Search Results (500)

The CRISPR/Cas system has attracted increasing attention in accurate nucleic acid detection. Herein, we reported a CRISPR/Cas12a-chemiluminescence cascaded bioassay (CCCB) for the amplification-free and sensitive detection of human papillomavirus type 16 (HPV-16) and parvovirus B19 (PB-19). A magnetic bead (MB)-linking single-stranded DNA (LssDNA)-alkaline phosphatase (ALP) complex was constructed as the core component of the bioassay. During the detection process, the single-stranded target DNA was captured and enriched by LssDNA and then activated the trans-cleavage activity of Cas12a. Due to the Cas12a-mediated cleavage of LssDNA, ALP was released from the MB, subsequently catalyzing the substrate to generate a chemiluminescence (CL) signal. Given the cascade combination of CRISPR/Cas12a with the CL technique, the limits of detection for HPV-16 and PB-19 DNA were determined as 0.14 pM and 0.37 pM, respectively, and the whole detection could be completed within 60 min. The practicality and reliability of the platform were validated through target-spiked clinical specimens, and the recovery rate was 93.4–103.5%. This dual-amplification strategy—operating without target pre-amplification—featured high specificity, low contamination risk, facile preparation, and robust stability. It provides a novel approach for sensitive nucleic acid detection, with the potential for rapid extension to the diagnosis of various infectious diseases. Full article

Perennial ryegrass (Lolium perenne), an important forage and turfgrass species, can establish a mutualistic symbiosis with the fungal endophyte Epichloë festucae var. lolii. Although the physiological and ecological impacts of endophyte infection on ryegrass have been extensively investigated, the response of the soil microbial community and nitrogen-cycling gene to this relationship has received much less attention. The present study emphasized abundance and diversity variation in the AOB-amoA, nirK and nosZ functional genes in the rhizosphere soil of the endophyte–ryegrass symbiosis following litter addition. We sampled four times: at T₀ (prior to first litter addition), T₁ (post 120 d of 1st litter addition), T₂ (post 120 d of 2nd litter addition) and T₃ (post 120 d of 3rd litter addition) times. Real-time fluorescence quantitative PCR (qPCR) and PCR amplification and sequencing were used to characterize the abundance and diversity of the AOB-amoA, nirK and nosZ genes in rhizosphere soils of endophyte-infected (E+) plants and endophyte-free (E−) plants. A significant enhancement of total Phosphorus (P), Soil Organic Carbon (SOC), Ammonium ion (NH₄⁺) and Nitrate ion (NO₃⁻) contents in the rhizosphere soil was recorded in endophyte-infected plants at different sampling times compared to endophyte-free plants (p ≤ 0.05). The absolute abundance of the AOB-amoA gene at T₀ and T₁ times was higher, as was the absolute abundance of the nosZ gene at T₀, T₁ and T₃ times in the E+ plant rhizophere soils relative to E− plant rhizosphere soils. A significant change in relative abundance of the AOB-amoA and nosZ genes in the host rhizophere soils of endophyte-infected plants at T₁ and T₃ times was observed. The experiment failed to show any significant alteration in abundance and diversity of the nirK gene, and diversity of the AOB-amoA and nosZ genes. Analysis of the abundance and diversity of the nirK gene indicated that changes in soil properties accounted for approximately 70.38% of the variation along the first axis and 16.69% along the second axis, and soil NH₄⁺ (p = 0.002, 50.4%) and soil C/P ratio (p = 0.012, 15.8%) had a strong effect. The changes in community abundance and diversity of the AOB-amoA and nosZ genes were mainly related to soil pH, N/P ratio and NH₄⁺ content. The results demonstrate that the existence of tripartite interactions among the foliar endophyte E. festucae var. Lolii, L. perenne and soil nitrogen-cycling gene has important implications for reducing soil losses on N. Full article

