Methods (San Diego, Calif.)[JOURNAL]
Sun
200 papers
Low-Potential Osmium-Based PVI redox polymer mediators for Diffusion-Controlled electrochemical glucose quantification.
Chung SY, Jeon WY, Seo TW … +1 more , Choi YB
Methods · 2026 Jul · PMID 41974238 · Publisher ↗
Osmium-based redox polymer mediators are attractive candidates for low-potential electrochemical biosensing, but a systematic evaluation of polymer-ligand coordination effects under identical electrode configurations is...
Osmium-based redox polymer mediators are attractive candidates for low-potential electrochemical biosensing, but a systematic evaluation of polymer-ligand coordination effects under identical electrode configurations is limited. In this study, an osmium-based polymer redox mediator, PVI-Os(dma-bpy)Cl, was synthesized, and its quantitative electrochemical performance for glucose detection was evaluated on an identical electrode platform. The formation of the polymer-metal complex and its physicochemical properties were confirmed by UV-Vis spectrophotometer, Fourier transform infrared spectroscopy, H-nuclear magnetic resonance spectroscopy, and zeta potential analyses, indicating strong positive surface charge and good aqueous dispersibility. Electrochemical characterization using cyclic voltammetry and scan rate analysis showed diffusion-controlled redox behavior on carbon screen-printed electrodes immobilized with FAD-dependent glucose dehydrogenase (FAD-GDH), indicating efficient charge transport within the polymer network. The measurement conditions were optimized with respect to pH and enzyme loading, and the highest sensitivity and signal stability were obtained in phosphate-buffered saline (PBS, pH 7.4) with an enzyme concentration of 40 mg mL. Under multi-potential step (MPS) conditions, the PVI-Os(dma-bpy)Cl-modified electrode exhibited stable and stepwise current responses to increasing glucose concentrations. Quantitative analysis revealed a wide linear range from 1 to 20 mM glucose with a sensitivity of 7.4190 µA cm mM at a low operating potential of 0.3 V and excellent linearity (R = 0.99643). Negligible current responses were observed in the presence of common electroactive interferents, including ascorbic acid, dopamine, and uric acid, confirming the high selectivity under the applied conditions. Overall, this study presents a systematic evaluation of osmium-based polymer redox mediators and shows that ligand-tuned PVI-Os systems allow stable, low-potential, and interference-resistant electrochemical glucose detection. The proposed mediator platform is suitable for integration into electrochemical glucose sensors and enzymatic biofuel cell systems.
Renovating the Barnes maze for mouse models of dementia with STARR FIELD: A 4-day protocol for learning rate, retention, and cognitive flexibility.
Bertolli AX, Halhouli O, Weber MA … +19 more , Liu-Martínez Y, Wendt LH, Blaine B, Thangavel R, Smadi S, Pellatz PJ, Liu D, Bornhoft KN, Gupta K, Dean R, Tish MM, Kerr G, Heiney SA, Emmons EB, Zhang Q, Gumusoglu SB, Narayanan NS, Geerling JC, Aldridge GM
Methods · 2026 Jul · PMID 41966430 · Publisher ↗
Land-based spatial mazes are a low-stress method to evaluate learning in rodent models of dementia. By using innate exploratory and hiding behavior, the Barnes maze requires fewer trials, allowing examination of early le...
Land-based spatial mazes are a low-stress method to evaluate learning in rodent models of dementia. By using innate exploratory and hiding behavior, the Barnes maze requires fewer trials, allowing examination of early learning rate and retention, as well as executive and motivational features that can be characteristic of non-amnestic dementias. However, unwanted odor cues may disrupt interpretation of acquisition rate during typical learning trials. We designed and tested our Barnes FIELD protocol (Find the Invisible Exit to Locate the Domicile) to improve reproducibility, allow evaluation of learning trials, and limit experimenter influence. The protocol uses 3D-printed escape shuttles and docking tunnels, allowing mice to exit the maze to the home cage. We show evidence that our shuttles mitigate the possibility of undesired cues. We demonstrate the feasibility of our protocol across several models of cognitive impairment and aging, and develop an additional stage, the STARR (Spatial Training And Rapid Reversal) maze, to better challenge behavioral flexibility. By examining commonly used outcome measures we identify important considerations for interpretation. These insights are used to evaluate several models of cognitive change, including deficits in an Alzheimer's disease mouse model and behavioral flexibility in a model of brainstem dysfunction. This work provides comprehensive instructions to build, perform, and analyze a robust spatial maze that expands the range of behavioral and motivational outcomes that can be identified and screened. Our findings will aid interpretation of traditional protocols, enhance rigor and reproducibility, and provide an updated method to screen for cognitive changes in mice.
