Real-time Fluorescent PCR Research Articles

3D printed β-TCP scaffolds loaded with SVVYGLR peptide for promoting revascularization and osteoinduction

It is crucial for the successful transplantation of large segmental bone defects to achieve rapid vascularization within bone scaffolds. However, there are certain limitations including uncontrolled angiogenesis and inadequate vascular function. Therefore, there is an urgent need to develop bone scaffolds with functional vascular networks. In our study, porousβ-tricalcium phosphate (β-TCP) scaffolds with varying pore sizes were prepared by 3D printing technology, loaded with osteopontin derived peptide Ser-Val-Val-Tyr-Gly-Leu-Arg (SVVYGLR) to induce osteoinduction and angiogenesis.In vitro, the proliferation and migration behaviors of human umbilical vein endothelial cell on scaffolds were assessed by Cell Counting Kit-8, confocal laser scanning microscopy and scanning electron microscopy. And the osteogenic ability of bone marrow mesenchymal stem cells was assessed using alkaline phosphatase staining and Alizarin Red S staining. The messenger ribonucleic acid (mRNA) expression levels of cell adhesion molecule (CD31), vascular endothelial growth factor and hypoxia inducible factor-1αin each group were detected by quantitative real-time fluorescence polymerase chain reaction (PCR) analysis.In vivo, cube scaffolds were subcutaneously implanted on the right hips of Sprague-Dawley (SD) rats for 6 weeks. Hematoxylin and Eosin staining, Masson's trichrome staining, and immunohistochemical analysis of osteocalcin and CD31 were performed on slices for every sample with three sections to explore the effect of SVVYGLR-loaded scaffolds on angiogenesis and osteogenic induction for bone reconstruction. The results indicate that 3D printedβ-TCP scaffolds loaded with the SVVYGLR peptide offer superior revascularization and osteoinduction to the scaffolds without the SVVYGLRin situ. Moreover, scaffolds with a pore size of 400 µm demonstrate higher effectiveness compared to those with a 150 µm pore size. The distinct hollow channel scaffolds and the specific SVVYGLR peptide substantially improve cell adhesion, spreading, and proliferation, as well as promote angiogenesis and bone formation. Furthermore, scaffolds with a pore size of 400 µm may exhibit greater efficacy compared to those with a pore size of 150 µm. The results of this study provide an idea for the development of practical applications for tissue-engineered bone scaffolds.

Influence of COVID-19 public health restrictions on community-acquired pneumonia pathogens in children in Henan, China: a multicenter retrospective study

ObjectiveTo investigate the distribution of pathogens and epidemiological changes in children hospitalized with community-acquired pneumonia (CAP) during and after the COVID-19 pandemic public health restrictive measures. aiming to provide a foundation for clinical diagnosis, treatment, and policy formulation.MethodsThis multicenter retrospective study spanned January 2019 to December 2023. The study included 78,256 children hospitalized for CAP in four hospitals in Henan, China, among which 27,580 cases (35.2%) were tested for pathogens using multiplex real-time fluorescent polymerase chain reaction (PCR). The pathogens detected include Streptococcus pneumoniae (SP), Staphylococcus aureus (SA), Haemophilus influenzae (HI), respiratory syncytial virus (RSV), influenza A virus (Flu A), influenza B virus (Flu B), and Mycoplasma pneumoniae (MP).ResultsPathogens were identified in 18,690 of the 27,580 children, resulting in a 67.8% positive detection rate. Of these cases, 15,105 (54.8%) were single pathogen infections and 3,585 (13%) were mixed pathogen infections. The pathogen positivity rate was lowest in the first year of the COVID-19 pandemic (2020), at 54.7%, and peaked at 79.1% in 2023, after public health restrictions were lifted. During the COVID-19 pandemic (2020–2022), seasonal variation in pathogen prevalence was disrupted. Post-restriction, there was a significant increase in RSV and MP cases. SP remained the leading bacterial cause of CAP, especially in young children. RSV was the predominant viral pathogen, particularly affecting infants. MP showed a rising trend with age, yet it also affected younger individuals.ConclusionThe COVID-19 pandemic altered the epidemiological characteristics of pathogens in children with CAP. This impact is likely to persist, necessitating enhanced surveillance of CAP pathogens to mitigate the healthcare burden in children.Clinical trial numberNot applicable.

Effect of WW-domain transcription regulator 1 on aging regulation of human dental pulp stem cells

