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Compound-specific isotopic analysis of amino acids (CSIA-AA) is increasingly used in ecological and biogeochemical studies tracking the origin and fate of nitrogen (N). Its advantages include the potential for resolving finer-scale trophic dynamics than possible with standard bulk SIA and for reconstructing historical changes in the food webs of consumers from analyses of specimens in preserved sample archives. For the latter, assessing the effects of chemical preservatives on δ15NAA has been inconclusive because the conventional CSIA approach for derivatized AAs by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) has analytical errors (0.4-1.0 parts per thousand) in the range expected from chemical preservation. Here, we show improved analytical precision (0.15 ± 0.08‰) for 11 underivatized AA standards analyzed by high-pressure liquid chromatography followed by offline elemental analysis-IRMS (HPLC/EA-IRMS). Using this method, we report the first high-precision tests of preservation effects on δ15NAA in Northern Anchovy (Engraulis mordax) kept 1.5-yr in ethanol and up to 27-yr in formaldehyde. We found minimal methodological induced fractionation for eight AAs, and preservation effects on δ15N were similar regardless of duration and preservative used. Although some of the AAs differed significantly from frozen control samples (AA average +1.0 ± 0.8‰), changes in δ15N in the source AA phenylalanine and trophic position estimates were statistically insignificant. Our results are encouraging for resolving fine-scale natural variability using HPLC/EA-IRMS on chemically preserved specimens and for ultimately reconstructing biogeochemical records and trophic dynamics over long time scales.

Objective: This study tested the feasibility of a self-help intervention based on Compassion-Focused Theory (CFT), and estimated treatment effects in a population of adults with skin conditions and...

Cyclin-dependent kinases (CDKs) are the master regulators of the eukaryotic cell cycle. To become activated, CDKs require both regulatory phosphorylation and binding of a cognate cyclin subunit. We studied the activation process of the G1/S kinase Cdk2 in solution and developed a thermodynamic model that describes the allosteric coupling between regulatory phosphorylation, cyclin binding, and inhibitor binding. The results explain why monomeric Cdk2 lacks activity despite sampling an active-like state, reveal that regulatory phosphorylation enhances allosteric coupling with the cyclin subunit, and show that this coupling underlies differential recognition of Cdk2 and Cdk4 inhibitors. We identify an allosteric hub that has diverged between Cdk2 and Cdk4 and show that this hub controls the strength of allosteric coupling. The altered allosteric wiring of Cdk4 leads to compromised activity toward generic peptide substrates, and comparative specialization toward its primary substrate Retinoblastoma (RB).

The Schild site in west-central Illinois comprises two Mississippian cemeteries (ca. AD 1030), one of which includes the burial of a young male (SA117) without a left hand who was buried on the periphery of the cemetery. We suggest a complicated scenario including that the forearm was burned, resulting in loss of the hand, and we argue for a deviant social identity for this unfortunate young man. SA117 was not provided with grave goods—he was buried in a semi-flexed position, and his grave is unusual in that a fire was built over the grave fill. His marginalization and his disability may reflect outsider status; in fact, based on principal components analysis, his skull differs significantly from other Mississippian males. Several American Indian groups marked unusual causes of death in mortuary practices, and survivors of some kinds of trauma—for example, scalping—were treated as if dead. The use of fire in ordeals marked the transformation of captives into slaves or adopted members of the captor community; SA117 may have been such a person.

Skin shame plays an important role in the psychological distress of dermatological patients. However, it is rarely examined in research or clinical practice due to the lack of availability of a robust measure of skin shame. This study sought to adapt and validate the Skin Shame Scale (SSS-24) for use in the German-speaking population.The SSS-24 questionnaire was completed by 488 dermatologically healthy (DH) individuals (66.6% women) and 339 dermatological patients (DP; 66.7% women). Participants also completed measures of general shame (SHAME) and of psychological distress (BSI[Brief Symptom Inventory]-18).The SSS-24 showed adequate psychometric properties and a high internal consistency in both samples (DH: α = 0.91; DP: α = 0.95). DP reported more skin shame than DH (p 0.001) but in both samples more skin shame was associated with more general shame and more psychological distress (p 0.001).Overall, the psychometric properties of the English original version were replicated in the German version of the SSS-24. Due to the strong associations with general shame and psychological distress, skin shame should be considered an important parameter in psychodermatology. The SSS-24 enables further research into the role of shame in skin conditions and provides a useful tool for identifying patients who might benefit from psychosocial interventions.

BACKGROUND There is a lack of psychological support for individuals with skin conditions, and few low-intensity self-help interventions are available. OBJECTIVE This study aimed to test the acceptability and usability of a support website and its embedded self-help resources. METHODS A mixed methods approach was utilized. A total of 583 participants (426 with a skin condition, 97 relatives and friends, and 60 dermatology professionals) viewed the British Association of Dermatologist’s SkinSupport website and then completed a survey about their well-being and the usability of the website. A comparison group comprising 816 participants also completed the well-being measures. In total, 37 participants (19 living with a skin condition, and the relatives and friends of individuals with a skin condition, and 18 dermatology professionals) viewed the SkinSupport website and then took part in focus groups. Participants were recruited via social media, professional networks, and volunteer lists. Data from the survey were analyzed using descriptive and inferential statistics and qualitative content analysis. RESULTS Both quantitative and qualitative responses suggest that the SkinSupport website was viewed positively by both patients and health professionals. Overall, 79.8% (417/523) of individuals with a skin condition, and the relatives and friends of individuals with a skin condition, said that they would use the website again; and 86.7% (52/60) of dermatology professionals said that they would recommend the site to somebody with a skin condition. Qualitative responses related to the website fell into 4 key themes: (1) appearance, (2) use and navigation, (3) information, and (4) areas for development. CONCLUSIONS The SkinSupport website was considered acceptable and usable. A range of areas requiring modification were identified. The website provides a useful resource that patients can access freely. Given the lack of services available to patients with skin conditions, health care professionals could routinely inform patients of this resource at assessment.