Label-free naked-eye aptasensor for detection of Cytochrome c- mediated apoptosis drug screening.
Karimi MS, Borghei YS, Hosseinkhani S
Methods · 2026 Jul · PMID 41956334 · Publisher ↗
Apoptosis is a tightly regulated biochemical process essential for cellular homeostasis, in which the mitochondrial release of Cytochrome c (Cyt c) represents an early and irreversible hallmark. Reliable detection of Cyt...
Apoptosis is a tightly regulated biochemical process essential for cellular homeostasis, in which the mitochondrial release of Cytochrome c (Cyt c) represents an early and irreversible hallmark. Reliable detection of Cyt c is therefore of great importance in apoptosis-based drug screening and cancer research. In this study, we report a label-free colorimetric aptasensor for Cyt c detection based on the in-situ synthesis of gold nanoparticles (AuNPs) on a polyguanine (poly-G)-extended aptamer, without any chemical modification or DNA labeling. The aptamer-templated AuNPs (Apt@AuNPs) act as both a recognition element and signal transducer, where specific binding of Cyt c induces nanoparticle aggregation, leading to a distinct localized surface plasmon resonance (LSPR) red shift that is visible to the naked eye. The proposed sensing platform exhibits a linear response toward Cyt c in the concentration range of 1 to 1000 µM, with a calculated detection limit of 0.47 µM. In addition the intrinsic nanozyme activity of Apt@AuNPs was exploited to develop a dot blot assay for selective Cyt c recognition. Importantly, the assay successfully differentiated apoptotic and non-apoptotic cancer cells based on a clear color change (purple vs. red). Overall, this work introduces a simple, modification-free, and visually interpretable biosensing strategy for Cyt c detection, highlighting its potential application in apoptosis assessment and apoptosis-based drug screening, particularly in settings requiring rapid and instrument-free analysis.
Design principles for LAMP-CRISPR molecular diagnostics.
Silva CS, Nascimento GR, Cruz PEO … +5 more , Arancibia RH, Andrade Belitardo EMM, Castro TLP, Villar LM, Pacheco LGC
Methods · 2026 Jul · PMID 41932456 · Publisher ↗
Nucleic acid detection methods leveraging Cas9, Cas12, and Cas13 enzymes have recently been widely integrated with isothermal amplification techniques, particularly Loop-Mediated Isothermal Amplification (LAMP), to devel...
Nucleic acid detection methods leveraging Cas9, Cas12, and Cas13 enzymes have recently been widely integrated with isothermal amplification techniques, particularly Loop-Mediated Isothermal Amplification (LAMP), to develop CRISPR-based diagnostic assays for a broad range of pathogens. Coupling these systems with portable result-readout platforms such as lateral flow devices, microfluidics, and smartphones offers a promising pathway for deploying LAMP-CRISPR diagnostics at the point-of-care (PoC), especially in settings where conventional, resource-intensive methods like real-time PCR are not feasible. However, the development of LAMP-CRISPR assays presents unique challenges not typically encountered in real-time PCR workflows. These include the need for a larger number of oligonucleotides, the complexity of integrating multiple biochemical conditions, and a heightened risk of false-positive results. Despite the growing number of bioinformatics tools designed to aid assay development, establishing a robust and reproducible workflow for LAMP-CRISPR remains a significant hurdle. In this review, we critically examine current strategies for designing LAMP-CRISPR assays and offer a detailed, step-by-step guide to achieving high-performance diagnostic tools using this approach. We cover key aspects of target sequence selection, oligonucleotide and CRISPR system design, and the strategic choice of readout methods. We further discuss available tools for LAMP primer and CRISPR guide RNA design, providing practical recommendations for optimizing sequence selection. Various probe formats for Cas-mediated trans-cleavage detection are summarized, and we present best practices for assay standardization and minimizing false-positive signals. Finally, we highlight the current limitations and outline future directions for LAMP-CRISPR diagnostics in decentralized and PoC testing environments.
Digital PCR as a potential reference measurement procedure to support monkeypox virus/Orthopoxvirus external quality assessment schemes.
Falak S, Beheim-Schwarzbach J, Hübner A … +11 more , Kammel M, Martin A, Grunert HP, Dühring U, Zeichhardt H, Ehret R, Obermeier M, Groß A, Schellenberg I, Kummrow A, Valiente E
Methods · 2026 Jul · PMID 41921633 · Publisher ↗
Since May 2022, a wave of several thousand cases of monkeypox Virus (MPXV) infections has been reported across Europe. In the beginning of this outbreak, reference material (RM) and reference measurement procedure (RMP)...
