Methods (San Diego, Calif.)[JOURNAL]
Sun
200 papers
Viral-mediated fluorescent labelling of activated hippocampal memory engrams to study epigenetic dynamics associated with gene expression.
Pola-Véliz V, Arredondo SB, Arancibia Y … +9 more , Ahumada J, Estay S, Vidal N, Haeger PA, Fuenzalida M, Varela-Nallar L, Bustos FJ, Montecino M, van Zundert B
Methods · 2026 Mar · PMID 41512978 · Publisher ↗
Memory formation activates a relatively sparse population of engram cells that store long-term memories. Changes in the epigenetic landscape and 3D chromatin architecture have been proposed as key candidate regulators of...
Memory formation activates a relatively sparse population of engram cells that store long-term memories. Changes in the epigenetic landscape and 3D chromatin architecture have been proposed as key candidate regulators of transcriptional waves that control gene expression in engram cells; however, isolating chromatin efficiently from engram cells has remained challenging. Double-transgenic Targeted Recombination in Active Populations (dTRAP) mice have enabled indelible EYFP labeling of hippocampal engram cells expressing the immediate-early gene (IEG) Arc when ArcCreER mice are crossed with R26R-STOP-floxed-EYFP mice and exposed to learning paradigms. A major limitation of dTRAP mice is that labeling of activated hippocampal Arc neurons with soluble EYFP compromises the efficiency of fluorescence-activated nuclear sorting (FANS) of engram nuclei, and hence isolation of chromatin. Here, we used viral-mediated delivery of GFP-KASH (AAV-PHP.eB-FLEX-EGFP-KASH) to ArcCreER mice -generating vkTRAP mice- to enable precise and robust endogenous perinuclear fluorescent tagging of activated hippocampal neurons following contextual fear conditioning (CFC). At 24 h post-CFC (24 h-CFC), vkTRAP mice exhibited a robust freezing behavior. Electrophysiological recordings in CA1 hippocampal slices showed occluded long-term potentiation (LTP). Efficient FANS-based isolation of hippocampal engram nuclei enabled chromatin immunoprecipitation (ChIP) assays (detecting H3K4me3, H3K9ac and H3K27ac) at promoters of immediate-early (Egr1) and plasticity-related (Dlg4/PSD95) genes. Expression peaks of both Egr1 and Dlg4/PSD95 transcripts during memory acquisition (1 h-CFC) and consolidation (24 h-CFC) were accompanied by active epigenetic histone mark profiles. We conclude that vkTRAP provides a robust model to study epigenomic regulation in engram cells.
ERAPID: an end‑to‑end RNA‑seq analysis pipeline for integrative candidate biomarker discovery with applications to neuropsychiatric disorders.
Park J, Youn S, Kang K … +1 more , Kang K
Methods · 2026 Mar · PMID 41500484 · Publisher ↗
RNA sequencing datasets in the gene expression omnibus (GEO) increasingly include NCBI-generated count matrices, enabling streamlined signature gene discovery. We present ERAPID, a computational framework that automatica...
RNA sequencing datasets in the gene expression omnibus (GEO) increasingly include NCBI-generated count matrices, enabling streamlined signature gene discovery. We present ERAPID, a computational framework that automatically processes this public data for robust differential expression analysis. The pipeline integrates metadata harmonization, surrogate variable analysis (SVA) to capture latent technical variation for batch correction, dual differential expression methods (DESeq2 and dream), gene set enrichment analysis (GSEA), and evidence-based gene prioritization via automated literature mining. ERAPID delivers interactive dashboards (volcano/MA plots, heatmaps, enrichment reports, searchable DEG tables) and supports an optional meta‑analysis step. Applied to a neuropsychiatric cohort (GSE80655), ERAPID completed analysis on a standard laptop in under an hour, recapitulating the reported association of EGR1 with schizophrenia. In an Alzheimer's disease (AD) case study integrating four GEO datasets, ERAPID identified 17 DEGs consistently altered across all AD‑versus‑control comparisons (e.g., ADCYAP1, PPEF1, VGF, and CRH), with KEGG Alzheimer's disease and oxidative phosphorylation pathways showing negative enrichment. Thus, ERAPID lowers the barrier to reusing public transcriptomes for signature gene discovery and biological interpretation.
MetaChrome: an open-source, user-friendly tool for automated metaphase chromosome analysis.
Sagar MAK, Dalal Y, Pegoraro G … +1 more , Arunkumar G
Methods · 2026 Mar · PMID 41475630 · Publisher ↗
DNA Fluorescence In Situ Hybridization (DNA FISH) is an essential technique to study chromosome biology and genetics, enabling precise visualization of specific genomic loci to study structural abnormalities, gene mappin...
