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Transition from pediatric to adult care for adolescents and young adults (AYAs) with chronic illness affects the entire family. However, little research has compared AYA and parent experiences of transition. Using Sandelowski and Barroso’s method, the aim of this metasynthesis was to summarize findings of qualitative studies focusing on the transition experiences of AYAs and their parents across different chronic physical illnesses. PubMed, EMBASE and CINAHL were searched followed by forward and backward citation searching. Two authors completed a two-step screening process. Quality was appraised using Guba’s criteria for qualitative rigor. Study characteristics and second order constructs were extracted by two authors and an iterative codebook guided coding and data synthesis. Of 1,644 records identified, 63 studies met inclusion criteria and reflect data from 1,106 AYAs and 397 parents across 18 diagnoses. Three themes were synthesized: transition is dynamic and experienced differently (differing perceptions of role change and growth during emerging adulthood), need for a supported and gradual transition (transition preparation and the factors which influence it) and liminal space (feeling stuck between pediatric and adult care). While AYAs and parents experience some aspects of transition differently, themes were similar across chronic illnesses which supports the development of disease agnostic transition preparation interventions. Transition preparation should support shifting family roles and responsibilities and offer interventions which align with AYA and family preferences.

Measuring transition readiness is important when preparing young people with chronic illness for successful transition to adult care. The Expanded Socioecological Model of Adolescent and Young Adult Readiness to Transition (Expanded SMART) offers a holistic view of factors that influence transition readiness and outcomes. The aim of this study was to examine conceptual congruency of transition readiness instruments with the Expanded SMART to determine the breadth and frequency of constructs measured.

Behavioural time scale plasticity (BTSP) is non-Hebbian plasticity induced by integrating presynaptic and postsynaptic components separated by a behaviourally relevant time scale (seconds) 1 . BTSP in hippocampal CA1 neurons underlies place cell formation. However, the molecular mechanisms that enable synapse-specific plasticity on a behavioural time scale are unknown. Here we show that BTSP can be induced in a single dendritic spine using two-photon glutamate uncaging paired with postsynaptic current injection temporally separated by a behavioural time scale. Using an improved Ca 2+ /calmodulin-dependent kinase II (CaMKII) sensor, we did not detect CaMKII activation during this BTSP induction. Instead, we observed dendritic, delayed and stochastic CaMKII activation (DDSC) associated with Ca 2+ influx and plateau potentials 10-100 s after BTSP induction. DDSC required both presynaptic and postsynaptic activity, which suggests that CaMKII can integrate these two signals. Also, optogenetically blocking CaMKII 15-30 s after the BTSP protocol inhibited synaptic potentiation, which indicated that DDSC is an essential mechanism of BTSP. IP 3 -dependent intracellular Ca 2+ release facilitated both DDSC and BTSP. Thus, our study suggests that non-synapse-specific CaMKII activation provides an instructive signal with an extensive time window over tens of seconds during BTSP., (© 2024. The Author(s).)

Preventative treatment for Alzheimer's Disease (AD) is dire, yet mechanisms underlying early regional vulnerability remain unknown. In AD, one of the earliest pathophysiological correlates to cognitive decline is hyperexcitability, which is observed first in the entorhinal cortex. Why hyperexcitability preferentially emerges in specific regions in AD is unclear. Using regional, cell-type-specific proteomics and electrophysiology in wild-type mice, we uncovered a unique susceptibility of the entorhinal cortex to human amyloid precursor protein (hAPP). Entorhinal hyperexcitability resulted from selective vulnerability of parvalbumin (PV) interneurons, with respect to surrounding excitatory neurons. This effect was partially replicated with an APP chimera containing a humanized amyloid-beta sequence. EC hyperexcitability could be ameliorated by co-expression of human Tau with hAPP at the expense of increased pathological tau species, or by enhancing PV interneuron excitability in vivo. This study suggests early interventions targeting inhibitory neurons may protect vulnerable regions from the effects of APP/amyloid and tau pathology., (© 2024. The Author(s).)

