Methods (San Diego, Calif.)[JOURNAL]
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200 papers
An accessible, absorbance-based plate reader assay to assess cumulative exposure of blood plasma & serum to thawed conditions.
Jehanathan N, Quansah IA, Kremer S … +2 more , Kapuruge EP, Borges CR
Methods · 2026 Jul · PMID 42392415 · Publisher ↗
The integrity of human plasma and serum (P&S) is crucial for reliable biomarker discovery and validation, but it is affected by preanalytical exposure to thawed conditions (temperatures > -30 °C). The limited number of e...
The integrity of human plasma and serum (P&S) is crucial for reliable biomarker discovery and validation, but it is affected by preanalytical exposure to thawed conditions (temperatures > -30 °C). The limited number of existing quality control methods for assessing plasma or serum (P/S) integrity often require resource-intensive techniques like mass spectrometry. We have developed and validated an accessible, cost-effective absorbance-based plate reader assay based on the oxidative ex vivo consumption of most small molecule thiols & disulfides (SMT&D) by albumin. The assay separates SMT&D from proteins, then uses tris(2-carboxyethyl)phosphine to reduce SMT&D, with subsequent quenching by 4-azidobenzoic acid, followed by reaction of thiols with Ellman's reagent. The method was optimized for yield, including steps to remove trace metals and minimize thiol re-oxidation. Due to inter-individual matrix effects, it was determined that measurements of each specimen before and after intentional expiration (which produce a single Δ-absorbance value, calibrated to Δ-SMT&D) are required to forensically evaluate prior exposure to thawed conditions. The assay demonstrated robust linearity, precision, and accuracy across diverse inter-individual matrices. Stability assessments at 23 °C, 4 °C and -20 °C revealed consistent exponential decay patterns in SMT&D, similar to those observed in the established LC/MS-based ΔS-Cys-Albumin assay. Estimated population reference ranges for P&S demonstrated sufficient dynamic range for the analysis of single samples with unknown histories. This potential was verified with blinded challenges. The assay provides a practical, readily accessible tool for quantifying P&S exposure to thawed conditions, facilitating improved quality control for investigators working with P&S for clinical or research purposes.
EC-isHCR: A rapid method for in situ hybridization chain reaction in diverse animal samples.
Kozono Y, Mikami K, Yorimoto S … +15 more , Hayashi T, Okamura H, Qian Q, Hoshino R, Kamiyama T, Sanaki Y, Asaoka M, Ohsawa S, Azuma S, Iwasaki YW, Hayashi M, Hayashi Y, Shigenobu S, Niwa R, Kobayashi S
Methods · 2026 Jun · PMID 42372957 · Publisher ↗
The in situ hybridization chain reaction (isHCR) visualizes RNA across multiple spatial scales, from organs to subcellular structures, in diverse samples. We previously proposed a rapid protocol, EC-isHCR, for Drosophila...
The in situ hybridization chain reaction (isHCR) visualizes RNA across multiple spatial scales, from organs to subcellular structures, in diverse samples. We previously proposed a rapid protocol, EC-isHCR, for Drosophila embryos and ovaries. Whether EC-isHCR retains the features of conventional isHCR, including wide-spatial-scale analyses in various samples, however, has remained unclear. Here, we show that EC-isHCR enables robust RNA detection in a broad range of samples, such as whole-mount fruit fly, parasitoid wasp, and aphid preparations; paraffin sections of trout; frozen mouse sections; and human cultured cells. Moreover, EC-isHCR enabled detection of subcellular RNA localization. EC-isHCR also visualized association of RNA with phase-separated condensates in fruit fly embryos and detected the protrusion-enriched mRNA in HeLa cells. To broaden the applicability of EC-isHCR, we developed an automated probe design tool (https://github.com/ShuntaYorimoto/hcrkit). By combining this tool with EC-isHCR, we provide a fast and versatile framework to visualize mRNAs. This framework will help reduce the barrier to using fast isHCR and thereby facilitate research across diverse areas of the life sciences.
Single-Molecule methods to investigate mechanisms of transcription by RNA polymerase of Mycobacterium tuberculosis.
Lin W, Herrera-Asmat O, Tong AB … +2 more , Kong T, Bustamante C
Methods · 2026 Jun · PMID 42372956 · Publisher ↗
Single molecule methods have become prevalent tools in elucidating molecular processes across various life science fields over the past three decades, driving breakthroughs in understanding their underlying molecular mec...
