BACKGROUND: Detection of serum allergen-specific IgE antibodies (sIgE) that can cross-link the high-affinity IgE receptor (FcεRI) is crucial for diagnosing IgE-mediated food allergy (FA). We previously developed an ampli...BACKGROUND: Detection of serum allergen-specific IgE antibodies (sIgE) that can cross-link the high-affinity IgE receptor (FcεRI) is crucial for diagnosing IgE-mediated food allergy (FA). We previously developed an amplified luminescence proximity homogeneous assay involving cross-linking (AlphaCL) to detect functionally relevant sIgE. This study was intended to enhance analytical sensitivity of AlphaCL and evaluate its utility in detecting functional allergen-sIgE in sera from rats and IgE-mediated FA patients. METHODS: Serum samples were collected from ovalbumin (OVA)-sensitized rats and from 10 patients each with allergies to ovomucoid (OVM), ω5-gliadin (ω5G), or casein. To enhance sensitivity of conventional AlphaCL, allergens were desthiobiotinylated and immobilized on streptavidin plates, allowing serum components to be washed out before assaying. sIgE levels were measured using AlphaCL (in both rats and patients), ELISA (rats), and ImmunoCAP™ (patients). AlphaCL signals were compared with those from unsensitized controls and analyzed the correlation with ImmunoCAP™ results. RESULTS: The improved AlphaCL showed significantly higher sensitivity than the conventional method and retained 66% of the signal observed in serum-free samples. AlphaCL signals were elevated in all OVA-sensitized rats, all patients with OVM or ω5G allergy, and 8 of 10 with casein allergy. Strong correlations were observed between AlphaCL and ImmunoCAP™ for OVM (r = 0.98), ω5G (r = 0.93) and casein (r = 0.86). AlphaCL detected allergen-sIgE corresponding to ImmunoCAP™ Class ≥ 3 for OVM and ω5G, and Class ≥ 4 for casein. CONCLUSIONS: The improved AlphaCL can detect functional allergen-sIgE with FcεRIα-cross-linking ability, supporting its potential clinical utility in diagnosing IgE-mediated FA.
Preserving DNA integrity, defined as intact strands and correct sequence context, is crucial to the reliability of measurements and interpretations in molecular methods. The denaturation temperature of genomic DNA is inf...Preserving DNA integrity, defined as intact strands and correct sequence context, is crucial to the reliability of measurements and interpretations in molecular methods. The denaturation temperature of genomic DNA is influenced by several factors, including GC content, ionic strength, and the presence of stabilizing or destabilizing agents. In this work, we report the investigation of human genomic DNA denaturation behavior under low salt and high temperature conditions. We used digital PCR as the primary analytical tool for DNA stability which could quantify the extent of DNA strand separation. To ensure that observed changes reflected structural alterations rather than DNA degradation or evaporation, we employed complementary analytical approaches including quantitative PCR and isotope dilution liquid chromatography with tandem mass spectrometry. Despite the widespread use of heat inactivation protocols in restriction enzyme workflows, there remains a significant gap in our understanding of how these treatments affect genomic DNA structure. Our findings have important implications for the reliability and reproducibility of molecular biology procedures, particularly those that depend on precise DNA quantification or structural integrity.
Three-dimensional (3D) tumour spheroids are widely used as physiologically relevant in vitro models to study tumour biology, therapeutic responses, and the distribution of drugs and nanomaterials. However, experimental w...Three-dimensional (3D) tumour spheroids are widely used as physiologically relevant in vitro models to study tumour biology, therapeutic responses, and the distribution of drugs and nanomaterials. However, experimental workflows for spheroid-based studies remain highly heterogeneous, with limited standardization across spheroid generation, processing, and quantitative analysis, hindering reproducibility and accessibility. Here, we present TEAMSTER, an integrated, end-to-end pipeline for the generation, cryosectioning, immunostaining, and quantitative image analysis of tumour spheroids. The workflow combines optimized protocols for reproducible spheroid formation that minimize variability, a liquid nitrogen-free, colour-coded OCT embedding strategy improving spheroid localization during cryosectioning, standardized fixation and staining procedures compatible with multiplex fluorescence imaging, and a semi-automated, GUI-based CellProfiler pipeline enabling unbiased quantitative analysis without coding skills, machine learning, or dedicated computing hardware. TEAMSTER is experimentally validated in two distinct tumour spheroid models treated with fluorescently labelled nanoconjugates. Quantitative performance metrics for spheroid preparation (intraplate and interplate coefficients of variation) and image segmentation benchmarking (Dice and IoU coefficients) support the robustness of single-cell-resolved measurements across cryosectioned spheroid models and imaging conditions. By prioritizing experimental robustness, standardization, and usability, TEAMSTER provides a practical methodological resource for reproducible quantitative spheroid-based studies in cancer biology and nanomedicine.
