Search Results (349)

Background/Objectives: Cxbladder^® Triage Plus is a multimodal urinary biomarker assay that combines reverse transcription-quantitative analysis of five mRNA targets and droplet-digital polymerase chain reaction (ddPCR) analysis of six DNA single-nucleotide variants (SNVs) from two genes (fibroblast growth factor receptor 3 (FGFR3) and telomerase reverse transcriptase (TERT)) to provide risk stratification for urothelial carcinoma (UC) in patients with hematuria. This study evaluated the analytical validity of Triage Plus. Methods: The development dataset used urine samples from patients with microhematuria or gross hematuria that were previously stabilized with Cxbladder solution. Triage Plus was evaluated for predicted performance, analytical criteria (linearity, sensitivity, specificity, accuracy, and precision), extraction efficiency, and inter-laboratory reproducibility. Results: The development dataset included 987 hematuria samples. Compared with cystoscopy (standard of care), Triage Plus had a predicted sensitivity of 93.6%, specificity of 90.8%, positive predictive value (PPV) of 46.5%, negative predictive value of 99.4%, and test-negative rate of 84.1% (score threshold 0.15); the PPV increased to 74.6% for the 0.54 score threshold. For the individual FGFR3 and TERT SNVs, the limit of detection (analytical sensitivity) was a mutant-to-wild type DNA ratio of 1:440–1:1250 copies/mL. Intra- and inter-assay variance was low, while extraction efficiency was high. All other pre-specified analytical criteria (linearity, specificity, and accuracy) were met. Triage Plus showed good reproducibility (87.9% concordance between laboratories). Conclusions: Cxbladder Triage Plus accurately and reproducibly detected FGFR3 and TERT SNVs and, in combination with mRNA expression, provides a non-invasive, highly sensitive, and reproducible tool that aids in risk stratification of patients with hematuria. Full article

Cardioprotection against ischemia is achieved using openers of mitochondrial ATP-sensitive K⁺ (mitoKATP) channels such as diazoxide (DZX), leading to pharmacological preconditioning (PPC). We previously reported that PPC decreases the abundance of ventricular Cav1.2 channels, but PPC’s effects on other channels remain largely unexplored. In this study, we hypothesized that DZX regulates the expression of hyperpolarization-activated cyclic nucleotide potassium channel 4 (HCN4) channels in sinoatrial node cells (SANCs), the specialized cardiomyocytes that generate the heartbeat. DZX increased the heart rate in intact adult rats. Patch-clamp experiments revealed an increase in the magnitude of ionic currents through HCN4 channels, which was abolished by the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) and the selective mitoKATP channel inhibitor 5-hydroxydecanoate (5-HD). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot assays showed that DZX increased HCN4 channel expression at the mRNA and protein levels. Immunofluorescence analyses revealed that PPC increased HCN4 fluorescence, which was abolished by NAC. DZX increased nuclear translocation of c-Fos and decreased protein abundance of RE1 silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF), suggesting the involvement of these factors. Our results suggest that PPC increases the heart rate by upregulating HCN4 channel expression through a mechanism involving c-Fos, REST, and ROS. Full article

Background/Objectives: Inflammatory disorders contribute to the pathogenesis of numerous diseases and are known to markedly reduce quality of life. Although anti-inflammatory drugs approved by the Food and Drug Administration are available, their prolonged use is frequently associated with adverse effects. In this study, we evaluated the pharmacological properties of araliadiol, a naturally occurring polyacetylene compound, as a novel anti-inflammatory agent. Methods: An in vitro hyperinflammatory model was established by stimulating RAW 264.7 cells with lipopolysaccharide (LPS). Dexamethasone (DEX) was used as a positive control to compare anti-inflammatory efficacy. The protective effects of araliadiol against LPS-induced cytotoxicity were assessed using adenosine triphosphate content and crystal violet staining assays. The anti-inflammatory activity was further examined by quantitative reverse transcriptase–polymerase chain reaction, Western blotting, cell fractionation, immunofluorescence staining, a nitric oxide assay, and an enzyme-linked immunosorbent assay. Results: Araliadiol significantly attenuated cytotoxicity and cell death in LPS-stimulated RAW 264.7 cells. It suppressed the expression of cell death markers Cleaved caspase-3 and Cleaved PARP-1. In addition, araliadiol downregulated key pro-inflammatory mediators, including inflammasome-related genes, cytokines, chemokines, and inducible nitric oxide synthase. It also reduced the expression of Cox-2 and PGE₂, indicating potential anti-hyperalgesic effects. Moreover, araliadiol inhibited the activation of Nfκb and Stat1 signaling pathways in LPS-stimulated macrophages. Conclusions: Araliadiol demonstrated robust anti-cytotoxic, anti-inflammatory, and anti-hyperalgesic activities in LPS-induced RAW 264.7 cells, with efficacy comparable to DEX. These findings support its potential as a plant-derived therapeutic candidate for the management of inflammatory conditions. Full article

