Methods and Protocols

Epidermal development and regeneration rely on the stepwise differentiation of keratinocytes, the primary constituent cells of the epidermis. In the integrated epidermis, keratinocyte differentiation (KD) occurs in response to the increasing calcium gradient. Reflecting this, high-calcium incubation remains a popular approach for inducing KD in vitro. The outcome is primarily assessed by differential expression of differentiation markers compared to proliferative cells. However, it is significantly influenced by the cell models used, calcium concentration and incubation time, which often vary between studies. Such differences compromise reproducibility and comparability. In this article, we overview major parameters that shape the outcome of high-calcium induced KD. Comparative experimental data between high-calcium incubation and high-density induced differentiation underscore the need for combining additional parameters, such as microscopy based visualization with marker protein analysis for unbiased assessment of KD. These recommendations are intended to guide future research in keratinocyte biology. Full article

Large-scale, multi-center projects have become common in the era of rapid technological development, but protocol standardization remains challenging. In whole-exome sequencing (WES), various exome enrichment kits exhibit variable efficiency across genomic regions, leading to systematic, non-biological batch effects, much stronger than other technical factors. We propose a workflow to minimize the effect of WES capture inconsistencies in single-nucleotide variation (SNV) data. The pipeline consists of quality control, mapping to the genome, SNV calling, joint genotyping, and imputing genotypes using reference haplotypes. SNVs are then aggregated into gene-level features measuring the burden of deleterious variants. Finally, a gene-level imputation is performed using a customized algorithm. Namely, if the detection rate of a gene is low in samples enriched with a given capture kit but high in samples enriched with other kits, missing values in the former group are imputed, as such differences are unlikely to reflect true biology. As a benchmark, we conducted a study on over a thousand breast cancer cases across 11 cohorts, using eight exome capture kits. We demonstrated that the proposed pipeline leads to a considerable decrease in the batch effect signal, potentially increasing the likelihood of finding true biological signals. Full article

Cell-free DNA (cfDNA) has emerged as a promising liquid biopsy biomarker; however, differences in analytical methods may influence cfDNA quantification and integrity assessment. This study compared plasma cfDNA concentration and integrity between treatment-naïve breast cancer (BC) patients (n = 100) and healthy controls (HC; n = 70) using complementary analytical platforms. cfDNA concentration was measured by Qubit fluorometry, TapeStation (TS) electrophoretic profiling, and ALU-based quantitative PCR (qPCR). An exploratory genomic DNA (gDNA) correction of Qubit-derived cfDNA concentration was performed using TS-derived fragment-size information. cfDNA integrity was assessed using the ALU-derived DNA Integrity Index (DII (ALU); ALU247/ALU115) and the TS-derived DNA Integrity Index (DII (TS)), and methods were compared for their correlation and agreement. Qubit- and TS-based cfDNA measurements were significantly higher in BC patients than in HC. Among the ALU-based qPCR measures, although ALU115 did not differ significantly between groups, ALU247 and DII (ALU) were significantly higher in BC patients, indicating an enrichment of longer cfDNA fragments. Exploratory gDNA correction did not substantially alter the observed differences. Significant positive correlations were observed among the cfDNA quantification methods. Supporting these findings, agreement analysis demonstrated improved agreement following gDNA correction. In contrast, cfDNA integrity assessment was method-dependent: although DII (ALU) was significantly higher in BC patients, DII (TS) did not differ significantly between groups, and no significant correlation or agreement was observed between the two indices, indicating that the two methods evaluate different lengths of cfDNA fragments. These findings demonstrate that complementary analytical platforms consistently differentiated BC patients from HC based on cfDNA concentration, whereas cfDNA integrity assessment depended on the analytical method employed. Together, these findings provide methodological insights into cfDNA quantification and fragmentation assessment and support the complementary use of fluorometric, electrophoretic, and ALU-based qPCR approaches for comprehensive cfDNA characterization. Full article

