Search Results (3,054)

Early confirmation of leptospirosis is essential for prompt antimicrobial treatment, and PCR-based diagnosis has been reported as a highly sensitive method during the acute phase in the first week since the symptom’s onset. We evaluated the diagnostic performance of two commercial real-time PCR assays—Viasure Leptospira Real-Time PCR (Certest Biotec, Spain) and Genesig Advanced Leptospira spp. (Primerdesign, UK) against an in-house qPCR assay targeting lipL32 as the reference method. A retrospective comparative evaluation was conducted on 235 human EDTA-blood samples obtained during the acute phase of clinical presentation suspected of leptospirosis. The in-house qPCR reference assay detected 55 positive and 180 negative samples, and both commercial kits accurately classified every specimen, achieving 100% sensitivity (95% CI: 93.5–100), 100% specificity (95% CI: 98.0–100), and 100% overall accuracy. In conclusion, both commercial qPCR kits offer high accuracy for the early detection of pathogenic Leptospira in human blood samples. Full article

Background: Terahertz (THz) waves exhibit both photon-like and electron-like properties, showing emerging potential in biomedical applications. Cutaneous squamous cell carcinoma (CSCC) is one of the most common skin tumors. Studies have reported that THz waves can induce apoptosis in cancer cells or ablate tumor tissues. Our previous studies also confirmed that 0.1 THz radiation could significantly promote apoptosis in cutaneous melanoma cells, while it had no apparent effect on fibroblast viability, proliferation, migration, and apoptosis. However, the effects of 0.1 THz radiation on CSCC cells have not yet been explored. Furthermore, there remains a lack of investigation into the structural and functional effects on fibroblasts. Therefore, it is necessary to conduct a systematic study to evaluate the influence of 0.1 THz radiation on both CSCC cells and fibroblasts in order to better understand its potential therapeutic applications in the treatment of skin cancer. Purpose: This study aims to explore the biological effects of 0.1 THz radiation on SCC-7 cells and to uncover the molecular mechanisms underlying THz-induced apoptosis, as well as its potential effect on L-929 cells. Methods: Cell viability was evaluated through the CCK-8 assay, while cell cycle distribution was analyzed with the DNA content detection kit. Wound healing assays were performed to assess cell migration, and Annexin V-FITC staining was used to detect apoptosis. Caspase-3 activity was measured using the caspase-3 activity assay kit. Cell morphology was observed using the Atomic Force Microscope (AFM) and the Transmission Electron Microscopy (TEM). Alterations in membrane potential were detected with the M09 membrane potential probe kit, and intracellular Ca²⁺ levels were quantified using the Fluo-8 AM fluorescent probe. Mitochondrial permeability transition pore (mPTP) opening was assessed with the MPTP detection kit, mitochondrial membrane potential changes were measured using the JC-1 probe kit, and cellular ATP levels were measured with the enhanced ATP assay kit. Subsequently, proteomic analysis was performed. Intracellular reactive oxygen species (ROS) levels were quantified with the ROS detection kit, and cytochrome c (Cyt c) release was quantified using the mouse Cyt c ELISA kit. Apoptosis-inducing factor (AIF) expression was analyzed at both mRNA and protein levels by quantitative real-time PCR (qPCR) and Western blot. AIF expression in CSCC tissues was further evaluated based on the GSE42677 and GSE45164 databases. Finally, cyclosporin A (CsA) was used to inhibit mPTP, and in combination with the iMAC inhibitor, the Aifm1 expression and Cyt c release were examined. Results: Our results showed that THz waves significantly disrupted the membrane integrity of SCC-7 cells and induced mitochondrial structural and functional damage. This resulted in a significant increase in ROS levels and the activation of mPTP and the mitochondrial apoptosis channel (MAC). THz radiation promoted the release of Cyt c and AIF from mitochondria, triggering a noncanonical caspase-3-dependent apoptosis pathway. Notably, L-929 cells did not show significant phenotypic or apoptotic changes under the same irradiation conditions. Bioinformatics analysis of the Gene Expression Omnibus (GEO) database revealed that AIF expression was significantly altered in CSCC tissues compared to normal skin tissues. Conclusions: These findings indicated that 0.1 THz radiation effectively induced apoptosis in SCC-7 cells by triggering mitochondrial dysfunction and ROS generation, which led to the release of AIF. Furthermore, the dysregulation of AIF in CSCC tissues suggested its potential as a promising biomarker. These results provided important molecular insights into the therapeutic potential of THz radiation, particularly for the treatment of cutaneous squamous cell carcinoma. Full article