Accurate prediction of MHC-I antigen presentation is central to neoantigen discovery and immunotherapy development. Although recent deep-learning based predictors achieve high accuracy, most operate as black-box models,...Accurate prediction of MHC-I antigen presentation is central to neoantigen discovery and immunotherapy development. Although recent deep-learning based predictors achieve high accuracy, most operate as black-box models, which limits interpretability, and consequently trustworthiness of these predictors. We present MHCCBM, a gray-box framework that decomposes antigen presentation into a set of pathway-level intermediate concepts. Each step, i.e., proteasomal cleavage, TAP transport, peptide-MHC binding affinity, and chaperone dependency is treated as an independent predictive module, allowing different computational or experimental estimators to be substituted without altering the overall model. This architecture naturally supports representational multimodality, permitting the integration of sequence-based, structure-informed, or empirical predictors. In our reference implementation, ESM-2-derived models estimate peptide processing and chaperone dependency, while peptide-MHC binding affinity is predicted using MHCflurry. The concept outputs are combined using logistic regression. Our accuracy is comparable to that of state-of-the-art accuracy while providing MHC-I pathway level interpretability consistent with known cellular mechanisms.
Currently, there are very few standardized and developed analysis methods for detecting the deposition of black carbon (BC) in human tissues, and they have certain limitations. A non-destructive and robust method has bee...Currently, there are very few standardized and developed analysis methods for detecting the deposition of black carbon (BC) in human tissues, and they have certain limitations. A non-destructive and robust method has been developed for identifying BC deposits in soft biological tissue using placental tissue matrix as a case study. Placental tissues were obtained from 50 pregnant women in their third trimester. The method employs a correlative workflow, utilizing a scanning electron microscope (SEM) for high-resolution morphological screening of suspected BC particulates and energy-dispersive X-ray fluorescence (EDXRF) for specific elemental confirmation. The method's specificity was first tested in a preliminary experiment conducted by externally embedding charcoal in two placental tissues, which confirmed its ability to distinguish the exogenous BC particles from the native biological matrix based on definitive elemental analysis. Subsequently, this method was applied to 48 original placental tissues without external embedding of charcoal. The deposition of BC was identified on the surface of placental tissue, specifically at the foetal side of the placenta. This correlative SEM-EDXRF method provides a robust and effective framework for identifying exogenous BC in biological tissues, as demonstrated by the clear distinction between the elemental signatures of BC particles and the native biological matrix. The method has limitations in terms of quantification.
A classical pathological hallmark of many neurodegenerative diseases is the formation of protein-rich aggregates and inclusions. In Parkinson's disease (PD), α-synuclein (α-syn) constitutes a major protein component of p...A classical pathological hallmark of many neurodegenerative diseases is the formation of protein-rich aggregates and inclusions. In Parkinson's disease (PD), α-synuclein (α-syn) constitutes a major protein component of pathological inclusions, termed Lewy bodies. These α-syn aggregates are hypothesized to spread throughout the nervous system by cell-to-cell transmission acting as templates to amplify aggregate formation. In vitro generated α-syn aggregates, commonly called preformed fibrils (PFFs), have been used to investigate a number of aspects related to α-syn mediated pathology across different model systems. Here we describe a semi-automated assay to screen for small molecules that interfere with the cellular uptake and accumulation of PFFs. The assay uses dopaminergic progenitor cells (DPCs), derived from human induced pluripotent stem cells (hiPSCs). In an initial screen, we tested 1520 small molecules and identified several molecules that strongly reduce intracellular PFF load in DPCs. From these hits, candidate compounds were validated in dopaminergic neurons (DNs) to demonstrate the utility of the assay. This assay provides a robust, scalable and adaptable tool to screen for molecules that affect PFF uptake in hiPSC-derived cell models. Within the scope of this screen, it led to the identification of a set of compounds with diverse annotated targets that effectively reduce the accumulation of α-syn aggregates in DPCs and DNs.
