BioTechniques

Assessing reproducibility across different molecular profiling studies is a persistent methodological challenge (Zhang et al., 2009; Sweeney et al., 2017; Ioannidis, 2005). Differences in platform technology, cohort composition, analytical pipelines, and feature definitions often make it difficult to interpret cross-study comparisons based solely on gene-identity overlap. In this study, we conducted a retrospective computational analysis of seven publicly available analytical datasets (including alternative analytical pipelines applied to the same cohort) derived from five biologically independent peripheral blood transcriptomic and DNA methylation cohorts, comprising 3,487 samples (1,824 Parkinsons disease cases and 1,663 controls). Reproducibility was evaluated using gene-identity overlap, enrichment-based comparisons, and a permutation-based framework to assess directional consistency of effect estimates across datasets. We also tested the robustness of results by varying false discovery rate thresholds and applying alternative probe-to-gene collapsing strategies. All analyses were performed using reproducible workflows implemented in R and Python with fixed random seeds. Across independent cohorts, gene-identity overlap was generally limited, with enrichment ratios close to one, especially when datasets were generated using different platforms. In several datasets, limited numbers of statistically significant features further constrained overlap-based comparisons. In contrast, directional consistency showed greater stability. High levels of directional consistency were observed across independent cohort comparisons when restricted to overlapping statistically significant features and remained stable across statistical thresholds (90.0% at FDR < 0.05 and 82.8% at FDR < 0.10). When evaluated across the full shared gene universe without conditioning on statistical significance, directional consistency was substantially lower ([~]30 to 32%) but remained significantly above permutation-based null expectations. Permutation testing confirmed that the observed directional consistency exceeded what would be expected by chance. A combined analysis including methodological replicates (n [&ge;] 3 datasets) showed 98.3% directional consistency; however, this estimate includes non-independent analytical pipelines applied to the same cohort and reflects analytical stability rather than independent biological replication. Rather than introducing a new statistical method, this study examines how commonly used reproducibility metrics behave under crossstudy heterogeneity and identifies their practical limitations and appropriate use boundaries.

Coenzyme A is an essential cofactor synthesized from pantothenate, cysteine, and ATP, and is involved in numerous processes of cellular metabolism through its ability to carry activated acyl groups. Coenzyme A participates in catabolism of carbohydrate, fat and amino acids; biosynthesis of fatty acids, cholesterol and heme; and protein modification including acetylation and 4-phosphopantetheinylation. Despite CoAs critical functions, the regulation of CoA levels and the rate of CoA synthesis in different cell types and disease states are not well understood. One reason for this gap is that many acyl-CoA species are analytically challenging to measure due to factors including instability, poor ionization, and the wide range of biochemical properties conferred by different acyl chain lengths. In addition, most current methods do not support analysis of CoA isotopic labeling, which is required to quantify CoA synthesis rate or to measure absolute concentration using isotope-labeled internal standards. Here, we describe a method to quantify the concentration and isotopic labeling of total CoA, defined as the sum of CoASH plus all acyl-CoA species. Acyl-CoA species are hydrolyzed using sodium hydroxide to remove acyl chains, then CoA is derivatized on the thiol with N-ethylmaleimide (NEM). Following protein precipitation and solid phase extraction, samples are analyzed by liquid chromatography-mass spectrometry. This method is linear in a wide range that captures mouse tissue CoA levels, with accuracy within 15% error and precision below 15% relative standard deviation for both pure standards and tissue samples. We applied this method to measure total CoA concentration in five tissues from male and female mice, and total CoA synthesis rate in mouse liver via infusion of 13C-15N-pantothenate. Overall, this method offers a tractable approach to measure total CoA concentration and isotopic labeling to enable study of total CoA synthesis rates and concentrations in health and disease.

