This observation establishes low leaf K+ as a new phenotype for loss-of-function alleles of CPR5. We investigate the factors affecting this low leaf K+ in cpr5 using double mutants defective in salicylic acid (SA) and jasmonic acid (JA) signalling, and by gene expression analysis of various channels and transporters. Reciprocal grafting between cpr5 and Col-0 was used to determine the relative importance of the shoot and root in causing the low leaf K+ phenotype of cpr5. Our data show that loss-of-function of CPR5 in shoots primarily determines the low leaf K+ phenotype of cpr5, though the roots also contribute to a lesser degree. The low leaf K+ phenotype of cpr5 is independent of the elevated SA and JA known to occur in cpr5. In cpr5 expression of genes encoding various Cyclic Nucleotide Gated Channels (CNGCs) are uniquely elevated in leaves. Further, expression of HAK5, encoding the high affinity K+ uptake transporter, is reduced in roots of cpr5 grown with high or low K+ supply. We suggest a model in which low leaf K+ in cpr5 is driven primarily by enhanced shoot-to-root K+ export caused by a constitutive activation of the expression of various CNGCs. This activation may enhance K+ efflux, either indirectly via enhanced cytosolic Ca2+ and/or directly by increased K+ transport activity. Enhanced shoot-to-root K+ export may also cause the reduced expression of HAK5 observed in roots of cpr5, leading to a reduction in uptake of K+. All ionomic data presented is publically available at www.ionomicshub.org.Here, we demonstrate that the reduction in leaf K It participates in numerous physiological processes including regulation of enzyme activity, cell expansion, stomata movement and defence towards pathogens + homeostasis, and many K+ channels and transporters have been identified. In plants K+ is highly mobile. Its movement into the xylem is driven by transpiration, and in the phloem by the specific requirements of tissues and organs +. For example, under drought stress the concentration of K+ in the xylem decreases, possibly because of the closure of stomatal pores induced by abscisic acid (ABA) Potassium  plays a central role in the response of plants to K+ shortage + is beneficial or not in conferring resistance towards pathogens is controversial + is clearer. Plant-pathogen interactions cause an increase in cytosolic Ca2+ triggering anion channel activation, plasma membrane depolarization, activation of +K permeable efflux channels leading to enhanced K+ efflux and the initiation of the hypersensitive response (HR) The effects of low KA. thaliana mutant with a 10–30% reduction in leaf K+ which was previous identified in an ionomic screen of fast neutron mutagenized plants CPR5, a gene originally identified in two independent screens for altered response to pathogens cpr5 has a high content of salicylic acid (SA) and shows constitutive expression of pathogenesis related genes (PR), as well as plant defensin PDF1.2 which is a marker of the JA-dependent pathway. Therefore, it has been suggested the CPR5 is a negative regulator of local defence response to pathogens CPR5 is also implicated in cell senescence CPR5 is also associated with K+ homeostasis possibly via modulation of expression of various CNGCs and HAK5.In this study, we describe the characterization of an A. thaliana seeds were purchased from Lehle Seeds  and plants screened for their leaf elemental profile by ICP-MS cpr5-2 (CS3770) were provided by the Arabidopsis Biological Resource Center (The Ohio State University).Fast neutron-mutagenized M2 −2 sec−1) and bottom watered at regular intervals with a solution containing 0.25× Hoagland's macro and micronutrients For non axenic conditions, plants were grown in pots containing moist soil  in a climate-controlled room  and solidified with 10 g L−1 pure agarose , which contains negligible amounts of K+ . K+ was added as KCl at the final concentration of 10, 50 and 100 µM and the pH adjusted to 5.8 with Ca(OH)2. K+ content in root and shoot was measured in plants grown for 2 weeks on solidified medium containing 1/20th MS salts accordingly to Cheong et al.,For plants grown in axenic conditions, surface sterilized seeds were stratified at 4°C in the dark for five days before sowing. To measure the expression of cpr5-3 was outcrossed to A. thaliana Landsberg erecta (Ler-0) accession, F1 seeds were planted on 0.5× MS medium solidified with 10 g L−1 of agar and seedlings visually screened for hyponastic and early yellowing cotyledons. All seedlings of F1 generation looked wild-type and were transferred to soil and grown to produce F2 seeds. F2 plants grown on soil were visually selected for small size and yellow early senescing leaves and used to map the mutation with a positional cloning approach with single strand length polymorphism (SSLP) markers.http://www.qiagen.com) from five weeks old plants grown on soil or from two week old plants grown on plates. DNase digestion on column was performed to eliminate possible contamination with DNA. Two micrograms of total RNA were used as a template to synthesize first-strand cDNA with SuperScript VILO cDNA Synthesis Kit . Quantitative real-time PCR was performed with the SYBR Green reagent mix in a StepOnePlus instrument according to the manufacturer's instructions . The expression of K+ channel/transporter genes was detected with the following set of primers: HAK5, 147 (5′-TGCTGATCTAGGTCACTTCAGTGTT-3′) and 148 (5′-AAAGCAGGATATGCGACACATG-3′); AKT1, 170 (5′-TCTAAATTGTGTTCTTCTTCTGTTGGA-3′) and 171 (5′-CCTTCCGCGTCTCTGCAA-3′); CNGC19, 137 (5′-TTCTCACTTGGTGCCTCTCTTCT-3′) and 138 (5′-AATCCCTTTGGTGGCATCTTT-3′); CNGC10, 139 (5′-TCATCATTGATCTACTCTCTATCCTTCCT-3′) and 140 (5′-TGGTTGACGCTTGGAATAACG-3′); CNGC12, 