Search Results (1,850)

For accurate and reliable monitoring, compliance monitoring devices (CMDs) in Port State Control must meet strict and uniform quality standards. This study evaluates how effectively CMDs, using variable fluorescence (VF) and fluorescein diacetate (FDA) technologies, detect live organisms in the 10–50 μm size range. Employing a detailed analytical framework, we analyzed key performance indicators, including accuracy, precision, sensitivity, specificity, trueness, detection limits, and reliability by comparing CMD outputs to those of traditional microscopic methods. Reliability assessments revealed that VF-type CMD and FDA-type CMD performed robustly, with a stability rate of 99% for both, surpassing the 90% verification threshold. Precision analysis indicated an average CV exceeding 0.25; however, some samples, especially those below the D-2 standard, achieved a CV of less than 0.25. Concordance evaluations revealed that VF-CMDs and FDA-CMDs achieved rates of 63% and 55%, respectively, falling short of the 80% verification standard and underscoring the need for further calibration or optimization. Structural equation modeling shows that organism density significantly influences CMD performance. These findings underscore the challenges of accurately detecting low organism concentrations, further complicated by biological diversity and environmental variability. Despite their limitations in assessing ballast water compliance, CMDs are effective initial screening tools. Full article

Gamma-hydroxybutyric acid (GHB) is a short-chain fatty acid with both endogenous and exogenous origins, complicating its detection in clinical and forensic toxicology. Due to its rapid metabolism and short detection window in conventional biological matrices, identifying reliable biomarkers of GHB exposure is crucial. This literature review aims to assess current knowledge on potential GHB biomarkers that may extend the detection window or improve specificity. A systematic search of scientific databases was conducted to identify studies investigating GHB metabolites, conjugates, and related biochemical markers using advanced analytical techniques such as LC-MS/MS and GC-MS. The review highlights promising candidates, including glycolic acid, carnitin-GHB, and glycin-GHB, as well as 3,4-dihydroxybutyric acid, which show potential for distinguishing exogenous intake. However, significant interindividual variability and limited validation studies hinder their widespread implementation. Despite promising findings, further research is needed to confirm the specificity, stability, and reproducibility of these biomarkers. This review underscores the importance of developing standardized protocols to enhance GHB exposure detection in both clinical and forensic settings. Full article

Root-knot (Meloidogyne spp.) and false root-knot (Nacobbus aberrans) nematodes limit greenhouse tomato productivity. The effectiveness of integrating pre-plant biofumigation with post-plant chemical, biological, and botanical inputs was assessed under commercial conditions. A split-plot trial (2019) contrasted biofumigated and non-biofumigated whole plots (50 t ha⁻¹ of sorghum residues plus poultry and sheep manures) and 13 subplot treatments (fluopyram, Purpureocillium lilacinum, Pochonia chlamydosporia, Trichoderma viride, Tagetes erecta, and plant oil formulations). Nematodes were sampled 0, 60, and 120 days after transplanting, and the area under the nematode population curve (AUNPC), area under the root-damage curve (AURDC), and yield were analyzed. Biofumigation reduced pre-transplant N. aberrans populations by 86% and lowered the AUNPC by 39% relative to the non-biofumigated treatment; the whole-plot yields did not differ. Meloidogyne incognita remained at a very low density throughout. Among the subplot treatments, fluopyram decreased the AURDC by ≈22% and more than doubled the yield (63 vs. 26 t ha⁻¹; +142%), while the AUNPC of N. aberrans was unchanged. Biological and botanical packages reduced damage indices in some cases but did not increase the yield. No whole-plot × subplot interaction was detected for the yield. The results indicate that sorghum-based biofumigation, complemented by a low-risk nematicide at transplanting, can be embedded in integrated nematode-management programs for greenhouse tomato. Full article