Terrestrial environmental DNA (eDNA) approaches are rapidly expanding, yet robust, field-ready substrates for detecting insect DNA remain limited in forest ecosystems. Tree sap is a localized microhabitat that attracts diverse insects and may provide a useful substrate for surface eDNA sampling, but its potential for insect monitoring has rarely been evaluated. Here, we present a pilot proof-of-concept study testing naturally exuding tree sap and sap-mimicking traps as terrestrial eDNA substrates. We collected swab samples from sap and trap surfaces at two forest sites in Japan (Fujisawa and Minamisanriku) and performed metabarcoding using COI and an arthropod-focused 16S marker (gInsect). Reads were processed into amplicon sequence variants and assigned by BLAST top hits against NCBI nt, with high-confidence detections defined at identity [&ge;]98%. Across sites, sap and trap swabs yielded multiple high-confidence insect detections spanning several orders, including sap-associated stag beetles (Dorcus spp.). Overlap with contemporaneous conventional monitoring was limited, suggesting that sap-surface eDNA and conventional surveys capture partly different components of sap-associated insect assemblages. In a targeted 2024 spot survey, actively fermenting sap yielded multiple insect eDNA detections, whereas inactive, non-fermented sap yielded no high-confidence insect detections. Although limited by small sample size and the absence of dedicated process controls, these findings support the feasibility of tree sap as a localized terrestrial eDNA substrate and provide a basis for future replicated studies of sap-associated insect monitoring.

Osteocytes play a central role in bone remodeling, mineral metabolism, and skeletal homeostasis, but direct molecular analysis of human osteocytes remains technically challenging because they are embedded within the mineralized bone matrix. Surgically obtained human bone specimens provide valuable material for studying human bone biology; however, surface-associated cells, marrow-derived cells, and adherent soft tissues can confound downstream transcript analysis. Here, we describe a bone fragment-based protocol for preparing surgically obtained human bone specimens for molecular analysis of osteocyte-associated transcripts. The protocol consists of mechanical trimming, mincing into small bone fragments, repeated washing, and five sequential rounds of collagenase digestion to reduce non-osteocytic cellular components associated with the bone surface and marrow spaces. The remaining mineralized bone fragments are then frozen in liquid nitrogen, cryogenically pulverized, and lysed in TRIzol reagent for total RNA extraction. Histological validation using residual maxillary bone specimens showed that sequential collagenase digestion markedly reduced adherent soft tissue and extra-matrix nuclei while preserving osteocyte lacunar occupancy. This protocol provides a practical workflow for bone fragment-based RNA analysis focused on osteocyte-associated transcripts in human bone specimens. Specifications table O_TBL View this table: org.highwire.dtl.DTLVardef@1cec618org.highwire.dtl.DTLVardef@2f746forg.highwire.dtl.DTLVardef@1854247org.highwire.dtl.DTLVardef@1c26c1aorg.highwire.dtl.DTLVardef@1473a88_HPS_FORMAT_FIGEXP M_TBL C_TBL

Background: Surgical site infection (SSI) is the leading cause of perioperative morbidity following oral cancer surgery, yet the role of the oral microbiota in SSI pathogenesis remains poorly defined. This study prospectively investigated microbiota dynamics in relation to SSI occurrence in patients undergoing resection for oral squamous cell carcinoma (OSCC). Methods: A total of 172 oral swab samples were collected from 45 OSCC patients across four longitudinal time points: baseline (~29 days pre-surgery), immediately pre-surgery (hospital admission), early post-surgery (within 5 days), and late post-surgery (6 to15 days). Bacterial composition was profiled by 16S-rDNA V3-V4 sequencing (172 successfully sequenced samples), and bacterial/human DNA ratios were quantified by qRT-PCR (170 samples evaluated). SSI was assessed within 30 days post-surgery using adapted CDC criteria. Results: Fourteen of 45 patients (31.1%) developed SSI. Younger age was significantly associated with SSI occurrence (median age 53.2 years in SSI group vs. 67.4 years in non-SSI group; p=0.011), with each one-year decrease in age conferring a 7% increased risk. Notably, younger patients presented with larger and more advanced tumors (T3/T4: median age 57.2 vs. 72.9 years for T1/T2; p=0.033), leading to more extensive surgical procedures. Across all 172 samples, surgery induced a marked post-operative reduction in bacterial load and diversity. However, at the late post-surgery time point (collection IV), patients with SSI exhibited significantly higher alpha-diversity compared to non-infected patients (p<0.05 for Observed, Shannon, and Simpson indices). Beta-diversity also differed significantly between groups at this time point (weighted UniFrac, p=0.043). Prevotella and Porphyromonas dominated SSI patients at infection, together accounting for ~40% of reads versus 9.5% in non-infected patients. Among the 172 samples analyzed longitudinally, Aggregatibacter abundance at the early post-surgery time point (collection III) emerged as a significant predictor of subsequent SSI (OR per 1% increase: 1.10; p=0.012), with frequencies >0.044% conferring a 5.7-fold higher risk. Conclusions: Our longitudinal analysis demonstrate that while OSCC surgery profoundly disrupts the oral microbiota, non-SSI patients restore their preoperative profile within 12 days. In contrast, SSI is characterized by persistent dysbiosis dominated by Prevotella and Porphyromonas. Younger patients with advanced tumors are at particular risk. Early post-surgical Aggregatibacter abundance may serve as a novel risk indicator for SSI, potentially enabling timely preventive interventions in high-risk patients.