156 (5′-CAACGAACATTCAGGTTATACTCACA-3′) and 157 (5′-GCCGCTTGAATGAAGAATGC-3′); CNGC20, 160 (5′-TGCCTCGAACGCTCTTCTG-3′) and 161 (5′-CCTTTGATGGCATCCTTATCCT-3′); CNGC11, 196 (5′-GATAAAAACATGAATCTTCAGAGGAGAA-3′) and 197 (5′-CTAACACTTTTCAATTTTCCATCAACTC-3′). As an internal reference the expression of PP2A (At1g13320) was used since it showed stable expression throughout the experimental series of development, shoot and root abiotic stress, hormones, nutrient stress, light and biotic stress CPR5. In addition to PP2A the expression of HAK5 and AKT1 was also normalized to UBQ10 (At4g05320). UBQ10 also shows stable expression in cpr5. The average value from real-time PCR measurements from at least three independent biological replicates was used to evaluate transcript abundance. Biological replicates were composed of tissues harvested from between 5 and 10 plants for analysis of shoot tissue, and between 25 and 30 plants for analysis of root tissue, from plants grown on plates. For plants grown in soil 2–3 leaves from at least 3 individual plants were used. Steady state mRNA levels were calculated relative to a reference gene and presented based on the 2Ct−Δ method Total RNA was extracted with the Qiagen RNeasyPlant Mini Kit , rinsed with 18 MΩ water, placed into Pyrex digestion tubes and dried at 92°C for 20 hours. Alternatively, shoots and roots were harvested from plants grown on 1/20Total SA content was quantified in leaves of five weeks old plants using a Waters Alliance HPLC system equipped with Millenium software, 2695 Separation Module, 2475 Fluorescence Detector, and 2996 Photodiode array detector. A Nova-Pack C-18 column was used with a flow rate and methanol gradient as described previously cpr5-2 and Col-0 seeds were germinated in the dark on 0.5× MS plates containing 1 mL L−1 of MS Vitamins , 3 mg L−1 Benomyl (methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate; Sigma), 0.04 mg L−1 BA , 0.02 mg L−1 IAA , and 12 g L−1 agar. Plates were held vertically and after seven days seedlings were grafted as previously described ANOVA was conducted using the software CoStat 6.2 . Separation of means was performed using LSD test at P = 0.05 significance level.A. thaliana Col-0 plants were previously screened for altered leaf elemental composition 12645 was identified as a low K+ mutant with a reduction in leaf K+ of approximately 20% compared to wild-type Col-0   . In addi12645 was out-crossed to Ler-0, and F1 and F2 populations scored for mutant like plants. All F1 plants from this cross looked wild-type, while in the F2 small plants with early senescent leaves segregated with a ratio of 3:1 (wild-type to mutant phenotype), indicating that 12645 is inherited as a single recessive nuclear mutation. Small hypersenescent plants from the F2 population also showed reduced K+ content (38.3 µg g−1 dry weight) compared to those that looked wild-type (42.8 µg g−1 dry weight), indicating that the traits of early senescence and small size co-segregate with reduced leaf K+ (P<0.001).To map and define the Mendelian character of the mutation, 12645 mutation was determined using a positional cloning approach with SSLP markers in 171 F2 plants from the outcross with Ler-0. The recombinant population scored for markers in the region between nucleotides 25,500,000 and 26,100,000 on chromosome 5 revealed that only one plant had Ler-0 alleles on the BAC clone MUB3 and two plants on F15O5, which placed the mutation on the BAC clone MXK3  were grown together, along with the wild-type Col-0 and the concentration of Li, B, Na, Mg, P, S, K, Ca, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Mo, Cd in leaves of each genotype determined by ICP-MS (n = 10–22 replicate plants per genotype). Of all the elements measured only K+ was found to be significantly different between the wild-type Col-0 and all the cpr5 alleles (P<0.005 after Bonferroni correction for multiple testing). Wild-type Col-0 had a leaf K+ concentration of 40,067±6222 µg g−1 dry weight, compared to 27,490±3575 µg g−1 dry weight for cpr5-1, 29,751±2616 µg g−1 dry weight for cpr5-2, and 27,324±2502 µg g−1 dry for cpr5-3 .The chromosomal position of the one MXK3 . DNA seq930 gene . This inn cpr5-3 . All cprCPR5 acts as a negative regulator of local resistance to pathogens by partially repressing the accumulation of SA, so that cpr5 mutants have elevated SA content cpr5-3 .t cpr5-3 . This ra+ of cpr5 is dependent on the elevated JA in this mutant jasmonate resistant 1 (jar1) mutant is unable to conjugate JA with isoleucine to form the active jasmonoyl-L-isoleucine form of JA that is required to elicit the JA response cpr5jar1, prepared from cpr5-1 and jar1-1cpr5-1, cpr5-2 and cpr5-3 all share a similar reduction of leaf K+ which is not observed in the single jar1-1 mutant . From this we conclude that the low leaf K+ of cpr5 is not dependent on JA signalling. Taken together these experiments support the conclusion that the reduced leaf K+ of cpr5 is independent of both SA and JA.We also followed a similar approach to determine if the low leaf Kcpr5 is easily distinguishable from wild-type plants from the presence of necrotic and chlorotic spots on the leaves, a trait which characterizes plants infected by bacteria and/or fungi, as well as mutants with a constitutively active response to pathogens. Hypothesizing that low leaf K+ was associated with the response to pathogens, we measured K+ content in leaves of lesion mimic mutants (LMM) selected from the two major classes of initiation  and propagation  of lesion mutants +  that may be directly or indirectly involved in K+ efflux during the response to pathogens in A. thaliana. An initial survey of transcriptional data publically available in experiment 175 on