Objective: Investigating the changes of phenotype and moleculars associated with aging with the increase of passage times of human dental pulp stem cells (hDPSC), to explore the role of WW-containing transcriptional regulator 1 (WWTR1) in the aging mechanism. Methods: hDPSCs were cultured by tissue block method, and were divided into 4 groups according to the age, algebra, cell knockdown and overexpression of WWTR1 in hDPSCs. Group I: hDPSCs from human teeth were further divided into youth group (15-25 years old) and group middle-aged group (40-50 years old)] according to different ages. Group Ⅱ: according to different passage, hDPSCs were divided into young cells group (hDPSCs were transmitted to P3 generation), and old cells group (hDPSCs were transmitted to P10 generation). Group Ⅲ: hDPSCs were knocked down of WWTR1, which were further divided into knockdown group (sh-WWTR1) and knockdown carrier group (sh-NC). Group Ⅳ: hDPSCs were overexpressed of WWTR1, which were further divided into overexpression group (OE-WWTR1) and overexpression carrier group (OE-NC). Real-time quantitative fluorescent PCR (RT-qPCR) was used to detect the changes of WWTR1 expression in groups Ⅰ and Ⅱ, and cell counting kit-8 (CCK-8) was used for groups Ⅱ, Ⅲ, and Ⅳ. Cell proliferation capacity was detected by CCK-8 assay. The ability of osteogenic differentiation was detected by alizarin red staining. Cell senescence positive rate was detected by age-related β-galactosidase staining. The expression levels of age-related genes p53 and p21 were detected by RT-qPCR. Results: The proportion of senescent cells increased gradually with continuous culture. The proliferation and osteogenic differentiation of hDPSCs in the old group were significantly lower than those in the young group (P<0.001). The expression levels of senescence related genes p53 (2.09±0.24) and p21 (4.91±0.54) in old cell group were higher than those in young cell group respectively [p53: (1.08±0.09) and p21: (1.09±0.08)] (P<0.01, P<0.001). The WWTR1 expression levels of hDPSCs in middle-aged group and old cells group were both decreased compared with those in young group and young cells group (P<0.01). The proportion of senescent cells in knockdown group (44.50±2.42) was higher than that in knockdown carrier group (22.27±0.56) (P<0.001). After knocking down WWTR1 in hDPSCs, the expression levels of age-related genes p53 and p21 were up-regulated (P<0.001), and the abilities of proliferation and osteogenic differentiation in the knockdown group were lower than those in the knockdown carrier group (P<0.001). The proportion of senescent cells in overexpression empty carrier group (20.4±0.79) was higher than that in overexpression group (10.07±0.61) (P<0.001). After WWTR1 overexpression ins hDPSCs, the expression levels of age-related genes p53 and p21 were down-regulated, and the proliferation and osteogenic differentiation ability in overexpression group were higher than those in OE-NC group (P<0.001). Conclusions: WWTR1 can inhibit the expression levels of age-related genes p53 and p21, thus delaying the aging process as well as promoting the proliferation and osteogenic differentiation of hDPSCs.

Genome-Wide Identification of the ClpB Gene Family in Tomato and Expression Analysis Under Heat Stress.

Tomato is a widely grown horticultural crop, and its growth process is often affected by high temperatures. Caseinolytic Protease B (ClpB), a homologous protein to heat shock protein 101 (HSP101), plays a vital role in plant heat adaptation and development. In this study, we identified six SlClpB genes in tomatoes, distributed across four chromosomes. Collinearity analysis revealed that the gene pairs SlClpB-2 and SlClpB-3A, as well as SlClpB-3C and SlClpB-12, resulted from segmental duplication events. Phylogenetic and motif analyses showed that ClpB proteins possess highly conserved domains across different species. We used RNA-seq data to analyze the expression patterns of the ClpB family. Among them, SlClpB-3A and SlClpB-12 exhibited increased expression in multiple tissues under heat stress. Specifically, SlClpB-2, SlClpB-3A, and SlClpB-3C were highly expressed in the fruit orange stage and in flower buds under heat treatment, while in seedlings, SlClpB-2 and SlClpB-3A exhibited heat-induced expression. Real-time quantitative fluorescent PCR (qRT-PCR) results showed that the expression of SlClpB-2 and SlClpB-3A was significantly increased under heat stress in the leaves and buds of Ailsa Craig, Micro-Tom, and M82. Overall, our findings provide valuable insights into the regulatory mechanisms of SlClpB genes in response to heat stress.

Bio-characteristics, tissue expression of miR-375 in hypothalamic-pituitary-ovarian axis and its regulation in reproduction-related diseases

Our study concentrated on the expression of miRNA-375 in the hypothalamic-pituitary-gonadal axis of female Hu sheep. The investigation involved cloning the precursor sequence of miR-NA-375, followed by comparison with database entries and subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. In our approach, we obtained ovaries, thalamus, cerebellum, brain, uterus, pituitary gland, hypothalamus, and pineal gland from fertile but nonpregnant Hu ewes. MiRNA extraction kit was used to extract miRNA from the above eight tissues. Real-time fluorescent quantitative polymerase chain reaction was used to evaluate the role of miR-375 in the hy-pothalamic-pituitary-gonadal axis. The results of miR-375 precursor sequence cloning were compared with those of Anopheles gambiae, Apis mellifera, Bos taurus, Drosophila melanogaster, Danio rerio, Fugu rubripes, Gallus gallus, Homo sapiens, Monodelphis domestica, Macaca mulatta, Mus musculus, Pan troglodytes, Rattus norvegicus, Tetraodon nigroviridis, Xenopus tropicalis miR-375 in miRBase database. It was found that oar-miR-375 was highly conserved. Notably, miR-375 expression in the pineal gland was significantly higher (p < 0.01) than that in the ovaries, thalamus, cerebellum, brain, uterus, pituitary gland, hypothalamus. The study also involved predicting miR-375 target genes. GO and KEGG enrichment analyses of these predicted target genes revealed that miR-375 is involved in 182 biological processes, affects 186 cellular components, and participates in 184 molecular functions. In terms of pathway enrichment, miR-375 was linked to nine pathways, including the Hippo, Wnt, and mTOR signaling pathways. This study has validated the interaction between miR-375 and its target gene FZD4, which can be recognized and bound to produce effects. These findings lead to the inference that miR-375 may play a crucial regulatory role in sheep reproduction through the Hippo pathway and Wnt pathway, laying a foundation for further exploration of miR-375’s role in this domain.

