Search Results (220)

Background and Objectives: Pseudoexfoliative glaucoma (PEXG) is a secondary glaucoma caused by the deposition of abnormal flaky protein-like material inside the eye, leading to blockage of the drainage system, increased intraocular pressure (IOP), and optic nerve damage. This study aimed to evaluate the effects of latanoprost and timolol–dorzolamide therapy on the concentrations of interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12p70, and tumor necrosis factor-alpha (TNF-α) in the aqueous humor and serum samples of patients with PEXG. Materials and Methods: This prospective observational study included 60 patients allocated to three age-matched groups (n = 20 per group). Group 1 received latanoprost therapy, Group 2 received timolol–dorzolamide therapy, and Group 3 comprised patients with cataracts without PEXG who were not receiving antiglaucoma medication (control group). All participants underwent a comprehensive ophthalmological examination, including visual acuity assessment, specular microscopy, IOP measurement, and intraocular lens (IOL) measurement. Peripheral venous blood was taken from each patient before the surgical procedure and aqueous humor samples were collected during cataract surgery. Concentrations of IL-1β, IL-6, IL-8, IL-10, IL-12p70, and TNF-α were determined using a Cytometric Bead Array and flow cytometry. Results: No significant differences were observed in the aqueous humor and serum concentrations of IL-6 and IL-8 between the treatment groups, while IL-1β, IL-10, IL-12p70, and TNF-α did not reach detection. A significant positive correlation was found between IOL and aqueous humor IL-6 concentration in group 1 and between IL-6 and IL-8 serum concentration and IOP in controls. Conclusions: Latanoprost and timolol–dorzolamide therapy were not associated with significant differences in aqueous humor or serum IL-6 and IL-8 concentrations in patients with PEXG. IL-1β, IL-10, IL-12p70, and TNF-α were not detectable in the analyzed samples. These findings support the presence of low-grade inflammation in patients with PEXG that does not differ according to the therapy administered. Corneal endothelial alterations are disease-related rather than treatment-induced. Full article

Campylobacter jejuni and Campylobacter coli are major foodborne pathogens whose accurate species-level discrimination is important for outbreak investigations as well as rapidly assessing putative antimicrobial resistance profiles and the pathogenic potential of the bacterium. To facilitate species differentiation, a novel assay that integrates the nucleic acid-sensing capability of the oligo-Alpha with the multiplexing capacity of the AlphaPlex bead chemistries was developed. This wash-free system (designated as oligo-Plex) enables the detection and differentiation of C. jejuni and C. coli within a single reaction and can be completed in approximately 75 min. It works by using custom oligonucleotides modified for bead attachment, which hybridize sequentially along Campylobacter’s glyA gene and ultimately bridge the donor and acceptor beads. Improvements in assay stringency were made by increasing incubation temperatures, thus allowing the resolution of target from non-target. Comparisons of FITC–europium and DIG–terbium labeling systems revealed superior performance by the FITC–europium pair and suggested that helical positioning and steric accessibility likely influence donor–acceptor efficiency. Maximized signal separation was seen when using terbium for the detection of C. coli and europium for the detection of C. jejuni. Testing was performed in a Tris-based buffer and milk to confirm matrix tolerance, with potential areas for further optimization identified. The oligo-Plex presented here establishes a streamlined, adaptable platform suitable for high-throughput screening of multiple nucleic acid analytes that is readily extendable to a diverse array of pathogens through appropriate oligo selection. Full article

