Current Protocols

Efficient protein synthesis in eukaryotic cells typically requires a 5' cap structure on messenger RNAs (mRNAs). However, under stress conditions or in viral infection, translation can also occur independently of the cap via internal ribosomal entry sites (IRES). IRES elements are therefore key regulators of protein expression in both viral and cellular contexts. Here we describe a cell-free protocol to quantitatively assess IRES-mediated translation using wheat germ extract (WGE) and a firefly luciferase (FLuc) reporter. The protocol includes template preparation, RNA synthesis and luminescence measurement following in vitro translation in WGE. This method enables rapid and robust comparison of IRES activity under controlled conditions and can additionally be applied to evaluate mRNA modifications designed to enhance translation efficiency. Key featuresO_LIStringent in vitro workflow from DNA template preparation through RNA synthesis and protein synthesis to reporter readout, including quality controls. C_LIO_LIEvaluation of IRES-driven translation suitable for testing combinations of IRES and CDS. C_LIO_LItranslation analysis without radioactive labeling. C_LI Graphical overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/716985v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1457b00org.highwire.dtl.DTLVardef@8e7405org.highwire.dtl.DTLVardef@6303eforg.highwire.dtl.DTLVardef@974d71_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical AbstractPipeline for the production and evaluation of IRES-firefly luciferase constructs using wheat germ extract. (1-4) Preparation: IRES-firefly luciferase constructs are amplified in E. coli and isolated from bacterial cells. Plasmids are linearized to prepare for in vitro transcription. (5-6) Transcript synthesis and verification: In vitro transcription is followed by electrophoretic validation to confirm integrity and correct molecular weight. (7-8) Translation and detection: Translation is executed in wheat germ extract and quantified by measuring reporter activity in a luminometer.

RNase R is a processive 3 to 5 exoribonuclease that degrades a broad array of linear RNA species while preserving RNA lariats and circular RNAs (circRNA). In recent years, this enzyme has become pivotal for the field of circRNA research, serving as a key step for circRNA enrichment, purification, and identification. Despite this growing importance, the effects of mutations and truncations in RNase R have been incompletely studied. We make several point mutations and assay their effects on the ability of RNase R to bind and/or degrade RNA substrates. Our data show that selected active site mutations have varying effects on RNA binding and degradation. Furthermore, the increasing interest in circRNA-based RNA therapeutic platforms highlights an urgent need for RNase R in RNA molecular biology labs. However, the substantial cost of commercial RNase R remains a bottleneck, particularly for large-scale studies or the development of circRNA-based technologies. In this protocol, we offer a solution to that problem, namely a more accessible and cost-effective means of purifying high-quality and low-cost RNase R. We provide a highly detailed yet simplified, high-yield protocol that produces recombinant RNase R from Escherichia coli. The method uses a single-step Ni-NTA affinity chromatography procedure without proteolytic tag removal and is optimized for entry-level FPLC systems such as the AKTA Start, ensuring that high-purity enzyme production does not require specialized, high-end instrumentation. A second key feature is the establishment of an optimized reaction framework, including specific buffer compositions and defined enzyme-to-substrate ratios for the purified RNase R. The protocol achieves functional equivalence to premium commercial RNase R, ensuring complete linear RNA digestion without compromising the integrity of circRNA. The combination of a simplified purification workflow and a robust reaction protocol provides an accessible, cost-effective, and reliable solution for any molecular biology laboratory requiring high volumes of RNase R. Key FeaturesO_LIRNase R mutations can block RNase activity, RNA binding or both C_LIO_LIThis protocol purifies [~]40 mg of active RNase R per liter of E. coli culture C_LIO_LIThe protocol avoids medium and high end FPLC systems C_LIO_LIRNase R expression constructs (WT and mutants) will be available on Addgene C_LIO_LIThe protocol includes an optimized reaction buffer to pair with this RNase R C_LIO_LIOptional endotoxin removal step is also included C_LI