Recent investigation of a constitutively active ADAMTS13 variant (caADAMTS13) in murine models of acute ischaemic stroke (AIS) have revealed a potential anti-inflammatory mechanism of action contributing to its protective effect. However, it remains unclear whether these observations are a direct result of VWF proteolysis by caADAMTS13. We have implemented state of the art in vitro assays of neutrophil rolling and transmigration to quantify the impact of caADAMTS13 on these processes. Moreover, we have tested caADAMTS13 in two in vivo assays of neutrophil migration to confirm the impact of the treatment on the neutrophil response to sterile inflammation. Neutrophil rolling, over an interleukin-1β stimulated hCMEC/D3 monolayer, is directly inhibited by caADAMTS13, reducing the proportion of neutrophils rolling to 9.5 ± 3.8 % compared to 18.0 ± 4.5 % in untreated controls. Similarly, neutrophil transmigration recorded in real-time, was significantly suppressed in the presence of caADAMTS13 which reduced the number of migration events to a level like that in unstimulated controls (18.0 ± 4.5 and 15.8 ± 7.5 cells/mm 2 /h, respectively). Brain tissue from mice undergoing experimental focal cerebral ischaemia has indicated the inhibition of this process by caADAMTS13. This is supported by caADAMTS13's ability to reduce neutrophil migration into the peritoneal cavity in an ischaemia-independent model of sterile inflammation, with the VWF-dependent mechanism by which this occurs being confirmed using a second experimental stroke model. These findings will be an important consideration in the further development of caADAMTS13 as a potential therapy for AIS and other thromboinflammatory pathologies, including cardiovascular disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Adolescents with cystic fibrosis (CF) and their parents must navigate changing roles and responsibilities within the family including transfer of disease management responsibilities., Aim/objective: The aim of this qualitative study was to explore how families share and transfer CF management responsibility from the perspectives of adolescents with CF and their parents., Methods: Guided by qualitative descriptive methodology, we purposively sampled adolescent/parent dyads. Participants completed two surveys measuring family responsibility (Family Responsibility Questionnaire [FRQ]) and transition readiness (Transition Readiness Assessment Questionnaire [TRAQ]) We conducted semistructured video or phone interviews, used a codebook to guide team coding and analyzed qualitative data using both content analysis and dyadic interview analysis., Results: Thirty participants (15 dyads) enrolled (7% Black; 33% Latina/o; 40% female; adolescent age 14.4 ± 2 years; 66% prescribed highly effective modulator therapy; 80% of parents were mothers). Parent FRQ and TRAQ scores were significantly higher than their adolescent indicating differing perceptions of responsibility and transition readiness. We inductively identified four themes: (1) CF management is a delicate balance (CF management is a routine which is easily disrupted), (2) Growing up and parenting under extraordinary circumstances (the burden of CF weighs on families as they navigate adolescence), (3) Differing Perceptions of risk and responsibility (adolescent and parent perceptions of treatment responsibility and the risks of nonadherence do not always align), and (4) Balancing independence and protection (families must weigh the benefits and risks of allowing adolescents increased independence)., Conclusions: Adolescents and parents demonstrated differing perceptions of CF management responsibility, which may be related to a lack of communication between family members about this topic. To help facilitate alignment of parent and adolescent expectations, discussion of family roles and responsibility for CF management should begin early during the transition process and be discussed regularly during clinic visits., (© 2023 Wiley Periodicals LLC.)

Blood-brain barrier (BBB) dysfunction occurs in many brain diseases, and there is increasing evidence to suggest that it is an early process in dementia which may be exacerbated by peripheral infection. Filter-exchange imaging (FEXI) is an MRI technique for measuring trans-membrane water exchange. FEXI data is typically analysed using the apparent exchange rate (AXR) model, yielding estimates of the AXR. Crusher gradients are commonly used to remove unwanted coherence pathways arising from longitudinal storage pulses during the mixing period. We first demonstrate that when using thin slices, as is needed for imaging the rodent brain, crusher gradients result in underestimation of the AXR. To address this, we propose an extended crusher-compensated exchange rate (CCXR) model to account for diffusion-weighting introduced by the crusher gradients, which is able to recover ground truth values of BBB water exchange (k in ) in simulated data. When applied to the rat brain, k in estimates obtained using the CCXR model were 3.10 s -1 and 3.49 s -1 compared to AXR estimates of 1.24 s -1 and 0.49 s -1 for slice thicknesses of 4.0 mm and 2.5 mm respectively. We then validated our approach using a clinically relevant Streptococcus pneumoniae lung infection. We observed a significant 70 ± 10% increase in BBB water exchange in rats during active infection (k in  = 3.78 ± 0.42 s -1 ) compared to before infection (k in  = 2.72 ± 0.30 s -1 ; p = 0.02). The BBB water exchange rate during infection was associated with higher levels of plasma von Willebrand factor (VWF), a marker of acute vascular inflammation. We also observed 42% higher expression of perivascular aquaporin-4 (AQP4) in infected animals compared to non-infected controls, while levels of tight junction proteins remain consistent between groups. In summary, we propose a modelling approach for FEXI data which removes the bias in estimated water-exchange rates associated with the use of crusher gradients. Using this approach, we demonstrate the impact of peripheral infection on BBB water exchange, which appears to be mediated by endothelial dysfunction and associated with an increase in perivascular AQP4., (© 2023. The Author(s).)