A naive Bayesian classifier (NBC) combined with principal component analysis (PCA) effectively differentiates the dose-dependent effects of certain groups of psychoactive drugs based on their impact on the amplitude–spectral characteristics of electrocorticograms (ECoG) in rats. This approach has been shown to be useful for pharmacological screening of agents with unknown or poorly understood activity. Despite previously obtained optimistic results, classification determination for some drugs was inaccurate, necessitating the search for possible ways to improve the predictive effectiveness of the proposed algorithm. One possible approach would be to use as input quantitative data not only the impact of the psychoactive drugs studied on the amplitude–spectral characteristics of ECoG but also connectivity changes, including the average coherence power of different pairs of leads. The aim of this study was to compare the accuracy of NBC in classifying the pharmacological mechanism of action of agents with well-known mechanisms (test set) using pharmaco-EEG data on changes in the amplitude–spectral characteristics of ECoG, coherence, and the combined use of two data sets. Materials and methods. Experiments were performed on Wistar rats with chronically implanted ECoG electrodes. The training set, relative to which the effects of the pharmacological agents from the test set were classified, were the matrices of effects of 12 pharmacological agents: the NMDA antagonist dizocilpine, the D2/D3 antagonists haloperidol and sulpiride, the M-anticholinergic tropicamide, the H1/5HT2A receptor blocker hydroxyzine, the acetylcholinesterase inhibitor galantamine, the alpha-2 adrenergic agonist dexmedetomidine, the alpha-2 adrenergic antagonist atipamezole, the adenosine receptor blocker caffeine and the GABA-mimetics aminophenylbutyric acid (phenibut), bromdihydrochlorophenylbenzodiazepine (phenazepam) and 5-ethyl-5-phenyl-2,4,6(1H,3H,5H)-pyrimidinetrione. The test set included various drugs with tropism for the targets of the training set drugs: dopamine receptor antagonists chlorpromazine, droperidol, tiapride and raclopride, H1-histamine blockers diphenhydramine and promethazine, 5-HT2-receptor blockers ritanserin and glemenserin, acetylcholinesterase inhibitor ipidacrine, alpha2-adrenergic receptor antagonist yohimbine, alpha2-adrenergic agonists medetomidine and xylazine, GABA-mimetics 5-ethyl-5-(1-methylbutyl)-2,4,6(1H,3H,5H)-pyrimidinetrione and chloral hydrate. The analysis of the ECoG signal included the calculation of 132 amplitude–spectral characteristics and 75 coherence indicators, which, using the PCA, led to new integrative indicators used for further classification of the NBC. Results and discussion. For each drug in the test set, the median similarity probability with a particular group from the training set was calculated, which was used to assess the classification quality. It was found that, when using the amplitude–spectral characteristics of ECoG, the proposed methodological approach allows for the identification of the ECoG effects of several groups of psychoactive drugs, including D2/D3-dopamine, M-cholinergic, H1-histamine, and 5-HT2-serotonin receptor blockers, AChE inhibitors, GABA-mimetics, and alpha-2-adrenergic receptor agonists and antagonists. This approach enabled the correct classification of 18 of 24 groups in the test set. When using changes in coherence indices as the initial data, the classification accuracy also amounted to 18 of 24 groups. When combining the two data sets, the number of correctly identified NBC groups was 20 of 24 groups. When comparing the classification during training (confusion matrix), it was found that coherence data or adding coherence data to the data based on changes in amplitude–spectral characteristics leads to a statistically significant (p < 0.01 in both cases) increase in accuracy. Conclusions. The obtained data demonstrated high accuracy in classifying the pharmacological activity of the test sample drugs using any of the three compared approaches. Despite the lack of statistically significant differences between them, classification based on the combined dataset demonstrated a higher number of “correct” similarities. This allows us to recommend the approach based on combined data of drug effects on amplitude–spectral characteristics and coherence as the most promising for further studies using pharmaco-EEG screening. Full article

A comprehensive assessment of allograft quality is essential for predicting successful transplantation and graft survival. Elevated levels of flavin mononucleotide (FMN) in perfusate obtained during hypothermic oxygenated perfusion (HOPE) may serve as a promising predictor of early graft dysfunction and post-transplant complications. The aim of this study was to evaluate the feasibility of applying an FMN determination method to HTK-based perfusion solutions used during liver machine perfusion. The spectrofluorimetric method for FMN determination in modified HTK perfusion solution (modHTK) was evaluated for the following parameters: linearity, accuracy, repeatability, reproducibility, and stability. The study demonstrated the suitability of the quantitative FMN determination method for modHTK solution used during HOPE of liver grafts. Full article

