MBNL splicing factors regulate the microtranscriptome of skeletal muscles.

Muscleblind like splicing regulators (MBNLs) govern various RNA-processing steps, including alternative splicing, polyadenylation, RNA stability and mRNA intracellular localization. In myotonic dystrophy type 1 (DM1), the most common muscular dystrophy in adults, MBNLs are sequestered on toxic RNA containing expanded CUG repeats, which leads to disruption of MBNL-regulated processes and disease features of DM1. Herein, we show the significance of MBNLs in regulating microtranscriptome dynamics during the postnatal development of skeletal muscles and in microRNA (miRNA) misregulation observed in mouse models and patients with DM1. We identify multiple miRNAs sensitive to MBNL proteins insufficiency and reveal that many of them were postnatally regulated, which correlates with increases in the activity of these proteins during this process. In adult Mbnl1-knockout mice, miRNA expression exhibited an adult-to-newborn shift. We hypothesize that Mbnl1 deficiency influences miRNA levels through a combination of mechanisms. First, the absence of Mbnl1 protein results in alterations to the levels of pri-miRNAs. Second, MBNLs affect miRNA biogenesis by regulating the alternative splicing of miRNA primary transcripts. We propose that the expression of miR-23b, miR-27b and miR-24-1, produced from the same cluster, depends on the MBNL-sensitive inclusion of alternative exons containing miRNA sequences. Our findings suggest that MBNL sequestration in DM1 is partially responsible for altered miRNA activity. This study provides new insights into the biological roles and functions of MBNL proteins as regulators of miRNA expression in skeletal muscles.

Novel function of U7 snRNA in the repression of HERV1/LTR12s and lincRNAs in human cells.

U7 snRNA is part of the U7 snRNP complex, required for the 3' end processing of replication-dependent histone pre-mRNAs in S phase of the cell cycle. Here, we show that U7 snRNA plays another function in inhibiting the expression of a subset of long terminal repeats of human endogenous retroviruses (HERV1/LTR12s) and LTR12-containing long intergenic noncoding RNAs (lincRNAs), both bearing sequence motifs that perfectly match the 5' end of U7 snRNA. We demonstrate that U7 snRNA inhibits LTR12 and lincRNA transcription and propose a mechanism in which U7 snRNA hampers the binding/activity of the NF-Y transcription factor to CCAAT motifs within LTR12 elements. Thereby, U7 snRNA plays a protective role in maintaining the silencing of deleterious genetic elements in selected types of cells.

CANTATAdb 3.0: An Updated Repository of Plant Long Non-Coding RNAs.

CANTATAdb 3.0 is an updated database of plant long non-coding RNAs (lncRNAs), containing 571,688 lncRNAs identified across 108 species, including 100 Magnoliopsida (flowering plants), a significant expansion from the previous version. A notable feature is the inclusion of 112,980 lncRNAs that are expressed specifically in certain plant organs or embryos, indicating their potential role in development and organ-specific processes. In addition, CANTATAdb 3.0 includes 74,886 pairs of evolutionarily conserved lncRNAs found across 47 species and inferred from genome-genome alignments as well as conserved lncRNAs obtained using a similarity search approach in 5,479 species pairs, which would further aid in the selection of lncRNAs for functional studies. Interestingly, we find that conserved lncRNAs with tissue-specific expression patterns tend to occupy the same plant organ across different species, pointing toward conserved biological roles. The database now offers extended search capabilities and downloadable data in popular formats, further facilitating research on plant lncRNAs.

Evaluation of oropharyngeal deglutitive pressure dynamics in patients with Parkinson's disease.