Background/Objectives: Pyrogens, fever-inducing substances from biological or environmental sources, are recognized by Toll-like receptors (TLRs) predominantly expressed by human monocytes and represent a critical quality attribute (CQA) for pharmaceutical safety. The rabbit pyrogen test (RPT), widely used for pyrogen assessment, suffers from high variability, limited accuracy, and poor reproducibility, particularly for vaccines containing inherent pyrogens such as outer membrane protein complex (OMPC)-based vaccines. Existing in vitro alternatives using peripheral blood mononuclear cells (PBMCs) are challenged by donor-to-donor variability and the operational complexity of ELISA readouts. To support the 3Rs (Refinement, Reduction, Replacement) and provide a more reliable quality control (QC) method, we developed a reporter cell–based monocyte activation test (MAT) suitable for release testing. Methods: We screened human monocytic reporter cell lines engineered with NFκB-responsive promoter elements driving a luminescent reporter. Reporter cells were treated with diverse endotoxin and non-endotoxin pyrogens and luminescence was quantified after stimulation. Selected THP-1-derived reporter cells were used to develop an MAT for OMPC. Assay performance was evaluated following validation guidelines: linearity, accuracy, precision, analytical range (relative to a reference lot), and robustness under deliberate parameter variations. Results: The THP-1 reporter cells could detect a wide range of pyrogens via simple luminescence readouts. For OMPC testing, the MAT demonstrated strong linearity (R² ≥ 0.99), accuracy with relative bias within ±10.3%, and high precision (overall %RSD ≤ 6.9%) across the 25–300% range. Deliberate variations in assay parameters did not materially affect performance, indicating robustness appropriate for routine release testing. Conclusions: The implementation of reporter cell-based MAT assays enhances consistency, reliability, and efficiency in evaluating the pyrogenicity and safety of drug products, supporting global initiatives to minimize animal testing while ensuring regulatory compliance. Full article

Aldehyde dehydrogenase 1A1 (ALDH1A1) has emerged as a significant biomarker associated with tumor progression, chemoresistance, and poor prognosis in various cancers, including breast, lung, prostate, and lymphoma. Current diagnostic methods for ALDH1A1, such as flow cytometry and ELISA, are limited by long detection times, the need for labeling, and a reduced sensitivity in complex biological matrices. This study presents a novel optical fiber biosensor based on magnesium silicate nanoparticle-doped fibers for the label-free detection of ALDH1A1. The biosensor design incorporated protein G for enhanced antibody orientation and binding efficiency and anti-ALDH1A1 antibodies for specific recognition. Several sensor configurations were fabricated using a semi-distributed interferometer (SDI) format, and their performances were evaluated across a wide concentration range (10 fM–100 nM) in both phosphate-buffered saline (PBS) and fetal bovine serum (FBS). Our findings demonstrated that the inclusion of protein G significantly improved sensor sensitivity and reproducibility, achieving a limit of detection (LoD) of 172 fM in PBS. The sensor also maintained a positive response trend in FBS, indicating its potential applicability in clinically relevant samples. This work introduces the first reported optical fiber biosensor for soluble ALDH1A1 detection, offering a rapid, label-free, and highly sensitive approach suitable for future use in cancer diagnostics. Full article

Background/Objectives: Critical limb ischemia (CLI) is a severe manifestation of peripheral arterial disease with limited treatment options. Mesenchymal stromal cell (MSC) therapy has shown promise, but variability in efficacy suggests that paracrine mechanisms, particularly extracellular vesicle (EV)-associated microRNAs (miRNAs), may play a central role. Methods: We analyzed angiogenesis-related miRNAs in bone marrow-derived MSCs (BM-MSCs) and their EVs. Five angiomiRs (miR-9, miR-105, miR-126, miR-135b, miR-210) were examined; only miR-126, miR-135b, and miR-210 were consistently detected in EVs. Expression variability was assessed across donor age and individuals. Functional evaluation was performed using co-culture of BM-MSCs with human umbilical vein endothelial cells (HUVECs) and by transfecting synthetic miRNAs into HUVECs. Tube formation assays quantified angiogenesis, and angiogenesis-related protein expression (VEGF, FGF, Endoglin, uPA) was analyzed. Biological replicates (multiple donors) and technical replicates (duplicate assays) were clearly defined to ensure reproducibility. Results: Co-culture of BM-MSCs and HUVECs significantly enhanced angiogenesis in a dose-dependent manner. EVs selectively packaged angiogenic miRNAs, with expression levels varying according to donor age and inter-individual variability. Transfection of miR-126, miR-135b, and miR-210 individually enhanced tube formation, while the miR-126 + miR-135b combination and triple transfection elicited the strongest effects. Protein analysis confirmed upregulation of VEGF, FGF, and Endoglin. Notably, miR-210 did not further enhance angiogenesis beyond miR-126 + miR-135b but may exert context-dependent effects. Conclusions: This study demonstrates that BM-MSC-derived EV miRNAs promote angiogenesis via combinatorial mechanisms, providing mechanistic support for ongoing CLI therapy. Our findings highlight the translational potential of EV-based nucleic acid therapeutics for ischemic disease. Full article