Comparative analysis of the NF-Y transcription factor family identifies VaNF-YA6 as a positive regulator of salt and drought tolerance in grapevine

Nuclear factor Y transcription factors (NF-Y TFs) play crucial roles in plant responses to abiotic stress. However, there is a lack of research on the comparative analysis of evolutionary relationships, real-time quantitative fluorescence PCR (RT-qPCR), and functions of NF-Y TFs to screen key NF-Y TFs that are resistant to salt and drought stresses between Vitis vinifera (V. vinifera) and Vitis amurensis (V. amurensis). In this study, 27 and 26 NF-Y TFs were identified in V. vinifera and V. amurensis, respectively, and were divided into three subgroups. Subcellular localization prediction revealed that NF-Ys TFs were mainly located in the nucleus. Interestingly, the NF-YA protein sequence of ‘NTKKLDWEFWGCCDDCEKWFGGCC’ was lost in the V. vinifera compared to V. amurensis, whereas the sequence ‘SSVYSQPWWGHSIVCVA’ was gained, thus, these sequences might be closely related to the functions performed. RT-qPCR analysis of ‘Pinot Noir’ (cultivated variety) and ‘Zuoyouhong’ (wild variety) plantlets demonstrated that the expression levels of VaNF-YA6, VaNF-YB5, VvNF-YA3, VvNF-YA5, and VvNF-YC2 were significantly upregulated under 400 mmol·L-1 NaCl and 10% PEG treatments for 24 h Subcellular localization showed that the VaNF-YA6-GFP fusion protein was functioned primarily in the nucleus. Overexpression of VaNF-YA6 in grapevine leaves and Arabidopsis thaliana (Arabidopsis) could significantly enhance tolerance to salt and drought stresses by improving VvSOS2, VvSOS3, VvABF3, VvCPK6 expression levels, enzyme activities, and other protective substances. In summary, our study provides a theoretical basis for the further use of VaNF-YA6 to improve salt and drought resistance in grapevines.

VEGFB ameliorates insulin resistance in NAFLD via the PI3K/AKT signal pathway

BackgroundNon-alcoholic fatty liver disease (NAFLD) is one of the most universal liver diseases with complicated pathogenesis throughout the world. Insulin resistance is a leading risk factor that contributes to the development of NAFLD. Vascular endothelial growth factor B (VEGFB) was described by researchers as contributing to regulating lipid metabolic disorders. Here, we investigated VEGFB as a main target to regulate insulin resistance and metabolic syndrome.MethodsIn this study, bioinformatics, transcriptomics, morphological experiments, and molecular biology were used to explore the role of VEGFB in regulating insulin resistance in NAFLD and its molecular mechanism based on human samples, animal models, and cell models. RNA-seq was performed to analyze the signal pathways associated with VEGFB and NAFLD; Palmitic acid and High-fat diet were used to induce insulin-resistant HepG2 cells model and NAFLD animal model. Intracellular glucolipid contents, glucose uptake, hepatic and serum glucose and lipid levels were examined by Microassay and Elisa. Hematoxylin-eosin staining, Oil Red O staining, and Periodic acid-schiff staining were used to analyze the hepatic steatosis, lipid droplet, and glycogen content in the liver. Western blot and quantitative real-time fluorescent PCR were used to verify the expression levels of the VEGFB and insulin resistance-related signals PI3K/AKT pathway.ResultsWe observed that VEGFB is genetically associated with NAFLD and the PI3K/AKT signal pathway. After VEGFB knockout, glucolipids levels were increased, and glucose uptake ability was decreased in insulin-resistant HepG2 cells. Meanwhile, body weight, blood glucose, blood lipids, and hepatic glucose of NAFLD mice were increased, and hepatic glycogen, glucose tolerance, and insulin sensitivity were decreased. Moreover, VEGFB overexpression reduced glucolipids and insulin resistance levels in HepG2 cells. Specifically, VEGFB/VEGFR1 activates the PI3K/AKT signals by activating p-IRS1Ser307 expression, inhibiting p-FOXO1pS256 and p-GSK3Ser9 expressions to reduce gluconeogenesis and glycogen synthesis in the liver. Moreover, VEGFB could also enhance the expression level of GLUT2 to accelerate glucose transport and reduce blood glucose levels, maintaining glucose homeostasis.ConclusionsOur studies suggest that VEGFB could present a novel strategy for treating NAFLD as a positive factor.Graphical

Establishment and validation of a real-time fluorescent PCR freeze-dried type assay for 11 sheep and goats pathogens