Single molecule methods have become prevalent tools in elucidating molecular processes across various life science fields over the past three decades, driving breakthroughs in understanding their underlying molecular mechanisms. In our study, we employed two single-molecule methods, Förster Resonance Energy Transfer (smFRET) and high-resolution optical tweezers, to investigate the transcription of Mycobacterium tuberculosis RNA polymerase (MtbRNAP) from initiation through to termination. We aim to provide a set of comprehensive biophysical tools to deepen our current understanding of MtbRNAP and its transcription factors. These experimental assays represent an important step towards unraveling the molecular dynamics and interactions that support transcription in Mycobacterium tuberculosis.
Detection and sequencing of Usutu virus during mosquito surveillance: Use of multiple assays and techniques for identification at low levels.
Findlay-Wilson S, Everall I, Nettlemill A … +7 more , Crossley L, Johnston C, Abbott A, Medlock J, Vaux A, Pullan S, Dowall S
Methods · 2026 Jun · PMID 42320777 · Publisher ↗
BACKGROUND: Usutu virus (USUV) is a medically important flavivirus linked to significant mortalities within bird populations and an increasing association with human infections, particularly within immunocompromised indi...
BACKGROUND: Usutu virus (USUV) is a medically important flavivirus linked to significant mortalities within bird populations and an increasing association with human infections, particularly within immunocompromised individuals. It's genetic and etiological similarity with West Nile virus (WNV) means that it is often detected within the same ecological niche, consequently the detection of USUV is a good indicator that the conditions are suitable for the introduction of WNV. METHODS: Surveillance approaches often require detection of low viral loads within arthropod vectors. Although useful for straightforward detection, strain and genetic details can be missed which provide important information on autochthonous survival and/or reintroduction. We evaluated a pan-flavivirus screening process followed by tiled amplicon sequencing for the detection and analysis of USUV within mosquito pools. RESULTS: RT-PCR screening of 270 mosquito pools from a range of species revealed one positive (0.37%) in a pool of Culex modestus. Further analysis using pan-flavivirus end-point PCR produced DNA amplicons which sequencing confirmed to differ from the positive reference control. Tiled amplicon-based whole genome amplification using USUV specific primers followed by GridION sequencing produced a near full-length sequence which phylogenetic analysis placed within the African 3.2 clade. CONCLUSION: Using targeted mosquito surveillance followed by an array of molecular techniques we were able to detect and phylogenetically analyse USUV from low viral loads within infected mosquito pools. This system is useful for the initial screening and subsequent taxonomy of viruses in samples with low viral loads and high host genomic background, providing an important tool for surveillance and the analysis of circulation dynamics.
Experimental validation of an AI-driven digital healthcare platform for oral health behavior and plaque assessment among vietnamese children.
Lim JN, Nhu Ngoc VT, Vu TM … +1 more , Lee JY
Methods · 2026 Jun · PMID 42314814 · Publisher ↗
BACKGROUND: Oral health among children in developing countries, including Vietnam, remains a significant public health concern. Innovative approaches leveraging artificial intelligence AI-based digital health platforms m...
BACKGROUND: Oral health among children in developing countries, including Vietnam, remains a significant public health concern. Innovative approaches leveraging artificial intelligence AI-based digital health platforms may offer effective strategies for managing dental plaque and promoting better oral hygiene behaviors among school-aged children. This study aimed to evaluate the effectiveness of an AI-driven oral healthcare platform (Denti-i Vietnam) in improving oral hygiene and behavioral outcomes among Vietnamese primary school students. METHODS: A total of 204 primary school students aged 8-10 years in Hanoi, Vietnam, participated in this experimental study. Participants were randomly assigned to an intervention group (n = 107), which used the AI-driven oral healthcare platform, and a comparison group (n = 97), which received traditional oral health education via pamphlets. Oral health behaviors, dental plaque levels (Simplified Oral Hygiene Index; OHI-S), and caries indices (dft/DMFT) were assessed at baseline and after the intervention period. RESULTS: The intervention group demonstrated a significant reduction in the OHI-S score compared to baseline (2.49 ± 0.60 to 1.70 ± 0.76, p < 0.001), particularly in the debris component, indicating enhanced plaque control. Notable improvements were also observed in oral hygiene behaviors, including increased frequency of toothbrushing before and after breakfast (p < 0.01) and more frequent parental assistance during brushing (p = 0.03). Furthermore, parental awareness of dental caries significantly increased in the intervention group (p = 0.001). CONCLUSIONS: The AI-driven oral healthcare platform significantly improved both oral hygiene behaviors and plaque control among Vietnamese primary school children. These findings suggest that AI-driven digital health tools can serve as practical and scalable solutions for promoting oral health in developing countries.