Double-stranded RNA (dsRNA) is recognized by cellular receptors as a sign of viral infection, triggering the innate immune response. Increasing evidence shows that cellular dysregulation, for example in immune disorders and neurodegenerative diseases, can also lead to accumulation of endogenously produced dsRNA that stimulates a viral-like immune response. Additionally, dsRNA contamination in RNA therapeutics can lead to harmful side effects via a similar pathway. Despite the clinical relevance of dsRNA, reliable tools for its detection remain limited. At present, dsRNA detection relies almost exclusively on the monoclonal antibodies J2 and K1, which suffer from sequence bias and low sensitivity, limiting their reliability. To address this challenge, we aimed to repurpose naturally occurring dsRNA-binding domains (dsRBDs) to produce reliable, pan-specific affinity reagents for dsRNA. We first systematically screened the dsRBDs of the three human adenosine deaminases acting on RNA (ADARs). This analysis identified ADAR3 dsRBDs as promising candidates due to their reduced sequence dependence compared to the dsRBDs of ADAR1 and ADAR2. We then engineered ADAR3-derived dsRBD constructs having varying linker lengths and domain combinations, allowing us to specifically vary the length cutoff of dsRNA detected, thus creating dsRNA accumulation detected by ADAR3 RBDs (dsRADAR) affinity reagents. Finally, we demonstrate the superior performance of dsRADAR over currently available dsRNA antibodies in a cell model of viral infection and a tissue model of gastric inflammation. Together, dsRADAR provides a sensitive and reliable approach for imaging and quantifying diverse dsRNA structures in a variety of biological contexts. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/724404v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1d89c30org.highwire.dtl.DTLVardef@1f64fc1org.highwire.dtl.DTLVardef@1ee391forg.highwire.dtl.DTLVardef@e834a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Citrus yellow vein clearing virus (CYVCV) is the causal agent of an emerging disease representing a potentially high-impact threat for citrus production. Despite remaining outside Europe for decades, CYVCV has now expanded towards two important European citrus producers, Italy and, more recently, Spain. The presence of this virus in the EPPO region represents a current threat with unpredictable and potentially devastating consequences for European citriculture. Therefore, urgent protective measures need to be taken to prevent CYVCV spread and minimize its impact. Diagnostics is a key measure in the management of viral diseases, highlighting the need for harmonized methods suitable for reliable routine detection of the currently known CYVCV diversity. In this study, an inclusive, efficient and highly sensitive real-time RT-qPCR for the detection of CYVCV in plant material and transmission vectors has been developed and validated according to EPPO standards. Moreover, the validated method has been successfully adapted to both PCR digital platforms, that allow high-sensitive absolute quantitative detection, essential in the diagnostics at low viral concentrations; and PCR portable tools, that can be applied in a real diagnostic context for on-site detection. This versatility combines standard validated performance, absolute sensitive quantitation and real on-site detection. The study has also addressed sampling strategies to support reliable molecular diagnostic performance. Our results represent an improvement in the detection of CYVCV to be applied in epidemiological studies and different real diagnostic contexts for the containment of this important citrus pathogen.

