Tribolium castaneum, Herbst) population growth. Flours infested with RFB and kept in sealed PET bottles experienced much less weight loss over a three-month storage period than infested flour kept in unsealed bottles. RFB populations in wheat flour kept in sealed bottles did not increase, while populations in unsealed bottles grew about 50-fold during the same three-month period. Flour in sealed bottles had lower levels of oxygen and moisture than flour stored in unsealed bottles. Similar trends were observed for oxygen and moisture levels in maize flour held in hermetically sealed bottles. Hermetically-sealed bottles were effective in preventing RFB population growth and preserving maize and wheat flour. Farmers, consumers and food processors can safely store grain flour in hermetic sealed containers.Hermetic storage is used to protect grain against insect pests, but its utility is not limited to whole grains. We evaluated hermetically-sealed, polyethylene terephthalate (PET) bottles for preserving wheat and maize flour against red flour beetle (RFB,  Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), is a major economic pest of flour and other processed food products [Red flour beetle (RFB), products –3. In deproducts . Maize aproducts ,6. Insecproducts .Fumigants have been routinely used to control insect populations in stored products. The major fumigants used worldwide have been phosphine and methyl bromide. However, these chemical control methods are becoming less effective due to the development of resistance and are subject to environmental concerns. New management alternatives are needed –13.2 (hypercarbia). With inadequate oxygen available, insect development and reproduction is limited, mortality rates increase, and damage to the stored grain is prevented [Modified atmospheres (MA) are effective and non-chemical alternatives to fumigants for controlling insect pests in stored grains ,15. Hermrevented ,16–18. Mrevented ,19,20.Given its effectiveness in protecting grain against insect pests, hermetic storage has not been tested on wheat and maize flour in a controlled setting. Specifically, its effectiveness in preventing destructive RFB populations from developing in flours has not been demonstrated, although there is evidence from short-term studies that suggests hermetic conditions would be effective. In the present study, we assessed the effectiveness of hermetic storage using polyethylene terephthalate bottles (PET) in protecting maize and wheat flour against the RFB.Tribolium castaneum Herbst) were obtained from colonies maintained on wheat flour by the Purdue Improved Crop Storage (PICS) Laboratory at the Department of Entomology, Purdue University. The colonies were kept in a Conviron™ Environmental Chamber (C710)  at 25°C and 40% R.H. The wheat flour used in this study was purchased from Monarch ; the maize flour from Bob’s Red Mill . Both flours are commercial grade products of fine quality with initial moisture contents at 4.79% for wheat and 5.59% for maize.Red flour beetles  were attached to the inside wall of each bottle. The upper Oxydot was placed near the top, on the tapered portion of the bottle approximately 2 cm from the neck. The remaining two oxydots were placed at 6 cm (middle) and 24 cm (bottom) from the neck of the bottle.Transparent water bottles- 26 cm tall and one liter capacity each- made of polyethylene terephthalate  were purchased from Wal-Mart Corp. Three fluorescent yellow Oxydots  and three control groups  for each flour type. There were three replicates of each treatment. Each bottle was loaded with 400g of either maize flour or wheat flour. The flour covered all but the uppermost Oxydot. Infested bottles were each infested with 125 adult RFB per kg of flour. Sealed bottles had their caps tightly screwed into place and then the caps were wrapped with three layers of Parafilm around the outer edge to ensure a tight seal. The mouths of unsealed bottles were covered with cloth mesh secured with rubber bands. All treatments were held for three months (91 days) at 25°C and 40% R.H. in an environmental chamber.Oxygen concentrations in each bottle were recorded daily for the first two weeks and weekly thereafter. At the end of three months, the following assessments were carried out: (1) the number of adult RFB present; (2) percentage loss of flour; (3) the moisture content of the flour.At the end of three months, we sealed all bottles with three layers of Parafilm to ensure there was no moisture loss and froze the bottles at -20°C for two days to kill any insects present. We then separated the adult insects and flour portions from the infested bottles with two #20 sieves (Opening size: 0.84 mm) and one collecting pan stacked together, all purchased from Cole Parmer . Two sieves were used to ensure that larvae were excluded from the sieved flour as much as possible. Samples were collected and weighed separately. We also counted the number of adult RFB. Immature stages were not quantified due to their range in sizes and the difficulty of separating them completely from the flour.To determine moisture content of the flour, we used a modified methodology based on AACC method 44–15.02 . We fillStatistics were performed using GraphPad Prism 7.01 . Two-factor ANOVA was used for all statistical comparisons except where noted otherwise. = 454.5, P < 0.0001; maize flour: F = 374.3, P < 0.0001)  . When thOxygen levels in unsealed, infested bottles were different for the two flour types  . In the Oxygen levels were different depending upon the position of the Oxydot in the bottle. Oxygen levels recorded from the upper dots were higher than those recorded from the middle (Mean difference = 0.94% (v/v), paired t-test: t = 14.66, df = 207, P < 0.0001) and bottom dots (Mean difference = 1.07% (v/v), paired t-test: t = 14.66, df = 207, P < 0.0001) that were surrounded by flour. Oxygen levels between the middle and bottom dots were significantly different (Mean difference = 0.12% (v/v), paired t-test: t = 4.74, df = 207, P < 0.0001). = 144, P < 0.001; Flour type: F = 143.7, P < 0.0001)  . In seal = 10.79, P = 0.0111; Flour type: F = 2.35, P = 0.1638) ) relative to the other treatment groups  . Flour w = 147.3, P < 0.0001; Flour type: F = 132.5, P < 0.0001)  . MoisturT. castaneum larvae and adults (wheat) and larvae, though not adults (maize). The presence of active insect populations reduced flour mass. When the bottles were hermetically sealed, the infesting population of beetles never developed beyond the first generation.Storage in airtight containers arrests flour mass loss and the buildup of moisture that otherwise occurs when insects and a supply of oxygen are present in the bottles. When oxygen could pass freely in and out of the bottles, as was the case with the unsealed ones, we observed greatly increased populations of T. castaneum adults survived for some days after the bottles were sealed, all were immobile or dead within two weeks. This observation is consistent with Zhang et al. [The rapid decline in oxygen is a major factor contributing to the protective effect of sealed bottles. These sealed bottles function similarly to larger hermetic containers such as hermetic bags . Patterng et al.  who obseg et al. ,20,25. Tg et al. . While tThe change in oxygen levels in the unsealed bottles over time reflects the substantial oxygen demands of large insect populations. High rates of population growth significantly reduce oxygen levels even within bottles that have continuous but restricted access to fresh air. High oxygen demand coupled with restricted supply of oxygen probably explains the marked drop we observed in oxygen levels in unsealed/infested bottles containing wheat flour . RespiraThe presence of RFB also appears to affect the distribution of oxygen in the bottles. Oxygen levels recorded from different locations  along the bottles’ length were significantly different. Oxygen molecules must diffuse between grain particles in order to penetrate into the grain mass . BecauseThe high rates of insect respiration also likely caused the higher flour moisture content observed in unsealed infested bottles. During the experiment, we saw evidence of high moisture in the form of water droplets accumulating on the inside of the unsealed infested wheat flour bottles as well as areas of discolored flour where microbes carried by the beetles were presumably growing . In the T. castaneum populations in the unsealed maize bottles was unexpected. While RFB is a known secondary pest of maize [The observed lack of growth of of maize , its faiof maize . PerhapsCollectively, our results demonstrate that storage of wheat and maize flour in hermetic containers stops the growth of RFB populations and in the case of wheat flour reduces the amount of damage. In sealed bottles, oxygen levels drop rapidly, and no population growth occurs. In conclusion, hermetic containers can be used effectively to protect wheat and possibly other types of flour against insect damage. Farmers, consumers and food processors can safely store grain flour in hermetic sealed containers.