Small ruminants are economic mainstay in developing countries because of its direct contribution to food security, but the occurrence of epidemics poses a continuous threat. Early diagnosis can strengthen the prevention of zoonosis. Therefore, a high-sensitivity real-time fluorescent PCR freeze-dried type assay that can be stored and transported at room temperature was developed for 11 sheep and goats pathogens in this study. The results showed that there was no non-specific amplification in systems containing different pathogen positive controls; The areas under the ROC curves were all in the range of 0.99–1.00; The LOD counted by Digital PCR were 194, 96, 84, 40, 265, 68, 208, 236, 118, 53 and 723 copies/mL for M. ovis, Pathogenic Leptospira, C. burnetii, BTV, PPRV, Brucella, exJSRV, C. abortus, ORFV, FMDV and CPV, respectively. The coefficients of variation of both intra-group and inter-group replicate tests were less than 5.00 %; The accelerated thermostatic lyophilization test was used to predict the validity period; The LOD of each positive pathogen was still detected after 6 days at 56 ℃, suggesting that the validity period can be at least 567 days at room temperature. In this study, a high-sensitivity real-time fluorescent PCR freeze-dried type assay that can be stored and transported at room temperature was developed for 11 sheep and goats pathogens, which were Mycoplasma ovis (M. ovis), Pathogenic Leptospira, Coxiella burnetii (C. burnetii), Bluetongue virus (BTV), Peste des pestis ruminants virus (PPRV), Brucella, exogenous jaagsiekte sheep retrovirus (exJSRV), Chlamydia abortus (C. abortus), Orf virus (ORFV), Foot-and-mouth disease virus (FMDV), and Capripox virus (CPV).

Screening of key genes related to M6A methylation in patients with heart failure

ObjectiveThis study aims to identify m6A methylation-related and immune cell-related key genes with diagnostic potential for heart failure (HF) by leveraging various bioinformatics techniques.MethodsThe GSE116250 and GSE141910 datasets were sourced from the Gene Expression Omnibus (GEO) database. Correlation analysis was conducted between differentially expressed genes (DEGs) in HF and control groups, alongside differential m6A regulatory factors, to identify m6A-related DEGs (m6A-DEGs). Subsequently, candidate genes were narrowed down by intersecting key module genes derived from weighted gene co-expression network analysis (WGCNA) with m6A-DEGs. Key genes were then identified through the Least Absolute Shrinkage and Selection Operator (LASSO) analysis. Correlation analyses between key genes and differentially expressed immune cells were performed, followed by the validation of key gene expression levels in public datasets. To ensure clinical applicability, five pairs of blood samples were collected for quantitative real-time fluorescence PCR (qRT-PCR) validation.ResultsA total of 93 m6A-DEGs were identified (|COR| > 0.6, P < 0.05), and five key genes (LACTB2, NAMPT, SCAMP5, HBA1, and PRKAR2A) were selected for further analysis. Correlation analysis revealed that differential immune cells were negatively associated with the expression of LACTB2, NAMPT, and PRKAR2A (P < 0.05), while positively correlated with SCAMP5 and HBA1 (P < 0.05). Subsequent expression validation confirmed significant differences in key gene expression between the HF and control groups, with consistent expression trends observed across both training and validation sets. The expression trends of LACTB2, PRKAR2A, and HBA1 in blood samples from the qRT-PCR assay aligned with the results derived from public databases.ConclusionThis study successfully identified five m6A methylation-related key genes with diagnostic significance, providing a theoretical foundation for further exploration of m6A methylation’s molecular mechanisms in HF.

CRISPR/Cas12 System-Based Assay for Rapid, Sensitive Detection of Rotavirus in Food Samples.

Foodborne viruses have become an important threat to food safety and human health. Among the foodborne viruses, group A rotavirus is the most important pathogen of diarrhea in autumn and winter. The field detection of rotavirus is crucial for the early control of infection and patient management. Quantitative real-time reverse transcription-polymerase chain reaction is the most widely used in virus detection. However, the technique relies on high-cost instruments and trained personnel, which limit its use in field detection. In this study, we developed accurate, realizable, and simple detection methods by combining optimized CRISPR (clustered regularly interspaced short palindromic repeats) Cas12 and reverse transcription loop-mediated isothermal amplification (RT-LAMP) (reverse transcription loop-mediated isothermal amplification) to reduce the requirements for temperature control and costly real-time fluorescence polymerase chain reaction instruments. We investigated two nucleic acid detection systems combining RT-LAMP with CRISPR Cas12a and RT-LAMP with CRISPR Cas12b and compared them with reverse transcription-quantitative polymerase chain reaction. The resulting detection system only needs a reaction temperature and in single tube to react for 60 min with the detection sensitivity of 38 copies/μL. Overall, this study developed an innovative method for the rapid detection of rotavirus in food samples, which will help to effectively identify food contaminated by pathogens and prevent human infections and economic losses caused by disease outbreaks.