Zeta potential: An efficient and cost-effective alternative for investigating cell-surface interactions.
Gupta A, Wang W, Zhao J … +1 more , Cheng Q
Methods · 2026 Jun · PMID 42303009 · Publisher ↗
BACKGROUND: Monitoring and quantifying cell-surface interactions is important for drug discovery and understanding the physiological and pathological activities of cells. While techniques like Enzyme-linked immunosorbent...
BACKGROUND: Monitoring and quantifying cell-surface interactions is important for drug discovery and understanding the physiological and pathological activities of cells. While techniques like Enzyme-linked immunosorbent assay (ELISA), Fluorescence resonance energy transfer (FRET), and Atomic force microscopy (AFM) have proven effective in this area, these technologies are associated with the cost of capital equipment, extended turnaround time, and specialized trained personnel. Moreover, they may not accurately reflect what is happening in living systems, e.g., live cells. METHODS: To mitigate these concerns, we introduce Zeta potential analysis, which offers a cost-effective, real-time, and label-free quantitative method to assess the shift of surface charge induced by molecular interactions. This approach is particularly valuable for evaluating receptor ligand binding dynamics. In this study, we provided two examples of measuring biological response to the external stimuli in living systems using zeta potential. The first example demonstrated how per- and polyfluoroalkyl substances (PFAS) interact with human mammary cells, MCF10A and MDA-MB-231. The second example determined how nanoparticles (NPs) interact with bacteria, Escherichia coli, Salmonella typhi, and Bacillus subtilis. These interactions were benchmarked by paired zeta potential results with immunofluorescent staining using standard antigen-antibody staining on live cells. RESULTS: Our results of zeta potential to prove cell surface interactions are obtained within a 15 min time period. The supply cost of a single experiment is <$5. We demonstrated that zeta potential analysis is a powerful and versatile technique for investigating cell-surface interactions. It enables real-time monitoring of molecular binding events and is broadly applicable across living systems, including mammalian and bacterial cells. DISCUSSION: The benchmarking results showed that even subtle biological responses that shift the surface charge of living cells can be clearly detected. To conclude, this capability of detecting subtle changes allows for confirmation of ligand-receptor interaction mechanisms and provides early and reliable insights to justify further investment in costly research methods during pre-clinical drug development.
An automated workflow for quantifying the formation of synuclein aggregates in human dopaminergic neurons.
Han C, Nguyen-Renou E, Benaliouad F … +11 more , Luo W, Chen CX, Alluli A, Villegas L, Beitel LK, Shlaifer I, Reintsch WE, Krahn AI, Cid Pellitero ED, Fon EA, Durcan TM
Methods · 2026 Jun · PMID 42297199 · Publisher ↗
Parkinson's disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (α-syn) aggregates termed Lewy bodies. To model PD pathology in vitro, preformed fibrils of α-syn (PFFs), which can be taken up by...
Parkinson's disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (α-syn) aggregates termed Lewy bodies. To model PD pathology in vitro, preformed fibrils of α-syn (PFFs), which can be taken up by cells, provide a seed that drives misfolding and aggregation of endogenous α-syn, with new aggregates amplifying this process. External application of PFFs to dopaminergic neurons (DNs) increases aggregate formation, marked by α-syn phosphorylation at serine 129 (pS129-syn), a pathological PD marker. Building on this, we developed an automated synuclein seeding assay to quantify new α-syn aggregates in iPSC-derived DNs. Using pS129-syn as a readout, we show that PFFs elicit a time- and dose-dependent increase in pS129-syn aggregates. Our automated seeding assay further revealed that aggregate formation depends on endogenous α-syn levels. Treatment with PFFs produced a greater increase in pS129-syn aggregates in iPSC DNs derived from a PD patient with a triplication in the SNCA gene, which encodes the α-syn protein and which elevates total α-syn levels, relative to DNs from an isogenic iPSC line from the same individual, in which the SNCA gene mutation had been corrected by CRISPR/Cas9. In contrast, no pS129-syn signal was detected in neurons in which all copies of the SNCA gene had been knocked out (KO). This proof-of-principle automated high-content imaging workflow for synuclein seeding has been validated using isogenic cell lines with defined SNCA copy number variants and it offers a platform for assessing compounds and therapeutics that may impede α-syn aggregate formation.