Compound specific isotopic analysis of amino acids (CSIA-AA) is increasingly used in ecological and biogeochemical studies tracking the origin and fate of nitrogen (N). Its advantages include the potential for resolving finer-scale trophic dynamics than possible with standard bulk SIA and for reconstructing historical changes in the food webs of consumers from analyses of specimens in preserved sample archives. For the latter, assessing the effects of chemical preservatives on δ15NAA has been inconclusive because the conventional CSIA approach for derivatized AAs by gas chromatography – combustion – isotope ratio mass spectrometry (GC-C-IRMS) has analytical errors (0.4 - 1.0 ‰) in the range expected from chemical preservation. Here, we show improved analytical precision (0.15 ± 0.08 ‰) for 11 underivatized AA standards analyzed by high pressure liquid chromatography followed by offline elemental analysis – IRMS (HPLC/EA-IRMS), an approach originally developed by Broek and McCarthy (2014). Using this method, we report the first high-precision tests of preservation effects on δ15NAA in Northern Anchovy (Engraulis mordax) kept 1½-year in ethanol and up to 27-years in formaldehyde. We found minimal methodological induced fractionation for 8 AAs, and preservation effects on δ15N were similar regardless of duration and preservative used. Although some of the AAs differed significantly from frozen control samples (average +1.0 ± 0.8 ‰), changes in δ15N in the source AA Phenylalanine and trophic position estimates were statistically insignificant. Our results are encouraging for resolving fine-scale natural variability using HPLC/EA-IRMS on chemically preserved specimens and for ultimately reconstructing biogeochemical records and trophic dynamics over long time scales.

Burns patients often suffer severe pain during interventions such as dressing changes, even with analgesia. Virtual Reality (VR) can be used to distract patients and reduce pain. However, more evidence is needed from the patients and staff using the technology about its use in clinical practice and the impact of different VR strategies. This small-scale qualitative study explored patient and staff perceptions of the impact and usability of active and passive VR during painful dressing changes. Five patients took part in three observed dressing changes—one with an active VR scenario developed for the study, one with passive VR, and one with no VR—following which they were interviewed about their experiences. Three nurses who performed the dressing changes participated in a focus group. Thematic analysis of the resulting data generated four themes: “Caution replaced by contentment,” “Distraction and implications for pain and wound care,” “Anxiety, control and enjoyment,” and “Preparation and communication concerns.” Results suggested that user-informed active VR was acceptable to burn patients, helped manage their perceived pain, and was both usable and desirable within the clinical environment. Further testing with larger samples is now required.

Background\ud Individuals with visible differences can experience appearance anxiety that is distressing and disruptive to daily functioning. Understanding psychological factors that maintain appearance anxiety related to scarring is important in developing theoretical understanding of adjustment to injury, and in identifying targets for psychological therapies. This study aimed to investigate whether psychological flexibility, a key element underpinning acceptance and commitment therapy (ACT), was associated with appearance anxiety. It was hypothesised that reduced psychological flexibility (lower acceptance, cognitive defusion, mindfulness, and committed action) would be related to increased appearance anxiety. The role of psychological flexibility in the maintenance of appearance anxiety was investigated using a cross-sectional quantitative questionnaire study.\ud \ud Method\ud Seventy-eight burns patients (47 female, 31 male; M age = 45.2 years) completed the Derriford Appearance Scale (DAS-24), the Acceptance and Action Questionnaire (AAQ-II), the Cognitive Fusion Questionnaire (CFQ), the Five Facet Mindfulness Questionnaire (FFMQ), and the Committed Action Questionnaire (CAQ-8).\ud \ud Results\ud As hypothesised, increased appearance anxiety was related to reduced acceptance (rs(76) = 0.80, p < 0.001, one-tailed) and cognitive defusion (rs(76) = 0.76, p < 0.001). Reduced levels of mindfully describing (r(72) = −0.39, p < 0.001), acting with awareness (r(72) = −0.57, p < 0.001), non-judging (r(72) = −0.61, p < 0.001) and non-reactivity (r(72) = −0.28, p < 0.01) as well as reduced committed action (r(72) = −0.57, p < 0.001) were also related to increased appearance anxiety.\ud \ud Conclusions\ud Individuals experiencing appearance anxiety associated with a burn injury may struggle with accepting difficult emotions, stepping back from distressing thoughts, being mindful and engaging in valued action. These findings suggest that ACT may be useful in treating appearance related anxiety and concerns associated with conditions causing a visible difference.

Females often receive autism spectrum condition diagnoses later than males, leaving needs misunderstood. This study aimed to explore the lived experiences of female adults diagnosed with an autism spectrum condition in middle to late adulthood. Eleven autistic females diagnosed over the age of 40 years completed semi-structured interviews, analysed using Interpretative Phenomenological Analysis. Four superordinate themes emerged: A hidden condition (pretending to be normal and fitting in; mental health and mislabelling), The process of acceptance (initial reactions and search for understanding; re-living life through a new lens), The impact of others post-diagnosis (initial reactions; stereotyped assumptions), and A new identity on the autism spectrum (negotiating relationships, connections and community; changing well-being and views of the self; the meaning of diagnosis). Findings highlight several factors not previously identified that affect late diagnosis in females, including widespread limited understandings of others. Diagnosis was experienced by several participants as facilitating transition from being self-critical to self-compassionate, coupled with an increased sense of agency. Participants experienced a change in identity that enabled greater acceptance and understanding of the self. However, this was painful to adjust to at such a late stage.

Summary Background Those affected by alopecia are at risk of experiencing a range of psychosocial consequences. Alopecia UK provides online peer support via facilitated Facebook pages. Online peer support has been found to provide a variety of benefits in other long‐term conditions, such as providing information and emotional support. Objectives This study sought to gain an experiential account as to how online support provides benefit to people living with alopecia. Methods A purposive sample of 12 participants was recruited directly from Alopecia UK's Facebook group. A total of 11 were diagnosed with alopecia and one participant was a family member of someone with alopecia. The qualitative approach of Interpretative Phenomenological Analysis was used to guide data collection and analysis. Participants took part in online synchronous interviews, which drew on review of participant‐selected screenshots of online activity. Results Four themes were identified, including ‘gradual healing’, ‘image concern’, ‘belonging’ and ‘new identity and self‐acceptance’. The participants made use of the group through expressing emotion, finding practical advice, and also as a place to connect and share experiences. Participants also reported that the online support facilitated the return of social confidence. Conclusions This study provides a detailed understanding as to how online peer support groups can be beneficial. Online groups have the potential to create a feeling of belonging, which can develop a sense of being accepted and understood and as such can be important in maintaining psychological well‐being. The findings indicate that online groups have the potential to be used as a platform for assisting people in developing both effective coping styles and feeling understood, and such platforms warrant further investment by healthcare professionals. What's already known about this topic? Alopecia may be associated with psychosocial distress due to both the individual impact and the reactions of others.Online peer support is known to be a beneficial method of providing psychosocial support for people living with a range of health conditions. What does this study add? An in‐depth view of the benefits experienced through belonging to an online peer support group for those diagnosed with alopecia, in addressing the psychosocial effects experienced with the condition.This research highlights the need for further investigation of the utility of supporting the development of online peer support for people living with skin conditions.Online peer support groups could be highly effective to use in combination with existing psychological therapies to connect others with shared experience. What are the clinical implications of this work? People living with alopecia can benefit from peer support, and healthcare professionals should ensure that patients know where they can gain access to both psychological support and peer support., https://www.bjdonline.com/article/investigating-the-beneficial-experiences-of-online-peer-support-for-those-affected-by-alopecia-an-interpretative-phenomenological-analysis-using-online-interviews/ Linked Comment: https://doi.org/10.1111/bjd.18354. https://doi.org/10.1111/bjd.18498 available online

