Spatiotemporal and functional characterisation of transient receptor potential vanilloid 4 (TRPV4) in the murine intervertebral disc

The molecular regulators of mechano-transduction in intervertebral disc (IVD) cells are not well understood. The aim of the present study was to characterise the expression and function of the mechano-sensitive ion channel TRPV4 in the IVD. A novel transgenic reporter mouse, in which the endogenous Trpv4 locus drove the expression of LacZ, was used to localise Trpv4 expression at specific stages of spine development [embryonic day (E) 8.5, 12.5, 17.5, postnatal day 1] and time points following skeletal maturity (2.5, 6, 9 and 12 months). The TRPV4-specific agonist GSK1016790A and antagonist GSK2193874 were used to assess the functional response of annulus fibrosus (AF) cells using epifluorescence imaging with Ca2+-sensitive Fura-2 dye and F-actin staining. The effects of TRPV4 agonism and antagonism in mechanically stimulated AF cells were quantified by gene expression analysis. Trpv4 expression was specific to the developing notochord and intervertebral mesenchyme at E12.5. At 2.5, 6 and 9 months, Trpv4 expression was detected in the nucleus pulposus, inner AF, cartilage endplate and vertebral growth plate. AF cells treated with GSK1016790A demonstrated heterogeneity in TRPV4-dependent Ca2+ responses (no response, calcium oscillation or sustained response). TRPV4-induced Ca2+ signalling was associated with Rho/ROCK-dependent actin cytoskeleton remodelling and stress-fibre formation. In AF cells, cyclic-tensile-strain-induced changes in Acan and Prg4 expression were mediated by TRPV4 channel activation. These data establish TRPV4 as an important mechano-sensor regulating IVD mechano-biology

An integrative SDT-based investigation of the relation between financial incentives, motivation, and performance

To this day, researchers are debating the adequacy of using financial incentives to bolster performance in work settings. Our goal was to contribute to current understanding by considering the moderating role of distributive justice in the relation between financial incentives, motivation, and performance. Based on self-determination theory, we hypothesized that when bonuses are fairly distributed, using financial incentives makes employees feel more competent and autonomous, which in turn fosters greater autonomous motivation and lower controlled motivation, and better work performance. Results from path analyses in three samples supported our hypotheses, suggesting that the effect of financial incentives is contextual, and that compensation plans using financial incentives and bonuses can be effective when properly managed

A multi-target antisense approach against PDE4 and PDE7 reduces smoke-induced lung inflammation in mice

<p>Abstract</p> <p>Background</p> <p>Recent development in the field of COPD has focused on strategies aimed at reducing the underlying inflammation through selective inhibition of the phosphodiesterase type IV (PDE4) isoform. Although the anti-inflammatory and bronchodilator activity of selective PDE4 inhibitors has been well documented, their low therapeutic ratio and dose-dependent systemic side effects have limited their clinical utility. This study examined the effect of 2'-deoxy-2'-Fluoro-β-D-Arabinonucleic Acid (FANA)-containing antisense oligonucleotides (AON) targeting the mRNA for the PDE4B/4D and 7A subtypes on lung inflammatory markers, both <it>in vitro </it>and <it>in vivo</it>.</p> <p>Methods</p> <p>Normal human bronchial epithelial (NHBE) cells were transfected with FANA AON against PDE4B/4D and 7A alone or in combination. mRNA levels for target PDE subtypes, as well as secretion of pro-inflammatory chemokines were then measured following cell stimulation. Mice were treated with combined PDE4B/4D and 7A AON via endo-tracheal delivery, or with roflumilast via oral delivery, and exposed to cigarette smoke for one week. Target mRNA inhibition, as well as influx of inflammatory cells and mediators were measured in lung lavages. A two-week smoke exposure protocol was also used to test the longer term potency of PDE4B/4D and 7A AONs.</p> <p>Results</p> <p>In NHBE cells, PDE4B/4D and 7A AONs dose-dependently and specifically inhibited expression of their respective target mRNA. When used in combination, PDE4B/4D and 7A AONs significantly abrogated the cytokine-induced secretion of IL-8 and MCP-1 to near baseline levels. In mice treated with combined PDE4B/4D and 7A AONs and exposed to cigarette smoke, significant protection against the smoke-induced recruitment of neutrophils and production of KC and pro-MMP-9 was obtained, which was correlated with inhibition of target mRNA in cells from lung lavages. In this model, PDE AONs exerted more potent and broader anti-inflammatory effects against smoke-induced lung inflammation than roflumilast. Moreover, the protective effect of PDE4B/4D and 7A AON was maintained when a once-weekly treatment schedule was used.</p> <p>Conclusion</p> <p>These results indicate that inhaled AON against PDE4B/4D and 7A have unique effects on biomarkers that are believed to be important in the pathophysiology of COPD, which supports further development as a potential therapy in this disease.</p