A novel methodological framework for the assessment of the neural control of the shoulder using high-density surface electromyography.
Inglis JG, Rio S, Cabral HV … +5 more , Cosentino C, Pagani R, Lulic-Kuryllo T, Dickerson CR, Negro F
Methods · 2026 Jun · PMID 42269733 · Publisher ↗
BACKGROUND: Complex shoulder function relies on coordinated activation of small and large muscles, including the deltoid, pectoralis major, trapezius, and latissimus dorsi. However, detailed knowledge of their neuromuscu...
BACKGROUND: Complex shoulder function relies on coordinated activation of small and large muscles, including the deltoid, pectoralis major, trapezius, and latissimus dorsi. However, detailed knowledge of their neuromuscular control remains limited. This feasibility study aimed to develop a methodological framework to investigate the neural control of larger superficial shoulder muscles by combining a six-degree-of-freedom load cell, robotic arm and high-density surface electromyograms (HDsEMG). METHODS: Six healthy participants performed 30 % maximal voluntary isometric contractions (abduction, adduction, flexion, extension) at 30° and 65° of lateral abduction shoulder positions. HDsEMGs were recorded and analysed at the global activation, spatial distribution of activation and motoneurone levels. Global activation was quantified using averaged normalized root-mean-square (RMS) and spatial distribution using coefficient-of-variation of topographic maps. HDsEMGs were decomposed into individual motor unit spike trains using convolutive blind source separation. Motor unit (MU) behaviour was characterized by mean discharge rate and spatial distribution of MU action potentials (MUAPs). RESULTS: RMS maps revealed action-specific activation within and between muscles, with the trapezius active across all tasks, while the anterior, middle, and posterior deltoid, pectoralis major, and latissimus dorsi predominantly activated during abduction, flexion, and extension. MU discharge rate showed task-dependent activity. MUAP spatial distributions showed distinct MU territories within arrays, suggesting region-specific recruitment strategies across actions. CONCLUSION: The present framework provides a promising methodological approach for the non-invasive assessment of individual MU activity in superficial shoulder muscles. The approach provides a methodological basis for novel incorporation of neural control information into biomechanical models of shoulder function.
A fluorescence-based high-content imaging workflow for multi-parametric lipid droplet phenotyping via Oil Red O fluorescence.
Yan S, Yao Y, Liu C … +4 more , Zhu H, Wu X, Lyu C, Yang Y
Methods · 2026 Jun · PMID 42259461 · Publisher ↗
UNLABELLED: Progression of Metabolic-associated Fatty Liver Disease (MAFLD) involves excessive accumulation of intrahepatic lipid droplets (LDs). However, cost-effective, high-throughput imaging for detailed, multi-param...
UNLABELLED: Progression of Metabolic-associated Fatty Liver Disease (MAFLD) involves excessive accumulation of intrahepatic lipid droplets (LDs). However, cost-effective, high-throughput imaging for detailed, multi-parametric LDs quantification at the single-cell level remains difficult. MATERIALS AND METHODS: Here, we established a high-content screening (HCS) platform that utilizes the fluorescence of Oil Red O (ORO) to quantify LD phenotypes. This method enables the simultaneous extraction of multi-parametric morphological features, including LD number, size, intensity, and spatial dispersion, in free fatty acid (FFA)-induced AML12 hepatocytes. As a proof-of-concept, we systematically screened and validated a library of 36 lipid-lowering compounds derived from the literature. RESULTS: The ORO fluorescence-based HCS platform demonstrated adequate sensitivity, low background interference, and spatial resolution for single-cell quantification compared to bright-field analysis. Data clustering classified monomeric compounds into distinct lipid-regulatory patterns. For instance, pentacyclic triterpenoids decreased the number and size of LDs, while diterpene quinones reduced LD density. CONCLUSION: This study establishes a credible, visualizable, and economical HCS approach for assessing LD dynamics. By correlating multidimensional imaging metrics with specific cellular phenotypes, this platform provides a practical method for evaluating lipotoxicity and for high-throughput discovery of anti-steatotic drug candidates from natural product libraries.
Streamlined isolation of enriched axoplasm from primary dissociated neuronal cultures.