In Parkinson's disease (PD), oropharyngeal dysphagia is common and clinically relevant. The neurophysiology of dysphagia in PD is complex and incompletely understood. The aim of the study was to determine the changes in oropharyngeal deglutitive pressure dynamics in PD and to correlate these with clinical characteristics including dysphagia and PD severity. In prospective consecutive series of 64 patients with PD [mean age: 66.9 ± 8.3 (SD)], we evaluated dysphagia severity clinically as well as with Sydney Swallow Questionnaire (SSQ) and Swallow Quality-of-Life Questionnaire (SWAL-QOL). PD severity was assessed with Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS). We used high-resolution pharyngeal impedance manometry (HRPIM) to objectively evaluate swallow function and compared data from 23 age-matched healthy controls [mean age 62.3 ± 9.1 (SD)]. Metrics assessed were upper esophageal sphincter (UES), integrated relaxation pressure (IRP), relaxation time (RT), maximum opening (MaxAdm), and pharyngeal intrabolus pressure (IBP) and pharyngeal contractility (PhCI). Mean MDS-UPDRS score was positively associated with dysphagia severity on SSQ and SWAL-QOL. HRPIM in PD compared with controls showed impaired UES relaxation parameters, with shorter RT, and elevated IRP and IBP. MaxAdm was not affected. The overall pharyngeal contractility was significantly higher in PD. Only the IBP and IRP were associated with PD severity and only IBP was significantly associated with dysphagia severity. UES dysfunction leading to increased flow resistance is common in patients with PD and correlates with dysphagia severity. Increased flow resistance may suggest impaired UES relaxation and/or impaired neuromodulation to bolus volume.NEW & NOTEWORTHY In Parkinson's disease, objective assessment of swallow function with high-resolution impedance manometry identifies upper esophageal sphincter dysfunction leading to increased flow resistance.

SyntDB: defining orthologues of human long noncoding RNAs across primates.

SyntDB (http://syntdb.amu.edu.pl/) is a collection of data on long noncoding RNAs (lncRNAs) and their evolutionary relationships in twelve primate species, including humans. This is the first database dedicated to primate lncRNAs, thousands of which are uniquely stored in SyntDB. The lncRNAs were predicted with our computational pipeline using publicly available RNA-Seq data spanning diverse tissues and organs. Most of the species included in SyntDB still lack lncRNA annotations in public resources. In addition to providing users with unique sets of lncRNAs and their characteristics, SyntDB provides data on orthology relationships between the lncRNAs of humans and other primates, which are not available on this scale elsewhere. Keeping in mind that only a small fraction of currently known human lncRNAs have been functionally characterized and that lncRNA conservation is frequently used to identify the most relevant lncRNAs for functional studies, we believe that SyntDB will contribute to ongoing research aimed at deciphering the biological roles of lncRNAs.

lncEvo: automated identification and conservation study of long noncoding RNAs.

BACKGROUND: Long noncoding RNAs represent a large class of transcripts with two common features: they exceed an arbitrary length threshold of 200 nt and are assumed to not encode proteins. Although a growing body of evidence indicates that the vast majority of lncRNAs are potentially nonfunctional, hundreds of them have already been revealed to perform essential gene regulatory functions or to be linked to a number of cellular processes, including those associated with the etiology of human diseases. To better understand the biology of lncRNAs, it is essential to perform a more in-depth study of their evolution. In contrast to protein-encoding transcripts, however, they do not show the strong sequence conservation that usually results from purifying selection; therefore, software that is typically used to resolve the evolutionary relationships of protein-encoding genes and transcripts is not applicable to the study of lncRNAs. RESULTS: To tackle this issue, we developed lncEvo, a computational pipeline that consists of three modules: (1) transcriptome assembly from RNA-Seq data, (2) prediction of lncRNAs, and (3) conservation study-a genome-wide comparison of lncRNA transcriptomes between two species of interest, including search for orthologs. Importantly, one can choose to apply lncEvo solely for transcriptome assembly or lncRNA prediction, without calling the conservation-related part. CONCLUSIONS: lncEvo is an all-in-one tool built with the Nextflow framework, utilizing state-of-the-art software and algorithms with customizable trade-offs between speed and sensitivity, ease of use and built-in reporting functionalities. The source code of the pipeline is freely available for academic and nonacademic use under the MIT license at https://gitlab.com/spirit678/lncrna_conservation_nf .

Clinical utility of cricopharyngeal distensibility measurements during endoscopic myotomy for Zenker's diverticulum.

