To identify factors influencing clinicians decision-making about ongoingstroke rehabilitation for people with pre-existing dementia/cognitiveimpairment and the impact on clinical practice.Qualitative semi-structured interviews with stroke specialist healthcareprofessionals analysed using thematic analysis.Acute stroke unit, inpatient stroke rehabilitation units, and communitystroke services.Twenty three professionals from six multidisciplinary stroke teams involvedin decision-making about stroke patients’ rehabilitation potential andclinical pathways.Factors influencing decision-making about ongoing rehabilitation were (1)gaining understanding of the individual patient, (2) clinician’s knowledgeof dementia/cognitive impairment, (3) predicting rehabilitation potential,(4) organizational constraints, and (5) clinician’s perceptions of theirrole within the team. Decision-making led to two outcomes, eitheraccommodating the pre-existing dementia/cognitive impairment within deliveryof rehabilitation or ending rehabilitation for that patient to allocatelimited resources where they were perceived more likely to be effective.Participants felt that patients with pre-existing dementia/cognitiveimpairment had difficulty demonstrating the required rehabilitationpotential within the short timescales available in the current model ofservice delivery. Participants identified a need for training to improvetheir knowledge and confidence for decision-making and delivery ofrehabilitation for this growing population.Clinicians’ decision-making about ongoing rehabilitation for patients withprestroke dementia/cognitive impairments is influenced by gaps in theirknowledge and by service constraints. Increased training and more flexible,patient-centred services would enable clinicians to better accommodate thesepatients in rehabilitation. If inadequate rehabilitation is a contributory factor, thenthat is modifiable through service reorganization. Increasing rehabilitation couldimprove life after stroke because, although patients with pre-existingdementia/cognitive impairment often start at lower functional baselines, evidencesuggests that they benefit from rehabilitation.7Debates about the suitability of stroke rehabilitation for patients with pre-existingor current cognitive deficits occur regularly in clinical practice and the literature.9The term rehabilitation potential sits uncomfortably with many but is understood asthe ability to benefit from rehabilitation;10 a broad process which aims to reduce impairment, increase independence andautonomy, and enhance well-being.10 Rehabilitation potential is difficult to predict due to the fact that somepatients demonstrate their potential later than others.10 Enderby et al.10 call for research into decision-making about rehabilitation potential afterstroke. It is unclear whether, and if so how, pre-existing dementia/cognitiveimpairment influences decision-making about rehabilitation potential and treatmentplans after stroke.10UK models of stroke care require professionals to make early predictions about aperson’s ‘rehabilitation potential’ for initiating or continuing withrehabilitation.The present research aimed to identify (1) factors influencing the clinicians makingdecisions about rehabilitation for people with pre-existing dementia/cognitiveimpairment and (2) how these factors influence clinical practice.Online Appendix 2),11 which was approved by the University of Manchester Ethics Committee(reference no. 16438) and relevant UK National Health Service (NHS) bodies.Clinicians working in stroke services as part of multidisciplinary teams (MDTs) andwho were involved in making decisions about rehabilitation were eligible forinclusion. Teams were approached with information about the study, and cliniciansvolunteered to participate. We sought a purposive sample to include a range ofsettings and disciplines, for example, hyper acute, rehabilitation, and community intwo different NHS trusts, in order to gain as wide a range of perspectives aspossible and cover the entire stroke pathway.The consolidated criteria for reporting qualitative research (COREQ) checklist wasused to develop and report this study (see Online Appendix 1). The guide was initially piloted on twoclinicians working in different services to those sampled from and was reviewed andrefined throughout the interview process to ensure it was as relevant as possible.Field notes were made following each interview to aid the topic guide. The choice offace-to-face or telephone interviews were offered to minimize logistical challengesfor healthcare professionals.12 Informed consent was obtained prior to interview. Interviews were conductedby V.L., an Occupational Therapist (OT) with experience of delivering clinicalservices to people with stroke and dementia, and of research. This was disclosed toparticipants prior to interview, and participants were unknown to the interviewer.With consent, interviews were audio recorded and transcribed verbatim by auniversity-approved transcription service. Transcripts were checked for accuracyprior to deletion of audio recordings.One-off individual semi-structured interviews using open and closed questions wereundertaken in a private room in the participant’s workplace and followed a topicguide  were removed from the transcripts, which were then read repeatedly inorder to increase familiarity. Themes were derived using an iterative process ofdata familiarization, generation of initial codes, identification of themes,reviewing themes, definition of themes, and report production.13 V.L. analysed the transcripts and generated initial codes. A subset oftranscripts were analysed by the co-authors, an MDT of health service researcherswith expertise in stroke rehabilitation and dementia. Emerging themes were thendiscussed by all co-authors at each stage of analysis to agree final themes.Data were analysed using thematic analysis.n = 11) due to often having the role ofassessing cognition in stroke settings. Four physiotherapists, one Speech andLanguage Therapist (SLT), one Assistant Psychologist, one Clinical Psychologist,three Nurses, and two Physicians were also recruited.Six multidisciplinary stroke teams across two NHS trusts in the north of England wereapproached. Twenty three clinicians from six professions volunteered to participatein the study Participants discussed the complexity of identifying cognitive impairments, andthe importance of teasing out prestroke and post-stroke impairments in order toidentify rehabilitation needs. Participants discussed how patients with existingimpairments have different rehabilitation needs to those with new post-strokecognitive impairments. However, participants from some professions revealed amore nuanced view than others: To be honest, in three months we’ve not really had a real dementia. We’vehad a few mild dementias but not had a proper dementia with a stroke.Decisions around ongoing rehabilitation for patients with pre-existingdementia/cognitive impairment were influenced by participants’ own knowledge ofdementia. While most participants observed that many of the patients they seehad pre-existing dementia/cognitive impairments, some were unable to identifypatients with dementia: While this view was not commonplace, it implies that some participants understooddementia as a singular condition rather than a complex one with differingpresentations.At uni I think it was quite limited, I’ve learnt most of what I know fromcognitive impairment in placements at uni or from work, just shadowingsenior staff and things like that. In contrast to post-stroke cognitive impairments, participants expressed feelingthat they had a lack of knowledge of dementia, which affected their ability tomake decisions about ongoing rehabilitation for these patients. Mostparticipants recognized their limited understanding of dementia, attributingthis to a lack of training. Instead they relied on ‘common sense’  and opportunistic learning: Participants also highlighted the lack of formal structure and priority availablefor extra training, especially for ward nurses. Some participants had hadspecialist dementia training funded by their workplace; however, it wasacknowledged that working within stroke services requires a broad spectrum ofknowledge, some of which was perceived to be best gained through experience.It would be a disservice to say to someone, because you’ve got a dementiayou can’t possibly have stroke rehab. Participants’ knowledge about dementia influenced decision-making aboutrehabilitation potential. Participants initially described giving all patientsthe opportunity to have a ‘fair chance’  at rehabilitation, but balanced this with their perceptions aboutthe individual patient’s potential to benefit from rehabilitation: If there’s pre-existing cognitive impairment there that might be memoryrelated … I would probably then start to think, well what’s thisperson’s capacity for learning and improving? … the thing we should bedoing for that person is discharge planning. Participants discussed their belief that patients need to possess carry-over inorder to benefit from rehabilitation: ‘the idea of rehab is that you can buildon something and carry over [to the next] session’ . Carry-over was viewed as an area in which patientswith pre-existing dementia/cognitive impairments have difficulty, andparticipants had lower expectations of the patient’s ability to change: I wouldn’t expect anything to improve [if a patient has a pre-existingcognitive impairment] … I just wouldn’t expect [them] to change. While participants expressed the desire to provide fair chances atrehabilitation, these perceptions were associated with a broad belief thathaving a diagnosis of dementia equated to lack of rehabilitation potential orcapacity to change, unless the patient demonstrated otherwise: Some also expressed difficulty in determining whether patients possessedrehabilitation potential and were mindful that ‘people have also completelybucked the trend’ . Participantsdescribed a lack of rehabilitation potential as patients being unable to achievetherapy goals and ‘starting to plateau’  with progress. However, rehabilitation potential was typically assessedwithin the first few days of admission and participants felt obliged ‘to make adecision pretty quickly and I find that can be quite hard as well’ .But, yeah, I think it’s been so difficult for me to describe who has gotrehab potential, it is really difficult. Sometimes it is a gut instinctas well and, yeah, I think it would be good if we did have maybe a bitmore guidance on that, you know, what to look out for. That’s what is scary with the rehab potential part, so if you say thisperson’s got no rehab potential anymore, then they could essentiallyhave things decided for them … it’s difficult, isn’t it, how long is apiece of string. Previous experience of working with patients with dementia or pre-existingcognitive impairments was used to inform decision-making about rehabilitationpotential. Junior participants with less experience in particular highlighteddifficulty when determining rehabilitation potential and acknowledged that thesedecisions held a great deal of weight if they resulted in a patient beingdischarged into residential care: Nobody’s ever sat down and said, this is how you decide if somebody’s gotrehab potential. You kind of get taught that if something’s not working,if you’ve tried it three times and it’s not improving then try somethingelse. Participants also expressed concern that rehabilitation potential isunpredictable. Accurately determining an individual’s rehabilitation potentialwas viewed as an impossible task and one in which training was lacking: [It is difficult] when we are trying to do our first assessments and thenmaybe someone with a dementia that’s quite advanced