An explorative paper to describe how family carers, through the caregiving journey, reaffirm and promote the agency of people with dementia. Agency is an important concept in dementia care and is crucial to the promotion of wellbeing and the delivery of person-centred care. This article is based on one of the key findings of a study that explored family carers’ experiences of engaging their relatives in daily activities in domestic settings. Following research governance and ethical approval, 30 in-depth interviews  were carried out with 15 resident-carers of people with dementia who were recruited via local community mental health teams. Then five focus groups were conducted with 21 participants accessed through carers support groups. Interviews and focus groups were transcribed, coded and analysed using a grounded theory method. Findings showed the process in which family carers encouraged and sustained a sense of autonomy and control (agency) in their relative’s daily activities. Key strategies used by carers included: being non-judgemental; facilitating a sense of worth; taking calculated risks; maintaining the continuity of their relative’s identity; enhancing a sense of connection with their relative’s role and identity using enjoyable activities; preventing inactivity and attending to the bodily source of the agency. Lack of support for carers could ultimately pose a risk to the maintenance of the agency of people with dementia. This study provides a deeper insight into the process used by home carers to support the agency of people with dementia. This is essential if practitioners are to identify and develop more realistic intervention strategies and to work in effective partnership with family carers. The implications for the creation of dementia-friendly communities are discussed. Dementia is a syndrome that can be caused by a variety of progressive illnesses that affect memory, thinking, behaviour and the ability to perform everyday activities . Grounded theory was initially developed by Glaser and Strauss in the 1960s. In the next few decades, they both acknowledged some differences between their versions of grounded theory Glaser, . StraussIn the larger study, participants were co-resident carers who had specific experience of caregiving and responsibilities for their relative. An open sampling method  in order to ensure easy access  was used to indicate pauses. Names of people and places were removed for reasons of anonymity. Transcripts were entered into the Nud*ist 6 computer software package to ease the management of such a large amount of data. Each transcript was read and re-read many times and coded to develop concepts further until all core concepts were identified. These concepts were developed by constant comparison until no new categories emerged  the felt importance of maintaining a sense of stability and continuity in daily life and (2) and the all-encompassing nature of supporting everyday activity and agency.My husband used to work in the high street, I think he probably missed the people coming to and fro… for some reason he likes to go down to the town…I think that is habit…I send him a little notes, and then he goes off and fetches them—I mean sometimes he got the wrong things but as long as it is something he is used to—that’s his routine.Routines and meaningful activities were one way of maintaining some stability and continuity, and were of fundamental importance for the carers. In the face of their relative’s progressive memory decline, carers considered it very important to maintain stability in their relationship by supporting their relative’s sense of control (or agency) through occupations that still had some value or meaning for their relative. Family carers did not just “look after” and “care” for their relative, they continually set and adapted the everyday activities for their relative. By encouraging and supporting the usual activities and routines of their relative as far as possible, the carers maintained their relative’s (and to some extent their own) sense of self and personhood. They created a link to the “usual” previous life that they had led with their family members  and created a sense of continuity with the past. This is an important aspect in anyone’s life McAdams, , but espYou have to take on board what your partner wants to do and just guide them, you know, even to make a cup of tea—You know, if I stood in the kitchen and perhaps my husband would like to make us a cup of tea, you would agree that he couldn’t find the spoon, to find the tea bag, to find the kettle, to pour the cup…you need to offer support and guide them through.Over time, the sense of agency of those with dementia was gradually challenged, they lost the ability to control their environment because of cognitive deterioration, and their carers took on responsibility for making things happen, acting according to their relative’s wishes. This can be seen in a quote by Mavis (Phase 2), who set up the environment for her husband to engage in activity and was guided by her perceptions of her husband’s intention:Through supporting self-initiated everyday activities, carers provided relatives with an opportunity to act as they wished and to implement their chosen activities with support. This promoted a sense of autonomy in them by respecting their wishes.Involving relatives in everyday activities was all-pervasive and was not just carrying out a variety of tasks, but was part of a complex pattern of daily interactions requiring constant decision making.In the beginning, when I said to my husband, “You are not really able to work out your accounts, are you?” He said, “Oh yes, I am”, and he’d get quite annoyed—But I mean, he went into the newspaper shop—I thought he was capable of going and buying a Radio Times…and I showed him the picture on the front—and—I said, “Go in and ask for one of this”. He said, “Fine.” Well, one of the girls served him and he gave her £75, just like that. They’re 45p. Luckily they phoned me up and said, “We made him put it back in his purse.”…So I’d to persuade him not to put too much money in his purse—say £10, and then if he wants any more he’ll have to ask me. In the beginning, he was reluctant. Now, he sort of doesn’t mind.All the carers attempted to encourage their relative to take an active part in everyday activities that had specific meaning to that person, both in their past and present life situation. The activities that carers supported were largely related to self-management, household management and entertainment. The types of activities that carers talked about ranged from navigating for their relative when driving, helping with paying bills, taking a short holiday break, going out for a walk, watching television together, listening to music, assisting with toileting and persuading their relative to have a regular bath. The list of activities was endless and sometimes mundane. In order to ensure that each of these activities was meaningful and appropriate to the specific needs of their relative, carers made constant decisions in four key areas: (1) setting meaningful goals for activities, (2) identifying changes (retained abilities) in their relatives’ performance of them, (3) developing subsequent adjustments to accommodate changes in the relative’s ability and (4) negotiating with them. The nature of this decision making was complex and constantly evolved during the course of caregiving. This was especially so when the family carers felt increasing difficulty in communicating with their relative concerning the latter’s wishes and preferences. An example of this is illustrated by Lilian (Phase 1) below:The strategies that carers used to support agency were wide ranging and included: being non-judgemental; facilitating a sense of worth; taking calculated risks; maintaining the continuity of their relative’s identity; enhancing a sense of connection with their relative’s role and identity; using enjoyable activities; preventing inactivity and attending to the bodily source of the agency. These will be discussed in more detail below. The association between positive aspects of caregiving, agency and identity preservation, as well as the consequences of lack of support for carers and following loss of agency of their relative, will also be discussed.When you first notice the little things…you don’t really want to sort of draw their attention to it because at the early stages you still think…I will get you over this sort of thing. Although perhaps deep in your mind you don’t realise it is, but you don’t want to bring it to their attention so that it sort of knocks their confidence or self-esteem back, you know.Initially carers often went along with or agreed to something that they may not have been completely sure of in order to help their relative maintain, as far as possible, their pre-existing role and responsibility within any usual routine activity. This was often seen as a necessary adjustment to support and respect a family member who was going through some “bad days”. This can be seen in the quote below by Rob (Phase 2), who talked about how he accommodated his wife’s problem in the beginning:By gradually taking over or compensating for their relative’s role with or without their realisation, many participants began to re-establish the boundaries of involvement with their relative at the same time maintaining some continuity in their relative’s routine and relationship. Most of the participants considered their relative’s wishes to make decisions for themselves even though they may have perceived that such decisions were unwise at the time.My husband will go to the shop, the shops that he uses that he knows, but I will give him one thing written on a piece of paper. If I give him more than one he might manage to—but he may get confused and he certainly can’t remember…I have tried not to take away from him anything that he possibly could do.Carers promoted their relative’s sense of success by enabling them to maximize their retained ability, which, carers considered, linked with the past self and routine activity of their relative before the onset of dementia. Through engaging their relative in a modified activity, carers facilitated a sense of worth in their relative. This could be illustrated in the quote from Liz (Phase 1):Hence, stability was also maintained by controlling the parameters of an activity so that their relatives could successfully continue routine activities/roles that were usual for them. This was felt to enhance a sense of continuity of self and security for their relative.My husband used to go out with the dog and he would be gone perhaps for three hours, and the dog always brought him home…I suppose he was just wandering…I used to worry like hell all the time he was out, but I didn’t want to take that little bit of independence away from him…I thought it would be good for him because his concentration was such that you couldn’t get him to do anything.Often, carers encouraged agency by developing a strategy that enabled their relative to take part in “risky” activities that their relative initiated. In such a situation, carers felt that the benefit outweighed the risk. Through engaging their relative in activities of their own choice, carers promoted a sense of independence and autonomy in their relative. This could be illustrated by a quote from Lorna (Phase 2):The doctor asked my husband’s opinion, on how he felt about driving. And he said, “Well although I am still capable of driving perfectly alright I am frightened that if there is an accident, because I have got Alzheimer’s, I will get the blame even if it is not my fault.”…He did in fact change his mind and drive a bit after that when we were out in the country. I said, “Yes go for it”, and then we would get to a main road and I would take over again.Some carers continued to help their relative carry out certain daily activities even though they may have been considered by others as risky . They tried hard to get the balance right to enable a sense of autonomy and agency. For example, Joy (Phase 2), following the confirmation of a diagnosis of dementia given to her partner, was reluctant to cooperate with a medical consultant who advised her to stop him driving, she said:This reflected that, even after the diagnosis, many carers continued to respect their relative as an agent who was capable of making decision and could negotiate for their own needs. In Joy’s case, she realized that her husband missed his driving very much because he used to go on holidays with her in his car until he obtained the diagnosis. Hence, she was prepared to compromise her own needs.These examples illustrated that taking risks was an inevitable aspect of carers’ daily lives. This was often at a cost to carers in terms of their own psychological and emotional wellbeing.My father is a gardener and I have a patio with pots so…I let him do whatever he likes and then I go round afterwards and sort it out again because he does some weird things now’…he’s still got the mind that he had before—it just doesn’t always show.Often carers supported an activity, even if it was not perceived to have been “successful” in terms of outcome, because they felt it maintained some stability in terms of continuity of identity. Through engaging their relative in activities that aimed at enhancing a sense of identity, carers enabled their relative to express themself in a particular way that reflected the latter’s value and interest. This in turn enhanced a sense of agency and personhood. This can be illustrated in this quote from Carole (Phase 1):It is interesting to see how Carole stated, “my father is a gardener”. This was despite the fact that her father had been experiencing memory loss for many years. By maintaining her father’s routine in gardening she was also maintaining her father’s identity as a gardener. She enabled her father to act according to his will and to use his remaining abilities to carry out a familiar activity within a supportive environment. Through taking the action to go round afterwards and sort things out things in the garden for her father, Carole showed an awareness of the need to adapt the environment in order to minimize its demand on her father’s performance; and to enable her father to achieve a sense of productivity and satisfaction. There was a sense of wishing to reaffirm a sense of capacity and meaningful purpose in everyday living in their relative.As can be seen, the perceived benefit of activity engagement often focused on supporting those with dementia in taking part in the process of meaningful doing, not just producing a tangible end product.My husband liked to watch the television, but erm, he couldn’t follow a story, so I wouldn’t put on a programme that had a continuous story because he’d be losing the plot…but something—very old programmes—sort of comedy, you know. He liked that, and it is funny…but I knew it was no good putting on, you know some murder mystery because he wouldn’t know what it was about. So he wouldn’t enjoy it—I could leave him there on his own for a while.Carers often tried to support enjoyable activities that still held a value for their relative. Even when severe cognitive decline was apparent, carers tried to respond to their relative’s wishes (or positive choices) and to promote their wellbeing by adjusting the physical and social environment; thus maintaining feelings of stability and connection with the past. For some carers, this could mean that, despite memory and functional loss, they could enable and share a sense of fun and enjoyment through doing things which were still meaningful to their relative, as illustrated in a quote from Gill (Phase 1):It seemed that, in the face of progressive cognitive decline, the need for carers to maintain a sense of enjoyment and happiness in their relative became increasingly