The mechano-response of murine annulus fibrosus cells to cyclic tensile strain is frequency dependent

The intervertebral disk (IVD) is a composite structure essential for spine stabilization, load bearing, and movement. Biomechanical factors are important contributors to the IVD microenvironment regulating joint homeostasis; however, the cell type-specific effectors of mechanotransduction in the IVD are not fully understood. The current study aimed to determine the effects of cyclic tensile strain (CTS) on annulus fibrosus (AF) cells and identify mechano-sensitive pathways. Using a cell-type specific reporter mouse to differentiation NP and AF cells from the murine IVD, we characterized AF cells in dynamic culture exposed to CTS (6% strain) at specific frequencies (0.1 Hz, 1.0 Hz, or 2.0 Hz). We demonstrate that our culture model maintains the phenotype of primary AF cells and that the bioreactor system delivers uniform biaxial strain across the cell culture surface. We show that exposure of AF cells to CTS induces cytoskeleton reorganization resulting in stress fiber formation, with acute exposure to CTS at 2.0 Hz inducing a significant yet transient increase ERK1/2 pathway activation. Using SYBPR-based qPCR to assess the expression of extracellular matrix (ECM) genes, ECM-remodeling genes, candidate mechano-sensitive genes, inflammatory cytokines and cell surface receptors, we demonstrated that exposure of AF cells to CTS at 0.1 Hz increased Acan, Prg4, Col1a1 and Mmp3 expression. AF cells exposed to CTS at 1.0 Hz showed a significant increase in the expression of Acan, Myc, and Tnfα. Exposure of AF cells to CTS at 2.0 Hz induced a significant increase in Acan, Prg4, Cox2, Myc, Fos, and Tnfα expression. Among the cell surface receptors assessed, AF cells exposed to CTS at 2.0 Hz showed a significant increase in Itgβ1, Itgα5, and Trpv4 expression. Our findings demonstrate that the response of AF cells to CTS is frequency dependent and suggest that mechanical loading may directly contribute to matrix remodeling and the onset of local tissue inflammation in the murine IVD

The role of panx3 in age-associated and injury-induced intervertebral disc degeneration

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. Pannexin 3 (Panx3) is a mechanosensitive, channel-forming glycoprotein implicated in the progression of post-traumatic osteoarthritis. Despite evidence for Panx3 expression in the intervertebral disc (IVD), its function in this cartilaginous joint structure remained unknown. Using Panx3 knockout mice, this study investigated the role of Panx3 in age-associated IVD degeneration and degeneration induced by annulus fibrosus (AF) needle puncture. Loss of Panx3 did not significantly impact the progression of age-associated histopathological IVD degeneration; however, loss of Panx3 was associated with decreased gene expression of Acan, Col1a1, Mmp13 and Runx2 and altered localization of COLX in the IVD at 19 months-of-age. Following IVD injury in the caudal spine, histological analysis of wild-type mice revealed clusters of hypertrophic cells in the AF associated with increased pericellular proteoglycan accumulation, disruptions in lamellar organization and increased lamellar thickness. In Panx3 knockout mice, hypertrophic AF cells were rarely detected and AF structure was largely preserved post-injury. Interestingly, uninjured IVDs adjacent to the site of injury more frequently showed evidence of early nucleus pulposus degeneration in Panx3 knockout mice but remained healthy in wild-type mice. These findings suggest a role for Panx3 in mediating the adaptive cellular responses to altered mechanical stress in the IVD, which may buffer aberrant loads transferred to adjacent motion segments

Genome-wide transcriptional response of an avian pathogenic Escherichia coli (APEC) pst mutant