DNA Fluorescence In Situ Hybridization (DNA FISH) is an essential technique to study chromosome biology and genetics, enabling precise visualization of specific genomic loci to study structural abnormalities, gene mapping, and chromosomal rearrangements. High-Throughput Imaging (HTI) can automate the analysis of DNA FISH chromosome images, but the accurate and automated segmentation of mitotic chromosomes and simultaneous colocalization of DNA FISH signals remains a challenge. While several commercial automated karyotyping tools partially solve these issues, open-source software that effectively combines robust chromosome segmentation with comprehensive colocalization analysis capabilities remains necessary. To address this unmet need, we developed MetaChrome, an open-source software platform built around a graphical user interface and explicitly designed for automated metaphase chromosome analysis. MetaChrome leverages fine-tuned deep learning models to automate metaphase chromosome segmentation, together with colocalization analysis of chromosome-specific FISH probes and immunofluorescent-labeled proteins. Importantly, MetaChrome achieves enhanced segmentation accuracy compared to traditional image processing methods by adopting a Cellpose segmentation model fine-tuned with manually annotated metaphase chromosome datasets. The fine-tuned model ensures the precise assignment of DNA FISH spots to individual chromosomes in an automated manner. This facilitates rapid identification of chromosomal abnormalities, reduces human error, and advances high-throughput chromosome analysis workflows, addressing a key bottleneck in chromosome biology research.
Korean longitudinal study on digitally optimized mental healthcare: a cohort profile.
Kim O, Choi KH, Kim TH … +13 more , Son S, Lee J, Kim K, Chung US, Cheon EJ, Lee I, Jung H, Kwon HJ, Han X, Choi J, Kim JW, Shin AL, Lee JJ
Methods · 2026 Mar · PMID 41455494 · Publisher ↗
The Korean Longitudinal Study on Digitally Optimized Mental Healthcare is an innovative multicenter trial-ready cohort study. It aims to develop a digitally integrated mental healthcare platform that integrates robots, a...
The Korean Longitudinal Study on Digitally Optimized Mental Healthcare is an innovative multicenter trial-ready cohort study. It aims to develop a digitally integrated mental healthcare platform that integrates robots, artificial intelligence, and local community services. A total of 3,100 participants, including 1,000 from the previous Chungnam Province cohort and 700 each from Chungnam, Gangwon, and Daegu, will be recruited between December 2024 and the end of 2027. Sociodemographic factors and physical health data are collected at enrollment using questionnaires. Every four months, formal tools are used to conduct psychiatric diagnoses and determine participants' mental health condition, such as depressive symptoms, anxiety symptoms, suicidality, stress, insomnia, loneliness, quality of life, and disability. We assessed smartphone overdependence at regular intervals. Passive data on phone usage, life patterns, and health monitoring from a smart band are collected in real time. Weekly ecological momentary assessments monitor daily life and mood and detect risk situations early. A subset of 500 participants engages in a Living Lab, collecting multi-modal data through robots and sleep radar sensors while evaluating the usability of and satisfaction with these devices. This study will show how tailored digital applications and web-based platforms can facilitate personalized self-help interventions, enhance expert-user interaction, and promote active user engagement. This approach can potentially reduce stigma and improve public awareness of mental healthcare by shifting from a treatment-centered model to a community-based prevention framework.
Cross-modal image generation with uncertainty quantification from echocardiogram to MRI.
Choudhury ZZ, Dey S, Wang H … +3 more , Coffey S, Chakraborti T, McCane B
Methods · 2026 Mar · PMID 41448444 · Publisher ↗
Medical imaging is fundamental to cardiovascular diagnostics, with modalities such as Transthoracic Echocardiography (TTE) and Cardiac Magnetic Resonance (CMR) offering complementary strengths. TTE provides real-time, no...
Medical imaging is fundamental to cardiovascular diagnostics, with modalities such as Transthoracic Echocardiography (TTE) and Cardiac Magnetic Resonance (CMR) offering complementary strengths. TTE provides real-time, non-invasive visualization of cardiac function but is often limited by operator dependency and incomplete views. In contrast, CMR delivers comprehensive, high-resolution structural assessments, although it comes with greater time and cost burdens. To address these limitations, this study explores cross-modal generative modeling techniques for synthesizing CMR-like images directly from TTE. We propose a novel architecture that combines a UNet backbone with a vision transformer, utilizing the UNet for feature extraction and the transformer for global attention to improve image synthesis quality. Quantitative and qualitative evaluations demonstrate the model's ability to produce realistic and anatomically consistent CMR images, with strong potential to improve diagnostic accuracy and clinical decision-making across multiple image modalities.
Gold nanoparticles for salivary-based sensors: A review.