Clinical image publications represent an important educational resource across healthcare disciplines, providing concise visual demonstrations of diseases, diagnostic findings, and therapeutic outcomes. Despite their widespread use, reporting practices remain highly heterogeneous, with substantial variability in the description of clinical context, image acquisition, ethical considerations, and educational content. Existing reporting guidelines, including the CARE Guidelines, do not specifically address the unique methodological and technical aspects of image-based publications. The aim of this study was to develop the IMAGE Statement (Improving Clinical Image Reporting Standards), a reporting guideline specifically designed for clinical image publications as an extension of the CARE Guidelines. The development process was informed by recommendations from the EQUATOR Network and included a literature review, analysis of journal instructions for authors, generation of candidate reporting items, and expert consensus. The resulting IMAGE Statement comprises 15 essential reporting items covering the title, patient information, clinical context, diagnostic assessment, image acquisition, image quality, image selection, image annotation, image description, diagnostic interpretation, final diagnosis, educational message, ethics and consent, artificial intelligence use disclosure, and figure legend. The IMAGE Statement provides a structured framework intended to improve completeness, transparency, reproducibility, educational value, and ethical reporting of clinical image publications across healthcare disciplines. Full article

This study optimised and validated a microplate Ferric-Reducing Antioxidant Power (FRAP) assay for honey that incorporates a honey-specific matrix-matched blank—a paired absorbance measurement lacking the chromogenic probe 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ)—to separate true ferric reduction from non-reductive Fe³⁺–matrix interactions that otherwise inflate apparent antioxidant capacity. A 30 min incubation at 37 °C in the dark was selected as the optimal compromise between reaction completeness and reagent/matrix stability, using 20 µL sample and 180 µL FRAP reagent per well (absorbance at 620 nm). The assay was validated according to International Council for Harmonisation (ICH) principles, showing linearity for FeSO₄·7H₂O from 200–1200 µM (R² ≥ 0.9988), Limit of detection/limit of quantification (LOD/LOQ) of 0.28/0.94 mmol Fe²⁺ equivalents kg⁻¹, recoveries of 101–103%, and precision ≤ 5% relative standard deviation (RSD). All tested individual sugars except maltodextrin and the artificial honey matrix produced FRAP responses below the LOQ. Flavonoids, however, showed pronounced, time-dependent, non-reductive Fe³⁺ interactions and were markedly overestimated under conventional water blanking, while phenolic acids were less affected. Applied to 47 Western Australian honeys, the validated assay yielded FRAP activities of 2.80–9.52 mmol Fe²⁺ kg⁻¹; matrix-matched blanking gave consistently lower, more chemically specific values than conventional water blanking, with corrections ranging up to >30% depending on honey type. Matrix-matched blanking is therefore essential, not optional, for accurate FRAP measurement in honey, and the same principle should apply directly to other phenolic-rich foods like wine, tea, and fruit extracts—wherever iron–polyphenol interactions confound conventional antioxidant assays. Full article

While the recently developed tissue clearing protocols pathoDISCO and activeDISCO significantly accelerate the clearing of large tissue specimens through active chemical dehydration using 2,2-dimethoxypropane, the final step of refractive index (RI) matching with viscous organic solvents as dibenzyl ether (DBE) remains restricted by slow passive diffusion. To overcome this bottleneck, we applied 40 kHz ultrasound using a standard, cost-effective laboratory bath to significantly enhance the diffusion kinetics of the clearing medium into large specimens. Our investigation on multi-centimeter-sized porcine muscle and human earlobe samples demonstrates that 40 kHz acoustic oscillations generated by a standard ultrasound cleaning device not only accelerate the clearing process but also yield superior and stable long-term optical transparency. We also tested 1 MHz high-frequency ultrasound but it offered no kinetic advantages and tended to induce tissue micro-fractures, an artifact we have not observed at 40 kHz at comparable energy levels. We therefore propose that standard 40 kHz ultrasound baths, ubiquitous in laboratories for cleaning purposes, represent an ideal and accessible tool for optimizing solvent-based tissue clearing. Full article