maybe can’t followinstruction, can’t participate with your assessment, maybe is just notvery engaged with you … It makes it hard because then you can’t justgive them an instruction to follow. Participants described how assessing patients with pre-existingdementia/cognitive impairments was challenging and that patients neededincreased time to demonstrate progression in rehabilitation which was limitedwithin current service delivery models: Rehab as a journey in terms of weeks with people getting better takeslonger, but equally individual sessions take longer because you oftenhave to repeat commands, take things really slowly, give people time fordelayed processing, so I think it definitely takes more time. Examples were given of how participants worked with patients with pre-existingdementia/cognitive impairment. Participants observed that such patientssometimes required longer to make equivalent progress in rehabilitation thanpatients without pre-existing impairments: We’re a service that’s very much based around potential to improve, we’renot really a management service and we’ve only got 6 weeks, with thosepatients I think it’s only fair that we try and get them into the rightservice. Participants described an awareness of management strategies for working withpeople with dementia, but were struggling to provide these due to servicelimitations and expressed the opinion that stroke services were not necessarilythe most appropriate service for patients with pre-existing dementia/cognitiveimpairments: I know a lot of the people with vascular dementia get put on the strokepathway and it’s actually not the right place for them and they’re notgetting the service that they need. They’re also getting a potentiallyincorrect message in terms of you’re on a rehabilitation ward whichmeans that you’re going to get better, and people with dementias … arepotentially going to deteriorate and we can do what we can to supportthem but we’re not able to rehabilitate them in the sense that they’regoing to improve. In addition to this, participants expressed the opinion that the model ofrehabilitation they were working within, that is, that rehabilitation led toimprovement in function, was not suited to patients with pre-existingdementia/cognitive impairments. It was acknowledged that dementia is aprogressive condition that requires a different approach to rehabilitation: Sometimes they’ll think it’s [our] role to psychologically analyse thatperson and to provide a full treatment plan for cognitive impairment andto be a psychiatrist and be a psychologist. I think that can besometimes quite frustrating because they look at us and go, so what wethinking then, do you think they’ve got dementia? And it’s like I’mlooking at it in terms of function, do they remember to take theirmedication, I’m not looking at it to diagnose. Participants’ perception of their own role within the team influenceddecision-making for patients. Collaborative working was frequently cited as animportant factor in decision-making; ‘not just a single thing that we do, we doit as an MDT’ . Somedisciplines perceived a lack of understanding within the team about the scope oftheir role when working with patients with pre-existing cognitive impairments: I think us as therapists – physio and OT, we do work obviously veryclosely on stroke, so it is good because you’re bouncing ideas off eachother, but I think [medical staff] can be quite quick to be like, she’snot got rehab potential, they’ve had this stroke and that’s it kind ofthing. Teamwork and gaining specialist knowledge from others was an important factorwhen making decisions about rehabilitation, and participants used opinions fromother disciplines to inform decisions: ‘[I] don’t feel that confident to makethat decision on my own at all, and I think it is meant to be an MDT decision aswell’ . This was notwithout difficulty; some professions sampled perceived their colleagues ashaving different attitudes about rehabilitation potential to themselves whichlimited decision-making: The previous five themes reveal the factors influencing whether patients wouldreceive ongoing rehabilitation. As shown in For people where already you’re starting to get a feel that it’s moreabout a management approach, it’s more about long term potentialdeterioration rather than improvement. There might be some level ofimprovement but that will normally be environmental or compensatoryrather than doing rehab. Participants described focussing on compensatory strategies in order toaccommodate patients with pre-existing dementia/cognitive impairments, tomaintain function and address safe discharge instead of attempting to improveabilities: Participants also talked about strategies they used to tailor their approach torehabilitation for individuals with pre-existing dementia/cognitive impairmentssee .Trying to get strategies to work on [the deficit], so whether that’scompensatory or teaching or equipment or further practice or things likethat, … it’s going to be trial and error I think really on yourtreatment, but trying to think what might be useful to help themovercome that deficit, and then just keep trying until you findsomething that works or you get to a point where you think, I don’tthink this person’s going to be able to get that back. While participants expressed feeling that they had a lack of knowledge and skillsfor working with patients with pre-existing dementia/cognitive impairment, theyactually described a variety of methods that they used to support patients.These were acquired through trial and error and would be reviewed alongside thedecision to continue with rehabilitation: Ultimately, participants described how rehabilitation would have to end for somepatients with pre-existing dementia/cognitive impairments. Participantsdiscussed how they would give priority to patients making faster progress inrehabilitation because working with patients with pre-existingdementia/cognitive impairments ‘does add pressure onto the staffing levels’.So if I have 18 patients on one ward and I have half of me for that day …if somebody’s got quite significant cognitive problems and I feel thatthey are not going to make massive difference by […] giving dailytherapy, then I will de-prioritise them over … somebody who wouldbenefit from daily input. When you have such a flow of patients through, all requiring suchdemanding input … all requiring equal rights and access to this service,there has to be a point when you look at rehab potential and outcome andwho was best placed. Participants discussed how decisions had to be based on outcomes from previouspatients in order to facilitate continuing or ending rehabilitation: Participants talked about service pressures and a reduced ability to provideextensive support when having a large number of patients; treatment becamereactive rather than focussed on long-term outcome. This ultimately impacted onhow patients who are taking longer to improve would be prioritized within theservice. Participants working within time-limited services adapted theirrehabilitation to fit within their limits, that is, taking a compensatoryapproach rather than focussing on improvement within a short timeframe.I think it’s difficult really and I think sometimes that pressure we’vegot this [six week] window, we know we’ve got the provision to do morebut then if we’ve got lots of referrals and we’ve got a big caseloadthen we’re kind of reduced to what we can do with our patients. Time limits on services intend to focus interventions and enable prioritization;however, this means that some individuals are not given the opportunity todemonstrate progression within the timeframe. Participants discussed how theyfelt current stroke pathways were ill-suited for patients with pre-existingdementia/cognitive impairments due to this.The findings demonstrate the way information, confidence, service models, andteam-working inform decision-making about stroke rehabilitation for people withpre-existing dementia/cognitive impairment and their impact on clinical practice.Clinicians attempted to distinguish prestroke from post-stroke cognitive impairmentsin order to determine rehabilitation needs and potential when working to a somewhatnarrow concept of rehabilitation  and towards goals thatappeared service-led rather than patient-centred. Often this was based oninformation from family members and intuition rather than systematic assessment.This identification was influenced by participants’ own knowledge and understandingof dementia, often acknowledged to be limited. Participants expected patients withdementia to have difficulty demonstrating rehabilitation potential, which wasconfounded by limitations of the model of rehabilitation they were working within.Participants reported patients needed longer to progress with rehabilitationcompared to those without prestroke cognitive impairments, but clinicians wererequired to make early decisions about potential to progress. In addition,misconceptions over roles limited shared decision-making.The decision of whether a patient will receive ongoing rehabilitation was expressedin two ways. Participants described positive strategies of how they would supportpatients with pre-existing dementia/cognitive impairments and engaged in aniterative process of reviewing their decision to continue with rehabilitation,shifting their focus from improvement to maintenance. Participants also describedhow they would have to end rehabilitation for patients with pre-existingdementia/cognitive impairments due to service constraints.This study has strengths and limitations. A qualitative approach to this topicallowed a relatively unexplored area of clinical practice to be investigated, andthis was done from the perspectives of clinicians from a range of relevantdisciplines and stroke services. The contextual issues highlighted in this studyaround working in high pressured environments could be applicable to servicesnationally. The level of self-reflection from staff demonstrates an awareness ofareas for improvement while attempting to work to the best of their currentabilities given the imposed service constraints.8 47% of participants still came from one profession (OT). This was expecteddue to their role in assessing cognition; however, greater representation from otherprofessions could improve transferability of the findings. The overrepresentation ofOTs could underestimate the training needs of this population, because while as adiscipline they had received the most training of those sampled and have a role inassessing cognition,15 some still lacked confidence. In addition, the fact the lead author (V.L.) isan OT could introduce bias to the study, although every attempt was made to mitigatethis. Participant’s awareness of the researcher’s role can affect results; however,the mutual understanding of services may have allowed participants to speak more freely.16This was a difficult topic to discuss in some cases due to sensitivity around sharingworking practices caring for vulnerable patients and in an area in whichparticipants felt they lacked skills, knowledge, and resources to provide idealservices for patients. Sampling from two trusts within one geographical area maylimit the transferability of the findings; however, the six sampled services were asdifferent as possible and covered a large population. While the sample was moreprofessionally diverse than related studies,17 Education on dementia has been found to be inadequate for adult nursing,occupational therapy, and social work courses in some UK Higher Education Institutions,18 as highlighted by clinicians in this study. Clinical experience has beenfound to be one of the most important factors influencing decision-making in strokerehabilitation, therefore training to support those with less experience