Genevestigator CNGCs was up-regulated in cpr5 leaves. We confirmed the differences initially observed in the database using qRT-PCR, and performed similar measurements in root tissue. Our analysis revealed that steady state levels of CNGC10, CNGC11, CNGC12, CNGC19 and CNGC20 mRNA are all elevated in leaves of both cpr5-2 and cpr5-3 compared to wild-type Col-0  + channel AKT1+ uptake at both low and intermediate K+ supply (0.01–5 mM) + the transport processes involved in K+ uptake are currently undefined HAK5 and AKT1 in K+ uptake in A. thaliana we used qRT-PCR to quantify the steady state levels of HAK5 and AKT1 mRNA in cpr5 roots to test if altered expression of these genes may be involved in the low leaf K+ phenotype we observe in cpr5. Steady state levels of AKT1 mRNA in cpr5 where found to be the same as wild-type Col-0 after growth on medium supplemented with either high (20 mM) or low (100 µM) K+  K+ . A sligh+ supply . Further in cpr5 . Interesild-type . However similar .4+ given that NH4+ is known to suppress activity HAK5. Pure agarose was used to solidify the growth medium to avoid any extra K+ supply HAK5 mRNA were observed to increase as external K+ was reduced , with the root playing a significant but smaller role (19%). Interestingly, the presence of the cpr5 allele in both roots and shoots is required to produce the full low leaf K+ phenotype, suggesting that feedback between both organs is needed. In investigating the factors that in cpr5 cause the reduction of leaf K+, we surveyed the experiment AT-175 performed on cpr5 shoots (www.genevestigator.ethz.ch) and this revealed that genes belonging to the CNGC family are highly expressed in cpr5. Using qRT-PCR we confirmed that CNGC10, CNGC11, CNGC12, CNGC19, CNGC20 are indeed highly expressed in leaves, but not roots, of cpr5. The expression of CNGC1 and CNGC13 which showed a milder increase in the AT-175 array did not show any difference when expression was analyzed by qRT-PCR. CNCG2 and CNGC4, null mutants of which (dnd1 and dnd2) have enhanced disease resistance, also did not show any misregulation when analyzed by qRT-PCR. In the interaction between plant and pathogens the recognition of factors of avirulence by the host plant induces fluxes of Ca2+ that initiate the immune response and leads to enhanced K+ efflux CNGCs are believed to be the channels that deliver the Ca2+ signal required for pathogen recognition dnd1, dnd2 and the gain of function chimeric CNGC11/12 mutant cpr22 strongly support this conclusion CNGCs provides direct evidence of Ca2+ transport activity cpr5 the constitutively high level of expression of CNGCs we observe leads to a persistent activation of this Ca2+ initiated signalling cascade that in turn leads to constitutively enhanced K+ efflux + efflux could increase shoot-to-root K+ export, and may explain the leaf-driven portion of the reduced leaf K+ observed in cpr5. The transporters/channels involved in this K+ efflux are currently unknown. However, these is evidence that CNGC10 transports K+CNGC10 mRNA are elevated in cpr5, making it possible that CNGC10 could play a role in the proposed enhanced K+ efflux and shoot-to-root export in cpr5.The data presented here establishes that the wild-type allele of HAK5 encodes the primary high affinity root K+ transporter in A. thaliana+ supply cpr5 roots show a consistent reduction in the steady state levels of HAK5 mRNA under both high and low K+ supply. Though HAK5 primarily plays a role in K+ uptake at low K+ supply, it is possible that the reduced steady state levels of HAK5 mRNA in cpr5 roots we observe even at high K+ supply is responsible for the small (18%) but significant contribution of roots to the reduced leaf K+ phenotype of cpr5. It is also interesting to speculate that expression of HAK5 in cpr5 roots is suppressed by an increased flux of K+ arriving in the roots from the shoots driven by elevated expression of CNGCs in shoots.CPR5 encode a membrane protein with five transmembrane domains and a nuclear localization signal (NLS). CPR5 appears to localize to the nucleus CPR5 from the membrane to allow its participation in transcriptional processes TPR1 and EDS1 that direct repression of expression of CNGC2 and CNGC4CPR5 may play a similar role in the nucleus to negatively regulate the expression of the various CNGCs affected on the cpr5 mutant.Based on sequence analysis + phenotype for cpr5 that is primarily driven by the shoot, and is independent of the elevated levels of SA and JA found in this mutant. We suggest that the reduced leaf K+ of cpr5 is caused by the elevated expression of various CNGCs in shoots and the reduced expression of HAK5 in roots, driving both an enhanced K+ export from shoots and a reduced K+ uptake in roots. Our observation of altered K+ homeostasis in cpr5 may also be an important piece of evidence linking the function of CPR5 as a negative regulator of local defence responses to pathogens with the role K+ efflux plays in these responses; likely through the direct or indirect regulation of CNGCs by CPR5.In summary, we have identified a new low leaf KFigure S1CPR5 expression and salicylic acid (SA) levels in A. thaliana leaves and roots of wild-type Col-0 and cpr5.A. Steady state levels of CPR5 mRNA in wild-type Col-0 and cpr5-3 quantified using qRT-PCR. RNA was extracted from leaves of five-week old plants grown in soil. Data represents the mean of measurements of four independent biological replicates. Each biological replicate consisted of 2-3 leaves from individual plants. Errors bars represent standard deviation. B. Steady state levels of CPR5 mRNA in root and shoots of wild-type Col-0 quantified using qRT-PCR. RNA was extracted from shoots and roots of two week old wild-type Col-0 plants grown on 0.5× MS media solidified with 1% agar (w/v). Values represent mean of measurements from at least three independent replicates. Errors bars represent standard deviation. Steady state mRNA levels (A & B) are presented as 2Ct−Δ. UBQ10 (At4g05320) was used as an endogenous reference gene for normalization across samples. C. SA content (mg g−1 of fresh weight) in leaves of wild-type Col-0, cpr5-2 and cpr5-3. Data represents the mean of three independent leaf samples harvested from individual plants grown in soil for five weeks. Error bars represent the standard error.(TIF)Click here for additional data file.


