Establishment of A triplex TaqMan qPCR assay for EHP, VpTPD, VpAHPND with optimized efficacy using methyl cellulose.

Simulated construction of tilmicosin nucleic acid aptamers based on molecular docking and molecular dynamics techniques.

Development of dual-mode recombinase polymerase amplification assays integrated with fluorescence and biosensor for rapid Monkeypox detection.

A PtRu nanozyme and aggregation-induced emission nanoparticle mediated novel fluorescent and colorimetric dual-mode immunoassay for xylazine detection.

Clostridioides difficile (C. difficile) is the leading cause of hospital-acquired life-threatening diarrhea. C. difficile toxins TcdA and TcdB contain a glucosyltransferase domain (GTD) that glucosylates and inactivates host GTPases, disrupting the actin cytoskeleton and compromising epithelial integrity. TcdB, the most potent virulence factor, drives disease progression and is a high-priority target forC. difficiletreatment and prevention. The iminosugar isofagomine has been shown to inhibit the GTD activity of TcdB by an uncompetitive inhibition mechanism, but requires the uridine 5'-diphosphate (UDP) reaction product. Compound classes synthesized here, ranging from isofagomine analogues to acyclic mimics, probe which modifications can tap into UDP-binding energy to enhance inhibition. Structure-activity relationship studies of isofagomine derivatives demonstrate remarkable specificity for isofagomine and limited advantage in accessing the UDP-binding site. Fluorescence and absorbance assays allowed facile inhibition assessment of TcdB's UDP-glucose hydrolysis. The molecules reported here guide scaffolds for future catalytic site inhibitors.

Cascade catalysis-based signal amplification for colorimetric detection of β-galactosidase activity and its application.

Chickpea-derived antimicrobial peptides compromise Escherichia coli O157:H7 membrane biophysics through lipid-specific interactions.

Vitamin D deficiency in Mongolian pregnant women.

Rabies remains a major neglected disease, causing tens of thousands of deaths annually in endemic regions. Cell culture-based assays are central to rabies diagnostics and antibody quantification, yet interpretation relies on manual fluorescence image reading, which is labour-intensive, subjective, and difficult to scale. Here, we present, what is to our knowledge, the first AI-based workflow for detecting rabies virus infection in BHK-21 cell cultures and quantifying antibody titres. Three state-of-the-art architectures-ResNet, Swin Transformer V2, and MambaOut-were trained on a large dataset of 2344 fluorescence images (36,822 annotated patches) capturing biological and technical variability. Evaluation at patch, image, and well levels showed MambaOut consistently outperformed other models, achieving 0.996 accuracy, 0.993 F1-score, and 0.999 AUC-ROC at the well level. To assess applicability, models were tested on unseen images from the WHO/WOAH-recommended fluorescent antibody virus neutralisation assay. MambaOut demonstrated complete agreement with ground truth in predicting antibody concentrations, confirming workflow reliability for vaccine assessment and pet travel schemes. These results establish a foundation for AI-assisted rabies diagnostics and open opportunities for applying deep learning to other cell culture-based viral assays. AI-driven automation for high-throughput image analysis offers a scalable and time-efficient solution for diagnostic laboratories, particularly in endemic countries.

Droplet digital enzyme-linked immunosorbent assay (ddELISA) enables ultrasensitive protein quantification; however, its performance is often limited by low single-bead encapsulation efficiency due to Poisson statistics and significant background interference from empty droplets. We present an integrated microfluidic system that overcomes both limitations through synergistic magneto-inertial manipulation (SMIM). By coupling an external magnetic field with laminar inertial focusing, the system applies coordinated hydrodynamic lift and magnetophoretic forces on immunomagnetic beads, thereby enabling three interdependent functions within a continuous workflow: ordered single-file bead focusing, high-throughput single-bead encapsulation, and active sorting of bead-containing droplets. Under optimized conditions (30 μL min-1; 300 mT), magnetic beads are focused into a stable single-file train with a lateral deviation of only 0.95 ± 1.21 μm. This deterministic ordering prior to droplet generation fundamentally overcomes the Poisson limit, achieving single-bead encapsulation efficiency of 81.31%─a 2.21-fold improvement over stochastic loading─and a total encapsulation efficiency of 91.11%. The integrated magnetic sorting module generates localized high-gradient fields, capturing bead-containing droplets with 93.97 ± 1.18% efficiency while eliminating >91% of empty droplets. The platform's quantitative capability was validated using a dual-color fluorescent bead assay (R2 = 0.9977; 0.50 - 1.75 × 107 beads mL-1) and an E. coli O157 ddELISA (R2 = 0.9999; LOD = 18.23 cells mL-1). This work establishes a fully integrated microfluidic platform that addresses the long-standing challenges of encapsulation inefficiency and empty-droplet interference in ddELISA by transforming bead encapsulation from a stochastic to a deterministic process, thereby providing a broadly applicable framework for high-throughput, single-particle-resolved digital bioanalysis.