Stem cell-derived secretome and exosomes present a promising cell-free strategy for tissue repair and wound healing. This study aimed to isolate and characterize, for the first time, exosomes derived from rat hair follicle stem cells (rHFSCs) and to evaluate their wound-healing potential alongside rHFSC secretome. Exosomes were isolated via ultracentrifugation and characterized using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), biomarker profiling and protein quantification. Scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDS) confirmed their spherical morphology, diameter and elemental composition. Protein quantification showed higher protein content in the secretome than in exosomes. RT-PCR and biomarker profiling highlighted the therapeutic relevance of the exosomal cargo compared to parent rHFSCs. Functional analysis of 30 wound-healing biomolecules validated their pro-regenerative potential. Cytocompatibility was confirmed via the PrestoBlue™ viability assay, while scratch assays demonstrated significant wound closure in the treated groups, both with and without mitomycin C. These findings highlight the potential of rHFSC-derived exosomes and secretome as innovative, cell-free therapeutic agents for cutaneous regeneration. This study advances our understanding of their role in wound healing and underscores their broader applicability in regenerative medicine. Full article

Circular RNAs (circRNAs) are increasingly recognized as significant regulators in multiple cancers, such as hepatocellular carcinoma (HCC), frequently affecting microRNA (miRNA) expression. The diagnostic and prognostic roles of circRNAs, specifically circ_0009910 and circ_0027478, in conjunction with miR-1236-3p, in HCC, have not yet been fully investigated. In this pilot study, we assessed the expression levels of circ_0009910, circ_0027478, and miR-1236-3p in plasma samples from 100 patients diagnosed with HCC and 50 healthy controls through reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR). The diagnostic performance was evaluated using receiver operating characteristic (ROC) curve analysis, and correlations with clinicopathological features were examined. Circ_0009910 and circ_0027478 exhibited significant upregulation in patients with HCC (p < 0.05), whereas miR-1236-3p demonstrated downregulation (p < 0.05). Circ_0009910 demonstrated significant diagnostic accuracy (area under the curve [AUC] = 0.90), effectively differentiating HCC from controls and showing a correlation with tumor size, metastasis, and alpha-fetoprotein (AFP) levels (p < 0.05). Both circ_0009910 and circ_0027478 exhibited a positive correlation with clinicopathological features, whereas miR-1236-3p demonstrated an inverse correlation. Logistic regression validated the diagnostic and prognostic capabilities of these biomarkers. The results indicate that circ_0009910, circ_0027478, and miR-1236-3p, in conjunction with AFP three, present a promising diagnostic and prognostic profile for HCC. Additional validation in larger cohorts is required to establish their clinical utility. Full article

Background/Objectives: This study aimed to analyze optic nerve parameters, retinal nerve fiber layer thickness (RNFLT), ganglion cell layer thickness (GCLT), and subfoveal choroidal thickness (ChT) in patients who have recovered from coronavirus disease 2019 (COVID-19). Methods: This comparative study included 78 recovered COVID-19 patients (16 men, 62 women) and 56 age- and sex-matched healthy controls (18 men, 38 women). COVID-19 was confirmed in all patients, either through the detection of viral RNA in nasopharyngeal swabs via reverse transcriptase polymerase chain reaction or by serological testing for SARS-CoV-2 antibodies. Spectral-domain optical coherence tomography (SD-OCT) was used to assess optic nerve parameters, RNFLT, GCLT, and ChT. Results: The mean age was 35.0 ± 8.3 years in the COVID-19 group and 31.5 ± 8.3 years in the control group, with no statistically significant differences in age or sex distribution between groups (p = 0.41 and p = 0.16, respectively). Optic nerve parameters and RNFLT (overall and across the four peripapillary quadrants) did not differ significantly between the COVID-19 and control groups. However, the mean ganglion cell–inner plexiform layer (GC-IPL) thickness was significantly reduced in all quadrants in the COVID-19 group compared to the controls. No significant difference was observed in mean subfoveal ChT between groups. Conclusions: A significant reduction in ganglion GCLT was observed in recovered COVID-19 patients compared to healthy controls, suggesting a potential neurodegenerative effect of the disease on the optic nerve. Full article