may bebeneficial.20 Clinicians inthis study indicated their own desire to further develop their knowledge; however,there is limited guidance on working with this patient group, as highlighted in theUK National Clinical Guidelines for Stroke.21While this is to our knowledge the first study exploring decision-making for strokerehabilitation for people with pre-existing dementia/cognitive impairment, somecomparisons to other studies can be drawn. In this study, there was inconsistencyover the amount of training and experience participants had about dementia among alldisciplines. A lack of knowledge about the aetiology of dementia may impact on thesuccess of interventions in the long term.22 Adapting to loss could be thought of as maintaining function.10 The UK National Clinical Guidelines21 even state over time stroke rehabilitation will shift from a restorative tocompensatory approach due to the evolving needs for people post-stroke. It seems,therefore, that acknowledgement of different approaches for some patients should bemade clear from services delivering rehabilitation. In fact, rehabilitation taking acompensatory approach has been found to be effective for people with dementia23 and cognitive rehabilitation can be used to facilitate management of acondition, which is a growing field in dementia care.24 In addition, no definitive literature has been identified about specificpatient groups who do not benefit from stroke rehabilitation.25 Maintenance and management of function remains a vital part of therehabilitation process, and stroke services need to make provision for people withpre-existing dementia/cognitive impairments who may require more of a managementapproach.Some clinicians in this study saw rehabilitation as an active process leading toimprovement in function rather than maintenance of function or well-being; ifpatients did not demonstrate functional improvement, then they were unable toprogress with rehabilitation. This is a somewhat narrow interpretation ofrehabilitation, which is defined as restoring, or adapting to loss of, physical andpsychological functions.26 Clinicians working in inpatient environments suggested theywere not always conducive to the demonstration of rehabilitation potential forpatients with dementia, and therefore, this needs to be considered when deciding ifa patient has potential in these settings.8 Studies have highlighted challenges in devising meaningful interventions inclinical environments, which particularly impact on patients with cognitivedifficulties;28 and thus, deferring decisions about rehabilitation potential may be more appropriate for these patients.8The current rehabilitation delivery model in the study settings, therefore, appearsto have a number of constraints which influence clinical practice. As identified inthis study, patients requiring longer to progress in rehabilitation or todemonstrate their potential to change become deprioritized due to limitations aroundavailability of services, which is similar to findings in the widerliterature.29 The concept of giving patientsa fair chance at rehabilitation is framed by resource availability and patientabilities, which requires resilience on the part of clinicians.20 It is clear more support andrevised service models are required in order to deliver care that clinicians feel isin patients’ best interests.One aspect to emerge from the study findings is the difficulty clinicians have injudging rehabilitation potential for patients with pre-existing dementia/cognitiveimpairments. The emotional element of decision-making, with clinicians sometimesfulfilling a need to act or feeling ‘torn’ about seeing patients perceived as alower priority, has been highlighted in the literature.30 Improvements in stroke care mean higher survival rates, and anincrease in older patients who are surviving strokes.31 Staff in this study identified a lack of training even at university level inworking with people with dementia; therefore, it is clear that improvements ineducation and training are required in order to ensure clinicians possess theappropriate skills to work alongside patients with pre-existing dementia/cognitiveimpairments, particularly as patient numbers with dementia are likely to increase.3Stroke and dementia are associated with age and incidence of both isincreasing.Changes to the current model of stroke rehabilitation are required to better suit theneeds of stroke patients with pre-existing dementia/cognitive impairments. Patientsare currently required to demonstrate their potential for change early in the acutephase of their treatment; however, this is not always appropriate for this patientgroup and more flexibility may be required.32 and exploring how to better involve them in decision-making in the earlystages of stroke rehabilitation could potentially facilitate care planning.Throughout this study, there was little mention of how patients were included in thedecision-making process. This could be an area for future study; people withdementia are often excluded from decisions about their careClinicians should have access to training in order to increase knowledgeof dementia and accommodate cognitive problems in rehabilitation.Timeframes need to be more flexible for patients to demonstraterehabilitation potential.Click here for additional data file.Supplemental material, cre-2017-6705-File001 for What influences decisions aboutongoing stroke rehabilitation for patients with pre-existing dementia orcognitive impairment: a qualitative study? by Verity Longley, Sarah Peters,Caroline Swarbrick and Audrey Bowen in Clinical Rehabilitation


The effects of different buffer structures, such as a single buffer layer, compositionally graded buffer layers, and superlattice buffer layers, on the crystalline quality and property were investigated. Double-crystal X-ray diffraction (DC-XRD) measurement, Raman scattering spectrum, and Hall measurements were used to evaluate the crystalline quality and electrical property. Scanning electron microscope (SEM), atomic force microscope (AFM), and transmission electron microscope (TEM) were used to characterize the surface morphology and microstructure, respectively. Compared with the In0.82Ga0.18As epitaxial layer directly grown on an InP substrate, the quality of the sample is not obviously improved by using a single In0.82Ga0.18As buffer layer. By introducing the graded InxGa1−xAs buffer layers, it was found that the dislocation density in the epitaxial layer significantly decreased and the surface quality improved remarkably. In addition, the number of dislocations in the epitaxial layer greatly decreased under the combined action of multi-potential wells and potential barriers by the introduction of a In0.82Ga0.18As/In0.82Al0.18As superlattice buffer. However, the surface subsequently roughened, which may be explained by surface undulation.In It is a direct band gap semiconductor of the III–V family with a sphalerite structure, making it a suitable choice for creating short-wavelength infrared detectors [xGa1−xAs materials have attracted much research attention and have been widely used in the fields of civil, military, space remote sensing, spectroscopy, and so on [0.53Ga0.47As epitaxial material covering a wavelength of 0.9–1.7 μm is lattice-matched to an InP substrate, which has been developed mainly for fiber communication applications. However, as there is no lattice-matched substrate for In0.82Ga0.18As materials, the large lattice mismatch between the In0.82Ga0.18As epitaxial layer and the substrate will result in misfit dislocations or other defects, which will destroy the material quality and further weaken the performance of devices [0.8Ga0.2As buffer layer was introduced between an In0.8Ga0.2As epitaxial layer and an InP (100) substrate and showed that the heterostructure with a buffer thickness of 100 nm had the optimum properties [0.8Ga0.2As photodiodes grown on InP substrates were investigated by using a linearly graded InxAl1−xAs buffer and an excellent performance was demonstrated [0.9Al0.1Sb superlattice between InSb thin films and a GaAs (001) substrate by the molecular beam epitaxy (MBE), and deduced that the superlattice (SL) structure could effectively prevent dislocation propagating to the upper InSb thin films [Inetectors ,3,4. Becnd so on ,6,7,8. T devices , molecul devices , or low- devices  with dif devices , a compo devices ,15,16,17d an InP 0 substranstrated . Moreovenstrated ,21,22,23in films . Obviousin films ,26. 0.82Ga0.18As epitaxial layer grown on an InP (100) substrate by a low-pressure metalorganic chemical vapor deposition (LP-MOCVD) technique. The mechanism of reducing dislocations is also explored. Hence, a comprehensive understanding of dislocation-mediated strain relaxation during III–V heteroepitaxial growth is very necessary. One purpose of this article is to compare and evaluate the effect of different buffer structures, such as a single buffer layer, compositionally graded buffer layers, and superlattice buffer layers, on the crystalline quality, surface morphology, and structure of an In0.82Ga0.18As epitaxial layers were grown on semi-insulating InP (100) substrates by a low pressure metalorganic chemical vapor deposition technique. Trimethylindium (TMIn), trimethylgallium (TMGa), and 10% arsine (AsH3) in H2 were used as group III and V source materials, respectively. Phosphine (PH3) was employed as a protective atmosphere. Prior to the growth, the InP (100) substrates on the graphite susceptor were heated at 630 °C for 10 min to remove the oxide layer from the surface using inductively coupled radio frequency power in the phosphine atmosphere. The reactor pressure was maintained at 1 × 104 Pa. When the temperature in the reaction chamber fell to 530 °C, the growth of the heterostructure for sample A was carried out and was directly grown on the InP (100) substrate. For sample B, when the temperature of the reaction chamber was reduced to 450 °C, the 100 nm thick In0.82Ga0.18As buffer layer was grown on the InP (100) substrate and then the In0.82Ga0.18As epitaxial layer was grown when the temperature rose to 530 °C. For sample C and sample D, the two heterostructures were grown with different buffer structures at 530 °C. Sample C was grown with InxGa1−xAs buffer layers that In composition x graded from 0.53, 0.62, 0.72, to 0.82 in which every layer of the structure was 25 nm; sample D with a 9-pair In0.82Ga0.18As/In0.82Al0.18As superlattice where the thickness ratio between the In0.82Ga0.18As layer and In0.82Al0.18As layer was fixed to 1:1 and the total thickness of one pair was 10 nm, in addition to a 25 nm thick In0.53Al0.47As layer inserted between the InP substrate and superlattice which was to ensure a smooth surface for the subsequent layer growth. In these samples, all the In0.82Ga0.18As epitaxial layers were also grown at 530 °C by LP-MOCVD with a thickness of 1500 nm. In0.82Ga0.18As/InP heterostructures. Hall measurements  were carried out to characterize and evaluate the properties of the samples.The surface morphologies were evidenced and analyzed by atomic force microscopy  and scanning electron microscope . Double-crystal X-ray diffraction (DC-XRD) and Raman scattering measurements  were used to investigate the composition and crystal quality of the epitaxial layers. Transmission electron microscopy  was performed to examine the microstructure and the distribution of dislocations in the In2 were performed to investigate the surface morphology of the In0.82Ga0.18As epitaxial layers of four samples. Slightly disordered corrugation patterns were observed, as shown in 0.82Ga0.18As materials is 5.985 Å, and thus the lattice mismatch between In0.82Ga0.18As and the InP substrate is 2%. For sample A, the In0.82Ga0.18As epitaxial layer directly grown on the InP substrate was subjected to the action of the larger mismatched strain which brought lots of defects into the epitaxial layer, resulting in a poorer surface quality. However, various buffer structures in samples B, C, and D reduced the defects in the epitaxial layer, so the surface qualities of sample B–D were improved by the introduction of a buffer layer; these surface qualities being better than that of sample A.AFM scans over 15 × 15 μm0.82Ga0.18As epitaxial layer and InP substrate, respectively, are clearly observed in 0.82Ga0.18As epitaxial layer and the InP (100) substrate, no other diffraction peaks were detected, which confirms that the In0.82Ga0.18As epitaxial layer has an uniform (100) rientation. According to the DC-XRD curves, the full width at half maximum (FWHM) values of the In0.82Ga0.18As epitaxial layer are calculated and summarized in  density . The dis0.82Ga0.18As epitaxial layer was also examined by the scanning electron microscope. 