The SOX9 Dimerization Domain And Its Role in Cooperative DNA Binding

Nucleic acid binding proteins are key regulators in developmental processes and controlling the onset of diseases. Human SOX9 is a group E member of the SOX transcription family of proteins characterized by a conserved amino acid region first identified as the high mobility group (HMG) with high affinity for DNA. In parallel, a 38 amino acid region preceding the HMG domain described as the dimerization domain supports the formation of cooperative dimers on promoters with inverted sites, by binding to the HMG domain. Cooperative binding occurs when the binding at a first site increases the rate at which binding occurs at a second site between identical or non-identical proteins. Using mutagenesis and electrophoretic mobility shift assays (EMSA) the transdimerization model of dimeric SOX9 binding to DNA was studied. The molecular model of the SOX9 dimeric complex is characterized as a dimer by way of the amphipathic helix of the dimerization domain of one SOX9 protein docks on the HMG domain of an identical SOX9 protein in the DNA-bound state. Cooperative binding of one protein to the consensus-binding site may facilitate binding to the non-consensus site. This model provides insight to the characterized mutations associated with Campomelic dysplasia, an embryonic malformation of the skeletal and reproductive system

Practicum Experiences of Prospective Teachers: The Inevitable Adaptation to Teaching Online

Prospective teachers (students) in the Bachelor of Education (BEd.) programme at the University of Trinidad and Tobago, Year Three, would have been required to conduct ‘field’ practicum at various schools throughout the country, under normal circumstances. However, due to the pandemic and the closure of all schools, only online classes were conducted and adaptations had to be made to acclimatize to the situation. Prospective teachers were required to engage in teaching to their peers online, together with micro-teaching which complemented the process. Instructors as well as classmates and presenters engaged in critique, rigorous analysis, reflection and feedback. The sample included forty (40) full-time students, generally between the ages of 18 to 25, who had no previous classroom teaching experiences. A mixed-method approach was deemed as suitable to investigate how prospective teachers adapted to online teaching and learning to implement the content of the prescribed curriculum. Online questionnaires, focus group interviews, via Zoom and reflective writings were used to collect data. Both qualitative and quantitative methods were integrated for data analyses. The findings revealed that most prospective teachers were very proficient with the use of the Zoom platform and were very creative in their approaches for teaching using videos, online games, power point presentations and voice-overs. However, there were instances of major problems involving internet connectivity and instability, particularly for those who live in remote villages. In addition, some students preferred online-teaching, while others indicated a preference for a blended approach. The results have serious connotations for the health and well-being of students. Those who are constrained by a lack of internet feel disadvantaged, vulnerable and marginalized

A Role for Amyloid in Cell Aggregation and Biofilm Formation

Cell adhesion molecules in Saccharomyces cerevisiae and Candida albicans contain amyloid-forming sequences that are highly conserved. We have now used site-specific mutagenesis and specific peptide perturbants to explore amyloid-dependent activity in the Candida albicans adhesin Als5p. A V326N substitution in the amyloid-forming region conserved secondary structure and ligand binding, but abrogated formation of amyloid fibrils in soluble Als5p and reduced cell surface thioflavin T fluorescence. When displayed on the cell surface, Als5p with this substitution prevented formation of adhesion nanodomains and formation of large cellular aggregates and model biofilms. In addition, amyloid nanodomains were regulated by exogenous peptides. An amyloid-forming homologous peptide rescued aggregation and biofilm activity of Als5pV326N cells, and V326N substitution peptide inhibited aggregation and biofilm activity in Als5pWT cells. Therefore, specific site mutation, inhibition by anti-amyloid peturbants, and sequence-specificity of pro-amyloid and anti-amyloid peptides showed that amyloid formation is essential for nanodomain formation and activation