Mosanaee B, Mazareei S, Fattahiyan I … +2 more , Ziyazadeh M, Ehtesabi H
Methods · 2026 Feb · PMID 41429341 · Publisher ↗
Advancements in nanotechnology integrated with biomedical diagnostics have facilitated substantial progress in non-invasive health monitoring, with salivary biosensors utilizing gold nanoparticles (AuNPs) emerging as a n...
Advancements in nanotechnology integrated with biomedical diagnostics have facilitated substantial progress in non-invasive health monitoring, with salivary biosensors utilizing gold nanoparticles (AuNPs) emerging as a novel approach for rapid, sensitive, and user-friendly point-of-care testing. This review article demonstrates how researchers utilize the distinctive physicochemical qualities of AuNPs, specifically their plasmonic responsiveness and bioconjugation ability, to improve diverse biosensing techniques. Rather than traditional blood-based diagnostics, AuNP-based devices utilize saliva as a molecular reservoir to monitor a variety of biomarkers such as hormones, metabolites, nucleic acids, or other analytes. The development of these biosensors extends beyond technological innovation to their incorporation into affordable, user-friendly devices that address clinical needs. Still, converting these biosensors into standardized health care diagnostic tools encounters similar challenges as other bioassays pertaining to biofluid-based complexity, burden of reproducibility, and regulatory approval. In addition to summarizing the extent of scientific and engineering accomplishments to date, this review highlights the opportunity for future implementation of AuNP-based biosensor tests in personalized medicine, advocating synergistic interdisciplinary approaches that integrate materials science, digital analytics, and device engineering to fully realize AuNP-based salivary diagnostics.
Microplate assay for the quantification of catalase activity in biological samples.
Montejo U, Beldarrain G, Olza S … +6 more , Alart JA, Chillida M, García-Alonso I, Alonso-Alconada D, Alonso-Varona A, Herrero de la Parte B
Methods · 2026 Feb · PMID 41422878 · Publisher ↗
Catalase is a key antioxidant enzyme that protects cells from oxidative stress by decomposing hydrogen peroxide (HO). Traditional methods for quantifying catalase activity, such as the Aebi UV method and the dichromate a...
Catalase is a key antioxidant enzyme that protects cells from oxidative stress by decomposing hydrogen peroxide (HO). Traditional methods for quantifying catalase activity, such as the Aebi UV method and the dichromate assay, are limited by low sensitivity, interference from biological molecules and require large sample and reagent volumes. Here, we present an optimized, microplate assay based on the oxidation of cobalt(II) to cobalt(III) by HO, followed by complexation with ethylenediamine-tetraacetic acid (EDTA) to form a highly colored Co(III)-EDTA complex measurable at 570 nm. This method demonstrates superior sensitivity and dynamic range (10-0.039 U/mL) compared to previous colorimetric assays, while minimizing reaction volume and avoiding interference from common biological substances. The assay has been validated using standard bovine liver catalase, liver homogenates, serum and cell lysates, showing strong linearity and specificity. This approach offers a rapid, cost-effective, and robust alternative for catalase activity measurement in biomedical research.
Establishment and application of a fully automated serum total free sulfhydryl assay based on 2,2'-DTDP.
Zeng J, Shao Z, Wang S … +9 more , Dong J, Zhou W, Zhao H, Zhang T, Zhang J, Wang J, Zheng H, Chen W, Zhang C
Methods · 2026 Feb · PMID 41422877 · Publisher ↗
BACKGROUND: Serum free thiols (FTs) are sensitive biomarkers of oxidative stress, closely associated with aging and chronic disease risk. Existing DTNB-based detection methods suffer from product instability, affecting a...
BACKGROUND: Serum free thiols (FTs) are sensitive biomarkers of oxidative stress, closely associated with aging and chronic disease risk. Existing DTNB-based detection methods suffer from product instability, affecting analytical reliability. A more stable and high-throughput method is needed. METHODS: We developed an automated FT assay using 2,2'-dithiodipyridine (2,2'-DTDP) on a clinical chemistry analyzer. Serum samples reacted with 2,2'-DTDP under optimized conditions, and absorbance at 340 nm was used to quantify FT levels. Method performance, stability, and associations with cardiovascular disease (CVD) risk factors were assessed in 341 healthy individuals. RESULTS: FTs reacted with 2,2'-DTDP to generate 2-TP, a stable product with maximum absorbance at 340 nm. The method demonstrated excellent linearity (0-1200 μmol/L), low imprecision (1.8 %-3.3 %), and reasonable recovery (≥80 %). Serum FTs were stable for at least one year at -70 °C, 12 h at 2-8 °C, and 2 h at room temperature. In the study population, FT levels were positively correlated with serum albumin, a major thiol carrier, and negatively correlated with age, triglyceride, glucose and creatinine, suggesting links to redox status and CVD risks. These associations support the potential of FTs as indicators of systemic oxidative balance and aging-related health decline. CONCLUSION: This simple and robust 2,2'-DTDP-based method enables accurate, high-throughput measurement of serum FTs. It is well suited for exploring thiol antioxidant function and evaluating age-related and cardiovascular risk in clinical and research settings.
