This study developed a detection method for antisense oligonucleotides (ASOs) by ligase detection reaction (LDR) using T4 RNA ligase 2 (T4Rnl2). The LDR was applied to detect a nusinersen-based ASO, an 18-mer 2'-O-methoxyethyl-modifiedoligonucleotide with phosphorothioate linkages. The method enabled detection of the ASO at concentrations of 0.08-10 nM in aqueous samples. In addition, when combined with mixed-mode solid phase extraction for ASO spiked samples, the method detected 0.8-100 nM in serum and 0.04-25 nmol/g tissue in spinal tissue. The method was further applied to determine ASO levels in plasma and spinal tissue following intrathecal injection in mice. These results demonstrated that LDR using T4Rnl2 was a simple and sensitive approach for detecting ASOs in biological samples.

In laboratory mice, the H2-L[d] gene confers resistance to encephalitis upon infection with Toxoplasma gondii, a foodborne parasite capable of infecting all endotherm animals tested. H2-L[d] is carried by inbred strains with major histocompatibility complex (MHC) haplotypes H-2[a] (e.g., A/J) and H-2[d] (e.g., BALB/c), but not by those with haplotypes H-2[b] (e.g., C57BL/6) and H-2[k] (e.g., C3H/He). As H2-L[d] is typically detected by serological typing of ex vivo spleen samples, testing is usually terminal. Here we present a polymerase chain reaction (PCR)-based method to reliably detect the H2-L[d] gene in ear biopsies from live mice, thus allowing researchers to ascertain the H2-L[d] genotype for breeding or other in vivo procedures.

This protocol describes a methodology combining TurboID - a recently developed proximity biotinylation technique - with conventional epitope-tag based co-immunoprecipitation (Co-IP) to analyze protein-protein interactions (PPIs) in cell culture systems. This integrated approach allows the targeted examination of both transient and stable protein interactors, enhancing our understanding of protein dynamics. TurboID captures transient interactions often missed by Co-IP, which captures high affinity, stable interactors. Combination of both techniques enables direct comparison of interaction strengths, providing insights into the dynamic nature of protein interactions within cells. The rapid biotinylation capability of TurboID reduces background noise and false positives while Co-IP enriches stable interactors, together improving data quality and interpretation of the interactomic landscape. Demonstrating the efficacy of this methodology, proteins relevant to the pathology of Spinal Muscular Atrophy were utilized to explore variations at the interactome level. The use of identical starting cell lysates for both TurboID and Co-IP minimized variability and ensured datasets were comparable, allowing for consistency and enhancing the reliability of findings regarding the nature and strength of protein interactions. This novel framework effectively combines both innovative and classical techniques while maintaining consistency in sample handling, advancing our understanding of the intricate networks that govern cellular processes. Full data-set available at https://www.ebi.ac.uk/pride/ reference ID PXD066858.

Paraformaldehyde-fixed mouse liver samples are sectioned on a vibratome at 100 µm. The free-floating sections are permeabilized, followed by exposure to one of two heat-induced antigen retrieval methods selected based on the antigen of interest. Antigen retrieval is carried out in a mechanical stabilization apparatus developed here to prevent free-floating sections from deforming. The immunostaining is then carried out with noted modifications to previously established protocols.

Cell line stability of mammalian cell lines is an important concern for manufacturing of biopharmaceuticals. Characterization of the master cell bank (MCB) for monoclonal antibody A (Mab A) via multiplex fluorescence in situ hybridization (M-FISH) analysis identified two distinct karyotypic cell populations. As a result, a regulatory agency requested additional analysis to support a single clonal lineage of the MCB and demonstrate control of the two cell populations in subsequently derived cell banks. Next Generation Sequencing (NGS) revealed 11 transgene integration site sequences. A multiplex (measure two amplicons) Droplet Digital PCR (ddPCR) method was developed and characterized those 11 integration site copy numbers in the MCB. Analysis of 12 subclones from the MCB displayed that among 7 subclones there was loss of at least one of five specific integration sites. The characterization of these five unstable sites in the end production cells (EPCs) of four manufacturing batches are consistent with the MCB. The conservation of five other integration sites among all clones supported that these cells were derived from the same population and that loss of the unstable integration sites contributed to the two distinct karyotypic populations.

Long interspersed nuclear element-1 (LINE-1 or L1) is the only autonomously active retrotransposon in the human genome and produces both sense and antisense transcripts from its 5' untranslated region (5'UTR) of L1Hs, a human-specific L1 subfamily. Among these, ORF0 is an antisense transcript-derived protein implicated in retrotransposition activity, yet its mRNA expression has been difficult to quantify because strand discrimination is required for accurate detection. Here, we developed a strand-specific quantitative polymerase chain reaction (qPCR) method incorporating multiplex gene-specific tagged primers in the reverse transcription (RT) step, which enables simultaneous quantification of antisense ORF0 and sense 5'UTR and ORF2 transcripts. Validation in 5-aza-2'-deoxycytidine-treated neuroblastoma cells confirmed dose-dependent increases in expression of ORF0 and sense transcripts, demonstrating the strand specificity and functionality of this method. Application to human postmortem prefrontal cortex and cerebellum samples revealed stable expression of ORF0 and sense transcripts. This assay provides a robust and scalable tool for the precise quantification of strand-specific L1 transcription, complementing locus-specific analyses, and offering a platform for future studies on the role of ORF0 and L1 antisense transcription.