Objective: To explore experiences of symptoms of suspected or confirmed COVID-19 illness among women using the CovidWatcher mobile citizen science app., Design: Convergent parallel mixed-methods design., Participants: Twenty-eight self-identified women consented for follow-up after using CovidWatcher. Participants' ages ranged from 18 to 83 years old., Methods: We collected data via semistructured, virtual interviews and surveys: the COVID-19 Exposure and Family Impact Survey and Patient-Reported Outcomes Measurement Information System measures. We used directed content analysis to develop codes, categories, themes, and subthemes from the qualitative data and summarized survey data with descriptive statistics., Results: We derived five themes related to symptom experiences: (a) Physical Symptoms, (b) Mental Health Symptoms, (c) Symptom Intensity, (d) Symptom Burden, and (e) Symptom Trajectories. Subthemes reflected more nuanced experiences of suspected or confirmed COVID-19 disease. For those without COVID-19, anxiety and mental health symptoms were still present. Of those who attested to one of the PROMIS-measured symptoms, all but one had at least mild severity in one of their reported symptoms., Conclusion: This study demonstrates the cross-cutting impact of the COVID-19 pandemic on individuals who identify as women. Future research and clinical practice guidelines should focus on alleviating physical and mental health symptoms related to the ongoing pandemic, regardless of COVID-19 diagnosis. Furthermore, clinicians should consider how patients can use symptom reconciliation apps and tracking systems., (Copyright © 2022 AWHONN. Published by Elsevier Inc. All rights reserved.)

Objectives: Evaluate the efficacy of interventions to improve symptoms for ICU surrogates at highest risk of developing psychologic distress: those facing end-of-life care decisions., Data Sources: MEDLINE, CINAHL, PsycInfo, Embase, Cochrane Central Register of Controlled Trials, and ClinicalTrials.gov were searched through April 16, 2022., Study Selection: Following an a priori protocol, randomized trials of interventions delivered to surrogates of adult ICU patients who died or had high likelihood of mortality evaluating surrogate symptoms were identified., Data Extraction: Two reviewers performed screening and data extraction and assessed risk of bias (Cochrane Risk of Bias [RoB] 2 tool). Trials were eligible for meta-analysis if group mean symptom scores were provided at 3 or 6 months. Pooled effects were estimated using a random effects model. Heterogeneity was assessed (Cochrane Q, I2 ). Certainty of evidence was assessed (Grading of Recommendations Assessment, Development and Evaluation)., Data Synthesis: Of 1,660 records, 10 trials met inclusion criteria representing 3,824 surrogates; eight were included in the meta-analysis. Overall RoB was rated Some Concerns. Most ( n = 8) interventions focused on improving communication and enhancing psychologic support in the ICU. All trials measured anxiety, depression, and posttraumatic stress. Significant improvement was seen at 3 months (depression, mean difference [MD], -0.68; 95% CI, -1.14 to -0.22, moderate certainty; posttraumatic stress, standardized MD, -0.25; 95% CI, -0.49 to -0.01, very low certainty) and 6 months (anxiety, MD, -0.70; 95% CI, -1.18 to -0.22, moderate certainty). Sensitivity analyses suggest significant findings may be unstable. Subgroup analyses demonstrated differences in effect by trial location, interventionist, and intervention dose., Conclusions: Communication and psychological support interventions in the ICU yielded small but significant improvement in psychological symptoms with moderate to very low certainty evidence in a prognostically-enriched sample of ICU surrogates facing end-of-life care decisions. A new approach to interventions that extend beyond the ICU may be needed., Competing Interests: Ms. DeForge’s institution received funding from the National Center for Advancing Translational Sciences; she received funding from Society of Critical Care Medicine (2022 Critical Care Congress Scholarship Grant). Ms. DeForge and Dr. Baldwin received support for article research from the National Institutes of Health. Dr. George received funding from AstraZeneca, Genetech, Sanofi Regeneron, and Teva. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2022 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.)

