Methods (San Diego, Calif.)[JOURNAL]
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200 papers
An Affordable paper based platform for eDNA Filtration, Concentration, and nucleic acid extraction.
Pham CH, Hønsvall BK, Jothinarayanan N … +2 more , Johannessen EA, Ta BQ
Methods · 2026 Jun · PMID 41734876 · Publisher ↗
Environmental DNA (eDNA) analysis is a non-invasive method for discovering and identifying rare and endangered species in a variety of ecosystems including aquatic environments. However, conventional eDNA analysis tends...
Environmental DNA (eDNA) analysis is a non-invasive method for discovering and identifying rare and endangered species in a variety of ecosystems including aquatic environments. However, conventional eDNA analysis tends to be time consuming and labor-intensive, which limits their efficiency. In this study, a novel eDNA capture system is made from stacked layers of membranes with a binding pad of borosilicate glass paper embedded with a triple layer of high molecular weight chitosan and silica nanoparticles. The system is designed to enhance DNA capture efficiency and is part of a portable system for on-site eDNA extraction that measures approx. 22 x 70 mm using sample volumes up to 3 mL and enables DNA extraction under 10 min. The DNA recovery rate of 86% is comparable to silica bead-based laboratory methods at a significantly lower cost, and the Ct values from the nucleic acid amplification step are reached within 18 to 36 min. Real water samples were tested, yielding results comparable to those obtained with the silica bead-based laboratory methods. Thus, enables downstream applications, such as species detection through a portable system that has the potential to improve the accessibility of eDNA analysis for effective environmental monitoring and conservation practices.
Electric field stimulation in Caenorhabditis elegans as a novel approach to investigate mitochondrial Ca homeostasis during in vivo muscle aging.
Gabrijelčič S, Bresilla D, Mossegger F … +4 more , Malle E, Đerek V, Hirtl M, Madreiter-Sokolowski CT
Methods · 2026 May · PMID 41722631 · Publisher ↗
Mitochondrial calcium ([Ca]) homeostasis is a key regulator of cellular physiology, controlling signal transduction, energy metabolism, and cell survival. To examine how these processes change with age in vivo, we used t...
Mitochondrial calcium ([Ca]) homeostasis is a key regulator of cellular physiology, controlling signal transduction, energy metabolism, and cell survival. To examine how these processes change with age in vivo, we used the pharyngeal muscle of Caenorhabditis elegans (C. elegans) as a tractable model for studying [Ca] dynamics. We introduced electric field stimulation as a robust trigger for [Ca] uptake that overcomes limitations of compound stimulation, which relies on pharyngeal pumping and endoplasmic reticulum store filling and is typically confined to single-stimulus protocols. In contrast, electric field stimulation enables physiologically relevant repeated excitation that challenges [Ca] uptake and recovery. Stimulation with 10 V for 10 s reliably evoked reproducible [Ca] transients in the pharyngeal muscle of C. elegans and produced higher responder rates than compound stimulation. Basal [Ca] increased with age, and the first field-evoked transient was significantly larger in aged animals, an effect not detected with pharmacological triggers. Repeated pulses unmasked cumulative [Ca] loading and incomplete recovery in aged pharynx, indicative of [Ca] overload, which was attenuated by inhibition of the mitochondrial Ca uniporter (MCU) with mitoxantrone. Regional analyses identified the corpus as a hotspot for electric field-evoked [Ca] uptake and overload, while MCU inhibition reduced repeated responses in both corpus and posterior bulb. Electric field stimulation enables precise, repeated in vivo probing of [Ca] uptake and recovery, revealing overload in the C. elegans pharynx. This approach identified age-enhanced, MCU-dependent, and region-specific [Ca] loading, providing a pathophysiologically relevant readout of impaired Ca handling that may contribute to age-related muscle dysfunction.
Advancements in ex vivo bone biomechanics: multimodal technologies and their integration with artificial intelligence.
Unal S, Unal M
Methods · 2026 May · PMID 41720279 · Publisher ↗
Bone biomechanics is essential for understanding how bone structure, composition, and mechanical function interact in healthy and diseased states. Conventional approaches such as mechanical testing, micro computed tomogr...