0.82Ga0.18As epitaxial layers of the four samples. It was found that the mounds elongated to form long ridges on the surface for sample A, sample B, and sample D, indicative of the misfit dislocation array at the interface [The surface morphology of the Innterface . For sam0.82Ga0.18As epitaxial layers for samples A–D. Two-mode Raman peaks are observed in each spectrum which correspond to longitudinal optical (LO) phonon modes of InAs and GaAs, respectively [0.82Ga0.18As epitaxial layer. Furthermore, the asymmetry ratio (Γa/Γb) of Raman scattering spectra can be used to characterize the crystalline quality of samples [0.82Ga0.18As materials, the asymmetric ratios (Γa/Γb) of GaAs-like LO-phonon peaks are selected to characterize the crystalline quality of samples, which is displayed in the inset of 0.82Ga0.18As epitaxial layers can be improved by the introduction of buffer layers with different structures, which is in accord with the DC-XRD measurements.In addition to DC-XRD measurement, Raman scattering is an indirect way to characterize the crystalline quality of materials. ectively . Because samples . The clo0.82Ga0.18As/InP heterostructures with or without buffer structures. As is well known, the variation of microstructure can affect the quality of materials to a great extent. Because of the large lattice mismatch of about 2% and ae > as , the In0.82Ga0.18As epitaxial layer of sample A is a positive mismatch to the InP substrate and is under compression strain. It can be seen in 0.82Ga0.18As buffer layer was inserted between the epitaxial layer and InP substrate. The In0.82Ga0.18As buffer layer grown on the substrate was in favor of the growth of the In0.82Ga0.18As epitaxial layer because they matched well with each other. Furthermore, both the growth of the buffer layer and epitaxial layer are a positive mismatch to the InP substrate. By comparing this with previous reports performed by Zhao Liang et al. [0.82Ga0.18As buffer layer provides a nucleation center with the same substrate orientation, which acts like a template of the epitaxial layer and provides a nuclear surface for the subsequent growth for In0.82Ga0.18As epitaxial layer. When the growth of the buffer layer is completed, the temperature of the reaction chamber is gradually increased to the growth temperature of the epitaxial layer (530 °C), which is a process of high temperature annealing for the buffer layer. Then, the surface of the buffer layer is recrystallized, which makes the surface of the buffer layer smooth and can effectively release the thermal strain caused by the mismatch of coefficient of thermal expansion between In0.82Ga0.18As and InP. It can clearly be seen in 0.82Ga0.18As epitaxial layer and InP substrate and accommodate the dislocations induced by the strain, and the high In content buffer layer produce more dislocations which might extend to the epitaxial layers. Therefore, the differences in the surface morphology and roughness are very small for sample A and sample B, as listed in xGa1−xAs buffer layers inserted between the top In0.82Ga0.18As epitaxial layer and InP substrate grades from 0.53 to 0.82, in which the first layer with an In content of 0.53 and the last layer with an In content of 0.82 match well with the InP substrate and In0.82Ga0.18As epitaxial layer, respectively. Furthermore, every layer (except for the first layer) in the graded buffer and the top In0.82Ga0.18As epitaxial layer are positive mismatching to the InP substrate and under compression strain. At the same time, the thicknesses of the InxGa1−xAs buffer layers of each component are larger than the critical thickness, so a large number of dislocations and defects are restricted in the buffer layers. The graded buffer layers give play to three functions: releasing stress, restraining surface undulation, and restricting dislocations. It can be clearly seen from xGa1−xAs buffer layer is that the dislocations annihilation cannot only occur between different dislocations, but can also occur between different layers, which gives much more possibilities for dislocation annihilation. Therefore, the dislocation density of sample C reduced significantly and the surface quality improved remarkably, as listed in 0.82Ga0.18As/In0.82Al0.18As superlattice was employed as buffer to reduce the dislocation density. For In0.82Ga0.18As/In0.82Al0.18As superlattice structure, In0.82Ga0.18As layers and In0.82Al0.18As layers form alternate potential wells and potential barriers, respectively. On the one hand, the defects such as dislocations are limited in the In0.82Ga0.18As potential wells and have a lower energy state, thus In0.82Ga0.18As layers play a trapping role in the structure. On the other hand, since the defects already have a lower energy state in the potential wells, they do not possess enough energy to pass through the barriers, therefore the barriers formed by In0.82Al0.18As layers have a blocking effect [0.82Ga0.18As/In0.82Al0.18As superlattice buffer area under the combined action of multi-potential wells and potential barriers. Although there are still a few dislocations passing through the buffer area and extending to the In0.82Ga0.18As epitaxial layer, the number of dislocations reduced in the epitaxial layer and these dislocations did not propagate to the surface. Therefore, the In0.82Ga0.18As/In0.82Al0.18As superlattice buffer area can effectively play the role of “dislocation filter”. However, the elongated mounds forming long ridges appear again on the surface of sample D  and the highest Hall mobility value (3.21 × 103 cm2/VS). Meanwhile, the calculated dislocation density in the In0.82Ga0.18As epitaxial layer of sample D is the least. Therefore, sample D has the best quality in epitaxial growth. Therefore, it can be concluded that the higher the mobility and the lower the carrier concentration of the material, the better the quality of the epitaxial layer. In addition, the quality of the In0.82Ga0.18As epitaxial layers were grown on InP (100) substrates by a low pressure metalorganic chemical vapor deposition (LP-MOCVD) technique. The effect of different buffer structures, such as a single buffer layer, compositionally graded buffer layers, and superlattice buffer layers, on the crystalline quality and surface morphology was investigated. When the In0.82Ga0.18As epitaxial layer was directly grown on the InP substrate, a large number of dislocations were generated and extended to the surface, resulting in a particularly poor quality sample. Using a single In0.82Ga0.18As buffer layer, the dislocation density reduced slightly. However, there were still some dislocations extending to the epitaxial layer and even propagating to the surface This was because the misfit strain between the In0.82Ga0.18As epitaxial layer and InP substrate could not compensate the single buffer layer. By introducing the graded InxGa1−xAs buffer layer, it was be shown that the dislocations were mainly distributed at the interior of the graded buffer layers, the dislocation density reduced significantly and the surface quality improved remarkably. The In0.82Ga0.18As/In0.82Al0.18As superlattice buffer can effectively play the role of "dislocation filter". The number of dislocations in the epitaxial layer greatly decreased under the combined action of multi-potential wells and potential barriers of the In0.82Ga0.18As/In0.82Al0.18As superlattice buffer. However, the surface subsequently roughened, which may be explained by the other strain relaxation mechanism known as surface undulation.In this work, In


Individuals who have experienced stroke are often described as apathetic, having lost of interest, and unmotivated. This might be a problem in achieving treatment results. It is still unclear what impact age and gender have on the motivation.  To evaluate motivation influence on the effectiveness of occupational therapy in patients with stroke. Study included 30 patients who experienced acute stroke. Multidimensional Health Locus of Control (MHLC) scale has been used for motivation assessment. Internal persons' motivation was evaluated to determine how much a person believes that his recovery depends on his own behaviour and external persons' motivation—how a person relates his state of health to the impact of the surrounding people. Performance of daily activities was assessed using Functional Independency Measure (FIM). At the beginning of rehabilitation of the patients with stroke, external motivation was greater than the internal one. At the end of rehabilitation internal motivation has increased by 1.8±0.4 points, while the external motivation decreased by 2.4±0.6 points (p<0.05). At the beginning of rehabilitation patients independence in activities of daily living assessed with FIM was 70.0±2.9 points. At the end of rehabilitation their ability to perform daily activities improved by 26.9±1.9 points and reached 96.9±2.7 points (p<0.05). We found statistically significant (p<0.05) moderate correlation (r=0.72) between patients internal motivation at the beginning of the rehabilitation and increase of independence after rehabilitation. Older patients had lower internal motivation than younger ones, and independence in daily activities improvement was influenced by younger age and by brain damage depth. The aim of research is to evaluate motivation influence on the effectiveness of occupational therapy in patients with stroke.Individuals who have experienced stroke are often described as apathetic, having lost of interest, and unmotivated. This might be a problem in achieving treatment results. Factors associated with rehabilitation success include patients assertiveness, goal directedness, and presence of an intimate relationship. Motivation is the most important problem facing the rehabilitation professional. To foster motivation, therapists must be effective psychosocial advocates as well as functional coaches. Practitioners are just beginning to develop systematic methods to evaluate and enhance motivation in rehabilitation settings . It is sMortality from stroke is decreasing; however, rate of patients experiencing long-term effects of stroke increases. Different sources indicate that stroke causes death up to 20 percent patients, more than 50 percent stroke survivors remain temporarily or permanently disabled, and about 10 percent affected individuals require long-term care. Only 20 percent of the working age individuals return back to work . Individ t-test for dependent and independent samples. Associations between two variables were estimated using Pearson's correlation coefficient. Differences were accepted as statistically significant for values of p<0.05.Statistical analysis was performed using the software packages IBM SPSS 17. Quantitative variables were described using the mean, standard deviation (SD), and percentages (%). Comparison of two means was made