Alginate oligosaccharides (AOSs) are natural marine oligosaccharides with diverse physiological functions, which have broad application prospects in both the cosmeceutical and food industries. This study systematically investigated the inhibitory mechanisms of AOSs against tyrosinase and elastase, two key enzymes associated with skin aging, by combining multi-spectroscopic approaches with zebrafish in vivo evaluation. Results demonstrated that AOSs acted as a reversible mixed-type inhibitor for both enzymes, exhibiting moderate inhibitory activities with IC50 values of 7.37 ± 0.15 mM for tyrosinase and 10.05 ± 0.17 mM for elastase. Fluorescence quenching assays revealed that AOSs interacted with the two enzymes through a static quenching mechanism. Further investigations via synchronous fluorescence and ANS-binding assay confirmed that AOS binding induced conformational rearrangements of both enzymes, altered the microenvironments around tyrosine and tryptophan residues, and decreased their surface hydrophobicity. CD spectra results indicated that AOSs could cause changes in the secondary structures of the two enzymes, which was closely related to the loss of enzymatic activity. Zebrafish experiments further verified that AOSs could effectively inhibit melanin formation and alleviate oxidative stress in vivo. Collectively, these findings provide a comprehensive mechanistic basis for the dual skin-whitening and anti-wrinkle efficacy of AOSs as a multifunctional active ingredient in cosmeceutical formulations.

Cell-free protein synthesis (CFPS) has the potential to reduce the cost of biologics manufacturing through simplified lysates, high-throughput workflows, and streamlined downstream purification; however, high reagent costs and inconsistent product yields remain significant barriers to industrial adoption. A key limitation is the lack of cost-effective, high-throughput methods to monitor metabolite dynamics during the CFPS optimization. To address this challenge, we developed a fluorescent assay that enables the real-time monitoring of transcription, translation, and key metabolite pools. The fluorescent assay was used to identify and address technical challenges and produce insights that were subsequently used to engineer a minimal CFPS platform that costs nearly 97.5% less than commercial systems but still attains comparable protein yields. More broadly, our work introduces a generalizable framework for interrogating and engineering CFPS systems through the real-time observation of transcription and metabolism.

Primordial germ cells (PGCs) exhibit molecular heterogeneity during development, yet whether this reflects functionally distinct states remains unclear. We investigated the functional significance of DND1 translation heterogeneity in human PGC-like cell (hPGCLC) extended culture, using dual fluorescent reporters, transcriptomic analysis and functional assays. Single-cell RNA-sequencing revealed that DND1 + hPGCLCs are enriched in RNA regulatory genes including those associated with P-bodies and translational control. DND1 + cells exhibit reduced proliferation, cell cycle exit, and elevated nuclear expression P27 (CDKN1B). These cells also maintained stable expression of genes essential for germ cell differentiation. Notably, BMP2 supplementation stabilized DND1 translation without altering mRNA levels or proliferation rates. Together, these findings demonstrate that post-transcriptional regulation by DND1 coordinates cell cycle dynamics with distinct cellular state in human germ cells, highlighting critical translational control mechanisms in germline development.

