Search Results (293)

Background/Objectives: Adalimumab and Infliximab are biologics used to treat autoimmune diseases. Monitoring drug and anti-drug antibody (ADA) levels in patients helps optimize treatment. However, current quantitation methodologies for drug and total (free and drug-bound) ADAs often involve multi-step workflows. Automated systems can streamline the process. The i-Tracker chemiluminescent immunoassays (CLIA) are cartridge-based kits for quantifying serum levels of drugs such as Adalimumab, Infliximab, and associated ADAs. Herein, we aimed to establish performance characteristics of the i-Tracker Adalimumab, Infliximab, and total ADAs in serum on the random-access analyzer IDS-iSYS and to compare patient results with an electrochemiluminescent immunoassay (ECLIA)-based reference method. Methods: Remnant serum specimens, calibration material, or spiked serum were used to evaluate assay linearity, precision, functional sensitivity, and accuracy on the IDS-iSYS analyzer and to perform the method comparison. Results: The assays displayed linearity, accuracy, and up to 8% imprecision across clinically relevant analyte ranges. Compared to the reference method, the drug assays exhibited a strong linear fit (correlation coefficient > 0.95) with <±1.0 µg/mL mean bias. The total anti-Adalimumab assay demonstrated over 85% qualitative agreement. The total anti-Infliximab assay, however, showed higher detection rate of ADAs in Infliximab-treated patient specimens, yielding < 60% negative agreement with the reference method. Although i-Tracker total ADA assays exhibited drug sensitivity, they still detected ADAs in supratherapeutic drug concentrations. Conclusions: The i-Tracker assays demonstrated robust analytical performance, suggesting potential for clinical application. The method comparison underscored functional differences with the reference method, an important consideration when transitioning assay formats for monitoring Adalimumab- and Infliximab-treated patients. Full article

This study investigated the morphometric characteristics of adenohypophyseal thyrotrophs and circulating thyroid hormone profiles in dromedary camels (Camelus dromedarius) in relation to age and lactation status. Clinically healthy Brela breed camels were divided into lactating female, and non-lactating female groups across two age categories (5–10 years and ≥11 years), with fifty animals per group. Blood samples were collected before slaughter and pituitary glands were collected post-slaughter and processed for immunohistochemical detection of thyroid-stimulating hormone (TSH) using anti-porcine TSHβ antibody, while morphometric measurements of thyrotrophs were conducted through image analysis. Plasma concentrations of TSH, triiodothyronine (T₃), and thyroxine (T₄) were quantified using validated ELISA and enzyme immunoassay kits. Group differences were analyzed using one-way ANOVA followed by post hoc comparisons, with statistical significance set at p < 0.05. Morphometric analysis revealed that lactating female camels exhibited significantly higher thyrotroph counts compared with non-lactating counterparts, whereas non-lactating females displayed larger cell and nuclear dimensions. Age influenced these patterns, with older camels showing hypertrophied thyrotrophs but reduced functional plasticity compared to younger animals. Plasma hormone assays demonstrated that non-lactating camels had higher TSH and T₄ concentrations, while lactating camels maintained elevated T₃ levels, suggesting enhanced peripheral conversion of T₄ to T₃ during milk production. Additionally, younger camels exhibited higher T₃ concentrations than older animals, indicating age-related decline in thyroidal activity. These findings highlight the dynamic regulation of the hypothalamic–pituitary–thyroid axis in camels, demonstrating how lactation and age shape thyroidal morphology and function to meet diverse physiological demands. These findings not only broaden the comparative endocrinology of underexplored species but also provide physiopathological insights relevant to farm animal management, lactation efficiency, and adaptive metabolism in harsh environments. Full article

