Fic-mediated AMPylation tempers the unfolded protein response during physiological stress.

The proper balance of synthesis, folding, modification, and degradation of proteins, also known as protein homeostasis, is vital to cellular health and function. The unfolded protein response (UPR) is activated when the mechanisms maintaining protein homeostasis in the endoplasmic reticulum become overwhelmed. However, prolonged or strong UPR responses can result in elevated inflammation and cellular damage. Previously, we discovered that the enzyme filamentation induced by cyclic-AMP (Fic) can modulate the UPR response via posttranslational modification of binding immunoglobulin protein (BiP) by AMPylation during homeostasis and deAMPylation during stress. Loss of fic in Drosophila leads to vision defects and altered UPR activation in the fly eye. To investigate the importance of Fic-mediated AMPylation in a mammalian system, we generated a conditional null allele of Fic in mice and characterized the effect of Fic loss on the exocrine pancreas. Compared to controls, Fic-/- mice exhibit elevated serum markers for pancreatic dysfunction and display enhanced UPR signaling in the exocrine pancreas in response to physiological and pharmacological stress. In addition, both fic-/- flies and Fic-/- mice show reduced capacity to recover from damage by stress that triggers the UPR. These findings show that Fic-mediated AMPylation acts as a molecular rheostat that is required to temper the UPR response in the mammalian pancreas during physiological stress. Based on these findings, we propose that repeated physiological stress in differentiated tissues requires this rheostat for tissue resilience and continued function over the lifetime of an animal.

A Delphi survey to determine a definition and description of hyperacusis by clinician consensus.

OBJECTIVE: There is currently no singularly accepted definition of hyperacusis. The aim of this study was to determine a definition and description of hyperacusis by clinician consensus. DESIGN: A three-round Delphi survey involving hearing healthcare professionals built towards clinical consensus on a definition of hyperacusis. Round 1 involved three open-ended questions about hyperacusis. Seventy-nine statements were generated on descriptions, impact, sounds, and potential features of hyperacusis. Agreement on the relevance of each statement to defining or describing hyperacusis was then measured in Rounds 2 and 3. General consensus was defined a priori as ≥70% agreement, or ≥90 for clinical decision making. STUDY SAMPLE: Forty-five hearing healthcare professionals were recruited to take part in this study. Forty-one completed Round 1, 36 completed Round 2, and 33 completed Round 3. RESULTS: Consensus was reached on 42/79 statements. From these a consensus definition includes "A reduced tolerance to sound(s) that are perceived as normal to the majority of the population or were perceived as normal to the person before their onset of hyperacusis". A consensus description of hyperacusis was also determined. CONCLUSIONS: This consensus definition of hyperacusis will help to determine the scope of clinical practice guidelines and influence needed research on hyperacusis.

Enzymes Involved in AMPylation and deAMPylation.

Posttranslational modifications are covalent changes made to proteins that typically alter the function or location of the protein. AMPylation is an emerging posttranslational modification that involves the addition of adenosine monophosphate (AMP) to a protein. Like other, more well-studied posttranslational modifications, AMPylation is predicted to regulate the activity of the modified target proteins. However, the scope of this modification both in bacteria and in eukaryotes remains to be fully determined. In this review, we provide an up to date overview of the known AMPylating enzymes, the regulation of these enzymes, and the effect of this modification on target proteins.

The TinMan study: feasibility trial of a psychologically informed, audiologist-delivered, manualised intervention for tinnitus.