Bone biomechanics is essential for understanding how bone structure, composition, and mechanical function interact in healthy and diseased states. Conventional approaches such as mechanical testing, micro computed tomography, and histology provide valuable information but cannot fully capture the multiscale heterogeneity that governs bone strength and fragility. This review summarizes recent progress in advanced ex vivo bone biomechanical techniques that address these limitations, including synchrotron-based phase contrast imaging, digital volume correlation, three dimensional bioprinting, acoustic emission testing, and the combined use of nanoindentation with Raman spectroscopy. These methods allow detailed evaluation of bone microarchitecture, mineral organization, local mechanical behavior, and biochemical composition, thereby improving the detection of micro damage and early structural deterioration. The review also highlights the growing role of artificial intelligence in bone biomechanics. Machine learning approaches enhance the interpretation of complex imaging and mechanical datasets, improve the prediction of mechanical properties, and support more accurate evaluations of fracture risk. By integrating high resolution experimental techniques with data driven computational models, researchers can achieve a more complete understanding of bone quality. Together, these developments provide a focused framework for studying bone fragility and for guiding individualized diagnostic and therapeutic strategies in conditions such as osteoporosis and diabetes related bone disease.
First report of CRISPR/Cas13a-based rapid detection of groundnut bud necrosis virus without amplification.
Shashikala T, Yogi D, Akshay K … +6 more , Nagesh SN, Manamohan M, Venkataravanappa V, Jha GK, Ashok K, Asokan R
Methods · 2026 May · PMID 41713565 · Publisher ↗
Globally, the groundnut bud necrosis virus (GBNV) (Bunyaviridae), pose a serious threat to solanaceous and leguminous crops causing serious crop loss. This requires a rapid and sensitive diagnostics for initially identif...
Globally, the groundnut bud necrosis virus (GBNV) (Bunyaviridae), pose a serious threat to solanaceous and leguminous crops causing serious crop loss. This requires a rapid and sensitive diagnostics for initially identifying at the earliest stage and further to initiate disease management. This study presents the first report of a CRISPR/Cas13a-based diagnostic assay for GBNV detection without amplification. GBNV was maintained in cowpea cv. C152 through mechanical inoculation, further viral RNA was isolated to clone the two target genes viz. nucleocapsid (NP) and movement protein (MP) genes. These genes were ligated to pTZ57R/T vector and sequenced. Similarly, the LshCas13a gene was cloned from pUC19 into pET28a, expressed in E. coli BL21, and purified using Ni-NTA affinity chromatography. Guide RNAs targeting conserved regions of NP and MP genes were synthesized by in vitro transcription and mixed with Cas13a protein to form ribonucleoprotein (RNP) complex. Target RNA, obtained either by in vitro-transcription or crude extract of infected tomato was used to detect GBNV, using a fluorescence-based reporter assay. This method found to be highly sensitive that could detect GBNV at as low as 0.01 ng. From the field perspective, GBNV could be detected from the crude extract of the GBNV infected tomato leaves using an alkaline PEG buffer. Thus CRISPR/Cas13a-based assay provides a rapid, amplification-free, and field-deployable diagnostic platform for GBNV. This lays the groundwork for a field adoptable CRISPR diagnostics for other plant RNA viruses also.
Interpretable multimodal machine learning model for predicting health risks of patients with heart failure.
Chae R, Zhou J, Chou OHI … +7 more , Yang B, Pu H, Tse G, Cheung BMY, Zhu T, Car J, Lu L
Methods · 2026 May · PMID 41698516 · Publisher ↗
Heart failure (HF) is one of the major causes of morbidity and mortality globally, necessitating accurate tools for health outcome prediction and risk stratification. In this study, we propose an interpretable multimodal...
Heart failure (HF) is one of the major causes of morbidity and mortality globally, necessitating accurate tools for health outcome prediction and risk stratification. In this study, we propose an interpretable multimodal machine learning framework integrating four clinical data modalities (i.e., demographics, medications, laboratory tests, and electrocardiograms [ECGs]) to predict 30-day all-cause mortality and hospital readmission in HF patients. Using clinical data from 2868 HF patients across 43 local hospitals in Hong Kong, we trained and evaluated ten machine learning models for HF risk prediction, with the best performing model achieving an area under the receiver operating characteristic curve (AUC) of 0.881 for mortality and 0.709 for readmission. Notably, laboratory tests and ECG features dominate predictive power, and their combination alone yielded near-optimal results (AUC: 0.872), suggesting that these two modalities may be adequate for effective risk prediction in resource-constrained settings. The SHapley Additive exPlanations (SHAP) analysis identified serum albumin, high-sensitivity troponin I, lactate dehydrogenase, and QT interval dispersion as key predictors. Feature redundancy analysis further revealed strong correlations within laboratory tests and ECG features, suggesting opportunities for model simplification. To the best of our knowledge, this is the first study that comprehensively evaluates diverse configurations of four data modalities for HF risk prediction through ablation analysis, quantifying the marginal gains of each data modality and their combinations. Our findings demonstrate that an interpretable multimodal machine learning model can enhance risk prediction in HF patients, supporting personalized management and scalable deployment across diverse healthcare settings.