Fused filament fabrication (FFF) has become an important additive manufacturing technique for producing functional polymer-composite components. The tensile performance of carbon fiber-reinforced polyethylene terephthalate glycol (PETG/CF) fabricated by FFF depends on multiple printing parameters. This study presents an integrated experimental and predictive framework for investigating the effects of extrusion temperature, printing speed, layer height, infill pattern, and infill density on the tensile strength of PETG/CF containing 15 wt.% carbon fiber. A mixed-level Taguchi L36 orthogonal array was employed, comprising 36 experimental runs with three independently printed specimens per run, resulting in 108 ASTM D638 Type V specimens. Analysis of variance showed that the printing speed had the largest contribution to tensile strength (20.51%), followed by layer height (18.29%). The highest tensile strength of 33.225 MPa was obtained using grid infill, 60% infill density, 270 °C extrusion temperature with 40 mm/s printing speed, and 0.3 mm layer height. An artificial neural network (ANN) was developed for the tensile-strength prediction, achieving R = 0.9801, R² = 0.9569, and MAPE = 1.52% for the overall dataset. Scanning electron microscopy qualitatively revealed bead-interface defects, fiber pullout, and localized void-like features. The proposed framework provides a systematic approach for evaluating process-parameter effects and predicting tensile strength within the investigated PETG/CF parameter domain. Full article

Fluorescence-based immunoassays are widely used for sensitive and specific biomarker detection; however, further improvements in sensitivity remain desirable for detecting low-abundance analytes without increasing assay complexity. In this work, we present a proof-of-concept demonstration of guided electrokinetic assembly of functionalized latex beads as a post-assay signal enhancement strategy using alternating-current electro-osmosis (ACEO). Carboxyl-modified 1 μm polystyrene beads conjugated with Alexa Fluor 647-labeled anti-IgG were localized within lithographically defined windows on carbon interdigitated electrode arrays, producing localized fluorescence enhancement through physical bead localization without enzymatic amplification or additional labeling chemistries. Compatibility of the approach with fluorescence-based immunoassays was demonstrated through adaptation of a TNF-α ELISA workflow. Electro-osmotic localization of functionalized bead conjugates was achieved within 120 s, producing an approximately 12-fold increase in corrected total fluorescence relative to the corrected signal of the pre-electro-osmosis condition while demonstrating negligible enrichment of unbound fluorescent protein. Application of the platform to a TNF-α sandwich immunoassay yielded an approximately 5.5-fold enhancement in fluorescence signal, and robust bead localization was maintained across anti-IgG concentrations ranging from 1 to 4 μg/mL. These findings demonstrate that guided electrokinetic bead localization provides an effective signal enhancement strategy for fluorescence-based immunoassays and represents a promising approach for improving the detection of low-abundance analytes. Full article

Accurate identification and enumeration of microscopic particles are important for microfluidic analysis, electrokinetic studies, and microscopy-based characterization of microfabricated systems. This study presents an optimized Hough Circle Transform (HCT) workflow for automated particle detection, sizing, and counting. Gold interdigitated electrode arrays (IDEAs) were fabricated on wafer substrates to generate electroosmotic flow, and 3 μm and 5 μm polystyrene microbeads were used as model particles. The final workflow incorporates parallelized multicore parameter optimization and composite statistical metrics based on detection accuracy and frame-to-frame standard deviation, enabling a small manually counted calibration set to be converted into locked detection parameters. In the final validation workflow, 10 manually counted calibration frames were used to optimize HCT parameters for each of four scenarios, and the locked parameters were then validated on 50 new frames per scenario (200 validation frames total) with two independent annotators. Mean validation success rates were 85.1% for 3 μm beads, 90.0% for 5 μm beads, 86.1% for 3 μm beads in mixed suspensions, and 87.2% for 5 μm beads in mixed suspensions, corresponding to object-level error rates of 17.9%, 10.9%, 19.9%, and 13.7%, respectively. Compared with the historical Generation I serial workflow, the optimized workflow reduced parameter-selection time from 24–48 h to 1–2 h, and the runtime image-processing time was approximately 45 ms per frame during offline analysis. These results show that parameter optimization is essential for robust HCT-based particle enumeration and that the workflow provides a practical analytical tool for microfluidic device characterization and electrokinetic experiments. Full article