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by the SFTS virus (SFTSV). A rapid and cost-effective point-of-care testing detection system is important for the early diagnosis of SFTS. Herein, we developed a ready-to-use dried reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the direct detection of SFTSV in clinical samples. The assay enables simple, RNA-extraction-free detection using heat-treated serum or plasma, followed by a 30 min incubation at 65 °C. The results are visually interpreted through the color emitted, which can be observed under LED light. The established assay demonstrated detection sensitivity for SFTSV at 10⁴ copies/µL and was effective in identifying infections in cats. Despite being less sensitive than real-time RT-PCR, this dried RT-LAMP method offers a rapid, cost-effective alternative suitable for point-of-care use, particularly in remote or resource-limited settings. The simplified workflow and visual readout make it a practical tool for the early detection and daily surveillance of SFTSV in animals. Full article

Investigating the seismic damage mechanism of large underground complexes is essential for the safe development of urban underground space. This paper examines a five-story and three-span underground complex situated in a soft soil site. Shaking table tests were designed and conducted on both the free field and the soil–underground complex interaction system. The time–frequency evolution of the free field under various seismic motions was investigated. A combined experimental and numerical simulation approach was employed to examine the seismic response of the soil–underground complex interaction system. The structural deformation evolution, stress distribution, and development process of plastic damage under different seismic motions were analyzed. The results reveal that soft soil exhibits a significant energy amplification effect under far-field long-period ground motions. Structural deformation is mainly governed by horizontal shear. Under strong seismic excitation, plastic damage first initiates at the end of the bottom-story columns and extends to column-to-slab and wall-to-slab connections, where abrupt stiffness changes occur. Under the far-field long-period ground motion, the structural deformation, stress distribution, and plastic damage are significantly greater than those under the Shanghai artificial wave. These findings provide valuable insights for the seismic design of large underground complexes in soft soil sites. Full article

Accurate and sensitive detection of protein biomarkers is critical for advancing in vitro diagnostics (IVD), yet conventional enzyme-linked immunosorbent assays (ELISA) often fall short in terms of sensitivity compared to nucleic acid-based tests. Bridging this sensitivity gap is essential for improving diagnostic accuracy, particularly in diseases where protein levels better reflect disease progression than nucleic acid biomarkers. In this review, we present strategies developed to enhance the sensitivity of ELISA, structured according to the sequential steps of the assay workflow. Beginning with surface modifications, we then discuss the methodologies to improve mixing and washing efficiency, followed by a summary of recent advances in signal generation and amplification techniques. In particular, we highlight the emerging role of cell-free synthetic biology in augmenting ELISA sensitivity. Recent developments such as expression immunoassays, CRISPR-linked immunoassays (CLISA), and T7 RNA polymerase–linked immunosensing assays (TLISA) demonstrate how programmable nucleic acid and protein synthesis systems can be integrated into ELISA workflows to surpass the present sensitivity, affordability, and accessibility. By combining synthetic biology-driven amplification and signal generation mechanisms with traditional immunoassay formats, ELISA is poised to evolve into a highly modular and adaptable diagnostic platform, representing a significant step toward the next generation of highly sensitive and programmable immunoassays. Full article

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression and have emerged as critical biomarkers in various diseases, including cancer. Their stability in bodily fluids and role as oncogenes or tumor suppressors make them attractive targets for non-invasive diagnostics. However, conventional detection methods, such as Northern blotting, RT-PCR, and microarrays, are limited by low sensitivity, lengthy protocols, and limited specificity. Electrochemical biosensors offer a promising alternative, providing high sensitivity, rapid response times, portability, and cost-effectiveness. These biosensors translate miRNA hybridization events into quantifiable electrochemical signals, often leveraging redox-active labels, mediators, or intercalators. Recent advancements in nanomaterials and signal amplification strategies have further enhanced detection capabilities, enabling sensitive, label-free miRNA quantification. This review provides a comprehensive overview of the recent advances in electrochemical biosensing of miRNAs, emphasizing innovative redox-based detection strategies, probe immobilization techniques, and hybridization modalities. The critical challenges and future perspectives in advancing electrochemical miRNA biosensors toward clinical translation and point-of-care diagnostics are discussed. Full article