The current study outlines a synthetic method for creating a new class of indazol-pyrimidine derivatives 4a–h and 5a–h. The new derivatives were evaluated as in vitro cytotoxic agents against three types of cancer cell lines (MCF-7, A549 and Caco-2), utilizing the MTT assay. Compounds 4a, 4c, 4d, 5a and 5f demonstrated potent cytotoxic activity against MCF-7 cell line, showing higher activity than the reference drug Staurosporine. Among the examined compounds, 5f showed a strong cytotoxic effect against all three tested cancer cells (MCF-7, A549 and Caco-2), with IC₅₀ values of 1.858, 3.628 and 1.056 µM, respectively. In comparison, the reference drug exhibited IC₅₀ values of 8.029, 7.354 and 4.202 µM respectively, indicating promising anti-proliferative potential of compound 5f. On the other hand, Compound 4b demonstrated the greatest potency against Caco-2 cell line, with an IC₅₀ of 0.827 µM, markedly outperforming reference compound’s IC₅₀ of 4.202 µM. Furthermore, compound 5h revealed significant anti-proliferative activity against A549 cell line, with an IC₅₀ value of 1.378 µM, compared to the reference drug, with an IC₅₀ value of 7.354 µM. Additionally, the molecular docking study revealed a strong binding affinity of compound 5f within the binding site of the c-Kit tyrosine kinase protein, and the molecular dynamics study confirmed its stability. Full article

Nucleic acid amplification methods are widely used in science, medicine and forensics for molecular biological assays and for the detection of genetic material. The newly developed strand displacement chain reaction (SDCR) method is a hybrid amplification technique based on polymerase chain reaction (PCR) and isothermal nucleic acid amplification. Here, we compared conventional PCR, the “gold standard” for molecular diagnostic assays, with the SDCR method by performing real-time amplification assays using human, bacterial and viral genetic materials. In the assays, SDCR demonstrated very high sensitivity and amplification efficiency. We found that the SDCR method provided an amplification factor above three, which noticeably outperformed that of PCR amplification and enabled a marked reduction in the number of cycles in comparison with PCR. Therefore, the new hybrid amplification technique could be extremely useful for the detection of genetic material and the development of new diagnostic kits. Full article

Background/Objectives. Multi-drug-resistant (MDR) microorganisms pose a significant challenge in healthcare settings, particularly with beta-lactam-resistant Gram-negative bacteria and glycopeptide-resistant enterococci. Culture represents the most reliable technique in determining their presence within surveillance swabs. However, it requires a long time-to-result (TTR) and shows low sensitivity. Molecular techniques integrate diagnostic procedures, allowing TTR reduction and precise identification of genes. Methods. During our usual surveillance campaign, we had the opportunity to evaluate the Allplex Entero-DR assay (Seegene Inc., Seoul, Republic of Korea) and the Entero-DR Plus assay (Arrow Diagnostics srl, Genova, Italy) molecular kits for the detection of extended-β-lactamases (ESBL), carbapenem- and vancomycin-resistant genes, as well as Acinetobacter spp. and Pseudomonas aeruginosa spp. identification directly from rectal swabs. A comparison between these tests and the culture-based routine completed the study. Results. The analysis included 300 rectal swabs from the University Hospital Policlinico (Catania, Italy). One hundred and eighty-eight samples (62.6%) resulted as positive for at least one Allplex™ target, reaching optimal sensitivity and negative predictive value (100%). Our results underlined the ubiquitous blaCTX-M and van genes presence and demonstrated the diffusion of double-carbapenemases genes and metallo-β-lactamases-producing strains. In our epidemiological setting, few data were collected about carbapenem-resistant P. aeruginosa and Acinetobacter spp., which require further evaluations on simultaneous respiratory colonization and higher sample numbers. Conclusions. Our analysis highlighted the importance of combining conventional and advanced diagnostic methods in investigating MDR pathogens. The right approach should be based on the prevalence and variability of resistance mechanisms within a specific epidemiological area. Remarkably, molecular screenings may exclude negative samples within high-risk areas due to a significant negative predictive value. Full article