Objective: Intracranial infection is a severe complication that can occur following neurosurgery, and early diagnosis is crucial for improving patient prognosis. In this study, we aimed to investigate, from an exploratory perspective, whether the immune microenvironment of intraoperative cerebrospinal fluid (CSF) is associated with postoperative intracranial infection (PII) in patients undergoing craniotomy for benign brain tumors. Methods: A total of 134 patients undergoing neurosurgery for benign brain tumors were included and categorized into an infection group (n = 18) and a non-infection group (n = 116). CSF samples were collected aseptically immediately after dural opening during surgery. The concentrations of 16 cytokines, including monocyte chemoattractant protein-1 (MCP-1); macrophage inflammatory protein-1α (MIP-1α) and MIP-1β; interleukin (IL)-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, and IL-17; interferon (IFN)-α and IFN-γ; tumor necrosis factor-α (TNF-α); and granulocyte colony-stimulating factor (G-CSF), were quantified using Cytometric Bead Array (CBA) technology. An independent samples t-test was used for normally distributed data, while the Mann–Whitney U test was applied for non-normally distributed data. Group comparisons were performed using independent-samples t-tests or Mann–Whitney U tests for continuous variables and χ² tests or Fisher’s exact tests for categorical variables. The Benjamini–Hochberg false discovery rate (FDR) correction was applied to all 16 cytokines to control for multiple testing. Receiver operating characteristic (ROC) curve analysis was performed to assess discriminatory capacity. Statistical significance was defined as p < 0.05. Results: PII developed in 18 of 134 patients (13.4%). Age (47.78 vs. 54.86, p = 0.028) and operative duration (390 vs. 244 min, p = 0.005) showed differences in unadjusted analyses. In the unadjusted comparisons, MCP-1 and IL-4 levels were found to be significantly lower in the infection group (MCP-1: 57.78 vs. 116.03 pg/mL, p = 0.003; IL-4: 24.38 vs. 28.18 pg/mL, p = 0.032). No cytokine remained significant after FDR correction. The ROC analysis showed that age and IL-4 demonstrated mild discriminatory performance, with AUC values of 0.665 (95% CI 0.526–0.803, p = 0.025) and 0.657 (95% CI 0.540–0.774, p = 0.032), while MCP-1 and operative duration demonstrated modest discriminatory performance, with AUC values of 0.716 (95% CI 0.595–0.838, p = 0.003) and 0.708 (95% CI 0.578–0.838, p = 0.002). Conclusions: In this study, single-point intraoperative CSF cytokines were not significantly associated with PII after stringent correction for multiple testing, and did not provide a validated clinical prediction tool. The unadjusted and direction-corrected findings for MCP-1 and IL-4 remain exploratory and require validation. Full article

Three-dimensional (3D) endothelium–stromal co-cultures were established using human endometrial cells from biopsy of healthy women (n = 13) and serum samples from both healthy and endometriotic women (n = 5). For 3D construction, stromal cells were mixed with extracellular matrix components, followed by endothelial cell seeding. Morphological analysis confirmed the organization of tissue-like structures. Immunofluorescence and flow cytometry verified the expression of specific stromal and endothelial markers (Cytokeratin, Vimentin, Insulin-like growth factor-binding protein 1, and von Willebrand factor). Cell viability and proliferation increased over time, with minimal cell death. To test functional responsiveness, these co-cultures were exposed to inflammatory serum from endometriotic patients. After 48 h, cytometric bead array showed elevated levels of IL-1β, IL-6, and IL-8 in cultures treated with inflammatory serum, indicating preserved functional activity and responsiveness. By allowing detailed investigation of functional endometrial states within a physiologically relevant cellular network, this approach provides a valuable organoid-like tool to explore conditions such as implantation failure and infertility and to study the cellular interactions underlying reproductive pathologies. Full article