PurposeCarrier screening for hereditary conditions is challenged by genes with complex genomic architecture, where short-read sequencing can fail to detect clinically relevant variants. This study evaluated a unified, amplification-based nanopore sequencing workflow across multiple laboratories for comprehensive analysis of such loci. MethodsA modular long-read sequencing assay was evaluated across five laboratories using targeted PCR enrichment, Oxford Nanopore sequencing, and automated variant analysis. The workflow interrogated genes associated with spinal muscular atrophy, thalassemia, cystic fibrosis, fragile X syndrome, congenital adrenal hyperplasia, Gaucher disease, and hemophilia A. Performance was assessed against orthogonal methods for single nucleotide variants (SNVs), indels, copy-number variants, repeat expansions, and structural rearrangements. ResultsAcross 882 unique samples (1,266 tests), overall agreement with comparator methods exceeded 96% for variant-level detection and 97% for genotype status classification. Long-read sequencing enabled phasing of paralogous loci, integrated sizing and interruption analysis for FMR1 repeats, and simultaneous detection of SNVs and structural variants in globin loci and CYP21A2-TNXB region, reducing reliance on multiple workflows. ConclusionThis multisite evaluation suggests that targeted long-read sequencing can consolidate complex variant detection into a single workflow, improving analytical completeness and operational efficiency for carrier screening.

BackgroundDirect conversion of human somatic cells into functional neurons could offer a faster way to generate patient-specific neurons for use in regenerative medicine, disease modelling, and drug development. Although it has been reported that neuronal direct reprogramming bypasses the intermediate pluripotent state, no reports have included time-lapse experiments, potentially overlooking transient intermediate states. Recent studies have shown that the conversion of human mesenchymal stromal cells (hMSCs) into neuron-like cells involves a transition through a transient intermediate state. Therefore, further research is needed to fully understand the process by which human somatic cells can become neurons without cell division. In this study we investigates whether direct neuronal reprogramming of human bone marrow-derived MSC (hBM-MSCs), dental pulp-derived MSC (hDP-MSCs), and adult human dermal fibroblasts (HDFa), involves a transient intermediate state, and sought to further validate the neuronal identity of hMSC-derived induced neurons. MethodsIn this study, we conducted time-lapse experiments to observe the transformation of hBM-MSCs, hDP-MSCs and HDFa, into neurons using a small-molecule-based direct reprogramming protocol. Cellular and ultrastructural changes were further characterized by confocal and electron microscopy. ResultsDirect conversion of hBM-MSCs, hDP-MSCs and HDFa into neuron-like cells occurred rapidly and in absence of cell division. Time-lapse analyses revealed that reprogramming proceeds through a transient intermediate state characterized by distinct morphological changes and dynamic nuclear remodelling. Furthermore, we found that neuron-like cells derived from hBM-MSCs and hDP-MSCs exhibit neuronal polarization, expressed specific neuronal and synaptic markers, formed interconnected cellular networks, and exhibited functional plasticity, providing further evidence that hMSCs can become functional neurons. ConclusionsThis study provides clear evidence that the direct neuronal reprogramming process involves a transition through an intermediate, transient state. Our findings also provide further evidence that hMSCs can become functional neurons. In summary, our work provides new insights into the direct neuronal reprogramming process, which is essential for advancing both developmental biology and regenerative medicine.

Freshwater algae represent an underexplored source of naturally occurring bioactive metabolites with potential applications in pharmaceutical and biomedical research. This study investigated the phytochemical composition, antioxidant capacity, and preliminary cytotoxic potential of ethanolic and n-hexane extracts of freshwater algal species collected at Jilani Park, Lahore, Pakistan. Algal species were identified morphologically by Dr. Ghazal Yasmeen (Institute of Botany, Punjab University, Lahore). Extracts were analyzed using gas chromatography-mass spectrometry (GC-MS) and qualitative phytochemical screening. Antioxidant activity was evaluated using DPPH radical scavenging, hydrogen peroxide scavenging, and reducing power assays. Cytotoxic potential was assessed using MTT and cell adhesion assays on HeLa and SF767 cell lines as preliminary indicators of bioactivity. GC-MS analysis identified 25 compounds, including sterols, fatty acid esters, terpenoids, phenolic compounds, and volatile metabolites. Phytochemical screening confirmed the presence of flavonoids, phenolics, tannins, and terpenoids in the extracts. Among the tested extracts, the n-hexane fraction demonstrated comparatively higher antioxidant activity across multiple assays. Ethanolic extracts showed moderate reductions in HeLa cell viability, whereas limited effects were observed in SF767 cells. These findings suggest that freshwater algae are promising natural reservoirs of antioxidant metabolites with potential relevance for future isolation and characterization of bioactive compounds for biomedical applications. Further purification and mechanistic studies are required to identify specific active constituents.