Since May 2022, a wave of several thousand cases of monkeypox Virus (MPXV) infections has been reported across Europe. In the beginning of this outbreak, reference material (RM) and reference measurement procedure (RMP) were lacking. We developed a measurement procedure using digital PCR (dPCR) for the absolute quantification of the viral genome of MPXV. This study investigated dPCR as potential reference method for MPXV quantification and evaluated performance in the External quality assessment (EQA) and clinical samples. This included DNA extraction performance using two commercial kits, MPXV target assays performance in duplex format and assay characteristics to differentiate the MPXV to Orthopoxvirus (OPXV). The analytical performance and validation on different dPCR platforms were examined. The developed dPCR assay was applied and supported extensive EQA schemes for mokeypox/Orthopox virus genome detection program and assigned reference values to the corresponding EQA materials within the range of 103 copies/mL to 10 copies/mL. The candidate reference method was applied to clinical MPXV patients' samples. The estimated viral DNA quantity in clinical samples using quantitative real time PCR (qPCR) showed good correlation and agreement with dPCR reference value. Thus, the dPCR-based method enabled reliable value assignment of MPXV reference materials. This method offers a calibration-free alternative for labs without access to certified standards. Our findings suggest that dPCR could be used for reference measurement value assignment of MPXV reference materials to support calibration of MPXV viral load testing in molecular diagnostic testing laboratories.
A simple and accurate high throughput evaluation method for larval zebrafish xenografts.
Liu T, Liang Y, Cai AQ … +7 more , Liu YW, Zhao YJ, Liu L, Liu YJ, Yi XF, Zou XZ, Huang P
Methods · 2026 Jun · PMID 41921632 · Publisher ↗
The advantages of short assay duration, low cost, and high-throughput make larval zebrafish xenografts a popular in vivo model in cancer research, especially in large-scale screening of anti-tumor drug and target identif...
The advantages of short assay duration, low cost, and high-throughput make larval zebrafish xenografts a popular in vivo model in cancer research, especially in large-scale screening of anti-tumor drug and target identification. Recently, the great potential of larval zebrafish xenografts in precision cancer therapy has also been demonstrated. Establishing an accurate high-throughput analytical method is now the main challenge of this model. In this study, we, for the first time, compared the current mainstream analytical methods. Calculating the volume of xenografts based on confocal imaging was accurate but labor-intensive and time-consuming. Although measuring tumor area is an effective method to achieve high-throughput analysis, its accuracy was low. Based on our experience, we found that analyzing the maximum fluorescence intensity (mFI) avoided manual error, reduced the intra-group variability, and enable high-throughput analysis. Moreover, mFI accurately reflected the change of xenograft size and significantly reduced the sample size required for one test. This study provided a simple and accurate high-throughput analytical method for assessing larval zebrafish xenografts, and the development of an automatic high-throughput analysis platform based on this method will further promote anti-tumor drug development and precision cancer therapy.
Cell labeling approaches for tracking stromal vascular fraction fate.
Dadashizadeh G, Wojtowicz-Piotrowski S, Khalaf F … +2 more , Vlavcheski F, Jeschke MG
Methods · 2026 Jun · PMID 41887345 · Publisher ↗
The stromal vascular fraction (SVF) of adipose tissue is a heterogeneous cell population with significant regenerative, paracrine, and immunomodulatory potential, positioning it as a promising therapeutic platform in reg...
The stromal vascular fraction (SVF) of adipose tissue is a heterogeneous cell population with significant regenerative, paracrine, and immunomodulatory potential, positioning it as a promising therapeutic platform in regenerative medicine and oncology. As SVF-based strategies advance toward preclinical and clinical application, robust methods to label and track transplanted cells have become essential for elucidating their biodistribution, engraftment, survival, differentiation, and functional roles in vivo. A wide spectrum of SVF labeling approaches has been developed, including fluorescent dyes, magnetic and inorganic nanoparticles, radiotracers, and genetically encoded reporters. While these techniques have enabled important biological insights, they differ markedly in sensitivity, stability, biocompatibility, detection modality, and translational feasibility, and each presents distinct methodological limitations such as signal dilution, cytotoxicity, or challenges in distinguishing viable from non-viable cells. In this review, we provide a comprehensive and critical synthesis of current SVF labeling strategies, detailing their mechanistic principles, technical workflows, and applications in in vitro and in vivo tracking studies. Moreover, we highlight the advantages, limitations, and appropriate uses of each modality. By consolidating and evaluating the existing literature, this review aims to support informed selection of labeling techniques and to facilitate the design of rigorous, reproducible, and clinically relevant SVF tracking studies.
The transformative role of single-cell analysis in multifactorial disorders research.
Wang CY, Ko CC, Kumar S … +6 more , Xuan DTM, Lin HR, Lee YK, Nguyen NUN, Yang PM, Solomon DD
Methods · 2026 Jul · PMID 41862065 · Publisher ↗
Multifactorial inherited disorders (MIDs) arise from complex interactions between polygenic risk and environmental exposures, presenting major challenges for mechanistic discovery, patient stratification, and targeted th...