Precise measurements of small molecule-protein interactions are critical for drug discovery. However, most biochemical profiling platforms measure binding using recombinant kinase domains or proteins in cell lysates, which can miss conformational regulation present in intact living cells. Here, we used flow cytometry-based fluorescent probe cellular binding assays (FPCBA) to demonstrate that the anticancer drug dasatinib binds native, untagged ABL1 kinase with 3-6-fold higher affinity than NanoLuc- or mVenus-tagged constructs in living cells. We further used this method for in-cell profiling of 25 native kinases, revealing conformational regulatory mechanisms, including SRC autoinhibition and membrane-dependent conformational states of DDR1, DDR2, and EPHA4 that are absent or attenuated in biochemical assays. For these studies, coumarin-dasatinib probes spanning a range of fluorophore acidity (pKa 4.1-7.3) were optimized for intracellular target engagement. To enhance sensitivity of detection, we found that uptake of acidic probes can be promoted by expression of the organic anion transporter OATP1B3. Quantitative flow cytometry with NIST-standardized beads established that intracellular concentrations of an intermediate-acidity 6FC-dasatinib probe approximated extracellular concentrations in HEK293T cells at equilibrium. Cellular Ki values of dasatinib and imatinib for 25 kinases by FPCBA were broadly concordant with kinobead LC/MS measurements in cancer cell lysates but diverged substantially from recombinant KINOMEscan values, with divergences attributable to competition with ATP, autoinhibition, and membrane-dependent conformational states in living cells. FPCBA enables profiling of native protein-small molecule interactions in a physiologically relevant cellular context.

The adulteration of dairy products with melamine has created an urgent need for reliable, sensitive, and convenient detection methods. Since melamine can form extensive hydrogen bonding and stacking interactions with DNA bases, aptamers represent an attractive platform for melamine sensing. In this work, capture-SELEX was employed using two DNA libraries containing either 30 or 36 randomized nucleotides. The longer library generated a greater diversity of sequence families, including a dominant family featuring more than 30 conserved nucleotides, a structural motif unattainable with the shorter library. Comparison was made with previous length-dependent SELEX experiments. The highest affinity aptamers exhibited a dissociation constant (Kd) of ∼60 nM, as determined by isothermal titration calorimetry. Screening aptamer responses using a thioflavin T fluorescence assay revealed a pair of aptamers with opposite signal readouts, enabling melamine detection with limits of detection of 61 nM and 208 nM, respectively. In 5% (v/v) milk samples, limits of detection of 89 and 315 nM were achieved, respectively. Overall, this study advances understanding of the impact of library length on aptamer selection and provides a set of high affinity aptamers along with a robust, label-free strategy for melamine detection.

One of the main obstacles in Cholangiocarcinoma (CCA) treatment is drug resistant to the standard therapy, Cisplatin combined with Gemcitabine (Cis/Gem). Brusatol (Bru) has shown its potential in enhancing the effects of other chemotherapeutic drugs. Therefore, this study investigates the anti-cancer effects of Bru alone or combined with chemotherapeutic drugs, Cis and Gem, in human CCA cells (KKU-100), and explores the underlying mechanisms. Cell viability was determined using MTT assay, while intracellular reactive oxygen species (ROS) levels were measured using a DCFDA fluorescence assay. Apoptosis was assessed by caspase-3, -8, and -9 activity and annexin-V-FITC/propidium iodide (PI) double staining. Mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining. Combination of Bru and Cis or Gem increased intracellular ROS levels, caspase-3, -8, and -9 activities, apoptotic cell population and the loss of ΔΨm in KKU-100 cells. This indicated the enhanced apoptotic effects of the combination therapy through the activation of both intrinsic and extrinsic pathways. Additionally, combination of Bru with Cis or Gem significantly reduced colony formation, and suppressed cell migration and invasion in KKU-100 cells. These findings support the potential of Bru as a promising anti-cancer agent or adjunct therapy to enhance current chemotherapy in CCA treatment.

A real-time FRET ubiquitin transfer assay for quantitative characterization of ternary complexes in targeted protein degradation.