MOTIVATIONAdult cardiomyocytes are difficult to profile by whole-cell single-cell RNA sequencing because of their large size and fragility, which make them poorly compatible with standard workflows. Current approaches for adult cardiomyocyte transcriptomics often require a trade-off between data quality and throughput, thus, studies instead rely heavily on sequencing of nuclei alone. Therefore, we set out to develop a high-quality and scalable workflow for adult heart cells using in-cell ligation and split-pool barcoding strategies to address this methodological gap. This workflow may be further generalisable to other large cell types or samples containing cell populations with highly unequal RNA content. SUMMARYAdult cardiomyocytes are difficult to profile by whole-cell single-cell RNA sequencing (scRNA-seq). Here, we developed a high-quality and scalable workflow for adult heart cells using in-cell ligation and split-pool barcoding. We identified per-cell RNA content as a significant variable that must be accounted for. Separation of cardiomyocytes (large cells) and non-cardiomyocytes (small cells) before library construction, and allocation of deeper sequencing to cardiomyocytes, produced high-quality whole-cell datasets for both compartments. Compared with single-nucleus RNA sequencing, whole-cell cardiomyocyte profiling better recovered metabolic, mitochondrial, cytoplasmic translational, and contractile gene programs. This workflow provides a practical method for scalable, high-quality cardiomyocyte whole-cell scRNA-seq and offers general strategies for other large cell types or samples containing cell populations with highly unequal RNA content.

This protocol enables rapid CRISPR-Cas9 genome editing in Saccharomyces cerevisiae by replacing restriction/ligation guide cloning with PCR-based protospacer installation and seamless plasmid recircularization. It describes in silico HDR donor and SgRNA design, install guide sequences into cas9 plasmid by PCR and seamless assembly, plasmid cloning and sequence verification in E. coli, and LiAc/PEG co-transformation of yeast with Cas9-sgRNA plasmid plus HDR donor. The workflow selects yeast colonies on G418 and confirms edits by PCR and sequencing.

Currently there is a lack of high-throughput, low material-input methods to screen early-stage product quality of viral and non-viral gene therapy products. Here we propose using multiplex droplet digital PCR (dPCR) to screen and characterize vector sequences. We describe the adaptation of a Poisson-multinomial model to quantitate integrity of any combination of 4 targets in multiplexed ddPCR. We show the success and limitations of model employment and provide some suggested best practices.

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

Genetically encoded biosensors are GFP-based tools that can visualize the dynamics and spatial features of cellular processes. The design of a genetically encoded biosensor dictates the method that is used to measure the response. Common read-outs use some sort of fluorescence intensity measurement, which is subject to both technical and biological perturbations, including sample drift, excitation power fluctuations, changes in sample size/volume, or a change in expression level. Yet, the fluorescence lifetime of a fluorophore is not affected by the aforementioned perturbations. Therefore, biosensors that respond with a large lifetime change offer a more robust method of detecting cellular processes. Here, we report on protocols for calcium imaging using fluorescence lifetime imaging microscopy (FLIM) to measure the response of a genetically encoded lifetime biosensor. The protocols include details on biosensor production and purification, calibration of purified biosensor with FLIM, introduction of the plasmid in HeLa and endothelial cells, and timelapse analysis of FLIM data. In this chapter we use the green fluorescent biosensor G-Ca-FLITS as an example but the protocols can be generally applied to biosensors with lifetime contrast. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/717680v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@167f612org.highwire.dtl.DTLVardef@4c5603org.highwire.dtl.DTLVardef@1a2eb6borg.highwire.dtl.DTLVardef@10ddc63_HPS_FORMAT_FIGEXP M_FIG C_FIG