important. Hence, carers attempted to identify meaningful activities that prevented their relative from feeling a failure and being distressed by demanding experiences.I asked my husband if he wanted to do something, or go out, he just said, “No, I’m all right here”…Well, I felt I ought to stimulate him a bit more, but err, he just sits there all day and doesn’t want to be stimulated…If he doesn’t sit he will be lying and sleeping…so, I talk with him, we talk together…talk about programmes probably, and events, family events.Carers faced enormous challenges when their relative was not interested in any activities that kept either their mind or body active. They considered inactivity to be an unhealthy state, which could lead to rapid deterioration in their relative’s wellbeing. They felt it necessary to find strategies to continuously engage their relative in activities even as dementia progressed, as Ann (Phase 1) said:I try to keep my husband as active as possible, because I believe that if you sit down and let it happen, you stagnate…he’s not allowed to stagnate, which I think is helpful to him.Being inactive was considered by carers to be a state of stagnation. Carers perceived that, in such a state, their relative ceased to act on their own volition or to express who he or she was. This created a barrier for carers, preventing them from connecting with the past self of their relative and challenging the continuity of their relative’s and their own agency. The link to the past or the person they knew appeared to be lost. Carers felt that this had an adverse impact on the relationship with their relative and so they made an effort to prevent their relative from sitting idly by encouraging some kind of action as a way of expressing themselves. This in turn enabled carers to support their relative to act on their wishes and to relate to them in a meaningful manner. Lillian (Phase 1) said:It appeared that recognition of the continuity of the previous identity and agency of those with dementia could be best understood when a person was engaging in meaningful activities with the support of a carer who endeavoured to connect their relative’s past and present unique characteristics. This process, whereby carers sustained continuity of the current self of their relative, through bringing back the past self when the person was no longer able to do that alone, has been described by Jennings  as “memoMy wife would sit in her arm chair with a chest of drawers and magazines and newspapers and, she loves, rather than sit forward she loves to take the papers, tear them apart—then, fold and refold, fold and refold, and it keeps her occupied…you know, you do study your loved one very intently…they are slumping like that, they really want something to occupy their hands…Their mind is going back to when they were a wife you know what I mean, putting things away, folding, ironing, and if she is doing something she thinks she is doing the housework.It seemed evident that as family carers experienced increasing difficulty in communicating verbally with their relative, they attended more to the bodily source of agency of their relative as a means to help them understand the wishes and preferences of their relative. Such bodily sources may manifest themselves as nonverbal  or verbal behaviours . Carers often found clues in these behaviours to make the link between their relative’s present behaviour and their past routines, habits and preferences. Carers created tactics to understand the bodily source of agency of their relative. This can be seen in the quote below by Norman (Phrase 2), who said:Implicit in this quote was that some carers tried to find the symbolic meaning of their relative’s action as a way to make sense of how they could support their relative to engage in activities that appeared to be meaningful to the individual. By doing so, carers also maintained a connection with the old self and meaningful relationship with the relative.Caring for a relative with dementia can be a very difficult experience due to the loss of personhood and relationship as well as the related responsibilities. Carers faced enormous challenges when their relative was not interested in any activities that might keep either their mind or body active. The findings of this study showed that maintaining previous identity through encouraging activities and reaffirming agency of their relative could benefit the carers’ psychological wellbeing. This was shown in several ways: firstly, as carers’ lives and sense of self were intimately entwined with that of their relative, the more they maintained the “usual” life of their relative, the more they could maintain the stability of their own daily life and routine and less distressing for them, as shown in the earlier example by Susan who continued to support her husband to “go down to town” and “fetch something” from the shops.We are able to get out and of course…my wife can use a walking stick and she can walk out to the car with a walking stick and walk from the car to one of the benches and then I either get the folding chair out for her or the three-wheeler, so she is able to have a bit of activity. And then of course we sit on the bench and we have an ice-cream cornet. We sit there with a cornet in our hands like a couple of kids, licking our ice creams. That is fun.Secondly, despite the stresses and strains from caring responsibility, many carers continued to enjoy moments when they experienced shared activities with their relative, as Tony (Phase 1) said:By taking his wife on a ride in a car to a familiar location, this carer achieved a sense of being relaxed and sharing companionship with his wife. There was also a sense of having a pleasurable experience in terms of making the right decision to engage his wife in a positive experience.I try to encourage my husband to dance and sing. I have a limited amount of success. He stands up and I put my arms around him, I know he is not very steady but he remembers what he has to do more or less and I do a lot of singing you know, to cheer myself up and I will say come on, you know this.Some carers also encouraged activities that supported and maintained intimacy such as singing or dancing, as Nora (Phase 2) said:Implicit in this quote was that some carers attempted to identify familiar and safe activities that still had value to their relative as a way of maintaining a sense of connection, bringing pleasure to both.At first when the consultant psychiatrist said about the dementia…I thought “Ah! I’ve got a label…Now I can understand why my mother is behaving like this.”…But then it was like, that didn’t help me at all, because it didn’t explain, really. No, no. And it wasn’t like, you know, she’s got this physical thing, and means that this is how she will behave, and how she will progress, and this is what we can do, you know, this is what we can do to help…and I’m trying to manage it, and as time’s going by, I’m doing it less and less well.Carers attempted to continue to engage their relative in daily routine activities that enabled them, to as far as practicable, to respect their will and exercise their sense of control. Nevertheless, many acknowledged that they often felt uncertain of how to behave or respond over a range of everyday situations and found themselves struggling with the everyday management of the everyday care, even though their relative had received a diagnosis. This is reflected in this quote by Julie (Phase 1):The staff in the day centre said, “you must fetch him [my husband] and take him home.” I thought if he is sensible enough to walk home, let him! To me, I believe in rehabilitation…he gets into a habit where he’s got used to doing what he used to do [walk home from a day centre by himself], and then suddenly he isn’t allow to do it, it’s like…the staff are worried thinking why can’t me come and pick him up, you know.Engagement in their relative’s everyday activities was a time-consuming and demanding process. Many carers worked in isolation. The findings also suggested that carers themselves often did not receive any helpful feedback from others  in the decisions that they had made. Hence, they often live with a lot of uncertainty and had to learn by trial-and-error. Many were living with a sense of criticism from others. As Linda (Phase 1) said:This carer explained that she used to worry a lot when her husband first insisted on walking back home from the day centre on his own. The journey took about 45 min from an isolated country house through a country lane (a journey he was familiar with before prior to the diagnosis). On numerous occasions, she had tried to persuade her husband not to go out on his own without success. As a result, she had to give in to her husband’s argument and supported him to carry on walking to the property on foot. Hence, she tried to comfort herself that by supporting her husband to do what he wanted to do, she helped him get some physical exercise and therapy at the very least. Nevertheless, she resented the judgment of her by staff concerning her perceptions of risk-taking with her husband’s daily routines. Moreover, she worried that she was blamed for the perceived negative consequences of her decision making.Even though carers faced a sense of uncertainty and criticism, many carried on developing strategies to support their relative, demonstrating how important activity engagement was for them in their lives. The findings showed that carers had determination and perseverance and devoted the time to facilitate their relative’s activity, highlighting their conviction that there was a fundamental need to maintain everyday routines and connections to the past.Well the carer [home care assistants] comes in at 8am–9am. She gets my wife up, gives her a shower, and gets her dressed. That helps me a lot you see; but from then on, it is all over to me you see and I have to do everything…We have to carry on until something happens, you know. Until I can’t do it anymore…But she would do the same for me if the situation was reversed. I am sure she would look after me as best she could.As John (Phase 1) explained below, even though he received a range of outside resources to support him to care for his wife at home, he felt that he had to devote all his time, day and night, to the care of his wife because the social services care package did not provide sufficient cover for his wife’s agency needs:Carers’ decisions related to their caring role was often motivated by love, passion, reciprocity and interdependency and positive relationships between the past and present. Nevertheless, family carers often faced the challenge of whether they were making the right decisions with limited support from professional services.It is worth noting that those carers who were caring for a relative with severe loss of cognition and functioning were often able to give accounts of episodes of positive interactions with their relative, for example, dancing and singing with them and validating their “usual” behaviours with personal meaning. Hence, by recognising the uniqueness of their relative’s individual characteristics, carers played a key role in maintaining the personhood and continuity of their relative’s identity and agency.Agency is crucial to the promotion of wellbeing and the delivery of person-centred care . The basis of Kitwood’s person-centred approach is that when positive interactions have been applied consistently, and over a long period, many psychological needs of the person with dementia are met Kitwood, , 1997b. This shows that it is crucial to support family carers in meeting the continuing activity needs of those with dementia if person-centred care in dementia care is to be adopted fully in a home setting. This could be achieved through recognising the agency and identity work that carers have already been carrying out effectively and using this as a basis for understanding. Using a non-judgmental approach is crucial as it can be seen that carers already feel under pressure, often feel like failures and are less likely to try new strategies that they have found useful if they feel they are going to be judged. If support for carers is not provided, it is possible that they will lose touch with the needs of those with dementia (or unwittingly deprive them of their autonomy and dignity) because they themselves do not have the energy, confidence and/or skills to assess and act within the situation. This in turn may lead to incidents of ignoring, disempowerment, objectification, banishment and invalidation, termed Malignant Social Psychology by Kitwood , 1993, aIt is crucial for practitioners to recognize the individual strategies that carers are currently using, to check with the carers if they feel that the strategies they using are useful or not, and to be able to suggest other strategies (proposed by family carers) that could lead to a successful intervention. It is also important to help carers recognize those strategies that might lead to failure or frustration. A model that highlights the strategies that family carers use successfully at different phases of dementia care has been drawn up by Chung et al.  and may This study provides some evidence for and highlights the importance of professionals working collaboratively with family carers, especially when assessing the actual performance of those with dementia. This is because family carers often have practical experience about the embodied knowledge of their relative. Moreover, such tactic knowledge enables professionals to gain insight into the mechanisms of support that family carers offer to their relative and how some family carers maintain a connection with the old self of and meaningful relationship with the relative. This insight and understanding is crucial if professionals are to work collaboratively with carers to develop a personalized intervention plan for those with dementia, as promised in the government policy  of their relative through engaging them in daily activities. Due to the fact that they were a self-selecting participants , the experience of such a group of co-resident carers might not be considered to be typical of co-resident carers. However, from the researchers’ clinical experience it was felt that although these carers were not specifically skilled, they were definitely committed to care for their relative and wished to enable their relative to stay at home for as long as practical. The researchers feel that this would be the case for most carers of people with dementia. The results may not be applicable to non-resident carers and relationships within residential care due to the different contexts involved, as Strauss and Corbin  stated tIn conclusion, the findings support many of Kitwood’s ideas and have provided empirical evidence of and illuminated how the agency and personhood of those with dementia is maintained by family carers at home. This study provided further insight into how carers played an important role in sustaining the sense of autonomy and control (agency) of their relative through engaging them in everyday activities and the centrality of the carer-relative relationship in dementia care in family care setting. Carers play a key role in creating an enabling environment to support their relative’s agency in many ways and develop strategies that are constantly reassessed and modified over time. Lack of timely and ongoing support for carers could ultimately pose a risk to the maintenance of the agency of persons with dementia. The successful strategies used by the carers to reaffirm agency can be adopted to guide the development of not only assessment and intervention strategies for people with dementia, but also training resources for carers (both paid and unpaid) to enable those with dementia to stay active and connected and feeling in control in dementia-friendly communities.