Increasing incidences of cancer combined with prolonged survival have raised the need for developing community based rehabilitation. The objectives of the analysis were to describe and interpret the key issues related to coordination and coherence of community-based cancer rehabilitation in Denmark and to provide insights relevant for other contexts.Twenty-seven rehabilitation managers across 15 municipalities in Denmark comprised the sample. The study was designed with a combination of data collection methods including questionnaires, individual interviews, and focus groups. A Grounded Theory approach was used to analyze the data.A lack of shared cultures among health care providers and systems of delivery was a primary barrier to collaboration which was essential for establishing coordination of care. Formal multidisciplinary steering committees, team-based organization, and informal relationships were fundamental for developing coordination and coherence.Coordination and coherence in community-based rehabilitation relies on increased collaboration, which may best be optimized by use of shared frameworks within and across systems. Results highlight the challenges faced in practical implementation of community rehabilitation and point to possible strategies for its enhancement. Cancer survivors can be defined as persons ‘living with a cancer diagnosis following primary cancer treatment for cancer through the end of life’ . The numConsequently, coordination and quality of follow-up care are needed for rehabilitation after hospital-based interventions. Although community-based cancer rehabilitation is being developed around the world, we have yet to discover what key issues are being encountered in implementation and how they might best be solved. More specifically, we need to identify how community-based rehabilitation can be efficiently organized and how adequate coordination and coherence between health care providers and across sectors can be optimized in practice. This paper begins to address these gaps based on a national study from Denmark utilizing data from the perspective of the health care professionals involved in community-based cancer rehabilitation.In recent years there has been an increasing international interest among health care administrators and providers of cancer care in developing community-based rehabilitation ,5,6. In While prior research has addressed the organization and provision of cancer rehabilitation and survivor care ,10,11, iIn Denmark, municipalities and local communities have a long history of providing services for people who are limited in their ability to manage on their own in their home environment. Community-based rehabilitation of cancer survivors has, however, not been common. Following changes in the Danish Healthcare Law in 2007, we witnessed an increasing focus on community-based rehabilitation for people surviving cancer. This legal act placed the primary responsibility for providing rehabilitation onto the municipalities, including the obligation to provide coordinated and coherent rehabilitation care. With a focus on coordination and coherence, the Danish healthcare system emphasized securing coordination of services among disciplines and across sectors in rehabilitation services as well as coherence in the rehabilitation trajectory.In order to meet these demands, funding was given to 11 projects in 15 municipalities across Denmark to explore how community-based rehabilitation for cancer survivors could be optimally developed with regard to its organization and services. Three areas of rehabilitation were prioritized: (1) coordination and coherence in the transition from hospital to community based services, (2) physical activity, and (3) returning to life, including coping with changed life conditions and the return to work. The first of these areas focused on organizational aspects of the provision of rehabilitation, while the other two pointed to specific intervention areas in relation to the cancer survivors.We focus on the first area in this paper. The objective of the study was to explore and describe the key issues, such as structural as well as inter-professional challenges related to coordination and coherence of community-based cancer rehabilitation in the Danish case. Taking into account that the communities in this study did not have any established professional and collaborative patterns for this task, we chose to focus on the perspectives of rehabilitation managers involved in the organization and delivery of care. A subsequent goal of the analysis was to provide insights that might be relevant not only for cancer rehabilitation in Denmark but in other contexts as well.The study was part of a large multicenter program aiming to develop community-based rehabilitation in Denmark as well as to evaluate that development and the resulting services. In order to explore and understand ways in which such a program can be implemented, we employed a qualitative methodology to gain access to social processes involved in organization and collaboration in rehabilitation. We