Introduction: Quantitative assessment of lung tissue architecture is essential for evaluating disease progression in experimental models of acute lung injury (ALI). However, conventional methods for measuring alveolar septum thickness rely on manual procedures that are time-consuming, labor-intensive, and, observer-dependent, compromises comparability across studies and reproducibility in translational biomedical research and drug development.Method: We present a novel, fully automated approach based on two-dimensional spatial autocorrelation function (2D-ACF) analysis to quantify septum thickness from hematoxylin and eosin (H&E)-stained lung sections.

Catalytic DNA molecules (DNAzymes) offer a programmable means to manipulate RNA without genomic modification. Although DNAzymes have been thoroughly characterized in culture and in various animals, their utility in the prevalent model system, Caenorhabditis elegans, has not been explored. Here, we examine the feasibility of catalytic DNA-mediated RNA targeting in C. elegans. Cholesterol-conjugated 8-17 DNAzymes were efficiently delivered to intestinal cells following ingestion, with no visibile toxic effects on growth or health, reducing targeted reporter fluorescence and endogenous gene function. The observed dependence on cholesterol tagging was consistent with a requirement for cellular uptake. Our findings provide a proof-of-principle demonstration of catalytic DNA activity in C. elegans, and highlight considerations for design and optimization. This exploratory study lays the foundation for expanding catalytic nucleic acid technologies in nematodes, supporting future development of programmable RNA tools in a powerful model system.

Antibiotic resistance genes (ARGs) are important contaminants in water systems, and their detection depends strongly on methodological sensitivity. This study compared three molecular platforms, high-throughput quantitative polymerase chain reaction (HT-qPCR), hydrolysis probe-based qPCR, and droplet digital PCR (ddPCR), for detecting ARGs in wastewater, river water, and seawater in Thailand. HT-qPCR enabled broad resistome profiling, detecting 325-336 ARGs out of 373 targets (87.1-90.1%), with aminoglycoside, beta-lactam, macrolide-lincosamide-streptogramin B, sulfonamide, mobile genetic element, and integron genes most prevalent. qPCR quantified selected ARGs using standard curves constructed from plasmid standards whose absolute copy numbers were calibrated by ddPCR, yielding high efficiency and strong linearity. ddPCR detected the same target genes as qPCR with comparable concentration estimates and additionally identified blaIND, which was not observed by HT-qPCR or qPCR. Quantifications from qPCR and ddPCR showed high rank-based concordance across matrices. Combining HT-qPCR with qPCR improved coverage by about 1% relative to HT-qPCR alone, while HT-qPCR with ddPCR offered nearly identical values. In conclusion, HT-qPCR was most effective for comprehensive profiling, qPCR ensured reliable quantification, and ddPCR enabled detection of rare targets, supporting a tiered and cost-effective framework for smart ARG monitoring in tropical aquatic environments.

The presence of 5-methylcytosine at CpG sites in mammalian DNA plays a significant role in various biological processes, both normal and aberrant. Similarly, CpG site methylation status of therapeutic DNAs can affect activity and therapeutic efficacy, as is the case for ADI-100, an immune tolerance-inducing drug candidate for the treatment of type 1 diabetes and other GAD-associated autoimmune diseases. To assess the methylation characteristics of ADI-100 and other therapeutic DNA product candidates, we developed a simple and versatile method, methylation-sensitive restriction enzyme digest and agarose gel electrophoresis (MSRE-AGE), using methylation-sensitive restriction enzyme digest, agarose gel electrophoresis, and custom in silico band pattern analysis for quantitative measurement of CpG methylation levels and patterns. We compared MSRE-AGE with bisulfite pyrosequencing methylation analysis of plasmid DNA and a synthetically-produced closed-linear DNA, Doggybone DNA (dbDNA), demonstrating that MSRE-AGE produces results consistent with bisulfite pyrosequencing but without structure and sequence-dependent artifacts. These findings demonstrate that MSRE-AGE is a robust and practical method for methylation analysis of therapeutic DNA products.

BackgroundLong single-stranded DNA (ssDNA; >200 nucleotides) is valuable for DNA nanotechnology, precision medicine, and as a CRISPR-Cas9 knock-in donor template, but existing preparation methods are laborious, low-yield, or difficult to scale. MethodsWe developed a workflow combining enzymatic digestion with high-temperature reversed-phase high-performance liquid chromatography (RP-HPLC) to purify kilobase-length ssDNA. The method was evaluated across analytical and semi-preparative formats. ResultsThe approach enables clean resolution of linear and circular ssDNA species ranging from 1.5 to 4.5 kb and is scalable across formats. A 1.5 kb ssDNA donor supported efficient CRISPR knock-in at the T-cell receptor alpha constant (TRAC) locus in primary human CD8⁺ T cells, without adversely affecting viability or expansion. ConclusionsThis RP-HPLC workflow provides a scalable and reproducible method for generating high-purity long ssDNA suitable for genome engineering applications.