Childhood skin conditions can affect the quality of life of children, adolescents, and families. As such, paediatric dermatological conditions have been the focus of a number of qualitative studies and there is now a need to integrate the findings. A meta-ethnography was carried out with the existing 12 studies, which included nine studies examining parental experiences and three studies of child experiences. Meta-ethnographic analysis of the studies identified themes focused on the child's sense of stigmatisation and the challenges for families that arose from this. Common experiences across studies were feelings of difference relating to the appearance of the skin. The results highlight that children and adolescents can experience negative social reactions and that parents may struggle with some aspects of the physical management of the condition. The studies indicate the need to examine in more detail the psychosocial aspects of childhood skin conditions and the role played by stigmatisation.

Changes in medical school curricula often require educators to develop teaching strategies that decrease contact hours while maintaining effective pedagogical methods. When faced with this challenge, faculty at the University of Cincinnati College of Medicine converted the majority of in-person histology laboratory sessions to self-study modules that utilize multiple audiovisual modalities and a virtual microscope platform. Outcomes related to this shift were investigated through performance on in-house examinations, results of the United States Medical Licensing Examination® (USMLE® ) Step 1 Examination, and student feedback. Medical School College Admissions Test® (MCAT® ) scores were used as a covariate when comparing in-house examinations. Results revealed no significant change in performance on in-house examinations when the content being assessed was controlled (F(2, 506) = 0.676, P = 0.51). A significant improvement in overall practical examination grade averages was associated with the self-study modules (F(6, 1164) = 10.213, P

Understanding the processes that affect fish larval survival and recruitment is a fundamental tenant of fisheries science. Small, isolated fjords are ideal study systems for elucidating early life history processes, as population dynamics are well traced in these partially closed systems. We examined the distribution, growth and mortality of eggs and larvae of a fjord population of cod during a 5 mo field campaign in the fjord Kapisigdlit, West Greenland. Cod mainly spawnedearly in the season in the innermost shallow region of the fjord. Egg survival was generally high in the fjord. The high survival may have been driven by relatively high temperature and/or low predation in the inner region. Early in the season, the distribution of eggs and young larvae was mostly restricted to the spawning area. Later in the season, larger larvae had become more evenly distributedin the fjord. This shift in distribution was observed after the seasonal pulse in freshwater outflow following the ice break-up in Kapisigdlit River. There was a positive correlation between the amount of food in a larval stomach and growth, and larval growth was greater in the outer fjord where prey availability was higher. The timing between spawning and freshwater input may be essential for survival and recruitment, this ensuring low dispersal of eggs and younger stages and high dispersal of older, actively feeding stages. Therefore, cod in this area could be vulnerable to future climate change affecting the timing and magnitude of freshwater outflow, by changes in precipitation, temperature or prey availability

Gross anatomy practical examinations have traditionally been composed of primarily free-response identification questions. Because student learning and study habits are influenced by testing, it is important that curricular design and objectives are reflected in all types of assessments, including those that take place in a laboratory setting. The use of higher-order and discipline-integrated questions on gross anatomy practical examinations reinforces the importance that anatomy knowledge has in clinical reasoning and assesses students’ ability to apply foundational information from an early point in their medical career. In this study, gross anatomy practical examinations were analyzed to assess the impact that higher-order and discipline-integrated testing modalities have on various measures of student performance. Two years of examinations (N = 338 questions) were reviewed and categorized by question type, Bloom’s taxonomy and discipline. Difficulty, discrimination index (DI) and point-biserial correlations were calculated for each question. These statistics were then compared between categories using Kruskal-Wallis tests with post-hoc pairwise comparisons (Dunn’s method). Results demonstrate that higher-order and discipline-integrated questions were significantly more difficult than traditional identification questions and tended to have higher mean DIs and point-biserials. Questions that targeted higher Bloom’s taxonomic levels were also found to be more difficult. This study demonstrates that integrated examinations can be incorporated into the gross anatomy laboratory, but suggests that this be done carefully as these changes can impact student performance.

The California Undercurrent transports Pacific Equatorial Water (PEW) into the Southern California Bight from the eastern tropical Pacific Ocean. PEW is characterized by higher temperatures and salinities, with lower pH, representing a source of potentially corrosive (aragonite, Ω

The Beaded Burial central to F101 within Cahokia’s mound 72Sub1 has been fundamental to some cosmological explanations of the founding of this North American precolumbian polity. The central burial, identified as two males surrounded by retainers, has been interpreted as paradigmatic of a paramount chiefdom, or conversely, as a mythic cosmogram. Recent bioarchaeological reanalysis and two independent osteological studies of F101 and associated burials have identified the presence of male/female pairs, numerous females, and at least one child, suggesting that previous explanations privileging the male Red Horn association should be reexamined. We suggest that 72Sub1 is most likely correlated with ritual practices promoting world creation, renewal, and fertility symbolism.

We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electron-electron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiac myosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0-C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein's flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein-protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility.

Background There is a lack of psychological support for individuals with skin conditions, and few low-intensity self-help interventions are available. Objective This study aimed to test the acceptability and usability of a support website and its embedded self-help resources. Methods A mixed methods approach was utilized. A total of 583 participants (426 with a skin condition, 97 relatives and friends, and 60 dermatology professionals) viewed the British Association of Dermatologist’s SkinSupport website and then completed a survey about their well-being and the usability of the website. A comparison group comprising 816 participants also completed the well-being measures. In total, 37 participants (19 living with a skin condition, and the relatives and friends of individuals with a skin condition, and 18 dermatology professionals) viewed the SkinSupport website and then took part in focus groups. Participants were recruited via social media, professional networks, and volunteer lists. Data from the survey were analyzed using descriptive and inferential statistics and qualitative content analysis. Results Both quantitative and qualitative responses suggest that the SkinSupport website was viewed positively by both patients and health professionals. Overall, 79.8% (417/523) of individuals with a skin condition, and the relatives and friends of individuals with a skin condition, said that they would use the website again; and 86.7% (52/60) of dermatology professionals said that they would recommend the site to somebody with a skin condition. Qualitative responses related to the website fell into 4 key themes: (1) appearance, (2) use and navigation, (3) information, and (4) areas for development. Conclusions The SkinSupport website was considered acceptable and usable. A range of areas requiring modification were identified. The website provides a useful resource that patients can access freely. Given the lack of services available to patients with skin conditions, health care professionals could routinely inform patients of this resource at assessment.

