IRE1α recognizes a structural motif in cholera toxin to activate an unfolded protein response.

IRE1α is an endoplasmic reticulum (ER) sensor that recognizes misfolded proteins to induce the unfolded protein response (UPR). We studied cholera toxin (CTx), which invades the ER and activates IRE1α in host cells, to understand how unfolded proteins are recognized. Proximity labeling colocalized the enzymatic and metastable A1 segment of CTx (CTxA1) with IRE1α in live cells, where we also found that CTx-induced IRE1α activation enhanced toxicity. In vitro, CTxA1 bound the IRE1α lumenal domain (IRE1αLD), but global unfolding was not required. Rather, the IRE1αLD recognized a seven-residue motif within an edge ß-strand of CTxA1 that must locally unfold for binding. Binding mapped to a pocket on IRE1αLD normally occupied by a segment of the IRE1α C-terminal flexible loop implicated in IRE1α oligomerization. Mutation of the CTxA1 recognition motif blocked CTx-induced IRE1α activation in live cells, thus linking the binding event with IRE1α signal transduction and induction of the UPR.

Mechanisms underlying distinct subcellular localization and regulation of epithelial long myosin light-chain kinase splice variants.

Intestinal epithelia express two long myosin light-chain kinase (MLCK) splice variants, MLCK1 and MLCK2, which differ by the absence of a complete immunoglobulin (Ig)-like domain 3 within MLCK2. MLCK1 is preferentially associated with the perijunctional actomyosin ring at steady state, and this localization is enhanced by inflammatory stimuli including tumor necrosis factor (TNF). Here, we sought to identify MLCK1 domains that direct perijunctional MLCK1 localization and their relevance to disease. Ileal biopsies from Crohn's disease patients demonstrated preferential increases in MLCK1 expression and perijunctional localization relative to healthy controls. In contrast to MLCK1, MLCK2 expressed in intestinal epithelia is predominantly associated with basal stress fibers, and the two isoforms have distinct effects on epithelial migration and barrier regulation. MLCK1(Ig1-4) and MLCK1(Ig1-3), but not MLCK2(Ig1-4) or MLCK1(Ig3), directly bind to F-actin in vitro and direct perijunctional recruitment in intestinal epithelial cells. Further study showed that Ig1 is unnecessary, but that, like Ig3, the unstructured linker between Ig1 and Ig2 (Ig1/2us) is essential for recruitment. Despite being unable to bind F-actin or direct recruitment independently, Ig3 does have dominant negative functions that allow it to displace perijunctional MLCK1, increase steady-state barrier function, prevent TNF-induced MLCK1 recruitment, and attenuate TNF-induced barrier loss. These data define the minimal domain required for MLCK1 localization and provide mechanistic insight into the MLCK1 recruitment process. Overall, the results create a foundation for development of molecularly targeted therapies that target key domains to prevent MLCK1 recruitment, restore barrier function, and limit inflammatory bowel disease progression.

Maximum tolerable daily dose of mirror movement therapy ankle exercises after stroke: an early phase dose screening study.