Background/Objectives: The induction of anti-SARS-CoV-2 antibodies by COVID-19 vaccination reduces morbidity and mortality, but immune responses may be compromised in people living with HIV (PLWH). The aims of the current study were to determine whether viral suppression (VS) or immune reconstitution (IR) in PLWH directly affected their ability to produce effective levels of anti-SARS-CoV-2 antibodies in mucosal secretions or blood induced by vaccination. Methods: Anti-SARS-CoV-2 spike IgG, IgA and secretory IgA (SIgA) antibodies and their avidities were measured by ELISA in HIV-negative healthy controls (HC; n = 49) and PLWH (n = 94) using stimulated oral fluid (SOF) and serum. Frequencies of CD4/CD8 T cells and their expression of exhaustion/senescence were determined by flow cytometry. Cytokine levels were measured by cytokine bead arrays. Results: We showed that higher HIV burden negatively impacted the levels of systemic and mucosal anti-SARS-CoV-2 spike IgG antibodies produced. This differential IgG antibody production was unaffected by IR status, antiretroviral therapy duration or T cell exhaustion/senescence. PLWH elicited higher anti-SARS-CoV-2 spike IgA antibodies both in peripheral blood and oral mucosa and highr secretory IgA (SIgA) antibodies in the oral mucosa. PLWH with higher HIV RNA copies elicited lower IgG avidity but the IgA avidity indices remained unaffected. PLWH expressed higher levels of innate immunity cytokines in the oral mucosa, irrespective of the HIV RNA copies. Conclusions: Significantly fewer breakthrough infections in PLWH compared with HC, along with high IgA/SIgA antibodies and increased innate immunity cytokines in the SOF, suggest a potential role for mucosal immunity in the immunopathogenesis of COVID-19. Full article

This study evaluates a preliminary multi-objective optimization framework for a permanent magnet mechanical clutch designed for automated curtain actuators. To analyze the highly non-linear trade-off between disengagement capability (Y-direction magnetic resistance) and positional stability (Z-direction magnetic attraction force), a numerical approach combining three-dimensional (3D) magnetostatic finite element method (FEM) simulations, Taguchi L9 orthogonal arrays, and regression modeling was implemented. Three magnetic bead diameters were introduced as noise factors to investigate the sensitivity of the magnetic forces within a controlled simulation environment. A multiplicative composite objective function was employed to assess the competing performance criteria without masking single-factor failures. Statistical analysis indicates that within the investigated design space, the axial distance (Factor D) is the primary geometric parameter influencing the force distributions, followed by the outer diameter (Factor B) and inner diameter (Factor A). The identified parameter configuration (A = 8 mm, B = 10.5 mm, C = 0.5 mm, D = 1.5 mm) demonstrated an improved composite objective value and narrower standard deviations under the designated simulation boundaries compared to the initial discrete trials. These exploratory findings suggest that the proposed workflow was validated using a physical prototype based on one of the Taguchi configurations. Full article

Background/Objectives: The emergence of SARS-CoV-2 variants and breakthrough infections underscores the need for next-generation vaccines capable of protecting from natural infection and/or preventing virus transmission. Intranasal vaccination offers a promising approach by eliciting local immune responses in the nasal mucosa, the primary site of infection and reservoir for transmissible virus. We evaluated two live-attenuated, respiratory syncytial virus-vectored vaccines in which the RSV F and G surface glycoproteins were replaced with a chimeric SARS-CoV-2 Spike protein from the ancestral USA/WA-1/2020 strain (MV-014-212) or the Delta variant (MV-014-212-delta). Methods: K18-hACE2 mice and LVG Syrian hamsters were vaccinated with a single intranasal dose of MV-014-212 or MV-014-212-delta. Systemic and mucosal immunity were assessed following vaccination, and protection was evaluated following Delta SARS-CoV-2 challenge. In vaccinated hamsters, morbidity, viral shedding, and lung inflammation and injury were also assessed following natural exposure to infected cagemates. Results: A single intranasal dose of either vaccine elicited systemic and mucosal immunity in K18-hACE2 mice, including serum neutralizing antibodies, Spike-specific memory B cells and plasmablasts, and Spike-specific CD8⁺ lung-resident memory T cells. Although MV-014-212-delta vaccination provided the best protection against the Delta variant virus challenge, both vaccines decreased viral loads in nasal discharge, lung, and brain, and reduced weight loss and mortality. In naturally acquired infection studies, vaccinated hamsters exposed to infected cagemates exhibited minimal weight loss, limited viral replication within the nasal mucosa, and attenuated lung pathology. Conclusions: Intranasal RSV-vectored vaccines can elicit broad protective respiratory immunity, suggesting that this platform could be leveraged for other respiratory pathogens. Full article