OBJECTIVE: To develop a manualised psychological treatment for tinnitus that could enhance audiologist usual care, and to test feasibility of evaluating it in a randomised controlled trial. DESIGN: Feasibility trial, random allocation of patients to manualised treatment or treatment as usual, and mixed-methods evaluation. Study sample: Senior audiologists, and adults with chronic tinnitus. RESULTS: Recruitment reached 63% after 6 months (feasibility pre-defined as 65%). Only nine patients (47%) were retained for the duration of the trial. Patients reported that the treatment was acceptable and helped reassure them about their tinnitus. Audiologists reported mixed feelings about the kinds of techniques that are presented to them as 'psychologically informed'. Audiologists also reported lacking confidence because the training they had was brief, and stated that more formal supervision would have been helpful to check adherence to the treatment manual. CONCLUSIONS: The study indicate potential barriers to audiologist use of the manual, and that a clinical trial of the intervention is not yet feasible. However, positive indications from outcome measures suggest that further development work would be worthwhile. Refinements to the manual are indicated, and training and supervision arrangements to better support audiologists to use the intervention in the clinic are required. Trial Registration: ISRCTN13059163.

Fic-mediated deAMPylation is not dependent on homodimerization and rescues toxic AMPylation in flies.

Protein chaperones play a critical role in proteostasis. The activity of the major endoplasmic reticulum chaperone BiP (GRP78) is regulated by Fic-mediated AMPylation during resting states. By contrast, during times of stress, BiP is deAMPylated. Here, we show that excessive AMPylation by a constitutively active FicE247G mutant is lethal in Drosophila This lethality is cell-autonomous, as directed expression of the mutant FicE247G to the fly eye does not kill the fly but rather results in a rough and reduced eye. Lethality and eye phenotypes are rescued by the deAMPylation activity of wild-type Fic. Consistent with Fic acting as a deAMPylation enzyme, its activity was both time- and concentration-dependent. Furthermore, Fic deAMPylation activity was sufficient to suppress the AMPylation activity mediated by the constitutively active FicE247G mutant in Drosophila S2 lysates. Further, we show that the dual enzymatic activity of Fic is, in part, regulated by Fic dimerization, as loss of this dimerization increases AMPylation and reduces deAMPylation of BiP.

Patients' and Clinicians' Views of the Psychological Components of Tinnitus Treatment That Could Inform Audiologists' Usual Care: A Delphi Survey.

OBJECTIVES: The aim of this study was to determine which components of psychological therapies are most important and appropriate to inform audiologists' usual care for people with tinnitus. DESIGN: A 39-member panel of patients, audiologists, hearing therapists, and psychologists completed a three-round Delphi survey to reach consensus on essential components of audiologist-delivered psychologically informed care for tinnitus. RESULTS: Consensus (≥80% agreement) was reached on including 76 of 160 components. No components reached consensus for exclusion. The components reaching consensus were predominantly common therapeutic skills such as Socratic questioning and active listening, rather than specific techniques, for example, graded exposure therapy or cognitive restructuring. Consensus on educational components to include largely concerned psychological models of tinnitus rather than neurophysiological information. CONCLUSIONS: The results of this Delphi survey provide a tool to develop audiologists' usual tinnitus care using components that both patients and clinicians agree are important and appropriate to be delivered by an audiologist for adults with tinnitus-related distress. Research is now necessary to test the added effects of these components when delivered by audiologists.

A therapeutic skating intervention for children with autism spectrum disorder.

PURPOSE: The purpose of this study was to evaluate the effects of a highly structured therapeutic skating intervention on motor outcomes and functional capacity in 2 boys with autism spectrum disorder aged 7 and 10 years. METHODS: This multiple-baseline, single-subject study assigned participants to three 1-hour skating sessions per week for 12 weeks focusing on skill and motor development. Multiple data points assessed (a) fidelity to the intervention and (b) outcomes measures including the Pediatric Balance Scale, Timed Up and Go, floor to stand, Six-Minute Walk Test, goal attainment, and weekly on-ice testing. RESULTS: Improvements were found in balance, motor behavior, and functional capacity by posttest with gains remaining above pretest levels at follow-up. CONCLUSIONS: Therapeutic skating may produce physical benefits for children with autism spectrum disorder and offer a viable, inexpensive community-based alternative to other forms of physical activity.

How to build resiliency in autistic individuals: an implication to advance mental health.