Using fluorescent in vitro amphibian cell infection models to quantify pathogenicity of Batrachochytrium dendrobatidis.
Sumanasekera KK, Berger L, Vu AL … +5 more , Robert J, Akram N, Skerratt LF, Jesús Andino F, Webb RJ
Methods · 2026 May · PMID 41692369 · Full text
Chytridiomycosis is a devastating amphibian fungal disease and methods to understand host pathogen interactions and test novel mitigation strategies are urgently needed. A recently developed in vitro frog cell infection...
Chytridiomycosis is a devastating amphibian fungal disease and methods to understand host pathogen interactions and test novel mitigation strategies are urgently needed. A recently developed in vitro frog cell infection model offers an efficient and ethical approach for chytridiomycosis research, allowing precise manipulation of host and fungal traits. However, reliable assays are needed to quantify disease outcomes. Here, we validate methods to quantify chytridiomycosis severity in an in vitro model by measuring pathogen loads and cytopathic effects on host cells. We assessed the ability of methods to distinguish fungal loads by comparing results of exposure to various Batrachochytrium dendrobatidis (Bd) infectious doses incubated under optimal and suboptimal conditions for fungal growth. Using a genetically modified strain of fluorescent Bd allowed quantification of fungal burden via microscopy, spectrophotometry and flow cytometry. For host cell effects, a DAPI staining protocol quantified cell damage and a modified MTT assay quantified cell viability. Our work provides a toolbox of methods to utilise in vitro cell infection models to investigate the function and effect size of fungal virulence and host resistance factors, enabling diverse research aimed at understanding and mitigating chytridiomycosis.
Refolding-assisted purification of native full-length TDP-43 compatible with BSL-2 safety regulations.
Dehury S, Tiwari S, Los Rios P
Methods · 2026 Apr · PMID 41692368 · Publisher ↗
TAR DNA-binding protein 43 (TDP-43) is a prion-like RNA-binding protein that plays a key role in amyotrophic lateral sclerosis and frontotemporal dementia. Producing full-length TDP-43 consistently is thus relevant for i...
TAR DNA-binding protein 43 (TDP-43) is a prion-like RNA-binding protein that plays a key role in amyotrophic lateral sclerosis and frontotemporal dementia. Producing full-length TDP-43 consistently is thus relevant for its in vitro studies and yet it remains challenging, especially with the current requirement to work under biosafety level-2 (BSL-2) containment due to new safety regulations for Prion-like and amyloidogenic proteins. Here we describe a refolding-assisted purification protocol for TDP-43 from soluble fraction that can be implemented with basic equipment in standard BSL-2 laboratories. Expression in Escherichia coli is followed by IMAC-capture on an EDTA/DTT-tolerant Ni-NTA resin under 4 M urea, then on-column refolding via a gradient urea wash using resin-limiting conditions that favour the binding to high-affinity His-tagged protein. After removal of the SUMO solubility tag, the preparation is monitored by a robust quality-control pipeline: SDS-PAGE and immunoblotting for integrity and purity, mass photometry for oligomeric state, far-UV circular dichroism for secondary structure, fluorescence anisotropy for native functional assays, and light-scattering for stability and aggregation propensity measurements. A concise BSL-2 standard operating procedure specifies containment, decontamination, and waste handling for prion-like proteins. This protocol enables safe, cost-effective, and reproducible access to native-like full-length TDP-43 and is readily adaptable to other prion-like aggregation-prone proteins.
Novel methods for the discovery of disease-associated T cell epitopes in autoimmunity.
Mertens E, Willekens B, Derdelinckx J … +1 more , Cools N
Methods · 2026 Apr · PMID 41687796 · Publisher ↗
A detailed understanding of the pathology of autoimmune diseases hinges on the identification of self-antigens and epitopes targeted by the immune system. While the characterization of autoantibodies is now well-establis...