The translation of mRNA into protein is tightly regulated by both cellular trans-factors and cis-regulatory elements encoded within transcripts. Although transcript fate can be measured by transcript abundance or translation efficiency, separating the contribution of each individual cis-element within a single transcript is an ongoing challenge. Current massively parallel reporter assay (MPRAs) approaches enable systematic interrogation of cis-regulatory elements that control transcript stability, but translation-focused MPRAs remain technically limited and often inaccessible. Here we present Nascent Peptide Translating Ribosome Affinity Purification (NaP-TRAP), a reporter-based approach that simultaneously measures translation and mRNA abundance. Unlike previous methods, NaP-TRAP captures translation directly through the immunoprecipitation of epitope-tagged nascent peptide chains, providing instantaneous, frame-specific readouts without specialized instrumentation. The method is highly scalable from single reporters to complex libraries, and adaptable across in vivo and in vitro systems. NaP-TRAP is versatile, allowing assessment of cis-regulatory impact of elements distributed throughout the mRNA, from cap-to-tail. This protocol covers experimental design, reporter construction, sample processing, and computational analysis for both low- and high-throughput applications. Bench work can be completed in 4- 5 days, with qPCR-based readouts requiring only basic Excel skills for data processing. Sequencing-based readouts require skills in command-line tools and Python scripting and add an additional 2-3 days. NaP-TRAP thus offers an accessible, robust, and quantitative platform to decode the regulatory logic of mRNA translation and stability in diverse biological contexts. Basic Protocol 1Design, assembly, and synthesis of NaP-TRAP reporter libraries. Support Protocol 1Design, assembly, and synthesis of NaP-TRAP individual reporters and spike-ins. Basic Protocol 2NaP-TRAP delivery by micro-injection in zebrafish embryos. Alternate Protocol 1NaP-TRAP delivery by transfection in cultured mammalian cells. Basic Protocol 3NaP-TRAP pulldown and RNA extraction. Basic Protocol 4Preparation of NaP-TRAP cDNA Sequencing Libraries. Alternate Protocol 2NaP-TRAP-qPCR module for low-cost validation. Basic Protocol 5Computational analysis of NaP-TRAP MPRA data.

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

This protocol details the extraction of high-molecular-weight genomic DNA from grapevine tissues (wild and cultivated Vitis spp., including pathogen-infected samples) and the subsequent preparation of Illumina(R) whole-genome sequencing libraries using bead-bound Tn5 transposase. It is designed to overcome challenges from polyphenolic compounds and secondary metabolites in wild plants, providing a cost-effective workflow for large-scale population genomics. It includes recipes for buffers, incubation times, critical notes, and troubleshooting tips to maximize yield and library quality. Although designed for the grapevine DNA, this protocol is potentially applicable to other similar wild plant species HighlightsO_LIOptimized CTAB-PTB DNA extraction protocol for field-collected wild plant tissues. C_LIO_LIEffective removal of polyphenols and secondary metabolites associated with DNA using PTB. C_LIO_LICost-effective Illumina DNA Prep library preparation using bead-bound Tn5 transposase (Tagmentation). C_LIO_LIScalable workflow suitable for large-scale population genomics in Vitis species. C_LIO_LIValidated method for high-molecular-weight DNA and high-quality sequencing data. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/713680v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@b637d4org.highwire.dtl.DTLVardef@10b563aorg.highwire.dtl.DTLVardef@14a32caorg.highwire.dtl.DTLVardef@4c9577_HPS_FORMAT_FIGEXP M_FIG C_FIG

DNA labelling fluorescent dyes such as ethidium bromide have long been considered to be highly mutagenic during DNA replication. While recent studies have pushed back on this narrative, the intercalative nature of these dyes continues to raise the possibility that these dyes can induce mutations. The iconPCR instrument by n6tec uses fluorescent dyes to measure amplification in real time and to adjust cycling conditions. However, since this use of qPCR is preparative and not analytical, mutations introduced by fluorescent dyes would be propagated into the sequencing reaction. To address the impact of these dyes on downstream analyses, we have performed routine mutation calling as well as mutational signature analysis on samples amplified using the iconPCR in the presence of either SYBR or EvaGreen. Sequence analysis revealed very minimal impacts of dyes on the reactions, largely within the noise regimen with only subtle changes in mutation rates seen. Mutational signature analysis was unable to identify any key signatures assignable to the dyes in either substitutions or indel domains. The mutational impact of intercalating dyes during fluorescence-guided amplification is therefore minimal and can be disregarded in all but the most sensitive NGS applications.