Burns patients often suffer excruciating pain during clinical procedures, even with analgesia. Virtual Reality as an adjunct to pharmacological therapy has proved promising in the management of burn pain. More evidence is needed regarding specific forms of Virtual Reality. This mixed-method study examined the impact of active and passive Virtual Reality scenarios in experimental conditions, gathering data relating to user experience, acceptability and effectiveness in managing pain. Four scenarios were developed or selected following a consultative workshop with burns survivors and clinicians. Each was trialled using a cold pressor test with 15 University students. Data were gathered regarding pain threshold and tolerance at baseline and during each exposure. Short interviews were conducted afterwards. The two active scenarios were ranked highest and significantly extended participants pain threshold and tolerance times compared to passive and baseline conditions. Passive scenarios offered little distraction and relief from pain. Active scenarios were perceived to be engaging, challenging, distracting and immersive. They reduced subjective awareness of pain, though suggestions were made for further improvements. Results suggested that active Virtual Reality was acceptable and enjoyable as a means of helping to control experimental pain. Following suggested improvements, scenarios should now be tested in the clinical environment.

Introduction: People with a visible difference, such as scarring or a skin condition, can experience anxiety and intrusive reactions from others when in social situations. The use of products to conceal marks on the skin is provided in a number of different hospital services and by charities. However, there are relatively few psychosocial interventions available for these individuals. Objectives: To examine the views of skin camouflage users and practitioners on the acceptability, usability and need for a specifically developed cognitive behavioural therapy (CBT) self-help booklet. Methods: A think-aloud protocol and descriptive form of thematic analysis were used to ascertain participants’ views of this novel psychosocial intervention. Nine participants took part in think-aloud interviews that were analysed using thematic analysis. Six skin camouflage users and three skin camouflage practitioners participated in the study. Results: Support for the relevance, acceptability and usability of the booklet was found from both participants who used camouflage and those who provided it. However, some participants reported that they would envisage that some people would need additional support so as to be able to use the techniques described within the booklet. Conclusions: This study represents an important step towards developing a brief self-help intervention for people with living with visible skin conditions or scars and demonstrates the importance of seeking feedback from experts by experience on theoretically informed psychological interventions for this patient group., Lay summary Individuals with visible difference can experience anxiety, depression and decreased quality of life. However, there are relatively few psychosocial interventions available to help improve the lives of individuals with visible difference. This study provides preliminary evidence for the feasibility and usability of a novel psychosocial intervention, designed to improve social confidence and reduce social distress, for individuals with visible difference. Specifically, we sought to examine the views of skin camouflage users and practitioners on the acceptability, usability and need for a specifically developed cognitive behavioural therapy (CBT) self-help booklet. An interview which included a guide to facilitate reflection on the materials was used to ascertain participants’ views of our novel psychosocial intervention. Nine participants took part, six skin camouflage users and three skin camouflage practitioners. The participants indicated that the self-help intervention had relevance, acceptability and usability. However, some participants reported that they would envisage that some people might need additional support to be able to use the techniques contained within the booklet. The findings from this study highlights the need for further development and testing of this psychosocial intervention, which holds promise for improving the lives of individuals with visible difference.

We have measured the orientation of the myosin light chain domain (lever arm) elements in demembranated muscle fibers by electron paramagnetic resonance (EPR), using a bifunctional spin label (BSL), with angular resolution of 4 degrees. Despite advances in X-ray crystallography and cryo-electron microscopy (cryo-EM), and fluorescence polarization, none of these techniques provide high-resolution structural information about the myosin light chain domain under ambient conditions in a muscle fiber. Two cysteines, 4 residues apart, were engineered on two α-helices in the myosin regulatory light chain (RLC), permitting stereoselective site-directed labeling with BSL. One labeled helix (helix E) is adjacent to the myosin lever arm, the other helix (helix B) is located farther apart from the motor domain beyond the “hinge” of the myosin. By exchanging BSL-labeled RLC onto oriented muscle fibers, we obtained EPR spectra that determined angular distributions of BSL with high resolution, which enabled the accurate determination of helix orientation of individual structural elements with respect to the muscle fiber axis. In the absence of ATP (rigor), each of the two labeled helices exhibited both ordered (σ ~ 9-11 degrees) and disordered (σ > 38 degrees) populations. We used these angles to determine the orientation of the myosin lever arm, concluding that the oriented population has lever arms that are perpendicular to the muscle fiber axis. This orientation is ~33 degrees different than predicted from a standard “lever arm down” model based on cryo-EM of actin decorated with isolated myosin heads, but it is compatible with fluorescence polarization and EM data obtained from muscle fibers. The addition of ATP, in the absence of Ca2+, shifted the orientation to a much more disordered distribution.SummaryWe used electron paramagnetic resonance to determine the orientation of elements within the myosin regulatory light chain in skinned skeletal muscle fibers. A bifunctional spin label provided sufficient resolution to detect an ordered population of lever arms perpendicular to actin.

The Improving Access to Psychological Therapies (IAPT) programme started in 2008, but it contained little provision for specifically meeting the needs of Black, Asian and minority ethnic (BAME) groups. The purpose of this evaluation was to describe the experience of transition from BAME community mental health worker (CMHW) to IAPT low-intensity psychological wellbeing practitioner (PWP) in order to identify possible gains and losses for the former communities served, and the factors that might contribute to successful training of people with BAME expertise. Four former CMHWs who had transitioned into working as PWPs were interviewed. Semi-structured interviews were used. The data were analysed using thematic analysis. Six major themes were identified with the benefits of training emerging as an important factor for the participants in enhancing their role. Three of the themes interconnected and focused on the impact for BAME communities in terms of access to service and barriers. Evident in the interviews were descriptions of adaptations that were made as a result of CMHW having access to both new and old skills. Finally, two themes focused on the participant recommendations as to how IAPT services might become more culturally responsive. The findings suggest that there can be significant benefits for services to provide IAPT training to people already providing culturally specific services. The participants reported that low-intensity cognitive behavioural therapy (LICBT) was effective, but only when cultural sensitive adaptations were made. The evaluation has some clear recommendations as to how IAPT services might seek to offer culturally responsive CBT. Suggestions for carrying out further practice-based evaluations are made.