Mountain biking over rough terrain exposes riders to vibrations that may affect comfort, health, bicycle control, and performance. This study analyzed the effect of wheel diameter on vibration transmission and performance during a short uphill and downhill test over rocky terrain. Forty-nine highly trained male mountain bikers completed repeated trials using two equivalent hardtail mountain bikes with 26- and 29-inch wheels. Vibrations were recorded with eight triaxial accelerometers placed on the bicycle and rider, and performance was assessed using an electronic photocell timing system. Both wheel sizes showed a similar vibration pattern, with lower root mean square (RMS) acceleration values at the helmet and coccyx and higher values at the wrists and ankles. However, wheel diameter significantly influenced vibration transmission. During the uphill test, the 26-inch bicycle produced higher accelerations at the coccyx and rear hub, whereas during the downhill test, the 29-inch bicycle transmitted greater vibrations to the rider’s wrists and ankles. No significant differences were observed between wheel sizes in the time required to complete either the uphill or downhill tests. These findings suggest that, in short and highly irregular uphill and downhill sections, 29-inch wheels do not necessarily improve performance over 26-inch wheels, although they modify the distribution of vibrations transmitted to the rider. Full article

Osteocalcin (OC), a bone protein, is evaluated by its proteoforms, which are implicated in various illnesses. Rapid, clinically used immunoassays are well-established. Epitope dependence, fragment cross-reactivity, uneven identification across carboxylation states, and lack of standardization restrict their efficacy. This study aims to characterize various OC proteoforms in patients with type 2 diabetes mellitus (T2DM) and vitamin D insufficiency and to develop a mass spectrometric immunoassay (MSIA) for their detection. The first MRM-MSIA test to specifically detect OC proteoforms in plasma with vitamin D deficiency-induced alterations in T2DM patients was developed and validated. A precise and selective MRM-MSIA was developed to assess OC fragments in plasma from T2DM patients with normal (DN) or deficient (DD) vitamin D levels. The approach examined OC-1 (aa64–71), OC-2 (aa64–70), and OC-3 (aa72–93) proteoforms. It met international linearity, precision, and accuracy criteria with variability <15% and intra/inter-day accuracy of 88.8% to 110.1%. The DD group had significantly higher OC-1 levels than the DN group. The receiver operating characteristic (ROC) curve for OC-1 produced an AUC of 0.8263 (95% confidence interval [CI], p = 0.0004). The developed MRM-MSIA measures plasma OC proteoforms with good specificity and sensitivity, making it a promising clinical diagnostic tool. Full article

Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal malignancy, highlighting the need for reproducible experimental systems that model pancreatic injury and subsequent neoplastic progression. Adult-onset genetically engineered models in which oncogenic Kras and mutant Trp53 are induced in the pancreas provide a relevant platform for studying PDAC pathogenesis, but efficient neoplastic progression often requires concomitant pancreatic injury. Cerulein-induced pancreatitis is widely used to provide an injury-associated inflammatory stimulus that promotes PDAC progression; however, repeated-injection regimens are labor-intensive and increase cumulative handling stress in animals. Here, we describe a dose-optimized, single-dose L-Lysine protocol designed to induce acute pancreatitis and characterize subsequent pancreatic neoplastic progression in adult-onset Kras/Trp53-driven mice. Tamoxifen-treated Ptf1a^CreER/+; Kras^LSL-G12D/+; Trp53^LSL-R172H/+; Rosa26-RFP mice received a single intraperitoneal injection of L-Lysine, followed by biochemical, histological, and immunohistochemical assessment of pancreatic injury and tumor development. A single 2.0 g/kg L-Lysine injection induced sublethal acute pancreatitis characterized by increased serum pancreatic enzymes, interstitial edema, inflammatory cell infiltration, acinar cell necrosis, and rapid mitochondrial alterations. During 3–6 months of follow-up, mice developed multifocal acinar-to-ductal metaplasia, PanIN lesions, invasive PDAC, and peritoneal dissemination. This protocol provides a simple, synchronized, and less labor-intensive model for studying pancreatic injury and subsequent neoplastic progression in adult-onset Kras/Trp53-driven mice. Full article