Nuclei isolation from myelin-rich adult mouse brain regions remains challenging for single-nucleus RNA sequencing because myelin and debris can reduce nuclei quality. We describe an optimized protocol for mouse hippocampi and cerebella using tube-and-pestle homogenization and low-volume sucrose-gradient pelleting with a standard benchtop centrifuge, with optional magnetic enrichment of nuclei to reduce debris/non-nuclear carryover. Under the tested conditions, the workflow produces intact, debris-reduced nuclei and supports downstream 10x Genomics Flex and PARSE WT library preparation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/716374v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@ccbd87org.highwire.dtl.DTLVardef@1aef4bcorg.highwire.dtl.DTLVardef@14569a8org.highwire.dtl.DTLVardef@1bc261_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIBenchtop sucrose-gradient pelleting enables rapid nuclei purification from myelin-rich adult mouse brain C_LIO_LIScales across tissue inputs (e.g., hippocampus [~]15-20 mg; cerebellum [~]50-70 mg) without ultracentrifugation or 15 mL gradients C_LIO_LIMagnetic enrichment as the recommended final cleanup step further reduces myelin/debris carryover and is compatible with 10x Flex and PARSE WT workflows. C_LI

Conventional semen cryopreservation involves equilibration at 4{degrees}C and optimum freezing rates. We hypothesized that a cholesterol-based semen extender obviates the need for equilibration, minimizing total processing time for semen cryopreservation. Experiments were conducted to determine the effects of semen extender (egg yolk- or cholesterol-based) and freezing method (routine or fast) on post-thaw sperm characteristics and fertility of beef and bison semen. In Experiment 1, beef semen diluted in tris-egg yolk-glycerol (TEYG) or cholesterol-cyclodextrin tris-glycerol (CCTG) extender underwent routine or fast freezing method. Cholesterol from animal and plant origins were compared. The routine method included 90-min equilibration at 4{degrees}C and routine freezing (RE-RF, total time 97 min) whereas the fast method included no equilibration and fast freezing (NE-FF, total time 14 min). Post-thaw sperm quality was assessed by CASA, and in vitro fertilization. Post-thaw sperm motility was not affected by the origin of cholesterol (animal or plant), but was lowest in the TEYG NE-FF group (24% vs 43-51%, P < 0.05). In vitro cleavage and blastocyst development rates did not differ between RE-RF and NE-FF groups. In Experiment 2, bison semen was diluted in TEYG or plant-CCTG extender and frozen as in Experiment 1. Post-thaw sperm motility was lowest in the TEYG NE-FF group (10% vs 39-51%, P < 0.05). In Experiment 3, beef semen diluted in TEYG or plant-CCTG extender underwent either a routine (RE-RF) or modified freezing (NE-RF, total time 25 min) method. Post-thaw sperm characteristics did not differ between extenders but were greater using routine freezing (RE-RF) compared to the modified method of freezing (NE-RF). Pregnancy rates were similar between extenders (TEYG vs plant-CCTG) using the modified freezing method without equilibration and insemination at 72 h after progesterone device removal. In conclusion, beef and bison semen diluted in cholesterol-based extender may be cryopreserved without equilibration.

BackgroundThe optic nerve serves as a vital conduit for visual signaling, and its degeneration in optic neuropathy results in irreversible vision loss. It is also a widely used model for studying central nervous system (CNS) injury and repair. Although adeno-associated virus (AAV) and lentivirus are extensively applied in CNS research, their transduction efficiency and cell-type specificity within the optic nerve remain poorly characterized. This study aimed to identify the most effective viral vector, serotype, and promoter for direct gene delivery to the adult rat optic nerve. MethodsSprague-Dawley rats (7-10 weeks) received intra-optic nerve injections of lentiviral or AAV vectors encoding GFP under different promoters (CAG, CMV, or GFAP). Two to three weeks post-injection, optic nerves were collected for immunohistochemistry with markers of oligodendrocytes (Olig2), astrocytes (GFAP, Sox9), and microglia (IBA1). Transduction efficiency and cell-type specificity were assessed using confocal microscopy. ResultsAAV2, AAV5, and lentivirus showed minimal transduction, with only sparse GFP-positive cells observed near injection sites. In contrast, AAV-PHP.eB carrying the CAG promoter yielded robust and widespread GFP expression near the injection site. Quantitative analysis revealed that approximately 90% of transduced cells were Olig2-positive oligodendrocytes, indicating strong tropism for this glial population. ConclusionAAV-PHP.eB driven by the CAG promoter enables efficient gene delivery to the optic nerve, with a predominant tropism for oligodendrocytes. This targeted intra-optic nerve injection approach offers a reliable platform for manipulating oligodendrocytes and investigating mechanisms of CNS development, injury, and repair relevant to both optic neuropathies and other CNS diseases.

