly reduced.

Phenotypic and genomic analysis of the hypervirulent methicillin-resistant Staphylococcus aureus ST630 clone in China.

Methicillin-resistant Staphylococcus aureus (MRSA) sequence type 630 (ST630) is a rarely reported lineage worldwide. This study aimed to trace the dissemination of the emerging MRSA ST630 clones in China and investigate their virulence potential. We collected 22 ST630-MRSA isolates from across China and performed whole-genome sequencing analysis and virulence characterization on these isolates. Epidemiological results showed that MRSA ST630 isolates were primarily isolated from pus/wound secretions, mainly originating from Jiangxi province, and carried diverse virulence and drug resistance genes. Staphylococcal cassette chromosome mec type V (SCCmec V) predominated (11/22, 50.0%) among the MRSA ST630 isolates. Interestingly, nearly half (45.5%) of the 22 ST630-MRSA isolates tested lacked intact SCCmec elements. Phylogenetic analysis demonstrated that ST630-MRSA could be divided into two distinct clades, with widespread dissemination mainly in Chinese regions. Five representative isolates were selected for phenotypic assays, including hemolysin activity, real-time fluorescence quantitative PCR, western blot analysis, hydrogen peroxide killing assay, blood killing assay, cell adhesion and invasion assay, and mouse skin abscess model. The results showed that, compared to the USA300-LAC strain, ST630 isolates exhibited particularly strong invasiveness and virulence in the aforementioned phenotypic assays. This study described the emergence of a highly virulent ST630-MRSA lineage and improved our insight into the molecular epidemiology of ST630 clones in China.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA) sequence type 630 (ST630) is an emerging clone with an increasing isolation rate in China. This study raises awareness of the hypervirulent MRSA ST630 clones in China and alerts people to their widespread dissemination. ST630-staphylococcal cassette chromosome mec V is a noteworthy clone in China, and we present the first comprehensive genetic and phenotypic analysis of this lineage. Our findings provide valuable insights for the prevention and control of infections caused by this emerging MRSA clone.

Pollution status and spatial distribution of antibiotic resistance genes in urban surface water and surrounding soil media

The pollution of urban rivers by antibiotic resistance genes (ARGs) is affected by the production of antibiotics and the activities of residents in the regions, posing a threat to the health of the population around the regions. A detailed investigation of the spread of ARGs in urban rivers and the surrounding soil media provides both data support and basic framework for tracking the spread and for the elimination of ARGs. In this study, real-time fluorescence quantitative PCR (qPCR) was used to investigate the contamination status and spatial distribution patterns of ARGs around the entire basin of urban river, namely the Minxin river, and the city ring water system in Shijiazhuang, China. The contamination of ARGs in midstream and downstream of the Minxin river was more severe. No obviously spatial distribution differences were found in the soil samples. Water bodies are more susceptible to human activities than soil. It was identified that sul1, sul2, and blaSHV were the dominant genes of the ARGs. Significant correlations exist between ARGs and int1 and tnpA-IS4. Mobile genetic elements (MGEs) play a key role in the horizontal gene transfer of ARGs, facilitating their spread and dispersal among different species. This work revealed the geospatial characteristics of the distribution of ARGs in urban river basins, concluding that the production and living activities could accelerate the spread and diffusion of ARGs in urban river environments, and that an attention should be given to the use control and discharge of antibiotics.

Establishment and performance analysis of a new multiplex detection method for influenza an and B virus antigen.