Effects of Age and Sex on Inspiratory Muscle Activation Patterns During Exercise

Purpose: Characterize the effects of age, sex, and their interaction on inspiratory muscle activation patterns during exercise. Methods: Twenty younger (20-30y, n=10 women) and twenty older (60-80y, n=10 women) subjects performed an incremental cycle exercise test. Electromyography of the scalene (EMGsca) and sternocleidomastoid (EMGscm) muscles were measured using skin surface electrodes, while diaphragm electromyography (EMGdi) and esophageal and transdiaphragmatic pressures were measured using an esophageal catheter. Electromyography data were transformed into root-mean-square with a 100ms time constant. Esophageal (PTPes) and diaphragmatic (PTPdi) pressure-time products were used as indices of total inspiratory muscle pressure production and diaphragmatic pressure production, respectively. Results: At absolute minute ventilations (V E), women and older subjects had greater EMGdi than men and younger subjects, respectively (all p<0.05), but no differences were noted when V E was expressed in relative terms (all p>0.05). Women had greater EMGsca activity than men at absolute and relative levels of V E (all p<0.05). Older subjects had greater EMGsca than younger subjects when V E was expressed relative (all p<0.05) but not absolute terms (all p>0.05). At absolute and relative levels of V E, women and older subjects had greater EMGscm than men and younger subjects, respectively (all p<0.05). Women and older subjects had a greater PTPdi/PTPes at a V E of 70 l·min-1 than men and younger subjects, respectively (both p<0.05), but no differences were noted when V E was expressed in relative terms (all p>0.05). No significant interactions between age and sex were noted (all p>0.05). Conclusion: Age and sex significantly affect inspiratory muscle activation patterns during exercise; however, the extent of the effects depends on whether comparisons are made at a solute or relative V E.This study was supported by the British Columbia Lung Association (BCLA). YMS, PBD, and AHR were supported by graduate scholarships from the Natural Sciences and Engineering Research Council of Canada (NSERC). PBD and MRS were supported by fellowships from the University of British Columbia and BCLA. JAG was supported by a Scholar Award from the Michael Smith Foundation for Health Research, a New Investigator Award from the Providence Health Care Research Institute and St. Paul’s Hospital Foundation, and a Canadian Institutes of Health Research Clinical Rehabilitation New Investigator Award

Manipulation of mechanical ventilatory constraint during moderate intensity exercise does not influence dyspnoea in healthy older men and women

© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society. KEY POINT SUMMARY:The perceived intensity of exertional breathlessness (i.e. dyspnoea) is higher in older women than in older men, possibly due to sex-difference respiratory system morphology. During exercise at a given absolute intensity or minute ventilation, older women have a greater degree of mechanical ventilatory constraint (i.e. work of breathing and expiratory flow limitation) than their male counterparts, which may lead to a greater perceived intensity of dyspnoea. Using a single-blind randomized study design, we experimentally manipulated the magnitude of mechanical ventilatory constraint during moderate-intensity exercise at ventilatory threshold in healthy older men and women. We found that changes in the magnitude of mechanical ventilatory constraint within the physiological range had no effect on dyspnoea in healthy older adults. When older men and women perform submaximal exercise at a moderate intensity, mechanical ventilatory constraint does not contribute significantly to the sensation of dyspnoea. We sought to determine the effect of manipulating mechanical ventilatory constraint during submaximal exercise on dyspnoea in older men and women. METHODS:Eighteen healthy subjects (60-80 y; 9 men, 9 women) completed two days of testing. On Day 1, subjects performed pulmonary function testing and a maximal incremental cycle exercise test. On Day 2, subjects performed three 6-min bouts of cycling at ventilatory threshold, in a single-blind randomized manner, while breathing: i) normoxic helium-oxygen (HEL) to reduce the work of breathing (Wb ) and alleviate expiratory flow limitation (EFL); ii) through an inspiratory resistance (RES) of ∼5 cmH2 O·l-1 ·s-1 to increase Wb ; and iii) ambient air as a control (CON). Oesophageal pressure, diaphragm electromyography, and sensory responses (using the category-ratio 10 Borg scale) were monitored throughout exercise. RESULTS:During the HEL condition, there was a significant decrease in Wb (men: -21 ± 6%, women: -17 ± 10%) relative to CON (both p < 0.01). Moreover, if EFL was present during CON (4 men, 5 women), it was alleviated during HEL. Conversely, during the RES condition, Wb (men: 42 ± 19%, women: 50 ± 16%) significantly increased relative to CON (both p < 0.01). There was no main effect of sex on Wb (p = 0.59). Across conditions, women reported significantly higher dyspnoea intensity than men (2.9 ± 0.9 vs. 1.9 ± 0.8 Borg scale units, p < 0.05). Despite significant differences in the degree of mechanical ventilatory constraint between conditions, dyspnoea intensity was unaffected, independent of sex (p = 0.46). CONCLUSION:When older men and women perform submaximal exercise at a moderate intensity, mechanical ventilatory constraint does not contribute significantly to the sensation of dyspnoea. This article is protected by copyright. All rights reserved.Natural Science and Engineering Research Council of Canada (NSERC) British Columbia Lung Association (BCLA