Globodera rostochiensis and G. pallida, and are characterized by a single SPRY domain, a non-catalytic domain present in modular proteins with different functions. The SPRY domain is wide-spread among eukaryotes and thought to be involved in mediating protein–protein interactions. Thus far, the SPRY domain is only reported as a functional domain in effectors of plant-parasitic nematodes, but not of other plant pathogens. SPRYSEC effectors have been implicated in both suppression and activation of plant immunity, but other possible roles in nematode virulence remain undefined. Here, we review the latest reports on the structure, function, and sequence diversity of SPRYSEC effectors, which provide support for a model featuring these effectors as a versatile protein-binding platform for the nematodes to target a wide range of host proteins during parasitism.Persistent infections by sedentary plant-parasitic nematodes are a major threat to important food crops all over the world. These roundworms manipulate host plant cell morphology and physiology to establish sophisticated feeding structures. Key modifications to plant cells during their transition into feeding structures are largely attributed to the activity of effectors secreted by the nematodes. The SPRYSEC effectors were initially identified in the potato cyst nematodes  Plant-parasitic nematodes are microscopic roundworms that can infect thousands of different plant species, causing severe damage to food crops all over the world . Annual Globodera and Heterodera) and root-knot nematodes (genus Meloidogyne)  . In the idogyne) . Cyst neidogyne) .The massive molecular and cellular changes associated with permanent feeding site establishment in plants are most likely brought about by nematode-secreted effectors . In otheHeterodera schachtii  in plant-nematode interactions. The SPRYSEC effectors were initially identified in the potato cyst nematodes proteins . While tproteins . HoweverBefore the introduction of new generation sequencing technologies, identifying nematode effectors was a challenging and lengthy process . In thisG. rostochiensis .The application of two differential display approaches using these criteria resulted in the cloning of the first SPRYSEC effectors from chiensis  and G. p pallida . The gen pallida . Furthernscripts . Recent families . The seqG. rostochiensis incubated in root diffusates of host plants . Because nematode effectors lack sequence similarity to other proteins with SPRYSEC architectures and because no functions have been assigned to other SPRYSEC proteins, it is not clear if the use of a secreted SPRY domain to promote virulence is exclusive to nematodes.Proteins with a SPRYSEC architecture seem to be rare in nature. The Pfam protein domain database includes around 9000 SPRY domain-containing proteins (PF00622), fifteen percent of which harbor no other functionally annotated domain(s) while about four percent of the latter are predicted to be secreted. Proteins with SPRYSEC architectures are predicted in different eukaryotes, including a number of pathogens and parasites  from the soil-inhabiting slime mold Dictyostelium discoideum as well as in three mammalian ryanodine receptors  characterize B30.2/SPRY proteins in protein domain databases, with the LDP being absent in the ‘SPRY-only’ group , but not of the LDP motif.The SPRY domain in SPRYSEC effectors was initially characterized as a sequence repeat in tyrosine kinase eceptors . Concurreceptors . Some asy’ group . The SPRThe LDP motif is present in proteins carrying a ∼60 amino acid extension at the N-terminus of the SPRY domain. This extension is cause for debate about the functional boundaries of the domain. In short, the B30.2 configuration is defined by a SPRY domain and an N-terminal extension, the PRY domain , which was initially suggested as a distinct structural element of the B30.2 domain . ‘SPRY-oG. pallida . Furthermore, two highly conserved residues, a tryptophan and a leucine, are found in the N-terminal extensions of all SPRYSEC effectors studied so far . Other amino acids in a region of 20 amino acids around these two conserved residues also show high levels of conservation. Protein database searches using only this region suggest that it may be a unique signature sequence of SPRYSEC effectors of nematodes .The SPRY domains in SPRYSEC effectors carry an N-terminal extension with lengths varying between 60 and 120 amino acids, depending on the SPRYSEC effector variant. These N-terminal extensions have no significant sequence similarity to the PRY domain or other N-terminal extensions known to be associated with SPRY domains. A PRY domain(s) was initially described in the N-terminus of the SPRYSEC effector GpRbp-1 from  pallida . HoweverThere is ample evidence showing that the SPRY/B30.2 domain functions as a versatile platform to selectively mediate physical protein–protein interactions . For insDrosophila melanogaster, also predicts a core β-sandwich joined by interspersed flexible loop regions that create exposed surfaces radiating from the β-sandwich core virulence.The selective forces favoring non-synonymous mutations in SPRYSEC effectors are not fully understood. Changes in amino acid residues that betray the presence of the nematodes to the plant innate immune system can have significant fitness benefits and they seem to contribute to the sequence diversity in SPRYSEC effectors. Position 187 is one of several positively selected sites on the hypervariable surface A of the SPRY domain in GpRbp-1 . Multiplthamiana . The intognition . This chG. pallida  genes that recognize pathogen effectors and activate effector-triggered immunity , and a C-terminal Leucine-Rich Repeat domain. Two major NB-LRR classes are further distinguished based on N-terminal extensions of either a coiled-coil domain (CC-NB-LRR) or a Toll/interleukin 1-like receptor (TIR-NB-LRRs) (M. incognita (Heterologous expression and identification of host targets of SPRYSEC effectors in plants suggest that they may function as suppressors of innate plant immunity. An important line of defense in plants relies on intracellular immune receptors encoded by host specific resistance (immunity . Most inNB-LRRs) . Activatncognita .G. rostochiensis selectively suppress the cell death phenotype triggered by a group of closely related CC-NB-LRRs  also selectively suppress the characteristic cell death induced by Gpa2  lack the ability to suppress cell death induced by either Gpa2 or Rx1 in N. benthamiana  in N. benthamiana leaves. Co-infiltration of N, p50, PVX-GFP with various SPRYSEC effectors results in enhanced PVX-GFP accumulation in N. benthamiana . Other members of this cluster of highly conserved CC-NB-LRR proteins are involved in resistance to tomato spotted wilt virus [TSWV; . Similarly, the transcriptomes of the cereal cyst nematode H. avenea (Pratylenchus coffea (Another important question that remains to be addressed is if only potato cyst nematodes exploit the versatility of the SPRY domain to modify host targets. The large expansion of SPRY domain-containing proteins in nematode genomes could be a tell-tale sign to their importance in nematode–plant interactions. At present, it is not possible to assess if similar expansions of the SPRY domain have occurred in related nematode species, given the availability of the genome sequences of only a small number of plant parasitic nematodes. Homologs of SPRYSEC effectors have not been identified in the genome sequence of the root-knot nematodes . Several. avenea  and migrs coffea  also incAll authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although pressured metallic glass (MG) has been reported in the literature; there are few studies focusing on pressure effects on the structure; dynamics and its plastic deformation. In this paper; we report on and characterize; via molecular dynamics simulation, the structure and dynamics heterogeneity of pressured MGs, and explore a causal link between local structures and plastic deformation mechanism of pressured glass. The results exhibit that the dynamical heterogeneity of metallic liquid is more pronounced at high pressure, while the MGs were less fragile after the release of external pressure, reflected by the non-Gaussian parameter (NGP). High pressure glass shows better plastic deformation; and the local strain zone distributed more uniformly than of in normal glass. Further research indicates that although the number of icosahedrons in pressured glass was much larger than that in normal glass, while the interpenetrating connections of icosahedra (ICOI) exhibited spatial correlations were rather poor; In addition, the number of ‘fast’ atoms indexed by the atoms’ moving distance is larger than that in normal glass; leading to the sharp decreasing in number of icosahedrons during deformation. An uniform distribution of ‘fast’ atoms also contributed to better plastic deformation ability in the pressured glass. These findings may suggest a link between the deformation and destruction of icosahedra with short-range order. Since their discovery several decades ago, metallic glasses (MGs) have attracted extensive attention in academia due to their excellent mechanical properties ,2,3,4. ISince the icosahedron is the typical cluster in the liquid and glass, the number and connectivity type may be connected with the increasing fragility under pressures. Strong evidence has been found to suggest that the icosahedral clusters often interpenetrate one another in Cu-Zr liquids, so that five atoms coincide on two icosahedral shells. These interpenetrating connections of icosahedra (ICOI) exhibit strong spatial correlations ,20 and pThe high pressure processed MG has a substantially enhanced glass transition temperature, and increased density and hardness. The enhanced stability of the bulk MG originates from the change of the local atomic structure and the bonding states between atoms under high pressure. Icosahedral clusters are responsible for the increment of shear strength in MGs. In fact, icosahedra represent short-range-order which is a key feature of microstructure in MGs ,23,24. ITo address the aforementioned important issues, we perform extensive computer simulations to study structural and dynamical heterogeneity of binary metallic glass subjected to high pressures by calculating the number, connectivity of icosahedra, and non-Gaussian parameters (NGP). In addition, the structures and macroscopic mechanical properties of glass are also discussed in the paper by comparing stress-strain curve of glass at 0 GPa and 25 GPa.50Zr50 MGs designed by pressurized quenching to provide the relationship between the structure and dynamics under pressures. By analyzing the change of the FI and their connection type, the influence of the pressure on structure is known. In the second part, the stress-strain curves are obtained by simulated loading and the difference of stress-strain curve between MGs obtained under different pressures can be explained. Previous theoretical and experimental works suggest that the low temperature relaxation of MGs could be attributed to ‘fast’ atoms which correlate with spatial heterogeneity [In this paper, the molecular dynamics (MD) simulations are introduced in