Background: Allergic rhinitis (AR) is a common disease that requires more convenient, safe, and effective therapy. This study aimed to investigate the therapeutic effect of Forkhead box protein3 (Foxp3) introduced with poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles (Foxp3 NPs) in an AR mouse model. Methods: A murine model of allergic rhinitis was established using BALB/c mice through initial sensitization by intraperitoneal administration of ovalbumin (OVA), followed by repeated intranasal OVA challenges. Foxp3 plasmid-loaded PLGA nanoparticles were subsequently administered via either the intranasal or intraperitoneal route to evaluate therapeutic efficacy. Episodes of sneezing and nose rubbing were counted. The serum total IgE, OVA-specific IgE, and cytokine levels in nasal lavage fluid (NALF) were determined by ELISA (Enzyme-Linked ImmunoSorbent Assay). Nasal mucosa from each group were analyzed using protein, reverse transcriptase–polymerase chain reaction (RT-PCR), and histological analyses. Result: Rubbing and sneezing symptoms improved in the Foxp3 NPs intranasal administration group. Foxp3 NPs intranasal administration markedly ameliorated OVA-induced nasal allergic inflammation. The total IgE and OVA-specific IgE serum level and IL-4, IL-13 expression levels of NALF were significantly decreased in the treated Foxp3 NPs group. The histopathological results of nasal mucosa were also normal, with no cellular infiltration and no inflammation in the Foxp3 NPs group. Conclusions: These results suggest that Foxp3 NPs alleviate nasal allergic inflammation and may have therapeutic value in the treatment of AR. Full article

The COVID-19 pandemic has highlighted the need for rapid and accurate tests to detect SARS-CoV-2. Objectives: This study evaluates the effectiveness of the Panbio™ COVID-19 Antigen Self-Test rapid test compared to reverse transcriptase polymerase chain reaction (RT-PCR). Methods: A prospective diagnostic testing study was performed. Patients with respiratory symptoms who provided informed consent were included. Results: We included 205 patients with COVID-19 symptoms who underwent both tests. The mean age was 35.55 ± 12.62 years, and 64% of the participants were female. Sensitivity and specificity were 71.2% (95% CI: 62.5–79.9%) and 100% (95% CI: 96.4–100%), respectively. Conclusions: If a test is positive within the first 72 h after the onset of symptoms, we can be sure that it is a case of COVID-19; however, when the antigen test is negative, confirmation with RT-PCR is necessary. Its ease of use and results with moderate precision make it a valuable tool for early detection. Full article

Groundnut bud necrosis orthotospovirus (GBNV), a tripartite single-stranded RNA virus, poses a significant threat to United States agriculture. GBNV is a quarantine pathogen, and its introduction could lead to severe damage to economically important crops, such as groundnuts, tomatoes, potatoes, peas, and soybeans. For the rapid and accurate detection of GBNV at points of entry, TaqMan reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR) assays were developed and the results validated using conventional reverse transcriptase–polymerase chain reaction (RT-PCR) followed by Sanger sequencing. These assays target highly conserved regions of the nucleocapsid (NP) and movement (MP) proteins within the viral genome. Multiplex GBNV detection assays targeting the NP and MP genes, as well as an internal control plant gene, ACT11, showed efficiency rates between 90% and 100% and R² values of 0.98 to 0.99, indicating high accuracy and precision. Moreover, there was no significant difference in sensitivity between multiplex and singleplex assays, ensuring reliable detection across various plant tissues. This rapid, sensitive, and specific diagnostic assay will provide a valuable tool at ports of entry to prevent the entry of GBNV into the United States. Full article