As the COVID-19 pandemic moves towards endemic disease, it remains of key importance to identify groups of individuals vulnerable to severe infection and understand the biological factors that mediate this risk. Stroke patients are at increased risk of developing severe COVID-19, likely due to stroke-induced alterations to systemic immune function. Furthermore, immune responses associated with severe COVID-19 in patients without a history of stroke parallel many of the immune alterations induced by stroke, possibly resulting in a compounding effect that contributes to worsened disease severity. In this review, we discuss the changes to systemic immune function that likely contribute to augmented COVID-19 severity in patients with a history of stroke and the effects of COVID-19 on the immune system that may exacerbate these effects., Competing Interests: None declared., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Immunology.)

Purpose: The experience of healthcare transition from pediatric to adult care in cystic fibrosis (CF) remains poorly understood, particularly among racially and ethnically diverse adolescents and young adults (AYAs) with CF. The objective of this qualitative study was to explore the perspectives of a diverse sample of AYAs with CF at one urban academic medical center regarding healthcare transition., Design and Methods: Guided by qualitative descriptive methodology, we purposively selected AYAs who represented the pre and post transition experience: some AYAs had experienced the transition preparation program CF R.I.S.E. Demographic information and responsibility for self-management behaviors were collected using an online survey. Semi-structured video interviews were conducted following an iterative interview guide. A codebook directed inductive coding. QSR NVivo Version 12 software was used to organize the data., Results: 12 AYAs with CF were enrolled (25% female, 25% Black AYA, 33% Hispanic/Latina/o AYA, 50% White AYA; mean age 20.8 years). Three themes were identified: independent care of the whole self, preparing for change and the unknown and transition experiences vary., Conclusions: Not all participants experienced a smooth transition. Participants identified suggestions for the development of transition preparation interventions, specifically around involving AYAs in transition decisions and beginning transition preparation early in adolescence., Practice Implications: Participants expressed uncertainty about transition when they felt little control over the process or lacked sufficient information about adult care. Therefore, comprehensive early transition preparation for all AYAs with CF with a focus on involving AYAs in transition decisions is recommended., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Advances in our understanding of ADAMTS13 structure, and the conformation changes required for full activity, have rejuvenated the possibility of its use as a thrombolytic therapy. We have tested a novel Ala1144Val ADAMTS13 variant (constitutively active [ca] ADAMTS13) that exhibits constitutive activity, characterized using in vitro assays of ADAMTS13 activity, and greatly enhanced thrombolytic activity in 2 murine models of ischemic stroke, the distal FeCl3 middle cerebral artery occlusion (MCAo) model and transient middle cerebral artery occlusion (tMCAO) with systemic inflammation and ischemia/reperfusion injury. The primary measure of efficacy in both models was restoration of regional cerebral blood flow (rCBF) to the MCA territory, which was determined using laser speckle contrast imaging. The caADAMTS13 variant exhibited a constitutively active conformation and a fivefold enhanced activity against fluorescence resonance energy transfer substrate von Willebrand factor 73 (FRETS-VWF73) compared with wild-type (wt) ADAMTS13. Moreover, caADAMTS13 inhibited VWF-mediated platelet capture at subphysiological concentrations and enhanced t-PA/plasmin lysis of fibrin(ogen), neither of which were observed with wtADAMTS13. Significant restoration of rCBF and reduced lesion volume was observed in animals treated with caADAMTS13. When administered 1 hour after FeCl3 MCAo, the caADAMTS13 variant significantly reduced residual VWF and fibrin deposits in the MCA, platelet aggregate formation, and neutrophil recruitment. When administered 4 hours after reperfusion in the tMCAo model, the caADAMTS13 variant induced a significant dissolution of platelet aggregates and a reduction in the resulting tissue hypoperfusion. The caADAMTS13 variant represents a potentially viable therapeutic option for the treatment of acute ischemic stroke, among other thrombotic indications, due to its enhanced in vitro and in vivo activities that result from its constitutively active conformation., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Anecdotal reports and clinical observations have recently emerged suggesting a relationship between COVID-19 disease and stroke, highlighting the possibility that infected individuals may be more susceptible to cerebrovascular events. In this review we draw on emerging studies of the current pandemic and data from earlier, viral epidemics, to describe possible mechanisms by which SARS-CoV-2 may influence the prevalence of stroke, with a focus on the thromboinflammatory pathways, which may be perturbed. Some of these potential mechanisms are not novel but are, in fact, long-standing hypotheses linking stroke with preceding infection that are yet to be confirmed. The current pandemic may present a renewed opportunity to better understand the relationship between infection and stroke and possible underlying mechanisms.