BACKGROUND AND AIMS: Flexible endoscopic cricopharyngeal myotomy (FECM) allows minimally invasive treatment of patients with Zenker's diverticulum (ZD); however, retreatment rates are substantial. We hypothesized that the functional lumen imaging probe (FLIP) may provide insight into ZD pathophysiology and serve as an intraprocedural guide to adequacy of myotomy. METHODS: We prospectively evaluated 11 ZD patients undergoing FECM and compared the baseline cricopharyngeal (CP) distensibility with 16 control subjects. Intraprocedural CP distensibility was measured immediately pre- and postmyotomy. The CP distensibility index (CP-DI) was defined as a ratio of the narrowest cross-sectional area (nCSA) and the corresponding intrabag pressure at 40 mL distension. Same-procedure myotomy extension was undertaken in a subgroup if threshold distensibility changes were not met. RESULTS: ZD patients had reduced baseline nCSA and CP-DI compared with control subjects, (169.6 vs 227.5 mm2 [P < .001] and 3.8 vs 7.6 mm2/mm Hg [P < .001], respectively). After CP myotomy, both nCSA and CP-DI increased significantly by an average of 74.2 mm2 (95% confidence interval [CI], 35.1-113.3; P = .002) and 2.2 mm2/mm Hg (95% CI, .6-3.8; P = .01), respectively. In the subgroup with no significant change in CP distensibility after initial myotomy (n = 6), myotomy extension resulted in significant increases in both mean nCSA and CP-DI of 66.6 mm2 (95% CI, 16.4-116.8; P = .03) and 1.9 mm2/mm Hg (95% CI, .4-3.3; P = .015), respectively. There were no adverse events. CONCLUSIONS: CP distensibility is reduced in ZD patients and is partially reversible by FECM. An intraprocedural FLIP CP distensibility measurement is safe and sensitive in detecting myotomy-induced changes. These findings support using FLIP to optimize FECM outcome. Further studies are required to derive precise metrics predictive of clinical response.

A chromatin-associated splicing isoform of OIP5-AS1 acts in cis to regulate the OIP5 oncogene.

A large portion of the human genome is transcribed into long noncoding RNAs that can range from 200 nucleotides to several kilobases in length. The number of identified lncRNAs is still growing, but only a handful of them have been functionally characterized. However, it is known that the functions of lncRNAs are closely related to their subcellular localization. Cytoplasmic lncRNAs can regulate mRNA stability, affect translation and act as miRNA sponges, while nuclear-retained long noncoding RNAs have been reported to be involved in transcriptional control, chromosome scaffolding, modulation of alternative splicing and chromatin remodelling. Through these processes, lncRNAs have diverse regulatory roles in cell biology and diseases. OIP5-AS1 (also known as Cyrano), a poorly characterized lncRNA expressed antisense to the OIP5 oncogene, is deregulated in multiple cancers. We showed that one of the OIP5-AS1 splicing forms (ENST00000501665.2) is retained in the cell nucleus where it associates with chromatin, thus narrowing down the spectrum of its possible mechanisms of action. Its knockdown with antisense LNA gapmeRs led to inhibited expression of a sense partner, OIP5, strongly suggesting a functional coupling between OIP5 and ENST00000501665.2. A subsequent bioinformatics analysis followed by RAP-MS and RNA Immunoprecipitation experiments suggested its possible mode of action; in particular, we found that ENST00000501665.2 directly binds to a number of nuclear proteins, including SMARCA4, a component of the SWI/SNF chromatin remodelling complex, whose binding motif is located in the promoter of the OIP5 oncogene.

Waterlogging-Stress-Responsive LncRNAs, Their Regulatory Relationships with miRNAs and Target Genes in Cucumber (Cucumis sativus L.).

Low oxygen level is a phenomenon often occurring during the cucumber cultivation period. Genes involved in adaptations to stress can be regulated by non-coding RNA. The aim was the identification of long non-coding RNAs (lncRNAs) involved in the response to long-term waterlogging stress in two cucumber haploid lines, i.e., DH2 (waterlogging tolerant-WL-T) and DH4 (waterlogging sensitive-WL-S). Plants, at the juvenile stage, were waterlogged for 7 days (non-primed, 1xH), and after a 14-day recovery period, plants were stressed again for another 7 days (primed, 2xH). Roots were collected for high-throughput RNA sequencing. Implementation of the bioinformatic pipeline made it possible to determine specific lncRNAs for non-primed and primed plants of both accessions, highlighting differential responses to hypoxia stress. In total, 3738 lncRNA molecules were identified. The highest number (1476) of unique lncRNAs was determined for non-primed WL-S plants. Seventy-one lncRNAs were depicted as potentially being involved in acquiring tolerance to hypoxia in cucumber. Understanding the mechanism of gene regulation under long-term waterlogging by lncRNAs and their interactions with miRNAs provides sufficient information in terms of adaptation to the oxygen deprivation in cucumber. To the best of our knowledge, this is the first report concerning the role of lncRNAs in the regulation of long-term waterlogging tolerance by priming application in cucumber.