using Student'sStudy was approved by the clinic's Bioethics Center. All of the participants provided their written informed consent prior to their participation in the study, in accordance with the ethical principles of the Declaration of Helsinki. The study involved 30 people who experienced acute stroke .The study included patients who at the beginning of rehabilitation were evaluated using Mini-Mental State Examination (MMSE) and scored 21 points or more. To evaluate patients' motivation Multidimensional Health Locus of Control (MHLC) scale was used. All evaluations were made by researchers who applied occupational therapy. Occupational therapist evaluated each patient before treatment and after 20 occupational therapy procedures. Evaluated internal person's motivation determines how much a person believes that his recovery depends on his own behaviour and external person's motivation—how a person relates his state of health to the impact of the people surrounding him. Performance of daily activities was assessed using Functional Independency Measure (FIM). Subjects were evaluated at the beginning and at the end of rehabilitation. Special rehabilitation programs were created for patients based on their functional abilities and were administered by a team of rehabilitation specialists: physical medicine and rehabilitation physician, physical therapist, occupational therapist, speech therapist, social worker, psychologist, orthosis specialist, and nurses. The patient was encouraged to participate actively in the treatment program alongside with his family members. All subjects underwent individual occupational therapy 5 times a week. The length of single treatment was 30–40 minutes. Each patient in both groups received 20 occupational therapy procedures. The aims of occupational therapy were (1) hand movement training; (2) training of meaningful everyday activities; (3) promotion of motivation to perform activities. Hand movement training was carried out at the occupational therapy cabinet, using occupational therapy techniques and equipment. Bilateral arm movements training had been applied to patients with hemiplegia. Patients with hemiparesis performed tasks with affected hand. Patients were taught to perform self-care and mobility tasks, including correct hand movements while eating; specific movements performing dressing and personal hygiene. Developing mobility skills, balance was trained and patients were trained to perform safe transfers to bed, wheelchair, etc. Motivation to activities was promoted during the occupational therapy sessions by ensuring feedback.During occupational therapy procedures, therapist encouraged patients to express and manage emotional upset related to illness and identified and addressed immediate concerns and patient's priorities and needs. Concern, responsiveness, and collaborative effort in the first few minutes set the tone and structure for all the treatment. Occupational therapist together with patient after stroke tried to formulate the main problem, its coping resources, and available solutions. The patient's hopes, ability to accept help and collaborate, and his own goal directed actions and self-evaluations were discussed, at the same time promoting realistic appraisal of rehabilitation possibilities. Occupational therapist emphasized the impact of personal effort on outcome in the treatment process and also invited the patient to express goals for treatment and for life after discharge. Concrete ADL objectives and broader life roles were discussed. Patients were encouraged to recognize that less is not necessary meaningless, defining the specific challenges of rehabilitation, promoting active coping and learning strategies, and at the same time promoting realistic progress appraisal. Positive feedback and reinforcement are essential therapeutic tools. Occupational therapist selected activities and interactions that helped patients in expressing and managing painful feelings; in sustaining performance of previously beneficial occupations and routines; in learning new skills for functional improvement, communication, stress management, and safety; in selecting activities that are remotivating by demonstrating the person's ability to accomplish goals and take care of himself or herself.At the beginning of rehabilitation, assessing the internal motivation, most of the patients agreed with the following statements: “if I get sick, it is my own behaviour which determines how soon I get well again” and “the main thing which affects my health is what I myself do”. At least patients agreed with the following statement: “When I get sick, I am to blame” . At the An analysis of the relationship between internal motivation and age showed statistically significant (p<0.05) and medium correlation (r=-0.41) between age and the size of internal motivation at the beginning of rehabilitation .In elderly people, the internal motivation in the beginning of rehabilitation was lower. We found that the age of one-year internal motivation decreases by 0.2 points. At the beginning of rehabilitation, the average of subjects' external motivation was 30.2±0.8 points out of 36 possible points. Most of the subjects agreed with the following statement: “whenever I don't feel well, I should consult a medically trained professional”. At least people agreed with this statement: “my family has a lot to do with me becoming sick or staying healthy” .At the end of rehabilitation external motivation decreased (p<0.05). The statement “whenever I don't feel well, I should consult a medically trained professional” was valued at the beginning of rehabilitation (5.7±0.1). The approach to the statement “whenever I recover from an illness, it's usually because other people have been taking good care of me” has not changed significantly (p> 0.05). All other statements reflecting external motivation assessed during the rehabilitation statistically significantly decreased (p<0.05). Most has changed the attitude to the following statements: “health professionals control my health” (decreased by 0.7±0.2 points) and “having regular contact with my physician is the best way for me to avoid illness” (decreased by 0.6±0.2 points) .Prior to treatment patients' average FIM test results were 70.0 (±2.9). Most of the problems caused ability to perform daily activities: bathing, lower body dressing, and climbing the stairs. During these activities, individuals were required for maximal assistance. Moderate assistance was necessary for dressing the upper body, grooming, toileting, transferring at toilet and at shower or bath, and walking or using a wheelchair. Minimal assistance was sufficient while eating and transferring from bed, chair, or wheelchair. During the rehabilitation patients' ability to perform daily activities significantly improved by 26.9 ± 1.9 points (p <0.05). Activities, which at the beginning of rehabilitation required maximal assistance, at the end of rehabilitation were sufficient of minimal assistance. .There was no statistically reliable difference between men and women average FIM scores prior to treatment (p>0.05). Both men and women required maximal assistance for the same activities. At the end of the rehabilitation men's and women's independence in daily activities significantly (p<0.05) improved . StaticaAn analysis of the relationship between the change of patients' independence during the rehabilitation and patients' age showed a moderate negative (r=- 0.41) statistically significant (p<0.05) correlation .Independence of older patients during rehabilitation increased less than the younger ones. We found that one year of age reduces the independence by 0.4 points. An analysis of data according to brain damage depth showed that, at the beginning of the rehabilitation individuals who had hemiplegia, FIM score average was 56.2 ± 1.5 points, while, for those who had hemiparesis, FIM score average was 64.5 ± 3.0. However statistically significant difference was not found (p>0.05). In both groups, activities of daily living at the end of rehabilitation statistically significant improved (p <0.05). For individuals who had hemiplegia, independence during rehabilitation course increased by 28.3 ± 3.8 points from 56.2 ± 1.5 to 84.5 ± 3.7 points, while the individuals who had hemiparesis – 26.5 ± 2.3 points from 64.5 ± 3.0 to 101.0 ± 2.7 points. A statistically significant difference (p<0.05) was obtained between independence scores at the end of the rehabilitation and brain damage depth.After analysing the test data according to the affected side of the body, it was found that, for those who had damaged left side of the body, the average FIM score at the beginning of the rehabilitation was 73.9 ± 4.0 and those who had damaged right body side – 65.0 ± 4.1. But there was no statistically significant difference (p> 0.05). However, in the course of rehabilitation, a statistically significant improvement was observed in independence of daily activities (p <0.05). At the end of rehabilitation for patients who had damaged left side of the body FIM scores increased by 26.7 ± 2.6 points from 73.9 ± 4.0 to 100.6 ± 3.4 points, while for those who had damaged the right side of the body the FIM scores increased by 27.1 ± 2.8 points from 65.0 ± 4.1 to 92.1 ± 4.2 points. Still statistically significant difference between the affected side of the body and independence has not been determined (p<0.05). Assessing correlation between internal motivation scores at the beginning of rehabilitation and the change of independence during the course of rehabilitation, a significant and statistically important (p<0.05) moderate (r=0.72) correlation was observed. For people whose internal motivation points at the beginning of the rehabilitation were higher, performance of daily activities in the course of rehabilitation has improved more than those with lower internal motivation scores at the beginning of rehabilitation. We found that one score of the inner motivation at the beginning of rehabilitation improves patients' performance of daily activities by 1.5 points. The relationship between external motivation scores and change of FIM scores in the course of rehabilitation has not been established.Stroke causes various consequences, which disrupts the daily human life and functional independence and changes his life-sufficiency. After evaluation of subjects' motivation at the beginning of the rehabilitation we found that the average internal motivation score was 26.6 ± 0.9 points, while external motivation average was – 30.2 ± 0.8 points. They thought that their recovery is more dependent on doctors and other people than the impact of their own behaviour. At the end of rehabilitation internal motivation mean score statistically significant increased p<0.05) by 1.8 ± 0.4 points, while external motivation mean score statistically significant decreased (p<0.05) by 2.4 ± 0.6 points. The survey data of MHLC questionnaire showed that patients believe that recovery depends on their own efforts, in the course of rehabilitation, increased, and the believe that physicians and others are responsible for their recovery decreased. According to the literature the increase in patients' age is associated with low motivation  by 1.8 ±. Our stuFor individuals who have suffered stroke, external motivation was greater than the internal. In the course of rehabilitation, internal motivation increased and the external decreased. There was a statistically significant moderate correlation between individuals' internal motivation at the beginning of rehabilitation and improvement in activities of daily living during the rehabilitation process. Older patients' internal motivation was lower than younger ones, and the improvement of daily activities was influenced by younger age and lesion depth.