Despite high-resolution crystal structures of both inactive and active G protein-coupled receptors (GPCRs), it is still not known how ligands trigger the large structural change on the intracellular side of the receptor since the conformational changes that occur within the extracellular ligand-binding region upon activation are subtle. Here, we use solid-state NMR and Fourier transform infrared spectroscopy on rhodopsin to show that Trp265 6.48 within the CWxP motif on transmembrane helix H6 constrains a proline hinge in the inactive state, suggesting that activation results in unraveling of the H6 backbone within this motif, a local change in dynamics that allows helix H6 to swing outward. Notably, Tyr301 7.48 within activation switch 2 appears to mimic the negative allosteric sodium ion found in other family A GPCRs, a finding that is broadly relevant to the mechanism of receptor activation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Chronic thromboembolic pulmonary hypertension (CTEPH) is an important consequence of pulmonary embolism that is associated with abnormalities in haemostasis. We investigated the ADAMTS13-von Willebrand factor (VWF) axis in CTEPH, including its relationship with disease severity, inflammation, ABO groups and ADAMTS13 genetic variants.ADAMTS13 and VWF plasma antigen levels were measured in patients with CTEPH (n=208), chronic thromboembolic disease without pulmonary hypertension (CTED) (n=35), resolved pulmonary embolism (n=28), idiopathic pulmonary arterial hypertension (n=30) and healthy controls (n=68). CTEPH genetic ABO associations and protein quantitative trait loci were investigated. ADAMTS13-VWF axis abnormalities were assessed in CTEPH and healthy control subsets by measuring ADAMTS13 activity, D-dimers and VWF multimeric size.Patients with CTEPH had decreased ADAMTS13 (adjusted β -23.4%, 95% CI -30.9- -15.1%, p<0.001) and increased VWF levels (β +75.5%, 95% CI 44.8-113%, p<0.001) compared to healthy controls. ADAMTS13 levels remained low after reversal of pulmonary hypertension by pulmonary endarterectomy surgery and were equally reduced in CTED. We identified a genetic variant near the ADAMTS13 gene associated with ADAMTS13 protein that accounted for ∼8% of the variation in levels.The ADAMTS13-VWF axis is dysregulated in CTEPH. This is unrelated to pulmonary hypertension, disease severity or markers of systemic inflammation and implicates the ADAMTS13-VWF axis in CTEPH pathobiology., Competing Interests: Conflict of interest: M. Newnham reports education support (travel, registration and accommodation) to attend conferences from MSD and GSK, outside the submitted work. Conflict of interest: K. South has nothing to disclose. Conflict of interest: M. Bleda has nothing to disclose. Conflict of interest: W.R. Auger reports grants and non-financial support for advisory board work (travel support only) from Bayer, outside the submitted work. Conflict of interest: J.A. Barberà has nothing to disclose. Conflict of interest: H. Bogaard has nothing to disclose. Conflict of interest: K. Bunclark has nothing to disclose. Conflict of interest: J.E. Cannon reports grants for educational meetings from Actelion, GSK and MSD, outside the submitted work. Conflict of interest: M. Delcroix has nothing to disclose. Conflict of interest: C. Hadinnapola has nothing to disclose. Conflict of interest: L.S. Howard reports grants from Bayer PLC, during the conduct of the study. Conflict of interest: D. Jenkins reports grants and personal fees from Bayer, and personal fees from Actelion, outside the submitted work. Conflict of interest: E. Mayer reports speaker and consultancy fees from Actelion, Bayer and MSD, and speaker fees from Pfizer, outside the submitted work. Conflict of interest: C. Ng has nothing to disclose. Conflict of interest: C.J. Rhodes has nothing to disclose. Conflict of interest: N. Screaton has nothing to disclose. Conflict of interest: K. Sheares reports educational support (travel, registration and accommodation) to attend conferences from Actelion, Bayer, MSD and GSK, and has been on an advisory board for Actelion, outside the submitted work. Conflict of interest: M.A. Simpson has a contract of service with Genomics PLC, outside the submitted work. Conflict of interest: M. Southwood has nothing to disclose. Conflict of interest: L. Su has nothing to disclose. Conflict of interest: D. Taboada reports speaker honoraria and education/travel grants from Actelion, Bayer, GlaxoSmithKline, Lilly, MDS and Pfizer, outside the submitted work. Conflict of interest: M. Traylor has nothing to disclose. Conflict of interest: R.C. Trembath has nothing to disclose. Conflict of interest: S.S. Villar has nothing to disclose. Conflict of interest: M.R. Wilkins has nothing to disclose. Conflict of interest: J. Wharton reports personal fees for advisory board work from Actelion Pharmaceuticals Ltd, outside the submitted work. Conflict of interest: S. Gräf has nothing to disclose. Conflict of interest: J. Pepke-Zaba (or her institution) has received research/educational grants and has been serving on advisory boards for Actelion, Bayer, Merck and GSK. Conflict of interest: M. Laffan reports grants from British Heart Foundation during the conduct of the study; and support to attend conferences from Shire, outside the submitted work. Conflict of interest: D.A. Lane has nothing to disclose. Conflict of interest: N.W. Morrell has nothing to disclose. Conflict of interest: M. Toshner reports grants and personal fees from Bayer, Merck and Actelion, personal fees from GSK, and grants from Roche, during the conduct of the study., (Copyright ©ERS 2019.)