the first part. We present a computer simulation study of Cuogeneity ,27. In o50Zr50 MG to present our study. Molecular dynamics simulations were performed by utilizing the open source code-LAMMPS [50Zr50 MG containing 115,200 atoms, with the periodic boundary conditions (PBC) applied in all three directions. The simulation samples were obtained in four separate stages as illustrated in 12 K/s, and finally (IV) the samples obtained by different pressures were equilibrated at 100 K temperature and zero pressure for 1 ns.We used molecular dynamics (MD) simulations of a Cue-LAMMPS .The embee-LAMMPS  was empl−1 and the maximum strains were 20% in the compressions.After the samples quenched under different pressures were obtained, the schematic of the uniaxial compression testing is shown in 12 K/s is shown in The difference between the total energy as a function of temperature for various MGs obtained under different pressures at the cooling rate of 10To examine the differences in local structures in more detail for the metallic liquids, we studied volution of Cu-centered full icosahedra with a Voronoi index <0, 0, 12, 0> using Voronoi tessellation analysis to identify the topological packing of nearest neighbors. As in Moreover, ISRO WAs found to undergo a pronounced increase during quenching close to the corresponding glass-transition temperature Tg. In More importantly, Cu-Zr liquids became more fragile with higher pressure, due to the steeper rate of increase of ISRO upon undercooling, as shown in As shown in 50Zr50 samples under compression. In the samples formed at 0 GPa, yielding μAs shown in ms (SAs) . The FAsThe Chi-square value of the sample quenched at 25 GPa was quite a bit lower than that of the sample at 0 GPa, which indicated more homogeneous distribution of FAs at high pressure. This led to the conclusion that more uniform plastic deformation originated from the number of FAs and their distribution in the MGs. The FAs were closely related to the initiation and evolution of localized deformation unites in MGs; therefore, the research is beneficial for understanding the plastic behavior of MGs obtained at high pressure.To examine the deformation mechanism at pressured MGs, we analyzed the connectivity of the network formed FIs. This analysis addresses the important question of how the clusters are connected and packed before deformation. Clusters can link together to form extended clusters. These extended clusters are made of linked clusters that are interconnected to neighbor clusters by sharing their vertex (VS), edge (ES), face (FS) or volume (TS), thereby forming different types of connections . The dis50Zr50 MGs. The full icosahedra <0, 0, 12, 0> were connected and overlapped to fill the space and formed a medium range order (MRO). The MRO clusters were characterized by an increased packing density, and high shear resistance. They became the backbone of the MGs, and were responsible for poorer plastic deformation ability.Although the number of FIs was much larger at 25 GPa, the number of clusters with a volume connection of FIs at 25 GPa is lower than that at 0 GPa. The volume connectivity is enhanced at 0 GPa. The interpenetrating connections of icosahedra, especially the full icosahedra, produced stable string-like networks of icosahedra, which were uniquely responsible for the dynamical features of the CuWe also showed the changes of main polyhedron during compressive deformation in In conclusion, we reveal here atomic-level dynamical heterogeneity-plastic deformation relationships for MGs prepared through a pressure-mediated processing path. The isothermal compressing makes glass-forming liquids become increasingly viscous, and glass formation ability is enhanced. Meanwhile, high pressure was also found to promote more heterogeneous dynamics from NGP measurement, which is related to the more fragile state of the liquid, while the pressured MGs become less fragile after pressure release. The pressured MGs have a lower elastic limit and lower yielding strength; beyond yielding, the corresponding stress did not drop sharply, implies that the samples in our case could show multiple shear location states during deformation. Further research indicates that the distribution of the local shear strains is more homogeneous in pressured samples, which is attributed to the more uniform distribution of FAs, and which are closely related to the initiation and evolution of localized deformation units in MGs. Although the number of FI is larger, the connectivity of the icosahedrons network is weaker, the number of FI decreases sharply, and the backbone of the network is prone to destruction during deformation. Since the high pressure was in favor of the formation of glass, our findings provided some fundamentals for further investigating the structure and dynamic behaviors of metallic glass-formers related to the plastic deformation, which may shed light on understanding the mechanical properties of pressured MGs.


The polycomb group (PcG) proteins are a large and diverse family that epigenetically repress the transcription of key developmental genes. They form three broad groups of polycomb repressive complexes (PRCs) known as PRC1, PRC2 and Polycomb Repressive DeUBiquitinase, each of which modifies and/or remodels chromatin by distinct mechanisms that are tuned by having variable compositions of core and accessory subunits. Until recently, relatively little was known about how the various PcG proteins assemble to form the PRCs; however, studies by several groups have now allowed us to start piecing together the PcG puzzle. Here, we discuss some highlights of recent PcG structures and the insights they have given us into how these complexes regulate transcription through chromatin. The development of a multicellular eukaryotic organism is a highly complex process that requires exact control of specific transcriptional programs in a spatially and temporally regulated manner. To facilitate such control, the eukaryotic genome is packaged into nuclei as chromatin; a nucleoprotein complex that can be physically remodeled in response to epigenetic modifications on DNA and histone proteins, either restricting or permitting access to the DNA for all DNA-templated processes ,2.Drosophila : ,10: 9,10While our knowledge of the biological importance of the PcG family and the modifications these complexes perform has been expanding, obtaining detailed structural information of these complexes had proved challenging, severely limiting our understanding of their regulation and the targeting to their biological substrate — nucleosomes. Recently, however, several new structures of PcG components and subcomplexes –15, or hDrosophila), non-enzymatic compaction of chromatin and possibly by direct interaction with the transcription machinery . Th. Th13]. Recent crystal structures of minimal PRC2 core complexes showed that EZH2 possesses many domains that act as interaction platforms for EED and SUZ12 VEFS, facilitating the formation of the active enzyme  12,14,1,114,15. The complex of EZH2, SUZ12 VEFS and EED activates the SET domain; however, further catalytic stimulation is achieved upon the binding of its product, H3K27me3, and is thought to be important for the propagation of the repressive mark. H3K27me3 is recognized by an aromatic cage on the WD40 domain of EED . The stiChaetomium thermophilum minimal PRC2 complex suggested an alternative mode of H3K27M binding in the EZH2 active site [The structures of the minimal PRC2 complex were also capable of shedding light on how an oncogenic point mutation (H3K27M) in a single histone H3 allele can bind and inhibit PRC2, leading to a global reduction in H3K27me3 and the development of pediatric glioblastomas ,64. Althive site , it is nive site . The entive site ,64.Drosophila histone-binding protein Nurf55, a WD40 repeat protein homologous to the interchangeable RbAp46/RbAp48 subunits in mammals, binds to the WD40-binding domain in the N-terminus of Su(z)12  and is positioned in the noncatalytic lobe of PRC2  and the PhoRC complex [Drosophila and humans, PRC2-mediated H3K27me3 targets PRC1 E3 ligase activity to H2AK118/119 and monoubiquitination of H2AK118/119 recruits PRC2 complexes that contain AEBP2 and JARID2, creating a positive feedback loop and further stimulating H3K27me3 catalysis and subsequent PRC1 recruitment [The recent structural data discussed above have gone a long way toward describing the activation and stimulation of the minimal PRC2 complex; however, the precise mechanisms of PRC2 recruitment to target loci in mammals remain contentious. In  complex –76. Howe complex  and otheruitment ,78–80. Aruitment . This suDrosophila genetic screens [in vitro [Drosophila [The PR-DUB complex is a histone deubiquitinase that removes the H2AK119ub1 mark deposited by PRC1. PR-DUB is a relatively recent addition to the PRCs; the gene encoding the catalytic subunit Calypso was first identified as a PcG gene in 2007 in  screens , and the screens . Calypsoin vitro . Deletioosophila ,10. Thisosophila .Drosophila, BAP1 is inactive against H2AK119ub1 nucleosomes in the absence of an ASXL protein [N-acetylglucosamine transferase, the forkhead box transcription factors FOXK1/2, MBD family proteins MBD5/6, transcription factor YY1 and ubiquitin-conjugating enzyme UBE20 [The mammalian Calypso homolog BAP1 (BRCA1-associated protein 1) also interacts with ASX homologs ASXL1–3; , and, li protein . Howeverme UBE20 ,83–87. Sme UBE20 –90.Figuin vitro H2AK119ub1 nucleosome deubiquitination assays [The interaction between BAP1 and the ASXL proteins is mediated by the ∼120-residue DEUBAD (DEUBiquitinase ADaptor) domain of the ASXL proteins , and thin assays . There in assays ,17,93,94n assays ,83,92.Activation of UCH-L5 by Rpn13 DEUBAD is mediated by allosteric effects that give the complex a greater affinity for ubiquitin than UCH-L5 alone. The active site crossover loop (ASCL), a feature shared by all UCH family members, is thought to restrict substrate access to the active site based on substrate size . Interesin vitro, although, somewhat surprisingly, experimental data suggest that neither the nucleosome acidic patch, nor the nucleosomal DNA, mediates this interaction [Key features of the BAP1/ASXL1 complex that cannot be modeled based on the UCH-L5/DEUBAD structures are the C-terminal extension (CTE) of BAP1  and the additional inserted region between the strands of the coiled coil . The CTEeraction .Drosophila Calypso, it may be required for the alternative, non-PcG functions of BAP1.The central region of BAP1 is predicted to be disordered. Sites within this central region have been identified as important for binding non-ASXL complex components; for example, the HCF-binding motif and the phosphorylated threonine residue that mediates BAP1's interaction with FOXK2 have been mapped to this region ,97. Addiin vitro, additional domains or proteins may be involved in targeting the complex in vivo. Structural predictions and phylogenetic analyses identify three globular regions of ASXL1: the DEUBAD domain, an N-terminal HARE-HTH (HB1 ASXL1 Restriction Endonuclease Helix-Turn-Helix) domain and a C-terminal atypical PHD finger [While BAP1 and ASXL1 DEUBAD alone are sufficient for nucleosome deubiquitination D finger ,98. HTH D finger ,96,99. WMammals have evolved a large complement of PcG proteins that could potentially combine to assemble hundreds of PRCs, each with distinct, overlapping or redundant functional roles in transcription regulation. Recent structural advances have meant that we are now beginning to develop a picture of the core components of these complexes and their primary enzymatic mechanisms, yet much remains to be discovered. While there appears to be a set of stable core subunits for each PRC, the accessory subunits of the complexes are highly dynamic . We stil