As software systems continue to increase in complexity and scale, the number of reported bugs also grows. Bug reports are essential artifacts in software maintenance, supporting critical tasks such as detecting duplicate reports, predicting bug severity, and assigning priority levels. Although stop word removal is a common text preprocessing step in natural language processing, its effectiveness in deep learning-based bug report analysis has not been thoroughly evaluated. This study investigates the impact of stop word removal on three core bug report classification tasks. The analysis uses a dataset containing over 1.9 million bug reports from eight large-scale open-source projects, including Eclipse, FreeBSD, GCC, Gentoo, Kernel, RedHat, Sourceware, and WebKit. Five deep learning models are applied: convolutional neural networks, long short-term memory networks, gated recurrent units, Transformers, and BERT. Each model is evaluated on its performance with and without stop word removal during preprocessing. The results show that the F1 score difference was less than 0.01 in over 85% of comparisons, so stop word removal has little to no effect on predictive performance in eight open-source projects. Average F1-scores remain consistent across all tasks and models, with 0.36 for duplicate detection, 0.33 for severity prediction, and 0.33 for priority prediction. Statistical significance tests confirm that the observed differences are not meaningful across datasets or model types. The findings suggest that stop word removal is not necessary in deep learning-based bug report analysis. Removing this step may simplify preprocessing pipelines without reducing accuracy, particularly in large-scale and real-world software engineering applications. Full article

The architecture, engineering, and construction (AEC) industry faces challenges related to inefficiencies and fragmentation that highlight the need for advanced construction technologies and drive interest in innovative solutions such as the platform approach to design. This study assessed platform-based building design through (1) interviews with practitioners from China, Jordan, and the UK, which helped to define the platform approach in the AEC industry and the challenges involved, and (2) a residential building design simulation conducted to evaluate the potential of the platform approach. The simulated design’s materials costs, energy efficiency, and construction time were compared with those of the traditional building design. The results of the comparison corroborate the interview findings concerning practitioners’ perspectives on platform definition, benefits, challenges, and implementation. The findings also demonstrate the potential of the platform approach to enhance productivity and scalability through modularization, kit-of-parts configuration, and standardization. This research bridges the gap between theory and practice by supporting shareholder perspectives on building design and construction with the results of a simulated platform approach to a real-world design project. This research addresses the urgent need to better understand and test the platform approach to achieve material, energy, and construction time savings through collaborative and practice-informed design. Full article

Background: Peyronie’s disease (PD) is a chronic inflammatory condition that affects the penile albuginea. Oxidative stress (OS) plays a crucial role in the development of the disease, prompting us to investigate OS levels at the site of the disease and in peripheral blood. This article presents our second study in which the OS was evaluated by calculating the OS index (OSI) in blood samples taken directly from the penile corpora cavernosa of patients with PD. Our innovative diagnostic method, which focuses on the analysis of oxidative stress (OS) in the corpora cavernosa of the penis, allows us to accurately identify the “chemical” signals (OS levels) of the pathology in the area where it is present. Methods: Our study included 102 PD patients from our Peyronie’s care center and 100 control cases. To conduct a comprehensive OS analysis, we measured both the total oxidant status (TOS) and total antioxidant status (TAS) and calculated the oxidative stress index (OSI) as OSI = TOS/TAS × 100. Blood samples were collected from the penis and a vein in the upper extremity, and OS was measured using d-ROMs and PATs (FRAS kit). Results: Pearson’s analyses revealed a significant statistical correlation between penile OSI values and PD plaque volumes (p = 0.003), while no correlation was found between systemic OSI values and plaque volumes (p = 0.356). Penile OSI values decreased significantly after PD plaque removal (p < 0.0001). A comparison of penile OSI values in PD patients (post plaque removal) and the control group showed no significant differences (p = 0.418). Conclusions: The lack of correlation between systemic OSI values and Peyronie’s plaque volume suggests that direct sampling from the site of the disease is preferable for OS studies. Conducting a penile OSI study could provide a precise oxidative marker dependent on plaque volume. In addition, the penile OSI study can biochemically monitor the therapeutic result, alongside penile ultrasound imaging. Full article