BACKGROUND: Mirror movement therapy may reduce lower limb motor impairment after stroke. The dose is unknown. OBJECTIVE: identify the maximum tolerable dose a day (MTD) of lower limb mirror movement therapy DESIGN: 3 + 3 cohort rule-based, dose escalation/de-escalation study. After undertaking baseline measures participants performed mirror movement therapy for 14 consecutive days. Participants then undertook outcome measures. Cohort One trained for 15 minutes daily. Subsequent cohorts exercised at a dose set according to pre-set rules and the modified Fibonacci sequence. The study stopped when the difference between set doses for consecutive cohorts was 10% or less. SETTING: Participants' homes (intervention) and a movement analysis laboratory (measures). PARTICIPANTS: Adults discharged from statutory stroke rehabilitation services. INTERVENTION: Mirror movement therapy ankle exercises. OUTCOME MEASURES: Motricity Index (primary) and bilateral time symmetry from movement onset to peak activation of Tibialis Anterior muscles during standardised sit-to-stand (secondary). RESULTS: Five cohorts of three participants were included (n = 15). Mean (SD) age and time after stroke were 61 (9) years and 35 (42) months respectively. Set daily doses for the five cohorts were: 15, 30, 50, 40 then 35 minutes. The set dose for a subsequent cohort (six) would have been 38 minutes thus the difference from cohort five would have been three minutes i.e., 9% different. Therefore, the study stopped CONCLUSION: The identified MTD of lower limb mirror therapy was 35 minutes daily when frequency was set at seven days a week and duration as two weeks. CLINICAL TRIAL REGISTRATION NUMBER: NCT04339803 (ClinicalTrials.gov) CONTRIBUTION OF THE PAPER: This early phase study found that the maximum tolerable dose per day (MTD) of mirror movement therapy ankle exercises was 35 minutes when frequency was set at seven days a week and duration as two weeks. The optimal therapeutic dose will therefore be somewhere in the range of 15 (starting dose) to 35 minutes per day. Further dose articulation studies are required to identify the optimal therapeutic dose before use of findings in clinical practice. This study is the first step in that research process.

p120 RasGAP and ZO-2 are essential for Hippo signaling and tumor-suppressor function mediated by p190A RhoGAP.

ARHGAP35, which encodes p190A RhoGAP (p190A), is a major cancer gene. p190A is a tumor suppressor that activates the Hippo pathway. p190A was originally cloned via direct binding to p120 RasGAP (RasGAP). Here, we determine that interaction of p190A with the tight-junction-associated protein ZO-2 is dependent on RasGAP. We establish that both RasGAP and ZO-2 are necessary for p190A to activate large tumor-suppressor (LATS) kinases, elicit mesenchymal-to-epithelial transition, promote contact inhibition of cell proliferation, and suppress tumorigenesis. Moreover, RasGAP and ZO-2 are required for transcriptional modulation by p190A. Finally, we demonstrate that low ARHGAP35 expression is associated with shorter survival in patients with high, but not low, transcript levels of TJP2 encoding ZO-2. Hence, we define a tumor-suppressor interactome of p190A that includes ZO-2, an established constituent of the Hippo pathway, and RasGAP, which, despite strong association with Ras signaling, is essential for p190A to activate LATS kinases.

Stressing out over mucus secretion.

In this issue of Cell Host & Microbe, Naama et al. show that autophagy controls mucus secretion in the colons of mice. They demonstrate that autophagy reduces ER stress in mucus-producing goblet cells to enhance mucus production, which shapes the gut microbial community and protects against colitis.

The epithelial-specific ER stress sensor ERN2/IRE1ß enables host-microbiota crosstalk to affect colon goblet cell development.

Epithelial cells lining mucosal surfaces of the gastrointestinal and respiratory tracts uniquely express ERN2/IRE1ß, a paralogue of the most evolutionarily conserved endoplasmic reticulum stress sensor, ERN1/IRE1α. How ERN2 functions at the host-environment interface and why a second paralogue evolved remain incompletely understood. Using conventionally raised and germ-free Ern2-/- mice, we found that ERN2 was required for microbiota-induced goblet cell maturation and mucus barrier assembly in the colon. This occurred only after colonization of the alimentary tract with normal gut microflora, which induced Ern2 expression. ERN2 acted by splicing Xbp1 mRNA to expand ER function and prevent ER stress in goblet cells. Although ERN1 can also splice Xbp1 mRNA, it did not act redundantly to ERN2 in this context. By regulating assembly of the colon mucus layer, ERN2 further shaped the composition of the gut microbiota. Mice lacking Ern2 had a dysbiotic microbial community that failed to induce goblet cell development and increased susceptibility to colitis when transferred into germ-free WT mice. These results show that ERN2 evolved at mucosal surfaces to mediate crosstalk between gut microbes and the colonic epithelium required for normal homeostasis and host defense.

Gut microbiota regulation of P-glycoprotein in the intestinal epithelium in maintenance of homeostasis.