Blood loss is prevented by the multidomain glycoprotein von Willebrand factor (VWF), which binds exposed collagen at damaged vessels and captures platelets. VWF is regulated by the metalloprotease ADAMTS13, which in turn is conformationally activated by VWF. To delineate the structural requirements for VWF-mediated conformational activation of ADAMTS13, we performed binding and functional studies with a panel of truncated ADAMTS13 variants. We demonstrate that both the isolated CUB1 and CUB2 domains in ADAMTS13 bind to the spacer domain exosite of a truncated ADAMTS13 variant, MDTCS ( K D of 135 ± 1 0.1 nm and 86.9 ± 9.0 nm, respectively). However, only the CUB1 domain inhibited proteolytic activity of MDTCS. Moreover, ADAMTS13ΔCUB2, unlike ADAMTS13ΔCUB1-2, exhibited activity similar to wild-type ADAMTS13 and could be activated by VWF D4-CK. The CUB2 domain is, therefore, not essential for maintaining the inactive conformation of ADAMTS13. Both CUB domains could bind to the VWF D4-CK domain fragment ( K D of 53.7 ± 2.1 nm and 84.3 ± 2.0 nm, respectively). However, deletion of both CUB domains did not prevent VWF D4-CK binding, suggesting that competition for CUB-domain binding to the spacer domain is not the dominant mechanism behind the conformational activation. ADAMTS13ΔTSP8-CUB2 could no longer bind to VWF D4-CK, and deletion of TSP8 abrogated ADAMTS13 conformational activation. These findings support an ADAMTS13 activation model in which VWF D4-CK engages the TSP8-CUB2 domains, inducing the conformational change that disrupts the CUB1-spacer domain interaction and thereby activates ADAMTS13., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Protein misfolding caused by inherited mutations leads to loss of protein function and potentially toxic 'gain of function', such as the dominant P23H rhodopsin mutation that causes retinitis pigmentosa (RP). Here, we tested whether the AMPK activator metformin could affect the P23H rhodopsin synthesis and folding. In cell models, metformin treatment improved P23H rhodopsin folding and traffic. In animal models of P23H RP, metformin treatment successfully enhanced P23H traffic to the rod outer segment, but this led to reduced photoreceptor function and increased photoreceptor cell death. The metformin-rescued P23H rhodopsin was still intrinsically unstable and led to increased structural instability of the rod outer segments. These data suggest that improving the traffic of misfolding rhodopsin mutants is unlikely to be a practical therapy, because of their intrinsic instability and long half-life in the outer segment, but also highlights the potential of altering translation through AMPK to improve protein function in other protein misfolding diseases., (© The Author 2016. Published by Oxford University Press.)