A detailed understanding of the pathology of autoimmune diseases hinges on the identification of self-antigens and epitopes targeted by the immune system. While the characterization of autoantibodies is now well-established, facilitating both mechanistic insights and clinical biomarker applications, the identification of autoreactive T cell epitopes remains considerably more challenging. This complexity is amplified by the presence of autoreactive T cells in healthy individuals, necessitating highly sensitive and specific methods to allow for the detection of subtle differences between the autoreactive T cell population in healthy controls and in patients. Fortunately, T cell epitope discovery is a rapidly advancing field, with new methods continually emerging to improve sensitivity, throughput, and resolution. In this review, we will provide a structured overview of the key methods used to identify T cell epitopes, spanning both foundational techniques thathave been instrumental in the early discovery of self-epitopes involved in autoimmunity as well as recent high throughput approaches that offer enhanced precision and scalability. In the second part, we give an overview of the techniques used in the validation of the role of self-peptides in the autoimmune disorder. We conclude by discussing future directions in the field, emphasizing the critical role of T cell epitope discovery in driving the development of targeted, antigen-specific therapies for autoimmune disorders. This review aims to provide a practical and conceptual framework for T cell epitope discovery in autoimmune diseases, integrating established experimental approaches with emerging computational and high-throughput methodologies to guide informed method selection in contemporary research.
Disentangling the events that constitute the main phase transition of predominantly neutral lipid bilayers containing anionic lipids by measuring their macroscopic properties.
Petko R, Marić I, Pem B … +1 more , Bakarić D
Methods · 2026 Apr · PMID 41666964 · Publisher ↗
The thinning of lipid bilayers during their main phase transition, as the outcome of the weakening of van der Waals interactions between their hydrocarbon chains and the hydration of the polar headgroup region, displays...
The thinning of lipid bilayers during their main phase transition, as the outcome of the weakening of van der Waals interactions between their hydrocarbon chains and the hydration of the polar headgroup region, displays a peak at the specific temperature (T). The most common technique used for T determination is differential scanning calorimetry (DSC), which provides the required information straightforwardly and rapidly, but it cannot provide any details on the molecular-level events that underlie the main phase transition. To uncouple the impact of hydrogen bonding and (de)protonation in the polar headgroup region on the measured T, in this work, we determined the latter of anionic lipid-containing predominantly neutral bilayers (10:90% molar ratio) in Britton-Robinson buffers with pH values 4, 7, and 9 using macroscopic techniques. Specifically, the temperature-dependent UV-Vis spectra combined with the measurement of refractive indices allowed the modeling of the bilayer thickness (d) change during the main phase transition. Significantly, the alteration in the hydrogen bonding pattern affected the change in d, unlike the deprotonation of anionic lipids. A molecular polarizability displayed an abrupt decrease upon the main phase transition, but its magnitude and associated uncertainty level were found to be acceptable only for a lipid mixture in which anionic lipid does not exchange protons with aqueous milieu, i.e., a pH-dependent (de)protonation obscures more precise determination of T. Besides the determination of T values, this new methodology enabled us to rank the contributions to T value: van der Waals interactions > hydrogen bonding > (de)protonation.
From wrinkles to malignancy: small-molecule-mediated stem cell approaches in skin aging.
Alkhammash A, Alotaibi G
Methods · 2026 Apr · PMID 41655728 · Publisher ↗
Skin aging is driven by the progressive exhaustion of stem cell niches, epigenetic drift, and accumulation of senescent cells, which together promote both aesthetic decline and a pro-tumorigenic microenvironment. This re...
Skin aging is driven by the progressive exhaustion of stem cell niches, epigenetic drift, and accumulation of senescent cells, which together promote both aesthetic decline and a pro-tumorigenic microenvironment. This review focuses on the emerging methodological theme of small-molecule-mediated reprogramming as a strategy to restore skin homeostasis. We evaluated the shift from traditional regenerative medicine toward targeted chemical modulation, focusing on the use of small-molecule cocktails to induce partial reprogramming and rejuvenate aged stem cell populations without erasing cellular identity. Central to this theme is the integration of high-throughput virtual screening and AI-driven predictive modeling to identify potent modulators of Wnt, Notch, and TGF-β pathways. We further bridge the gap between preclinical innovation and clinical application by analyzing "serious clinical studies" with proven efficacy, including randomized controlled trials of stem cell-derived secretomes and clinically validated small molecules, such as tretinoin and firming peptides. By contextualizing advanced delivery systems, including microneedles and stimuli-responsive nanoparticles, within this reprogramming framework, we demonstrate how spatially controlled interventions can optimize clinical outcomes. This review provides a unified perspective on how the intersection of computational drug discovery and niche-targeted pharmacology is moving small-molecule skin rejuvenation from theoretical potential to widespread clinical translation.