BACKGROUND: P-glycoprotein (P-gp) plays a critical role in protection of the intestinal epithelia by mediating efflux of drugs/xenobiotics from the intestinal mucosa into the gut lumen. Recent studies bring to light that P-gp also confers a critical link in communication between intestinal mucosal barrier function and the innate immune system. Yet, despite knowledge for over 10 years that P-gp plays a central role in gastrointestinal homeostasis, the precise molecular mechanism that controls its functional expression and regulation remains unclear. Here, we assessed how the intestinal microbiome drives P-gp expression and function. RESULTS: We have identified a "functional core" microbiome of the intestinal gut community, specifically genera within the Clostridia and Bacilli classes, that is necessary and sufficient for P-gp induction in the intestinal epithelium in mouse models. Metagenomic analysis of this core microbial community revealed that short-chain fatty acid and secondary bile acid production positively associate with P-gp expression. We have further shown these two classes of microbiota-derived metabolites synergistically upregulate P-gp expression and function in vitro and in vivo. Moreover, in patients suffering from ulcerative colitis (UC), we find diminished P-gp expression coupled to the reduction of epithelial-derived anti-inflammatory endocannabinoids and luminal content (e.g., microbes or their metabolites) with a reduced capability to induce P-gp expression. CONCLUSION: Overall, by means of both in vitro and in vivo studies as well as human subject sample analysis, we identify a mechanistic link between cooperative functional outputs of the complex microbial community and modulation of P-gp, an epithelial component, that functions to suppress overactive inflammation to maintain intestinal homeostasis. Hence, our data support a new cross-talk paradigm in microbiome regulation of mucosal inflammation. Video abstract.

User perspectives on the design and setup of lower limb mirror therapy equipment after stroke: a technical report.

OBJECTIVES: To co-design lower limb mirror therapy (MT) equipment and setup by working directly with stroke survivors and physiotherapists. DESIGN: Co-design approach through focus groups. PARTICIPANTS: Twenty-six participants. Sixteen stroke survivors and ten physiotherapists. DATA COLLECTION AND ANALYSIS: Data were collected in an iterative process through two sets of focus groups. Firstly, prototype one of the MT equipment was presented to the participants. They were encouraged to use and comment on it. Then, the key requirements for ankle exercise with MT were presented, and participants discussed whether the prototype one was able to deliver these requirements. These findings informed iterations to the device, and a second prototype was produced and discussed in the second set of focus groups. The final prototype was then produced based on the participants' feedback. All focus groups were audio-recorded, followed by verbatim transcriptions and thematic analysis. RESULTS: Main characteristics required of the lower limb MT device were found to be: the ability to produce MT ankle exercise from an upright sitting posture, an adjustable angle between 5 to 15 degree from the midline to allow clear lower limb reflection during seated exercise, and a lightweight device to enable easy use for stroke survivors. CONCLUSION: This work produced an iteratively co-design lower limb MT to be used with stroke survivors.

Evolution and function of the epithelial cell-specific ER stress sensor IRE1ß.

Barrier epithelial cells lining the mucosal surfaces of the gastrointestinal and respiratory tracts interface directly with the environment. As such, these tissues are continuously challenged to maintain a healthy equilibrium between immunity and tolerance against environmental toxins, food components, and microbes. An extracellular mucus barrier, produced and secreted by the underlying epithelium plays a central role in this host defense response. Several dedicated molecules with a unique tissue-specific expression in mucosal epithelia govern mucosal homeostasis. Here, we review the biology of Inositol-requiring enzyme 1ß (IRE1ß), an ER-resident endonuclease and paralogue of the most evolutionarily conserved ER stress sensor IRE1α. IRE1ß arose through gene duplication in early vertebrates and adopted functions unique from IRE1α which appear to underlie the basic development and physiology of mucosal tissues.

IRE1ß negatively regulates IRE1α signaling in response to endoplasmic reticulum stress.