A novel glucose biosensor is developed based on a tilted fiber Bragg grating (TFBG) functionalized with a pH-responsive polyelectrolyte multilayer membrane, onto which glucose oxidase (GOD) is immobilized. The sensing film is constructed via layer-by-layer self-assembly of poly(ethylenimine) (PEI) and poly(acrylic acid) (PAA), which undergoes reversible swelling and refractive index (RI) changes in response to local pH variations. These changes are transduced into measurable shifts in the resonance wavelengths of TFBG cladding modes. The catalytic action of GOD oxidizes glucose to gluconic acid, thereby modulating the interfacial pH and actuating the polyelectrolyte membrane. With an optimized (PEI/PAA)₄(PEI/GOD)₁ structure, the biosensor achieves highly sensitive glucose detection, featuring a wide measurement range (10⁻⁸ to 10⁻² M), a low detection limit of 27.7 nM, and a fast response time of ~60 s. It also demonstrates excellent specificity and robust performance in complex biological matrices such as rabbit serum and artificial urine, with recovery rates of 93–102%, highlighting its strong potential for point-of-care testing applications. This platform offers significant advantages in stability, temperature insensitivity, and miniaturization, making it well-suited for clinical glucose monitoring and disease management. Full article

Background: Accurate annotation of gene isoforms remains one of the major obstacles in translating genomic data into meaningful biological insight. Laminin-binding integrins, particularly integrin α6 (ITGA6), exemplify this challenge through their complex splicing patterns. The rare ITGA6 X1X2 isoform, generated by the alternative inclusion of exons X1 and X2 within the β-propeller domain, has remained poorly characterized despite decades of integrin research. Methods: We combined comparative genomics across primates with targeted re-alignment to assess exon conservation and annotation fidelity; analyzed RNA-seq for exon-level usage; applied splice-site prediction to evaluate inclusion potential; surveyed cancer mutation resources for exon-specific variants; and used structural/disorder modeling to infer effects on the β-propeller. Results: Exon X2 is conserved at the genomic level but inconsistently annotated, reflecting the limitations of current annotation pipelines rather than genuine evolutionary loss. RNA-seq analyses reveal low but detectable expression of X2, consistent with weak splice site predictions that suggest strict regulatory control and condition-specific expression. Despite its rarity, recurrent mutations in exon X2 are reported in cancer datasets, implying possible roles in disease. Structural modeling further indicates that X2 contributes to a flexible, disordered region within the β-propeller domain, potentially influencing laminin binding or β-subunit dimerization. Conclusions: Altogether, our results suggest that ITGA6 X1X2 could be a rare, tightly regulated isoform with potential functional and pathological relevance. Full article

The secretome of mesenchymal stromal cells (MSCs) can efficiently stimulate regeneration and therefore is a tempting remedy for “cell-free cellular therapy”. However, the usage of primary MSC cultures as secretome producers for translation studies has obvious obstacles, including the rapid aging of MSC cultures, the need for a large number of verified donors, and donor-to-donor variability of secretome content. MSCs immortalization makes it possible to overcome those limitations and to obtain secretome-producing cultures with a prolonged lifetime. However, the efficacy and safety of such secretomes are critical issues that limit their usage as therapeutic agents. In this study, we tested in large detail how the immortalization of MSC cultures affects the content, biological activity and safety of their secretome. MSCs immortalization via the overexpression of human TERT gene does not significantly alter the qualitative and quantitative composition of their secretome or its activity according to the results of proteomic analysis, ELISA, qPCR and functional tests in vitro. Moreover, we have demonstrated that the secretome of immortalized MSCs does not contain detectable amounts of telomerase and does not possess any transforming activity. Altogether, our data suggest that immortalized MSC cultures may become a reliable source for obtaining standardized active secretome in large-scale quantities for clinical use. Full article