Multifactorial inherited disorders (MIDs) arise from complex interactions between polygenic risk and environmental exposures, presenting major challenges for mechanistic discovery, patient stratification, and targeted therapy development. While traditional approaches like genome-wide association studies (GWAS) and bulk omics profiling have identified broad associations, they often struggle to resolve the cellular context in which these interactions drive pathogenesis.Emergingsingle-cell technologies now offer unprecedented resolution to dissect tissue heterogeneity, define rare or transient disease-relevant cell states, and map dynamic trajectories across tissues and disease stages. This reviewprovides a comprehensive synthesis ofcurrent single-cell methodologies including transcriptomic, epigenomic, proteomic, and spatial techniques and their application to MID research. We explore how these toolsare revealingcell-type-specific regulatory circuits,contextualizingthe functional impact of inherited risk variants, andelucidatingcellular responses to environmental perturbations.We propose thatintegrating single-cell multi-omics data is critical for illuminating the mechanistic basis of complex traits and for advancing biomarker discovery. However, significant challenges remain, including technical variability, limited cohort scalability, difficulties in multi-modal data integration, and a lack of standardized analytical workflows for polygenic diseases. Overcoming these barriers will require harmonized study designs, robust computational frameworks, and the incorporation of longitudinal and environmental exposure data.Ultimately, we conclude thatsingle-cell analysis is poised to transform MID research, offering a powerful new paradigm for mechanistic insight, therapeutic innovation, and the realization of precision medicine.
Digital choices as emotional Mirrors: Mapping psychosocial profiles through smartphone app usage patterns.
Lee MH, You KH, Cho J
Methods · 2026 Jun · PMID 41862064 · Publisher ↗
This study examines how distinct patterns of smartphone application usage relate to psychosocial indicators, specifically anger, depression, and self-esteem, while identifying user clusters based on behavioral profiles....
This study examines how distinct patterns of smartphone application usage relate to psychosocial indicators, specifically anger, depression, and self-esteem, while identifying user clusters based on behavioral profiles. A two-stage analytical approach was employed. K-means clustering was first used to group 523 participants based on usage patterns across 130 applications, and Principal Component Analysis (PCA) was subsequently applied to summarize covariance structures among the psychosocial variables. The PCA yielded a two-dimensional psychological space that accounted for 90.1% of the variance and provided a descriptive framework for organizing users into relative psychosocial profiles. Results indicated that participants with similar demographic backgrounds exhibited different psychosocial characteristics depending on their digital usage patterns. Users who more actively engaged with productivity, health, or analog-content applications tended to show relatively more favorable psychosocial indicators, whereas users with more limited engagement in socially interactive applications tended to report higher levels of anger and depression and lower self-esteem. These findings should be interpreted as descriptive associations rather than causal effects. Overall, the study provides an exploratory account of how heterogeneous smartphone usage patterns are associated with variation in psychosocial characteristics in a non-clinical population, and suggests that everyday digital behavior may function as an observable behavioral correlate of stable psychosocial tendencies.
Comparison of RNA extraction methods from kidney organoids encapsulated in alginate-norbornene.
Kearney H, Joris V, Moroni L … +1 more , Mota C
Methods · 2026 Jun · PMID 41850649 · Publisher ↗
The encapsulation of kidney organoids within hydrogels provides a biomimetic environment that enhances their structural and functional relevance for disease modeling and drug screening. However, the presence of hydrogel...
The encapsulation of kidney organoids within hydrogels provides a biomimetic environment that enhances their structural and functional relevance for disease modeling and drug screening. However, the presence of hydrogel matrices poses a major challenge for molecular analysis, particularly for RNA extraction, where residual material can interfere with yield, purity, and downstream applications. The objective of this study was to systematically evaluate RNA extraction methods for kidney organoids encapsulated in alginate-norbornene hydrogels and identify an optimized protocol suitable for reliable gene expression analysis. We compared commonly used extraction methods designed for mammalian tissues and plant-derived materials, with and without prior enzymatic digestion of the hydrogel. RNA yield and purity were assessed by spectrophotometry and fluorometry, while RNA integrity was analyzed by Bioanalyzer, and performance in downstream assays was evaluated by quantitative PCR of housekeeping genes. Our results showed that RNA yield was consistently lower in encapsulated organoids compared to suspension cultures, reflecting smaller organoid size and reduced metabolic activity in encapsulated conditions. Spectrophotometric purity ratios differed between suspension and encapsulated samples, but RNA integrity was preserved across all methods, with values within the acceptable range. Quantitative PCR revealed that TRIzol-based extractions introduced significant variability between suspension and encapsulated samples. Conversely, the protocol with alginate lyase digestion followed by the Maxwell RSC RNA kit produced the most reproducible results. Ct values for control and encapsulated samples were highly consistent, with inter-condition variability remaining below 0.5 standard deviations across replicates. These findings highlight the importance of adapting RNA isolation protocols to account for the presence of hydrogels. Alginate lyase digestion combined with a plant RNA extraction kit offers a reliable strategy for obtaining high-quality RNA from encapsulated kidney organoids within alginate-based hydrogels. While this approach enabled a robust gene expression analysis providing a foundation for transcriptomic studies, different hydrogels and organoid combinations might require additional adjustments underscoring the importance of adopting methods to ensure optimal RNA quality for downstream methods.
