Search Results (453)

Plant-derived extracellular vesicles (PDEVs) are nanoscale membrane vesicles released by edible plants that deliver proteins, lipids, nucleic acids, and small metabolites to recipient cells, thereby modulating inflammation, barrier function, metabolism, and intercellular signaling. In recent years, PDEV research has advanced from concept and in vitro observations to engineering-ready systems with validation in animal models, encompassing oral, transdermal, and intranasal delivery paradigms. Among edible plants, the apple has broad consumption and a favorable safety profile; however, studies on apple-derived extracellular vesicles (ADEVs) lag behind those on other plant EVs. Accordingly, this review systematically summarizes ADEV progress across extraction methods, characterization, molecular cargo, and roles in disease settings. We highlight evidence gaps in animal efficacy and translation, and propose priorities including process standardization, harmonized critical quality attributes, in vivo biodistribution, and long-term safety. Our aim is to provide a reference for ADEV research and to accelerate the development of safe, low-cost, scalable bionanocarriers for disease therapy. Full article

Protein–DNA and protein–RNA interactions are central to gene regulation and genetic disease, yet experimental identification remains costly and complex. Machine learning (ML) offers an efficient alternative, though challenges persist in representing protein sequences due to residue variability, dimensionality issues, and the risk of losing biological context. Traditional approaches such as k-mer counting or neural network encodings provide standardized sequence representations but often demand high computational resources and may obscure functional information. To address these limitations, a novel encoding method based on interpolation of physicochemical properties (PCPs) is introduced. Discrete PCPs values are transformed into continuous functions using logarithmic enhancement, highlighting residues that contribute most to nucleic acid interactions while preserving biological relevance across variable sequence lengths. Statistical features extracted from the resulting spectra via Tsfresh are then used for binary classification of DNA- and RNA-binding proteins. Six classifiers were evaluated, and the proposed method achieved up to 99% accuracy, precision, recall, and F1 score when amino acid highlighting was applied, compared with 66% without highlighting. Benchmarking against k-mer and neural network approaches confirmed superior efficiency and reliability, underscoring the potential of this method for protein interaction prediction. Our framework may be extended to multiclass problems and applied to the study of protein variants, offering a scalable tool for broader protein interaction prediction. Full article

Bidens pilosa L. extract (BPE), a traditional medicine known for its antimicrobial properties, has not been thoroughly investigated for its potential against avian pathogenic Escherichia coli (APEC), a major pathogen responsible for severe economic losses and high mortality in poultry. This study aimed to comprehensively assess the antibacterial activity of BPE against APEC through both in vivo and in vitro experiments and to explore its underlying mechanisms. In a chicken infection model, BPE treatment led to an 80% cure rate and 20% mortality, in contrast to the 90% diarrhea and 70% mortality observed in the untreated model group. BPE also significantly alleviated intestinal tissue damage and reduced serum levels of inflammatory cytokines IL-6 and IL-1β (p < 0.01). In vitro analyses revealed a minimum inhibitory concentration (MIC) of 625 mg/mL. BPE dose-dependently suppressed bacterial motility, swarming, and biofilm formation (p < 0.01) and markedly increased membrane permeability, indicated by elevated release of nucleic acids, proteins, and alkaline phosphatase (p < 0.001). Moreover, PCR results showed that treatment with BPE at 1/2 MIC for 24 h significantly downregulated multiple virulence-associated genes, including aatA, papC, ibeB, vat, ompA, iss, fyuA, and irp2 (p < 0.01). These results demonstrate that BPE exerts its anti-APEC effects by damaging cell membrane integrity, inhibiting biofilm formation and motility, and suppressing virulence gene expression. Our findings support the potential of BPE as a natural alternative for controlling APEC infections and contribute a scientific basis for the use of traditional herbal medicine in combating bacterial diseases. Full article