Background Despite increased understandings that the whole family system is influenced by a family member being diagnosed with an Autism Spectrum Condition (ASC), the experiences of siblings can be overlooked. However, recently understanding of siblings’ experiences has begun to develop via qualitative studies. Aims This review aimed to systematically identify, appraise and review the qualitative literature investigating the lived experience of siblings of autistic people. Methods and procedures Six databases were systematically searched for studies reporting empirical qualitative data on the lived experience of neurotypical siblings of autistic people. Eighteen studies met inclusion criteria and were analysed using Thematic Synthesis. Outcomes and results Data synthesis revealed four superordinate themes; Roles and responsibilities, different to what may be expected typically; Impact of behaviours, particularly aggressive behaviours; Process of adjustment, including learning and developing empathy and acceptance; Interpersonal experiences with others and between siblings. Conclusions and implications A narrative of love and empathy was present throughout. However, several participants also experienced significant difficulties affecting their mental health. Findings suggest a sense of agency, understanding ASC, time spent alone with parents, supportive environments and the chance to relate to other siblings as protective factors in facilitating positive psychological wellbeing for siblings of autistic people.

Significance Many drugs trigger changes to the structure of their target receptor upon binding. These conformational effects are thought to be an essential part of molecular recognition but have proven challenging to quantify. Using a high-throughput method for tracking structural changes in a protein kinase in solution, we discovered that many clinically important cancer drugs trigger substantial structural changes to their target protein kinase Aurora A, and that these effects systematically account for the ability of the drugs to differentiate between different biochemical forms of Aurora A. The results provide insight into mechanisms of drug selectivity and suggest strategies for tailoring inhibitors to target certain cancers in which Aurora A has been dysregulated in different ways., Protein kinases undergo large-scale structural changes that tightly regulate function and control recognition by small-molecule inhibitors. Methods for quantifying the conformational effects of inhibitors and linking them to an understanding of selectivity patterns have long been elusive. We have developed an ultrafast time-resolved fluorescence methodology that tracks structural movements of the kinase activation loop in solution with angstrom-level precision, and can resolve multiple structural states and quantify conformational shifts between states. Profiling a panel of clinically relevant Aurora kinase inhibitors against the mitotic kinase Aurora A revealed a wide range of conformational preferences, with all inhibitors promoting either the active DFG-in state or the inactive DFG-out state, but to widely differing extents. Remarkably, these conformational preferences explain broad patterns of inhibitor selectivity across different activation states of Aurora A, with DFG-out inhibitors preferentially binding Aurora A activated by phosphorylation on the activation loop, which dynamically samples the DFG-out state, and DFG-in inhibitors binding preferentially to Aurora A constrained in the DFG-in state by its allosteric activator Tpx2. The results suggest that many inhibitors currently in clinical development may be capable of differentiating between Aurora A signaling pathways implicated in normal mitotic control and in melanoma, neuroblastoma, and prostate cancer. The technology is applicable to a wide range of clinically important kinases and could provide a wealth of valuable structure–activity information for the development of inhibitors that exploit differences in conformational dynamics to achieve enhanced selectivity.

While it is widely acknowledged that people living with skin conditions can experience higher levels of psychosocial distress than the general population, access to psychologic support in dermatology is limited. Given the physical and psychosocial consequences of living with skin conditions, interventions used within physical and mental health may be beneficial. Mindfulness, defined as "paying attention in a particular way: on purpose in the present moment and non-judgmentally," has shown promise in improving outcomes in both mental and physical health populations, and studies have implicated a role for mindfulness in improving distress associated with skin conditions. The current review explores the theoretical underpinnings of mindfulness, in particular, the role it may play in reducing physiologic arousal and managing maladaptive thought processes. Although mindfulness interventions offer promise in reducing distress associated with skin conditions, further research is required to fully understand the underlying mechanisms of mindfulness and the active ingredient responsible for improving outcomes in dermatology patients. Mindfulness is one potential psychologic intervention and practitioners should be aware of the range of psychologic support options available. The current review also draws attention to the urgent need for further research into the effectiveness of psychologic interventions for dermatology patients.

We have employed two complementary high-resolution electron paramagnetic resonance (EPR) techniques with a bifunctional spin label (BSL) to test and refine protein structural models based on crystal structures and cryo-EM. We demonstrate this approach by investigating the effects of nucleotide binding on the structure of myosin’s catalytic domain (CD), while myosin is in complex with actin. Unlike conventional spin labels attached to single Cys, BSL reacts with a pair of Cys; in this study, we thoroughly characterize BSL’s rigid, highly stereoselective attachment to protein α-helices, which permits accurate measurements of orientation and distance. Distance constraints were obtained from double electron-electron resonance (DEER) on myosin constructs labeled with BSL specifically at two sites. Constraints for orientation of individual helices were obtained previously from continuous-wave EPR (CW-EPR) of myosin labeled at specific sites with BSL in oriented muscle fibers. We have shown previously that CW-EPR of BSL quantifies helix orientation within actin-bound myosin; here we show that the addition of high-resolution distance constraints by DEER alleviates remaining spatial ambiguity, allowing for direct testing and refinement of atomic structural models. This approach is applicable to any orientable complex (e.g., membranes or filaments) in which site-specific di- Cys mutation is feasible.

We have used high-resolution orientation and distance measurements derived from electron paramagnetic resonance of a bifunctional spin label (BSL) to build and refine atomistic models of protein structure. We demonstrate this approach by investigating the effects of nucleotide binding on the structure of myosin's catalytic domain while myosin is in complex with actin. Constraints for orientation of individual helices were obtained in a previous study from continuous-wave electron paramagnetic resonance of myosin labeled at specific sites with BSLs in oriented muscle fibers. In this study, new distance constraints were derived from double electron-electron resonance on myosin constructs labeled with a BSL specifically at two sites. Using these complementary constraints together, we thoroughly characterize the BSL's rigid, highly stereoselective attachment to protein α-helices, which permits accurate measurements of orientation and distance. We also leverage these measurements to derive a novel, to our knowledge, structural model for myosin-II in complex with actin and MgADP and compare our model to other recent actomyosin structures. The described approach is applicable to any orientable complex (e.g., membranes or filaments) in which site-specific di-Cys mutation is feasible.