IRE1ß is an ER stress sensor uniquely expressed in epithelial cells lining mucosal surfaces. Here, we show that intestinal epithelial cells expressing IRE1ß have an attenuated unfolded protein response to ER stress. When modeled in HEK293 cells and with purified protein, IRE1ß diminishes expression and inhibits signaling by the closely related stress sensor IRE1α. IRE1ß can assemble with and inhibit IRE1α to suppress stress-induced XBP1 splicing, a key mediator of the unfolded protein response. In comparison to IRE1α, IRE1ß has relatively weak XBP1 splicing activity, largely explained by a nonconserved amino acid in the kinase domain active site that impairs its phosphorylation and restricts oligomerization. This enables IRE1ß to act as a dominant-negative suppressor of IRE1α and affect how barrier epithelial cells manage the response to stress at the host-environment interface.

A modified cholera toxin B subunit containing an ER retention motif enhances colon epithelial repair via an unfolded protein response.

Cholera toxin B subunit (CTB) exhibits broad-spectrum biologic activity upon mucosal administration. Here, we found that a recombinant CTB containing an endoplasmic reticulum (ER) retention motif (CTB-KDEL) induces colon epithelial wound healing in colitis via the activation of an unfolded protein response (UPR) in colon epithelial cells. In a Caco2 cell wound healing model, CTB-KDEL, but not CTB or CTB-KDE, facilitated cell migration via interaction with the KDEL receptor, localization in the ER, UPR activation, and subsequent TGF-ß signaling. Inhibition of the inositol-requiring enzyme 1/X-box binding protein 1 arm of UPR abolished the cell migration effect of CTB-KDEL, indicating that the pathway is indispensable for the activity. CTB-KDEL's capacity to induce UPR and epithelial restitution or wound healing was corroborated in a dextran sodium sulfate-induced acute colitis mouse model. Furthermore, CTB-KDEL induced a UPR, up-regulated wound healing pathways, and maintained viable crypts in colon explants from patients with inflammatory bowel disease (IBD). In summary, CTB-KDEL exhibits unique wound healing effects in the colon that are mediated by its localization to the ER and subsequent activation of UPR in epithelial cells. The results provide implications for a novel therapeutic approach for mucosal healing, a significant unmet need in IBD treatment.-Royal, J. M., Oh, Y. J., Grey, M. J., Lencer, W. I., Ronquillo, N., Galandiuk, S., Matoba, N. A modified cholera toxin B subunit containing an ER retention motif enhances colon epithelial repair via an unfolded protein response.

Investigation into repetitive concussion in sport (RECOS): study protocol of a prospective, exploratory, observational cohort study.

INTRODUCTION: Sport-related concussion management remains a diagnostic dilemma to clinicians in all strata of care, coaching staff and players alike. The lack of objective diagnostic and prognostic biomarkers and over-reliance on subjective clinical assessments carries a significant health risk of undiagnosed concussive episodes and early return to play before full recovery increasing the risk of sustaining additional concussion, and leading to long-term sequelae and/or unfavourable outcome. OBJECTIVE: To identify a set of parameters (neuroimaging with neurophysiological, biological and neuropsychological tests) that may support pitch-side and outpatient clinical decision-making in order to objectively diagnose concussion, determine the severity of injury, guide a safe return to play and identify the potential predictors of the long-term sequelae of concussion. METHODS AND ANALYSIS: An exploratory, observational, prospective, cohort study recruiting between 2017 and 2020. The participants will have a baseline preseason screening (brain imaging, neuropsychological assessments, serum, urine and saliva sampling). If a screened player later suffers a concussion and/or multiple concussions then he/she will be assessed again with the same protocol within 72 hours, and their baseline data will be used as internal control as well as normative data. Inferential statistical analysis will be performed to determine correlations between biological, imaging techniques and neuropsychological assessments. ETHICS AND DISSEMINATION: This study was approved by the East of England-Essex Research Ethics Committee on 22 September 2017-REC 17/EE/0275; IRAS 216703. The results of this study will be presented at national and international conferences and submitted for publication in peer reviewed journals. TRIAL REGISTRATION NUMBER: ISRCTN16974791; Pre-results.

