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.

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.

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.

Specialist healthcare services for concussion/mild traumatic brain injury in England: a consensus statement using modified Delphi methodology.

OBJECTIVE: To establish a consensus on the structure and process of healthcare services for patients with concussion in England to facilitate better healthcare quality and patient outcome. DESIGN: This consensus study followed the modified Delphi methodology with five phases: participant identification, item development, two rounds of voting and a meeting to finalise the consensus statements. The predefined threshold for agreement was set at ≥70%. SETTING: Specialist outpatient services. PARTICIPANTS: Members of the UK Head Injury Network were invited to participate. The network consists of clinical specialists in head injury practising in emergency medicine, neurology, neuropsychology, neurosurgery, paediatric medicine, rehabilitation medicine and sports and exercise medicine in England. PRIMARY OUTCOME MEASURE: A consensus statement on the structure and process of specialist outpatient care for patients with concussion in England. RESULTS: 55 items were voted on in the first round. 29 items were removed following the first voting round and 3 items were removed following the second voting round. Items were modified where appropriate. A final 18 statements reached consensus covering 3 main topics in specialist healthcare services for concussion; care pathway to structured follow-up, prognosis and measures of recovery, and provision of outpatient clinics. CONCLUSIONS: This work presents statements on how the healthcare services for patients with concussion in England could be redesigned to meet their health needs. Future work will seek to implement these into the clinical pathway.

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.

Comparison of ultrasound-guided and palpation-inserted peripheral venous cannula in -patients before primary hip or knee arthroplasty: study protocol for a randomized controlled trial.

BACKGROUND: More than 2 billion peripheral vascular cannulas are introduced globally each year. It is the most frequently performed invasive procedure in medicine worldwide. There is a group of patients with difficult intravenous access (DIVA). In experts' hands, ultrasound-guided vascular access appears to be a significantly better method. Investigators hypothesize that UGVA is superior also in short-term patency of cannula and even for blood draw through cannula. Repeated cannula pricks in the operating room setting not only puts a lot of stress on the patient and medical staff, but they also waste OR time. METHODS: This investigator-initiated prospective randomized monocentric controlled trial is designed to randomly allocate 200 patients undergoing elective primary total joint arthroplasty of hip or knee to one of two groups as follows: Group C (control group) - peripheral venous cannula insertion by palpation or Group USG (intervention) - cannula insertion by ultrasound-guided vascular access. Our primary endpoint is to compare the number of attempts for ultrasound-guided insertion of the peripheral venous cannula with common palpation insertion of the peripheral venous cannula in overweight/obese patients (BMI ≥ 25). The secondary endpoint is a failure rate of the peripheral venous cannula to administer intravenous therapy up to 5 days postoperatively. Tertiary endpoints include a portion of long PVCs that are able to ensure blood draw up to 5 days postoperatively, time needed to insert PVC in each group, number of needle tip redirections in both groups, and reinsertion of PVC needed in both groups for any reason. DISCUSSION: This study is pragmatic and is looking for clinically relevant data. After completion, it will answer the question of whether it is clinically relevant to use ultrasound-guided vascular access in the context of not only short-term benefit of insertion, but also up to 5 days after insertion. Also, if this method can ensure blood draw through a peripheral vein cannula, it can save resources in the perioperative period - valuable especially considering the ongoing shortage of medical staff worldwide. If this hypothesis is confirmed, this finding could contribute to more widespread implementation of ultrasound-guided peripheral vascular access in the perioperative period. TRIAL REGISTRATION: ClinicalTrials.gov NCT05156008. Registered on 13.12.2021.

Biomedical research brings mTBI biomarkers a step closer to the bedside - implementation in clinical practice.

Research in the field of TBI (traumatic brain injury) has long been focused on severe brain injury, while the number of mild injuries far overweigh severe injuries. Mild head injuries constitute up to 95% of all traumatic head injuries. The purpose of this work is to identify mTBI (mild traumatic brain injury) patients who are unlikely to benefit from CT (computed tomography) scanning. Biomarkers capable of clearly discriminating between CT-positive and CT-negative subjects are needed. Biomarkers hold the potential to document whether a concussion occurred, especially when the history is unclear and neurocognitive sequelae persist. Recently, following advances in proteomics analysis, investigators have introduced ubiquitin C-terminal hydrolase-L1 (UCH-L1) and glial fibrillary acidic protein (GFAP) as two promising brain injury biomarkers. The authors provide an update on the current knowledge of TBI biomarkers, especially protein biomarkers for neuronal cell body injury (UCH-L1) and astroglial injury (GFAP, S100B), and a focused literature review dealing with implementation of mTBI biomarkers in clinical practice.