Purpose: The purpose of this study was to develop a murine model of allergic conjunctivitis induced by house dust mite (HDM) extract from Dermatophagoides pteronyssinus, a major allergen in humans., Methods: Forty BALB/c mice were divided into five groups, immunized with placebo, ovalbumin (10 μg), or HDM extract following a schedule. Twenty minutes after topical challenge, mice were examined clinically. Material collected from mice was used for measuring total and specific IgE, antigen-specific lymphocyte proliferation, and supernatant cytokine levels and for conjunctival histopathology and flow cytometric analysis of conjunctival cells., Results: This murine model showed similar clinical signs and laboratory findings to human allergy and the ovalbumin-induced allergic conjunctivitis model. Total IgE levels and conjunctival infiltration of mast cells and eosinophils in immunized mice were significantly higher than in the control group. Cervical lymphocyte proliferation was increased in antigen-stimulated cultures in immunized mice, concomitant with significantly higher IL-4 and IL-5 levels in the culture supernatant. The proportion of conjunctival CD4+ T cells expressing the ST2 receptor was increased, and conjunctival CD4+ST2+ T cells exhibited an increase in intracellular IL-5., Conclusions: House dust mite extract successfully induced allergic conjunctivitis in BALB/c mice. Ten micrograms of HDM extract was the optimal dose for systemic immunization in this model. This murine model is suitable for further studies on HDM-induced allergic conjunctivitis, and the data show that conjunctival CD4+ T cells expressing ST2 may play an important role in IL-5 secretion, recruiting eosinophils into conjunctiva on ocular allergen challenge.

A disintegrin and metalloprotease with thrombospondin motifs 13 (ADAMTS13) is a metalloprotease that regulates von Willebrand factor (VWF) function. ADAMTS13-mediated proteolysis is determined by conformational changes in VWF, but also may depend on its own conformational activation. Kinetic analysis of WT ADAMTS13 revealed ∼ 2.5-fold reduced activity compared with ADAMTS13 lacking its C-terminal tail (MDTCS) or its CUB1-2 domains (WTΔCUB1-2), suggesting that the CUB domains naturally limit ADAMTS13 function. Consistent with this suggestion, WT ADAMTS13 activity was enhanced ∼ 2.5-fold by preincubation with either an anti-CUB mAb (20E9) or VWF D4CK (the natural binding partner for the CUB domains). Furthermore, the isolated CUB1-2 domains not only bound MDTCS, but also inhibited activity by up to 2.5-fold. Interestingly, a gain-of-function (GoF) ADAMTS13 spacer domain variant (R568K/F592Y/R660K/Y661F/Y665F) was ∼ 2.5-fold more active than WT ADAMTS13, but could not be further activated by 20E9 mAb or VWF D4CK and was unable to bind or to be inhibited by the CUB1-2 domains, suggesting that the inhibitory effects of the CUB domains involve an interaction with the spacer domain that is disrupted in GoF ADAMTS13. Electron microscopy demonstrated a "closed" conformation of WT ADAMTS13 and suggested a more "open" conformation for GoF ADAMTS13. The cryptic spacer domain epitope revealed by conformational unfolding also represents the core antigenic target for autoantibodies in thrombotic thrombocytopenic purpura. We propose that ADAMTS13 circulates in a closed conformation, which is maintained by a CUB-spacer domain binding interaction. ADAMTS13 becomes conformationally activated on demand through interaction of its C-terminal CUB domains with VWF, making it susceptible to immune recognition.