Preeclampsia is a pregnancy complication marked by hypertension and proteinuria occurring after 20 weeks of gestation, and it is the primary cause of maternal and neonatal morbidity and mortality globally. The primary is...Preeclampsia is a pregnancy complication marked by hypertension and proteinuria occurring after 20 weeks of gestation, and it is the primary cause of maternal and neonatal morbidity and mortality globally. The primary issue in its management is the delayed diagnosis resulting from conventional procedures that depend on clinical symptoms, despite the earlier occurrence of pathological signs. Consequently, the identification of specific biomarkers and the advancement of precise, sensitive, and practical diagnostic tools are imperative. This review highlights recent advances in biosensor-based diagnostic strategies for preeclampsia by examining electrochemical, optical, mass-sensitive, and piezoelectric transducers used to detect angiogenic, anti-angiogenic, oxidative stress, and immunological biomarkers. The results show that biosensors, especially electrochemical platforms, offer advantages in sensitivity, specificity, short analysis time, and the possibility of miniaturization for point-of-care use. Biomarker combinations, such as the sFlt-1/PlGF ratio, further enhance diagnostic accuracy over a single biomarker approach. Notwithstanding these benefits, issues remain concerning bioreceptor stability, interference from biological matrices, and the necessity for extensive clinical validation. Ongoing progress in nanotechnology and materials engineering is anticipated to expedite the clinical application of biosensor-based diagnostics to facilitate early disease detection and clinical decision-making.
Accurate and timely assessment of burn depth is critical for determining effective treatment strategies and improving patient outcomes. However, traditional diagnostic methods, such as visual inspection and histological...Accurate and timely assessment of burn depth is critical for determining effective treatment strategies and improving patient outcomes. However, traditional diagnostic methods, such as visual inspection and histological analysis, are constrained by subjectivity, invasiveness, and diagnostic delays. Photoacoustic imaging (PAI), a hybrid modality combining optical contrast with ultrasonic resolution, has emerged as a promising tool for biomedical applications due to its high sensitivity to endogenous chromophores and capability for depth-resolved functional imaging. Although various PAI configurations have been investigated for non-invasive burn assessment, a comprehensive synthesis of this specific application is currently lacking. To address this gap, this review provides a detailed overview of the fundamental principles of PAI, evaluates its distinct advantages in burn depth assessment, and discusses current technological advancements, translational challenges, and future perspectives.
GPCR dimers and receptor mosaics are distinct pharmacological entities that can be targeted by specific ligands, including bivalent ligands, thus GPCR dimerization is a well-known concept. GPCR oligomerization can be stu...GPCR dimers and receptor mosaics are distinct pharmacological entities that can be targeted by specific ligands, including bivalent ligands, thus GPCR dimerization is a well-known concept. GPCR oligomerization can be studied with biochemical, biophysical, and computational methods. Here, we review classical and novel biochemical approaches to investigate this phenomenon. We discuss protomer co-expression, co-immunoprecipitation, heteromer-specific antibodies, proximity ligation assay, APEX2-MS proximity assay, receptor mutants and chimeras, radiation inactivation studies, cross-linking studies, and trans-complementation studies. For each approach, the basis of the technique is summarized, followed by an extensive list of its applications. This review is a comprehensive resource for biochemists and molecular biologists working in the field of GPCR oligomerization, as well as for scientists interested in GPCR drug design and discovery.