Prevalence of high-risk human papillomavirus genotypes and viral load correlated with squamous cell inflammation among women in Gabon

BackgroundHigh-risk genotypes of Human Papillomavirus are responsible for 90% of cases of cervical cancer worldwide. Inflammation of squamous cells is mainly linked to HPV. In Gabon, HPV is endemic and circulates among the female population. The study aimed to determine the prevalence of HR-HPV genotypes and to investigate the correlation between squamous cell inflammation and HPV viral load in infected women in Gabon.MethodsThe cross-sectional study was conducted at Libreville University Hospital Center (UHC) and National Public Health Laboratory from March to May 2024 among 399 women. Two cervical smears were taken. Genotype detection was carried out by multiplex fluorescence real-time PCR in the NPHL virology unit. Cytology was carried out in UHC’s anatomic-pathology laboratory. Data were analyzed by SPSS software. Graphs were plotted using Microsoft Excel 2016.ResultsThe prevalence of Human Papillomavirus was 26.1% (95% CI: 22-30.6). The prevalence of HR-HPV genotypes was 24.8%. The most common HR-HPV genotypes were HPV-16/52/18/35/56/58/53/68. The rate of multiple HPV infections was 29.8% and 95.2% for the HR-HPV infection rate. Viral load was significantly correlated with squamous cell inflammation (r = 0.977 and P = 0.001).ConclusionHR-HPV infection remains a concern in women, however early screening is necessary for optimal monitoring and management. HR-HPV viral load is a predictive marker of squamous cell inflammation.

Hsa_circ_0007718 facilitates the progression of colorectal cancer by regulating the miR-1299/PSMC2 axis

Colorectal cancer (CRC) represents one of the most prevalent forms of malignant tumors, characterized by a notably high rate of mortality among affected individuals. The primary objective of this investigation is to delve into the functional role of Hsa_circ_0007718 in the context of colorectal cancer and to elucidate its impact on the progression of CRC by modulating the interaction between the miR-1299 microRNA and its target gene, PSMC2. To assess the expression levels of Hsa_circ_0007718, along with miR-1299 and PSMC2, real-time quantitative fluorescent PCR (qRT-PCR) assays were meticulously performed using both CRC cell lines and clinical samples derived from patients. A cellular model was established to investigate the interactions occurring between miR-1299 and Hsa_circ_0007718, as well as the connections to PSMC2, thereby providing a comprehensive understanding of these molecular interactions. The findings of this research revealed a significant upregulation of Hsa_circ_0007718 in both colorectal cancer cell lines and tissue samples. Importantly, the data indicated that the suppression of Hsa_circ_0007718 led to a marked decrease in the proliferation rates, migratory potential, and invasive capabilities of CRC cells. Furthermore, the study confirmed that Hsa_circ_0007718 acts as a downstream target of miR-1299, exerting its regulatory effects by inhibiting miR-1299 and thereby promoting the expression of PSMC2.

Identification of neutrophil extracellular traps genes as potential biomarkers in psoriasis based on bioinformatics analysis

The epidermal infiltration of neutrophils is a hallmark of psoriasis (PSO) and its activation leads to the release of neutrophil extracellular traps (NETs). However, the molecular mechanism of NETs-related genes (NETRGs) has not been extensively studied in PSO. To define NETs-related-biomarkers for PSO. The GSE13355 and GSE78097 datasets, and NETRGs gene set were included in this study. The datasets used in this study were all microarray data. The weighted gene co-expression network analysis (WGCNA) and machine learning algorithms were used to mine key genes. Later on, single-gene gene set enrichment analysis (GSEA) and immune infiltration analysis were implemented. Finally, the expression of key genes was verified using quantitative real-time fluorescence PCR (qRT-PCR). A total of 3 key genes (S100A9, CLEC7A, and CXCR4) were derived, and they all had excellent diagnostic performance. The single-gene GSEA enrichment results indicated that the key genes were mainly enriched in the chemokine signaling pathway and humoral immune response in the high-expression group, while focal adhesion was enriched in the low-expression group. The correlation analysis indicated that all key genes were strongly negatively correlated with resting mast cells and TGF-β family member receptor, while they were strongly positively correlated with activated CD4 memory T cells and antigen processing and presentation. Lastly, the experimental results showed that the expression trends of key genes were consistent with public database. In this study, we successfully screened three potential PSO diagnostic genes (S100A9, CLEC7A and CXCR4) that were closely related to NETs, and these findings not only provided new molecular marker candidates for the precise diagnosis of PSO patients, but also revealed possible future therapeutic targets. However, further in-depth research and validation were necessary.

Study on the expression of lncRNA PRKCA-AS1 in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the most malignant tumors in the oral and maxillofacial region, with a poor prognosis. Previous studies have shown that long non-coding RNAs (lncRNAs) play a crucial role in tumor development by regulating the biological behavior of various cancer cells. The aim of this study is to explore the role and potential mechanisms of lncRNA PRKCA-AS1 in OSCC. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression levels of lncRNA PRKCA-AS1 in OSCC tissues and cell lines. Cell proliferation, migration and invasion were conducted to assess the biological functions of OSCC cell lines. The expression of lncRNA PRKCA-AS1 in OSCC tissues was higher compared to that of adjacent non-cancerous tissues, and its expression level was associated with the depth of tumor infiltration, lymph node metastasis, and tumor node metastasis (TNM) staging. Compared to the control group of normal human oral keratinocytes (HOK), the expression of lncRNA PRKCA-AS1 was also elevated in OSCC cell lines. Knockdown of lncRNA PRKCA-AS1 significantly affected the proliferation, migration, and invasion ability of OSCC cells. However, when lncRNA PRKCA-AS1 was further overexpressed, changes in cell proliferation and migration ability did not show statistical differences. LncRNA PRKCA-AS1 is highly expressed in OSCC, and its expression level is positively correlated with the depth of tumor infiltration, lymph node metastasis, and TNM staging. LncRNA PRKCA-AS1 is involved in regulating the proliferation, migration, and invasion of OSCC cells.