An optimized method for establishing experimental rat periodontitis using "double-ligature" technique.
Hu C, Wang H, Liao Y … +1 more , Hu X
Methods · 2026 Apr · PMID 41643797 · Publisher ↗
Establishing stable and dependable animal models of experimental periodontitis is critical in advancing our understanding of the etiology, clinical diagnosis, and treatment of periodontitis. However, conventional silk li...
Establishing stable and dependable animal models of experimental periodontitis is critical in advancing our understanding of the etiology, clinical diagnosis, and treatment of periodontitis. However, conventional silk ligation methods for inducing periodontitis in rats have limitations, with silk thread detachment being a major concern. In this study, we established a reliable rat periodontitis model using a novel "double-ligature" technique combining silk sutures with orthodontic wires, augmented by a high-sugar diet. Thirty rats were randomized into five groups: control, wire-only, silk-only, wire+silk, and wire+silk+sugar. After 2 weeks, the wire+silk+sugar group demonstrated significantly elevated clinical indices (sulcus bleeding index, probing depth, plaque index) versus controls (*p* < 0.05), alongside severe alveolar bone resorption quantified by micro-CT. Molecular analyses revealed upregulated inflammatory gene expression (e.g., TNF-α, IL-1β) in double-ligature groups. Histology confirmed extensive immune infiltration and periodontal ligament disruption. The combined approach induced robust periodontitis with 103% greater CEJ-ABC distance versus controls, while TRAP staining revealed 5-fold increased osteoclast activity. By this "double-ligature" technique, the pathogenesis of periodontitis can be studied on the one hand, and the therapeutic effect of biomaterials on the alveolar bone defects caused by periodontitis can be verified on the other hand. This reproducible method overcomes traditional silk ligature limitations (e.g., thread detachment) and provides a foundation for translational periodontitis research.
DNA-regulated structural engineering of metal nanomaterials: A strategy for advanced optical biosensing.
Li J, Cai L, Liu P … +5 more , Zhang R, Cai W, Wu D, Xu L, Kong Y
Methods · 2026 Apr · PMID 41643796 · Publisher ↗
The development of highly sensitive optical biosensors has emerged as a focal point in chemical research, exerting a profound influence on numerous fields related to the national economy and public welfare. Owing to thei...
The development of highly sensitive optical biosensors has emerged as a focal point in chemical research, exerting a profound influence on numerous fields related to the national economy and public welfare. Owing to their dual nanostructural and metallic properties, metal nanomaterials exhibit certain distinctive optical properties. Among them, localized surface plasmon resonance (LSPR), surface-enhanced Raman scattering (SERS), and fluorescence emission are particularly prominent. Therefore, metal nanomaterials possess significant potential to enhance the analytical performance of optical biosensors. Compared to peptides and proteins, DNA demonstrates remarkable superiority in terms of the diversity of length, sequence, backbone structure, and modification groups. Integrating DNA with metal nanomaterials provides a prerequisite for accurately identifying targets and precisely regulating metal nanomaterials. Effectively combining the superior properties of DNA and metal nanomaterials represents a critical scientific challenge in facilitating the development of highly sensitive optical analytical approaches. Exploring novel strategies to regulate the optical properties of metal nanomaterials can provide more opportunities for developing high-performance optical biosensors. In this review, the regulation modes of DNA with metal nanomaterials can be summarized into three parts i.e.: the morphological evolution of DNA-guided metal nanomaterials, the assembly of DNA with metal nanomaterials, and the formation of DNA-templated metal nanomaterials. For each part, typical applications have been displayed based on regulating the optical properties of metal nanomaterials via DNA. Furthermore, perspectives and challenges are also discussed at the end of the review.
Ultra-sensitive diagnostic platform utilizing gold nanoparticle integrated in a freestanding hydrogel matrix.
Rahman OM, Lan S, Pizano AMA … +3 more , Chung W, Kim Y, Hwang DK
Methods · 2026 Apr · PMID 41638460 · Publisher ↗
Fluorescence-based analysis for protein detection has been widely adopted; however, they fall short in achieving ultra-sensitive detection because of their inherently low signal-to-noise ratio at sub-picomolar concentrat...