The role of human dissection in modern medical curricula has been a topic of intense debate. In part, this is because dissection can be time-consuming and curricular hours are being monitored more carefully. This has led some to question the efficacy and importance of dissection as a teaching method. While this topic has received considerable attention in the literature, the question of how dissection impacts learning has been difficult to evaluate in a real-world, high-stakes setting since participation in dissection is often one of many variables. In this study, this challenge was overcome due to a change in the curriculum of a Special Master Program (SMP) that permitted a comparison between two years of students that learned anatomy using prosection only and two years of students that participated in dissection laboratories. Since each class of SMP students took courses in the medical school, and the medical school anatomy curriculum was constant, medical student performance served as a control throughout the study period. Results demonstrate that SMP students who learned through prosection had lower performance on anatomy practical and written examinations compared to medical students. When the SMP program changed and students started participating in dissection, there were measurable improvements in both practical and written examinations. These findings provide evidence of dissection's role in learning and applying anatomy knowledge both within and outside the gross anatomy laboratory.

WHAT IS KNOWN ON THE SUBJECT?: Health systems in many low- to middle-income countries (LMICs) are heavily underfunded, and staff training opportunities are limited. There is a lack of empirical data on the development and use of sustainable training programmes in aggression management skills in LMICs. WHAT DOES THIS PAPER ADD TO EXISTING KNOWLEDGE?: After RESPECT, participants showed greater agreement with statements about the role of environmental and relational factors as antecedents of aggression and described favouring the use of non-physical techniques to manage aggressive behaviour. Attitudes towards management practices of seclusion, medication and restraint remained unchanged after RESPECT training. WHAT ARE THE IMPLICATIONS FOR PRACTICE?: Training programmes like RESPECT may be useful to improve attitudes towards causes of aggression and its management in LMIC hospitals. However, further evidence is required to demonstrate whether these results can be sustained over time and whether these attitudinal changes are associated with reduction of workplace violence. Mental health nurses and other mental health professionals can play a key role in establishing hospital-wide training and sharing skills across geographical boundaries. Abstract Introduction Occupational violence has been demonstrated to impact negatively on the well-being of nurses and patients. Staff attitudes towards causes and management of patients' aggression influence their practice. Training is likely to influence attitudes towards aggression; however, Uganda's health system lacks adequate resources to provide aggression management training for staff. Aim To assess the impact of a training programme (RESPECT) on staff attitudes towards causes and management of patient's aggression in a Ugandan hospital. Methods This study used a mixed-methods convergent design. A convenience sample of nurses and support staff employed in the psychiatric ward and other services across the hospital (N = 90) completed the Management of Aggression and Violence Attitude Scale (MAVAS) pre- and post-training. The views of a smaller sample (n = 35) were captured via interviews and focus groups and analysed using thematic analysis. Results Participants reported greater agreement with patients' physical and social environment (external and situational causative models) as factors influencing patient's aggression. Qualitative findings substantiated the results identified in the survey. Attitudes towards seclusion, restraint and medication remained unchanged. Discussion and implications for practice RESPECT has the potential to change staff attitudes towards aggression in the short term. Further research is needed to investigate long-term effects and impact on incidents of aggression.

We have used electron paramagnetic resonance, with rigid and stereospecific spin labels, to resolve structural states in calmodulin (CaM), as affected by binding of Ca and a CaM-binding peptide (RyRp) derived from the ryanodine receptor (RyR), the Ca channel that triggers muscle contraction. CaM mutants containing a pair of cysteines in the N-lobe and/or C-lobe were engineered and labeled with a stereospecifically bound bifunctional spin label (BSL). RyRp was synthesized with and without TOAC (a stereospecifically attached spin-labeled amino acid) substituted for a single amino acid near the N-terminus. Intramolecular DEER distance measurements of doubly-labeled BSL-CaM revealed that CaM exists in dynamic equilibrium among multiple states, consistent with open, closed, and compact structural models. Addition of RyRp shifted the equilibrium partially toward the compact state in the absence of Ca, and completely toward the compact state in the presence of Ca, supporting a conformational selection model. Inter-protein distance measurements show that Ca stabilizes the compact state primarily by inducing ordered binding of the CaM N-lobe to RyRp, while only slightly affecting the C-lobe. The results provide insight into the structural mechanism of CaM-mediated RyR regulation, while demonstrating the power of using two types of rigidly and stereospecifically bound spin labels.

Introduction: There are no existing studies examining the psychological merits of using facial medical tattooing (MT) following burn injury. This study evaluated an MT service supported by The Katie Piper Foundation. It examined accessibility, satisfaction and whether there were improvements in quality of life (QoL). Methods: Thirty-five service-users were invited to participate in a cross-sectional online survey. Twenty-five (71%) responded (24 women; age range = 21–64 years), and of these five (4 women; age range = 26–59 years) also participated in telephone interviews, which were analysed using descriptive thematic analysis. Findings: The service was largely considered easy to access (22/25) and convenient (25/25). Most service-users (22/25) were satisfied with the results of MT. Some areas of dissatisfaction were described, by a minority of service-users, including: the procedure being painful (1/25); the tattoo being below expectation or fading over time (3/25). The majority reported that MT had improved confidence (22/25); mood (19/25); and ability to socialise (19/25). The procedure improved some service-users’ ability to carry out essential activities (14/25) and enjoyable activities (16/25). The qualitative responses provided during interview, indicated that all respondents found the procedure useful to their adjustment, although a minority (3/5) found it painful and also commented on fading (1/5). All described MT as contributing to a sense of increased normality. Conclusions: MT had the largest impact on emotional wellbeing and interpersonal domains of QoL. MT services should now improve awareness of the procedure, lobby for further support to provide wider access to the procedure, and routinely use measures assessing psychosocial outcomes., Lay summary As part of their support ’for’ individuals who have experienced burn injuries, The Katie Piper Foundation has offered to support medical tattooing (MT), a procedure which can enhance and restore the appearance of individuals’ eyebrows, eyes and lips following a burn injury. There are no known studies examining the psychological impact of MT for individuals with burn injuries. This evaluation asked its service-users (35 people) about their levels of satisfaction with the referral and assessment procedures, the procedure itself and its results, and whether medical tattooing had led to perceived improvements in quality of life. Twenty-five people agreed to complete a survey and five people agreed to be interviewed by telephone. Almost all respondents considered the service easy to access and convenient. Staff were perceived to be warm and friendly. Most respondents were satisfied with the results. Some areas of dissatisfaction were described, by a minority of respondents, including: the procedure being painful; the tattoo being contrary to expectation or fading over time. Large proportions of respondents reported that MT had improved their confidence, mood and ability to socialise. A small number of respondents reported functional benefits (e.g. ability to carry out essential daily tasks). All respondents reported finding the procedure helpful to their adjustment and that it had helped restore a sense of normality. The results of this evaluation suggest that medical tattooing has the potential to have a positive impact on emotional wellbeing of people who have experienced a burn injury. Providers of MT services could now consider ways to improve access to the the procedure and look to measure psychosocial outcomes in routine practice.