employed a combination of methods to generate data, and consistent with such a problem, we employed principles of Grounded Theory to analyze the data .Participants comprise 27 Rehabilitation Managers (RMs) who were hired by the 11 projects to manage and develop cancer rehabilitation in 15 diverse municipalities across Denmark. All RMs were included. RMs in this study are similar to what are often called case-managers or patient navigators. RMs coordinates rehabilitation-services and guide cancer survivors through the complex and multiple services needed. RMs were responsible for developing and organizing the delivery of community-based cancer rehabilitation in the municipalities. The RMs were distributed among the following professions: 12 nurses, 5 social workers, 3 medical doctors (MDs), 2 physiotherapists, 1 occupational therapist, 1 anthropologist, and 3 healthcare administrators.Data were gathered using a combination of methods including questionnaires with closed and open-ended questions, semi-structured interviews, and focus-group interviews ,14. The Using insights gleaned from the questionnaire data, two researchers (including the first author) conducted follow-up interviews with the RMs in each of the 15 communities. A generic interview guide based on the questionnaire insights was used. That guide included topics such as specification of procedures for inclusion of cancer survivors, organization and type of rehabilitation interventions. The interviews served to probe more deeply into the processes and contexts of organizational and delivery challenges in the projects. The interviews took place in the communities where the RMs were working. Each interview lasted between 1 – 2 hours. All interviews were recorded.To further expand RMs’ different perspectives and validate data and insights, focus group interviews were employed . All RMsThe qualitative data from questionnaires and the semi-structured interview recordings were transcribed verbatim. Based on careful listening to each focus group recording combined with the written memos from each session the lead evaluator (first author) wrote out extensive summaries of the focus group interviews including quotes of discussion parts central to the study objective. All qualitative data were analyzed using a constant comparative method according to the chosen Grounded Theory approach . Text frThe Study complied with the Helsinki Declaration, the Ethical committee System and was approved by the Danish Data Protection Agency identification number 2008-41-2417. Written consent was obtained from all study participants.The three main conceptual categories that emerged from the analysis were: (1) cultures that govern coordination, (2) insufficient pathways and collaborative keys to rehabilitation, and (3) advantages of team-based rehabilitation (see Overview of results). We present the categories below complemented by pertinent details and examples that help to contextualize the findings. To ensure anonymity all names of places and individuals are pseudonyms.Cancer care traditions by context and people.Relationships and steering committees as facilitators.Barriers for community-based cancer rehabilitation.Strategies for equal inclusion.Two ways of organizing rehabilitation-management.The analysis showed that several cultural dimensions of the rehabilitation process were of importance to intervention coordination. In particular we identified that culture expressed through discourses and traditions practiced in given contexts and by people of specific professions govern coordination. Collaboration was identified to be challenging interpersonally among healthcare providers as well as structurally between sectors within a given Municipality as well as with sectors and collaborating parties outside the individual Municipality. In addition, we found that coordination can be facilitated by informal and formal relationships within the care sector.According to the RMs in our study, collaboration is fundamental to the coordination of multi-modal therapies and specialized treatment. Because the projects were a new initiative, there were no traditions for collaboration about cancer rehabilitation in the municipalities involved. The RMs reported that there were no pre-established pathways of communication that they could follow. Nor did they find any guideline for coordinating the various interventions that cancer survivors could be in need of. In this regard it should be noted that, although cure was expected for the cancer survivors referred to the project of community-based rehabilitation, the citizens who participated in the rehabilitation programs were in varying stages of cancer diagnosis. The majority were suffering from a variety of physical and emotional problems that needed close collaboration and exchange of knowledge between hospital staff, social workers, general practitioners, dieticians, physiotherapists, occupational therapists, etc.As per pathways and traditions of care, within the individual communities as well as in the participating institutions  there was no traditional structure for how rehabilitation should be provided or what the community rehabilitation should entail. For instance, the RMs pointed to a variety of different foci of cancer rehabilitation  among the different disciplines involved. This fragmentation of disciplinary focus proved to be a major obstacle for collaboration as the basis for developing coordinated and coherent rehabilitation services to the individual citizen.A particular type of situation was highlighted by the RMs that different discourses dominate within different delivery subsystems and disciplines. For example a biomedical discourse was identified to dominate in