Cell-cell fusion is a fundamental biological process underlying diverse physiological and pathological phenomena, yet its quantitative analysis remains methodologically challenging due to its dynamic, heterogeneous, and...Cell-cell fusion is a fundamental biological process underlying diverse physiological and pathological phenomena, yet its quantitative analysis remains methodologically challenging due to its dynamic, heterogeneous, and multistep nature. Existing approaches to assess fusion largely rely on endpoint assays or manual scoring, limiting temporal resolution, scalability, and reproducibility. Here, we present a label-free, high-content live-cell imaging pipeline for real-time quantification of cell fusion dynamics, developed and validated using trophoblast syncytialization as a model system. The method integrates automated image acquisition with a reproducible, stepwise analysis workflow combining supervised texture-based segmentation, morphology-based measurements, and intensity-independent texture analysis. We define quantitative metrics, including the ratio of total cluster area to the number of detected clusters and cytoplasmic granularity features, that together discriminate bona fide fusion events from non-fusion-related cellular clustering or proliferation. Using canonical pharmacological inducers and inhibitors of fusion, we demonstrate the specificity and sensitivity of these parameters for detecting fusion-associated remodeling over time. We further demonstrate the scalability of the pipeline through high-throughput screening of biologically relevant growth factors, hormones, and inhibitors, enabling classification of modulators based on their independent, synergistic, or antagonistic effects on fusion dynamics. Consistent results obtained in an independent model further support its potential applications to additional fusion systems. By providing a robust, reproducible, and adaptable framework for time-resolved fusion analysis, this methodology bridges the gap between qualitative observation and quantitative kinetic assessment. Thus, the approach could be readily extended to other cell fusion systems following system-specific parameter optimization, offering a versatile platform for both mechanistic studies and discovery-driven screening applications.
Osteoclasts resorb bone and excessive osteoclast activity can lead to osteoporosis and increased risk of fractures. Methods are needed to measure osteoclast bone resorption activity in vitro to test potential therapeutic...Osteoclasts resorb bone and excessive osteoclast activity can lead to osteoporosis and increased risk of fractures. Methods are needed to measure osteoclast bone resorption activity in vitro to test potential therapeutics and to better understand osteoclast biology. Classical in vitro osteoclast activity assays use dentin wafers as a resorption substrate. As dentin is increasingly scarce, we formulated an alternative "bone mimetic" tissue culture plate (resorption assay plate) to reproducibly quantify osteoclast resorptive activity. The protocol requires an initial coating of untreated tissue cultures plates with bovine serum albumin (BSA) followed by serial treatment with calcium and other salt solutions to precipitate a mineral substratum on the plate surface. In cultures without cells or in cultures of RAW264.7 or mouse bone marrow monocytes cells (BMMs), no discernable resorption of the resorption assay plate substratum was observed after 14 days. However, when RAW264.7 or mouse BMMs were differentiated into osteoclasts, substantial resorption of the resorption assay plate substratum was observed after 10 days in RAW264.7-derived osteoclast cultures and after 14 days in BMM-derived osteoclast cultures. Treatment of osteoclast cultures with zoledronic acid, a bisphosphonate, significantly inhibited resorption. An ImageJ macro was developed to quantify resorption activity by identifying, counting, and measuring areas of resorption assay plate resorption from brightfield images. This protocol is a practical and consistent method to analyze osteoclast activity based on resorption of the resorption assay plate substratum.
Recently, smartphone-based sensing systems have gained significant attention due to their point-of-care detection potential. In this study, a smartphone-assisted detection strategy was developed for oxytetracycline (OTc)...Recently, smartphone-based sensing systems have gained significant attention due to their point-of-care detection potential. In this study, a smartphone-assisted detection strategy was developed for oxytetracycline (OTc) in fish muscle, utilizing an OTc-specific aptamer and the nanozyme activity of gold nanoparticles (AuNPs). The aptamer-functionalized AuNPs (Apt-AuNPs) catalyzed the oxidation of TMB via peroxidase-like activity, yielding a blue-colored oxidized TMB species. However, OTc inhibits peroxidase activity of Apt-AuNPs and blue color formation in a dose-dependent manner, enabling detection of OTc using image analysis based on RGB data. The aptamer-based assay preferentially responded to OTc, with only marginal interference effects arising from non-target antibiotics. With a detection range spanning 0.01-0.50 mg L, the method achieved a limit of detection of 0.011 mg L, which is significantly lower than the maximum permissible residue level (0.10 mg kg) defined by EU and FSSAI guidelines. The smartphone-based aptasensing technique showed great potential for fast and sensitive identification of OTc in food fish samples.