The global mpox outbreak has highlighted critical gaps in diagnostic capabilities, particularly the need for methods that can distinguish between the high-fatality Clade I and more transmissible Clade II of the mpox virus (MPXV). Current PCR-based approaches remain reliant on laboratory infrastructure, limiting their use in resource-limited settings. There is an urgent need for a versatile diagnostic platform that can provide both accurate clade discrimination and flexible deployment across diverse healthcare environments. We developed a dual-mode detection platform by integrating recombinase-aided amplification with Clustered Regularly Interspaced Short Palindromic Repeats-Cas12a technology, creating two distinct assays: a universal assay targeting OPG034, and a Clade I-discriminatory assay targeting OPG033. The platform achieved detection sensitivities of 1 copy/reaction for both fluorescence and visual readouts with OPG034, and 10 copies (fluorescence) and 1 copy/reaction (visual) with OPG033. Both assays demonstrated high specificity, successfully distinguishing MPXV from related orthopoxviruses and common viruses. Clinical validation using 23 Clade II samples and 10 healthy controls showed that, relative to quantitative PCR (qPCR) (cycle threshold ≤37), the OPG034 fluorescence assay detected all 13 qPCR-positive samples and 2 additional positives (100% sensitivity, 80.0% specificity), while the visual assay detected 12 of 13 positives (92.3% sensitivity, 100% specificity). For Clade I-specific detection, both OPG033 fluorescence and visual assays showed 100% specificity in Clade II samples (23/23). While the clade-discriminatory capability was established through sequence-specific design, further evaluation with authentic Clade I clinical specimens is warranted to confirm typing performance. The dual-mode design provides flexibility for both laboratory and field use, advancing mpox surveillance and outbreak response.IMPORTANCEMpox is a significant zoonosis. Accurate discrimination between its highly lethal Clade I and more transmissible Clade II is critical for clinical management and outbreak control, yet current methods primarily enable only general detection. To address this, we identified novel genetic markers (OPG034 for universal detection and OPG033 for Clade I specificity) and developed a dual-mode detection platform integrating Clustered Regularly Interspaced Short Palindromic Repeats-Cas12a with recombinase-aided amplification. Its key advantage is providing two result readouts: a sensitive fluorescence mode for laboratories and an instrument-free visual colorimetric mode for field use. The demonstrated excellent performance on clinical samples confirms that this platform meets the precision requirements of clinical laboratories while remaining suitable for resource-limited settings like field clinics. Thus, it offers a flexible and practical tool for enhancing mpox surveillance and control globally, particularly in regions with constrained medical resources.

<bold></bold> To enable differential detection of virulent and attenuated strains of duck short beak and dwarfism syndrome virus (SBDSV), we developed a fluorescent quantitative PCR (qPCR) assay based on locked nucleic acid (LNA)-TaqMan probes. This assay utilized a pair of universal primers and two strain-specific LNA-TaqMan probes (labeled with FAM and VIC, respectively) targeting the VP1 gene of SBDSV. Validation results showed that the assay specifically detected SBDSV virulent and attenuated strains without cross-reacting with other common waterfowl viruses. It exhibited high sensitivity, with a minimum detection limit of 6.0 × 10⁰ copies/μL, and excellent reproducibility, with intra- and inter-assay coefficients of variation (CVs) all < 3%. Additionally, viral titer estimates from this qPCR assay showed no significant difference from those obtained via the gold-standard 50% tissue culture infectious dose (TCID₅₀) assay (P > 0.05). In conclusion, this LNA-TaqMan qPCR assay provides a specific, sensitive, and reliable tool for the differential diagnosis of SBDSV virulent and attenuated strains in clinical practice, serving as strong technical support for the prevention, control, and eradication of SBDSV.

Blood group antigens are well known for their importance in transfusion medicine and transplant compatibility; however, their biological role extends beyond these functions and includes associations with the risk of several diseases. In this study, we investigated the relationship between ABO blood groups and the circulating levels of 73 different molecules. Fifty-six healthy donors were enrolled, including 24 individuals with blood group O, 19 with blood group A, and 13 with blood group B. Blood samples were collected and analyzed in a single laboratory using Luminex fluorescent bead-based assay panels to determine the concentrations of 73 circulating molecules. Depending on data distribution, ANOVA or Kruskal-Wallis tests and Student's t-test or Kolmogorov-Smirnov tests were applied to identify significant differences among groups. Associations were further assessed by binary logistic regression analysis. Subjects with blood group A showed significantly higher circulating levels of IL-1R1, IL-13, IL-23, PDGF-BB, VEGF-A, VEGF-D, soluble VEGF-R2 (KDR), soluble VEGF-R3 (FLT-4), VLA-4, CD141, MMP-1, syndecan-1 (SDC-1), and mannose-binding lectin (MBL) compared with the other blood groups. In contrast, individuals with blood group B exhibited significantly higher levels of IL-22, IL-23, PDGF-BB, CD62P (P-selectin), and amyloid β1-42. Several significant associations were identified by logistic regression analysis. Our findings indicate that ABO blood groups are associated with distinct circulating molecular profiles, supporting the existence of biological differences that may contribute to variations in disease susceptibility among individuals with different blood types. Nevertheless, given the exploratory's nature and limited sample size of this study, further investigations are required to validate these findings, confirm the observed associations, and clarify their potential clinical implications.