hospitals contexts whereas a psychosocial discourse characterized the contexts of community based services. In addition, different approaches to rehabilitation were identified across departments in a municipality. One of the RMs, Maria, shared that:In the community, where I am based, in the social welfare department we have great results with cancer survivors who have problems with returning to work, whereas health oriented needs are not met to the same extent.This example indicates how the particular context and distribution of labor  within a local cancer rehabilitation system creates unequal outcomes. Local cultures of cancer rehabilitation emerged based on who was there to provide care and from which departments influencing the discourse of care. It could be argued that a hierarchical structure among professions caused poor relations at times, or more simply it could be interpreted that communication is smoother within a profession. In addition to the challenges of dividing discourses, decisions about the content of cancer rehabilitation were complicated further as many of the required services had not been subject to sufficient and stringent research. Such lack of evidence was a continuous challenge for the RMs who reported that the lack of documentation of specific interventions made it difficult for them to build rehabilitation on solid ground.Personal relations among various parties involved in the rehabilitation process were central to good care. Relationships and continuous communication were noted by our participants as important for establishing and maintaining good collaboration, especially across delivery borders (between institutions). In turn, collaboration was suggested by RMs as essential for coordination and coherence in the provision of rehabilitation. Repeated visits by the RM to hospitals and other community administrative sites, and thus visibility to other involved parties were part of this process. In addition, cross-disciplinary steering committees proved to be of utmost importance in overcoming potential problems to coherence. As one of the RMs said:Many of us whom are now employed at the community rehabilitation centre have previously been working at the local hospital from where we recruit cancer clients and survivors. That means we know each other across systems and it makes it easier to just pass by the hospital wards and remind them to refer patients to us. In extension we have held several information meetings and lectures which in turn make the staff from the hospital come to us and benefit from the competencies we offer (Jesper).As seen in this example, community-based rehabilitation projects that recruited staff from hospitals found that this strategy eased coordination of rehabilitation, since the staff already had an established network within the hospital. Informal and personal relationships and networks across delivery systems were identified as the strongest vehicle for collaboration as a basis for coordinating services and creating coherence in patients’ rehabilitation course.Establishing steering committees that included representatives from institutions involved in rehabilitation across sectors in the delivery system was a common organizational strategy across the municipal projects. Besides the obvious advantage of cross-disciplinary assessment of cases, the formal mixed steering committees functioned as ‘door openers’ in regard to the various levels and agents of rehabilitation activities. For instance, leaders both in hospitals and community departments who were on steering committees would introduce rehabilitation projects at department meetings to facilitate cooperation by their staff. Another example was noted by participants who explained that representatives from different disciplines could more easily establish collaboration with other departments if they knew people in those departments from their own profession.Another important dynamic was the power relationship between hospitals and community-based services. RMs suggested that the hospital staff had low expectations of community-based services. In one of the projects, a local project leader conducted a few pilot interviews with hospital staff who distinctly expressed mistrust in community-based services being able to fulfill the task of providing rehabilitation. Therapists in the hospital did not expect that health care staff in the community could deliver an equally good intervention despite their shared professional background. These problems were in part related to the delivery context and the values and beliefs that go with them--the care cultures operating in municipalities; e.g., hospital services are to cure disease, and communities’ efforts are to support functioning in everyday life.Despite relationships, steering committees, and attempts to promote collaboration across professions and delivery systems involved in rehabilitation, communication for coordination purposes seemed to be an ongoing challenge in projects. In four of the municipalities, RMs reported that they used the International Classification system of Functioning (ICF) to providing a platform for mutual ‘language’ and shared understanding. One municipality in particular invested extra effort in disseminating knowledge to health care staff involved at all levels of rehabilitation by providing a scenario of continuing education seminars as part of their project.As alluded to above, the data brought attention to collaboration and insufficient pathways as prime factors for coordination of rehabilitation services as well as for making the course of rehabilitation coherent. Moreover, rehabilitation managers from multiple disciplinary backgrounds emphasized communication including