Streamlining bispecific antibody production in E. coli with scFv-Constant domain fusions.
Ch'ng ACW, Choong YS, Lim TS
Methods · 2026 Jun · PMID 41850648 · Publisher ↗
The single-chain Fragment variable (scFv) antibody format is a common format used for phage display. The solubility of identified scFv clones in phage format in instances does not translate the same solubility as indepen...
The single-chain Fragment variable (scFv) antibody format is a common format used for phage display. The solubility of identified scFv clones in phage format in instances does not translate the same solubility as independent recombinant proteins. To overcome this bottleneck, we propose the fusion of scFv clones to the antibody constant domains for improved solubility. It has been reported that immunoglobulins devoid of the heavy constant domain 1 (C1) will have reduced solubility allowing accumulation in Russell Bodies (RB). Here we propose the introduction of C1 and kappa constant domain (Cκ) with and without skp chaperone to improve soluble expression. Utilizing heavy and light constant domains facilitates the spontaneous assembly of a Fab-like structure. This configuration successfully presents two distinct scFVs on each chain, thereby achieving bispecificity. This system is specifically designed to optimize the expression of the scFv-constant domain fusion necessary for bispecific formation. Crucially, its modular design allows for simple and rapid scFv swapping, enabling the seamless assessment of different antibody pairings for effective bispecific antibody generation.
Protocol for partial heart transplantation of the pulmonary valve in a swine model: surgical technique, postoperative care, assessments and outcomes.
Furrukh A, Javed H, Zaghw MRA … +11 more , Rogers MO, Chung S, Contoro E, Steen L, Qasim A, Henrich Lobo R, Beach T, Lowery JD, Gee M, Irby D, Rajab TK
Methods · 2026 Jun · PMID 41839381 · Publisher ↗
Piglets serve as an excellent model for cardiovascular research due to the anatomic and physiological similarities between the porcine and human heart. This study establishes an improved protocol for performing partial h...
Piglets serve as an excellent model for cardiovascular research due to the anatomic and physiological similarities between the porcine and human heart. This study establishes an improved protocol for performing partial heart transplantation (PHT) in a swine model, addressing key technical and physiological challenges while optimizing perioperative management. Here we present the results of performing the PHT protocol in 70 piglets (35 donors and 35 recipients) using a standardized approach incorporating sternotomies, vascular anastomoses, and immunosuppression, and 6 unoperated controls. Intraoperative complications, primarily due to hemodynamic instability and arrhythmias, occurred in 22.9% of recipients (n = 8/35), while postoperative complications, primarily due to failure to thrive and infections occurred in 29.6% of the survivors (n = 8/27). Refinements such as prophylactic antiarrhythmic therapy, use of clamps to control bleeding in the field, and optimized selection of suture materials improved operative and post-operative outcomes over the course of the study. This protocol demonstrates the feasibility of PHT in a swine model and provides a framework for evaluating clinical partial heart transplant protocols.
The coefficient of variation by image correlation spectroscopy (CV-ICS) quantifies chromatin compaction from images with low photon counts.
Longo E, Paternò G, Lanzanò L
Methods · 2026 Jun · PMID 41831751 · Publisher ↗
Lower excitation light levels reduce phototoxicity in confocal and superresolution microscopy but generate images with lower photon counts. A well-established parameter that quantifies chromatin compaction from fluoresce...