This work aimed to establish a translationally viable, xeno-free, serum-free platform and protocol for the isolation and expansion of human salivary stem/progenitor cells (hS/PCs) suitable for regulatory qualification and future FDA-approved first-in-human autologous regenerative therapy trials for the treatment of hyposalivation disorders. Parotid gland specimens from non-cancerous regions/tissues were collected from consented surgical patients. Primary hS/PCs were isolated from tissue specimens, cultured in animal-component-free conditions, expanded to produce millions of cells, then enriched for CD44+ stem/progenitor cells by magnetic cell sorting. Normal epithelial purity was assessed using cytokeratins 5/14. Anti-CD133/PROM1 (cancer marker) and anti- fibroblast (clone TE-7) antibodies were used to demonstrate a lack of contaminating cells. Phenotype validation was performed by flow cytometry and immunocytochemistry on both CD44+ sorted and unsorted populations. Senescence-associated beta-galactosidase (SA-{beta}-gal) assays were performed across serial passages (P1-P6). Pluripotency was demonstrated by culture under conditions supporting lineage-specific differentiation. Primary hS/PCs demonstrated consistent expansion and epithelial morphology under serum-free conditions. CD44 expression remained high (>95%) throughout expansion, with negligible detection of CD133 or fibroblast markers, confirming epithelial purity and absence of tumorigenic or stromal contamination. Immunocytochemistry corroborated these expression profiles. SA-{beta}-gal staining revealed only a minor, passage-dependent increase (5-16%) in senescent cells from multiple donors, indicating retention of proliferative potential. Our defined, animal-free culture system supports stable expansion of pure low passage hS/PCs under conditions compatible with good manufacturing practice (GMP).

Variants affecting RNA splicing are a major contributor to human disease, yet the consequences of variants outside of the canonical splice motifs are often difficult to determine. Here, we present a protocol for minigene-based evaluation of candidate splice-altering variants. The methodology described includes locus-specific insert design, commercial gene fragment synthesis, and long-read sequencing. The combined approach enables rapid assay development and nucleotide level resolution of the effect on splice isoforms in vitro, providing a scalable framework for functional validation of predicted cryptic splice variants. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/723105v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1a88cb5org.highwire.dtl.DTLVardef@adda98org.highwire.dtl.DTLVardef@1ea587corg.highwire.dtl.DTLVardef@574a63_HPS_FORMAT_FIGEXP M_FIG C_FIG

1Sex steroid hormones are not exclusively localised in the circulation and can be found in numerous extragonadal tissues, in concentrations unrelated to the circulating fraction. Existing methodology to measure intramuscular steroid hormone concentrations includes both immune-based assays and liquid chromatography-mass spectrometry (LC-MS), the gold standard for hormone measurements. To date, no LC-MS based methods validation has been published on the measurement of intramuscular sex steroid hormones, despite clear biological relevance. Here, we describe the development and validation of a simple, high-throughput LC-MS Orbitrap method for the measurement of 10 intramuscular sex steroid hormones, including pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, epitestosterone, dihydrotestosterone, oestrone, oestradiol, and oestriol. In brief, isotope labelled standards were added to 5-6 milligrams of lyophilised muscle tissue, homogenised and extracted with ethyl acetate. The extracts were dried down and sequentially derivatised with 1-methylimidazole-2-sulfonyl chloride and hydroxylamine hydrochloride to target both the phenolic hydroxyl groups and ketone groups. The limit of detection was 1.0 {+/-} 1.0 pg/mg (range 0.36 - 3.26 pg/mg), with a R2 > 0.99 for all analytes. Matrix effects were 90-110% for all analytes except for dihydrotestosterone (143.6%), and precision was <10 CV% for all analytes in the presence of a muscle matrix. Our method allows for 20-40 samples to be prepared in [~]4 h, with a sample data acquisition time of 13 minutes. Moreover, our method provides the opportunity for specific analysis of steroid hormone concentrations in skeletal muscle, allowing target tissue specificity instead of relying on proxy measures from the circulation.