Interindividual Variability in Use-Dependent Plasticity Following Visuomotor Learning: The Effect of Handedness and Muscle Trained.

Motor learning has been linked with increases in corticospinal excitability (CSE). However, the robustness of this link is unclear. In this study, changes in CSE associated with learning a visuomotor tracking task were mapped using transcranial magnetic stimulation (TMS). TMS maps were obtained before and after training with the first dorsal interosseous (FDI) of the dominant and nondominant hand, and for a distal (FDI) and proximal (biceps brachii) muscle. Tracking performance improved following 20 min of visuomotor training, while map area was unaffected. Large individual differences were observed with 18%-36% of the participants revealing an increase in TMS map area. This result highlights the complex relationship between motor learning and use-dependent plasticity of the motor cortex.

False positives associated with responder/non-responder analyses based on motor evoked potentials.

BACKGROUND: A trend in the non-invasive brain stimulation literature is to assess the outcome of an intervention using a responder analysis whereby participants are di- or trichotomised in order that they may be classified as either responders or non-responders. OBJECTIVE: Examine the extent of the Type I error in motor evoked potential (MEP) data subjected to responder analyses. METHODS: Seven sets of 30 MEPs were recorded from the first dorsal interosseous muscle in 52 healthy volunteers. Four classification techniques were used to classify the participants as responders or non-responders: (1) the two-step cluster analysis, (2) dichotomised thresholding, (3) relative method and (4) baseline variance method. RESULTS: Despite the lack of any intervention, a significant number of participants were classified as responders (21-71%). CONCLUSION: This study highlights the very large Type I error associated with dichotomising continuous variables such as the TMS MEP.

StimTrack: An open-source software for manual transcranial magnetic stimulation coil positioning.

BACKGROUND: During Transcranial Magnetic Stimulation (TMS) experiments researchers often use a neuronavigation system to precisely and accurately maintain coil position and orientation. NEW METHOD: This study aimed to develop and validate an open-source software for TMS coil navigation. StimTrack uses an optical tracker and an intuitive user interface to facilitate the maintenance of position and orientation of any type of coil within and between sessions. Additionally, online access to navigation data is provided, hereby adding e.g. the ability to start or stop the magnetic stimulator depending on the distance to target or the variation of the orientation angles. RESULTS: StimTrack allows repeatable repositioning of the coil within 0.7mm for translation and <1° for rotation. Stimulus-response (SR) curves obtained from 19 healthy volunteers were used to demonstrate that StimTrack can be effectively used in a typical experiment. An excellent intra and inter-session reliability (ICC >0.9) was obtained on all parameters computed on SR curves acquired using StimTrack. COMPARISON WITH EXISTING METHOD: StimTrack showed a target accuracy similar to that of a commercial neuronavigation system (BrainSight, Rogue Research Inc.). Indeed, small differences both in position (∼0.2mm) and orientation (<1°) were found between the systems. These differences are negligible given the human error involved in landmarks registration. CONCLUSIONS: StimTrack, available as supplementary material, is found to be a good alternative for commercial neuronavigation systems facilitating assessment changes in corticospinal excitability using TMS. StimTrack allows researchers to tailor its functionality to their specific needs, providing added value that benefits experimental procedures and improves data quality.

Modifications in lower leg muscle activation when walking barefoot or in minimalist shoes across different age-groups.