Myopia, particularly high myopia, is a serious global public health issue. While CAMKMT is linked to axial length, its mechanistic role is unclear. In lens-induced myopic (LIM) guinea pigs, we performed intravitreal inje...Myopia, particularly high myopia, is a serious global public health issue. While CAMKMT is linked to axial length, its mechanistic role is unclear. In lens-induced myopic (LIM) guinea pigs, we performed intravitreal injection of shRNA CAMKMT-carrying AAV and measured ocular parameters. Non-invasive micro-test technology (NMT) revealed increased Ca outflow in LIM sclera, exacerbating endoplasmic reticulum (ER) stress. Protein docking and Co-IP confirmed ERNI-HSPA5 interaction. LIM animals showed increased axial length, decreased refraction, and thinner sclera. Masson staining, western blot, and immunofluorescence indicated altered scleral remodeling; flow cytometry and TUNEL staining showed elevated apoptosis. Importantly, CAMKMT knockdown reduced ER stress, suppressed apoptosis, ameliorated scleral remodeling, and delayed myopia progression. These findings suggest that CAMKMT is required for myopia-associated scleral remodeling and ER stress, supporting its involvement in myopia pathogenesis via Ca-mediated signaling.
Gene cloning, site-directed mutagenesis (SDM), and short gene synthesis are essential tools in molecular biology, yet existing approaches remain constrained by efficiency, flexibility, and cost. Here, we developed a vers...Gene cloning, site-directed mutagenesis (SDM), and short gene synthesis are essential tools in molecular biology, yet existing approaches remain constrained by efficiency, flexibility, and cost. Here, we developed a versatile, simple, and rapid (VSR) 3-in-1 method that integrates seamless cloning, mutagenesis, and short gene assembly in a single platform. This approach works on the principle of overlap extension polymerase chain reaction (O), uses high-fidelity DNA polymerase and short overlapping primers. Target genes can be directly amplified from genomic DNA or complementary DNA (cDNA) templates and cloned into vectors without intermediate purification, restriction enzyme digestion, or ligation. VSR supports the insertion of DNA fragments ranging from 80 bp to 33 kb at any plasmid locus while enabling the introduction of single or multiple nucleotide substitutions at one or more sites in a single reaction. Using this method, we cloned 35 genes of diverse lengths and two complete viral genomes with efficiencies ranging from 60 to 100% and introduced up to 17 substitutions in a single mutagenesis reaction. We further demonstrate efficient de novo synthesis of short genes from overlapping oligonucleotides. The VSR method is a reliable, rapid, and cost-effective approach with a wide range of applications for both routine and high-throughput workflows.
Protein folding is governed by the principle of free energy minimization, where a protein's native tertiary structure corresponds to the global minimum on an energy landscape shaped by quantum mechanical interactions suc...Protein folding is governed by the principle of free energy minimization, where a protein's native tertiary structure corresponds to the global minimum on an energy landscape shaped by quantum mechanical interactions such as hydrogen bonding, van der Waals forces, and electron delocalization. Despite significant advances in template-based modeling (TBM), ab-initio simulations, and deep learning approaches, classical methods continue to face challenges due to the exponential complexity of the conformational search space and the approximations involved in modeling molecular interactions. Although AlphaFold, a deep learning-based protein modeling tool, has achieved a remarkable score of 92.4 in the critical assessment of protein structure prediction (CASP), classical protein structure prediction (PSP) remains hindered by the computational limitations of conventional binary architecture in representing the physical constraints of biomolecular systems. By representing the combinatorial explosion of possible conformations as a more tractable optimization problem, quantum computing offers a fundamentally new paradigm for protein three-dimensional (3D) structure prediction. In this review, we explore how quantum computing (QC) techniques including quantum annealing, quantum optimization algorithms, and hybrid quantum-classical approaches can leverage quantum properties such as superposition, entanglement, and tunneling to more efficiently navigate the complex energy landscapes associated with protein folding. While current challenges, including limited qubit fidelity, error correction, and scalability, remain, the integration of quantum algorithms with classical strategies holds significant promise for advancing structural biology, with profound implications for drug discovery and the understanding of complex biomolecular systems.