Alarcón S, Fuentes A, Carrazana E … +4 more , Fabres K, Bahamonde P, Sanhueza M, Salvadores N
Methods · 2026 Jun · PMID 42248224 · Publisher ↗
Axons are highly specialized neuronal compartments that rely on local molecular regulation to support growth, maintenance, and synaptic function. The cytoplasmic contents of axons, collectively referred to as the axoplas...
Axons are highly specialized neuronal compartments that rely on local molecular regulation to support growth, maintenance, and synaptic function. The cytoplasmic contents of axons, collectively referred to as the axoplasm, constitute a biochemically distinct domain whose composition is central to neuronal development and disease. However, biochemical analysis of axoplasm has been technically challenging, particularly in dissociated neuronal cultures, where axons and somata are intermingled and axonal material is limited. As a result, most axoplasm-focused studies have relied on explant-based or peripheral neuron models, restricting direct biochemical access to axons from dissociated central nervous system neurons. Here, we describe a simple and robust culture-based approach that enables efficient physical separation of somata and axons from primary dissociated neuronal cultures and allows the recovery of enriched axoplasm fractions. Using a custom reaggregation device, neurons reorganize into compact somatic clusters that extend radially oriented axons, facilitating reliable manual separation of somatic and axonal material. Morphological and immunofluorescence analyses demonstrate robust neuronal polarization and effective spatial segregation of somatic and axonal components. Biochemical and molecular validation reveals that axonal fractions are enriched in axonal proteins and contain bona fide axon-associated transcripts, while being depleted of nuclear markers and genomic DNA, providing a practical and accessible method for axoplasm isolation from dissociated neuronal cultures. By enabling biochemical analysis of axonal material from neuron types that are otherwise difficult to access, this method expands the experimental toolkit for studying axon-specific molecular mechanisms in neuronal development, plasticity, and disease.
CRISPR-enhanced ELISA based on ssDNA-triggered Cas13a-Csm6 cascade for sensitive profiling of inflammatory biomarkers in sports-related osteoarthritis among martial arts athletes.
Li Y, Xu C, Li J
Methods · 2026 May · PMID 42214709 · Publisher ↗
Sports-related osteoarthritis (OA), which is common among martial arts athletes exposed to repetitive high-impact loading, is closely associated with complex inflammatory responses. Sensitive detection of inflammatory bi...
Sports-related osteoarthritis (OA), which is common among martial arts athletes exposed to repetitive high-impact loading, is closely associated with complex inflammatory responses. Sensitive detection of inflammatory biomarkers is therefore highly desirable for early diagnosis and disease monitoring. However, conventional enzyme-linked immunosorbent assays (ELISA) are often limited by insufficient sensitivity due to the lack of intrinsic signal amplification. Herein, we report a CRISPR-enhanced ELISA platform based on an ssDNA-triggered Cas13a-Csm6 cascade for ultrasensitive detection of OA-related inflammatory biomarkers. In this strategy, detection antibodies are conjugated with trigger ssDNA, which is retained in the sandwich immunocomplex after target binding and serves as an activator for Cas13a. Activated Cas13a subsequently cleaves the Csm6 activator precursor to generate secondary messengers, thereby initiating a Csm6-mediated amplification process and converting protein recognition into amplified nucleic acid fluorescence output under isothermal and amplification-free conditions. The developed platform enabled sensitive detection of multiple inflammatory biomarkers with low-picogram-per-milliliter limits of detection, including IL-6, IFN-γ, and MMP-3, while maintaining high specificity and good tolerance in simulated biological matrices. Well-resolved multi-analyte analysis was achieved through a spatially resolved ELISA format, allowing parallel detection of different targets in individual wells of the same microplate. The proposed method also showed good agreement with conventional ELISA, demonstrating its analytical reliability. This work provides a versatile and sensitive strategy for spatially resolved parallel protein analysis and offers promising potential for inflammatory biomarker monitoring and OA-related bioanalysis in martial arts athletes.
Modeling 3D Glioblastoma: A review of spheroid culture techniques and standardization challenges.
Castro LM, Paiva L, Lobo I … +4 more , Neves AR, Albuquerque T, Sousa Â, Costa D
Methods · 2026 Sep · PMID 42176913 · Publisher ↗
Glioblastoma (GB) is among the most aggressive and treatment-resistant cancers, highlighting the urgent need for physiologically relevant preclinical models. Traditional two-dimensional (2D) cultures fall short in replic...