The 16S rRNA gene is the most widely used genetic marker for microbial community profiling, but its limited sequence divergence often prevents species-level identification. The RNA polymerase {beta}-subunit gene (rpoB) offers higher sequence variability, single-copy occurrence, and stronger phylogenetic consistency, yet its adoption in metataxonomic studies has been constrained by the lack of universal primer sets. Here, we present a novel universal primer pair that amplifies an [~]1,800 bp rpoB region (rpoB_MV) compatible with long-read sequencing platforms. In silico evaluation across 17683 bacterial reference genomes demonstrated high universality, with over 86% of genomes predicted to amplify. Compared with full-length and partial 16S rRNA gene markers, the rpoB_MV amplicon exhibited significantly greater inter-species sequence divergence and improved phylogenetic concordance with core-genome trees. Sequencing of two complementary mock communities confirmed superior species-level identification accuracy, with misclassification rates below 0.01% and no reads assigned to unresolved species clusters. These results establish rpoB_MV as a robust alternative to 16S rRNA gene-based profiling for high-resolution metataxonomic applications. IMPORTANCEMicrobial community studies increasingly require species-level resolution because species within the same genus can differ substantially in pathogenicity, ecological function, and metabolic capacity. Current 16S rRNA gene-based methods frequently fail to distinguish closely related species, collapsing biologically distinct organisms into the same taxonomic assignment and obscuring community differences that matter for clinical diagnostics, food safety, and environmental monitoring. The rpoB_MV primer pair presented here overcomes this limitation by targeting a longer, more variable region of the rpoB gene, enabling accurate species-level identification across diverse bacterial phyla. Combined with advances in long-read sequencing, this approach provides researchers with a practical tool to resolve microbial communities at the species-level.

The sunflower sea star (Pycnopodia helianthoides) suffered a catastrophic population decline across its range from 2013 to 2017 due to the devastating Vibrio pectenicida FHCF-3 driven sea star wasting disease (SSWD) pandemic with minimal signs of population recovery. The functional extinction of this apex predator across substantial parts of its range has created a need to identify and track the remaining intact populations. Environmental DNA (eDNA) approaches provide a simple, cost-effective, and non-destructive method for monitoring occurrences, and in some cases abundances, of marine species, consistently outperforming visual occurrence monitoring efforts in sensitivity, speed, and cost. Here, we designed, developed, and validated a P. helianthoides-specific eDNA assay to identify refugia, using both quantitative and digital droplet PCR approaches. We first generated the most comprehensive sea star mitochondrial genome reference database to date (n=93 taxa, n= 15 novel). We then used unikseq and Geneious bioinformatics software to identify the unique nad5 gene region and design a highly specific hydrolysis probe-based PCR assay. We validated the performance of this assay through laboratory, mesocosm, and field testing, demonstrating a highly specific and sensitive assay. In a field application of the new assay across regions in British Columbia, Canada, we found a positive correlation between P. helianthoides eDNA concentrations and biomass density, especially when appropriately accounting for spatiotemporal integration scales (R2=0.67). The eDNA assay provides a rapid and scalable tool for monitoring the sunflower sea star which has been proposed for listing as threatened under the U.S. Endangered Species Act of 1973. Molecular tools like the one presented here enhance management and recovery efforts not only by identification and monitoring of remnant wild populations, but also by helping to assess population level response and recovery following reintroduction efforts.