Mast cell tumors are the most common skin neoplasm in dogs and may be influenced by reproductive status. A gonadectomy modifies gonadotropin levels and may affect the expression of hormonal receptors and proliferative factors; however, the evidence for this remains limited. The objective of this study was to evaluate the expression of VEGF, IGF-1, PCNA, Ki-67, c-KIT, LHR, FSHR, and ERα in intact and neutralized female dogs with and without MCTs. Gene expression in the skin samples was quantified by a real-time PCR, and four groups were included: intact controls (n = 10), neutralized controls (n = 10), intact MCT (n = 9), and neutralized MCT (n = 10). Tumor presence was associated with increased expression of LH and FSH receptors and c-KIT, while angiogenic and proliferative factors (PCNA, Ki-67, IGF-1, VEGF, and ERα) showed lower expression. In dogs with MCTs, a gonadectomy was associated with higher c-KIT and VEGF expression but lower LHR mRNA levels. PCNA expression was lower in the neutralized MCT dogs compared to the neutralized controls, whereas no differences were observed in the intact dogs. Additionally, ERα expression was higher in the neutralized control dogs than in the intact controls, with no differences detected in the MCT dogs. These findings suggest that reproductive status is associated with differential regulation of molecular pathways involved in canine MCT biology. Full article

The presence of human herpesviruses is frequently detected in the oral cavity, yet their disease-specific role in chronic inflammatory oral mucosal disorders remains uncertain. This comparative observational study investigated Epstein–Barr virus (EBV) and human herpesvirus-7 (HHV-7) genomic sequences in saliva and virus-specific antibodies in serum among patients with oral lichen planus (OLP; n = 35), aphthous stomatitis (AS; n = 31), and healthy controls (n = 34). Salivary viral loads were quantified using real-time PCR, while EBV and HHV-7-specific IgG and IgM antibodies were measured using ELISA-based assays. EBV and HHV-7 DNA in saliva were commonly detected across all groups, demonstrating high baseline shedding and marked interindividual variability. Although EBV IgG levels were higher in OLP compared with AS in univariate analysis, multivariate regression revealed that age, rather than disease status, was the primary determinant of EBV IgG levels. After adjustment for age, sex, and discomfort, neither EBV nor HHV-7 salivary loads showed independent associations with OLP or AS. HHV-7 salivary loads were uniformly distributed among groups. These findings suggest that salivary detection of EBV and HHV-7 reflects widespread latent infection rather than disease-specific activity in OLP or AS. Longitudinal and tissue-based studies integrating immunological profiling are warranted to clarify whether herpesvirus reactivation contributes to disease severity in defined patient subgroups. Full article

The aim of this study was to assess whether edible insects reared on substrates containing food allergens can carry these allergens into the final product, and to evaluate the effectiveness of a pre-harvest fasting period in reducing this risk to provide consumer protection. Hermetia illucens larvae, chosen as the model species, were grown on substrates containing 10% of each of the following food allergens: peanut, almond, soy, celery, and gluten. At the end of the feeding period, larvae were sampled at T0 (end of feeding), T1 (24 h fasting), T2 (48 h fasting) and tested by real-time PCR and ELISA to detect allergen residues. Positive results were observed by real-time PCR for soy (mean Ct: 28.84 at T0, 29.4 at T1, 30.95 at T2), celery (mean Ct: 26.74 at T0, 26.90 at T1, 29.77 at T2) and almond (Ct 33.96 at T0 and mean Ct: 34.01 at T1). Soy presence was also confirmed by ELISA test. Insects may represent an alternative food source; however, their use requires careful evaluation due to the potential presence of allergens. Our results showed that insects may contain allergens originating from their feeding substrates, potentially triggering a response in allergic consumers. Full article