INTRODUCTION: Individuals on the autism spectrum (ASD) often experience poor mental health and coping strategies compared to their peers due to social exclusion and co-occurring conditions. Resiliency has been identified as a key factor in preventing adverse outcomes and promoting mental health. Therefore, it is important to determine what strategies can be used to build resiliency among autistic individuals. The current paper is one of the first studies that aims to collect information from autistic individuals and their caregivers on potential strategies to enhance resiliency. METHODS: We interviewed 18 participants from various provinces in Canada, comprising of 13 autistic individuals and 5 parents. We used thematic analysis to identify patterns in the data. RESULTS: Thematic analysis revealed three themes to indicate strategies that could be used to enhance resiliency, including: (a) self-reliant strategies, (b) using community-based facilities, and (c) contextual and individual characteristics. CONCLUSION: Although the body of literature on resiliency is evolving, this paper provides a unique perspective as it is one of the few studies that considers the experiences of individuals on the spectrum. In addition, this study focuses on identifying and describing specific strategies that can be used to enhance resiliency and mental health, which consequently can help address the existing gaps in knowledge and practice.

FicD regulates adaptation to the unfolded protein response in the murine liver.

The unfolded protein response (UPR) is a cellular stress response that is activated when misfolded proteins accumulate in the endoplasmic reticulum (ER). The UPR elicits a signaling cascade that results in an upregulation of protein folding machinery and cell survival signals. However, prolonged UPR responses can result in elevated cellular inflammation, damage, and even cell death. Thus, regulation of the UPR response must be tuned to the needs of the cell, sensitive enough to respond to the stress but pliable enough to be stopped after the crisis has passed. Previously, we discovered that the bi-functional enzyme FicD can modulate the UPR response via post-translational modification of BiP. FicD AMPylates BiP during homeostasis and deAMPylates BiP during stress. We found this activity is important for the physiological regulation of the exocrine pancreas. Here, we explore the role of FicD in the murine liver. Like our previous studies, livers lacking FicD exhibit enhanced UPR signaling in response to short term physiologic fasting and feeding stress. However, the livers of FicD -/- did not show marked changes in UPR signaling or damage after either chronic high fat diet (HFD) feeding or acute pathological UPR induction. Intriguingly, FicD -/- mice showed changes in UPR induction and weight loss patterns following repeated pathological UPR induction. These findings show that FicD regulates UPR responses during mild physiological stress and may play a role in maintaining resiliency of tissue through adaptation to repeated ER stress.

FicD regulates adaptation to the unfolded protein response in the murine liver.

The unfolded protein response (UPR) is a cellular stress response that is activated when misfolded proteins accumulate in the endoplasmic reticulum (ER). Regulation of the UPR response must be adapted to the needs of the cell as prolonged UPR responses can result in disrupted cellular function and tissue damage. Previously, we discovered that the enzyme FicD (also known as Fic or HYPE) through its AMPylation and deAMPylation activity can modulate the UPR response via post-translational modification of BiP. FicD AMPylates BiP during homeostasis and deAMPylates BiP during stress. We hypothesized that FicD regulation of the UPR will play a role in mitigating the deleterious effects of UPR activation in tissues with frequent physiological stress. Here, we explore the role of FicD in the murine liver. As seen in our pancreatic studies, livers lacking FicD exhibit enhanced UPR signaling in response to short term physiologic fasting and feeding stress. However, in contrast to studies on the pancreas, livers, as a more regenerative tissue, remained remarkably resilient in the absence of FicD. The livers of FicD-/- did not show marked changes in UPR signaling or damage after either chronic high fat diet (HFD) feeding or acute pathological UPR induction. Intriguingly, FicD-/- mice showed changes in UPR induction and weight loss patterns following repeated pathological UPR induction. These findings indicate that FicD regulates UPR responses during mild physiological stress and in adaptation to repeated stresses, but there are tissue specific differences in the requirement for FicD regulation.

FicD Sensitizes Cellular Response to Glucose Fluctuations in Mouse Embryonic Fibroblasts.