Background/objectives: Although informative, current insights into the inflammatory nature of colorectal cancer (CRC) have yet to have a meaningful impact on the prevention of, and development of novel therapies for, the treatment of this prevalent and challenging disease. Accordingly, the current study was focused on identifying putative, key, systemic, mostly pro-inflammatory biomarkers of metastatic CRC (mCRC) prognosis and outcome. Methods: Patients with mCRC (n = 38) and matched healthy controls (n = 30) were recruited to the study. A multiplex magnetic bead array system and an ELISA procedure were used to measure the plasma concentrations of selected cytokines (n = 25) and that of C-reactive protein (CRP) by immunonephelometry. Systemic inflammatory indices (n = 5) were derived from the hematological data. Results: Plasma levels of 17/25 of the cytokine biomarkers and CRP were found to be significantly elevated, while the neutrophil/lymphocyte ratio proved to be the most useful of the various inflammatory indices. Subgroup analysis of the data derived from the group of mCRC patients revealed that the intensity of the systemic inflammatory response was mostly unaffected by tumor location, age, gender, and treatment line. The exception was time to progression, with a shorter time (<120 days) being associated with increased levels of IL-6, IL-8 and TNF-α. Hierarchical cluster analysis of the data revealed a possible association with a small group of four cytokines, comprising IL-1β, IL-13, IL-6/CRP and TGF-β1. Conclusions: This study confirms a strong association of established mCRC with cytokine-driven systemic inflammation. Four of these cytokines, IL-1β/IL-13 IL-6/CRP, and TGF-β1, appear prominent and are possibly indicative of novel targetable therapeutic options. Full article

Background/Objectives: Sporotrichosis is an emerging zoonotic subcutaneous fungal infection with limited therapeutic options, highlighting the need for improved immunomodulatory strategies. CTLA-4 is an inhibitory immune checkpoint that negatively regulates T-cell activation. In this study, we evaluated whether a CTLA-4 antisense oligonucleotide (CTLA-4 ASO) is associated with enhanced immune responses to an adjuvanted recombinant Sporothrix sp. enolase antigen (rSsEno) formulation. Methods: CTLA-4 ASO uptake, cytotoxicity, and gene-silencing activity were assessed in murine splenocytes in vitro. BALB/c mice were immunized with rSsEno formulated with Montanide Gel 01, either alone or in combination with 5 µg CTLA-4 ASO. Antigen-specific serum antibody responses were quantified by ELISA. Splenocytes from immunized mice were restimulated with enolase, and cytokine production (IFN-γ, IL-2, IL-17, and TNF-α) was measured using Cytometric Bead Array (CBA). Results: CTLA-4 ASO was efficiently internalized by splenocytes and was associated with reduced expression of CTLA-4 without detectable cytotoxicity in vitro. Mice receiving the ASO-supplemented formulation developed significantly higher anti-enolase antibody titers compared to those immunized with adjuvant alone. Upon antigen restimulation, splenocytes from ASO-treated mice produced higher levels of IFN-γ, IL-2, TNF-α, and IL-17, consistent with an enhanced recall response characterized by a mixed Th1/Th17 cytokine profile. Conclusions: CTLA-4 ASO was associated with an enhanced recall response characterized by a mixed Th1/Th17 cytokine profile. These findings suggest a potential immunomodulatory effect of CTLA-4 targeting. Further studies incorporating dose optimization, infection challenge models, and appropriate sequence controls are required to determine the specificity and relevance of these effects for protective immunity against sporotrichosis. Full article