Glioblastoma (GB) is among the most aggressive and treatment-resistant cancers, highlighting the urgent need for physiologically relevant preclinical models. Traditional two-dimensional (2D) cultures fall short in replicating the complex microenvironment of GB, whereas three-dimensional (3D) culture models offer improved structural, biochemical, and functional resemblance to in vivo tumors. However, the lack of a standardized protocol for GB spheroid formation leads to significant inter-study variability and limited reproducibility. This review summarizes the key variables affecting spheroid generation, such as cell line selection, passage number, seeding density, matrix composition, and culture duration, and compares the effectiveness of commonly used methods like low-attachment plates, scaffold-based systems, and hydrogel embedding. Additionally, emerging trends in co-culture strategies and tumor microenvironment (TME) simulation are also highlighted. By outlining current strengths, limitations, and gaps, we provide a framework for advancing standardized, high-fidelity GB spheroid systems to accelerate translational research and therapeutic discovery.
Virus-Like Particles: From Fundamental Concepts to Cutting-Edge Applications - A Comprehensive Review of Production, Engineering, and Clinical Translation.
Lotfi MS, Jafari S, Razmyeki M … +1 more , Hashemzadeh MS
Methods · 2026 Sep · PMID 42176912 · Publisher ↗
Virus-like particles (VLPs) are non-infectious nanoparticles that resemble native viruses, and are highly immunogenic because of their repetitive surface geometry and efficient lymph node drainage. Although their effecti...
Virus-like particles (VLPs) are non-infectious nanoparticles that resemble native viruses, and are highly immunogenic because of their repetitive surface geometry and efficient lymph node drainage. Although their effectiveness in prophylactic vaccines cannot be denied, the range of VLP applications has increased manifold. This is a comprehensive review of the whole VLP pipeline, including production and clinical translation. We present a critical comparison of expression systems, such as prokaryotic, yeast, baculovirus/insect cell (BEVS), mammalian, and emerging systems, with trade-offs in terms of yield, authenticity, and scalability. The article explores the more complex engineering approaches to functionalizing VLPs through genetic fusion and chemical conjugation, which allow functional antigen display, cell-specific targeting, and encapsulation of various cargo. In addition to vaccination, we discuss the latest uses of targeted drug delivery, cancer therapy, gene editing, and molecular imaging. Lastly, the review focuses on the critical issues of manufacturing scalability, analytical characterization, and regulatory pathways to clinical translation. This analysis combines the key principles with the leading-edge technologies to highlight the transformative nature of VLPs as versatile nanotechnologies capable of enhancing preventive and precision medicine.
Emerging frontiers in genome editing: From CRISPR to next-generation technologies.
Mishra S, Rehan S, Barekzai AM … +2 more , Sharma A, Raghav A
Methods · 2026 Sep · PMID 42173386 · Publisher ↗
Genome editing has revolutionized molecular biology. It offers precise modification of genetic material across diverse organisms. This review outlines the evolution of genome editing technologies from homologous recombin...
Genome editing has revolutionized molecular biology. It offers precise modification of genetic material across diverse organisms. This review outlines the evolution of genome editing technologies from homologous recombination to advanced Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (CRISPR-9) based systems that now dominate the field. Early methods, such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs), established the foundation for site-specific DNA cleavage. However, they were limited by complexity and cost. The advent of the CRISPR-Cas systems, particularly CRISPR-Cas9, transformed the landscape due to their simplicity, high efficiency, and adaptability. Variants such as CRISPR-Cas12a, base editors, and prime editors enhanced editing precision. They enable single-nucleotide modifications and targeted insertions without double-strand breaks. Emerging tools such as CRISPR-associated transposases, recombinase fusions, and RNA-targeting Cas13 enzymes expand the scope of manipulation beyond DNA to RNA. At the same time, epigenome editing, and gene drives present new therapeutic and ecological applications. Efficient delivery systems, both viral (Adeno-Associated Virus (AAV), lentivirus, adenovirus) and non-viral (lipid nanoparticles, gold nanoparticles, DNA nano clews), remain critical for clinical translation. Future directions emphasize artificial intelligence-guided design, retroelement-based integration, and novel biomimetic delivery vehicles to overcome current efficiency and safety barriers. These innovations can help overcome current efficiency and safety barriers. Together, they are propelling genome editing toward precise, programmable, and ethically responsible therapeutic applications. Despite unresolved challenges involving off-target effects, immunogenicity, and germline ethics, genome editing redefines biomedical research, drug development, and disease correction. CRISPR-derived technologies now stand at the forefront of next-generation genetic medicine.