Highlights • Newly qualified drivers’ crash risk declines over the first three months of independent driving. • The current study aimed to better understand the factors underpinning this safety improvement. • A longitudinal qualitative design identified possible contributing factors to this decline in crash risk. • Developments in skill, thrill-seeking and feelings of driving status were reported. • Implications for research and application are discussed., Drivers are at high crash risk when they begin independent driving, with liability decreasing steeply over the first three months. Their behavioural development, and other changes underlying improved safety are not well understood. We adopted an innovative longitudinal qualitative design, with thirteen newly qualified drivers completing a total of 36 semi-structured interviews, one, two and three months after acquiring a full UK driving license. The interviews probed high-risk factors for new drivers, as well as allowing space for generating novel road safety issues. Analysis adopted a dual deductive and inductive interpretative thematic approach, identifying three super-ordinate themes: (1) Improvements in car control skills and situation awareness; (2) A reduction in the thrill of taking risks when driving against a background of generally increasing driving speed; (3) Early concerns about their social status in the eyes of other road users during the early stages of driving, which may put pressure on them to drive faster than they felt comfortable with. The study provides important new leads towards understanding how novice driving becomes safer over the first few months of driving, including how well-studied concepts of driving skill and style may change during development of independent driving, and bringing the less rigorously studied concept of social status into focus.

Linked Article: Dixon et al. Br J Dermatol 2018; 178:951–957

2018 marks the ten year anniversary of Anatomical Sciences Education (ASE), which has become one of the premier journals in the field of scientific educational research. ASE provides a home for edu...

Many eukaryotic protein kinases are activated by phosphorylation on a specific conserved residue in the regulatory activation loop, a post-translational modification thought to stabilize the active DFG-In state of the catalytic domain. Here we use a battery of spectroscopic methods that track different catalytic elements of the kinase domain to show that the ~100 fold activation of the mitotic kinase Aurora A (AurA) by phosphorylation occurs without a population shift from the DFG-Out to the DFG-In state, and that the activation loop of the activated kinase remains highly dynamic. Instead, molecular dynamics simulations and electron paramagnetic resonance experiments show that phosphorylation triggers a switch within the DFG-In subpopulation from an autoinhibited DFG-In substate to an active DFG-In substate, leading to catalytic activation. This mechanism raises new questions about the functional role of the DFG-Out state in protein kinases., eLife digest The transfer of phosphate groups onto proteins (protein phosphorylation) is one of the most important methods used to send signals inside cells. The enzymes that catalyze this process, called protein kinases, are themselves controlled by the phosphorylation of a flexible region called the activation loop. For many years it had been thought that the purpose of activation loop phosphorylation was to clamp the otherwise flexible activation loop in an active state that allows molecules that need to be phosphorylated to bind to the kinase. This assumption was based on static pictures of protein kinases obtained by X-ray crystallography, in which individual states are trapped and visualized in a crystal lattice. However, new methods and approaches now mean it is possible to visualize how the position of the activation loop changes as it moves in solution. By applying these techniques, Ruff et al. show that the static model is incorrect in a protein kinase called Aurora A. In this enzyme, the phosphorylated activation loop continues to switch back and forth between active and inactive states. Phosphorylation instead enhances the catalytic activity of the active state. Aurora A regulates several important steps in cell division, and plays important roles in several kinds of cancer. The discovery that activated forms of Aurora A can have different dynamic properties raises the possibility that inhibitor molecules could be designed to exploit these differences and block specific activities of Aurora A in cancer cells. To realize this goal we need to better understand how a kinase switching between active and inactive states affects the ability of inhibitors to interact with it.

Background\ud People with a visible skin conditions can experience anxiety and intrusive reactions from others when in social situations. The use of products to conceal marks on the skin is provided in a number of different hospital services and by charities. However, little is known about the experience of people using camouflage as a tool for enhancing quality of life.\ud \ud Aim\ud This study aimed to examine the experience of using skin camouflage to cover a visible mark on the skin. It sought to gain a detailed account of the issues associated with camouflage use and the needs of people using it.\ud \ud Methods\ud Interpretative Phenomenological Analysis (IPA) was used to gain a detailed understanding of the meaning of camouflage use for participants. Six skin camouflage service‐users completed semi‐structured interviews.\ud \ud Results\ud The IPA analysis revealed that the experience of using camouflage was influenced by socio‐cultural factors and the views of others. The use of camouflage was linked to investment in appearance maintenance and functioned to give a sense of control over social interactions, increase social confidence and manage concern about the responses of others.\ud \ud Conclusions\ud This study provides an in‐depth insight into the experience of using skin camouflage and suggests that camouflage can play a valuable role in maintaining wellbeing in those living with a visible skin condition.

Aim The frequency and severity of habitat alterations and disturbance are predicted to increase in upcoming decades, and understanding how disturbance affects population integrity is paramount for adaptive management. Although rarely is population genetic sampling conducted at multiple time points, pre- and post-disturbance comparisons may provide one of the clearest methods to measure these impacts. We examined how genetic properties of the federally threatened Coachella Valley fringe-toed lizard (Uma inornata) responded to severe drought and habitat fragmentation across its range. Location Coachella Valley, California, USA. Methods We used 11 microsatellites to examine population genetic structure and diversity in 1996 and 2008, before and after a historic drought. We used Bayesian assignment methods and F-statistics to estimate genetic structure. We compared allelic richness across years to measure loss of genetic diversity and employed approximate Bayesian computing methods and heterozygote excess tests to explore the recent demographic history of populations. Finally, we compared effective population size across years and to abundance estimates to determine whether diversity remained low despite post-drought recovery. Results Genetic structure increased between sampling periods, likely as a result of population declines during the historic drought of the late 1990s–early 2000s, and habitat loss and fragmentation that precluded post-drought genetic rescue. Simulations supported recent demographic declines in 3 of 4 main preserves, and in one preserve, we detected significant loss of allelic richness. Effective population sizes were generally low across the range, with estimates ≤100 in most sites. Main conclusions Fragmentation and drought appear to have acted synergistically to induce genetic change over a short time frame. Progressive deterioration of connectivity, low Ne and measurable loss of genetic diversity suggest that conservation efforts have not maintained the genetic integrity of this species. Genetic sampling over time can help evaluate population trends to guide management.