DNA methylation, as a critical epigenetic modification, plays a central role in gene regulation and has emerged as a powerful biomarker for early disease diagnostics, particularly in cancer. Owing to the limitations of traditional bisulfite sequencing—such as high cost, complexity, and chemical degradation—electrochemical biosensors have gained substantial attention as promising alternatives. This review summarizes recent advancements in electrochemical platforms for bisulfite-free detection of DNA methylation, encompassing direct oxidation strategies, enzyme-assisted recognition (e.g., restriction endonucleases and methyltransferases), immunoaffinity-based methods, and a variety of signal amplification techniques such as rolling circle amplification and catalytic hairpin assembly. Additional approaches, including strand displacement, magnetic enrichment, and adsorption-based detection, are also discussed. These systems demonstrate exceptional sensitivity, often down to the attomolar or femtomolar level, as well as high selectivity, reproducibility, and suitability for real biological matrices. The integration of nanomaterials and redox-active probes further enhances analytical performance. Importantly, many of these biosensing platforms have been validated using clinical samples, reinforcing their translational relevance. The review concludes by outlining current challenges and future directions, emphasizing the potential of electrochemical biosensors as scalable, cost-effective, and minimally invasive tools for real-time epigenetic monitoring and early-stage disease diagnostics. Full article

The use of analog radio-over-fiber (RoF) systems combined with power-over-fiber (PoF) systems has been proposed in recent years for applications involving remote sensors used in hazardous environments or where electrical wiring may be impractical. This article presents a hybrid architecture topology that combines PoF and RoF, using Raman amplification to obtain RF gain. The first emphasis is placed on the use of two types of high-power laser sources (HPLSs) for the PoF system: a 1480 nm Raman-based HPLS and a 1550 nm HPLS that is based on an erbium-doped fiber amplifier (EDFA). The second emphasis of this paper is on how these two HPLSs simulate Raman scattering (SRS) in the fiber, considering different lengths of SMF 28 for the link. Thus, a comparative analysis is proposed considering the effects induced on the RF signal, mainly focused on its RF power gain ($G_{RF}$), noise figure (NF), and spurious-free dynamic range (SFDR). The obtained results show that the architecture using a PoF system based on the 1550 nm HPLS benefits from a lower noise figure degradation, even when the noise generated by the optical amplification is considered. Full article

Background and Aim: Glioblastoma (GBM) is the most aggressive primary brain tumor in adults, with poor prognosis despite maximal surgical resection, radiotherapy (RT), and temozolomide (TMZ) per the Stupp protocol. IDH-wild-type GBM, the predominant molecular subtype, frequently harbors EGFR amplification and is resistant to therapy, while MGMT promoter methylation predicts improved TMZ response. This study aimed to assess the prognostic impact of EGFR and MGMT status on survival and recurrence patterns in IDH-wild-type GBM. Materials and Methods: We retrospectively analyzed 218 patients with IDH-wild-type GBM treated at the Azienda Ospedaliero-Universitaria Senese (2016–2024). All patients underwent maximal safe surgical resection whenever feasible. The cohort includes patients who received gross total resection (GTR), subtotal resection (STR), or biopsy only, depending on tumor location and clinical condition, followed by intensity-modulated RT (59.4–60 Gy) with concurrent and adjuvant TMZ. EGFR amplification was assessed via FISH/NGS and immunohistochemistry; MGMT promoter methylation was determined using methylation-specific PCR. Progression-free survival (PFS), overall survival (OS), and recurrence patterns (in-field, marginal, out-field) were evaluated using Kaplan–Meier, Cox regression, and logistic regression analyses. Results: Among patients (64.7% male; mean age 61.8), 58.7% had EGFR amplification and 49.1% showed MGMT methylation. Median OS and PFS were 14 and 8 months, respectively. EGFR non-amplified/MGMT methylated tumors had the best outcomes (OS: 22.0 months, PFS: 10.5 months), while EGFR-amplified/MGMT unmethylated tumors fared worst (OS: 10.0 months, PFS: 5.0 months; p < 0.001). MGMT methylation was an independent positive prognostic factor (HR: 0.48, p < 0.001), while EGFR amplification predicted worse survival (HR: 1.57, p = 0.02) and higher marginal recurrence (OR: 2.42, p = 0.01). Conclusions: EGFR amplification and MGMT methylation significantly influence survival and recurrence dynamics in IDH-wild-type GBM. Incorporating these biomarkers into treatment planning may enable tailored therapeutic strategies, potentially improving outcomes in this challenging disease. Prospective studies are needed to validate biomolecularly guided management approaches. Full article