Background: We evaluated the Sysmex Highly Integrated Single-Cartridge Luminescence Immunoassay System (HISCL) hs-cTnT assay, and compared its performance to the Roche assay, with derivation of 99th-percentile upper reference limits (99% URLs) for healthy subjects. We assessed the effect of increasing age/decreasing eGFR on the HISCL hs-cTnT. Methods: We verified assay limits of blank/detection, precision and the functional sensitivity. Samples were analyzed on both the Sysmex HISCL and Roche Elecsys analyzers for method comparison. Results: The HISCL assay limit of blank/detection was 1.3/1.9 ng/L, and concentrations corresponding to 20/10% CVs were 1.8/3.3 ng/L. Assay precision of kit controls at 3253 ng/L was 2.2% and at 106 ng/L was 2.5%. Linear regression analysis (n = 2151) showed good agreement (r = 0.95) with the Roche hs-cTnT. Bland–Altman (Roche/HISCL) analysis for samples with hs-cTnT ≤ 52 ng/L showed a mean absolute difference of 3.5 ng/L; for hs-cTnT > 52 ng/L, the mean difference was 2.8%. In a cardio-renal healthy population (n = 1004), the 99% URLs were 14.4/17.0/13.9 ng/L for overall/male/female, respectively; assay CV% was below 10% at these levels. More than 50% of the hs-cTnT in the healthy male and female subjects were measurable above the limit of detection. Hs-cTnT increased with increasing age and decreasing eGFR. Conclusions: In conclusion, the Sysmex HISCL hs-cTnT fulfils the criteria for a high-sensitivity assay, with specific 99th URLs for males and females. Expectedly, the baseline Sysmex hs-cTnT increases with age and decreasing eGFR. Full article

Background: Accurate differentiation between Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) is essential for proper diagnosis and treatment. This study compares the diagnostic performance of two commercial real-time PCR kits, AdvanSure^TM TB/NTM and Kogene PowerChek^TM MTB/NTM, for detecting MTB, NTM, and negative (no growth, NG) clinical specimens. Methods: A total of 390 clinical residual specimens were collected from patients between December 2022 and June 2023. The samples, including sputum, bronchoalveolar lavage, tracheal aspirate and body fluid, were initially tested with MGIT culture and then analyzed using both PCR kits. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall accuracy were evaluated. Discrepant results between the two PCR assays were further investigated using sequencing to identify the detected mycobacterial species, and final diagnoses were verified by culture results and review of electronic medical records. Results: Of the 390 specimens, both AdvanSure^TM and PowerChek^TM real-time PCR assays demonstrated 100% sensitivity for both MTB and NTM detection. For MTB detection, AdvanSure^TM demonstrated a specificity of 100%, with a PPV, NPV, and overall accuracy all reaching 100%. In comparison, PowerChek^TM showed a specificity of 98.62%, a PPV of 96.15%, an NPV of 100%, and an overall accuracy of 98.97%. For NTM detection, both AdvanSure^TM and PowerChek^TM exhibited identical performance metrics. The specificity was 99.58% for both assays, with a PPV of 99.34%, NPV of 100%, and an overall accuracy of 99.74%. Five discrepant results were finally confirmed as four NTM detection cases and one negative case by culture and clinical diagnosis which showed four cases of PowerChek^TM MTB+NTM detection and one case of NTM detection, respectively. Conclusions: The PowerChek^TM MTB/NTM real-time PCR kit demonstrated excellent diagnostic performance for the detection of MTB and NTM, with high sensitivity, specificity, and accuracy. Minor discrepancies, particularly in detecting MTB+NTM mixed infections, highlight the importance of complementary sequencing analysis for resolving uncertain results. These findings support the clinical utility of both PCR assays as reliable tools for rapid diagnosis of mycobacterial infections. PowerChek^TM showed occasional false positives, suggesting that optimizing the assay’s cutoff threshold or amplification parameters could enhance its specificity and reduce false-positive results in clinically ambiguous cases. Full article