referral pathways of patients from one delivery system to another as barriers for establishing rehabilitation while they pointed to collaboration and visibility strategies to overcome the problems encountered.Inclusion of cancer-survivors to community-based rehabilitation was affected by the way in which referrals from hospital and community-based services were organized. In Denmark patients who transfer from hospital to community-based services are supposed to be assessed for rehabilitation needs at the hospital. The patient’s oncologist can refer the patient to the community-based services, but referral to those services is not consistently provided by all hospitals and not for all cancer diagnosis.All of the RMs reported that despite intensive efforts to include cancer patients in need of rehabilitation, referrals were rarely received. The RMs therefore engaged in comprehensive and time-consuming activity to inform cancer survivors about rehabilitation options. Furthermore, the RMs reported that the majority of clients enrolled in the community-based rehabilitation programs were dominated by resourceful cancer survivors (often women with breast cancer). Cancer survivors who were either socially deprived , males, or people with a non-Danish ethnic background were hardly represented among the client group. These inclusion problems brought RMs’ attention to two central issues: referral procedures and equality of access for all cancer survivors. RMs therefore developed strategies and recommendations to overcome these issues.One of the primary strategies that RMs devised to increase inclusion in community-based rehabilitation was identification of cancer survivors while they were still in hospital. RMs recommended an initial contact at the time of diagnosis with an in-person meeting between a community-based RM and the cancer patient. RMs also suggested the alternative approach of initiating contact during early stages of treatment. The purpose of this type of intervention is to reach the patient after the shock of diagnosis but while increased attention to life at home has happened. Furthermore the RMs proposed local visibility and easy access to community-based services. For example one of the projects had a rehabilitation bus that was parked in different public places of the municipality to make the rehabilitation and survivor service well known among community citizens. In addition, RMs experiences from the projects revealed that socially deprived citizens could more easily access services when these were located in the vicinity of public transportation.As another strategy, one project developed successful collaboration with General Practice (GP) of the area through close collaboration with a member of the local GP network. This led to a raise in referrals from GPs’ to the community-based cancer rehabilitation project. Finally to bridge the gap and transition from hospital to community-based rehabilitation, the RMs proposed to base coordination on clear collaboration agreements between hospital and community-based services. Such experimentation with and refinement of novel approaches seemed to improve collaboration among rehabilitation providers and increase referral of cancer survivors.Once cancer survivors were included to rehabilitation the challenge was to coordinate the array of services needed for the individual as well as creating optimal coherence in the course of rehabilitation. Also in this endeavor collaboration was identified to be the key. Moreover, the RMs all found that realistic rehabilitation plans was depending on close collaboration with the individual cancer survivor. To enhance coherence in the cancer survivors rehabilitation process the RMs experienced that a minimum of three rehabilitation in-person meetings was required. One should be held as an initiating meeting based on motivational principles, to support the cancer survivor’s personal resources and for empowerment purposes. A second in-person meeting was recommended by the RMs to evaluate and if necessary adjust initial goals for the rehabilitation process. Finally the third in-person meeting should be to provide a future oriented evaluation.In addition to the challenges of identifying and including cancer survivors in the community-based rehabilitation programs the municipality projects had to develop ways to organize their services. In particular, team-based organization was identified to be of advantage both for collaborative purposes as well as for the coordination of and coherence in the cancer survivors’ rehabilitation course.Different ways of organizing the case management were represented in the 15 municipalities, and these turned out to have very different implications for the rehabilitation programs over time. Twelve municipalities based the coordination of rehabilitation on non-team approach .In response to Belinda – Peter from another Municipality contributed:In Sellow Municipality we have success with such an adhoc model. But all team-members are present at all meetings.Through the analysis of the data from the different projects and the focus group discussions we identified the team-based model to hold several advantages. This model seemed to provide optimal conditions for developing coordination of services delivered in concert by multiple providers within the community. First, the RMs involved in teams reported that there was a high degree of knowledge exchange among team professionals. This exchange gave all team members in-depth knowledge of the others’ professional competencies. That in turn made it easier for the individual RM to refer the cancer survivor to other interventions by other team members. Second, the team-based model was identified by the