Background/Objectives: Acute respiratory infections remain a major global public health concern affecting individuals across all ages. Accurate and rapid diagnosis of respiratory pathogens is crucial for effective patient management and infection control. Multiplex real-time polymerase chain reaction (PCR) assays have gained prominence over conventional methods for routine viral detection in clinical laboratories owing to their enhanced sensitivity and specificity; however, comparative performance data for PowerChek™ RVP remain limited. This study aimed to evaluate the diagnostic performance of the PowerChek™ Respiratory Virus Panel 1/2/3/4, which detects 16 respiratory viruses, including SARS-CoV-2, in nasopharyngeal swab (NPS) specimens. Methods: Overall, 336 NPS specimens were analyzed using the PowerChek™ RVP, BioFire^® RP 2.1plus, and Allplex™ RP assays, with nucleic acid extraction performed using the Advansure™ E3 system. The performance metrics were calculated using two-by-two contingency tables. Results: Among 336 NPS specimens (232 positive, 104 negative), PowerChek™ RVP detected 226 positives with minimal discrepancies, showing high concordance with BioFire^® RP 2.1plus (accuracy 94.6%, kappa 0.843–1.000). Fifteen discordant cases were identified in this study. Eleven could not be sequenced because of amplification failure and most had high Ct values (>30). Sequencing of four samples confirmed concordance with BioFire^® RP 2.1plus and PowerChek™ RVP, whereas Allplex™ RP showed false-negative results. Conclusions: The PowerChek™ RVP assay demonstrated a high level of relative sensitivity, specificity, accuracy, diagnostic predictive values and strong concordance with comparable reference assays in identifying its targets. This assay is a reliable and efficient diagnostic tool for clinical laboratories to facilitate the accurate identification of respiratory pathogens. Full article

Omsk hemorrhagic fever is an acute zoonotic disease caused by Omsk hemorrhagic fever virus, a member of the genus Flavivirus (family Flaviviridae), with a reported case-fatality rate of approximately 3%. Historically confined to southwestern Siberia, ecological changes raise concerns about possible spread to non-endemic regions. Although no Omsk hemorrhagic fever cases have been reported in the Republic of Korea, the risk of accidental importation highlights the importance of establishing a reliable diagnostic protocol. We established and validated an institutionally developed diagnostic protocol employing real-time reverse transcription polymerase chain reaction targeting the NS2A and C genes of Omsk hemorrhagic fever virus. Primers and probes were designed from all available genomes to ensure broad strain coverage. Human ribonuclease P was used as an internal control to verify nucleic acid extraction and amplification. Using synthetic deoxyribonucleic acid fragments and in vitro-transcribed ribonucleic acid, assay performance was optimized, and analytical sensitivity was determined using probit analysis. The limits of detection were 74.50 copies/µL (threshold cycle 32.99) for NS2A and 70.41 copies/µL (threshold cycle 35.38) for C. Specificity testing using representative flaviviruses (West Nile virus, Yellow fever virus, Zika virus, St. Louis encephalitis virus, and Tick-borne encephalitis virus) and an alphavirus (Venezuelan equine encephalitis virus) demonstrated no cross-reactivity. The assay demonstrated high sensitivity, specificity, and reproducibility, supporting its potential application in national and international Omsk hemorrhagic fever virus surveillance systems. Full article

Edible and medicinal plants provide a treasure trove of natural phytochemicals for mining the next generation of green food preservatives. Herein, we evaluated antibacterial activities of 55–95% ethanol extracts from the edible rhizome of Rumex madaio (RmEEs). The 75% ethanol extract displayed the strongest antibacterial activity, and its purified fraction 2 (RmEE-F2) blocked the proliferation of common pathogens Staphylococcus aureus and Vibrio cholerae, with minimum inhibitory concentrations (MICs) of 391 μg/mL. RmEE-F2 (1 × MIC) altered the bacterial cell surface biophysical parameters and impaired cell structure, resulting in intracellular nucleic acid and protein leakage. It manifested bacteriostatic rates of 88.21–91.17% against S. aureus and V. cholerae in spiked fish (Carassius auratus) and shrimp (Penaeus vannamei) during storage at 4 °C for 24 h. Meanwhile, RmEE-F2 effectively rendered the pH rising and reduced lipid oxidation and protein degradation of C. auratus and P. vannamei meat samples at 4 °C for 6 days. Additionally, RmEE-F2 (< 781 µg/mL) showed non-cytotoxicity to human colon Caco-2, liver HepG-2, and lung A549 cell lines, and rescued V. cholerae and S. aureus-infected Caco-2 cellcells with enhanced viability of 14.31–16.60% (1 × MIC). Comparative transcriptomic analysis revealed down-regulated protein synthesis, cell wall and cell membrane synthesis, and or DNA replication and repair in the tested bacteria triggered by RmEE-F2. The major antibacterial compounds in RmEE-F2 included melibiose (9.86%), 3-(N, N-dimethylaminomethyl) indole (7.12%), and citric acid (6.07%). Overall, this study underscores the promising potential of RmEE-F2 for aquatic product green preservation. Full article