Nearest neighbor parameters are widely used in software for estimating the conformational stability of an RNA sequence folding into a specific structure. Folding stability for RNA with canonical nucleotides A, C, G, and U has been widely studied, but the same is not true for most modified nucleotides. In this work, we present a comprehensive set of nearest neighbor parameters for estimating the folding stability of RNA including pseudouridine in helical or loop contexts. These parameters are derived from 210 optical melting experiments involving helices with pseudouridine-A and pseudouridine-G pairs and with pseudouridine in loop motifs. The experiments include sequences with pseudouridine and U in the same strand, including U-A and U-G pairs, allowing us to consider the folding stability of sequences with both U and pseudouridine. On average, pseudouridine stabilizes RNA folding compared to U in an analogous motif, although this effect is sequence-context dependent. These parameters improve the modeling of folding stability for RNA secondary structures containing pseudouridine. We demonstrate that these parameters successfully model the secondary structure change for Saccharomyces cerevisiae U2 snRNA when two additional inducible pseudouridines are present. These parameters are freely available and incorporated into the RNAstructure software package. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/725682v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@e1167aorg.highwire.dtl.DTLVardef@18ac7f0org.highwire.dtl.DTLVardef@4c909eorg.highwire.dtl.DTLVardef@aa8bca_HPS_FORMAT_FIGEXP M_FIG C_FIG

The coleoid cephalopods (octopus, cuttlefish, and squid) are emerging model organisms for neuroscience, development, and evolutionary biology. Determining their sex early in life is critical for population management and controlled experiments. Here, we present a protocol to non-invasively determine the sex of multiple cephalopod species as young as 3 hours post-hatching using a skin swab and quantitative PCR (qPCR). We describe steps for designing qPCR primers, swabbing live animals, extracting DNA, running the qPCR, and analyzing the results. For complete details on the use and execution of this protocol, please refer to Rubino et al.1 HighlightsO_LISwab live cephalopods as early as 3 hours post-hatching C_LIO_LIExtract DNA from cephalopod skin swabs C_LIO_LIPerform qPCR-based sex determination C_LIO_LIDesign and validate qPCR primers for new species C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/715692v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@3aa68dorg.highwire.dtl.DTLVardef@8c7e61org.highwire.dtl.DTLVardef@1bd45d9org.highwire.dtl.DTLVardef@134cc4d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Phenomic prediction (PP) is a breeding value prediction method using near infrared spectroscopy (NIRS). Spectra pre-processing is a key step in the analysis pipeline of PP and generally involves chemometrics methods. However, there is still little understanding in the genetics community of what pre-processing does and why it increases performances. Consequently, the choice of pre-processing is done either arbitrarily or through a search of the optimal set of methods and associated parameters. In this study, we propose a PCA-based pre-processing method where genetic values of spectra are estimated on a set of principal components instead of individual wavelengths. This way, estimations are based on a few informative and orthogonal features of spectra instead of many correlated, uninformative wavelengths. We tested this new pre-processing method on five data sets representing four plant species (maize, rice, sorghum and grapevine). Results show that it performs as good, or better than the best classical chemometric pre-processing methods in almost all cases. Combining PCA-based and classical chemometric pre-processing methods maximizes predictive ability. Moreover, this pre-processing method opens up possibilities of better understanding and selecting parts of the spectral information that are relevant for the prediction of breeding values. Indeed, components representing together about 1% of spectral variability were found to be responsible for most of PP predictive ability. Plain language summaryCultivated plants are the result of a breeding process during which their genetic values are used to select those to breed. Estimation of breeding values requires heavy experimental means and is time consuming. Phenomic prediction is a low cost and high throughput genetic value estimation method that is increasingly being used. It often uses near infrared spectroscopy measurements as predictors of genetic values that are easy to collect and thus routinely used in many species. However, near infrared spectra generally require pre-processing before being used in prediction. Currently used pre-processing methods arise from the chemometrics community, and still deserve a better in-depth appropriation by geneticists. In this study, we propose a new pre-processing approach that performs as good as or better than the best chemometric pre-processing generally used, reduces computation time, and allows for a better understanding of what parts of spectral information are relevant for prediction. Core IdeasO_LIWorking on principal components of spectra instead of wavelengths increases predictive ability of phenomic prediction and performs as good as or better than classical chemometrics pre-processing C_LIO_LIWorking on principal components of spectra requires less optimization of parameters than chemometrics pre-processing C_LIO_LIAbout 1% of spectral variance is responsible for most of the predictive power of phenomic prediction C_LIO_LIWorking on principal components of spectra pre-processed with classical chemometrics pre-processing can increase predictive ability even more C_LIO_LIPCA-based methods are valuable to optimize predictive ability of phenomic prediction and could be used more widely in the quantitative genetics field C_LI