How gene expression patterns change spatially as the embryo transitions from simple to complex structures remains a major developmental biology question. Recently developed imaging-based spatial transcriptomics (ST) enable mapping expression of multiple gene at a single-cell resolution. Although Xenopus is a key model in embryology there is no established ST pipeline, and commercially available techniques face many challenges (sample preparation, probe design, cell segmentation). Furthermore, the highly diverse cell shapes and sizes across developmental stages and between different tissues represent major hurdles to accurately defining cells. Here, we describe an optimized workflow for ST in blastula-to-tailbud-stage frog embryos using Merscope, commercial MERFISH (Multiplexed Error-Robust Fluorescence In Situ Hybridization) originally designed for standard mammalian tissues. With stringent quality control and tailored computational pipelines, we optimize this technology for robust, semi-quantitative profiling of spatial transcriptomic landscapes in non-mammalian embryos. Reliable tissue preservation and cell-segmentation enable high-resolution mapping of gene expression during the development of a complex multi-tissue organization. This versatile strategy applies broadly to various dynamic systems, from embryos of various model organisms to complex and heterogeneous organs in mammals. Summary statementThis Single-cell Spatial Transcriptomics pipeline and reference atlas in Xenopus - a model organism in embryology - overcome technical challenges and resolve dynamic changes in patterning during development.

Measles virus remains a significant global health threat, and despite the availability of an effective vaccine, measles cases continue to increase worldwide in recent years. Genomic surveillance has become an essential tool for monitoring virus circulation and investigating outbreaks. Here, we describe a wet-laboratory method for whole-genome sequencing of measles virus using a tiled amplicon approach and Illumina sequencing technology. A previously published Oxford Nanopore-based tiled primer scheme was adapted to include both circulating measles genotypes and for use on the Illumina platform. Two Illumina library preparation kits, Illumina DNA Prep (IDP) and Nextera XT (XT), were evaluated for performance. The IDP kit demonstrated more complete genomes and consistent genome coverage compared with XT. Using quantified reference genomes, the limit of detection was determined to be 10,000 genome copies for genotype B3 and D8. Sequence accuracy was evaluated using previously characterized clinical samples and showed high concordance. This method provides a reliable and sensitive approach for measles virus whole-genome sequencing using Illumina platforms and is suitable for genomic surveillance applications.

As protein structure prediction tools become widely adopted across biology, there is a growing need for accessible methods to assess and visualize predicted protein-protein interactions (PPIs). Here we present LIVIA (Local Interaction Visualization and Analysis), a browser-based tool that computes local PPI confidence metrics across multiple prediction platforms, identifies predicted interface residues, embeds an interactive Mol* 3D viewer, and generates visualization scripts for ChimeraX and PyMOL. The tool automatically detects prediction formats; all parsing and computation occur locally on the users machine. LIVIA is freely available at https://flyark.github.io/LIVIA.

CRISPR-Cas genome editing toolkits have expanded the scope of genetic studies in various emerging model organisms. However, their applications are limited mainly to knockout experiments due to technical difficulties in establishing knock-in strains, which enable in vivo molecular tagging-based experiments. Here, we investigated knock-in strategies in the harlequin ladybug Harmonia axyridis, a model insect for evolutionary developmental biology, which shows more than 200 color pattern variations within a species. We tested several knock-in strategies using synthetic DNA templates. We found that ssDNA templates generated founder knock-in strains efficiently (2.5-11%), whereas the 5 regions of ssDNA templates were frequently deleted when the insert length exceeded [~]40 bases. To overcome this limitation, we designed several 3 extended DNA templates. Fast-annealed 3-extended double-stranded DNA templates, which were designed for tagging endogenous proteins with epitope tags, showed high founder generation efficiency (9.9-20.9%) and accuracy (30.8-85.7%). This strategy is also applicable to the two-spotted cricket Gryllus bimaculatus, suggesting that the fast-annealed 3-extended dsDNA template is a versatile DNA template for generating knock-in strains in emerging model insects for developmental genetic studies. Summary statementFast-annealed 3-extended dsDNA templates facilitate efficient CRISPR-Cas9-mediated knock-in in emerging model insects.

Chlamydomonas reinhardtii is a model green alga extensively used to study photosynthesis and cilia using molecular biology and genetics. Electroporation is a very common technique to transform DNA into the nuclear genome, which is essential to generate mutant collections and express transgenes. Here, we describe a simple, fast, and efficient protocol to transform strains with an intact cell wall. It achieves a good transformation efficiency without cell wall digestion or use of commercial kits and is compatible with the widely available Gene Pulser electroporation system. Key featuresO_LIHigh transformation efficiency of Chlamydomonas reinhardtii strains with an intact cell wall. C_LIO_LIFaster than currently available electroporation protocols. C_LI