Objectives: Mound 72 at Cahokia figures prominently into interpretations of early Mississippian sociopolitical development. A previous study utilizing dental morphology concluded that the groups of mostly young adult females interred in four mass graves in Mound 72 were likely not from Cahokia and possibly reflect sacrificial offerings from outside communities. The purpose of this study is to reevaluate these findings using multiple indicators of biological relatedness and place of origin/migration. Materials and Methods: Biological relatedness in Mound 72 was examined using dental metrics and morphology. Four additional archaeological samples from nearby sites were included to better assess biological variation within Mound 72. Strontium isotope analysis (87Sr/86Sr) was also conducted on individuals from several burial features in Mound 72 to determine heterogeneity in place of origin. Results: Biodistance studies indicate that individuals in the four mass graves are phenotypically similar to other groups in the region, whereas F229-lower, a burial group with an aberrant mortuary context, is phenotypically distinct. Strontium isotope analyses show that mean Sr signatures for each feature investigated fall within the established local range for Cahokia. However, the range of Sr ratios for individuals in F229-lower is very narrow, suggesting they reflect a single population from a limited geologic region. Discussion: Collectively, these results question the long-standing idea that individuals in the four mass graves were non-local to Cahokia and suggest that F229-lower contained a biologically dissimilar group that either came from an outside region with a similar Sr signature to Cahokia, or represent a distinct and restricted group from the region. Am J Phys Anthropol 158:341–357, 2015. © 2015 Wiley Periodicals, Inc.

Objective To identify and critically appraise the literature on the psychosocial outcomes of orthognathic surgery, reflect on the clinical and theoretical implications, and suggest avenues for future research. Design A search of the literature was completed using the databases Web of Science, MEDLINE, and PsycINFO to identify English-language articles published since January 2001 that have reported a measure of psychosocial functioning posttreatment. Results A total of 38 articles were eligible for inclusion in the review. The studies reported improvements in areas such as satisfaction with facial appearance, self-confidence, self-esteem, anxiety, and social functioning. Small percentages of patients were left dissatisfied or had difficulty adjusting to appearance change despite the absence of treatment complications. Gains in psychosocial functioning were maintained over several years, and satisfaction increased over time. Conclusions There are consistent positive outcomes reported as a result of orthognathic surgery, but conclusions are limited by methodological issues in study design such as small sample sizes, limited use of control groups, and measures that fail to tap into relevant areas of psychosocial functioning. In addition, further exploration is required of processes such as adjustment to facial change and the role of psychological support during treatment.

Using electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) bound stereospecifically to Dictyostelium myosin II, we determined with high resolution the orientation of individual structural elements in the catalytic domain while myosin is in complex with actin. BSL was attached to a pair of engineered cysteine side chains four residues apart on known α-helical segments, within a construct of the myosin catalytic domain that lacks other reactive cysteines. EPR spectra of BSL-myosin bound to actin in oriented muscle fibers showed sharp three-line spectra, indicating a well-defined orientation relative to the actin filament axis. Spectral analysis indicated that orientation of the spin label can be determined within2.1° accuracy, and comparison with existing structural data in the absence of nucleotide indicates that helix orientation can also be determined with4.2° accuracy. We used this approach to examine the crucial ADP release step in myosin's catalytic cycle and detected reversible rotations of two helices in actin-bound myosin in response to ADP binding and dissociation. One of these rotations has not been observed in myosin-only crystal structures.

We have used pulsed electron paramagnetic resonance, calorimetry, and molecular dynamics simulations to examine the structural mechanism of binding for dystrophin’s N-terminal actin-binding domain (ABD1) and compare it to utrophin’s ABD1. Like other members of the spectrin superfamily, dystrophin’s ABD1 consists of two calponin-homology (CH) domains, CH1 and CH2. Several mutations within dystrophin’s ABD1 are associated with the development of severe degenerative muscle disorders Duchenne and Becker muscular dystrophies, highlighting the importance of understanding its structural biology. To investigate structural changes within dystrophin ABD1 upon binding to actin, we labeled the protein with spin probes and measured changes in inter-CH domain distance using double-electron electron resonance. Previous studies on the homologous protein utrophin showed that actin binding induces a complete structural opening of the CH domains, resulting in a highly ordered ABD1-actin complex. In this study, double-electron electron resonance shows that dystrophin ABD1 also undergoes a conformational opening upon binding F-actin, but this change is less complete and significantly more structurally disordered than observed for utrophin. Using molecular dynamics simulations, we identified a hinge in the linker region between the two CH domains that grants conformational flexibility to ABD1. The conformational dynamics of both dystrophin’s and utrophin’s ABD1 showed that compact conformations driven by hydrophobic interactions are preferred and that extended conformations are energetically accessible through a flat free-energy surface. Considering that the binding free energy of ABD1 to actin is on the order of 6–7 kcal/mole, our data are compatible with a mechanism in which binding to actin is largely dictated by specific interactions with CH1, but fine tuning of the binding affinity is achieved by the overlap between conformational ensembles of ABD1 free and bound to actin.

We have used electron paramagnetic resonance (EPR) to examine the structural impact of oxidizing specific methionine (M) side chains in calmodulin (CaM). It has been shown that oxidation of either M109 or M124 in CaM diminishes CaM regulation of the muscle calcium release channel, the ryanodine receptor (RyR), and that mutation of M to Q (glutamine) in either case produces functional effects identical to those of oxidation. Here we have used site-directed spin labeling and double electron-electron resonance (DEER), a pulsed EPR technique that measures distances between spin labels, to characterize the structural changes resulting from these mutations. Spin labels were attached to a pair of introduced cysteine residues, one in the C-lobe (T117C) and one in the N-lobe (T34C) of CaM, and DEER was used to determine the distribution of interspin distances. Ca binding induced a large increase in the mean distance, in concert with previous x-ray crystallography and NMR data, showing a closed structure in the absence of Ca and an open structure in the presence of Ca. DEER revealed additional information about CaM’s structural heterogeneity in solution: In both the presence and absence of Ca, CaM populates both structural states, one with probes separated by ~4 nm (closed) and another at ~6 nm (open). Ca shifts the structural equilibrium constant toward the open state by a factor of 13. DEER reveals the distribution of interprobe distances, showing that each of these states is itself partially disordered, with the width of each population ranging from 1.5 to 3 nm. Both mutations (M109Q and M124Q) decrease the effect of Ca on the structure of CaM, primarily by decreasing the closed-to-open equilibrium constant in the presence of Ca. We propose that Met oxidation alters CaM’s functional interaction with its target proteins by perturbing this Ca-dependent structural shift.