Lower excitation light levels reduce phototoxicity in confocal and superresolution microscopy but generate images with lower photon counts. A well-established parameter that quantifies chromatin compaction from fluorescence images of labeled nuclei is the coefficient of variation (CV), which is defined as the ratio between the standard deviation and the average of the pixel intensity values. Here, we show that when imaging is performed at low photon counts the standard deviation of the intensity is dominated by the shot noise fluctuations and the CV is not representative of the chromatin compaction state. To overcome this issue, we introduce an alternative calculation of CV based on image correlation spectroscopy (CV-ICS), where the CV-ICS parameter is extracted from the spatial autocorrelation function of the image. We demonstrate the use of CV-ICS on fast confocal (resonant) imaging of live HeLa cells labeled with Hoechst 33342. First, we show that the conventional CV calculation deviates already by about 50% from the correct value when the maximum photon counts per pixel are below 40. In contrast, the CV-ICS calculation is unaffected by the presence of shot noise. Next, we use imaging at low photon counts and CV-ICS to follow changes in the chromatin compaction state after switching the cells to a medium of higher (hyper-) or lower (hypo-) osmolarity. Finally, we use CV-ICS to investigate the time course of chromatin compaction after induction of laser-induced DNA damage. This work shows that CV-ICS can be a robust parameter for quantifying chromatin compaction in all imaging applications with a limited photon budget, including live cell imaging and superresolution microscopy.
Now You See Me: Visualizing nuclear complexity by selective staining.
Casali C, Cavallo M, Diaf A … +2 more , Milanesi G, Biggiogera M
Methods · 2026 Jun · PMID 41831750 · Publisher ↗
Transmission electron microscopy (TEM) enables nanoscale resolution of cellular structures, yet its biological interpretability in cell biology critically depends on the staining protocols. Here, we present an integrated...
Transmission electron microscopy (TEM) enables nanoscale resolution of cellular structures, yet its biological interpretability in cell biology critically depends on the staining protocols. Here, we present an integrated TEM workflow applied to mammalian erythropoiesis, an established model for the study of nuclear remodeling, to dissect chromatin condensation and large-scale nuclear reorganization. We systematically compare multiple staining strategies, including uranyl acetate and lead citrate for general morphology, EDTA regressive for ribonucleoproteins, osmium ammine for DNA, terbium citrate vapors for RNA, and immunogold labeling for the heterochromatin marker H3K9me3. Each method reveals distinct structural features and, when combined with segmentation tools, enables their high-resolution mapping. We demonstrate that the choice of protocol markedly influences image interpretation and semi-quantitative image analysis. To our knowledge, this is the first systematic study to integrate these cytochemical protocols into a standardized imaging pipeline with potential applicability across biological systems. Our findings establish a methodological reference for nanoscale nuclear imaging and offer insights into the spatial organization of chromatin at the ultrastructural level.
First report of CRISPR prime editing in a globally significant non-model organism, the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae).
Yogi D, Shashikala T, Subramanian G … +7 more , Kumar A, Kaninika V, Manamohan M, Jithesh MN, Jha GK, Asokan R, Ashok K
Methods · 2026 Jun · PMID 41812962 · Publisher ↗
The power of CRISPR/Cas9 mediated genome editing has been harnessed in different facets of entomological research, particularly useful in developing genetic pest management strategies. The edits thus obtained are robust...
The power of CRISPR/Cas9 mediated genome editing has been harnessed in different facets of entomological research, particularly useful in developing genetic pest management strategies. The edits thus obtained are robust and results in a loss-of-function of the target gene. Recently the development of newer editing approach called Prime editing is yet another addition in the insect editing tool-box. In this regard, the prime editing offers a transformative approach to precise genome manipulation by enabling targeted insertions, deletions, and nucleotide substitutions without double-strand break or donor template. While its application has been explored in mammalian system and plants, its deployment through the delivery of ribonucleoprotein complex (RNP) has been demonstrated for the first-time in the globally significant pest, Spodoptera frugiperda. Using a Cas9 (H840A)-reverse transcriptase fusion protein (PE2) and a customized prime editing guide RNA (pegRNA), we targeted exon 3 of the Tryptophan 2,3-dioxygenase (SfTO) gene to introduce a premature stop codon. Recombinant PE2 protein was expressed in E. coli, purified, and validated functionally through RT-PCR. The Ribonucleoprotein complex was microinjected into G eggs and subsequent genotyping revealed successful edits, including perfect and imperfect prime edits, as well as unintended mutations. Phenotyping revealed the mutants with altered eye pigmentation, and chromatographic analysis confirmed disruption in ommochrome biosynthesis, validating functional consequences of Prime editing. This study provides a foundational proof-of-concept for Prime editing in insect pests, opening new avenues for functional genomics and designing next-generation pest management strategies.
Concurrent quantification of novel cyclometalated iridium-based photosensitizer and 5-fluorouracil using RP-HPLC: Applications in nanoformulation analysis, drug release, and skin permeation studies.
Kumbhar PR, Desai VM, Sumit S … +3 more , Chowdhury R, Laskar IR, Singhvi G
Methods · 2026 Jun · PMID 41812961 · Publisher ↗
BACKGROUND: Combination therapies for skin cancer are emerging as effective strategies to overcome limitations of monotherapies, such as limited efficacy, resistance development, and recurrence. Chemo-photodynamic therap...