Ginseng Alternaria leaf and stem blight, caused by Alternaria panax, imposes substantial yield and economic losses to the ginseng cultivation industry. Current diagnostic methods for ginseng diseases primarily rely on pathogen isolation from infected tissues, a procedure that is laborious, time-consuming, and inherently low in sensitivity. This study has therefore developed a rapid, specific and sensitive SYBR Green-based quantitative real-time PCR (qPCR) assay for detecting A. panax in plants, seeds, and soil. The developed qPCR assay exhibited high sensitivity and repeatability, with a detection limit of 0.074 fg/μL of target amplicon DNA (0.619 ng/μL of genomic DNA) and a coefficient of variation below 2%. In artificially inoculated tissues (leaves, stems and seeds), Ct values decreased progressively with increasing incubation time, reflecting pathogen proliferation. Analysis of field-collected leaves and stems showed a strong overall correlation between Ct values and visual disease grades. Surveying of ginseng-growing areas revealed that A. panax was detected in asymptomatic leaves and stems at rates of 12.12% and 14.29%, respectively, and in 14.46% of soil samples and 23.73% of seed samples. This qPCR assay presented here provides a robust tool for forecasting early disease, tracking the primary inoculum of the pathogen and its transmission chains, and screening of both ginseng seed lots and candidate soils for ginseng Alternaria leaf and stem blight prior to planting. Full article

The aim of this study was to evaluate the nephrotoxicity and molecular mechanism of Evodiamine (EVO). We combined RNA sequencing (RNA-seq) and network toxicology (NT) screening of potential target genes and signaling pathways, used molecular docking to validate core targets, and detected the mRNA expression of the key genes through quantitative real-time polymerase chain reaction (qRT-PCR). After exposure to EVO, body weight of mice decreased significantly, and the levels of renal index, Blood Urea Nitrogen (BUN) and Creatinine (Cr) were significantly increased, with varying degrees of pathological damage to the kidneys. NT identified 125 intersecting targets of EVO exposure related to kidney injury, including AKT1, TNF, TP53, etc. Among the 2888 differentially expressed genes obtained from RNA-seq, 504 genes were up-regulated and 2384 genes were down-regulated. By integrating NT and RNA-seq, 24 intersecting targets were identified. Among them, TRPV1, NOS3, HSP90AA1, and PPARG were selected for molecular docking validation. The results indicated that EVO had the highest affinity for PPARG (−8.07 kcal/mol). The qRT-PCR results indicated that the expression of the Pparg and Hsp90aa1 genes was significantly down-regulated, and the expression of the Nos3 and Trpv1 genes was significantly up-regulated. Immunohistochemistry further confirmed that EVO inhibited the expression of HSP90AA1 and PPARG, while enhancing that of TRPV1 and NOS3. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis suggested that EVO-induced nephrotoxicity is related to signaling pathways such as inflammatory mediator regulation of TRP channels, the PPAR signaling pathway, and the Apelin signaling pathway. In summary, the nephrotoxic effect of EVO may be related to the inhibition of the PPARG signaling pathway, the activation of the TRPV1 channel, the reduction in HSP90AA1 expression, and the imbalance of the Apelin-NOS3 pathway. This study provides a theoretical reference for clarifying the potential mechanism of renal injury caused by EVO and guiding its safe use. Full article