Fluorescence-based analysis for protein detection has been widely adopted; however, they fall short in achieving ultra-sensitive detection because of their inherently low signal-to-noise ratio at sub-picomolar concentrations. To overcome this limitation, signal amplifying materials such as gold nanoparticles (AuNPs) have been integrated into various detection platforms to enhance fluorescence signals. However, fabrication of such AuNP integrated platforms remains complex, often requiring sophisticated fabrication steps, yet none approach the ultra-sensitive detection range of 10 fg mL. In this study, we enabled analysis of captured protein/antibody at 10 fg mL concentrations through fluorescence signals utilizing freestanding gold nanoparticle integrated freestanding hydrogel platforms. The platform is fabricated by integrating AuNPs into the hydrogel matrix using a single-step photolithographic technique. The integrated AuNPs significantly enhanced the fluorescence signals compared to controls. Notably, at fg mL levels of fluorophore, the AuNP integrated hydrogels produced a robust optical signal in contrast to negligible responses observed in controls. The platform's versatility was validated using tumor necrosis factor-alpha antibody (TNF-α Ab), achieving detection at 10 fg mL. By synergizing the hydrogels' porous structure with AuNPs' signal amplification, this platform successfully achieves fluorescence-based ultra-sensitive protein/antibody detection.
Syntenins at the crossroads of host-virus interactions.
Kharkivska Y, Shkel O, Kim YK
Methods · 2026 Mar · PMID 41611193 · Publisher ↗
Syntenin is a multifunctional PDZ-domain adaptor protein that orchestrates membrane trafficking, cytoskeletal remodeling, and exosome biogenesis. Initially identified as a syndecan-binding molecule, syntenin has since em...
Syntenin is a multifunctional PDZ-domain adaptor protein that orchestrates membrane trafficking, cytoskeletal remodeling, and exosome biogenesis. Initially identified as a syndecan-binding molecule, syntenin has since emerged as a central hub connecting membrane receptors to intracellular signaling pathways that regulate adhesion, motility, immune signaling, and cellular plasticity. While extensively studied in cancer and neural development, recent discoveries reveal that a wide range of viruses exploit syntenin to facilitate their replication, assembly, or dissemination. This review consolidates current evidence across diverse viral infections to elucidate the molecular mechanisms underlying the interaction between syntenin and viruses. Coronaviruses utilize syntenin to link PDZ-binding motifs to p38 MAPK-driven inflammation and endosomal entry. Papillomaviruses and Epstein-Barr virus hijack the CD63-syntenin-ALIX complex to control vesicle-mediated trafficking. Hepatitis C virus employs it to secrete E2-coated, antibody-resistant exosomes. Dengue virus harnesses its mosquito homolog AeSyntenin to package sfRNA for transmission. Human T-cell leukemia virus type 1 employs its Tax-1 oncoprotein to bind the PDZ domains of syntenin, remodel extracellular vesicle cargo, and promote viral spread. In contrast, during human immunodeficiency virus infection, syntenin restricts viral fusion at the plasma membrane, though the nucleocapsid mimics its PDZ tandem to promote virion release. Collectively, these findings establish syntenin as a dynamic regulator at the host-virus interface, capable of exerting both proviral and antiviral effects. Emerging pharmacological strategies targeting syntenin PDZ domains further underscore its potential as a broad-spectrum, host-directed antiviral target.
Nanomaterial-based electrochemical sensors for phenolic antioxidants in foods and beverages: From design to device translation.
Hermawan A, Aris A, Khalil M … +5 more , Wulan Septiani NL, Estiasih T, Ray HRD, Palma M, Setyaningsih W
Methods · 2026 Mar · PMID 41587572 · Publisher ↗
Nanomaterial-enabled electrochemical sensors are nearing the performance and practicality needed for routine, on-site monitoring of phenolic compounds in foods and beverages. Advances in nanomaterial dimensionality and h...
Nanomaterial-enabled electrochemical sensors are nearing the performance and practicality needed for routine, on-site monitoring of phenolic compounds in foods and beverages. Advances in nanomaterial dimensionality and hybrid architectures, from atomically doped nanoparticles and zero-dimensional clusters to two- and three-dimensional porous frameworks, have enhanced electron-transfer kinetics, expanded electroactive surface area, and enabled more selective surface chemistries. These gains align with progress in molecular recognition using enzymes, aptamers, molecularly imprinted polymers, and permselective antifouling coatings, as well as electrode-engineering strategies that translate nanoscale activity into reliable printed-electrodes. Although laboratory detection limits are often impressive (micromolar to low-nanomolar in controlled media), challenges remain in reproducibility, shelf life, and performance in complex matrices such as wine, olive oil, and fermented foods. Closing these gaps requires integrated solutions that unite printable, stable nanomaterial inks with simple on-cartridge sample conditioning, modular recognition layers, and robust on-board calibration and data-handling routines. To enable practical deployment, we propose a development pathway focused on scalable manufacturing and quality control of nanomaterial inks and electrodes, harmonized validation against chromatographic reference methods, durable antifouling and self-cleaning strategies, and an ecosystem approach that uses smartphone connectivity and cloud analytics to convert electrochemical signals into traceable, defensible decisions for industry, regulators, and consumers.