BACKGROUND: Combination therapies for skin cancer are emerging as effective strategies to overcome limitations of monotherapies, such as limited efficacy, resistance development, and recurrence. Chemo-photodynamic therapy represents a promising approach for this. In this study, novel cyclometalated iridium-based photosensitizer was investigated in combination with 5-fluorouracil. As the photosensitizer is novel, no analytical method has been reported for its simultaneous estimation with 5-FU. Such a method is essential for quantifying both analytes in nanoformulations, enabling pharmacokinetic and skin permeation studies critical for development of chemo-photodynamic therapy. METHOD: A simultaneous RP-HPLC method was developed using a C18-column with an isocratic mobile phase of acetonitrile and pH 4.0 buffer (80:20v/v) at a flow rate of 1.0 mL/min. Detection was performed at 265 nm. The method was validated in accordance with ICH guidelines, including forced-degradation studies, and applicability was evaluated using nanodispersion and skin permeation samples. RESULTS: Good resolution was achieved between CIBP and 5-FU, with retention times of 5.944 and 2.714 min, respectively. The method showed excellent linearity (r>0.999) at concentration range of 500-12,000 ng/mL. High accuracy was observed, with recoveries of 100.74-101.35% for CIBP and 100.94-101.05% for 5-FU. High sensitivity was demonstrated, with LOD/LOQ values of 6.89/20.90 ng/mL for CIBP and 5.62/17.04 ng/mL for 5-FU. Precision studies showed %RSD < 2% for both analytes. CONCLUSION: The validated method is reliable, economical, and suitable for routine quantification of both analytes in stability studies, drug-encapsulation, in-vitro release, and ex-vivo skin permeation samples, supporting quality control and dermatokinetics profiling of dual-drug-loaded nanodispersion.
Methodological approaches for dissecting ferroptosis in lens epithelial cells and its role in diabetic cataract progression.
Xu H, Wang W, Li J … +2 more , Zhao X, Zhang H
Methods · 2026 Jun · PMID 41794308 · Publisher ↗
Diabetic cataract has become the second-largest blind causing disease among the complications of diabetes mellitus in eyes. Ferroptosis is responsible for many complications associated with diabetes mellitus, but the rol...
Diabetic cataract has become the second-largest blind causing disease among the complications of diabetes mellitus in eyes. Ferroptosis is responsible for many complications associated with diabetes mellitus, but the role of ferroptosis in diabetic cataract has not yet been studied. Here, we first ascertained that ferroptosis was triggered during diabetic cataract, as identified by elevated levels of transferrin receptor protein 1 (CD71) and solute carrier family 7 member 11 (SLC7A11), lipid peroxidation , total iron and Fe under conditions of excessive glucose. Moreover, voltage-dependent anion channel 3 (VDAC3) was found to decrease under high levels of glucose both in vivo and in vitro. VDAC3 knockdown resulted in increased reactive oxygen species (ROS) levels and reduced nicotinamide adenine dinucleotide (NADH) levels, indicating that decreased VDAC3 modulates ferroptosis in diabetic cataract via ROS-induced lipid peroxidation. Furthermore, we verified that the level of ubiquitin-specific peptidase 47 (USP47) was decreased in high glucose-treated lens epithelial cells. We found that USP47 bound to and modulated the deubiquitination of VDAC3. This binding between USP47 and VDAC3 was weakened and VDAC3 was subsequently ubiquitinated and degraded under high glucose conditions, which ultimately resulted in ferroptosis and diabetic cataract. Overall, we verified that the USP47-VDAC3-ROS/NADH axis regulates ferroptosis in high glucose-treated lens epithelial cells (LECs) and in an animal model of diabetic cataract. Our findings increase the understanding of human diabetic cataract and provide important mechanistic insight into potential ferroptosis treatments in medicine and technology for treating diabetic cataract.
Clinical assessment of the diagnostic performance of the LIAISON® Legionella urinary antigen assay.
Cusato J, Menegatti G, Ravotto M … +7 more , Pighini A, Capuano F, Pícha D, Bianco G, Martino C, Simiele M, D'Avolio A
Methods · 2026 May · PMID 41771313 · Publisher ↗
Legionella pneumophila is the primary species responsible for Legionnaires' disease, a severe pneumonia. While culture remains the diagnostic gold standard, its slow turnaround time limits clinical usefulness. Rapid meth...