Background/Objectives: Neutralizing autoantibodies against type I interferons, particularly interferon-alpha (IFN-α), have been implicated in severe COVID-19 outcomes. This study investigated the prevalence and functional significance of anti-IFN-α autoantibodies (AAbs) in hospitalized unvaccinated COVID-19 patients and their association with COVID-19 disease severity. Methods: We retrospectively analyzed serum samples from 122 hospitalized COVID-19 patients (asymptomatic/mild: n = 69, moderate: n = 35, severe/critical: n = 18) and 32 healthy uninfected controls. Anti-IFN-α AAbs were quantified using a commercial enzyme-linked immunosorbent assay (ELISA) kit, with functional neutralization assessed via competitive ELISA and STAT1 phosphorylation inhibition. Statistical comparisons were performed using one-way ANOVA for parametric data and the Kruskal–Wallis test for non-parametric variables. Results: Anti-IFN-α AAbs were detected in 24.6% of COVID-19 patients, with all clinical subgroups showing significantly higher titers compared to healthy controls (p < 0.05). Although no significant differences in anti-IFN-α AAb levels were found between mild, moderate, and severe cases, patients with severe or critical COVID-19 had markedly higher mean titers (10,511.3 ng/mL) compared to non-severe (mild + moderate) cases (375.2 ng/mL, p < 0.001). Strongly neutralizing anti-IFN-α AAbs, with high titers (>20,000 ng/mL) and the ability to inhibit STAT1 phosphorylation, were identified in three severe COVID-19 cases. Anti-IFN-α AAb levels correlated positively with CRP (r = 0.80, p < 0.0001), LDH (r = 0.80, p = 0.001), and neutrophil count (r = 0.52, p = 0.003), and negatively with lymphocyte count (r = −0.59, p = 0.0006). Conclusions: Elevated and functionally neutralizing anti-IFN-α AAbs were associated with severe COVID-19. These findings support their role as a risk factor for poor outcomes and emphasize the importance of early COVID-19 vaccination. Screening may help identify high-risk individuals, particularly those unvaccinated or with immune vulnerabilities. Full article

Circulating cell-free nucleic acids (NAs), in particular plasma-derived cell-free DNA, have evolved into promising clinical analytes for prenatal diagnostics, cancer analysis, and cancer surveillance and therapy monitoring. Nevertheless, salivary extracellular and extracellular vesicle (EV)-derived DNA and microRNA have recently gained attention as potential non-invasive biomarkers for a variety of diseases, including cancer, cardiovascular, autoimmune, and infectious diseases. Our goal in this study was therefore to evaluate and optimize commercially available approaches for cell-free nucleic acid isolation, focusing specifically on DNA and miRNA present in cell-free saliva or saliva-derived EVs. Along these lines, we investigated various commercially available kits, which enable parallel isolation of cell-free DNA and RNA in separate fractions from cell-free saliva and salivary EVs, respectively, and compared them to single analyte extraction kits. The efficiency of all tested nucleic acid extraction methods was determined by comparing DNA and RNA fluorescence spectroscopy measurements and quantitative PCR values obtained from a selection of different DNA- and microRNA targets. We found the Norgen Plasma/Serum RNA/DNA Purification Mini kit in combination with the miRCURY exosome isolation kit to work best in our hands and to provide the highest yields of EV-derived nucleic acids. Having tested and identified effective protocols for isolating salivary extracellular nucleic acids, we present with this comparison study, among others, a sound basis for future circulating small nucleic acid and epigenetic biomarker research aiming for early disease diagnosis, prognosis, and prediction from cell-free saliva, representing an easy-to-collect and readily available diagnostic fluid. Full article