Background/Objective: Timely identification of bloodstream pathogens is critical for sepsis management; however, PCR inhibitors such as sodium polyanetholesulfonate (SPS) in blood culture broth compromise nucleic acid recovery and long read sequencing. We assessed whether coupling a benzyl alcohol SPS-removal step to the fully automated LabTurbo AIO extractor improves Oxford Nanopore-based pathogen detection. Methods: Thirteen positive blood culture broths were pre-treated with benzyl alcohol and divided: half volumes were purified on the LabTurbo AIO; paired aliquots underwent manual QIAamp extraction. DNA purity was evaluated by NanoDrop and Qubit. Barcoded libraries were sequenced on MinION R9.4.1 flow cells for 6 h. Results: Automated eluates showed a median A₂₆₀/A₂₈₀ of 1.92 and A₂₆₀/A₂₃₀ of 1.96, versus 1.80 and 1.48 for manual extracts. The automated workflow generated 1.69 × 10⁶ total reads compared with 3.9 × 10⁵ reads for manual extraction. The median N50 read length increased from 5.9 kb to 8.7 kb, and the median proportion of reads classified to species increased from 62% to 84%. The hands-on time was <5 min and the sample-to-answer turnaround was <8 h, compared with >9 h and 90 min for the manual protocol, respectively. Conclusions: Benzyl alcohol SPS removal integrated into the LabTurbo AIO extractor yielded purer, longer, and higher read counts, enhancing nanopore sequencing depth and accuracy while compressing diagnostic turnaround to a single working day. This represents a practical advance for rapid blood culture pathogen identification in critical care settings. Full article

Objective: This study aimed to validate the clinical utility of a salivary molecular platform (Oral Predict^®) for periodontal pathogen detection across preventive, therapeutic, and maintenance settings. Methods: A longitudinal randomized study was conducted involving 78 adults who provided saliva samples at baseline, one month, and three months after professional dental hygiene. Participants were randomized into two groups: control group (n = 39) and probiotic group with Oral Predict^® probiotic supplementation (n = 39). Crude saliva was processed directly without nucleic acid extraction and analyzed by multiplex real-time PCR using either the compact Real-time PCR system or standard thermocyclers. Results: At baseline, Fusobacterium nucleatum was the most prevalent pathogen (84.6%), followed by Tannerella forsythia (53.8%) and Porphyromonas gingivalis (46.2%). The Total Pathogen Burden Score (TPBS) showed progressive increases with age, smoking, and poor oral hygiene, and was significantly higher in participants with gingival bleeding. Among individual pathogens, no significant associations were observed with periodontitis staging or grading. Professional hygiene induced mean reductions of 1–2 logs across all pathogens, with TPBS decreasing from 8.7 ± 3.2 to 4.1 ± 2.8 (p < 0.001). At three months, 69.2% of the control group experienced bacterial rebound, whereas 85% of probiotic users sustained or improved bacterial reductions. Conclusions: Salivary molecular testing provides a robust, non-invasive approach for periodontal pathogen detection, treatment monitoring, and long-term maintenance assessment. The flexibility of the Oral Predict^® platform across point-of-care and laboratory settings, combined with automated interpretation, supports integration into preventive protocols and personalized periodontal care. These findings demonstrate the potential of saliva-based molecular diagnostics to shift periodontal management from reactive to predictive and precision-based strategies. Full article

Viral metagenomic next-generation sequencing (vmNGS) has transformed our capacity for the untargeted detection and characterisation of (re)emerging zoonotic viruses, surpassing the limitations of traditional targeted diagnostics. In this review, we critically evaluate the current landscape of vmNGS, highlighting its integration within the One Health paradigm and its application to the surveillance and discovery of (re)emerging viruses at the human–animal–environment interface. We provide a detailed overview of vmNGS workflows including sample selection, nucleic acid extraction, host depletion, virus enrichment, sequencing platforms, and bioinformatic pipelines, all tailored to maximise sensitivity and specificity for diverse sample types. Through selected case studies, including SARS-CoV-2, mpox, Zika virus, and a novel henipavirus, we illustrate the impact of vmNGS in outbreak detection, genomic surveillance, molecular epidemiology, and the development of diagnostics and vaccines. The review further examines the relative strengths and limitations of vmNGS in both passive and active surveillance, addressing barriers such as cost, infrastructure requirements, and the need for interdisciplinary collaboration. By integrating molecular, ecological, and public health perspectives, vmNGS stands as a central tool for early warning, comprehensive monitoring, and informed intervention against (re)emerging viral threats, underscoring its critical role in global pandemic preparedness and zoonotic disease control. Full article