The potential relationship between EasyNAT system Tt values and Cobas z480 Ct values in the detection of SARS-Cov-2

ObjectivesThis study aimed to evaluate the potential relationship between the time threshold (Tt) values of a commercial EasyNAT system, which is based on cross priming amplification (CPA) technology, and the cycle threshold (Ct) values of the Cobas z480 analyzer, which is based on a real-time fluorescence polymerase chain reaction (PCR) method, in the detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) from oropharyngeal swabs. Design and Methods: Data were retrospectively collected from a clinical laboratory between December 4, 2022 and July 1, 2024. ResultsA total of 277 EasyNAT-positive samples (Tt values from 3.83 to 29.5) were simultaneously investigated using the Cobas z480 analyzer (Ct values from 10.74 to 38.78). The concordance rate between the two systems was 100 %. Among the positive samples, the mean and maximum PCR Ct values of O and N genes increased in line with increasing Tt values of the left and right amplification areas of the EasyNAT system. The maximum Ct values of the O or N gene determined by the Cobas z480 analyzer were no more than 29.52 when the Tt values of the left or right amplification areas of the UC0116 analyzer were no more than 6. ConclusionsThe safe, simple, fast, accurate, and automatic EasyNAT system used in conjunction with a PCR system might be a better choice for the detection of SARS-CoV-2 in hospitals, especially in settings without sophisticated PCR facilities. The Tt value (≤6) of the EasyNAT system can be a reference index for estimating the maximum Ct value (29.52) in SARS-CoV-2-positive samples.

Canagliflozin can improve cardiac function in HFpEF rats partly by regulating ferroptosis

Objective: To explore the effects of canagliflozin on cardiac function and its regulation of ferroptosis in rats with heart failure with preserved ejection fraction (HFpEF). Methods: Thirty-two 7-week-old Dahl salt-sensitive rats were selected and randomly divided into four groups: the control group (fed with low-salt diet), the HFpEF group (fed with high-salt diet), the canagliflozin 20 group (fed with high-salt diet and 20 mg·kg-1·d-1 canagliflozin), and the canagliflozin 30 group (fed with high-salt diet and 30 mg·kg-1·day-1 canagliflozin). Body weight and blood pressure of the rats in each group were monitored. Metabolic cage tests were conducted at the10th week of the experiment, and echocardiography was performed at the 12th week, after which the rats were killed. Blood and left ventricular samples were collected. HE staining, Masson staining, Prussian blue iron staining, and reactive oxygen species staining were performed to observe the cardiomyocyte size and shape, degree of interstitial fibrosis, iron staining, reactive oxygen species production under optical microscope. The ultrastructure of cardiomyocytes was observed under electron microscope. Western blotting and real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to detect the expression levels of proteins and mRNA related to ferroptosis in left ventricular myocardial tissue of rats in each group. Results: After 1 week of adaptive feeding, all rats survived. Metabolic cage results showed that compared with control group, rats in the HFpEF group, canagliflozin 20 group and canagliflozin 30 group had more food intake, water intake and urine output, and lower body weight (all P<0.05). These changes were more pronounced in canagliflozin 20 group and canagliflozin 30 group than in HFPEF group, and only the body weight at the 12th week showed a statistically significant difference between canagliflozin 20 group and canagliflozin 30 group (P<0.05). The blood pressure of 6th week and 12th week, heart weight and left ventricular corrected mass of 12th week of rats in HFpEF group were higher than those in control group, canagliflozin 20 group and canagliflozin 30 group, while the ratio of early mitral valve peak velocity to late mitral valve peak velocity of 12th week was lower (all P<0.05). HE and Masson staining showed that compared to control group, the myocardial fibers in the left ventricular myocardial tissue of rats in HFpEF group were disordered, with larger cell diameter ((0.032±0.004) mm vs. (0.023±0.003) mm, P<0.05), irregular shape, obvious proliferation of interstitial collagen fibers, and higher collagen volume fraction (0.168±0.028 vs. 0.118±0.013, P<0.05). Compared with HFpEF group, rats in the canagliflozin 20 group and canagliflozin 30 had more orderly arranged myocardial fibers, more regular cardiomyocyte shape, smaller cell diameter, and lower collagen volume fraction (P<0.05). It was observed under electron microscopy that, compared to control group, most of the striated muscles in myocardial tissue of HFpEF group were broken, and the Z line and M line could not be clearly distinguished, some changes such as mitochondrial swelling, membrane thickening, cristae reduction or even disappearance occurred. In the canagliflozin 20 group and canagliflozin 30 group, the arrangement of striated muscles in the myocardial tissue of rats tended to be more regular, and the morphological changes of mitochondria were milder. Prussian blue iron staining results showed that the iron content in myocardial tissue of rats in HFpEF group was higher than that in control group, canagliflozin 20 group and canagliflozin 30 group. Reactive oxygen species staining results showed that the reactive oxygen species content in the myocardial tissue of rats in HFpEF group was higher than that of control group, canagliflozin 20 group and canagliflozin 30 group. Biochemical analysis of myocardial tissue showed that Fe2+ and malondialdehyde content in myocardial tissue of rats in HFpEF group were higher than those in control group, canagliflozin 20 group and canagliflozin 30 group, while glutathione content was lower (all P<0.05). Western blot and RT-qPCR detection results showed that compared to control group, rats in HFpEF group had higher expression levels of transferrin receptor 1 (protein relative expression level: 1.37±0.16 vs. 0.31±0.12), acyl-CoA synthetase long-chain family member 4 (protein relative expression level: 1.31±0.15 vs. 0.63±0.09) protein and mRNA, and lower expression levels of ferritin heavy chain 1 (protein relative expression level: 0.45±0.08 vs. 1.41±0.15) protein and mRNA (all P<0.05). There was no statistically significant difference in these indicators between canagliflozin 20 group and the canagliflozin 30 group (all P>0.05). There was no significant difference in levels of glutathione peroxidase 4 protein and mRNA expression in myocardial tissue of rats in four groups(P>0.05). Conclusion: Canagliflozin improves cardiac function in HFpEF rats by regulating the ferroptosis mechanism.