Identification of Novel miRNAs and Their Target Genes in the Response to Abscisic Acid in Arabidopsis.

miRNAs are involved in various biological processes, including adaptive responses to abiotic stress. To understand the role of miRNAs in the response to ABA, ABA-responsive miRNAs were identified by small RNA sequencing in wild-type Arabidopsis, as well as in abi1td, mkkk17, and mkkk18 mutants. We identified 10 novel miRNAs in WT after ABA treatment, while in abi1td, mkkk17, and mkkk18 mutants, three, seven, and nine known miRNAs, respectively, were differentially expressed after ABA treatment. One novel miRNA (miRn-8) was differentially expressed in the mkkk17 mutant. Potential target genes of the miRNA panel were identified using psRNATarget. Sequencing results were validated by quantitative RT-PCR of several known and novel miRNAs in all genotypes. Of the predicted targets of novel miRNAs, seven target genes of six novel miRNAs were further validated by 5' RLM-RACE. Gene ontology analyses showed the potential target genes of ABA-responsive known and novel miRNAs to be involved in diverse cellular processes in plants, including development and stomatal movement. These outcomes suggest that a number of the identified miRNAs have crucial roles in plant responses to environmental stress, as well as in plant development, and might have common regulatory roles in the core ABA signaling pathway.

Integration and Investigation of Selected On-Board Devices for Development of the Newly Designed Miniature UAV.

The article is a development of the topic generally presented in the conference proceedings issued after the 2019 IEEE International Workshop on Metrology for Aerospace. In contrast to topics presented in the conference, the article describes in detail avionic equipment and on-board systems integration process and their in-flight adjustment in regard to the newly designed miniature unmanned aerial vehicle (mini-UAV). The mini-airplane was constructed and assembled in the course of the research project, the purpose of which was to show implementation of a totally new mini-UAV design. The intention of the work was to develop a new unmanned system including an originally constructed small airplane with elements purchased from open market. Such approach should allow to construct a new aerial unmanned system, which technologically would not be very advanced but should be easy to use and relatively inexpensive. The demonstrator mini-airplane has equipment typical for such an object, i.e., electric propulsion, autopilot system, camera head and parachute device for recovery. The key efforts in the project were taken to elaborate an original but easy to use system, to integrate subsystem elements and test them so to prove their functionality and reliability.

Sequence-non-specific effects generated by various types of RNA interference triggers.

RNA interference triggers such as short interfering RNA (siRNA) or genetically encoded short hairpin RNA (shRNA) and artificial miRNA (sh-miR) are widely used to silence the expression of specific genes. In addition to silencing selected targets, RNAi reagents may induce various side effects, including immune responses. To determine the molecular markers of immune response activation when using RNAi reagents, we analyzed the results of experiments gathered in the RNAimmuno (v 2.0) and GEO Profiles databases. To better characterize and compare cellular responses to various RNAi reagents in one experimental system, we designed a reagent series in corresponding siRNA, D-siRNA, shRNA and sh-miR forms. To exclude sequence-specific effects the reagents targeted 3 different transcripts (Luc, ATXN3 and HTT). We demonstrate that RNAi reagents induce a broad variety of sequence-non-specific effects, including the deregulation of cellular miRNA levels. Typical siRNAs are weak stimulators of interferon response but may saturate the miRNA biogenesis pathway, leading to the downregulation of highly expressed miRNAs, whereas plasmid-based reagents induce known markers of immune response and may alter miRNA levels and their isomiR composition.

siRNA release from pri-miRNA scaffolds is controlled by the sequence and structure of RNA.

shmiRs are pri-miRNA-based RNA interference triggers from which exogenous siRNAs are expressed in cells to silence target genes. These reagents are very promising tools in RNAi in vivo applications due to their good activity profile and lower toxicity than observed for other vector-based reagents such as shRNAs. In this study, using high-resolution northern blotting and small RNA sequencing, we investigated the precision with which RNases Drosha and Dicer process shmiRs. The fidelity of siRNA release from the commonly used pri-miRNA shuttles was found to depend on both the siRNA insert and the pri-miR scaffold. Then, we searched for specific factors that may affect the precision of siRNA release and found that both the structural features of shmiR hairpins and the nucleotide sequence at Drosha and Dicer processing sites contribute to cleavage site selection and cleavage precision. An analysis of multiple shRNA intermediates generated from several reagents revealed the complexity of shmiR processing by Drosha and demonstrated that Dicer selects substrates for further processing. Aside from providing new basic knowledge regarding the specificity of nucleases involved in miRNA biogenesis, our results facilitate the rational design of more efficient genetic reagents for RNAi technology.