In vitro wound simulation: A high-throughput device for scratch assays.
Labovitz JE, Kulaga P, DuBois EM … +2 more , Li K, O'Shea TM
Methods · 2026 Feb · PMID 41421570 · Full text
Traumatic injury to the healthy central nervous system (CNS) causes mechanical tissue damage that results in localized cell death and blood-brain-barrier (BBB) disruption. CNS tissue damage stimulates a multicellular wou...
Traumatic injury to the healthy central nervous system (CNS) causes mechanical tissue damage that results in localized cell death and blood-brain-barrier (BBB) disruption. CNS tissue damage stimulates a multicellular wound response to limit the extent of damage but fails to reestablish the normal function of injured tissue. There is strong interest in developing new strategies to augment regeneration after CNS injury. To enable therapy development, reliable assays to screen and identify molecular approaches to augment glial-based wound responses over fibrotic scarring are needed. Scratch assays, which involve mechanically removing cells from an in vitro culture, allow for the simulation of wounds with high throughput and tight control over applied treatments to mechanistically study cell migration and proliferation functions that are critical to effective repair. Current methods require researchers to individually scratch each well with a pipette tip, resulting in low throughput as well as inconsistent scratch widths, straightness, and efficacy within and between wells. Here, we describe the design of a quickly assembled (<30 min), inexpensive (<$110) scratch assay rig that readily creates uniform scratches that are straight (average tortuosity < 1.1), have tunable widths (730-1100 µm), and fully remove damaged cells from the simulated wound region. Designed for a 24-well plate, the rig allows for high-throughput screening of varied experimental conditions or for testing many replicates. Application of the scratch assay device on an in vitro culture of neural progenitor cells (NPC) demonstrates the ability to detect differences in wound closure rate for three unique media conditions. These results support the implementation of this high-throughput scratch assay rig as a method to standardize and improve the efficiency of in vitro wound healing studies.
Development of a novel microsampling device to standardize the analysis of intranasal inflammatory biomarkers.
Lupancu T, Limpah J, Aydin E … +7 more , Fan T, Wong V, Rimmer J, Wang BS, Yen DM, Rostom E, Damry AM
Methods · 2026 Mar · PMID 41421569 · Publisher ↗
Nasal fluid biomarker analysis is an emerging technique for studying sinonasal pathophysiology, monitoring therapeutic efficacy, and discovering novel drug targets. Variability in biomarker results can be contributed to...
Nasal fluid biomarker analysis is an emerging technique for studying sinonasal pathophysiology, monitoring therapeutic efficacy, and discovering novel drug targets. Variability in biomarker results can be contributed to non-standardized collection methodology. To address this, a novel microsampler was developed, designed to enable precise site-specific sampling, consistent volume collection, and high analyte recovery. This study aims to evaluate the performance of this new microsampler device compared to commonly utilized flocked swab, and other absorbent materials. To do so, fixed volumes of a synthetic nasal fluid mimic were deposited onto the anterior region of the inferior turbinate of a 3D-printed sinus model to assess volumetric and collection site accuracy of the nasal microsampler, in comparison to a flocked swab. Additionally, protein biomarker recovery properties of the device's absorption membrane, Leukosorb, versus experimental proprietary absorbent materials, were assessed using ELISA. The microsampler, contrasting the flocked swab, demonstrated statistically significant lower coefficient of variation for collected nasal fluid volume and greater sampling site precision. The spike and recovery study indicated that the proprietary materials had statistically significant higher biomarker recovery rates than Leukosorb. Overall, the novel nasal microsampler offers significantly improved volumetric control and site-specific collection against flocked swab. All experimental proprietary absorbent materials displayed significantly higher protein recovery rates, comparing to widely accepted and utilized Leukosorb. Consistent use of the novel nasal microsampler device has the potential to standardize protein recovery processes and minimize variability across studies, leading to enhanced reliability and comparability of future findings.
Application of a 2A-peptide system for polycistronic human IgG production in Leishmania tarentolae.
Lai JY, Tan KY, Tan CH … +2 more , Choong YS, Lim TS
Methods · 2026 Feb · PMID 41407086 · Publisher ↗
Monoclonal antibodies are commonly expressed using mammalian cell lines due to their required post-translational modifications. However, this process can become costly and requires investment in specialized equipment and...