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.

Differential spatial distribution of white matter lesions in Parkinson's and Alzheimer's diseases and cognitive sequelae.

White Matter Lesions (WML) are a radiological finding common in aged subjects. We explored the impact of WML on underlying neurodegenerative processes. We focused on the impact of WML on two neurodegenerative diseases with different pathology. In this cross-sectional study of 137 subjects (78 female, 59 men, mean age 67.2; 43-87 years), we compared WML in healthy controls (HC; n = 55), patients with Alzheimer's disease and amnestic Mild Cognitive Impairment (aMCI), and Parkinson's disease patients with normal cognition and with MCI. Subjects with AD and aMCI were treated as one group (n = 40), subjects with PD and PDMCI were another group (n = 42). MRI T2_FLAIR sequences were analyzed. WML were divided into periventricular (pWML) or subcortical (sWML) depending on their distance from the ventricles. Subjects from the AD + aMCI group, had a significantly greater volume of WML than both HC and the PD + PDMCI group. The volume of WML was greater in the PD + PDMCI than in HC but the difference was not significant. In AD + aMCI subjects, sWML and not pWML were related to a decrease in global cognitive functioning despite greater volume of pWML. In PD + PDMCI, pWML correlate with decline in executive functions and working memory. In HC, pWML correlated with the multidomain decrease corresponding with the aging. This points to a difference between normal aging and pathological aging due to AD and PD brain pathology. The WML location together with underlying disease related neurodegeneration may play a role in determining the effect of WML on cognition. Our results suggest that the impact of WML is not uniform in all patients; rather, their volume, location and cognitive effect may be disease-specific.

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.

Decade of Patient Experience Improvement at a Tertiary Care Urban Hospital.

BACKGROUND AND OBJECTIVE: The purpose of this quality management study was to demonstrate how one hospital made a journey from average patient experience to become a regional leader in the experience of patient care for nationally recognized quality and safety metrics. METHODS: Saint Francis Hospital & Medical Center (SFHMC) located in Hartford, Connecticut, serves a diverse sociodemographic community as part of Trinity Health. "Recommend the Hospital" (RTH) has been the main marker of patient experience at SFHMC and Trinity Health across the United States as part of the Hospital Consumer Assessment of Healthcare Providers and Systems (HCAHPS). From 2010 to 2019, SFHMC implemented unit-based rounding hospital-wide, adopting charge nurse and executive leadership rounding as standard work. The intense support from senior leadership spurred the implementation of these changes across middle management and all frontline workers. The t test was used to determine differences between the mean RTH scores between SFHMC, Connecticut, and the United States. RESULTS: Patient experience at SFHMC was regularly assessed by Press Ganey surveys and HCAHPS, which demonstrated higher scores than averages for the state of Connecticut and the United States between 2010 and 2019 (both Ps < .001). SFHMC was the top performer with an RTH score of 83%, with the state average being 71% and the national average being 72%. In the years following the implementation of a multipronged low-cost strategy, hospital RTH scores rose linearly from the state and national average. SFHMC observed gains in patient safety and quality scores as measured by national benchmarks, including Leapfrog patient safety scores of 7 A's and 1 B over a 4-year period. SFHMC was the only hospital in Connecticut to receive an A grade 4 years in a row. CONCLUSION: A combination of nurse-led, unit-based rounding and executive team rounding with a consistent focus on patient experience resulted in significant improvement in RTH scores for a busy teaching urban hospital, with only a modest investment of resources. There was also improvement in quality and safety outcomes, which together with patient experience of care drove fiscal stability in an increasingly value-based health care environment.

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.

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.

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.

Impact of a hospital unit-based leadership triad on key performance metrics.