Microcapillaries are shown to be cheap but attractive alternatives to custom micromodels for the investigation of extremophile motility and tropism under reconstituted in situ conditions. We discuss practicalities and pi...Microcapillaries are shown to be cheap but attractive alternatives to custom micromodels for the investigation of extremophile motility and tropism under reconstituted in situ conditions. We discuss practicalities and pitfalls of their use in anoxic sample preparation and observation. Widefield transmission and fluorescence microscopy, combined with a simple particle tracking algorithm provide insights into motility and tropism. As an example, mono and co-cultures of Thermodesulfatator atlanticus and Methanocaldococcus villosus are examined at 70 °C and 100 bar under H:CO :: 4:1 (v:v), diluted here in 99 parts N to reproduce the standard headspace mixture for culture of methanogens. Surprisingly, despite 36 h incubation under these expected favourable conditions, we observe general mortality of both species, except for vigourous population growth of T. atlanticus at a point a few millimetres from the interface with the headspace. The space-time development of the concentrations of dissolved gases and of the pH are modelled in a numerical solution of the diffusion equations. It predicts quasi-stationary conditions at that particular point during the first ≈ 1/2h of incubation.
The vaginal microbiome is commonly classified into five Community State Types (CSTs) based on the dominance and relative abundance of Lactobacillus species, which play a central role in maintaining vaginal homeostasis th...The vaginal microbiome is commonly classified into five Community State Types (CSTs) based on the dominance and relative abundance of Lactobacillus species, which play a central role in maintaining vaginal homeostasis through the production of lactic acid and hydrogen peroxide. Disruption or depletion of Lactobacillus spp. Leads to elevated vaginal pH, thereby promoting the overgrowth of anaerobic bacteria associated with bacterial vaginosis (BV). Although next-generation sequencing (NGS)-based microbiome profiling has been widely used to characterize vaginal microbial communities, its routine clinical application remains limited by high cost, long turnaround time, and reduced suitability for rapid, species-specific quantification. In this study, we developed and validated species-specific quantitative real-time polymerase chain reaction (qRT-PCR) primer sets targeting four health-associated Lactobacillus species (Lactobacillus crispatus, Lactobacillus. gasseri, Lactobacillus. iners, and Lactobacillus. jensenii) and four BV-associated bacterial taxa (Atopobium vaginae, Clostridiales genomosp. BVAB1, Prevotella bivia, and Megasphaera type 1). Quantitative results obtained by qRT-PCR were systematically compared with relative abundance profiles derived from 16S rRNA gene V3-V4 sequencing, revealing a strong concordance between the two platforms in identifying key vaginal microbial signatures. Collectively, these findings demonstrate that qRT-PCR represents a rapid, cost-effective, and sensitive approach for targeted vaginal microbiome assessment and supports its potential utility as a molecular diagnostic tool for the precise detection of vaginal microbiota imbalance.
Tang Z, Irvine O, Duckworth E
… +11 more, Costanzo CM, Lillis K, Ren J, Anupama Bandaranayake PM, Al-Sarireh B, Mortimer M, Kanamarlapudi V, Higginbotham V, Chandrashekhara SH, Mora B, Roy D
Early detection of pancreatic cancer remains a critical challenge in oncology, with current diagnostic methods often failing to identify the disease until advanced stages. However, diagnostic accuracy alone may be insuff...Early detection of pancreatic cancer remains a critical challenge in oncology, with current diagnostic methods often failing to identify the disease until advanced stages. However, diagnostic accuracy alone may be insufficient for clinical adoption as regulatory frameworks and clinical workflows increasingly demand transparent, interpretable AI systems. This study investigates Fourier Transform Infrared (FTIR) spectroscopy combined with machine learning for non-invasive pancreatic cancer detection using urine and blood biofluids, augmented by a language-model-assisted transparency framework to bridge spectral feature attributions and biochemical interpretation. Five datasets were evaluated: urine ATR-FTIR (61.7% balanced accuracy), urine transmission FTIR (74.8%), filtered blood (<10 kDa; 89.8%), and two matched urine-blood fusion datasets. Transmission-mode urine combined with filtered blood achieved the highest performance (96.9% balanced accuracy), exceeding either biofluid alone. To support transparency, we developed an LLM-augmented explainability pipeline incorporating Monte Carlo Tree Search (MCTS) for structured hypothesis exploration, a curated retrieval-augmented knowledge base (RAG), and reliability-gated explanations that acknowledge disagreement between feature attribution methods. Explainability methods showed substantial disagreement (mean Spearman ρ = 0.23-0.28), motivating a tiered strategy: wavenumber-level interpretation when methods agree (ρ ≥ 0.3, with knowledge base verification) and zone-level interpretation otherwise. These results highlight both the potential and current limitations of transparent spectroscopic diagnostics.