Background and Objectives: The clinical value of HER2-low breast cancer (BC), defined by immunohistochemistry (IHC) scores of 1+ or 2+/ISH-negative without HER2 amplification, remains unclear in the neoadjuvant setting. This study aimed to determine whether HER2-low and HER2-zero tumors differ in pathological complete response (pCR) rates and disease-free survival (DFS) among early-stage breast cancer patients undergoing neoadjuvant chemotherapy (NAC). Materials and Methods: We retrospectively analyzed 134 early BC patients treated with NAC between 2017 and 2023. Patients were categorized as HER2-zero (IHC 0) or HER2-low (IHC 1+ or 2+/ISH–). The primary endpoint was total pCR (tpCR); secondary endpoints included breast (bpCR), nodal (npCR), and radiologic complete response (rCR), alongside DFS analysis stratified by hormone receptor (HR) status. Results: Of the cohort, 91 patients (67.9%) were HER2-zero and 43 (32.1%) were HER2-low. There was no statistically significant difference in tpCR (26.4% vs. 27.9%, p = 0.852), bpCR (28.6% vs. 30.2%, p = 0.843), npCR (37.4% vs. 32.6%, p = 0.588), and rCR (23.1% vs. 30.2%, p = 0.374) between HER2-zero and HER2-low groups. DFS did not significantly differ between HER2-zero and HER2-low groups overall (p = 0.714), nor within HR-positive (p = 0.540) or TNBC (p = 0.523) subgroups. Conclusions: HER2-low tumors demonstrated similar pathological responses and survival outcomes compared to HER2-zero tumors. While a HER2-low status does not appear to define a distinct biological subtype in early BC, it remains a relevant classification for emerging HER2-targeted therapies, needing further investigation in prospective studies. Full article

Background/Objectives: Intrachromosomal amplification of chromosome 21 (iAMP21) represents a rare and heterogeneous distinct cytogenetic subgroup of B-cell precursor acute lymphoblastic leukemia (ALL) initially associated with a poor prognosis. Treatment intensification with additional doses of methotrexate and asparaginase was associated with better treatment outcomes. Methods: In this retrospective single-center study, we evaluated the impact of iAMP21 on treatment outcome in a cohort of pediatric patients treated with an intensified asparaginase regimen and describe the genomic landscape of four patients with iAMP21. Results: Four out of 89 patients > 1 year old were classified as iAMP21 positive. Five-year event-free survival (EFS) was inferior in the iAMP21-positive group: 25% versus 85.6% (p = 0.001). The cumulative incidence of relapse and treatment-related mortality were 50% vs. 9.9% and 0% vs. 2.38%, respectively, in the iAMP21-positive and non-iAMP21 groups (p = 0.02 and 0.76, respectively). These results did not translate into a significant difference in overall survival: 100% vs. 93.7% (p = 0.6). The presence of iAMP21 (HR 7.68, 95% CI 2.04–29.05; p = 0.002) and a measurable residual disease ≥1% after induction on day +33 (HR 8.82, 95% CI 2.6–29.91; p = 0.001) retained significant negative impact on EFS in multivariate analysis. Conclusions: We found an independent significant prognostic impact of iAMP21 on EFS among pediatric patients with ALL, and clinical presentation and early treatment response did not classify these patients as HR. Diverse genetic backgrounds among iAMP21-positive patients might influence the treatment response and outcome of this heterogeneous disease. Full article

Background/Objectives: Lymph node metastases (LNM) undetected by standard hematoxylin and eosin (H&E) have been associated with unfavorable prognosis in colorectal cancer. The One-Step Nucleic Acid Amplification (OSNA) assay has demonstrated superior sensitivity in detecting LNM compared to H&E. We aimed to assess the performance of OSNA in detecting LNM, as well as its prognostic value in rectal cancer (RC) patients. Methods: Lymph nodes (LNs) of patients from 15 centers were analyzed by both H&E and OSNA. The total tumor load (TTL) was defined as the sum of cytokeratin 19 mRNA copies/µL in all LNs from a surgical specimen, using a threshold of 250 copies/μL for OSNA positivity. Cox proportional hazard regression was used to assess the effect of TTL ≥ 250 or 6000 copies/μL on cancer-specific survival (CSS) and recurrence-free survival (RFS), with Firth’s method applied to account for low event rate. Results: A total of 97 RC patients were included. Of these, 84 patients were eligible for survival analysis. The sensitivity and specificity of OSNA, compared to H&E, were 91.7% and 84.7%, respectively. TTL ≥ 6000 versus <6000 copies/μL was related to worse CSS and RFS. When dividing TTL into three groups: ≤250, 250–6000, and >6000 copies/μL, only TTL ≥ 6000 copies/μL was significantly associated with worse CSS and RFS. Conclusions: The OSNA assay is highly sensitive for detecting LNM in RC patients. A TTL of ≥6000 copies/μL could identify a subset of RC patients with worse CSS and RFS who might benefit from adjuvant treatment or intensive surveillance. Full article