Immunohistochemically, ameloblastomas often express CD56; however, novel neuroendocrine markers such as synaptophysin (SYP), insulinoma-associated protein 1 (INSM1), and chromogranin A (CgA) remain unexplored. We analyzed 36 ameloblastoma specimens for CD56, SYP, CgA, and clusterin (CLU) and examined limited samples for INSM1 expression by performing immunohistochemistry, transmission electron microscopy, and reverse transcriptase-polymerase chain reaction. Our findings indicate that the limited cells were positive for CD56, SYP, CgA, INSM1, and CLU expression in 72% (26/36), 14% (5/36), 0% (0/40), 80% (4/5), and 22% (8/36) of the cases, respectively. CD56 expression correlated with older age, but not with subtype, SYP, and CLU expression. However, SYP-positive cases were exclusively found in CD56- and CLU-positive cases, and SYP and CLU expression were significantly correlated. Selected cases had dense-core granules and NCAM1 and SYP mRNA expression. This study is the first to suggest neuroendocrine differentiation in ameloblastomas, as indicated by SYP and INSM1 immunoexpression and the presence of dense-core granules, which are consistent with the recent World Health Organization classification of Head and Neck Tumors guidelines. SYP-positive and CgA-negative phenotypes may characterize neuroendocrine differentiation in ameloblastoma. Although the underlying molecular mechanism remains unclear, CLU expression may be associated with neuroendocrine differentiation. Full article

Long non-coding RNAs (lncRNAs) play complex roles at multiple levels of gene regulation, thus modulating key cellular processes involved in the pathogenesis and progression of cancer. Aberrant expression of lncRNAs has been reported in various malignancies, including chronic lymphocytic leukemia (CLL). We investigated the expression of lnc-IRF2-3 and lnc-KIAA1755-4 in peripheral blood mononuclear cells of 112 previously untreated CLL patients by quantitative reverse-transcriptase polymerase chain reaction. Both lncRNAs were found to be overexpressed in CLL samples in comparison to healthy controls, and their high levels were associated with adverse clinico-biological characteristics of patients at diagnosis. High lnc-IRF2-3 expression was associated with high leukocyte and lymphocyte counts, high β2-microglobulin, advanced Binet stage, unfavorable cytogenetics, CD38-positivity and IGHV-unmutated status. Regarding lnc-KIAA1755-4, its high expression was associated with high leukocyte count, lymphocyte count, β2-microglobulin, lactate dehydrogenase and low hemoglobin, as well as with IGHV-unmutated status. In addition, we observed shorter time to first treatment and overall survival of patients expressing high levels of both lncRNAs in comparison to low-expressing patients. In summary, our study showed that high lnc-IRF2-3 and lnc-KIAA1755-4 expression at diagnosis predicts poor survival in CLL. The mechanisms of their upregulation, as well as their specific targets in CLL cells, remain to be elucidated. Full article

Reverse transcription polymerase chain reaction (RT-PCR) is an important tool for the detection of target RNA molecules and the assay of RNA pathogens. Coupled RT-PCR is performed with an enzyme mixture containing a reverse transcriptase and a thermostable DNA polymerase. To date, several biotechnological companies offer artificial thermostable DNA polymerases with a built-in reverse transcriptase activity for use in the coupled RT-PCR instead of the enzyme mixtures. Here, we compared the artificial DNA polymerases and conventional enzyme mixtures for the RT-PCR by performing end-point and real-time RT-PCR assays using severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV2) RNA and endogenous mRNA molecules as templates. We found that the artificial enzymes were suitable for different RT-PCR applications, including SARS-CoV2 RNA detection but not for long-fragment RT-PCR amplification. Full article