Monoclonal antibodies are commonly expressed using mammalian cell lines due to their required post-translational modifications. However, this process can become costly and requires investment in specialized equipment and facilities. The use of eukaryotic cell Leishmania tarentolae offers a cost-effective alternative while still allowing N-linked glycosylation to occur. The focus of this study explores the use of L. tarentolae as a platform for expressing a monoclonal IgG antibody against the long-chain neurotoxin (LNTX) from the venom of the Monocled Cobra, Naja kaouthia. The monoclonal antibody was isolated from a human naïve phage display library and expressed as single-chain fragment variable (scFv) in Escherichia coli with good specificity against the LNTX of Naja kaouthia. The scFv was then converted to the scFv-Fc format and full-length IgG for expression in L. tarentolae. The IgG expression was achieved using a 2A peptide-based bicistronic vector system with good expression yield. The expressed IgG demonstrated homogeneity in size and minimal degradation. Therefore, L. tarentolae can be considered a possible alternative platform for the expression of human scFv-Fc and IgG antibodies.
Sensitivity-improving CRISPR-Cas strategies for non-nucleic acid targets detection.
Qi H, Yang Y, Hou X … +2 more , Chen Y, Gong S
Methods · 2026 Feb · PMID 41407085 · Publisher ↗
CRISPR-Cas systems have revolutionized non-nucleic acid targets detection across diverse applications. Nevertheless, the relatively low enzymatic turnover rate of activated Cas nucleases during substrate cleavage remains...
CRISPR-Cas systems have revolutionized non-nucleic acid targets detection across diverse applications. Nevertheless, the relatively low enzymatic turnover rate of activated Cas nucleases during substrate cleavage remains a critical bottleneck, limiting the sensitivity of such detection methods. To address this challenge, numerous innovative strategies have been proposed to enhance the sensitivity of CRISPR-Cas systems, enabling high-sensitive non-nucleic acid targets detection. This review systematically summarizes the sensitivity-enhancing methodologies for non-nucleic acid targets detection using CRISPR-Cas technologies. We first delineate the working mechanisms of various CRISPR-Cas systems and the signal transduction pathways specific to non-nucleic acid targets. Subsequently, we detail diverse sensitivity-improving approaches, including nucleic acid amplification-facilitated strategies, multimolecular labeling techniques, dual-enzyme cascade methods, and multiplex amplification methodologies. Additionally, the current challenges and future perspectives in this field are discussed, aiming to inspire researchers to develop more ingenious solutions and facilitate real-world applications of CRISPR-Cas system for non-nucleic acid targets detection.
Denaturation, rapid dilution refolding, and single-step purification of the core histones using desalting size-exclusion chromatography.
Ige OO, Hendrickson-Rebizant T, Luo W … +8 more , Oyeyode M, Jacobson S, Ortiz Bada FM, Rowley MJ, Liu RW, Davie JR, Frankel A, Lakowski TM
Methods · 2026 Feb · PMID 41386538 · Publisher ↗
Nucleosomes, composed of DNA and histone octamers, regulate gene expression through histone modifications such as lysine acetylation and methylation. These modifications are added by writers, removed by erasers, and inte...
Nucleosomes, composed of DNA and histone octamers, regulate gene expression through histone modifications such as lysine acetylation and methylation. These modifications are added by writers, removed by erasers, and interpreted by readers to control gene expression, chromatin structure, and essential cellular processes such as differentiation and development. Accurate Post Translational modifications analysis requires high-purity histones due to the sensitivity of epigenetic assays. Recombinant histones are expressed in E. coli as inclusion bodies, requiring denaturation, refolding, and purification. These traditional purification methods involve complicated and lengthy protocols taking days and potentially exposing the histones to oxidation and proteolytic degradation. We developed a rapid method for refolding histones from inclusion bodies in a one-step purification using a desalting column achieving > 90 % purity. This method is compared to our previous High Performance Liquid Chromatography (HPLC)-based protocol. Our single-step desalting purification reduces purification time from multiple days to one day, lowers operational cost, and eliminates the need for reverse-phase HPLC, making high-purity histone production accessible to laboratories without specialized chromatography infrastructure.
Investigation on structure-property relationships of MgO-SrO containing silicate-based bioactive glasses: An experimental and molecular dynamics simulation study.
Moghanian A, Farmani R, Kolivand N … +2 more , Tayebi A, Safaee S
Methods · 2026 Feb · PMID 41365392 · Publisher ↗
Molecular dynamics (MD) simulations and experimental analysis were performed on MgO-SrO containing bioactive glasses (MSBGs) with the composition of 60SiO-(31-x)CaO-4PO-5MgO-xSrO (mol%) (x = 0, 1, 3, 5, 8, 10, 15, 20; M5...