In this study, we developed a multiplex one-tube nested real-time fluorescent quantitative PCR (mONRT-PCR) assay integrated with a portable, fully automated nucleic acid point-of-care testing (POCT) platform for the detection of Haemophilus influenzae (H. influenzae), Listeria monocytogenes (L. monocytogenes), and Cryptococcus neoformans (C. neoformans) in cerebrospinal fluid (CSF). The assay enables nested amplification within a closed system using conventional primers and probes, thereby reducing operational complexity and minimizing contamination risk. Analytical evaluation demonstrated limits of detection of 10⁰ copies/μL for H. influenzae and L. monocytogenes, and 10¹ copies/μL for C. neoformans using recombinant plasmids, as well as 10⁻⁷ to 10⁻⁶ ng/μL using genomic DNA. No cross-reactivity was observed when tested against a panel of 17 common non-target microorganisms encountered in clinical microbiology laboratories. In simulated CSF samples, the assay maintained detectable amplification at low pathogen concentrations. When implemented on the POCT platform, detection limits reached 5, 10, and 50 CFU/mL for the three pathogens, respectively. Clinical evaluation using 43 CSF samples showed almost perfect agreement with conventional qPCR (κ = 0.861, p < 0.001). Notably, additional C. neoformans detections were observed by mONRT-PCR-POCT compared with qPCR, suggesting improved sensitivity under clinical conditions. The assay yielded results within approximately 1 h and 47 min. These findings indicate that the proposed assay provides a rapid, sensitive, and integrated approach for meningitis pathogen detection, while maintaining a practical balance between analytical performance and operational simplicity. Further validation in larger cohorts is warranted. Full article

Background/Objectives: Down syndrome (DS) is characterised by a marked susceptibility to early-onset severe periodontitis, suggesting an intrinsic host predisposition. Interleukin-1 (IL-1) gene variants may influence inflammatory burden, yet DS-specific evidence is limited. Methods: Nineteen Caucasian adults with DS underwent a comprehensive periodontal examination and received a periodontal diagnosis according to the AAP/EFP 2018 classification. Buccal swabs were genotyped by real-time PCR for IL1A −889, IL1B +3954 and IL1RN +2018; the composite IL1A/B genotype was also evaluated. Results: All participants presented advanced, generalized periodontitis (Stage III/IV: 37%/63%; Grade B/C: 32%/68%). Variant alleles were detected in 63% for IL1A, 53% for IL1B and 37% for IL1RN, and the composite IL-1A/B genotype in 47%. Variant carriage showed associations with higher Clinical Attachment Loss (IL1A p = 0.03; IL1B p = 0.002; composite p = 0.012) and Bleeding on Probing (IL1A p = 0.02; IL1RN p = 0.05; composite p = 0.04). The composite genotype was associated with Stage IV (p = 0.027) and Grade C (p = 0.005), and tooth loss was greater among variant carriers for all polymorphisms (p = 0.01). Conclusions: In this DS cohort with advanced periodontitis, IL-1 variants (particularly the composite IL1A/B genotype) were frequently observed and were associated with greater periodontal severity and tooth loss. Full article

Galeruca daurica (Joannis) (Coleoptera: Chrysomelidae) is an oligophagous pest in which both adults and larvae prefer to feed on Allium forage grasses of the Liliaceae family. In this study, we identified gustatory receptor (GR) genes based on the transcriptome data of G. daurica; analyzed the expression profiles of these GR genes across different larval instars and various tissues of male and female adults using quantitative real-time PCR (qRT-PCR); detected the electrophysiological responses of the mouthparts of male and female G. daurica adults to flavonoids and carbohydrates using single sensillum recording (SSR); and recorded the changes in food consumption of G. daurica adults after feeding on six host plant-derived metabolites. A total of 26 GR genes were identified from the transcriptome data of adult and larval of G. daurica. Phylogenetic analysis was performed to screen candidate functional gustatory receptor genes, including four sugar receptors (GdauGR7, GdauGR10, GdauGR14 and GdauGR28), seven bitter receptors (GdauGR11, GdauGR16~17, GdauGR22, GdauGR25~26 and GdauGR30), and two CO₂ receptors (GdauGR15 and GdauGR20). Larval expression profiling of GdauGRs in G. daurica revealed that the relative expression levels of 17 genes exhibited dynamic changes during larval growth and development. GdauGRs were expressed to varying degrees in the antennae, mouthparts, brain, gut, and forelegs of adult G. daurica, with sex-specific differences. Notably, the expression levels of GdauGR4, GdauGR9 and GdauGR16 in the gut were extremely significantly higher than those in other tissues. In the SSR test, the six tested flavonoids and one carbohydrate were able to induce robust electrophysiological responses in the gustatory sensilla on the antennae and mouthparts of adult G. daurica at specific concentrations. In addition, the supplementation of several host-derived metabolites altered the food consumption of adult G. daurica. These findings lay a solid foundation for elucidating the molecular mechanisms underlying gustatory recognition and host adaptation in G. daurica. Full article