The seismic design of underground or aboveground structures is commonly based on the free-field assumption, which neglects the interaction between underground structures–soil–aboveground structures (USSI). This simplification may lead to unsafe or overly conservative, cost-intensive designs. To address this limitation, a series of shaking table tests were conducted on a coupled USSI system, in which the underground component consisted of a subway station connected to tunnels through structural joints to investigate the “city effect” on-site seismic response, particularly under long-period horizontal seismic excitations. Five test configurations were developed, including combinations of one or two aboveground structures, with or without a subway station. These were compared to a free-field case to evaluate differences in dynamic characteristics, acceleration amplification factors (AMFs), frequency content, and response spectra. The results confirm that boundary effects were negligible in the experimental setup. Notably, long-period seismic inputs had a detrimental impact on the field response when structures were present, with the interaction effects significantly altering surface motion characteristics. The findings demonstrate that the presence of a subway station and/or aboveground structure alters the seismic response of the soil domain, with clear dependence on the input motion characteristics and relative structural positioning. Specifically, structural systems lead to de-amplification under high-frequency excitations, while under long-period inputs, they suppress short-period responses and amplify long-period components. These insights emphasize the need to account for USSI effects in seismic design and retrofitting strategies, particularly in urban environments, to achieve safer and more cost-effective solutions. Full article

Cell-free DNA (cfDNA), a fragmented DNA circulating in blood, is a promising biomarker for cancer diagnosis and monitoring. Standardization of cfDNA isolation to enhance the sensitivity of molecular analyses in prostate cancer (PCa) is required. Towards this goal, we optimized existing methods to obtain a high quantity and quality of cfDNA from low volumes of plasma. The protocol was applied to samples from healthy males and three patient categories: radical prostatectomy (RP), disease-free (>6 years post-RP), and metastatic castration-resistant PCa (mCRPC). The yield was significantly higher in mCRPC cases, and the size of fragments was shorter. We compared for the first time library preparation using two cfDNA inputs and low vs. high sequencing depth. Clonal events were observed irrespective of input and depth, but lower input showed more subclonal events. The clinical application of the refined protocols to cfDNA samples from an mCRPC patient showed no tumor fraction before RP, while it increased to 25% at the advanced stage. Among chromosomal changes and mutations, the androgen receptor gene amplification was detected. Altogether, this comprehensive study on improved cfDNA procedures is highly promising to enhance the quality of liquid biopsy-based research for discoveries and much-needed clinical applications. Full article

Circulating tumour DNA (ctDNA) is a promising biomarker for personalised oncology. However, its clinical utility is limited by detection sensitivity, particularly in early-stage disease. T-Oligo Primed Polymerase Chain Reaction (TOP-PCR) is a commercial amplification approach utilising an efficient “half-adapter” ligation design and a single-primer-based PCR strategy. This study evaluated the clinical value and application of cell-free DNA (cfDNA) pre-amplification. cfDNA amplification with TOP-PCR preserved DNA size profiles and resulted in a 22 bp size increase due to the half-adaptor ligation. Gene target amplification rates varied, showing lower efficiency for the GC-rich TERT promoter amplicon and higher efficiency for the BRAF and TP53 amplicons. Optimised pre-amplification (20 ng cfDNA input and 5–7 cycles of PCR) enhanced ctDNA detection sensitivity and expanded sample availability for the detection of multiple tumour-informed mutations. Importantly, PCR errors emerged in pre-amplified cfDNA samples, underscoring the necessity for negative controls and the establishment of stringent mutation positivity thresholds. Full article