Molecular dynamics (MD) simulations and experimental analysis were performed on MgO-SrO containing bioactive glasses (MSBGs) with the composition of 60SiO-(31-x)CaO-4PO-5MgO-xSrO (mol%) (x = 0, 1, 3, 5, 8, 10, 15, 20; M5S0-M5S20) to evaluate the structural properties, ion clustering, and dissolution behavior as a function of SrO content. Simulation employed the Buckingham potential for short-range interactions and Coulombic potentials for long-range forces. MSBGs had Si-O and P-O bond lengths of 1.609 Å and 1.491 Å, with O-Si-O and O-P-O bond angles centered at ∼109.3° and 109.4°, respectively, confirming tetrahedral SiO/PO coordination. Across all compositions, Si-O-Si bonds dominated the majority of the distribution (88-89 %), with Si-O-P at 11-12 % and P-O-P negligible (∼0.3 %). Densities decreased from 2.913 g·cm (M5S20) to 2.631 g·cm (M5S0), reflecting network loosening with SrO substitution. Q distribution remained stable, with Q/Q fractions of 38-43 % and 26-30 % for Si-based tetrahedra. R-factor analysis revealed optimal homogeneity for M5S5 (R = 0.838252), balancing reduced cation clustering and moderate network stability. ICP-AES showed M5S5 with a sustained release of Si, Mg, and Sr over 24 h. Meanwhile, antibacterial study resulted in statistically significant increase in efficiency for M5S5 compared to M5S0 (***p < 0.001). The combined computational and experimental findings identify M5S5 as the most promising candidate for biomedical applications requiring structural benefits, controlled ion release, and antibacterial efficiency.
MDM2/p53-based live-cell quantitative FRET imaging for apoptosis drug discovery.
Xiao Z, Xie L, Wu Z … +3 more , Cai X, Zhuang Z, Chen T
Methods · 2026 Feb · PMID 41352426 · Publisher ↗
Targeting the interaction between P53 and MDM2 to re-activate P53 to induce apoptosis is an important strategy for cancer treatment. In this study, based on the unique advantages of in situ visualization, dynamic imaging...
Targeting the interaction between P53 and MDM2 to re-activate P53 to induce apoptosis is an important strategy for cancer treatment. In this study, based on the unique advantages of in situ visualization, dynamic imaging, and quantitative analysis of living cell FRET imaging, a method for screening apoptotic drugs targeting p53-MDM2 interaction was developed. A stable model of Nutlin-3-induced apoptosis was established in MCF-7 cells, which was verified by reducing mitochondrial membrane potential and increasing the proportion of nuclear chromatin condensation (from 9.16 % to 50.55 %). Biochemical methods such as WB analysis found that after activating P53, BAX expression was up-regulated through a Puma-independent pathway, which promoted BAX oligomerization. Live-cell quantitative FRET imaging found that the maximum donor center FRET efficiency (E) of CFP-p53 and YFP-MDM2 decreased from 0.50 to 0.22 after Nutlin-3 treatment, and the co-localization coefficient decreased significantly from 83 % to 22 %, confirmed that Nutlin-3 directly disrupted the interaction between P53/MDM2, promoting P53 nuclear translocation and apoptosis. This indicated that Nutlin-3 was a direct inhibitor of the P53/MDM2 interaction. Apoptosis drug screening was performed in MCF-7 cells, and we found that the E was 0.29 and 0.31 for the cells treated with DOX and RSV, respectively, and 0.48 for the IKE-treated cells and 0.43 for the SOR-treated cells, indicating that DOX and RSV, but not IKE and SOR, were potential P53/MDM2-dependent apoptotic drugs. In addition, Nutlin-3 treatment decreased the E value in p53 wild-type U2OS cells from 0.43 to 0.20. In summary, our method can identify p53-MDM2 interaction inhibitors in living cells, providing a quantitative in vivo supplement for traditional target-based drug discovery.
Development and characterization of a membrane-permeant GRASP65-mimetic peptide that inhibits Golgi unlinking and cell cycle progression.
Cervigni RI, Bonavita R, Barretta ML … +9 more , Spano D, Ayala I, Mascanzoni F, Iannitti R, Henklein P, Monti A, Renna M, Doti N, Colanzi A
Methods · 2026 Feb · PMID 41338296 · Publisher ↗
The Golgi complex is central to cellular homeostasis and serves as a key processing and sorting hub for protein trafficking. In many cell types, the Golgi complex is organized as interconnected stacks of cisternae, formi...