<p>Abstract</p> <p>Background</p> <p>Avian pathogenic <it>E</it>. <it>coli </it>(APEC) are associated with extraintestinal diseases in poultry. The <it>pstSCAB</it>-<it>phoU </it>operon belongs to the Pho regulon and encodes the phosphate specific transport (Pst) system. A functional Pst system is required for full virulence in APEC and other bacteria and contributes to resistance of APEC to serum, to cationic antimicrobial peptides and acid shock. The global mechanisms contributing to the attenuation and decreased resistance of the APEC <it>pst </it>mutant to environmental stresses have not been investigated at the transcriptional level. To determine the global effect of a <it>pst </it>mutation on gene expression, we compared the transcriptomes of APEC strain χ7122 and its isogenic <it>pst </it>mutant (K3) grown in phosphate-rich medium.</p> <p>Results</p> <p>Overall, 470 genes were differentially expressed by at least 1.5-fold. Interestingly, the <it>pst </it>mutant not only induced systems involved in phosphate acquisition and metabolism, despite phosphate availability, but also modulated stress response mechanisms. Indeed, transcriptional changes in genes associated with the general stress responses, including the oxidative stress response were among the major differences observed. Accordingly, the K3 strain was less resistant to reactive oxygen species (ROS) than the wild-type strain. In addition, the <it>pst </it>mutant demonstrated reduced expression of genes involved in lipopolysaccharide modifications and coding for cell surface components such as type 1 and F9 fimbriae. Phenotypic tests also established that the <it>pst </it>mutant was impaired in its capacity to produce type 1 fimbriae, as demonstrated by western blotting and agglutination of yeast cells, when compared to wild-type APEC strain χ7122.</p> <p>Conclusion</p> <p>Overall, our data elucidated the effects of a <it>pst </it>mutation on the transcriptional response, and further support the role of the Pho regulon as part of a complex network contributing to phosphate homeostasis, adaptive stress responses, and <it>E. coli </it>virulence.</p

Differences and similarities in instant countertransference towards patients with suicidal ideation and personality disorders.

Previous findings showed that suicidal patients elicit mostly negative countertransference such as distress, hopelessness, feelings of inadequacy, and apprehension, and that a concurrent personality disorder is associated with more feelings of entrapment and mistreatment, among other adverse reactions. No studies were however conducted on instant countertransference (iCT), i.e., after a single encounter, for example in an emergency setting. We aimed to evaluate the impact of suicidal ideations, self-harm and presence of personality disorders on instant Countertransference (iCT). Caregivers rated their iCT with two validated and standardized questionnaires after a first emergency or outpatient consultation. Suicidal ideation, self-harm and personality disorders were tested as predictors for iCT in a multivariate and multilevel analysis. Thirty caregivers rated their iCT towards 321 patients. Personality disorders and suicidal ideation, but neither recent nor past history of self-harm, predicted iCT. Common iCT included tension, lack of self-confidence and feeling of being tied. iCT specifically associated with suicidal ideation included distress, lack of hope, confusion, and sense that the patient's life had little worth. In contrast, iCT towards patients with personality disorders suggested tension in the therapeutic relationship (low affiliation with patient, anger, disappointment, devaluation). Caregiver's characteristics were not considered in the analysis. Furthermore, while countertransference also includes unconscious phenomena, only conscious iCT was assessed. Patients with suicidal ideation and personality disorders elicit common but also specific negative iCT. Mental health institutions need to devote specific resources (such as clinical supervision and training) to help caregivers manage their iCT

A chemoenzymatic route to chiral siloxanes

An approach employing two enzymes—toluene dioxygenase and immobilized lipase B from Candida antarctica (N435)—was explored as a potential biocatalytic method for the coupling of chiral diols with siloxane species. Analysis of reaction mixtures using1H NMR spectroscopy suggested that up to 66% consumption of the siloxane starting materials had occurred. Oligomeric species were observed and chiral products from the coupling of a cyclic diol with a siloxane molecule were isolated and characterized by MALDI-ToF MS and GPC. Immobilized lipases from Rhizomucor miehei and Thermomyces lanuginosus were also explored as potential catalysts for the coupling reactions, however, their use only returned starting material

A lower bound for the two-variable Artin conjecture and prime divisors of recurrence sequences

The final publication is available at Elsevier via https://dx.doi.org/10.1016/j.jnt.2018.06.017 © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/In 1927, Artin conjectured that any integer other than −1 or a perfect square generates the multiplicative group (Z/pZ)× for infinitely many p. In 2000, Moree and Stevenhagen considered a two-variable version of this problem, and proved a positive density result conditionally to the generalized Riemann Hypothesis by adapting a proof by Hooley for the original conjecture. In this article, we prove an unconditional lower bound for this two-variable problem. In particular, we prove an estimate for the number of distinct primes which divide one of the first N terms of a non-degenerate binary recurrence sequence. We also prove a weaker version of the same theorem, and give three proofs that we consider to be of independent interest. The first proof uses a transcendence result of Stewart, the second uses a theorem of Bombieri and Schmidt on Thue equations and the third uses Mumford's gap principle for counting points on curves by their height. We finally prove a disjunction theorem, where we consider the set of primes satisfying either our two-variable condition or the original condition of Artin's conjecture. We give an unconditional lower bound for the number of such primes.Natural Sciences and Engineering Research Council of CanadaFonds de Recherche du Québec - Nature et TechnologiesCanada Research Chair