Ageing is associated with a decline in muscle strength and impaired sensory mechanisms which contribute to an increased risk of falls. Walking barefooted has been suggested to promote increased muscle strength and improved proprioceptive sensibility through better activation of foot and ankle musculature. Minimalist footwear has been marketed as a method of reaping the suggested benefits of barefoot walking whilst still providing a protective surface. The aim of this study was to investigate if walking barefoot or in minimalist footwear provokes increased muscle activation compared to walking in conventional footwear. Seventy healthy adults (age range 20-87) volunteered for this study. All participants walked along a 7m walking lane five times in four different footwear conditions (barefoot (BF), minimalist shoes (MSH), their own shoes (SH) and control shoes (CON)). Muscle activity of their tibialis anterior (TA), gastrocnemius medialis (GCM) and peroneus longus (PL) were recorded simultaneously and normalised to the BF condition. MSH are intermediate in terms of ankle kinematics and muscle activation patterns. Walking BF or in MSH results in a decrease in TA activity at initial stance due to a flatter foot at contact in comparison to conventional footwear. Walking BF reduces PL activity at initial stance in the young and middle age but not the old. Walking in supportive footwear appears to reduce the balance modulation role of the GCM in the young and middle age but not the old, possibly as a result of slower walking speed when BF.

Intra and inter-session reliability of rapid Transcranial Magnetic Stimulation stimulus-response curves of tibialis anterior muscle in healthy older adults.

OBJECTIVE: The clinical use of Transcranial Magnetic Stimulation (TMS) as a technique to assess corticospinal excitability is limited by the time for data acquisition and the measurement variability. This study aimed at evaluating the reliability of Stimulus-Response (SR) curves acquired with a recently proposed rapid protocol on tibialis anterior muscle of healthy older adults. METHODS: Twenty-four neurologically-intact adults (age:55-75 years) were recruited for this test-retest study. During each session, six SR curves, 3 at rest and 3 during isometric muscle contractions at 5% of maximum voluntary contraction (MVC), were acquired. Motor Evoked Potentials (MEPs) were normalized to the maximum peripherally evoked response; the coil position and orientation were monitored with an optical tracking system. Intra- and inter-session reliability of motor threshold (MT), area under the curve (AURC), MEPmax, stimulation intensity at which the MEP is mid-way between MEPmax and MEPmin (I50), slope in I50, MEP latency, and silent period (SP) were assessed in terms of Standard Error of Measurement (SEM), relative SEM, Minimum Detectable Change (MDC), and Intraclass Correlation Coefficient (ICC). RESULTS: The relative SEM was ≤10% for MT, I50, latency and SP both at rest and 5%MVC, while it ranged between 11% and 37% for AURC, MEPmax, and slope. MDC values were overall quite large; e.g., MT required a change of 12%MSO at rest and 10%MSO at 5%MVC to be considered a real change. Inter-sessions ICC were >0.6 for all measures but slope at rest and MEPmax and latency at 5%MVC. CONCLUSIONS: Measures derived from SR curves acquired in <4 minutes are affected by similar measurement errors to those found with long-lasting protocols, suggesting that the rapid method is at least as reliable as the traditional methods. As specifically designed to include older adults, this study provides normative data for future studies involving older neurological patients (e.g. stroke survivors).

Exploring the quiet eye in archery using field- and laboratory-based tasks.

The 'quiet eye' (QE)-a period of extended gaze fixation on a target-has been reported in many tasks that require accurate aiming. Longer quiet eye durations (QEDs) are reported in experts compared to non-experts and on successful versus less successful trials. The QE has been extensively studied in the field; however, the cognitive mechanisms underlying the QE are not yet fully understood. We investigated the QEDs of ten expert and ten novice archers in the field and in the laboratory using a computer-based archery task. The computer task consisted of shooting archery targets using a joystick. Random 'noise' (visual motion perturbation) was introduced at high and low levels to allow for the controlled examination of the effects of task complexity and processing demands. In this computer task, we also tested an additional group of ten non-archers as controls. In both field and computer tasks, eye movements were measured using electro-oculography. The expert archers exhibited longer QED compared to the novice archers in the field task. In the computer task, the archers again exhibited longer QEDs and were more accurate compared to non-archers. Furthermore, expert archers showed earlier QE onsets and longer QEDs during high noise conditions compared to the novices and non-archers. Our findings show skill-based effects on QED in field conditions and in a novel computer-based archery task, in which online (visual) perturbations modulated experts' QEDs. These longer QEDs in experts may be used for more efficient programming in which accurate predictions are facilitated by attention control.