The Golgi complex is central to cellular homeostasis and serves as a key processing and sorting hub for protein trafficking. In many cell types, the Golgi complex is organized as interconnected stacks of cisternae, forming a structure known as the Golgi ribbon. This ribbon undergoes dynamic remodelling during physiological processes, such as cell division, and under pathological conditions, including cancer and neurodegeneration. A critical step in the unlinking of the Golgi ribbon involves the phosphorylation of the stacking protein GRASP65, which leads to the separation of the ribbon into individual stacks, a process necessary for the G2/M transition of the cell cycle. However, existing tools for selectively manipulating the GRASP65 role in ribbon organization are limited by non-specific effects or technical challenges. Here, we present the development and characterization of a membrane-permeable peptide, R-GRASP65-S277, derived from GRASP65 and containing the phosphorylation site Ser277, which is essential for Golgi unlinking. This peptide effectively inhibited Golgi unlinking and mitotic entry in several cell lines, including cancer models. In contrast, a control peptide with a non-phosphorylatable alanine substitution (R-GRASP65-S277A) showed no such effect, confirming the specificity of the tool. Furthermore, the R-GRASP65-S277 peptide reversed Golgi unlinking induced by the chemotherapeutic agent doxorubicin, demonstrating its utility in studying stress-induced Golgi disassembly. These findings establish the R8-GRASP65-S277 peptide as a specific, potent, and scalable tool for probing the molecular mechanisms of Golgi unlinking, its regulation of cell cycle progression, and its potential contributions to pathological states.
Sensitive and specific detection of ctDNA using Copper-Free click chemistry and magnetic bead Technology.
Didarian R, Kanarya D, Sahin S … +4 more , Özyurt C, Evran S, Odaci D, Yildirim-Tirgil N
Methods · 2026 Feb · PMID 41330520 · Publisher ↗
Liquid biopsy, particularly the analysis of circulating tumor DNA (ctDNA), offers immense potential for non-invasive cancer diagnosis and monitoring. It provides a less invasive alternative to traditional tissue biopsies...
Liquid biopsy, particularly the analysis of circulating tumor DNA (ctDNA), offers immense potential for non-invasive cancer diagnosis and monitoring. It provides a less invasive alternative to traditional tissue biopsies, enabling earlier detection and real-time assessment of disease progression. However, a significant hurdle in its widespread adoption is the extremely low concentration of ctDNA in biological samples, especially during the early stages of cancer, making sensitive and specific detection challenging. This work addresses the critical problem of developing a highly sensitive and specific method for low abundance ctDNA detection. We developed a novel, highly sensitive, and specific method for ctDNA analysis, employing copper-free click chemistry (strain-promoted azide-alkyne cycloaddition, SPAAC) for enzyme-free amplification, coupled with magnetic bead-assisted fluorometric detection. This enzyme-free approach significantly enhanced specificity and reduced background noise. We meticulously optimized parameters, including primer length and annealing temperature, finding that 30-base primers and a 50 °C annealing temperature yielded optimal amplification efficiency. Our method successfully detected ctDNA at concentrations as low as 10 pM (15 bp primer). Agarose gel electrophoresis confirmed highly specific amplification with minimal non-specific products, and the assay demonstrated excellent allelic discrimination, accurately distinguishing single-nucleotide mutations. Importantly, the method proved robust in complex human serum samples, demonstrating its practical applicability. This innovative, cost-effective, and enzyme-free platform overcomes many limitations of current ctDNA detection technologies. By enabling highly sensitive and specific detection of low abundance ctDNA, this methodology represents a significant leap forward for non-invasive cancer diagnostics, paving the way for earlier disease detection, improved treatment monitoring, and the broader implementation of personalized medicine.
Fabrication of advanced microneedle-based targeted intravaginal drug delivery devices: therapeutic opportunities and translational challenges.
Patil TS, Chaudhari DP, Bhamare UU … +3 more , Palkar MB, Mahajan MR, Nangare SN
Methods · 2026 Feb · PMID 41320146 · Publisher ↗
Millions of women worldwide suffer from a variety of health conditions, including vaginal bacterial and yeast infections, sexually transmitted infections (STIs), urinary tract infections, pelvic inflammatory disorders, a...
Millions of women worldwide suffer from a variety of health conditions, including vaginal bacterial and yeast infections, sexually transmitted infections (STIs), urinary tract infections, pelvic inflammatory disorders, and hormonal abnormalities. Despite major advances in biomedical research, traditional intravaginal drug administration systems such as gels, creams, and suppositories frequently encounter issues such as fast drug clearance, leakage, and uneven mucosal retention, reducing therapeutic effectiveness. Microneedles, a painless and less invasive drug delivery technology, represent a viable alternative to standard formulations because they allow for accurate, controlled, and localized drug administration. Therefore, the present review focuses on possibility of microneedle-based techniques for intravaginal medication administration. In brief, it covers the need for breakthroughs in vaginal drug administration. It provides an overview of several types of microneedles, including solid, hollow, dissolving, coated, and hydrogel-forming, and their manufacturing procedures. Then, it delves into their use in intravaginal drug administration, highlighting their capacity to improve drug penetration and retention. Finally, it discusses future problems, prospective advancements, and the larger implications of microneedle technology in vaginal therapy. Microneedle-based intravaginal medication delivery is a huge step forward in targeted vaginal infection treatment. Notably, microneedles easily cross the cervicovaginal mucus barrier, increasing drug absorption at the target region while being minimally invasive. Future studies should focus on improving microneedle formulations, assessing long-term safety, and investigating their potential for wider clinical applications.