Influenza A and B virus detection is pivotal in epidemiological surveillance and disease management. Rapid and accurate diagnostic techniques are crucial for timely clinical intervention and outbreak prevention. Quantum dot-encoded microspheres have been widely used in immunodetection. The integration of quantum dot-encoded microspheres with flow cytometry is a well-established technique that enables rapid analysis. Thus, establishing a multiplex detection method for influenza A and B virus antigens based on flow cytometry quantum dot microspheres will help in disease diagnosis. To establish a codetection method of influenza A and B virus antigens based on flow cytometry quantum dot-encoded microsphere technology, which forms the foundation for the assays of multiple respiratory virus biomarkers. Different quantum dot-encoded microspheres were used to couple the monoclonal antibodies against influenza A and B. The known influenza A and B antigens were detected both separately and simultaneously on a flow cytometer, and the detection conditions were optimized to establish the influenza A and B antigen codetection method, which was utilized for their detection in clinical samples. The results were compared with the fluorescence quantitative polymerase chain reaction (PCR) method to validate the clinical performance of this method. The limits of detection of this method were 26.1 and 10.7 pg/mL for influenza A and B antigens, respectively, which both ranged from 15.6 to 250000 pg/mL. In the clinical sample evaluation, the proposed method well correlated with the fluorescent quantitative PCR method, with positive, negative, and overall compliance rates of 57.4%, 100%, and 71.6%, respectively. A multiplex assay for quantitative detection of influenza A and B virus antigens has been established, which is characterized by high sensitivity, good specificity, and a wide detection range and is promising for clinical applications.

LAMP combined with Pyrococcus furiosus Argonaute for the ultrasensitive and highly specific point-of-care test platform for Listeria monocytogenes detection

Listeria monocytogenes is an important foodborne pathogen that causes serious infectious diseases in humans and animals. Rapid, accurate, and sensitive detection in the field is critical for the timely implementation of control measures and ensuring food safety. The study established a method that combines loop-mediated isothermal amplification (LAMP) technology and Pyrococcus furiosus Argonaute (PfAgo) reporter system, which can detect L. monocytogenes hly gene levels as low as 1.8 × 101 copies. To overcome the demands on instrumentation and equipment, lateral flow strip (LFS) technology was combined for the first time in a LAMP-PfAgo-based system to detect L. monocytogenes. The method was highly sensitive, with a limit of detection of 1.8 × 102copies, and specificity results showed no cross-reactivity with other pathogens. The performance of the method was comparable to that of real-time fluorescence quantitative PCR (qPCR) in the detection of 18 food origin samples. Moreover, the LAMP-PfAgo reaction was equipped a low-cost thin metal bath as a simple amplification device, allowing it to detect L. monocytogenes samples in shorter time, highlighting its potential as a portable, rapid, accurate, and visual nucleic acid assay. The method can facilitate the early detection of pathogenic infections and promoting food safety detection technologies.

Simultaneous Extraction of Bone Marrow RNA and DNA from Patients with Hematologic Diseases Using a Combined Magnetic Bead Method within 1 Hour.

TRIzolTM is widely used for RNA and DNA extraction. However, this method is laborious and time-consuming. The objective of this study was to validate a time-effective and labor-saving protocol. The TRIzol method was used to separate the aqueous phase, protein, and phenol layer of bone marrow samples from 12 patients with hematological diseases. Subsequently, RNA and DNA were extracted from the aqueous layer containing RNA and phenol layer containing DNA, respectively, using magnetic bead extraction kits. The quantity and purity of extracted RNA and DNA were examined using a NanoDrop spectrophotometer. Quantitative fluorescence PCR amplification of the ABL1 gene was performed to verify the effectiveness of the extracted RNA and DNA for downstream experiments. RNA and DNA from another 16 bone marrow samples were extracted to compare the performance of the two methods. Co-extraction of RNA and DNA was completed within 1 h. The data showed that RNA and DNA yield ranged from 13.1 to 204.5 ng/µL and 33.1 to 228.8 ng/µL, respectively. The A260/A280 ratios of RNA and DNA samples ranged from 1.82 to 2.01 and 1.73 to 1.91, respectively. RNA and DNA extracted using this scheme exhibited ideal performance in quantitative fluorescence PCR. The present protocol showed better quality and effectiveness in extracting RNA and DNA compared to the TRIzol method. This protocol for RNA and DNA co-extraction is fast, labor-saving, and high throughput. It can be adopted for routine molecular biology analyses, particularly for non-reproducible specimens.