Pediatric sleep-disordered breathing (SDB) is underdiagnosed despite its associations with adverse neurobehavioral, psychosocial, and cardiometabolic outcomes. Pediatric dental providers routinely evaluate craniofacial growth and maintain longitudinal contact with children, yet practical approaches for integrating SDB risk assessment into dental settings remain limited. This protocol describes a prospective, cross-sectional observational study that will enroll 60 school-aged children aged 8–13 years from a university-based pediatric dental clinic and classify them as low-risk or high-risk for SDB using the Pediatric Sleep Questionnaire (PSQ; threshold ≥ 0.33). The primary aim is to examine associations between PSQ-based risk classification and objective physiologic sleep parameters, including the Apnea–Hypopnea Index, Respiratory Disturbance Index, Sleep Apnea Indicator, and Sleep Quality Index, obtained from a U.S. Food and Drug Administration-cleared wearable home sleep monitor—SleepImage Ring (MyCardio LLC, Denver, CO, USA)—worn for a minimum of three consecutive nights. Secondary aims will evaluate associations between SDB risk classification and body mass index, Mallampati score, Brodsky tonsillar grade, and psychosocial functioning (anxiety, depression, perceived stress, and daytime sleepiness). Exploratory craniofacial analyses will be conducted among participants with clinically available lateral cephalometric radiographs. This protocol could position pediatric dental visits as an accessible touchpoint for early identification of children with unrecognized SDB and inform pathways for timely referral. Protocol Version: 1.4, dated 13 May 2026. Trial Registration: ClinicalTrials.gov NCT07581938. Full article

Column-based RNA extraction is time-consuming, involves cumulative losses during transfer, wash, and elution steps, and has reduced recovery from sub-microliter inputs. These limitations restrict gene-expression analysis in small cell populations and low-titre pathogen detection in clinical specimens. We developed the Direct Cell-to-PCR Lysis Buffer, a defined non-ionic detergent formulation containing Tween 20 (0.3%), Triton X-100 (0.1%), and NP-40 (0.1%), for RNA-extraction-free one-step RT-qPCR. Lysates are added directly to reactions without pretreatment, heating, or column purification. Non-ionic detergents produced Ct values comparable to detergent-free controls, whereas SDS, Sarkosyl, and sodium deoxycholate abolished amplification. The method was benchmarked against the standard column-based PureLink RNA Mini Kit using SaOS-2 cells, MCF7 cells, peripheral blood mononuclear cells, and nasal and throat swabs. Direct lysates showed Ct offsets of 0.6 to 2.85 cycles compared with purified RNA, depending on sample type. Ubiquitin-C mRNA was detected down to a single-cell-equivalent input, and plasmid templates were detected to approximately 100 copies per reaction. The buffer was compatible with multiplex ScriptTaq COVID PCR, with unchanged RdRP, N, and RPP30 Ct values, and tolerated sodium azide up to 0.1%, supporting rapid direct RT-qPCR workflows. Full article

Recombinant superoxide dismutase (SOD) protein is a promising antigen candidate for brucellosis vaccines, and its quantitative determination is essential for vaccine quality control, standardization, and regulatory compliance. Although enzyme-linked immunosorbent assay (ELISA) is widely used for antigen quantification, its application to vaccine formulations may be limited due to chemical modifications of antigenic determinants occurring during detoxification and adjuvantation processes. Therefore, alternative analytical methods, such as single radial immunodiffusion (SRID), which enable direct antigen quantification independent of antigenic determinant modifications, represent valuable tools for vaccine analysis. The aim of this study was to develop and validate a single radial immunodiffusion (SRID) assay for the quantitative determination of recombinant SOD protein and to obtain hyperimmune sera in sheep. Sheep were immunized with recombinant SOD protein formulated with aluminum hydroxide or AddaS03 adjuvants. Antibody responses were evaluated using agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA). Immunization with aluminum hydroxide induced higher antibody titers than AddaS03. By day 42, ELISA titers reached 1:25,600 in the aluminum hydroxide group compared with 1:3200 in the AddaS03 group. The obtained hyperimmune sera were used for SRID assay development. The optimized SRID assay demonstrated high reproducibility and specificity, with no cross-reactivity against unrelated proteins. A linear relationship was observed between the square of the precipitation ring diameter (D²) and antigen concentration (R² = 0.956–0.989). The assay showed a limit of detection of 0.312 μg/mL, a linear range of 0.625–10 μg/mL, high precision (CV < 2%), and robustness under varying analytical conditions. The developed SRID assay represents a specific, reproducible, and robust tool for quantitative control of recombinant SOD protein in vaccine formulations. Full article