Comprehensive evaluation of reference miRNA for extracellular miRNA quantification in CKD.
Srivastava A, Maienschein-Cline M, Pabalan A … +3 more , Shankaranarayanan D, Cao H, Raj DS
Methods · 2026 Sep · PMID 42167467 · Publisher ↗
MicroRNAs (miRNAs) are promising biomarkers and therapeutic targets; however, reliable extracellular miRNA quantification by RT-qPCR depends on appropriate normalization, and no universal endogenous reference has been es...
MicroRNAs (miRNAs) are promising biomarkers and therapeutic targets; however, reliable extracellular miRNA quantification by RT-qPCR depends on appropriate normalization, and no universal endogenous reference has been established. This study aimed to identify optimal endogenous control for extracellular miRNA analyses. Candidate reference miRNAs were first identified through next-generation sequencing of plasma (n = 289) and urine (n = 109) using samples from the Chronic Renal Insufficiency Cohort (CRIC). Candidates were prioritized using NormFinder algorithm combined with complementary stability metrics (including biological coefficient of variation, expression difference, mean expression, proportion of detectable samples) and evidence from literature. The top candidate miRNAs identified in discovery phase were subsequently validated by RT-qPCR in 250 plasma and 205 urine samples randomly selected from the remaining CRIC participants. Both descriptive statistical analysis and NormFinder identified hsa-miR-126-3p, hsa-miR-30e-5p, and hsa-miR-24-3p as most stably expressed miRNAs in plasma, whereas members of let-7 family, hsa-miR-423-5p, and hsa-miR-16-5p showed the highest stability in urine. RT-qPCR validation showed that hsa-miR-24-3p was detectable in 97% of the validation cohort and showed significant superior expression stability (p < 0.0001), identifying it as the most robust endogenous reference miRNA for plasma normalization. The addition of hsa-miR-30e-5p moderately improves expression stability if dual-reference normalization is considered. In urine, candidate reference miRNAs showed low detectability; although hsa-miR-16-5p was more stable than hsa-miR-423-5p, neither is recommended for routine normalization without substantial technical optimization. Overall, hsa-miR-24-3p represents a reliable and robust endogenous control for plasma miRNA normalization within this CKD cohort, whereas urinary miRNAs require rigorous, context-specific validation to ensure accurate and reproducible quantification.
Robust carotid artery perfusion protocol for turtle brain fixation enabling multiscale imaging from magnetic resonance to super-resolution microscopy.
Medlej Y, Lotfinia A, Klinkmann M … +2 more , Rigneault H, Lauterbach MA
Methods · 2026 Sep · PMID 42155642 · Publisher ↗
Reptiles, particularly turtles, are increasingly recognized as valuable models in comparative and evolutionary neuroscience; however, standardized methods for brain perfusion and fixation remain lacking. Here, we present...
Reptiles, particularly turtles, are increasingly recognized as valuable models in comparative and evolutionary neuroscience; however, standardized methods for brain perfusion and fixation remain lacking. Here, we present the first detailed, stepwise protocol for turtle brain perfusion via cannulation of the common carotid arteries after decapitation. This approach overcomes the anatomical and technical limitations of transcardial perfusion and enables rapid and uniform delivery of fixatives directly to the cerebrovascular system. The protocol consistently yields pale, blood-free brains with well-preserved gross morphology. Uniform parenchymal fixation was validated using high-contrast magnetic resonance imaging (MRI), consistent immunohistochemical labeling, label-free stimulated Raman histology (SRH), and preservation of fine neuronal structures revealed by super-resolution stimulated emission depletion (STED) microscopy. In addition, we describe a comprehensive procedure for brain dissection from the skull. Together, these methods provide a robust framework for homogeneous brain fixation compatible with advanced multiscale imaging.
Intelligent methods in bioinformatics and genomics.
Zheng P, Zeng X
Methods · 2026 Sep · PMID 42134756 · Publisher ↗
Abstract loading — click title to view on PubMed.
Reflectance-based optical sensor for real-time detection and quantification of biofilms in complex matrices.