Proteomics method for identifying POT1-associated complexes at telomeres using ChIP-Mass spectrometry.
Zade NH, Jain M, Garg M … +3 more , Checker R, Ghosh A, Khattar E
Methods · 2026 Mar · PMID 41577003 · Publisher ↗
POT1 is the only single stranded telomere binding protein in the shelterin complex. Together with TPP1, POT1 plays a crucial role in regulating telomere length and protecting telomeres from DNA damage repair proteins. Th...
POT1 is the only single stranded telomere binding protein in the shelterin complex. Together with TPP1, POT1 plays a crucial role in regulating telomere length and protecting telomeres from DNA damage repair proteins. The activation of DNA damage repair proteins at telomeres can be detrimental to cells, so their activity must be suppressed. POT1 interacts with other telomeric proteins (TRF2, TRF1, TIN2 and RAP1) via its association with TPP1. These proteins function together to protect and maintain the telomeres. Despite extensive knowledge of POT1's role within the shelterin complex, the full spectrum of its interactors at the single-stranded telomeric overhang remains poorly defined. To study these interactions, we generated an endogenous Flag-tag knock-in of POT1 using the CRISPR-Cas9 gene editing system. To address the risk of unintended gene disruption associated with this technique, we conducted an in-depth characterization of the endogenously Flag-tagged POT1 clone to ensure that its telomere and TPP1 binding functions remained intact. Further, we performed proteomic profiling of the Flag-tagged POT1 within the chromatin fraction using ChIP-MS to explore its proteome. Our analysis uncovered a novel set of POT1-associated proteins at the extremes of telomeres. Given that POT1 exclusively binds to the single-stranded 3' overhang of telomeres, the proteomic data obtained indicates POT1 interactions occurring at the extreme ends of telomeres. In conclusion, our study reveals previously uncharacterized POT1 associated proteins using ChIP mass spectrometric approach, paving the way for further investigations into telomere biology and potential therapies targeting telomere regulation.
The clinical potential and limitations of metal-organic frameworks (MOFs) in dentistry.
Hussein WS, Al-Hussainy AF, Menon SV … +9 more , Maharana L, Sabarivani A, Mukherjee G, Sinha A, Nigman K, Sameer HN, Salih RM, Adil M, Kalantari S
Methods · 2026 Mar · PMID 41525804 · Publisher ↗
Nanoparticles (NPs) are intentionally incorporated into dental products to enhance material quality. Recently, metal-organic framework (MOF) NPs have attracted significant attention owing to their unique characteristics...
Nanoparticles (NPs) are intentionally incorporated into dental products to enhance material quality. Recently, metal-organic framework (MOF) NPs have attracted significant attention owing to their unique characteristics absent in their larger-scale counterparts. These properties make MOFs suitable for various functional platforms. MOF-based nanomaterials exhibit high antibacterial potential, owing to their biocompatibility, biodegradability, and safety for living organisms. They have also become a prominent component in the development of anticancer agents and in the manufacture of commercial medical products. Consequently, MOFs are becoming increasingly important in dentistry. They show promising therapeutic potential for inhibiting oral infectious diseases, such as those caused by bacterial plaque, and have a broad range of other biomedical applications, including the diagnosis and treatment of dental diseases. However, significant challenges hinder their transition from laboratory testing to widespread clinical use in hospitals. Key issues include long-term toxicity and biocompatibility concerns, unpredictable pharmacokinetics that can lead to unintended accumulation in organs like the liver and spleen, and the potential for adverse immune reactions. Other major obstacles are particle aggregation in biological fluids, the complex formation of a "protein corona" that can alter the NPs' intended function, and the high cost of large-scale production under consistent Good Manufacturing Practices (GMP). This article discusses MOFs and their potential applications in identifying cancer biomarkers and treating tooth infections and oral cancers. It highlights their unique porous features, which help combat oral biofilms and detect microorganisms. The summary also addresses the primary clinical challenges that must be addressed to ensure the safe development of MOF-based therapies.
Navigating the frontier of human microglia isolation-standardization and translational insight.