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a global health problem. One of the characteristic features of mycobacteria is their exceptional resistance to environmental factors and their slow growth rate, both of which significantly prolong microbiological diagnostics. Due to the mortality rate and the rising prevalence of multidrug-resistant (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB), early detection and prompt initiation of treatment are extremely important. Traditional diagnostic methods, such as microscopic examination and culture on solid and liquid media, are still important, but are time-consuming and resource-intensive. However, the dynamic development of nucleic acid amplification techniques (NAATs), genotyping assays, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has accelerated the identification of mycobacteria and the detection of drug resistance. Early and precise diagnosis is essential for effective disease control and improved treatment outcomes. This paper reviews the current state of knowledge on tuberculosis; including biological and structural characteristics of mycobacteria; the epidemiology of the disease; and the role of the main diagnostic methods; with a particular focus on molecular methods and MALDI-TOF MS. This paper highlights their advantages and limitations and discusses their implications for the future of TB diagnosis and control Full article

The Centers for Disease Control and Prevention (CDC) classifies Bacillus anthracis as one of the most dangerous pathogens that may affect public health and national security. Due to its importance as a potential biological weapon, this bacteria has been classified in the highest category A, together with such pathogens as variola virus or botulinum neurotoxin. Characteristic features of this pathogen that increase its military importance are the ease of its cultivation, transport, and storage and its ability to create survival forms that are extremely resistant to environmental conditions. However, beyond bioterrorism, B. anthracis is also a naturally occurring pathogen. Anthrax outbreaks occur in livestock and wildlife, particularly in spore-contaminated regions of Africa, Asia, and North America. Spores persist for decades, leading to recurrent infections and zoonotic transmission through direct contact, inhalation, or consumption of contaminated meat. This work presents a new electrochemical method for detecting and quantifying B. anthracis in spore form using a selective immune reaction. The developed method is based on the thiol-modified electrodes that constitute the sensing element of the electrochemical system. Tests with the B. anthracis spore suspension showed that the detection limit for this pathogen is as low as 10³ CFU/mL. Furthermore, it was possible to quantify the analyte with a sensitivity of 11 mV/log (CFU/mL). Due to several features, such as low unit cost, portability, and minimal apparatus demands, this method can be easily implemented in field analyzers for this pathogen and provides an alternative to currently used techniques and devices. Full article

(1) Background: Medicinal plants are widely used in folk medicine. Hypericum perforatum L. (St. John’s wort) is a medicinal plant that is used domestically and exported to other countries. This study addresses the need to develop methods for determining the composition and content of St. John’s wort to determine its biological activity. (2) Methods: High-performance liquid chromatography (HPLC) equipped with an Electrochemical Detector (ECD) and a Photodiode Array Detector (PDA) was employed to identify and quantify major phenolic compounds—gallic acid, catechin, epicatechin, hyperoside, quercetin, and hyperforin—in extracted and lyophilized St. John’s wort flower; stem; and leaf samples. Key analytes exhibited linear responses across both detection systems, within a quantification range of 0.5–10 µg/mL. (3) Results: The PDA method, validated according to ICH Q2(R1) guidelines, demonstrated specificity, linearity, precision, and accuracy, with limits of detection (LOD) ranging from 0.24 to 0.61 µg/mL and limits of quantification (LOQ) between 0.26 and 0.62 µg/mL. PDA effectively identified gallic acid, epicatechin, hyperoside, quercetin, and hyperforin, although catechin was not detected. ECD yielded comparable compound levels across the samples. (4) Conclusions: The novelty of this study lies in identifying the influence of climatic factors associated with the altitude at which St. John’s wort is grown on the content and ratio of biologically active components. Overall, the chemometric approach demonstrates the utility of raw chromatographic data in distinguishing samples by plant part and geographic origin; even when traditional compound-based comparisons may be limited. Full article