Rana S, Upadhyay LSB, Kumar N
Methods · 2026 Sep · PMID 42107827 · Publisher ↗
Biofilm detection is essential for maintaining public health and environmental integrity. Conventional detection techniques, including tube assays, microtiter plate assays, and microscopy, though effective, exhibit sever...
Biofilm detection is essential for maintaining public health and environmental integrity. Conventional detection techniques, including tube assays, microtiter plate assays, and microscopy, though effective, exhibit several limitations such as the requirement for trained personnel, sophisticated instrumentation, and prolonged processing durations. These factors constrain their applicability for high-throughput or large-scale sample screening. In recent years, reflectance-based analytical platforms have emerged as promising alternatives due to their operational simplicity, adaptability, and economic feasibility. In this study, a portable and cost-efficient reflectance-based optical sensor was designed for bacterial biofilm detection utilizing methylene blue-mediated colorimetric analysis. The system integrates a paper-based analytical device (PAD) fabricated from Whatman Grade 1 filter paper with a microcontroller (Arduino® Mini Pro), a light-dependent resistor (LDR), a white LED, and an OLED display for on-site reflectance monitoring. The PAD, optimized with 0.4 mM methylene blue, demonstrated effective interaction with extracellular matrix (ECM) constituents produced by Pseudomonas beteli and Bacillus tequilensis. Comparative evaluation with microtiter plate assays revealed a strong correlation (R = 0.9946), confirming high analytical accuracy and sensitivity. Overall, the developed sensor provides a reproducible, rapid, and efficient approach for biofilm quantification, with potential utility across clinical diagnostics, environmental monitoring, and industrial microbiology.
Determining optimal sgRNA coverage and screening duration for pooled CRISPR screens: A quantitative framework.
Xu T, Guo Z, Li Y … +1 more , Lyu H
Methods · 2026 Sep · PMID 42107826 · Publisher ↗
CRISPR-based loss-of-function screening has emerged as a powerful tool for systematically characterizing gene functions. However, standardized quantification metrics for sgRNA coverage-a critical parameter determining ge...
CRISPR-based loss-of-function screening has emerged as a powerful tool for systematically characterizing gene functions. However, standardized quantification metrics for sgRNA coverage-a critical parameter determining genome-wide screening reliability and resource efficiency, remain undefined. In this study, we first conducted systematic sgRNA coverage tests in HeLa cells to determine the optimal coverage for CRISPR knockout libraries. Furthermore, we incorporated multiple timepoints to monitor sgRNA-mediated gene knockout dynamics. Here, balancing data quality, time efficiency, and cost, we identify 15 days and 800 × coverage as the optimal screening parameters for standard iBAR‑based CRISPR screens. Data from varying coverage levels can also serve as essential references for screening under different experimental conditions. Longitudinal analysis revealed that extending the screening period beyond 15 days maintains stable sgRNA distribution patterns within the cell population. This study establishes key parameter benchmarks to ensure CRISPR knockout screening efficacy and reproducibility, providing a solid foundation for downstream drug screening and target identification.
Targeted next-generation sequencing techniques: From primer design to sophisticated library construction.
Koryukov M, Mikheeva R, Kechin A
Methods · 2026 Sep · PMID 42107825 · Publisher ↗
Targeted Next-Generation Sequencing (tNGS) represents a cornerstone technology in modern clinical diagnostics and biological research. Widely adopted by clinical laboratories, tNGS provides a robust and cost-effective al...
Targeted Next-Generation Sequencing (tNGS) represents a cornerstone technology in modern clinical diagnostics and biological research. Widely adopted by clinical laboratories, tNGS provides a robust and cost-effective alternative to whole-genome sequencing. Despite its extensive applications, the most technically complex and variable stage of the tNGS workflow remains target enrichment, a critical step that ensures target molecules are present at sufficiently high concentrations relative to background sequences. Enrichment methodologies are broadly classified into PCR-based and hybridization-based strategies, each offering distinct advantages and limitations. This review critically examines both approaches within the context of the computational challenges inherent to tNGS assay design, and surveys the full spectrum of established and emerging enrichment techniques, highlighting their suitability across different areas of molecular research and clinical diagnostics. The use of non‑canonical nitrogenous bases, circularization of linear DNA fragments, unique molecular identifiers (UMIs), and partially double‑stranded primers are among the many sophisticated approaches described in the review. We hope this review will contribute to accelerating the development of diagnostic strategies with targeted next-generation sequencing in the rapidly evolving field of molecular diagnostics.
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