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

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

Acptp2,3 participates in the regulation of spore production, stress response, and pigments synthesis in Aspergillus cirstatus.

Aspergillus cristatus was a filamentous fungus that produced sexual spores under hypotonic stress and asexual spores under hypertonic stress. It could be useful for understanding filamentous fungi's sporulation mechanism. Previously, we conducted functional studies on Achog1, which regulated the hyperosmotic glycerol signaling (HOG) pathway and found that SI65_02513 was significantly downregulated in the transcriptomics data of ΔAchog1 knockout strain. This gene was located at multiple locations in the HOG pathway, indicating that it might play an important role in the HOG pathway of A. cristatus. Furthermore, the function of this gene had not been identified in Aspergillus fungi, necessitating further investigation. This gene's conserved domain study revealed that it has the same protein tyrosine phosphatases (PTPs) functional domain as Saccharomyces cerevisiae, hence SI65_02513 was named Acptp2,3. The function of this gene was mostly validated using gene knockout and gene complementation approaches. Knockout strains exhibited sexual and asexual development, as well as pigments synthesis. Morphological observations of the knockout strain were carried out under several stress conditions (osmotic stress, oxidative stress, Congo Red, and sodium dodecyl sulfate (SDS). Real-time fluorescence polymerase chain reaction (PCR) identified the expression of genes involved in sporulation, stress response, and pigments synthesis. The deletion of Acptp2,3 reduced sexual and asexual spore production by 4.4 and 4.6 times, demonstrating that Acptp2,3 positively regulated the sporulation of A. cristatus. The sensitivity tests to osmotic stress revealed that ΔAcptp2,3 strains did not respond to sorbitol-induced osmotic stress. However, ΔAcptp2.3 strains grew considerably slower than the wild type in high concentration sucrose medium. The ΔAcptp2,3 strains grew slower than the wild type on media containing hydrogen peroxide, Congo red, and SDS. These findings showed that Acptp2,3 favorably controlled osmotic stress, oxidative stress, and cell wall-damaging chemical stress in A. cristatus. Deleting Acptp2,3 resulted in a deeper colony color, demonstrating that Apctp2,3 regulated pigment synthesis in A. cistatus. The expression levels of numerous stress-and pigments-related genes matched the phenotypic data. According to our findings, Acptp2,3 played an important role in the regulation of sporulation, stress response, and pigments synthesis in A. cristatus. This was the first study on the function of PTPs in Aspergillus fungi.

Normal sperm quality despite partial deletion of sY84 in the AZFa region: A case report.

The complete absence of the azoospermia factor A (AZFa) region typically results in nonobstructive azoospermia. Partial deletions of the AZFa region are particularly noteworthy due to the limited and enigmatic reports of partial deletions in the AZFa region. Here, we present a rare case report of partial deletion of sY84 in the AZFa region but exhibiting normal sperm quality. The aim of this case report is to gain a deeper insight into the impact of AZFa region deletion on male fertility and to guide future clinical decisions and treatment strategies. A 25-year-old man presented to the hospital with his 25-year-old wife due to recurrent spontaneous abortions. Routine semen analysis, sperm morphology analysis, acrosomal enzyme analysis, sperm DNA fragmentation indexed, and peripheral blood karyotype analysis revealed no abnormalities. Y chromosome microdeletion was detected by real-time fluorescence polymerase chain reaction, which showed that sY84 could not be amplified and sY86 was amplified nonspecifically. The man was diagnosed with partial deletions in the AZFa region. The wife underwent in vitro fertilization treatment for tubal infertility and recurrent spontaneous abortions. The couple successfully delivered a healthy daughter weighing 2.7 kg at 39 weeks of gestation, following 2 assisted reproductive pregnancies. Our findings contribute to expanding our knowledge of the AZFa region. A sY84 deficiency in the AZFa region may not lead to spermatogenesis failure and may potentially be one of the factors causing recurrent miscarriages, which needs to be confirmed by further data.