During homeostasis, the endoplasmic reticulum (ER) maintains productive transmembrane and secretory protein folding that is vital for proper cellular function. The ER-resident HSP70 chaperone, BiP, plays a pivotal role in sensing ER stress to activate the unfolded protein response (UPR). BiP function is regulated by the bifunctional enzyme FicD that mediates AMPylation and deAMPylation of BiP in response to changes in ER stress. AMPylated BiP acts as a molecular rheostat to regulate UPR signaling, yet little is known about the molecular consequences of FicD loss. In this study, we investigate the role of FicD in mouse embryonic fibroblast (MEF) response to pharmacologically and metabolically induced ER stress. We find differential BiP AMPylation signatures when comparing robust chemical ER stress inducers to physiological glucose starvation stress and recovery. Wildtype MEFs respond to pharmacological ER stress by downregulating BiP AMPylation. Conversely, BiP AMPylation in wildtype MEFs increases upon metabolic stress induced by glucose starvation. Deletion of FicD results in widespread gene expression changes under baseline growth conditions. In addition, FicD null MEFs exhibit dampened UPR signaling, altered cell stress recovery response, and unconstrained protein secretion. Taken together, our findings indicate that FicD is important for tampering UPR signaling, stress recovery, and the maintenance of secretory protein homeostasis. Significance Statement: The chaperone BiP plays a key quality control role in the endoplasmic reticulum, the cellular location for the production, folding, and transport of secreted proteins. The enzyme FicD regulates BiP's activity through AMPylation and deAMPylation. Our study unveils the importance of FicD in regulating BiP and the unfolded protein response (UPR) during stress. We identify distinct BiP AMPylation signatures for different stressors, highlighting FicD's nuanced control. Deletion of FicD causes widespread gene expression changes, disrupts UPR signaling, alters stress recovery, and perturbs protein secretion in cells. These observations underscore the pivotal contribution of FicD for preserving secretory protein homeostasis. Our findings deepen the understanding of FicD's role in maintaining cellular resilience and open avenues for therapeutic strategies targeting UPR-associated diseases.

AMPylation of small GTPases by Fic enzymes.

Small GTPases orchestrate numerous cellular pathways, acting as molecular switches and regulatory hubs to transmit molecular signals and because of this, they are often the target of pathogens. During infection, pathogens manipulate host cellular networks using post-translational modifications (PTMs). AMPylation, the modification of proteins with AMP, has been identified as a common PTM utilized by pathogens to hijack GTPase signalling during infection. AMPylation is primarily carried out by enzymes with a filamentation induced by cyclic-AMP (Fic) domain. Modification of small GTPases by AMP renders GTPases impervious to upstream regulatory inputs, resulting in unregulated downstream effector outputs for host cellular processes. Here, we overview Fic-mediated AMPylation of small GTPases by pathogens and other related PTMs catalysed by Fic enzymes on GTPases.

Test-retest reliability of the 6-minute walk test in individuals with Down syndrome.

OBJECTIVES: To evaluate the reliability of the 6-minute walk test (6MWT) in individuals with Down syndrome and explore factors affecting walking distance. DESIGN: Four repeated walk tests in the span of 2 weeks including 2 practice walks. SETTING: All tests were carried out in a 40-meter corridor at a university sport complex. PARTICIPANTS: Adolescents and young adults with Down syndrome (N=55) aged 11 to 26 years. INTERVENTION: Not applicable. MAIN OUTCOME MEASURE: Participants were instructed to walk as far as possible for the duration of 6 minutes. Distance walked, heart rate, blood pressure, and perceived exertion were measured across 4 tests (t1, t2, t3, and t4). RESULTS: The walking distances for t1, t2, t3, and t4 averaged 395, 428, 433, and 436 meters, respectively. The 6-minute walk distance (6MWD) during t1 and t2 was significantly different from that during t3 and t4 (t(54)=-6.475, P<.001). Repeated analysis of variance showed no significant difference between the distance walked in t3 and t4 (433±64m vs 436±68m) (F(1,54)=2.439, P=.124). Body mass index as well as levels of intellectual disability and physical activity all affected the distance walked to different degrees. CONCLUSIONS: The 6MWT showed good test-retest reliability and increased the walking distance after 2 practice walks, emphasizing the need to account for a learning effect among people with Down syndrome. Reported 6MWD appears lower than that previously reported for individuals without Down syndrome.