Stomatin is an ancient, widely expressed, oligomeric, monotopic membrane protein that is associated with cholesterol-rich membranes/lipid rafts. It is part of the SPFH superfamily including stomatin-like proteins, prohibitins, flotillin/reggie proteins, bacterial HflK/C proteins and erlins. Biochemical features such as palmitoylation, oligomerization, and hydrophobic “hairpin” structure show similarity to caveolins and other integral scaffolding proteins. Recent structure analyses of the conserved PHB/SPFH domain revealed amino acid residues and subdomains that appear essential for the structure and function of stomatin. To test the significance of these residues and domains, we exchanged or deleted them, expressed respective GFP-tagged mutants, and studied their subcellular localization, molecular dynamics and biochemical properties. We show that stomatin is a cholesterol binding protein and that at least two domains are important for the association with cholesterol-rich membranes. The conserved, prominent coiled-coil domain is necessary for oligomerization, while association with cholesterol-rich membranes is also involved in oligomer formation. FRAP analyses indicate that the C-terminus is the dominant entity for lateral mobility and binding site for the cortical actin cytoskeleton. Stomatin is a 31 kDa monotopic integral membrane protein that is palmitoylated, forms homo-oligomers, and associates with cholesterol-rich membrane domains, also known as lipid rafts –5. Due tSTOM), stomatin-like proteins 1–3 , and podocin (NPHS2) photocholesterol was synthesized as described by Thiele et al. photocholesterol in DMEM at 37°C for 15 min and irradiated for 1 min with a 500 W beam of filtered UV-light (λ > 310 nm) of a high-pressure mercury lamp at 4°C to crosslink photocholesterol to cholesterol-binding proteins. The cells were lysed, stomatin was immunoprecipitated with anti-stomatin antibody GARP-50 and analyzed by SDS-PAGE and autoradiography. Protein expression levels were estimated by immunoblotting with GARP-50 and densitometry of stomatin bands.COS-7 cells were transiently transfected with WT and mutated stomatin constructs, respectively, with or without myc-tags, and subjected to photocholesterol cross-linking according to the method of Thiele et al. ,56. Brie™ Standards (Bio-Rad) were used as molecular weight markers. After blocking, the blots were incubated with anti-stomatin antibody GARP-50, anti-GFP, or anti-myc for 1 h. After washing, the blots were incubated with anti-mouse or anti-rabbit horseradish peroxidase conjugate and the SuperSignal™ chemiluminescent substrate (Pierce).Fractions collected from the sucrose gradients were analyzed by 10% SDS-PAGE and blotted onto nitrocellulose by standard methods as described previously . HighPreDensitometric values of Western blot or autoradiographic bands were obtained by using ImageJ software and analyzed statistically with the IBM SPSS program. Mean values, standard deviation, and standard error were calculated and p-values were determined by two-tailed t-test.To study the relevance of some prominent structural features of stomatin, we deleted parts of the molecule and exchanged single amino acids. The recent elucidation of the 3-dimensional structure of the highly conserved PHB/SPFH core of archaeal and mouse stomatin ,48 highlAll of the constructs shown in In a variety of cell lines, stomatin is localized to the plasma membrane (PM) and the late endosomal/lysosomal compartment ,11,19,57The localization in A431 cells of WT stomatin, Stom(1–288)GFP, and the deletion mutants Stom(22–288)GFP (ΔN), Stom(1–263)GFP (ΔC), and Stom(d204-241)GFP (ΔCC), depicted in 3H]-labeled photocholesterol and irradiated with UV light for crosslinking. The expressed proteins were immunoprecipitated with anti-stomatin antibody and the covalently bound [3H]photocholesterol was identified by autoradiography of SDS-PAGE gels. To estimate the expression levels of WT and mutant stomatin, immunoblotting with anti-stomatin antibody was performed. Radioactive bands were observed for WT stomatin and essentially all mutants  measurement of those mutants that were stably expressed at the PM . CompareStomatin binds cholesterol , generatIn general, treatment of WT and mutants with cytoD showed increased MF and recovery , Table 5Cholesterol depletion of WT and mutants showed little changes in MF , Table 5Cholesterol loading showed some reduction in MF, particularly of ΔC (-26%) and Arg62Ala (-17%). ΔC and Arg62Ala are mutants that do not associate with DRMs , Table 3In this study, we used targeted mutation to evaluate the structural and functional significance of prominent domains and amino acid residues of the monotopic membrane protein stomatin. We selected these domains and residues on the basis of recent NMR and crystal structures of the PHB/SPFH domain –49 and oWhen we expressed the GFP-tagged mutants, we surveyed the subcellular localization and found that several mutants were unable to target the PM . Other m3H]photocholesterol to WT stomatin and all mutants  resulted in the absence of oligomers thereby confirming the interacting function of this domain , whereasComparing the three phenotypes of stomatin mutants, expression at the PM, oligomerization, and DRM association, showed that there is some intricate correlation between them . SeveralThe in vitro biochemical characterization of oligomerization and DRM association does not directly reflect the situation of molecules in the living cell. Therefore, we complemented our biochemical data with Fluorescence Recovery After Photobleaching (FRAP) measurements to relate the lateral mobility of stomatin mutants to the phenotypes of varying oligomerization and/or DRM-association , Table 4To evaluate the effects of the membrane actin meshwork on lateral mobility of stomatin mutants, we treated the cells with cytoD to disrupt the microfilaments. Moreover, to estimate the influence of cholesterol-rich lipid domains, we manipulated membrane cholesterol levels , Table 5Depletion of cholesterol had little effect on MFs of mutants, except for the marked MF reduction of Pro245Ala and MF increase of ΔCC ; howeverTo sum up our data, we give here an overview of the induced structural changes and the resulting phenotypes . We put In conclusion, we have shown that stomatin is a cholesterol binding protein and that one binding site is located within the C-terminal region (residues 264–288). Two regions of stomatin, the putative cholesterol binding sites, CRAC/CARC (residues 55–68) and ORA/CARC (residues 263–273), are equally responsible for the association with cholesterol-rich membranes. We conclude that these two regions are either structurally connected or cooperating. Moreover, this association with cholesterol-rich membranes appears to be a condition for oligomer formation possibly by creating a membrane microenvironment that induces oligomerization. For oligomerization, the coiled-coil domain is clearly essential, while the CRAC/CARC-residues Tyr-60, Arg-62, and the ORA/CARC domain, as well as Phe-91, may play an indirect role in oligomerization by their primary association with cholesterol-rich domains. The interaction of stomatin with the cortical actin cytoskeleton has been linked to the C-terminal region (residues 264–288).S1 FigConfocal laser scanning microscopy (CLSM) of A431 cells stably expressing GFP-tagged wildtype (WT) stomatin shows the normal, dual localization to the plasma membrane (PM) and late endosomal/lysosomal compartment. Deletion of the N-terminus, C-terminus, or coiled-coil domain did not alter this localization and distribution. The point mutants Cys30Ser, Arg62Ala, Phe91Ala, and Phe269Ala, also showed the normal localization and distribution. Lack of PM staining or largely reduced staining was observed for Pro47Ser, Cys87Ser, Asp89Ala, Arg97Ala, Lys198Ala, and Pro270Ala, while weak staining of PM and preferential staining of cytoplasmic vesicles was visible in cells expressing Ile57Ala, Tyr60Ala, Pro200Ala, and Pro245Ala.(TIF)Click here for additional data file.S2 FigThe relative amounts of mono-/dimers (fractions 1–6), oligomers (fractions 7–18), and aggregates (fraction 19), as listed in (TIF)Click here for additional data file.S3 FigThe relative amounts of DRM-associated (fractions 1–3) and Triton X-100-soluble stomatin (fractions 4–9), as listed in (TIF)Click here for additional data file.S4 FigIllustration of the structural consequences of deletions and point mutations. The color code and marks apply as in (TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


NMR-derived backbone amide temperature coefficients for many residues show a sharp transition above 30–40 °C, indicating a temperature-dependent conformational change that is most prominent around the mutated EF-hand IV, as well as throughout the C-domain. Smaller, isolated changes occur in the N-domain. Cardiac skinned fibres reconstituted with D145E are more sensitive to Ca2+ than fibres reconstituted with wild-type, and this defect is amplified near body-temperature. We speculate that the D145E mutation destabilises the native conformation of EF-hand IV, leading to a transient unfolding and dissociation of helix H that becomes more prominent at higher temperatures. This creates exposed hydrophobic surfaces that may be capable of binding unnaturally to a variety of targets, possibly including the N-domain of cTnC when it is in its open Ca2+-saturated state. This would constitute a potential route for propagating signals from one end of TnC to the other.The hypertrophic cardiomyopathy-associated mutant D145E, in cardiac troponin C (cTnC) C-domain, causes generalised instability at multiple sites in the isolated protein. As a result, structure and function of the mutant are more susceptible to higher temperatures. Above 25 °C there are large, progressive increases in N-domain Ca The N-domain of cTnC has only one functional site (site II), where Ca2+ triggers conformational changes that lead to adjustments in the thin-filament proteins to permit actin-myosin interaction3. The C-domain anchors cTnC to the thin filament, and binds Ca2+/Mg2+ competitively at sites III and IV. It is now generally accepted that events in one domain can have an effect on the other, especially when the other proteins of the thin filament are present, but the mechanism is not clear6.Cardiac troponin C (cTnC) controls key events of systole and diastole through its ability to bind and release Ca7. Recently, four new mutations related to familial hypertrophic cardiomyopathy (HCM) were discovered in the human TNNC1 gene8. Among these, the D145E proband had a positive family