Clinical utility of a functional lumen imaging probe in management of dysphagia following head and neck cancer therapies.

Background and aims Chemoradiotherapy for head and neck cancer (HNC) with/without laryngectomy commonly causes dysphagia. Pharyngoesophageal junction (PEJ) stricturing is an important contributor. We aimed to validate a functional lumen imaging probe (the EndoFLIP system) as a tool for quantitating pretreatment PEJ distensibility and treatment-related changes in HNC survivors with dysphagia and to evaluate the diagnostic accuracy of EndoFLIP-derived distensibility in detecting PEJ strictures. Methods We studied 34 consecutive HNC survivors with long-term (> 12 months) dysphagia who underwent endoscopic dilation for suspected strictures. Twenty non-dysphagic patients undergoing routine endoscopy served as controls. PEJ distensibility was measured at endoscopy with the EndoFLIP system pre- and post-dilation. PEJ stricture was defined as the presence of a mucosal tear post-dilation. Results PEJ stricture was confirmed in 22/34 HNC patients (65 %). During distension up to 60 mmHg, the mean EndoFLIP-derived narrowest cross-sectional area (nCSA) in HNC patients with strictures, without strictures, and in controls were 58 mm2 (95 % confidence interval [CI] 22 to 118), 195 mm2 (95 %CI 129 to 334), and 227 mm2 (95 %CI 168 to 316), respectively. A cutoff of 114 mm2 for the nCSA at the PEJ had perfect diagnostic accuracy in detecting strictures (area under the receiver operating characteristic curve = 1). In patients with strictures, a single session of dilation increased the nCSA by 29 mm2 (95 %CI 20 to 37; P < 0.001). In patients with no strictures, dilation caused no change in the nCSA (mean difference 13 mm2 [95 %CI -4 to 30]; P = 0.13). Conclusions EndoFLIP is a highly accurate technique for the detection of PEJ strictures. EndoFLIP may complement conventional diagnostic tools in the detection of pharyngeal outflow obstruction.

CANTATAdb: A Collection of Plant Long Non-Coding RNAs.

Long non-coding RNAs (lncRNAs) represent a class of potent regulators of gene expression that are found in a wide array of eukaryotes; however, our knowledge about these molecules in plants is still very limited. In particular, a number of model plant species still lack comprehensive data sets of lncRNAs and their annotations, and very little is known about their biological roles. To meet these shortcomings, we created an online database of lncRNAs in 10 model plant species. The lncRNAs were identified computationally using dozens of publicly available RNA sequencing (RNA-Seq) libraries. Expression values, coding potential, sequence alignments as well as other types of data provide annotation for the identified lncRNAs. In order to better characterize them, we investigated their potential roles in splicing modulation and deregulation of microRNA functions. The data are freely available for searching, browsing and downloading from an online database called CANTATAdb (http://cantata.amu.edu.pl, http://yeti.amu.edu.pl/CANTATA/).

miRNEST 2.0: a database of plant and animal microRNAs.

Ever growing interest in microRNAs has immensely populated the number of resources and research papers devoted to the field and, as a result, it becomes more and more demanding to find miRNA data of interest. To mitigate this problem, we created miRNEST database (http://mirnest.amu.edu.pl), an integrative microRNAs resource. In its updated version, named miRNEST 2.0, the database is complemented with our extensive miRNA predictions from deep sequencing libraries, data from plant degradome analyses, results of pre-miRNA classification with HuntMi and miRNA splice sites information. We also added download and upload options and improved the user interface to make it easier to browse through miRNA records.

Treatment efficacy of sacral nerve stimulation in slow transit constipation: a two-phase, double-blind randomized controlled crossover study.

OBJECTIVES: Sacral nerve stimulation (SNS) is a potential treatment for constipation refractory to standard therapies. However, there have been no randomized controlled studies examining its efficacy. In patients with slow transit constipation, we evaluated the efficacy of suprasensory and subsensory SNS compared with sham, in a prospective, 18-week randomized, double-blind, placebo-controlled, two-phase crossover study. The primary outcome measure was the proportion of patients who, on more than 2 days/week for at least 2 of 3 weeks, reported a bowel movement associated with a feeling of complete evacuation. METHODS: After 3 weeks of temporary peripheral nerve evaluation (PNE), all patients had permanent implantation and were randomized to subsensory/sham (3 weeks each) and then re-randomized to suprasensory/sham (3 weeks each) with a 2-week washout period between each arm. Daily stool dairies were kept, and quality of life (QoL; SF36) was measured at the end of each arm. RESULTS: Between November 2006 and March 2012, 234 constipated patients were assessed, of whom 59 were willing and deemed eligible to participate (4 male; median age 42 years). Of the 59 patients, 16 (28%) responded to PNE. Fifty-five patients went on to permanent SNS implantation. The proportion of patients satisfying the primary outcome measure did not differ between suprasensory (30%) and sham (21%) stimulations, nor between subsensory (25%) and sham (25%) stimulations. There were no significant changes in QoL scores. CONCLUSIONS: In patients with refractory slow transit constipation, SNS did not improve the frequency of complete bowel movements over the 3-week active period.