The effects of swim training on respiratory aspects of speech production in adolescents with down syndrome.

Reduced respiratory muscle strength in individuals with Down syndrome (DS) may affect speech respiratory variables such as maximum phonation duration (MPD), initiation volume, and expired mean airflow. Researchers randomly assigned adolescents with DS (N = 28) to either 12 weeks of swim training (DS-ST) or a control group (DS-NT). Repeated measures MANOVA demonstrated a significant increase in MPD for DS-ST participants from pretest to posttest, t(11) = -3.44, p = 0.006, that was not maintained at follow-up, t(11) = 6.680, p < .001. No significant change was observed for DS-NT participants across time, F(2, 11) = 4.20, p = 0.044. The lack of long-term change in DS-ST participants may be related to the relatively short training period.

Independent living in adults with autism spectrum disorder: Stakeholders' perspectives and experiences.

Independent living is a basic human right that enables individuals with disabilities to determine where they live, who they live with and what kind of support that they receive. Limited research exists regarding the challenges that adults with autism spectrum disorder (ASD) may face when attempting to live independently. Given the importance of independent living for adults with ASD, this study aimed to examine the perspectives of stakeholders about independent living among adults with ASD. METHODS: We recruited a total of 19 stakeholders including adults with ASD and parents of adults with ASD from Canadian provinces. In-depth, semi-structured interviews were conducted to identify factors affecting independent living among adults with ASD. Interviews were transcribed and analyzed using thematic analysis to identify overarching themes. RESULTS: Three themes emerged in our findings, including: a) Psychophysical stability and daily living; b) Financial management and planning; and c) Integrated community living and independence. CONCLUSION: The findings from this study suggest that adults with ASD face several challenges related to independent living. Factors related to psychophysical stability and daily living, financial management, and integrated community living and housing were all found to influence the ability of adults with ASD to live independently. By exploring stakeholders' perspectives of independent living for adults with ASD, this study provides some insight that can help inform the development of programs and services to facilitate independent living for adults with ASD.

Manipulation of IRE1-Dependent MAPK Signaling by a Vibrio Agonist-Antagonist Effector Pair.