Octopus vulgaris and other cephalopods are of increasing interest as neurobiological model organisms. This protocol describes a method to record calcium activity from individual cells in acute brain slices from Octopus vulgaris hatchlings during exogenous application of neurotransmitters. Using this protocol, we characterized single-cell responses to specific neurotransmitters in the optic lobes, which process visual information. The approach is readily adaptable to other cephalopods and small invertebrate species. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=146 HEIGHT=200 SRC="FIGDIR/small/711860v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1564eaeorg.highwire.dtl.DTLVardef@147b682org.highwire.dtl.DTLVardef@11f3b85org.highwire.dtl.DTLVardef@17c9d70_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundLasers have wide applications in medicine and dermatology, but are associated with pain and anxiety, particularly in younger patients. Pain mitigation is often limited to topical anesthetics in the outpatient setting. Distraction techniques are limited by the need for ocular protection, which can include adhesive eye patches that can completely occlude vision. Virtual reality is effective at managing procedural pain and anxiety under other short medical procedures and is a promising tool for this population. ObjectiveThis trial aims to assess the safety, feasibility, and efficacy of Virtual Reality Pain Alleviation Therapeutic (VR-PAT) for pain management during outpatient laser procedures. Methods40 patients requiring outpatient laser therapy for at least two sessions will be recruited from a pediatric hospital in the midwestern United States for this crossover randomized, two-arm clinical trial with a 1:1 allocation ratio. During the first laser visit, the participant will be randomly assigned to either play the VR-PAT game during their procedure or wear the headset with a dark screen. Participants will answer questions about their pain (Numeric Rating Scale (NRS) 0-10), anxiety (State Trait Anxiety Inventory for Children, NRS 0-10, Modified Yale Preoperative Anxiety Scale (mYPAS)), and pain medication usage. Those playing the VR-PAT will additionally report simulator sickness symptoms and their experience playing the game. At their second laser visit, participants will crossover to the opposite intervention from their first visit. The primary outcomes are the difference in self-reported pain and anxiety between the two interventions. Feasibility outcomes include the proportion of screened patients who are eligible, consent, and complete both visits and adverse events reported. To evaluate the efficacy of pain reduction, composite scores of pain score, pain medication will be calculated for each laser visit. To evaluate the efficacy of anxiety reduction, the change of mYPAS scores will be compared between control and VR groups at each visit using Wilcoxon rank sum tests. All statistical analyses will follow the intention-to-treat principle in regard to intervention assignment at each visit. ResultsThe study was funded in January 2023 and began enrollment at that time. A total of n=44 participants were recruited and data collection was completed in November 2025, with n=40 subjects completing both visits. The sample was balanced with n=40 subjects using the intervention and participating in the control condition. The age range of the complete sample was 6 to 21 years at recruitment and was 55% female sex. Data analysis is in progress with final results planned for June 2026. ConclusionsFindings from this innovative randomized clinical trial will provide early evidence on the efficacy of the VR-PAT for reducing self-reported pain and anxiety during outpatient laser procedures. The results from this trial will inform a large-scale, multisite study. Trial RegistrationClinicalTrials.gov: NCT05645224 [https://clinicaltrials.gov/study/NCT05645224]

Transcriptional bursts regulate gene expression by altering burst size or burst frequency. Here, we present a protocol that integrates fixed-cell smFISH and live-cell single-molecule imaging to analyze estrogen-responsive transcriptional bursting of the TFF1 gene in human breast cancer cell lines. This workflow enables measurement of burst size, burst initiation, and active allele frequency to determine how endocrine disruptor chemicals modulate transcriptional bursting dynamics. For complete details on the use and execution of this protocol, please refer to Day, Yasar et al.1