Arachidonic acid (eicosatetraenoic acid) is the precursor of the eicosanoids, which include prostaglandins (PG). Methods based on GC-MS/MS are the Gold Standard for the quantitative analysis of eicosanoids in biological samples. After extraction and derivatization, biological F₂-prostaglandins are analyzed on quadrupole GC-MS/MS apparatus as pentafluorobenzyl (PFB) ester trimethylsilyl (TMS) ether derivatives, i.e., PFB-TMS. Negative-ion chemical ionization (NICI) in the ion source generates abundant anions due to [M-PFB]⁻, which are detected in the selected ion monitoring (SIM) mode. Collision-induced dissociation (CID) of [M-PFB]⁻ in the collision cell generates numerous product ions, which are suitable candidates for quantitative analyses in the selected reaction monitoring (SRM) mode. In this article, we report on investigations of gas-phase reactions of PFB-TMS derivatives of F₂-prostaglandins, which consist of PGF_2α, 8-iso-PGF_2α, and up to 62 further isomers, known as the F₂-isoprostanes. We performed a meta-analysis of previously reported CID mass spectra (32 eV) of PFB-(TMS)₃ of seven chemically closely related isomeric F₂-prostaglandins of the 15-F_2t-IsoP type. This unique dataset contains 19 product ions generated by CID of the common precursor at m/z 569 [M-PFB]⁻ in the m/z range of 150–600. All isomers produced the same product ions, which, however, greatly differed in their intensity. Principal Component Analysis (PCA) and Receiver Operating Characteristic (ROC) Analysis (ROCA) were performed. Two compounds, i.e., 8-iso-9β,11α-PGF_2α and 9α,11β-PGF_2α, and two product ions, i.e., m/z 299 [M-PFB-3×TMSOH]⁻ and m/z 215 [M-PFB-3×TMSOH-C₄H₈-C₂H₄]⁻, were noticeable. ROCA revealed the highest disagreement between PGF_2α and 8-iso-9β,11α-PGF_2α (AUC = 0.7075 ± 0.0834, p = 0.0248). PCA and ROCA are of limited value in the GC-MS/MS of closely chemically related F₂-prostaglandins. Fragmentation mechanisms were proposed for the formation of all 19 product ions generated by CID of common precursor anions due to [M-PFB]⁻. Full article

Microplastics (MPs) are increasingly recognized as widespread environmental contaminants, with confirmed presence in human tissues and biological fluids through ingestion, inhalation, and direct systemic exposure. Their potential impacts on human health have become an important subject of scientific investigation. The detection and quantification of MPs, particularly nanoplastics, in complex biological matrices remain challenging because of their low concentrations, diverse physicochemical properties, and interference from organic and inorganic matter. This review presents a critical assessment of current methods for the separation and detection of MPs from human-relevant samples. It examines pre-treatment, separation, and analytical approaches including physical filtration, density-based separation, chemical and enzymatic digestion, vibrational spectroscopy, thermal analysis, and electron microscopy, highlighting their principles, advantages, and limitations. Key challenges such as low sample throughput, absence of standardized procedures, and the difficulty of nanoplastic detection are identified as major barriers to accurate exposure assessment and risk evaluation. Recent advances, including functionalized adsorbents, improved anti-fouling membranes, integrated microfluidic systems, and artificial intelligence-assisted spectral analysis, are discussed for their potential to provide sensitive, scalable, and standardized analytical workflows. By integrating current challenges with recent innovations, this review aims to guide multidisciplinary research toward the development of reliable and reproducible detection strategies that can support MPs exposure assessment and inform evidence-based health policies. Full article

Background: Malignant solid tumors diagnosed during the first year of life represent a rare but clinically significant subgroup of pediatric cancers. Their biological behavior, treatment responses, and prognosis differ substantially from tumors diagnosed in older children due to developmental immaturity and age-related therapeutic limitations. Methods: We retrospectively analyzed 88 infants diagnosed with malignant solid tumors before 12 months of age at a single tertiary center between March 2011 and March 2023. Demographic, clinical, pathological, and treatment data were collected. Overall survival (OS) was estimated by Kaplan–Meier analysis, and prognostic factors were evaluated using univariate and multivariate Cox regression models. Results: Of the 98 initially screened patients, 88 were eligible for analysis. The median age at diagnosis was 7 months, with a median follow-up of 42 months. The most common tumor locations were intra-abdominal (64.7%), brain (20.5%), and bone/soft tissue (12.5%). Neuroblastoma was the leading diagnosis (30.7%), with spontaneous regression observed in 29.6% of cases. Atypical teratoid rhabdoid tumor (ATRT) was the most frequent brain tumor (9.1%). The 5-year OS for the entire cohort was 78.3%. Brain tumors were associated with significantly higher mortality (HR 4.32, p = 0.01), while intra-abdominal tumors predicted improved survival (HR 0.31, p = 0.02). Conclusions: Infantile malignant solid tumors display heterogeneous clinical behavior and outcomes. While favorable results can be achieved in neuroblastoma and soft tissue sarcomas, brain tumors, particularly ATRT, remain a therapeutic challenge. Age-specific, risk-adapted treatment strategies and earlier detection are critical to improving survival and reducing long-term sequelae in this vulnerable population. Full article