history for HCM, suggesting segregation of the mutation among family members as the cause. The mutation D145E is located in the C-domain of human cTnC (HcTnC), where it drastically reduces Ca2+-binding affinity9. Its capacity for causing diastolic dysfunction has been attributed to its effect at the N-domain site II, where it conversely increases the affinity for Ca2+ and consequently delays cardiac muscle relaxation10. This mutation also causes minor changes in certain structural parameters, including the α-helical content11.Over the last two decades, mutations in sarcomeric proteins that lead to cardiomyopathies have been studied extensively2+ sensitivity of the thin filaments  of the recombinant HCM protein to obtain the backbone assignment, and we evaluated its stability and structural features by NMR and circular dichroism (CD) at different temperatures. In a separate report, we have looked for evidence of changes in the internal dynamics of the N- and C-domains of the mutants18.In troponin and tropomyosin, most mutations that are related to HCM tend to increase Ca2+ sensitivity and the rate of sarcomere shortening20. Thus, a second aim was to evaluate the temperature dependence of stability and function for the HcTnC D145E mutant incorporated into skinned cardiac myofibrils and compare it with the WT protein using temperatures in the near-physiological range.Since in these experiments temperatures near the physiological range promoted substantial changes in HcTnC D145E structure that were not seen in the WT protein, we also investigated the effect of temperature on the function of HcTnC D145E. For both skeletal and cardiac muscle, it is well known that changes in temperature alter force development, Ca22, none matches exactly the entire primary sequence and buffer conditions used in this work. Thus, we recorded a complete experimental set of triple-resonance NMR spectra for HcTnC WT and D145E backbone assignments. Structural changes in HcTnC caused by the D145E mutation were identified by mapping the chemical-shift perturbations (CSP) between the 1H-15N HSQC spectra of WT and D145E at 25 °C   Fig. , showingm) values of 36.6 ± 1.5 and 70.9 ± 0.6 vs 32.9 ± 0.5 and 72.5 ± 0.4 °C, respectively . The second Tm values were very similar to data recorded for the isolated WT N-domain23 as well as for the full-length protein fitted to a single-transition equation24. With Ca2+/Mg2+ present (holo state), however, the two proteins diverged: the WT became very stable, with a single transition at a higher temperature (Tm 81.8 ± 0.6 °C), while the mutant essentially reproduced the result recorded in EGTA (Tm1 32.9 ± 0.5 and Tm2 72.5 ± 0.4 °C)  for WT and D145E proteins in EGTA (apo state) were not statistically different Fig. , with tw°C) Fig. . We know2+. However, the finding of similar signals from the mutant regardless of Ca2+  as a measure of binding of this probe to hydrophobic surfaces on HcTnC. The fluorescence of bis-ANS in our experiments increased by 68% upon conversion of the WT TnC from apo to holo state Fig. , indicat11. To probe more accurately for possible changes in secondary structure, based on NMR CSPs we compared deviations of α-carbons (Cα) and α-protons (Hα) in the backbone of each protein with values for the same residues in a set of model random-coil peptides27. The expected result of this residue-by-residue analysis is that α-helical segments show positive deviations for Cα and negative deviations for Hα, while β-strands and loops show the opposite. Overall, we observed significant differences between the WT and mutant secondary structure only in the G-helix  compared to the WT proteinlix Fig. , with li1HN) of each residue (see Methods). Temperature coefficients are inversely proportional to bond length, and a positive, linear coefficient denotes a simple process of thermal expansion, while a positive but non-linear coefficient can mean that a linkage is not only getting longer but is also sampling an alternative conformation29. Although conformational changes, aromatic residues and other factors unrelated to hydrogen bonding can also alter amide temperature coefficients29, values more negative than −4.5 ppb/°C are commonly involved in protein-solvent hydrogen bonds, while those less negative than −4.5 ppb/°C tend to be associated with intramolecular bonds, frequently observed in secondary contacts28. Additional experiments would be required to identify hydrogen bonds unambiguously. However, in this set of data, we were able to compare temperature coefficients of WT and D145E residues that displayed linear 1H shifts with temperature in {1H-15N} HSQC experiments 30. These residues drew our attention because they appear to be strategically located for an impact on transmission of signals from C- to N-domain. Thus the D145E mutation impacts temperature coefficients throughout the C-domain and the inter-domain linker, but not in the N-domain, except for a few scattered residues.Another clue to multiple changes in HcTnC caused by the D to E mutation in site IV emerges from measuring temperature coefficients (ppb/°C) for the chemical shift of the amide hydrogens ; at higher temperatures, the slope changed. Curves of this type were detected for residues in sites II, III and IV, with significant differences between WT and mutant found only in sites III and IV  or not    Fig. . These cck) Fig. , and the1HN chemical shifts and other aspects of HcTnC D145E structure were markedly affected by temperature between 30° and 40 °C. Accordingly, we analysed the effect of temperature on the Ca2+ affinity of the mutant in solution. In isolated HcTnC labeled with the fluorescent probe 2-(4′-(iodoacetamido)anilino)naphthalene-6-sulfonic acid (IAANS), D145E had a higher affinity (by 0.11 log units) than WT for Ca2+ binding to the N-domain site at room temperature (21 °C)11. Here we show that this difference increased to 0.83 log units when the temperature was raised to 45 °C  of the HcTnC Ca2+-binding curves was also altered by temperature. The increase in temperature from 15 to 30 °C significantly reduced the cooperativity in both sets of fibres (p < 0.05). Fibres reconstituted with the mutant had less cooperativity than those with WT at each of these temperatures  of cTnI unless a great excess of Ca2+ (1 mM) was present. However, a comparison among progressively more complex model systems reconstituted with the D145E mutant has shown that the incorporation of other myofibrillar proteins  contributes significantly to its ability to regulate the thin filament at room temperature. As a result, in fact it exhibits only a mildly defective functional profile in reconstituted skinned fibres, where the response to Ca2+ is dominated by the N-domain11. Thus, despite the remarkable array of dysfunctions in the C-domain, HcTnC D145E is capable of binding to myofibrils, reconstituting normal tensions and regulating full-fledged Ca2+ responses with only a slight departure from WT in Ca2+ off-rates from the N-domain site11. The question we have addressed is whether this is an accurate picture of its performance at higher temperatures, near the physiological range. A second question concerns interdomain communication: how does a defect in the C-domain produce a change in affinity for Ca2+ in the N-domain?In a previous report, Swindle and Tikunova analysed the C-domain changes wrought by three HCM mutations in isolated recombinant HcTnC1HN temperature coefficients and 1H-15N cross-peak intensity changes over a range of temperatures. The first set of observations was analyzed in conjunction with the presentation of results from Figs 33) the lines can become broader (lower intensity) or sharper (higher intensity), depending on the relaxation parameter Rex, which is the exchange contribution to the observed R2 (R2obs = R2 + Rex). The amplitude of Rex depends on the exchange regime, the exchange rate (kex) and the thermodynamic parameters of the conformational equilibrium. Larger values of Rex lead to broader lines and a decrease in intensity.We have used two NMR tools related to structural transitions to focus our analysis of changes in the D145E isoform, measuring ex. The DG residues (red lines), on the other hand, lost intensity with the increase in temperature. They are mostly responding to faster solvent exchange and are not involved in secondary structure. Some of them may also be increasing the exchange contribution (Rex).In Fig. More remarkable is the ML-DG group of residues, found only in the mutant. Between 15 and ~40 °C, intensities of these residues increased, followed by a decline at higher temperatures (~40 to 55 °C). This subgroup is certainly in conformational equilibrium between the native state and a higher-energy conformational state that was not present at the lower temperatures, and may be linked to an incipient unfolding event like that observed in the DG residues. Together with the CD data Fig. , these o18. Flexibility of the D/E linker was demonstrably maintained.In a related study of the WT and D145E proteins, experiments designed to measure Carr-Purcell-Melboom-Gill relaxation dispersion in the mutant identified a small, kinetically trapped excited state not found in the WT protein35. In terms of mechanism, the principal contribution of the present investigation has been to link the N-domain response to structural instabilities introduced by the C-domain mutation. As shown in Figs 36, the C-domain dominates the signal and is less stable, but does not necessarily unfold first even in the WT proteins36. However, since Ca2+ binding affinity for D145E C-domain is drastically reduced and only the curve for the Ca2+-bound mutant differs from WT, we infer that it is the C-domain that is most affected. The mass-spectrometry and bis-ANS experiments suggest that both C-domain sites bind Ca2+ poorly18, while site III can still bind Mg2+ 9. Based on CD data, the secondary structure of the Mg2+-loaded C-domain is similar for the WT and mutant proteins11. With regard to stability, the mutation renders the C-domain less stable than for WT, and this is reflected in the response of the N-domain when Ca2+ binds to site II. Thus temperatures near the physiological range (30° to 40 °C) significantly perturb the overall structure and function of D145E.Other studies of structural changes in cardiomyopathy-associated mutants of HcTnC have provided important information on flexibility, tertiary structure and interactions with troponin I, focusing primarily on the domain carrying the mutation2+ binding at site IV. Mutated residue 145 is located in site IV at the coordinating position z+. The exchange of aspartate for glutamate has been suggested as a destabilising element in the β-sheet structure that couples the two high-affinity sites but, as shown previously11, has only a small effect on the α-helical content overall. It is useful to compare the D145E mutation with D141A, analysed structurally and functionally by Putkey and co-workers38. Like D145E, D141A abolished Ca2+ binding to site IV. Their data showed clearly that loss of site-IV Ca2+ binding had virtually no effect on Ca2+ sensitivity or maximum force in skinned fibres. In marked contrast to D145E, however, there were essentially no global effects of the D141A