There are errors in the Author Contributions. The correct contributions are: Conceptualization: DJN SC NS VD. Data curation: DJN FG SC NS. Formal Analysis: DJN FG SC NS. Investigation: DJN FG SC VD. Methodology: DJN SC VD. Resources: VD. Supervision: VD. Validation: DJN SC. Writing–original draft: DJN NS. Writing–review & editing: DJN SC NS.


Background: The prevalence of allergic rhinitis (AR) has increased in recent decades. Accumulating evidence indicates that aberrant DNA demethylation modulated by enzymes of ten-eleven translocation (TET) promotes an imbalanced immune response.Objective: This study aimed to explore TETs on the activation of dendritic cells (DCs) in AR.Methods: The levels of TETs in peripheral blood mononuclear cells (PBMCs), peripheral myeloid DCs (mDCs), and plasmacytoid DCs (pDCs) from house dust mite (HDM)-sensitive AR patients and healthy volunteers (HC) were evaluated by qPCR and flow cytometry. The levels of 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) in PBMCs were determined by DNA-5hmC and DNA-5mC ELISA. The major HDM allergen, Dermatophagoides pteronyssinus (Der p 1), was used to stimulate atopic monocyte-derived DCs (moDCs) to assess its effect on the TETs. TET1 knockdown effect on the activation of non-atopic and atopic moDCs was investigated.Results: TETs and global 5hmC were higher in PBMCs of AR than HC. So was TET1 in peripheral mDCs and pDCs of AR. In vitro, TET1 in atopic moDCs was significantly decreased by allergen challenge. Knockdown of TET1 in moDCs tended to induce CD86, CD80, and CD40 in AR but not in HC. TET1-knockdown moDCs significantly decreased the differentiation of activated regulatory T cells in AR.Conclusion: DCs from AR patients express higher TET1 and are susceptible to be activated by TET1 decrease, which can be triggered by allergen challenge. Collectively, this suggests a role for TET in the pathogenesis of AR and potential for novel TET1-related, preventive, and therapeutic targets. The prevalence of allergic rhinitis (AR), which has become a global public health problem, is ~10–40%, and the annual cost of AR treatment in the United States alone is about $5 billion . AR is aDendritic cells (DCs) are critical antigen-presenting cells and known as the coordinators between innate and adaptive immune , 7. Grow+ T cells separated seasonal AR patients from healthy donors. After allergen challenge, hypermethylation of SLFN12 gene in human peripheral blood mononuclear cells (PBMCs) was induced and correlated with increased clinical rhinitis symptoms  enzymes, including TET1, TET2, and TET3, are a family of dioxygenases . They haIn this study, we wonder if the levels of TETs in the peripheral DCs from AR patients and healthy volunteers are different. Secondly, whether TET enzymes are involved in mediating Der p 1 challenge effect in monocyte-derived DCs (moDCs). Moreover, knockdown of TET1, which is predominant among TETs in DCs, was conducted to investigate its role in the phenotypic and functional activation of moDCs, and whether it's allergic status specific.Ninety-four diagnosed HDM-sensitive AR patients and 38 healthy volunteers were recruited in this study. The clinical characteristics of the participants were shown in The mRNA expression levels of TET1, TET2, and TET3 in PBMCs were evaluated using qPCR. PBMCs were isolated, from peripheral blood of the subjects, by Ficoll-Hypaque density gradient centrifugation with Lymphocyte Separation Medium . The detailed description is presented in the Genomic DNA was isolated from PBMCs by using E.Z.N.A. Genomic DNA isolation kit (Omega Bio-Tek). Global 5mC and 5hmC content in the PBMCs were quantified with 5mC DNA ELISA Kit and Quest 5hmC DNA ELISA kit . The detailed information is presented in the + cells and CD4+ cells were isolated from PBMCs using CD14 Microbeads and CD4 Microbeads . The cells were seeded into a 48-well plate at a density of 2.5 × 105 cells/well. Complete medium, which was made up of RPMI-1640 (Gibco), penicillin/streptomycin/L-glutamine (Gibco) and 10% fetal bovine serum , was used to culture CD4+ T cells for 7 days and the medium was changed every 2 days. To induce DCs from CD14+ cells monocytes, complete medium supplemented with recombinant human granulocyte-macrophage-colony-stimulating factor , and interleukin 4  was used. On day 5, the immature moDCs were stimulated by 1 μg/mL Der p 1 for 48 h. All experiments were conducted in a humidified 5% CO2 atmosphere at 37°C.PBMCs were isolated from 6 non-atopic healthy volunteers and 6 HDM-sensitive AR patients, as mentioned above. The clinical characteristics of the participants were shown in hTET1[shRNA#1]: CTTTGCTAGTGCAGTGTAT,hTET1[shRNA#2]: ACACAACTTGCTTCGATAATT,hTET1[shRNA#3]: CCTATATGTATGGCACAATAT.In order to knock down TET1 expression in moDCs, TET1 short hairpin RNA (shRNA) was packed in recombinant adenovirus GV119 vector constructed by GeneChem . TET1-shRNA sequences were as follows:4 cells/well). On day 5, immature moDCs were transfected with adenovirus at 300 multiplicity of infection (MOI) in the presence of polybrene . After another 48 h of culture, moDCs were collected for flow cytometric determination or further studies.For infection, moDCs were induced according to the protocols noted above, but in 96-well round bottom plate  in 96-well round bottom plates. Cells were co-cultured in RPMI-1640 (Gibco), penicillin/streptomycin/L-glutamine (Gibco) and 10% fetal bovine serum  at 37°C in 5% CO2 for another 3 days. The proliferation of CD4+ T cells was assessed by flow cytometry with carboxyfluorescein diacetate succinimidyl ester (CFSE)  labeling.The ability of TET1-inhibited moDCs to induce the proliferation of CD4To determine TET enzymes expression in human peripheral DCs, the following anti-human antibodies were used: anti-Lineage Cocktail FITC (CD3/14/16/19/20/56) (Biolegend), anti-HLA-DR Brilliant Violet 421™ (Biolegend), anti-CD11c PE (Biolegend), anti- CD123 PE/Cy7 (Biolegend), anti-TET1 , anti-TET2 , anti-TET3 , and Brilliant Violet 510™ Donkey anti-rabbit IgG (Biolegend). PBMCs were collected as mentioned above and incubated with surface marker antibodies at 4°C for 30 min. According to the manufacturer's instructions of Foxp3/Transcription Factor Staining Buffer Set (Thermofisher Scientific), cells were fixed at RT for 30 min and washed twice with 1X permeabilization buffer. The cells were incubated with intracellular primary antibodies  at RT for 30 min. After washing with 1X permeabilization buffer, the cells were incubated with Brilliant Violet 510™ Donkey anti-rabbit IgG antibody (1:300) at RT for 30 min. Cells were washed and examined by flow cytometry. Gating strategies are shown in in vitro, the following anti-human antibodies were used: anti-CD1a PE-Cyanine7 (eBioscience), anti-TET1, anti-TET2, anti-TET3, and Brilliant Violet 510™ Donkey anti-rabbit IgG. Staining process was followed, as mentioned previously. TET enzymes expression were analyzed in CD1a+ cells.To determine TET enzymes expression after allergen challenge To evaluate the phenotypic activation of moDCs, moDCs on day 7 were harvested and stained with the following anti-human antibodies: anti-CD1a PE-Cyanine7, anti-CD83 FITC, anti-CD80 (B7-1) PE-Cy5, anti-CD86 (B7-2) PE, and anti-HLA-DR-eFluro®450 (All from eBioscience). Cells were collected and incubated with surface marker antibodies at 4°C for 30 min.