miRNEST database: an integrative approach in microRNA search and annotation.

Despite accumulating data on animal and plant microRNAs and their functions, existing public miRNA resources usually collect miRNAs from a very limited number of species. A lot of microRNAs, including those from model organisms, remain undiscovered. As a result there is a continuous need to search for new microRNAs. We present miRNEST (http://mirnest.amu.edu.pl), a comprehensive database of animal, plant and virus microRNAs. The core part of the database is built from our miRNA predictions conducted on Expressed Sequence Tags of 225 animal and 202 plant species. The miRNA search was performed based on sequence similarity and as many as 10,004 miRNA candidates in 221 animal and 199 plant species were discovered. Out of them only 299 have already been deposited in miRBase. Additionally, miRNEST has been integrated with external miRNA data from literature and 13 databases, which includes miRNA sequences, small RNA sequencing data, expression, polymorphisms and targets data as well as links to external miRNA resources, whenever applicable. All this makes miRNEST a considerable miRNA resource in a sense of number of species (544) that integrates a scattered miRNA data into a uniform format with a user-friendly web interface.

mirEX: a platform for comparative exploration of plant pri-miRNA expression data.

mirEX is a comprehensive platform for comparative analysis of primary microRNA expression data. RT-qPCR-based gene expression profiles are stored in a universal and expandable database scheme and wrapped by an intuitive user-friendly interface. A new way of accessing gene expression data in mirEX includes a simple mouse operated querying system and dynamic graphs for data mining analyses. In contrast to other publicly available databases, the mirEX interface allows a simultaneous comparison of expression levels between various microRNA genes in diverse organs and developmental stages. Currently, mirEX integrates information about the expression profile of 190 Arabidopsis thaliana pri-miRNAs in seven different developmental stages: seeds, seedlings and various organs of mature plants. Additionally, by providing RNA structural models, publicly available deep sequencing results, experimental procedure details and careful selection of auxiliary data in the form of web links, mirEX can function as a one-stop solution for Arabidopsis microRNA information. A web-based mirEX interface can be accessed at http://bioinfo.amu.edu.pl/mirex.

The normative range for and age and gender effects on the Sydney Swallow Questionnaire (SSQ).

The Sydney Swallow Questionnaire (SSQ) is a validated measure of the symptomatic severity of oral-pharyngeal dysphagia. Up until now no normative ranges have been established for the questionnaire. This is a limitation in its utility as it makes it difficult to use the tool to identify the prevalence and burden of oral-pharyngeal dysphagia in the general population or within patient populations. The study's aim was to derive the normative range of dysphagia scores for the SSQ and to determine whether, in nondysphagic individuals, there are any age or gender effects on these scores. The questionnaire was administered to 73 eligible nondysphagic individuals who had been screened for any dysphagia or conditions that might predispose them to dysphagia. The frequency distribution of SSQ scores was first examined for normality and appropriate transformations performed before determining the upper limit of normal. Of the 73 healthy participants, 45 were male, and the cohort had a mean age of 58.6 years (range = 22.0-82.1 years). No statistically significant relationship between SSQ scores and either age (r s[73] = 0.140, p = 0.239) or gender (r pb[73] = 0.021, p = 0.857) was found. The mean total SSQ score (maximum possible score = 1,700) was 59.0 (SD = 56.7; range = 2-241). The frequency distribution of scores was non-normal and markedly skewed. After a Box-Cox transformation to normalise the distribution, the calculated upper limit of the reference interval was 234 with a 90 % CI of [193, 277]. The SSQ scores in a nondysphagic population are not influenced by age or gender. These data complement the existing reliability and validation data and thereby improve the overall utility of the SSQ in the context of future studies of oral-pharyngeal dysphagia prevalence, efficacy, and outcome.