Pal P, Sharma M, Gupta SK
Methods · 2026 Mar · PMID 41519496 · Publisher ↗
The expanding field of neuroscience has increasingly recognized the critical role of microglia, the resident immune cells of the central nervous system (CNS), in human health and disease. This review embarks on an in-dep...
The expanding field of neuroscience has increasingly recognized the critical role of microglia, the resident immune cells of the central nervous system (CNS), in human health and disease. This review embarks on an in-depth exploration of the current landscape of human microglia extraction techniques. With a focus on the unique challenges and opportunities presented by human tissue samples, we delve into various sources of human microglia, including primary brain tissue and stem cell-derived models, addressing the ethical and logistical considerations inherent to these methodologies. Laboratory techniques for microglia extraction are scrutinized, highlighting adaptations for human samples in enzymatic digestion, mechanical dissociation, density gradient centrifugation, and cell sorting, alongside innovations in cryopreservation and viability assessment. Critical perspectives are offered on technical hurdles, technique selection for human research, and the horizon of emerging technologies. Standardization efforts and international guidelines are discussed, underscoring their significance in fostering reproducibility and comparability across studies. The review also illuminates applications of human microglia in disease modeling, drug discovery, and understanding neurodevelopmental and neurodegenerative processes. Concluding remarks emphasize future directions of human microglia research, advocating for methodological precision and integration of next-generation technologies to unlock new therapeutic avenues. This review serves as a comprehensive guide for researchers navigating the complex terrain of human microglia extraction, aiming to catalyze advancements in neuroscience research through methodological innovation and standardization.
Rapid and improved surface passivation method for Single-Molecule experiments.
Gonneville AN, Ward AE, Naidoo NR … +2 more , Barrera FN, Lamichhane R
Methods · 2026 Mar · PMID 41512980 · Full text
Single-molecule fluorescence experiments are a powerful tool for studying biomolecular interactions, including protein dynamics and oligomerization, protein-protein interactions, and protein-nucleic acid interactions. Bi...
Single-molecule fluorescence experiments are a powerful tool for studying biomolecular interactions, including protein dynamics and oligomerization, protein-protein interactions, and protein-nucleic acid interactions. Biomolecules are commonly immobilized on the microscope surface to extend the observation time. However, non-specific interactions between biomolecules and the surface present a major challenge. The first critical step in these experiments is preparing the surface using polyethylene glycol (PEG) coated slides, which facilitate biomolecule immobilization while minimizing non-specific interactions. The surface treatment typically uses PEG-SVA (Succinimidyl Valerate) coated slides, and the protocol for the treatment is lengthy and time-consuming. To overcome this issue, we have developed a process that uses PEG-Silane to improve efficiency while maintaining reproducibility. Here, we present a one-step, rapid PEGylation methodology that can be completed in minutes rather than hours. We demonstrate its validity and feasibility through single-molecule fluorescence resonance energy transfer (smFRET) and single-molecule photobleaching experiments across various biological samples.
Precision mapping of NF-κB-DNA binding: high-resolution insights via dynamic UV-laser footprinting.
Lone IN, Çavdar G, Peshev Z … +2 more , Kale S, Angelov D
Methods · 2026 Mar · PMID 41512979 · Publisher ↗
Sequence-specific binding is at the core of all DNA-templated processes, including the initiation of DNA replication, gene expression and DNA repair. Yet the kinetics and precision of these interactions remain difficult...
Sequence-specific binding is at the core of all DNA-templated processes, including the initiation of DNA replication, gene expression and DNA repair. Yet the kinetics and precision of these interactions remain difficult to capture at high resolution. Here, we present Dynamic UV Laser Footprinting (DULF), a novel technique that integrates UV laser footprinting with stopped-flow mixing to probe transcription factor (TF)-DNA interactions at millisecond temporal and single base-pair spatial resolution. Using NF-κB as a model TF, we demonstrate the feasibility of DULF in visualizing the spatiotemporal details of a sequence-specific TF-DNA interaction. The high temporal resolution of our data reveals that TF-DNA binding proceeds through a rapid recognition step and a subsequent slower stabilization phase. DULF also revealed that the p50 homodimer binds specifically to DNA outside the canonical binding site, emphasizing the role of flanking sequences in these interactions. All-atom molecular dynamics simulations confirmed that DNA sequence context, including flanking base pairs, modulates NF-κB binding stability and induces local structural changes such as bending, groove widening, and base unstacking. DULF offers a unique opportunity to study DNA-protein interactions at unprecedented resolution, providing insights into the mechanism of sequence-specific binding and stabilization of chromatin interactors.
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