+ T cells at a ratio of 1:10 (1 × 104: 1 × 105) in 96-well round bottom plates at 37°C in 5% CO2 on day 7 for another 72 h. Anti-CD4 PerCP-Cy5.5, anti-CD45RA eFluor450, and anti-FoxP3 PE antibodies were used. Staining process was referred to the instruction manual of Foxp3/Transcription Factor Staining Buffer Set (Thermofisher Scientific). Samples were analyzed using a flow cytometer  and the CytExpert Software (Beckman Coulter).To explore the effect of TET1-knockdown moDCs on the differentiation of regulatory T cells (Tregs), transfected-moDCs were co-cultured with autologous CD4Western blot analysis was performed to analyze the protein levels of TET1, TET2, and TET3 Cells in PBMCs and moDCs, and verify the transfection efficiency of TET1-shRNA. TET1 , TET2 , TET3 , and HRP-conjugated GAPDH monoclonal antibody  were used in this experiment. The detailed description is presented in the t-tests were used to analyze the two groups of paired data. Friedman test and Dunn's multiple comparisons tests were used to analyze more than two groups of paired data. A p < 0.05 was considered to indicate a significant difference.Statistical analyses were carried out with GraphPad Prism 6 software . Data are presented as means ± SEM in the histogram. To compare TET enzymes expression between healthy volunteers and AR patients, the Mann-Whitney tests were conducted. Paired p = 0.04 and p = 0.0004, respectively) in the PBMCs of AR patients than that of healthy volunteers; TET2, although appearing higher in AR patients, was not significantly different between two groups (p = 0.07) (p = 0.03 and p = 0.01), while TET2 and TET3 were higher in pDCs (p = 0.04 and p = 0.001) of AR patients than healthy controls. Levels of TET2 and TET3 in mDCs of AR patients also appeared to be higher than that of healthy volunteers, but were not significantly so (p = 0.1 and p = 0.1) (p = 0.03 and p = 0.01) compared with those of healthy volunteers  . As DCs p = 0.1) . The mRNlunteers . These rp = 0.0003 and p = 0.01)  . These rp = 0.02, p = 0.2 and p = 0.4, To explore whether TET family enzymes are involved in the activation of atopic moDCs, we isolated monocytes from PBMCs of HDM-sensitive AR patients and induced them into moDCs. After challenged by allergen for 48 h, TET1 expression in atopic moDCs was significantly decreased by Der p 1 , but no such trends in HC (p < 0.05). After another 3 days of AMLR, CFSE-labeled CD4+ T cells were collected for analysis. The proliferation of CD4+ T cells show no significant changes by TET1 inhibition, neither in HC nor AR groups , CD45RA−FoxP3hi activated Treg cells (aTregs), and cytokine-secreting CD45RA−FoxP3lo non-suppressive T cells and proved that aTregs were immune suppressive in vitro  into three functionally distinct subsets, including CD45RAin vitro . We co-c subsets . Surprisshown in . Meanwhi3, SO2, and NO, have been widely proved to be associated with allergic disease exacerbation and development   but alsoSuperficially, our observation of higher baseline TET1 expression in AR group while also showing TET1 inhibition that induces DC activation in AR seem to be contradictory. However, the observation was based on a cross-sectional study. One should not assume that the higher TET1 expression in AR would be reflected similarly in the context of acute allergen challenge. Even though the levels of TET1, TET3, and 5hmC are higher in the PBMCs of AR patients, the difference between 5mC and 5hmC in the PBMCs of AR shown in  favors 5in vitro experiments. In the future, we will establish in vivo studies to investigate the environmental factors and allergen challenge effects on TET expression and its epigenetic regulation in the immune response.This study has several limitations. Firstly, since the restriction of obtaining the nasal mucous tissues from human volunteers, and the infiltration level of DCs in nasal mucosa is low, it is impractical to quantify the expression of TET in local infiltrated DCs in AR patients. Therefore, we sampled the PBMCs to explore TET expression in DCs by flow cytometry. Secondly, the roles of TET in other cell subtypes need to be further determined. Moreover, this study was based on clinical cross-sectional observation and In this study, TET1 and DNA hydroxymethylation were shown to come into play in the immune activation of AR. Revealing the role of TET enzymes and their specific target genes in particular cells or tissues will help researchers to clarify the molecular mechanism in the development of diseases. Furthermore, knowing the target sequences of TET enzymes, researchers can use epigenome editing tools, such as CRISPR/dCas9 system, to selectively regulate gene expression and biological activities .This study suggests that DCs from AR patients express higher TET1 and are susceptible to be activated by TET1 decrease, which can be triggered by allergen challenge. TET-mediated DNA demethylation is involved in the pathogenesis of AR, highlighting the epigenetic regulation in the development of allergic airway inflammation. These findings encourage us to explore the potential environmental elements that may mediate the epigenetic regulation in AR and targeting DNA demethylation regulation could help find more effective methods for AR prevention and treatment.This study was carried out in accordance with the recommendations of, and the protocol was approved by, the Ethics Committee of The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China . All subjects gave written informed consent in accordance with the Declaration of Helsinki.HL, WS, and WW designed the study. RM, DC, and YWen collected and processed the clinical specimens. HL, TL, and YWei conducted the experiments. HL and HZ prepared the figures and tables. HL and WS drafted the manuscript. WW and CC revised the manuscript. All authors read and approved the final manuscript.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.


C2-symmetrical “salen” ligand (+)-9 bearing two [5]ferrocenophane substituents has been prepared in five steps starting from readily available diacetylferrocene, p-hydroxybenzaldehyde and -N,N’-diphenylethylenediamine. Reaction of (+)-9 with Mn(OAc)3, Co(OAc)2 ZnEt2 or UO2(OAc)2 gave the corresponding metal-complexes which were characterised by spectroscopic methods. The  N,N’-bisethylendiamine  compounds are very popular ligands because of their easy formation and rich coordination chemistry with a large variety of metal ions, that has allowed their use as catalysts in different asymmetric reactions ferrocenophane 4 as a yellow solid, practically insoluble in all the common organic solvents, even after its O-deprotection. Deoxygenation of 4 by treatment with excess of borane in CH2Cl2 and removal of the benzyl group with hydrogen over Pd/C gave the alkylderivative 6 in 85% overall yield, this compound being soluble and more suitable for the following step of formylation on the aryl ring. The same reaction sequence was applied to prepare the isomeric [5]ferrocenophane 7 starting from 1 and 3 -9 by condensation of 8 with (+)--diphenylethylendiamine [-dpen] -dpen] .9 was confirmed by its 1H-NMR spectrum and diagnostic resonances at δ 8.41 and 13.16 for the iminic and phenolic protons, respectively, together with a singlet at δ 4.76 for the protons on the diimine bridge were observed. The methylenic protons on the ferrocenophane bridge gave rise to four separate multiplets in the range 1.8-2.4 ppm whereas the methinic signal was overlapped by the cycplopentadienyl resonances. Complete assignment of the carbon resonances by 2D-NMR spectra and molecular peaks at m/z 924.2 in the ESI-MS spectrum also supported the structure of ligand (+)-9.The symmetrical structure of (+)-9 with Mn(OAc)3 or Co(OAc)2 in EtOH gave the corresponding complexes (−)-10 and (+)-11 in high yield; due to their paramagnetic nature they were characterised by their ESI-MS and circular dichroism spectra (vide infra). The treatment of (+)-12, prepared by reacting the free ligand (+)-9 with Et2Zn in toluene, was mainly established by NMR analysis. The 1H-NMR spectrum of (–)-12 in CDCl3 was fully unresolved whereas in (CD3)2SO all the resonances could be assigned and the symmetric nature of the complex was evidenced; the main features of the spectrum are the absence of the phenolic signal and a slight upfield shift for both the resonances of azomethine and N-methinic protons compared with those of the free ligand. Complexes 10-12 -es 10-12  are quitSalen-uranyl(VI) complexes have been prepared as