Explainable machine learning-based prediction of psoriatic arthritis flares using heterogenous real-world data for personalised patient care.
Moon P, Li W, Chan A … +2 more , Wang B, Bazuaye E
Methods · 2026 Feb · PMID 41275993 · Publisher ↗
Psoriatic arthritis (PsA) is a chronic inflammatory disease characterised by unpredictable flare-ups that are difficult to forecast, particularly in patients without an acute phase response. In this paper, we propose and...
Psoriatic arthritis (PsA) is a chronic inflammatory disease characterised by unpredictable flare-ups that are difficult to forecast, particularly in patients without an acute phase response. In this paper, we propose and apply an explainable, multimodal machine learning framework that jointly leverages structured temporal electronic patient records (EPRs) - sequential blood tests, disease activity scores, comorbidity burden, medications, and demographics - and unstructured clinical referral letters pre-processed with large language models ((LLMs, (Qwen-2.5 family)) to predict PsA flares. Gradient boosting models, Light Gradient Boosting Machine (LGBM) and eXtreme Gradient Boosting (XGBoost) were used to predict PsA flares, achieving the highest predictive performance 3 months before a clinic visit (accuracy = 92.8 %, AUROC = 0.94). Model performance gradually declined for longer timeframes (6 months: 78.2 %, AUROC = 0.80; 9 months: 76.6 %, AUROC = 0.78; 12 months: 72.2 %, AUROC = 0.75). LLMs applied to unstructured GP referral letters had limited standalone predictive value, but enhanced sensitivity and specificity when combined with the structured models in an ensemble approach. SHapley Additive exPlanations (SHAP) helped explain the prediction and demonstrated comorbidity count, disease scores, and immunosuppressive medications as the top predictors. Our results show that integrating both structured longitudinal data with unstructured clinical narratives using interpretable multimodal artificial intelligence can enable time-sensitive, personalised management of PsA flares and early clinical intervention.
A smoothing method for DNA methylome analysis to enhance epigenomic signature detection in epigenome-wide association studies.
Oussalah A, Mousel L, Trégouët DA … +1 more , Guéant JL
Methods · 2026 Feb · PMID 41274433 · Publisher ↗
Epigenome-wide association studies (EWAS) are instrumental for mapping DNA methylation changes in human traits and diseases but often suffer from low statistical power and false positives, especially in small cohorts. We...
Epigenome-wide association studies (EWAS) are instrumental for mapping DNA methylation changes in human traits and diseases but often suffer from low statistical power and false positives, especially in small cohorts. We developed an EWAS smoothing method that exploits co-methylation of adjacent CpG probes within CpG islands via a sliding-window average and generalized it using Savitzky-Golay filtering. We applied the smoothing approach-with window widths of 1-3 CpGs and, for generalization, Savitzky-Golay filters of varying polynomial orders and window sizes-across five distinct EWAS settings. Performance was quantified by signal-to-noise ratio (SNR), noise-variance reduction, variance ratio (VR), Bayes factors, and sample-size sensitivity. In the MMACHC epimutation dataset, a 5-CpG window (width, w = 2) increased SNR by 90 %, reduced noise variance by 80 %, and elevated VR by 176 % at the target CpG island, with no genome-wide false positives. For MLH1, smoothing preserved the top association and suppressed background signals. In the aging EWAS, a "Polyepigenetic CpG aging score" was derived following smoothing. This score correlated strongly with chronological age in the discovery cohort (Spearman's ρ = 0.89; P = 3.0 × 10) and was independently validated in a separate dataset, significantly distinguishing newborns from nonagenarians (P = 3.4 × 10). Savitzky-Golay filtering of order 0 with a 5-CpG window yielded optimal SNR across bootstrap iterations, supporting this configuration as a robust choice for methylation array smoothing. As an extension of the Savitzky-Golay-based smoothing framework, reanalysis of a liver cancer dataset identified five top loci surpassing a smoothed P-value threshold of 1 × 10. Among these, MIR10A within the HOXB3 locus was the only previously reported functionally relevant site. In conclusion, the smoothing method improves EWAS performance by enhancing SNR, enabling detection of meaningful associations even in small cohorts, and offers a valuable tool for reanalyzing existing Infinium methylation array datasets to uncover previously undetected epigenomic signatures.
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