Accurate genealogical records are essential in livestock breeding for maintaining genetic diversity, preventing inbreeding, and mapping of economically important traits in beef production. This study aimed to assess parent–offspring relationships within South African Bonsmara and Nguni cattle populations using both traditional pedigree records and genomic data. Hair samples from 119 Nguni and 311 Bonsmara cattle were genotyped using the BovineSNP50 array, and these were imputed to Illumina BovineHD BeadChip using updated SNP coordinates from the assembly genome (ARC—UCSD 1.2). Quality control and data filtering were performed using PLINK v1.9, while relationship inference was conducted using KING v2.2.8 and PLINK v1.9 software for principal component analysis, IBD metrics and Mendelian error-based exclusion. Categories of relatedness through network relationship analysis revealed a predominance of half-sibling relationships in both breeds, with 2317 such relationships identified in Nguni and 1221 in Bonsmara. Inference of parent–offspring pairs showed discrepancies with the recorded pedigrees, with 49 inferred pairs compared to 47 recorded pairs in Nguni, and 62 inferred pairs compared to 75 pairs recorded in Bonsmara. Relationships based on IBD using PLINK with a ‘PI-HAT’ threshold greater than 0.45 revealed unique parent–offspring inferences that differed from those obtained using KING v2.2.8. Phylogenetic network analysis assigned each individual’s genomic origin independent of the pedigree records, supporting the efficiency of SNP data for genetic assignment. These results demonstrated that SNP-based pedigree verification can accurately identify parent–offspring and half-sibling relationships, providing a reliable foundation for recombination analysis and supporting precise trait mapping and informed selection in breeding programs. Full article

Accurate monitoring of internal particle motion in dense granular flows remains a significant challenge across various fields, ranging from geophysics to industrial processes. To address the limitations of existing observational techniques, this study presents a novel high-precision magnetic array positioning system based on magnetic dipole theory for dynamically tracking individual particles within opaque granular media. The system integrates an array of nine magnetic sensors with a hybrid optimization algorithm that combines Particle Swarm Optimization (PSO) and gradient-based local refinement, achieving a dynamic positioning accuracy within the maximum measurable range, with a maximum dynamic error of 2.5 ± 0.5 mm and a trajectory continuity exceeding 99%. Deployed in a quasi-two-dimensional rotating drum, the system enables detailed investigation of particle segregation mechanisms. Reconstruction and analysis of the trajectories of a high-density intruder (magnetic bead) allow quantification of the competition among segregation mechanisms through the Froude number. The results reveal three distinct motion phases with increasing rotational speed: a gravity-dominated percolation stage, a transitional collision–diffusion competition stage, and a centrifugal diffusion-dominated stage. Each phase exhibits unique kinematic signatures governed by the interplay of inertial, gravitational, and contact forces. This work not only establishes a robust and accurate sensor-based method for internal granular flow monitoring but also provides new mechanistic insights into segregation dynamics, with implications for understanding geological hazards such as debris flows. Full article

Flow cytometry, measuring light signals, can be used as a quantitative tool to appreciate the numbers of cell-surface molecules targeted with monoclonal antibodies, among other applications. This has been extensively used for blood cells and especially platelets. This review describes how techniques have evolved over time since the first developments of quantitative flow cytometry at the end of the 20th century. Technological issues are first described, applicable to all types of cells/molecules and largely relying on calibration beads with direct or, preferably, indirect immunofluorescence. The platelet field is then addressed with specific tools devoted to surface antigen quantitation. The array of commercially available kits is provided with their specificity. A panorama of platelet antigens quantified that can be used in the diagnosis workout of platelet disorders is then provided, accompanied by a reminder of the impressive stability of marker expression in normal individuals. Variations are then considered in the light of aging or genetic polymorphisms. Finally, the upcoming use of platelet antigen quantification as a monitoring tool for emerging targeted therapies is evoked. All in all, this review provides a comprehensive story of the evolution of the still too marginally used cell antigen quantification. Full article