models for the pseudo-octahedral salen Mn=O species responsible for the selective oxygen transfer in the asymmetric epoxidation of olefins  and rece9 with (UO2)(OAc)2 gave the uranyl complex (+)-13 as a brown solid, whose structure was fully assigned from its 1H- and 13C-NMR spectra registered in (CD3)2SO. The lack of phenolic resonance in the protonic spectrum of 13 and the marked deshielding of the azomethinic and aminic protons  were taken as an evidence of the nitrogen and oxygen coordination with the metal, which acts as an electron withdrawing group. In the 13C-NMR spectrum, the diagnostic resonances for the C-OH and C=N carbons were also shifted downfield upon complexation showing large Δδ values .The reaction of (+)-1H-NMR spectrum of 13 registered in CDCl3 showed broad resonances for all the protons, but two broad singlets in 1:1 ratio at 298 K were distinguishable for the azomethinic group and assigned to two diastereomeric conformers which slowly interconverted on the NMR time scale. By warming, the above resonances tended to collapse and at 338K a 80:20 ratio of the two conformers was measured. In the case of uranyl complex of 14, with the same aminic moiety of (+)-13 and taken as a reference, two conformers were also observed in CDCl3, but not in (CD3)2SO, with a coalescence temperature of 300K. The slower interconversion rate of (+)-13 compared with the (UO2)-14 complex could be ascribed to the presence of bulky ferrocenophane substituents.The 1H-NMR but no evidence of such coordination was obtained for (+)-13. The additional coordination of a molecule of ethanol to the uranium atom has been reported for some salen-uranyl complexes on the basis of elemental analyses data  and it cCircular dichroism (CD) spectroscopy has been applied as a useful tool in the assignment of absolute configuration as well as conformational analysis of chiral molecules and biological polymers ,37,38. T9 and the corresponding metal complexes were registered in CH3CN, except for (–)-12 that was unsoluble in this solvent, and no substantial differences were observed in CH3Cl. The electronic and CD spectra of the free ligand (+)-9 the azomethine π-π* transition band was observed at 338 nm and the benzene π-π* transitions gave rise to overlapping bands below 250 nm. Although two bands assigned to metal-ligand and intraligand charge transfer transitions could be expected for the ferrocene chromophore in the range 300-600 nm ; Anal. Calcd. For C21H22FeO: C, 72.85; H, 6.40. Found: C, 73.58; H, 6.47.To a solution of 6, the isomeric derivative 7 was obtained in 78% overall yield (referred to 1). 1H-NMR (CDCl3) δ: 2.00 , 2.28 , 2.54 , 4.08 , 4.10 , 4.22 , 4.26 , 4.61 , 4.88 , 6.78 , 6.95 , 7.10 , 7.31 ; 13C-NMR (CDCl3) δ: 24.08 (CH2), 31.71 (CH2), 32.00 (CH), 66.50 (CpH), 67.74 (CpH), 68.19 (CpH), 69.50 (CpH), 90.15 (Cp), 115.22 (Ar-H), 121.03 (Ar-H), 126.60 (Ar-H), 128.49 (Ar-H), 134.30 (Ar), 152.98 (Ar-OH); mp 134-135 °C, ESI-MS: m/z 346.6 [M+]; Anal. Calcd. For C21H22FeO: C, 72.85; H, 6.40. Found: C, 73.34; H, 6.46.Starting from salicylaldehyde and applying the same reaction sequence described for compound 6  was dissolved in CHCl3 (20 mL) and 13M KOH (2 mL) was added. The mixture was then refluxed for 24 h under vigorous stirring and the reaction monitored by TLC analysis (n-hexane:EtOAc 85:15). The reaction was then quenched at about 25% conversion of substrate, when 8 was detected as the sole product, by addition of H2O and CH3COOH to acidic pH. After extraction the organic layer was dried over MgSO4 and taken to dryness. Column chromatography on Si gel (n-hexane:EtOAc 85:15) gave pure 8  and the unreacted 6 , which was then recycled. mp 139-140 °C; 1H-NMR (CDCl3) δ: 1.96 , 2.06 , 2.22 , 2.48 , 4.04 , 4.09 , 4.21 , 6.99 , 7.49 , 7.53 , 9.95 , 10.90 ; 13C-NMR (CDCl3) δ: 23.82 (CH2), 32.45 (CH2), 39.36 (CH), 66.21 (CpH), 67.11 (CpH), 68.11 (CpH), 69.06 (CpH), 89.99 (Cp), 117.55 (Ar-H), 120.50 (Ar), 131.97 (Ar-H), 136.31 (Ar-H), 139.73 (Ar), 160.25 (Ar-OH) 196.61 (CHO); ESI-MS: m/z 374.3 [M+]; Anal. Calcd. For C22H22FeO2: C, 70.60; H, 5.93. Found: C, 70.98; H, 6.01.Compound 8  in absolute ethanol (100 mL) -1,2-diphenylethylenediamine  was added and the reaction mixture maintained under stirring at room temperature for 2 days. When the TLC analysis indicated the disappearance of the aldehyde, the solvent was removed in vacuo and the residue applied to a silica gel column (n-hexane:EtOAc 9:1) to give pure (+)-9  as a pale orange solid, mp 139-140 °C; [α]D = +20.2 ; 1H-NMR (CDCl3) δ: 1.85 , 1.96 , 2.16 , 2.41 , 3.98 , 4.00 , 4.07 , 4.20 , 4.76 , 6.95 , 7.11 , 7.24 , 8.41 , 13.15 ; 13C-NMR (CDCl3) δ: 23.88 (CH2), 32.50 (CH2), 32.56 (CH2), 39.35 (CH), 66.20 (CpH), 67.16 (CpH), 68.00 (CpH), 69.20 (CpH), 69.25 (CpH), 80.39 (CH-N), 89.20 (Cp), 116.77 (Ar-H), 118.40 (Ar), 127.58 (Ar-H), 127.94 (Ar-H), 128.36 (Ar-H), 130.64 (Ar-H), 132.00 (Ar-H), 138.45 (Ar), 139.48 (Ar), 159.08 (Ar-OH), 166.46 (C=N); ESI-MS: m/z 924.2 [M+], 462.4 [M++]; Anal. Calcd. For C58H56Fe2N2O2: C, 75.33; H, 6.10; N, 3.03. Found: C, 75.91; H, 6.16; N, 3.08.To a solution of aldehyde 9  in absolute EtOH (10 mL), the appropriate M(OAc)n·xH2O , Co(II) or [UO2]2+) was added and the mixture left to react at rt under stirring until TLC analysis showed the complete conversion of substrate. The complex was separated from the solution by filtration and air-dried (80–90% yield)To a solution of (+)-Mn(III)-complex (–)-10: [α]D = –642.1 ; ESI-MS: m/z 1023.3 [M + EtOH]+; CD: λ (Δε) 625 (+1.08), 370 (+2.08), 306 (–3.82), 268 (+0.38), 243 (–7.16); Anal. Calcd. for C60H57Fe2MnN2O4: C, 69.51; H, 5.54; N, 2.70. Found: C, 69.48; H, 5.50; N, 2.67.Co(II)-complex (+)-11: [α]D = +610.2 ; ESI-MS: m/z 981.2 [M] +, 490.8 [M] ++; CD: λ (Δε) 562 (+4.90), 470 (+1.83), 416 (–0.77), 372 (+0.42), 300 (–3.78), 257 (+2.07), 232 ( –8.31); Anal. Calcd. For C58H54CoFe2N2O2 : C, 70.69; H, 5.54; N, 2.85. Found: C, 70.66; H, 5.49; N, 2.82.2 complexUO (+)-13: [α]D = +142.7 ; 1H-NMR (6d-DMSO) δ: 1.91 , 2.03 , 2.12 , 2.42 , 4.07 , 4.10 , 4.19 , 6.22 , 6.97 , 7.20 , 7.49 , 7.62 , 7.66 , 9.44 ; 13C-NMR (6d-DMSO) δ: 23.94 (CH2), 32.98 (CH2), 38.81 (CH), 66.35 (CpH), 67.34 (CpH), 68.17 (CpH), 69.22 (CpH), 80.61 (CH-N), 89.00 (Cp), 120.70 (Ar-H), 123.22 (Ar), 127.60 (Ar-H), 127.79 (Ar-H), 128.68 (Ar-H), 133.84 (Ar-H), 135.08 (Ar-H), 136.06 (Ar), 142.2 (Ar), 168.21 (Ar-O), 171.99 (C=N); ESI-MS: m/z 1192.3 [M]+; CD: λ (Δε) 575 (+0.68), 398 (+1.16), 331 (–2.52), 287 (+8.60), 250 (+12.45), 228 (–11.48); Anal. Calcd. For C58H54Fe2N2O4U: C, 58.40; H, 4.56; N, 2.35. Found: C, 58.38; H, 4.49; N, 2.31.2Zn in toluene  was added to a solution of (+)-9  in toluene (5 mL) and the mixture stirred at rt overnight. The solvent was removed under vacuum, the residue suspended in CHCl3 and the solid removed by filtration. The final solution was taken to dryness to give pure complex in quantitative yield: [α]D = – 21.3 ; 1H-NMR (6d-DMSO) δ: 1.77 , 1.92 , 2.05 , 2.37 , 3.86 , 4.00 , 4.03 , 4.06 , 4.14 , 5.05 , 6.64 , 7.00 , 7.17 , 7.26 , 7.36 , 7.44 , 8.36 ; 13C-NMR (6d-DMSO) δ: 23.87 (CH2), 32.56 (CH2), 39.23 (CH), 66.20 (CpH), 67.22 (CpH), 68.02 (CpH), 69.33 (CpH), 73.13 (CH-N), 89.40 (Cp), 118.9 (Ar), 123.09 (Ar-H), 127.85 (Ar-H), 127.92 (Ar-H), 128.87 (Ar-H), 131.4 (Ar), 133.44 (Ar-H), 133.56 (Ar-H), 142.37 (Ar), 170.32 (Ar-O), 170.65 (C=N); ESI-MS: m/z 987.3 [M]+, CD: λ (Δε) 416 (+0.63), 389 (–2.74), 353 (3.58), 289 (–3.25); Anal. Calcd. For C58H54Fe2N2O2Zn : C, 70.50; H, 5.51; N, 2.83. Found: C, 70.46; H, 5.48; N, 2.80.A 2M solution of Et9 bearing two [5]ferrocenophane para-substituents on the salicylideneamine rings has been synthesized starting from readily available diacetylferrocene, p-hydroxybenzaldehyde and -dpen and its complexes with four different metals have been also prepared and characterised. The evaluation of the use of these complexes as ligands in asymmetric synthesis is currently under investigation.A novel salen ligand (+)-