Tumor cell invasion is a critical step in local tumor progression, recurrence, and metastasis. Conventional two-dimensional migration assays and many existing three-dimensional invasion models often assess cell migration, invasion into the extracellular matrix and matrix degradation as separate endpoints, although these processes are tightly coupled in vivo. Therefore, robust and reproducible in vitro models are needed to investigate tumor cell invasion under defined extracellular matrix conditions. We developed an agarose-based three-dimensional invasion assay, termed the Freiburg 3D invasion assay, for the simultaneous analysis of tumor cell migration, invasion, and extracellular matrix degradation. The system consists of a 2.8% agarose matrix containing defined microcavities connected by a common loading channel. Tumor cells are seeded into these microcavities, where they form compact cell aggregates. The cavities are subsequently filled with collagen type I or extracellular matrix gel. After polymerization, the matrix-containing agarose strips are transferred into parking pockets, cultured for several days, and monitored by microscopy. Invasion distance, single-cell migration, and ECM-cleared area are quantified from serial microscopic images using image analysis software. The system distinguished weakly invasive MCF7 breast cancer cells from highly invasive MDA-MB-231 cells. In addition, treatment with a protease inhibitor and irradiation reduced tumor cell invasion and extracellular matrix remodeling, demonstrating the suitability of the assay for pharmacological and radiation-response studies. The Freiburg 3D invasion assay provides a practical and reproducible three-dimensional in vitro model for analyzing tumor cell invasion and protease-associated extracellular matrix degradation. Full article

Viral diseases are one of the most destructive threats to global agriculture. Among plant viruses, tobacco mosaic virus (TMV) is a highly contagious pathogen infecting numerous economically vital crops. With no curable treatments, the only strategy to mitigate virus spread is early detection and plant removal. The gold standard for TMV detection is reverse transcriptase-polymerase chain reaction (RT-PCR), followed by agarose gel electrophoresis. To reduce TMV diagnosis time and eliminate the requirement for expensive equipment, this study developed and optimized a one-step RT-PCR-coupled cysteamine-functionalized gold nanoparticle assay for the colorimetric determination of the virus. By integrating cDNA synthesis and PCR amplification into a single tube and analyzing the results using cysteamine- functionalized gold nanoparticles (Au@Cys), the diagnosis turnaround time was significantly reduced. Furthermore, this AuNPs-based colorimetric assay enabled straightforward visual inspection with the naked eye, eliminating the need for costly optical devices. Additionally, our regression equation linking RT-PCR product color values, extracted as mean A value based on the CIELAB color space (green-red axis), and viral load allows for the accurate quantification of TMV infection levels in field samples. Our research lays the groundwork for the further development of more cost-effective, quantitative and rapid plant virus diagnosis methods. Full article

The development and validation of a sensitive, rapid, and specific gas chromatography method for the evaluation of 19 common Class 2 and Class 3 solvents frequently used in nanomedicine formulation is described. Method validation was performed using PerkinElmer’s headspace gas chromatograph system with flame ionization detection and an Elite 624 Crossbond 6% cyanopropylphenyl-94% dimethylpolysiloxane or DB-Fatwax-Ultra Inert column with helium as the carrier gas. Validation characteristics such as linearity, spike recovery, method precision, specificity, sensitivity, limit of detection/quantitation, and analyte stability were evaluated. The validated methods showed excellent linearity, with a correlation coefficient > 0.99, and good precision, with intra-day precision < 7.4% for all tested analytes. The percent recoveries ranged 83–104% within the method’s quantitation range. In comparison to previously reported methods, the current method has a much shorter equilibration time, higher sensitivity, better separation for many solvents, and a wide concentration detection range. The current method is also perfectly suitable to analyze short chain fatty acids such as formic acid, acetic acid, butyric acid, and valeric acid without requiring additional extraction or derivatization steps. Notably, the method was found to be suitable for analysis of formic acid—a common solvent in certain nanoformulations and one in which there is no prior gas chromatography method available which does not require this additional sample manipulation—down to approximately 75 ppm. Herein, the method is demonstrated using various nanoformulations, including the commercial Doxil formulation as well as several research nanoformulations, including polymeric, cross-linked polymeric, and dendrimer platforms. Full article