Microbial contamination in aquatic environments poses severe threats to aquaculture sustainability, ecological balance and public health. Traditional culture-based detection methods, while standardized, are time-consuming and labor-intensive, often failing to meet the urgent need for rapid on-site monitoring required to prevent disease outbreaks and manage water quality effectively. By integrating latest research advances (2020–2025), this study reviews advances in rapid detection technologies for aquatic microorganisms, including the evolution of nucleic acid amplification strategies, with a focused comparison of the analytical sensitivity and field deployability of quantitative polymerase chain reaction (qPCR) and mainstream isothermal amplification techniques (loop-mediated isothermal amplification, LAMP; recombinase polymerase amplification, RPA). Furthermore, this study reports on the emergence of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein (Cas) systems as next-generation diagnostic tools, highlighting their integration with microfluidic Lab-on-a-Chip (LOC) platforms to achieve attomolar sensitivity. We also consider the application of portable nanopore sequencing for real-time pathogen identification and the growing role of Artificial Intelligence (AI) in analyzing complex diagnostic datasets. Advanced molecular methods have achieved significant reductions in time consumption—from days to less than one hour—while challenges regarding sample preparation and environmental matrix inhibition remain. The future of aquatic monitoring lies in integrated, automated systems that combine the specificity of CRISPR-Cas diagnostics with the connectivity of IoT-enabled biosensors. Comparative analysis indicates that isothermal amplification methods (LAMP, RPA) coupled with CRISPR-Cas systems offer the optimal balance of sensitivity, speed, and field deployability for point-of-care aquaculture diagnostics, while qPCR/dPCR remain indispensable for quantitative regulatory applications. We propose a structured technology selection framework to guide researchers and practitioners in choosing appropriate detection modalities based on specific sensitivity, cost, throughput, and deployment requirements. Full article

Trichophyton indotineae is an emerging dermatophyte associated with extensive, chronic, recalcitrant, and frequently terbinafine-resistant dermatophytosis worldwide. In this study, 30 T. indotineae strains isolated in Italy were investigated. The isolates were obtained from patients originating from Asian countries, from patients from other countries, and from Italian patients who reported no travel outside Italy in the preceding years. Clinical isolates were identified by internal transcribed spacer (ITS) sequencing and analyzed to assess the occurrence and molecular basis of terbinafine resistance. Terbinafine resistance was detected in 18 strains (60%) using a real-time PCR assay. Sequencing of the squalene epoxidase (SQLE) gene revealed mutations associated with resistance, including L393S in nine strains and F397L in another nine strains. NGS analysis confirmed two terbinafine-resistant strains carrying the L393S and F397L mutations, respectively, and detected the A448T mutation in one terbinafine-susceptible strain. These findings demonstrate the presence of terbinafine-resistant T. indotineae across five regions of Italy and confirm the occurrence of SQLE mutations previously linked to antifungal resistance. Data obtained also support a link with endemic Asian areas, other than suggesting the possible occurrence of autochthonous transmission in Italy. Full article