Background/Objectives: Porcine circovirus type 2d (PCV2d) is the predominant genotype associated with porcine circovirus-associated disease (PCVAD), leading to significant economic losses. In South Korea, current vaccine lot-release testing relies on a T/C-ratio-based guinea pig assay, which lacks scientific justification and methodological robustness. This study aimed to develop and validate a statistically defined in-house ELISA using rabbit-derived polyclonal antibodies against PCV2d for the standardized evaluation of immunogenicity. Methods: Polyclonal IgG was generated by immunizing a rabbit with inactivated PCV2d, and it was purified through Protein A chromatography. Guinea pigs (n = 18) were immunized with IMMUNIS^® DMVac, an inactivated PCV2d vaccine candidate developed by WOOGENE B&G, at different doses. In-house ELISA parameters were optimized (antigen coating, blocking agent, and substrate incubation), and analytical performance was evaluated by ROC, linearity, reproducibility, and specificity. Sera from guinea pigs and pigs were analyzed under validated conditions. Results: The optimal performance was achieved using 10⁵ genomic copies/mL of the antigen coating and a 5% BSA blocking agent. The assay showed strong diagnostic accuracy (AUC = 0.97), reproducibility (CVs < 5%), and linearity (R² = 0.9890). Specificity tests with PCV2a, PCV2b, and PRRSV showed minimal cross-reactivity (<7%). The cross-species comparison revealed a positive correlation (R² = 0.1815) and acceptable agreement (bias = −0.21) between guinea pig and porcine sera. The validated cut-off (S/P = 0.4) enabled accurate classification across both species and aligned well with commercial kits. Conclusions: The in-house ELISA offers a robust, reproducible, and scientifically validated platform for immunogenicity verification, supporting its application in Korea’s national lot-release system. Homologous competition assays with PCV2d are planned to further confirm antigen specificity. Full article

The composition of the gut microbiome, defined by environmental factors, significantly affects research outcomes, with variations observed across animal facilities. Efforts to standardize led to the definition of the ‘Altered Schaedler flora’ (ASF), comprising eight bacterial groups. Our data highlights the variability of ASF under pathogen contact. Feces from two wild-type strains (C57Bl/6J and Balb/c mice) with and without proven infection was collected in two different animal facilities and analyzed. The data show a significant difference in the quantity (either reduction or increase) of the eight ASF bacterial groups when comparing infected and non-infected mice across different housing areas (SPF-specific pathogen-free, quarantine, and conventional-experimental areas) within a facility, as well as in comparison to another facility. Furthermore, strain-specific differences are also evident, with certain ASF groups showing a reduction in quantity at one facility but an increase at the other, comparing the same housing area. Comparative studies across facilities confirmed the necessity of baseline determination for accurate ASF analysis. Performing ASF analysis, facilitated by in-house qPCR (quantitative polymerase chain reaction) kits, offers prompt and precise microbiome profiling, enhancing experimental accuracy and health monitoring in animal research settings. Full article

Background/Objectives: Myelomeningocele (MMC) is the most severe form of spina bifida, often accompanied by impaired motor function due to paralysis of the lower limbs, as well as neurogenic bladder (NB). These factors may contribute to nutritional disorders and cardiovascular diseases (CVDs) in the future. High-sensitivity CRP (hsCRP) is a positive marker of unstable atherosclerotic plaques and is commonly used in the diagnosis of CVDs. Adiponectin has an opposite, anti-inflammatory function. The aim of this study was to assess the risk of CVDs in a group of children with NB and a control group, based on serum levels of adiponectin, hsCRP, and lipid profiles. Methods: A prospective clinical estimation based on 87 children (67 NB, 20 control group) was conducted. Data collected from medical histories included the following: sex, age, anthropometric parameters (height, weight, BMI), level of spinal lesion, and activity according to Hoffer’s scale. Lipid profile values (cholesterol, HDL, LDL, triglycerides) were assessed using standard blood sample tests. hsCRP and adiponectin were measured using an ELISA kit. Results: A comparison of adiponectin and hsCRP levels revealed statistically significant differences between the NB group and the control. Additionally, significant correlations were identified between BMI and the biomarkers: hsCRP was positively associated with BMI, whereas adiponectin exhibited a negative association. The highest concentrations of hsCRP were detected in MMC patients with a Th lesion level and in non-walker patients. Conclusions: Elevated hsCRP may reflect increased cardiovascular risk in children with NB. While adiponectin levels were also altered, their association with cardiovascular risk appears more complex and may involve additional metabolic mechanisms. Full article