Synthetic mRNA produced by in vitro transcription is the active pharmaceutical ingredient of approved vaccines and of many drugs under development. It typically contains a 3′ poly(A) tail required for optimal stabilization and translation of the mRNA. The average length of the poly(A) tail in the produced mRNA cannot be precisely predicted and consequently must be measured using complicated technologies such as reverse transcription and sequencing or cleavage of the poly(A) tail and analysis by capillary electrophoresis or mass spectrometry. We report an accelerated method to evaluate the average poly(A) tail length in synthetic mRNA, which benefits from the fact that thiazole orange is fluorescent only when in close proximity of nucleic acid. An oligo(dT)₁₂ oligonucleotide having thiazole orange at its extremities emits a fluorescence signal proportional to the amount of poly(A) sequence available. Using a titration curve made with known amounts of poly(A) RNA oligonucleotide, the thiazole orange oligo(dT)₁₂ oligonucleotide can instantly indicate the average length of the poly(A) tail in an mRNA sample. This fast and easy method can be used in any laboratory to determine the size of the poly(A) tail in in vitro-transcribed mRNA produced for research, pre-clinical studies and clinical trials. Full article

Background: Walking is a dynamic activity that relies on inputs from the multisensory system, i.e., somatosensory, vision, and vestibular. These inputs are processed and integrated in the central nervous system to produce motor impulses for efficient walking balance. The Sensory Organization Test (SOT) is established as the gold standard for assessing sensory contributions to standing balance. However, no comparable assessments have been developed for the clinical evaluation of balance during gait. This study evaluated the Gait Sensory Interaction Test (GaitSIT), a novel virtual reality (VR)-based assessment for characterizing sensory-condition-specific changes in walking balance. Methods: The GaitSIT comprises a VR environment with a physical compliant foam walking surface that evaluates gait–balance by systematically manipulating and evaluating the sensory systems. Twenty-nine healthy young adults (mean age 24.9 ± 6.4 years) were instructed to complete 6 m walking trials under six standardized conditions (C): eyes open, eyes closed/dark scene, and rotating visual scenes on a firm surface, then repeated on a foam surface. Wearing an Oculus VR headset, participants were instructed to walk in a straight line at their preferred speed, as naturally as possible, in two test sessions on the same day, followed by a third test session 24 h later. Headset-derived sway measures, including position, velocity, and acceleration data, were recorded, and the continuous trajectory deviation angle (i.e., directional control) and sensory ratios were calculated. Linear mixed-effects models included trial-level walking speed as a covariate. Additionally, participants completed the modified Clinical Test of Sensory Interaction on Balance (mCTSIB) as a clinical standing-balance reference measure; its concurrent-validity findings will be reported separately. Results: Significant condition effects were observed for position, velocity, acceleration, and CTDA after adjustment for trial-level walking speed (all $p<0.001$), indicating that the six sensory conditions elicited distinct gait–balance responses. Significant differences relative to the baseline condition (C1) were observed across conditions C2–C6 for position, C3–C6 for velocity, and C2 and C5 for acceleration. Session effects were not significant for any primary kinematic outcome after speed adjustment. A significant condition × session interaction was observed for position ($p<0.001$), whereas velocity, acceleration, and CTDA demonstrated no significant interactions. Walking speed was significantly associated with position, acceleration, and CTDA, but not velocity. Sensory-ratio analyses revealed larger visual and vestibular ratios relative to somatosensory ratios, with the visual and vestibular ratios generally decreasing across sessions. Conclusions: GaitSIT successfully manipulated sensory conditions during overground walking and produced significant changes in gait-related sway, directional control, and sensory-ratio measures. These findings support the feasibility of GaitSIT as a portable, low-cost, and immersive assessment framework for characterizing sensory-condition-specific gait–balance responses after accounting for walking speed and providing indirect behavioral indices related to sensory reweighting. Full article