Pulmonary neuroendocrine tumors (PULMONARY NETs) are heterogeneous tumors ranging from well-differentiated to highly aggressive neoplasms. The aim of this study is to prospectively test pre-operative circulating free DNA (cfDNA) in PULMONARY NET patients undergoing surgery and evaluate its relationship to clinicopathological features. From February to December 2024, 136 patients with suspected primary lung cancer underwent pre-operative blood sampling, of whom 21 were diagnosed with PULMONARY NETs. Total cell-free nucleic acid extraction was performed using the Genexus Purification System (Thermofisher). cfDNA was quantified using a fluorometric assay with the Qubit dsDNA HS Assay kit (Thermofisher) and a capillary electrophoresis-based assay (cell-free DNA ScreenTape kit) on the Tape Station 4200 systems (Agilent). A cfDNA quality assessment was also obtained (cfDNA sizing and % cfDNA). Most patients had Stage I (18/21.85.7%) typical carcinoids (16/21.76.2%). Nodal involvement was detected in one patient (0.5%). Six months after surgery, all patients were alive without recurrence. Larger tumors presented higher levels of cfDNA. The mean tumor size in patients with cfDNA > 40 ng was 266 mm (±16.7 mm), compared to 13.2 mm (±7.3 mm) for cfDNA < 40 ng (p-value = 0.018). Higher levels of cfDNA were observed in patients with pStages greater than IA (p-value = 0.007). Although limited by a small sample group and biases of a surgical series, we observed that larger/advanced PULMONARY NETs presented higher cfDNA levels pre-operatively. Full article

Background/Objectives: We have previously reported the engineering of a point-of-care (POC) system that fully automates the procedures for nucleic acid extraction and multiplexed real-time RT-PCR, with a major advantage of high-level multiplexing. In this study, we applied and validated the system in a respiratory tract infection setting. Methods: An automatic nested real-time RT-PCR assay was developed (POCm). It was a 40-plex assay that simultaneously detected 39 epidemiologically important respiratory pathogens in 1.5 h in the POC system. The analytical and clinical performance was evaluated. Results: The analytical sensitivities of the POCm assay were comparable to those of its single-plex counterparts performed manually on a bench-top. The minimum detectable concentrations ranged from 53 copies/mL to 5.3 × 10³ copies/mL for all pathogen targets except hCoV-NL63 (5.3 × 10⁴ copies/mL). The quantitative performance was demonstrated by the linear correlations between Ct values and input concentrations for all pathogen targets, with 24 of them demonstrating coefficients of correlation (r) greater than 0.9. The POCm assay was subsequently evaluated in 283 clinical samples. A high level of agreement (98.2–100%) was achieved for pathogen detection results between POCm and standard diagnostic methods. The POCm result was also fully concordant with the result of another commercial POC multiplex platform. For positive clinical samples, pairwise Ct values measured by POCm closely correlated with those of the bench-top reference method (r = 0.70). The feasibility of mutation genotyping of the viral subtype was further demonstrated. Conclusions: This study demonstrated the practicality of POCm for routine testing in clinical laboratories. Further clinical trials are being conducted to evaluate the clinical performance of the system. Full article

This study introduces a novel nucleic acid testing (NAT) protocol that integrates rapid deoxyribonucleic acid (DNA) extraction, isothermal amplification, and visual detection to enable efficient analysis of opportunistic pathogens. Polyethylenimine-functionalized iron oxide (PEI-Fe₃O₄) nanoparticles were prepared by combining PEI, acting as a stabilizing agent, with iron salt, which was utilized as the metal ion precursor by the ultrasonication-assisted co-precipitation method, and characterized for structural, optical, and magnetic properties. PEI-Fe₃O₄ exhibited cationic and anionic behavior in response to pH variations, enhancing adaptability for DNA binding and release. PEI-Fe₃O₄ enabled efficient extraction of E. faecium DNA within 10 min at 40 °C, yielding 17.4 ng/µL and achieving an extraction efficiency of ~59% compared to a commercial kit (29.5 ng/µL). The extracted DNA was efficiently amplified by loop-mediated isothermal amplification (LAMP) at 65 °C for 45 min. Pyrogallol-rich poly(tannic acid)-stabilized gold nanoparticles (PTA-AuNPs) served as colorimetric probes for direct visual detection of the DNA amplified using LAMP. The magnetic-nanogold (PEI-Fe₃O₄/PTA-AuNPs) hybrid system achieved a limit of quantification of 1 fg/µL. To facilitate field deployment, smartphone-based RGB analysis enabled quantitative and equipment-free readouts. Overall, the PEI-Fe₃O₄/PTA-AuNPs hybrid system used in NAT offers a rapid, cost-effective, and portable solution for DNA detection, making the system suitable for microbial monitoring. Full article