Force Sensitivity in Saccharomyces cerevisiae Flocculins

Many fungal adhesins have short, -aggregation-prone sequences that play important functional roles, and in the Candida albicans adhesin Als5p, these sequences cluster the adhesins after exposure to shear force. Here, we report that Saccharomyces cerevisiae flocculins Flo11p and Flo1p have similar -aggregation-prone sequences and are similarly stimulated by shear force, despite being nonhomologous. Shear from vortex mixing induced the formation of small flocs in cells expressing either adhesin. After the addition of Ca2, yeast cells from vortex-sheared populations showed greatly enhanced flocculation and displayed more pronounced thioflavin-bright surface nanodomains. At high concentrations, amyloidophilic dyes inhibited Flo1p- and Flo11p-mediated agar invasion and the shear-induced increase in flocculation. Consistent with these results, atomic force microscopy of Flo11p showed successive force-distance peaks characteristic of sequentially unfolding tandem repeat domains, like Flo1p and Als5p. Flo11p-expressing cells bound together through homophilic interactions with adhesion forces of up to 700 pN and rupture lengths of up to 600 nm. These results are consistent with the potentiation of yeast flocculation by shear-induced formation of high-avidity domains of clustered adhesins at the cell surface, similar to the activation of Candida albicans adhesin Als5p. Thus, yeast adhesins from three independent gene families use similar force dependent interactions to drive cell adhesion

Oral Ondansetron Administration in Emergency Departments to Children with Gastroenteritis: An Economic Analysis

Stephen Freedman and colleagues performed a cost analysis of the routine administration of ondansetron in both the United States and Canada and show that its routine administration to eligible children in such settings could provide substantial benefit

The GPI Anchor Signal Sequence Dictates the Folding and Functionality of the Als5 Adhesin from Candida albicans

Background: Proteins destined to be Glycosylphosphatidylinositol (GPI) anchored are translocated into the ER lumen completely before the C-terminal GPI anchor attachment signal sequence (SS) is removed by the GPI-transamidase and replaced by a pre-formed GPI anchor precursor. Does the SS have a role in dictating the conformation and function of the protein as well? Methodology/Principal Findings: We generated two variants of the Als5 protein without and with the SS in order to address the above question. Using a combination of biochemical and biophysical techniques, we show that in the case of Als5, an adhesin of C. albicans, the C-terminal deletion of 20 amino acids (SS) results in a significant alteration in conformation and function of the mature protein. Conclusions/Significance: We propose that the locking of the conformation of the precursor protein in an alternate conformation from that of the mature protein is one probable strategy employed by the cell to control the behaviour an

Oral ondansetron versus domperidone for symptomatic treatment of vomiting during acute gastroenteritis in children: multicentre randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Vomiting in children with acute gastroenteritis (AG) is not only a direct cause of fluid loss but it is also a major factor of failure of oral rehydration therapy (ORT). Physicians who provide care to paediatric patients in the emergency department (ED) usually prescribe intravenous fluid therapy (IVT) for mild or moderate dehydration when vomiting is the major symptom. Thus, effective symptomatic treatment of vomiting would lead to an important reduction in the use of IVT and, consequently, of the duration of hospital stay and of frequency of hospital admission. Available evidence on symptomatic treatment of vomiting shows the efficacy of the most recently registered molecule (ondansetron) but a proper evaluation of antiemetics drugs largely used in clinical practice, such as domperidone, is lacking.</p> <p>Objectives</p> <p>To compare the efficacy of ondansetron and domperidone for the symptomatic treatment of vomiting in children with AG who have failed ORT.</p> <p>Methods/Design</p> <p>Multicentre, double-blind randomized controlled trial conducted in paediatric EDs. Children aged from 1 to 6 years who vomiting, with a presumptive clinical diagnosis of AG, and without severe dehydration will be included. After the failure of a initial ORS administration in ED, eligible children will be randomized to receive: 1) ondansetron syrup (0,15 mg/Kg of body weight); 2) domperidone syrup (0,5 mg/Kg of body weight); 3) placebo. The main study outcome will be the percentage of patients needing nasogastric or IVT after symptomatic oral treatment failure, defined as vomiting or fluid refusal after a second attempt of ORT. Data relative to study outcomes will be collected at 30 minute intervals for a minimum of 6 hours. A telephone follow up call will be made 48 hours after discharge. A total number of 540 children (i.e. 180 patients in each arm) will be enrolled.</p> <p>Discussion</p> <p>The trial results would provide evidence on the efficacy of domperidone, which is largely used in clinical practice despite the lack of proper evaluation and a controversial safety profile, as compared to ondansetron, which is not yet authorized in Italy despite evidence supporting its efficacy in treating vomiting. The trial results would contribute to a reduction in the use of IVT and, consequently, in hospital admissions in children with AG. The design of this RCT, which closely reflect current clinical practice in EDs, will allow immediate transferability of results.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov: <a href="http://www.clinicaltrials.gov/ct2/show/NCT01257672">NCT01257672</a></p