mutation on structure, as analysed in solution by NMR37. The β-strands between Ca2+-binding loops III and IV formed normally in the presence of Ca2+, and the signals from the aromatic residues of the hydrophobic core were virtually identical to those for the WT protein. These results are particularly interesting because the NMR experiments were carried out at 40 °C, very near the physiological range and close to where our experiments have shown the greatest effects with D145E. While the D141A mutation eliminated Ca2+ binding, it would likely also decrease electrostatic repulsion within a native-like site-IV conformation, whereas the D145E mutation would exacerbate electrostatic repulsion in the native-like conformation.Alterations in the stability and function of the mutant could be related to lack of Ca39. How does the communication between N- and C-domains occur? The distance between the N- and C-domains of cTnC is about 48 Å40 and it has been suggested that the two domains may interact directly because of the bending of the flexible D/E linker in the troponin complex17. Conceivably, a mechanism based on flexibility of that link between N- and C-domains could be the main pathway for transmission of information from the C-terminus to the N-terminus. Nevertheless, even if the central D/E linker is in fact unfolded and mobile, the cTnI helix 1, which is embraced by cTnC C-domain, has several static segments. These might contribute to an additional pathway for signal transmission, one that passes along the core of the assembled thin filament41.Multiple studies of different TnC species and domain fragments provide evidence for interdomain communication2+ and temperature. The increase in Ca2+ affinity of cTnC upon incorporation into thin filaments is well documented, and can be attributed to interactions with other proteins of the regulatory complex as well as the presence of cross-bridges42. An increase in Ca2+ sensitivity of force development with temperature is also expected43. Of particular interest in our experiments was the differential response of the mutant, where the increment in Ca2+ sensitivity was greater than for the WT protein at both 25 °C and 30 °C , where cross-bridges detach from actin nearly twice as fast as Ca2+ dissociates from cTnC. This means that near the physiological temperature, Ca2+ dissociation from WT cTnC will clearly be the rate-limiting step for cardiac muscle relaxation. Thus a 2.9 fold increase in Ca2+ affinity, as we have found for D145E at 30 °C, may retard relaxation and contribute significantly to diastolic dysfunction, a characteristic of the HCM phenotype8.It is noteworthy that the Ca9. In another assay, Marques et al. have now shown a tighter binding of the mutant to the thin filament18. Altered interactions may also account for the significant difference in cooperativity  of the force vs pCa curves obtained in fibres with WT and mutant isoforms. In both cases the cooperativity decreased as the temperature was raised  for fibres reconstituted with the mutant. D145E is located in the C-domain, which makes contact with TnT and thus indirectly with tropomyosin. Thus it is in a position to affect tropomyosin function, thereby altering the cooperativity within or between regulatory units.As noted above, it has already been demonstrated that the structural problems of the mutant affect its ability to interact with its partnerssed Fig. , consist2+ binding to the isolated cTnC subunits . For NMR, CD and fluorescence experiments, protein batches were maintained in solution at −80 °C, were not subjected to thawing and freezing cycles, and were not used after a month of storage. Aliquots of fresh purified samples were dialysed or diluted in the specific buffer and centrifuged at 10,000× g, 10 min, 4 °C; concentration was measured by the Lowry method and adjusted for each experiment.The amino-acid substitution at residue 145 of human troponin C was inserted into HcTnC1H-15N HSQC, we judged it necessary to obtain a set of three-dimensional spectra NH, HN(CA)CO and HNCO) to complete the sequential HcTnC WT and D145E backbone assignment. The 15N,13C-labeled WT and D145E proteins were prepared as described previously46. Prior to NMR measurements, protein buffer was exchanged to 20 mM MOPS, 6 mM CaCl2, 1 mM MgCl2, 100 mM KCl, 10 mM DTT, pH 7.0 using a Vivaspin 20 10,000 MWCO  at 4 °C. Samples were prepared with a concentration range of 0.5–1 mM containing 10% D2O. The Computer Aided Resonance Assignment software  was used as assignment platform. For thermal susceptibility experiments we used a Bruker Avance 500 MHz equipped with a probe heater at the National Center of Nuclear Magnetic Resonance Jiri Jonas . 1H-15N HSQC experiments from WT and D145E were acquired at 15, 17.5, 20, 25, 30, 35, 40, 45, 50 and 55 °C and shift changes were monitored as a function of the temperature.NMR data were acquired at 25 °C using a Varian Inova 600 MHz with a triple resonance cryogenic probe system at the Brazilian Biosciences National Laboratory (LNBio), Campinas, Brazil. Because significant changes were detected for the WT and D145E 47. Data analysis was performed with CCPN (http://www.ccpn.ac.uk/) software using equation  analysis, data processing was carried out using the NMRPipe softwareequation , and con1H chemical shifts as a function of temperature for each assigned residue were fitted using a linear regression, and the temperature coefficients were obtained from the slope, in ppb/°C. Nonlinear shifts were fitted using a second-order polynomial equation or two linear equations, as required. NMR assignment of {1H-15N}HSQC spectra at different temperatures was done by tracking the systematic shifts, using the assigned {1H-15N}HSQC at 25 °C as a reference. For CSP and thermal susceptibility experiments, only cross-peaks not involved in overlapped signals were included. After heating to 55 °C, thermal reversibility was checked by returning to 25 °C. WT and D145E spectra were totally reversible.θ] in degrees.cm2.dmol−1 were calculated using Equation  1 K2EGTA, 20 MOPS and 100 KCl at pH 7.0, plus for apo cTnC, no further addition; and for Ca2+/Mg2+-bound, 2.075 MgCl2 and 1.096 CaCl2 (yielding a free [Ca2+] of 10−4 M and free [Mg2+] of 2 mM). Tm values were obtained by taking the first derivative of the mean residue ellipticity (MRE)θ=222vs temperature fitting. Data were fitted with a biphasic dose response function in OriginPro :A1 and A2 are the bottom and top plateau values, logx01 and logx02 are the temperatures at the first and second midpoint transitions, h1 and h2 are Hill slopes and p a proportion factor.Far-UV CD spectra were collected in a Chirascan spectropolarimeter (Applied Photophysics) using a 1-mm-path quartz cell. Each protein was heated from 10 or 20 °C up to 90 °C at a rate of 1 °C/min, and the ellipticity was recorded at 222 nm every 1 °C. At 20° and 90 °C full spectra were recorded  after temperature equilibration for 5 min. Three scans were averaged at each of these temperatures, and no numerical smoothing was applied. Immediately after reaching 90 °C, the temperature ramp was reversed in order to refold the protein. The optical activity of the buffer was subtracted from each protein spectrum. Molar ellipticities ) and 1 mM fresh DTT were added. Steady-state fluorescence measurements for Ca2+ binding at the N-domain of HcTnCIAANS (WT or D145E) were performed in a four-cell, Peltier-controlled Jasco FP-8300 spectrofluorimeter where IAANS fluorescence was excited at 330 nm and emission was detected at 450 nm. The cuvette containing the protein was equilibrated for 10 min at the experimental temperature before starting the titration. Calcium titrations at 30 °C and 45 °C (pCa 7 to 3.8) were carried out as previously described for 21 °C ref. 49. The data obtained previously at 21 °C were imported for comparison. At 21 °C, free Mg2+ increased from 0.45 mM (at pCa 7) to 0.61 mM (at pCa 3.8). At 30° and 45 °C, the free Mg2+ was lower by 0.12 mM and 0.29 mM, on average, at each pCa value.Both HcTnC isoforms were labeled with 2-(4′-(iodoacetamido)anilino)naphthalene-6-sulfonic acid  at their native cysteines, Cys 35 and Cys 84; in this situation the signal from pCa 6.8 to 3.8 is a function of the N-domain sites2EGTA (pH 7.0). Before each data set, 1 mM fresh DTT was added. For the holo state, Ca2+ and Mg2+ were added to obtain 0.1 mM free Ca2+ and 2 mM free Mg2+. Protein concentrations were 1 µM, mixed with buffer and 5 µM bis-ANS. Excitation was at 360 nm and emission at 400–600 nm.The bis-ANS fluorescence spectra were recorded at 20 °C on an ISSK2 spectrofluorometer . Each protein was dialysed into fluorescence buffer containing 120 mM MOPS, 100 mM KCl and 2 mM K8. Native cTnC was removed and replaced using a saturating concentration (109 µM) of HcTnC (WT or D145E) in relaxing solution. cTnC extraction and reconstitution were measured by recording residual tension at pCa 4.0, followed by pCa 8.0. The residual force, an index of remaining endogenous cTnC, was 16.9 ± 2.7% of P0 for fibres used in WT reconstitutions and 13.6 ± 1.2% of P0 for fibres with D145E (P > 0.05). Western blots have shown that 20% residual force at pCa 4.0 corresponds to an endogenous cTnC content of 14% and that ~70% recovery of P0 with HcTnC incorporation corresponds to a full complement of cTnC50.Cardiac fibre bundles were dissected from pig papillary muscle, skinned with Triton X-100 and prepared for recording as described0 at 21 °C  for WT and D145E, respectively. This corresponds to about 35 mN/mm2. Note that force recovery with the WT HcTnC reconstitution here was 10% higher than in Landstrom et al.8, most likely due to use of a ~4x higher concentration of cTnC to reconstitute the fibres. The reconstituted fibres were sequentially incubated with a range of [Ca2+] solutions containing 10−8 M to 10−4 M free [Ca2+], 1 mM free [Mg2+], 7 mM K2EGTA, 2.5 mM MgATP2−, 20 mM MOPS (pH 7.0), 20 mM creatine phosphate and 15 units/ml creatine phosphokinase; the ionic strength was adjusted to I = 0.15 using K propionate. The pCa values were re-calculated for 15 °C, 25 °C and 30 °C using pCa Calculator49. The variations in free Mg2+, MgATP2− and ionic strength were less than 10%. Data were analysed using equation  were imported for comparison. A separate set of fibres was used just for measurements of maximal force at 30 °C. In this case, a test for maximal force was performed at 21 °C, and then a Ca2+ curve ending at pCa 4.0 was carried out to provide a value for maximal force at 30 °C. This strategy was used because at 30 °C, considerable deterioration occurred in the maximal tension, different from the more stable tensions at 15 °C and 25 °C. The reconstituted Po values at 15, 25 and 30 °C were normalised to the Po recorded at 21 °C. In controls designed to evaluate fibre deterioration at 30 °C, the initial reconstituted Po was recorded at 21 °C and then temperature was raised directly to 30 °C, where Ca2+ sensitivity was measured on the same fibre 0, 10 and 20 min after reaching 30 °C (Supplementary Fig. where “[Cat-test paired or unpaired at p < 0.05 depending on the experimental design. The results are reported as means ± s.e.m.All data were analysed for significance using Student’s Supplementary Figure S1