In this study, the chemical composition of highland barley (HB), microwave fluidization HB (HB-1), extrusion and puffing HB (HB-2), and ultrafine pulverization HB (HB-3) were investigated based on untargeted metabolomics. In addition, RNA-seq transcriptomics, real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) analysis were used to investigate the lipid metabolism mechanism of HB-1, induced by a high fat and cholesterol diet (HFCD). The results indicated that a total of 1292 metabolites were detected and classified into 78 distinct classes in the untargeted metabolomics analysis including fatty acyls, carboxylic acids and derivatives, glycerophospholipids, organooxygen compounds, prenol lipids, and so on. HB-1, HB-2, and HB-3 all increased the levels of amino acids and their derivatives, phenols, and carboxylic acid and its derivatives compared with HB. Furthermore, RNA-seq transcriptomic results indicated that HB-1 significantly modulated key genes of Cyp2c38, Cyp2b13, and Cyp2b9 related to steroid hormone biosynthesis and CD36, Plin4, and Fabp4 related to the PPAR signaling pathway, which played key roles in lipid metabolism. Moreover, qRT-PCR and WB results indicated that HB-1 obviously enhanced ADIPOQ expression level, while it reduced SCD-1, CD36, Fabp4, and SREBP-1c expression levels, suggesting that the alleviation of lipid metabolic dysregulation by HB-1 in hyperlipidemia mice might be mediated via participating in the PPARγ pathway. This study provided essential theoretical insights for the development and utilization of HB. Full article

Rice blast, caused by the fungus Magnaporthe oryzae, is one of the most devastating threatening to global rice production. The narrow genetic background of modern rice cultivars exacerbates the shortage of durable resistance resources. In contrast, the wild rice species Oryza officinalis harbors abundant stress-resistance alleles and represents a valuable gene pool for identifying novel broad blast-resistance genes. The cloned resistance gene Pid2 is encoded in a receptor-like protein kinase conferring race-specific resistance against the M. oryzae isolate ZB15. In this study, three Pid2 homologs were isolated from O. officinalis. The special allele Pid2of-MD33 was transformed into “Yunjing 37(YG37), a blast-susceptible japonica rice cultivar” via Agrobacterium-mediated transformation. Quantitative real-time PCR analysis showed that Pid2of-MD33 was consistently expressed in various tissues of O. officinalis, with the highest transcript abundance detected in leaf mesophyll cells and plasma membranes. Inoculation with the M. oryzae isolate ZB15 revealed that transgenic YG37 lines expressing Pid2of-MD33 displayed significantly reduced lesion size and pathogen proliferation, suggesting recovered race-specific resistance. These results enrich the resistance gene resources for rice blast research and provide a promising candidate gene for rice blast resistance breeding. Full article

Background: Molecular diagnostics may detect several respiratory pathogens simultaneously with rapid turnaround times. The aim of this study was to determine the frequency and distribution of respiratory pathogens among symptomatic outpatients. Methods: All outpatients presented for testing due to suspected acute respiratory infection between 1 January and 31 December 2024 to the Teaching Institute for Public Health of Split-Dalmatia County, Croatia, and multiplex real-time PCRs for 13 respiratory pathogens were included. Results: Out of 15,437 analyzed panels, 8878 (57.5%) were positive. Single-pathogen infections dominated (82.6%), while co-infections were recorded in 17.4% of panels; therefore, a total of 10,546 individual pathogens were detected, which were mostly viruses (87.0%). The following distribution of pathogens was observed: rhinovirus/enterovirus in 38.9% of positive results, influenza A virus in 14.5%, SARS-CoV-2 in 9.5%, parainfluenza virus in 7.9%, respiratory syncytial virus in 7.3%, Mycoplasma pneumoniae in 4.9%, Bordetella pertussis in 4.6%, human metapneumovirus in 4.2%, adenovirus in 3.4%, Chlamydia pneumoniae in 3.4%, influenza B virus in 1.3%, Bordetella parapertussis in 0.1% and Legionella pneumophila had one positive result. The first trimester of the year had the highest number of positive test panels (47.0%). Conclusions: Our study demonstrates a predominance of viral pathogens across all age groups and seasons, further supporting guideline-based practice and highlighting the importance of confirming bacterial infection before initiating antibiotic therapy. This insight into the post-pandemic circulation of respiratory pathogens may help inform public health strategies, including improved surveillance, anticipation of seasonal outbreaks, and targeted interventions, thereby supporting future pandemic preparedness and mitigation efforts. Full article