We piloted a triad leadership model consisting of a unit-based hospitalist medical director, nurse manager, and case manager on five medical inpatient units. The purpose of this explanatory case study was to determine what, if any, impact the triad team would have on commonly measured operational and quality metrics: observed to expected length of stay, likelihood to recommend the hospital, hand-washing compliance, all-cause 30-day readmission rates, percent of discharges by noon, and percent of discharge to skilled nursing facilities. Over the course of a year triad units demonstrated improvement in most metrics in comparison to the baseline period. While trends for the metrics were favorable, most striking was a statistically significant improvement in the observed to expected length of stay ratio (1.25 to 1.15, p < 0.001) which is the organization's most widely used marker for efficient hospital patient flow.As a result of these sustained operational, safety, quality, and financial performance metrics the model is being generalized to other medical as well as surgical units, including our observation unit. Intangible benefits include creating leadership development path for hospitalist, nursing, and case management colleagues.

Interprofessional, multitiered daily rounding management in a high-acuity hospital.

PURPOSE: The purpose of this explanatory case study is to explain the implementation of interprofessional, multitiered lean daily management (LDM) and to quantitatively report its impact on hospital safety. DESIGN/METHODOLOGY/APPROACH: This case study explained the framework for LDM implementation and changes in quality metrics associated with the interprofessional, multitiered LDM, implemented at Saint Francis Hospital and Medical Center (SFHMC) at the end of 2018. Concepts from lean, Total Quality Management (TQM) and high reliability science were applied to develop the four tiers and gemba rounding components of LDM. A two-tailed t-test analysis was utilized to determine statistical significance for serious safety events (SSEs) comparing the intervention period (January 2019-December 2019) to the baseline period (calendar years 2017 and 2018). Other quality and efficiency metrics were also tracked. FINDINGS: LDM was associated with decreased SSEs in 2019 compared to 2017 and 2018 (p ≤ 0.01). There were no reportable central line-associated blood stream infection (CLABSI) or catheter-associated urinary tract infection (CAUTI) for first full calendar quarter in the hospital's history. Hospital-acquired pressure injuries were at 0.2 per 1,000 patient days, meeting the annual target of <0.5 per 1,000 patient days. Outcomes for falls with injury, hand hygiene and patient experience also trended toward target. These improvements occurred while also observing a lower observed to expected length of stay (O/E LOS), which is the organizational marker for hospital's efficiency. RESEARCH LIMITATIONS/IMPLICATIONS: LDM may contribute greatly to improve safety outcomes. This observational study was performed in an urban, high-acuity, low cost hospital which may not be representative of other hospitals. Further study is warranted to determine whether this model can be applied more broadly to other settings. PRACTICAL IMPLICATIONS: LDM can be implemented quickly to achieve an improvement in hospital safety and other health-care quality outcomes. This required a redistribution of time for hospital staff but did not require any significant capital or other investment. SOCIAL IMPLICATIONS: As hospital systems move from a volume-based to value-based health-care delivery model, dynamic interventions using LDM can play a pivotal role in helping all patients, particularly in underserved settings where lower cost care is required for sustainability, given limited available resources. ORIGINALITY/VALUE: While many hospital systems promote organizational rounding as a routine quality improvement process, this study shows that a dynamic, intense LDM model can dramatically improve safety within months. This was done in a challenging urban environment for a high-acuity population with limited resources.

Long-term motor skill training with individually adjusted progressive difficulty enhances learning and promotes corticospinal plasticity.

Motor skill acquisition depends on central nervous plasticity. However, behavioural determinants leading to long lasting corticospinal plasticity and motor expertise remain unexplored. Here we investigate behavioural and electrophysiological effects of individually tailored progressive practice during long-term motor skill training. Two groups of participants practiced a visuomotor task requiring precise control of the right digiti minimi for 6 weeks. One group trained with constant task difficulty, while the other group trained with progressively increasing task difficulty, i.e. continuously adjusted to their individual skill level. Compared to constant practice, progressive practice resulted in a two-fold greater performance at an advanced task level and associated increases in corticospinal excitability. Differences were maintained 8 days later, whereas both groups demonstrated equal retention 14 months later. We demonstrate that progressive practice enhances motor skill learning and promotes corticospinal plasticity. These findings underline the importance of continuously challenging patients and athletes to promote neural plasticity, skilled performance, and recovery.

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.