Diverse bacterial pathogens employ effector delivery systems to disrupt vital cellular processes in the host (N. M. Alto and K. Orth, Cold Spring Harbor Perspect Biol 4:a006114, 2012, https://doi.org/10.1101/cshperspect.a006114). The type III secretion system 1 of the marine pathogen Vibrio parahaemolyticus utilizes the sequential action of four effectors to induce a rapid, proinflammatory cell death uniquely characterized by a prosurvival host transcriptional response (D. L. Burdette, M. L. Yarbrough, A Orvedahl, C. J. Gilpin, and K. Orth, Proc Natl Acad Sci USA 105:12497-12502, 2008, https://doi.org/10.1073/pnas.0802773105; N. J. De Nisco, M. Kanchwala, P. Li, J. Fernandez, C. Xing, and K. Orth, Sci Signal 10:eaa14501, 2017, https://doi.org/10.1126/scisignal.aal4501). Herein, we show that this prosurvival response is caused by the action of the channel-forming effector VopQ that targets the host V-ATPase, resulting in lysosomal deacidification and inhibition of lysosome-autophagosome fusion. Recent structural studies have shown how VopQ interacts with the V-ATPase and, while in the ER, a V-ATPase assembly intermediate can interact with VopQ, causing a disruption in membrane integrity. Additionally, we observed that VopQ-mediated disruption of the V-ATPase activates the IRE1 branch of the unfolded protein response (UPR), resulting in an IRE1-dependent activation of ERK1/2 MAPK signaling. We also find that this early VopQ-dependent induction of ERK1/2 phosphorylation is terminated by the VopS-mediated inhibitory AMPylation of Rho GTPase signaling. Since VopS dampens VopQ-induced IRE1-dependent ERK1/2 activation, we propose that IRE1 activates ERK1/2 phosphorylation at or above the level of Rho GTPases. This study illustrates how temporally induced effectors can work as in tandem as agonist/antagonist to manipulate host signaling and reveals new connections between V-ATPase function, UPR, and MAPK signaling.IMPORTANCE Vibrio parahaemolyticus is a seafood-borne pathogen that encodes two type 3 secretion systems (T3SS). The first system, T3SS1, is thought to be maintained in all strains of V. parahaemolyticus to maintain survival in the environment, whereas the second system, T3SS2, is linked to clinical isolates and disease in humans. Here, we found that first system targets evolutionarily conserved signaling systems to manipulate host cells, eventually causing a rapid, orchestrated cells death within 3 h. We have found that the T3SS1 injects virulence factors that temporally manipulate host signaling. Within the first hour of infection, the effector VopQ acts first by activating host survival signals while diminishing the host cell apoptotic machinery. Less than an hour later, another effector, VopS, reverses activation and inhibition of these signaling systems, ultimately leading to death of the host cell. This work provides example of how pathogens have evolved to manipulate the interplay between T3SS effectors to regulate host signaling pathways.

Dual-energy x-ray absorptiometry to measure the influence of a 16-week community-based swim training program on body fat in children and adolescents with intellectual disabilities.

OBJECTIVE: To use dual-energy x-ray absorptiometry (DXA) to measure the effects of a 16-week community-based swim training program on percent body fat in children and adolescents with intellectual disability (ID). DESIGN: Convenience sample. SETTING: University sport complex and exercise science laboratory. PARTICIPANTS: Children and adolescents (n=8; mean age +/- SD, 13.1 +/- 3.4 y), 2 girls and 6 boys with ID, of varying fat levels (11%-35%). INTERVENTION: A swim training program lasting for the duration of 16 weeks with three 1-hour sessions held at a 25-m pool each week. MAIN OUTCOME MEASURE: Assessing percent body fat at pretest and posttest through the use of DXA. RESULTS: After the 16-week exercise training program, we observed a 1.2% median increase in body fat percentage with a range from -0.3% to 4.5%. Wilcoxon matched-pairs signed-ranks tests suggest that these results are statistically significant (P=.039; exact). CONCLUSIONS: Exercise training alone proved ineffectual in reducing percent body fat in 8 children and adolescents with ID. Further research should consider implementing a combined diet and exercise program. To gauge the effectiveness of intervention programs, valid methods and complex measurement tools such as DXA should be used to assess changes in percent body fat in such a heterogeneous population.

A distinct inhibitory mechanism of the V-ATPase by Vibrio VopQ revealed by cryo-EM.

The Vibrio parahaemolyticus T3SS effector VopQ targets host-cell V-ATPase, resulting in blockage of autophagic flux and neutralization of acidic compartments. Here, we report the cryo-EM structure of VopQ bound to the Vo subcomplex of the V-ATPase. VopQ inserts into membranes and forms an unconventional pore while binding directly to subunit c of the V-ATPase membrane-embedded subcomplex Vo. We show that VopQ arrests yeast growth in vivo by targeting the immature Vo subcomplex in the endoplasmic reticulum (ER), thus providing insight into the observation that VopQ kills cells in the absence of a functional V-ATPase. VopQ is a bacterial effector that has been discovered to inhibit a host-membrane megadalton complex by coincidentally binding its target, inserting into a membrane and disrupting membrane potential. Collectively, our results reveal a mechanism by which bacterial effectors modulate host cell biology and provide an invaluable tool for future studies on V-ATPase-mediated membrane fusion and autophagy.