Deregulation of micro-RNAs (miRNAs) may contribute to mechanisms of injury in the bicuspid aortic valve (BAV). Our objective was to investigate the expression of miRNAs in aortic tissue from patients who underwent aortic valve replacement for aortic stenosis and its relationship with aortic dilatation. The study included 78 patients, 40 with bicuspid aortic valve (BAV) and 38 with tricuspid aortic valve (TAV). The expression of miRNA-17-5p, hsa-let-7e, and miRNA-196a-5p in human aortic tissue was evaluated by a reverse transcriptase polymerase chain reaction (RT-qPCR). Comparative analysis between patients with BAV and controls with TAV explored the association between the miRNAs and aortic dilatation (AD), calcification, valve dysfunction, and stenosis. The results showed that the expression levels of miRNA-Let-7e-5p and miRNA-196-5p were mostly increased in patients with BAV and aortic dilatation (p = 0.01 and p = 0.01), respectively. In contrast, the levels of miRNA-17a-5p (p < 0.20) were lower but without a statistically significant difference. The downregulation of miRNA-17a-5p and the upregulation of miR-Let-7e-5p and miR-196-5p were related to an increased risk of AD risk. Subjects with BAVs with or without double aortic lesions had higher expression levels of Let-7e-5p and miRNA-17a-5p vs. TAV. In all patients, we found an inverse correlation of MiRNA-196-5p with High-Density Lipoprotein-Cholesterol (HDL-C) and indexed valvular area. In subjects with a higher expression of miRNA196, lower levels of HDL-C correlation (r²) [r² 0.27 (p = 0.02)] and a lower indexed valvular area [r² 0.28 (p = 0.05)] were observed. In the specific analysis for each patient group, it was found that in control subjects with tricuspid aortic valve (TAV), miRNA-196-5p had a positive correlation with valvular calcification (r² = 0.60, p = 0.02). Deregulation of miRNAs in the aortic tissue of a BAV may influence valvular stenosis, dysfunction, and concomitant aortic dilation. This information could help to define potential therapeutic target strategies to improve the prognosis and treatment of BAV. Full article

Background: The dysregulation of microRNAs (miRNAs) has been detected in patients with gastric cancer (GC), which inspired the use of miRNAs as a novel biomarker for GC. In this study, we investigated the previously reported miRNA dysfunction in cancer tissues as a potential plasma biomarker for GC using quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Methods: The published miRNA abnormalities were searched in the microRNA Cancer Association Database. Plasma samples were collected from patients with GC (n = 26) and controls (n = 17). The sensitivity and specificity of polyadenylation RT-PCR (PA-RT) and stem-loop RT-PCR (SL-RT) were compared. Statistical comparisons between patients with GC and controls were performed to identify miRNA biomarkers, and correlation analyses between the threshold cycle (Ct) values of miRNAs and various blood biochemical parameters were performed to elucidate the confounding factors. Results: mir-17, mir-21, mir-31, mir-99b, mir-222, and U6 were selected. PA-RT showed greater sensitivity and lower specificity than SL-RT (PA-RT vs. SL-RT, mean Ct: 19.6 vs. 29.2; coefficient of variation: 0.42 vs. 0.10). Adopting SL-RT owing to its higher specificity, only mir-222 was significantly upregulated in patients with GC (GC vs. control, miRNA expression: 15.4 vs. 5.27, p = 0.0098). Regarding the correlation between blood biochemical parameters and cells with miRNA expression, mir-31 and mir-99b were correlated with blood urea nitrogen, mir-17, mir-21, and mir-99b were negatively correlated with platelets, and mir-21 was correlated with neutrophils. No obvious correlations were noted between mir-222 expression and blood parameters. Receiver operating characteristic (ROC) curve analysis indicated that mir-222 identified GC patients with a maximum area under the curve (0.73, 95% confidence interval 0.57–0.89). Conclusions: Plasma mir-222 was confirmed to be dysregulated in patients with GC, irrespective of blood biochemical parameters. Full article

Nasal spray treatments that inhibit the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) entry into nose and nasopharynx at early stages can be an appropriate approach to stop or delay the progression of the disease. We performed a prospective, randomized, double-blind, placebo-controlled, parallel-group, multicentric, phase II clinical trial comparing the rate of hospitalization due to COVID-19 infection between azelastine 0.1% nasal spray and placebo nasal spray treatment groups. The study furthermore assessed the reduction in virus load in SARS-CoV-2-infected subjects estimated via quantitative reverse transcriptase polymerase chain reaction (RT-PCR) using nasopharyngeal swabs in both groups during the treatment period. A total of 294 subjects with mild COVID-19 infection were screened and randomized in a 1:1 ratio. There was no incidence of COVID-19-related hospitalization in either treatment group. Mean virus load was significantly reduced in both groups during the 11 treatment days as compared with baseline viral load values. The reduction in virus load in the azelastine 0.1% nasal spray group was significantly higher than the reduction in the placebo group at day 11 (log₁₀ 5.93 vs. log₁₀ 5.85 copies/mL, respectively, p = 0.0041). A total of 39 (32.0%) subjects in the azelastine 0.1% treatment group and 40 (31.0%) subjects in the placebo group reported 48 and 51 adverse events, respectively. It is therefore concluded that azelastine 0.1% nasal spray is an efficacious, safe, and well-tolerated treatment of mild COVID-19 infection. Full article