Nippostrongylus brasiliensis alleviates dextran sulfate sodium salt-induced ulcerative colitis in mice: a preliminary study

To investigate the alleviation of Nippostrongylus brasiliensis infection on dextran sulfate sodium salt (DSS)-induced ulcerative colitis in mice, and to explore the underlying mechanism. Thirty male C57BL/6J mice of the SPF grade, each weighing approximately 25 g, were randomly divided into three groups, including the blank control group (NC group), DSS modeling group (DSS group), and N. brasiliensis treatment group (Nb + DSS group), of 10 mice in each group. Mice in the DSS group were orally administered with 3.5% DSS daily since day 1 (D0) for 6 successive days, and given normal drinking water since D6, and animals in the Nb + DSS group were subcutaneously injected with the third-stage larvae of N. brasiliensis at a dose of 500 larvae per mice 5 days prior to D0, followed by oral administration with 3.5% DSS daily since D0 for 6 successive days and normal drinking water since D6, while mice in the NC group were given normal drinking water. Mouse body weight and stool were observed and the disease activity index (DAI) was scored in each group during the study period. All mice were sacrificed on D9. The mouse colon length was measured, and mouse colon specimens were subjected to hematoxylin-eosin (HE) staining and histopathological scoring. In addition, the mRNA and protein expression of interleukin (IL)-1β and IL-10 was quantified in mouse colon specimens using quantitative fluorescent real-time PCR (qPCR) assay and enzyme-linked immunosorbent assay (ELISA), and the mRNA and protein expression of mucosal repair-associated molecules zonula occludens-1 (ZO-1), mucin 2 (MUC2) and claudin-1 was detected in mouse colon specimens using qPCR assay and immunofluorescence assay. The mice body weights, DAI scores and colon lengths were (26.26 ± 1.93), (22.39 ± 1.65), (25.00 ± 1.58) g (F = 8.06, P < 0.01); (1.89 ± 0.34), (0.47 ± 0.39), 0 points (F = 57.61, P < 0.000 1); and (42.50 ± 5.75), (56.20 ± 5.96) mm and (61.17 ± 7.88) mm (F = 13.72, P < 0.001) in the NC, DSS and Nb + DSS groups on D9, respectively, and elevated mouse body weight (P < 0.05), reduced DAI score (P < 0.000 1) and increased colon length (P < 0.01) were observed in the Nb + DSS group relative to the DSS group on D9. Pathological examinations showed that the colonic crypts were relatively intact and the inflammatory cell infiltration was lower in the mouse colon specimens in the Nb + DSS group than in DSS the group. There was a significant difference in the histopathological scores of mouse colon specimens among the NC group (0 point), the DSS group [(2.00 ± 1.22) points] and the Nb + DSS group [(0.20 ± 0.45) points] (F = 10.71, P < 0.01), respectively, and the histopathological score of mouse colon specimens was significantly higher in the DSS group than in the NC and Nb + DSS groups (both P values < 0.01). qPCR assay quantified that the relative IL-10 and IL-1β mRNA expression was 1.25 ± 0.08, 0.44 ± 0.14 and 1.30 ± 0.45 (F = 10.66, P < 0.01), and 0.22 ± 0.13, 1.14 ± 0.31 and 0.41 ± 0.19 (F = 16.89, P < 0.001) in mouse colon specimens in the NC, DSS and Nb + DSS groups, respectively, and higher IL-10 mRNA expression and lower IL-1β mRNA expression were found in mouse colon specimens in the Nb + DSS group than in the DSS group (both P values < 0.01). The relative MUC2, claudin-1 and ZO-1 mRNA expression was 0.87 ± 0.25, 0.34 ± 0.26 and 4.21 ± 0.55 (F = 121.60, P < 0.000 1), 1.05 ± 0.41, 0.16 ± 0.09 and 0.22 ± 0.11 (F = 14.00, P < 0.01), and 1.03 ± 0.10, 0.60 ± 0.11 and 1.64 ± 0.28 (F = 32.16, P < 0.000 1) in mouse colon specimens in the NC, DSS and Nb + DSS groups, respectively, and significantly higher MUC2 and ZO-1 mRNA expression was quantified in mouse colon specimens in the Nb + DSS group than in the DSS group (both P values < 0.05). The mean fluorescence intensities of ZO-1 and claudin-1 were 17.18 ± 2.08, 12.38 ± 1.21 and 18.06 ± 2.59 (F = 8.95, P < 0.01) and 13.50 ± 1.63, 9.66 ± 2.03 and 13.61 ± 0.97 (F = 6.96, P < 0.05) in mouse colon specimens in the NC, DSS and Nb + DSS groups, respectively, and the mean fluorescence intensities of ZO-1 and claudin-1 were significantly greater in mouse colon specimens in the Nb + DSS group than in the DSS group (both P values < 0.05). N. brasiliensis infection may remarkably alleviate DSS-induced ulcerative colitis in mice through promoting expression of anti-inflammatory cytokines, inhibiting expression of pro-inflammatory cytokines and facilitating mucosal repair in colon tissues.