Legionella pneumophila is the primary species responsible for Legionnaires' disease, a severe pneumonia. While culture remains the diagnostic gold standard, its slow turnaround time limits clinical usefulness. Rapid methods like urinary antigen tests can improve timely detection. The LIAISON® Legionella Urinary Antigen test is a new chemiluminescence immunoassay designed to identify soluble antigens of Legionella in urine. This study evaluated its performance compared to existing CE-marked antigen tests. Samples were collected from six external laboratories and analyzed in singlicate using both the LIAISON assay and the corresponding comparison method. The LIAISON test demonstrated equivalent performance to reference assays, showing 100% diagnostic specificity and 97% sensitivity-statistically comparable to the claimed 98%. Notably, four samples showed discrepancies between tests, highlighting the diagnostic complexity of Legionella infections. A receiver operating characteristic (ROC) analysis was also conducted to determine the optimal threshold and confirm the assay's cutoff. Results from the LIAISON test were compared to expected outcomes based on consensus criteria. Overall, findings indicate that the LIAISON® Legionella Urinary Antigen assay provides reliable clinical performance for the qualitative detection of Legionella pneumophila and Legionella longbeachae antigens in urine samples from patients with suspected pneumonia in real-world settings.
Broad Screening of 2-Styrylchromones Identifies dual inhibitors of the COX-2 pathway.
Lucas M, Freitas M, Ribeiro D … +2 more , Silva AMS, Fernandes E
Methods · 2026 Jun · PMID 41765155 · Publisher ↗
Modulation of the cyclooxygenase (COX) pathway, particularly the regulation of COX-2 activity and expression, is central to anti-inflammatory therapy. In this study, the COX-2 inhibitory potential and COX-2/COX-1 selecti...
Modulation of the cyclooxygenase (COX) pathway, particularly the regulation of COX-2 activity and expression, is central to anti-inflammatory therapy. In this study, the COX-2 inhibitory potential and COX-2/COX-1 selectivity of a panel of 43 structurally related 2-styrylchromones (2-SC), a family of oxygen-containing heterocyclic compounds and vinylogues of flavones (2-arylchromones) were evaluated. COX-2 and COX-1 activities were assessed using in vitro enzymatic and ex vivo human whole blood assays, complemented by the analysis of COX-2 expression in lipopolysaccharide (LPS)-stimulated human leukocytes. Structure-activity relationships were established whenever possible. In the enzymatic assay, 2-SC B12 and B13 were the most active compounds, with IC values ≤ 1 μM. These results suggest that the presence of catechol groups at C-7 and C-8 on the A-ring, as well as at C-3' and C-4' on the B-ring, plays a key role in the inhibition of COX-2 enzymatic activity. Both compounds were selective for COX-2, though B12 showed the highest selectivity index. In the complex matrix of human whole blood, B1 was the only 2-SC that consistently displayed inhibitory activity in both enzymatic and whole blood assays. At the cellular level, several 2-SC significantly downregulated LPS-induced COX-2 expression in human leukocytes. Notably, 2-SC B6, B7 and B9 inhibited both COX-2 activity and expression, and also reduced prostaglandin E production, suggesting a dual modulatory effect on this inflammatory pathway. Overall, these findings identify the 2-SC scaffold as a promising and underexplored chemical platform for the rational design of selective COX-2 modulators and innovative anti-inflammatory agents.
Direct culture is as sensitive as bioassay to achieve in vitro isolation of Toxoplasma gondii from latently infected animal hearts.
Hamilton D, Hodgson K, McAllister MM
Methods · 2026 May · PMID 41763281 · Publisher ↗
Isolation of Toxoplasma gondii from latently infected animals is most frequently achieved using bioassay in mice, but we wondered if direct cell culture could be equally efficient. Paired heart and blood samples were obt...
Isolation of Toxoplasma gondii from latently infected animals is most frequently achieved using bioassay in mice, but we wondered if direct cell culture could be equally efficient. Paired heart and blood samples were obtained from slaughtered ewes and the proportion of isolates acquired using direct culture of acid-pepsin digested hearts was compared with the proportion of isolates acquired by bioassay followed by culture of infected mouse tissues. Isolation was successful from 8/30 seropositive ewes by direct culture and from 9/30 via bioassay. In a second trial using lambs experimentally infected with T. gondii for 3 months, organisms were re-isolated from 4/8 hearts by direct culture and from 3/8 via bioassay. Across both trials, 12/38 isolation attempts were successful for each method (p = 1.0). For naturally infected sheep, antibody titres ≥ 1,000 tended to favour isolation of organisms in comparison with titres between 60 and 400 (p = 0.018, α = 0.017). Isolation by direct culture was successful from 75% of qPCR positive heart digest suspensions in comparison with 0% of qPCR negative suspensions (p < 10). These results indicate that the most efficient and cost-effective method to culture T. gondii from latently infected animals is to select subjects with high antibody titres and maintain direct cultures up to five weeks for those acid-pepsin heart digests that have molecular confirmation of infection. Direct isolation of T. gondii into cell culture advances lab animal welfare and is as sensitive as mouse bioassay.
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