Background/Objectives: Grey zone serologic results in blood donor screening pose challenges for transfusion safety, donor management, and blood supply sustainability. In Saudi Arabia, standardized national protocols for managing grey zone outcomes remain lacking. This study aimed to evaluate the prevalence and follow-up outcomes of grey zone serologic results among blood donors at a Saudi hospital over a five-year period. Methods: Serological screening results of six transfusion-transmissible infections (TTIs) markers were extracted alongside nucleic acid testing (NAT) results for HBV, HCV, and HIV. The grey zone was defined as a signal-to-cutoff (S/CO) of 0.90–0.99. Repeat and follow-up results, including subsequent donations, were assessed for seroconversion. Results: A total of 48,241 donations from 38,524 donors were analyzed. Anti-HBc showed the highest reactivity (n = 2312; 4.8%), followed by HbsAg (n = 2292; 0.31%) and syphilis (n = 218; 0.5%). Grey zone results were rare, and most frequent in anti-HBc (n = 76; 0.16%), HCV (n = 39; 0.08%), and HBsAg (n = 28; 0.06%). Grey zone-to-reactive conversion upon subsequent donation was rare. Three donors who initially tested in the grey zone for anti-HBc later tested reactive in subsequent donations, but their HBV NAT remained negative. Conclusions: While grey zone outcomes were infrequent, a subset involving HBV markers showed low-level reactivity on repeat testing. For other TTIs markers, grey zone results likely reflected assay variability rather than true infection. We propose a six-month temporary deferral with follow-up serologic and NAT testing, allowing conditional re-entry for donors with consistently non-reactive results, supporting both transfusion safety and a more sustainable donor pool. Full article

Background: Bone tissue decalcification is essential for histopathological evaluation, but conventional methods using inorganic acids degrade nucleic acids, limiting molecular testing. EDTA is known to better preserve DNA, but its suitability for next-generation sequencing (NGS) in clinical settings remains to be validated. Methods: This retrospective study evaluated 752 formalin-fixed paraffin-embedded (FFPE) tissue samples undergoing NGS between January 2022 and October 2024. Of these, 31 were decalcified using EDTA (Osteosoft, Merck, Germany). DNA was extracted using the Qiagen AllPrep^® kit and quantified using Qubit and NanoDrop. Libraries were prepared with a custom 30-gene Ampliseq panel and sequenced on the Ion Torrent platform. Sequencing was deemed suboptimal if <95% of target regions reached ≥250X depth. Results were compared to 721 non-decalcified FFPE samples. Results: Suboptimal sequencing occurred in 9.7% of EDTA-decalcified and 9.0% of non-decalcified cases (p = 0.9). DNA concentration (Qubit) and NanoDrop 260/280 ratios were not significantly different (p = 0.4 and p = 0.8, respectively), though EDTA cases had lower DNA concentrations (NanoDrop, p = 0.006) and 260/230 ratios (p = 0.002). Mutation detection in decalcified samples was consistent with known mutation profiles for respective tumor types. Conclusions: EDTA-decalcified FFPE bone tissues produce NGS results comparable to non-decalcified specimens, with similar sequencing success rates and acceptable DNA quality. These findings support the use of EDTA as a suitable decalcification method for molecular diagnostics, enabling broader inclusion of bone specimens in clinical testing. Full article

Background: Circulating miRNAs have been identified as potential biomarkers for the early diagnosis and monitoring of cancers. However, limitations of polymerase chain reaction (PCR)-based methods are currently delaying the transition of miRNA research into clinical practice. These include labour-intensive workflows, exposure to errors and difficulties in detecting and quantifying low-abundance miRNAs. Objectives: This review emphasizes the need to develop amplification-free (“PCR-free”) technologies to improve the reliability, scalability and practicality of miRNA diagnostics in clinical settings. Methods: This review explores recent advances in PCR-free technologies developed over the past five years. It focuses on innovative methods, such as bead-based assays and sensor detection platforms, which serve as valuable alternatives to conventional PCR-based approaches. These emerging technologies have the potential to overcome the key limitations of PCR by offering streamlined workflows, reduced error rates and enhanced compatibility with a variety of clinical sample types. Crucially, they enable absolute quantification without the need for pre-nucleic acid extraction, reverse transcription or amplification, as well as the simultaneous detection of multiple miRNAs within a single assay. These provide cost-effective and scalable solutions for comprehensive biomarker profiling. The transition from PCR-based to PCR-free technologies is a significant step forward in miRNA diagnostics, overcoming long-standing technical barriers and paving the way for broader adoption of miRNA analysis in routine clinical settings. This shift supports the advancement of precision medicine and holds promises for improving early cancer detection. Full article