RMs to promote competence merging, in so far that team members could cover cases for each other . For example, RM, Margaret from one of the larger Municipalities shared,The team-based approach at our place has led to more fluent boundaries among the team. So for instance the social worker sometimes covers for our psychologist in providing supportive conversations and then if the problem is more persisting she refers to the team psychologist in case needed.Third, the team-based model supported an interdisciplinary approach to rehabilitation, increasing a mutual understanding among the team professions that in turn also seemed to support integration of services to the clients. Finally, the team-based model was found to be less vulnerable to issues such as staff shortages caused by, for example, sick leave or on other occasions.This study focused on community-based rehabilitation for cancer survivors in the Danish context, and it revealed significant health service issues related to the coordination and coherence of care. Coordination and coherence are concerns in a horizontal structure, such as among the services offered within an on-the-ground community care system. They also are concerns in the vertical articulation of systems, such as between a hospital system of services and on-the-ground community services. An array of professional services, both across horizontal and vertical structures, was involved in a community-based cancer rehabilitation project in Denmark. Rehabilitation managers participating in this study indicated that the encounters of various health-care cultures--both among different professions and across different delivery systems, hospital and community-based--was a key issue for the delivery of rehabilitation services. Furthermore, the RMs noted that for various reasons, many cancer survivors were not receiving needed services. In particular they identified unequal inclusion condition for cancer survivors with regard to gender, ethnicity, and socio-demographic background. Similar challenges such as organization of rehabilitation and equal inclusion are reported from research about community cancer rehabilitation around the world and highlight the need for organizational as well as procedural strategies to solve these problems ,19.In response to the issue of inclusion, RMs proposed that cancer survivors should be initially contacted by community-based rehabilitation as early as the time of diagnosis. This suggestion may not be the most appropriate for cancer-survivors who at the time of diagnosis may be in a state of shock and not oriented towards the future of returning to life in the community. Rather, the suggestion may be seen as an indication of the lack of a well-developed referral system, securing equal opportunities for rehabilitation for all cancer survivors identified to have rehabilitation needs. The results also suggest that barriers for developing coordination and coherence were in part due to the lack of prior cooperation across groups as well as insufficient knowledge among professions of discipline related competencies.The identified cultural barriers within systems and among rehabilitation professionals draw attention to underlying factors which may be pertinent to secure the overall quality of coordination and coherence in rehabilitation. From an organizational perspective, the development of increased dialogue between providers can make cultural barriers visible and raise awareness of existing and tacit knowledge supporting possibilities for change. By extension, if we relate the cultural barriers to Leavitt’s system oriented model , our finBased on the results, it appears necessary that rehabilitation staff within and across providers share a mutual language and understanding. The ICF may be a potential means for such purposes and for bridging cultural differences. Generally the ICF has received much attention in Scandinavia as a common conceptual classification system useful for rehabilitation and interdisciplinary collaboration ,21-23. HThe team-based model for rehabilitation case-management offered several advantages for coordination and coherence. This solution may not be an option for all municipalities, however, because of population, geography, and available RMs. Small communities may have to develop other approaches, such as team-based services that are not diagnosis specific. The results also showed that rehabilitation managers can come from a variety of professional backgrounds and should not be limited to a single profession. Required general competencies for community-based RMs included certain cancer specific knowledge integrated with a focus supporting cancer survivors in returning to contexts of everyday living.The complexity of organizations within health care systems, including hospitals and local provider groups, are often so complex that no single analytical tool can capture it adequately. Furthermore, organizations of health care services are pluralistic in form and in constant evolution. Because organizations are continuously subject to political shifts and other changes specific to time and place, approaches to community-based cancer rehabilitation must be based in principles that provide flexibility. In that light, the present study does not intend to suggest ‘final’ solutions to cancer rehabilitation but rather to reveal possibilities that can guide cancer care providers and organizations.The findings of this study extend existing knowledge by using the experiences of various professionals involved in establishing community-based cancer rehabilitation. Coordination and coherence in community-based rehabilitation relies on increased collaboration facilitated by informal relationships and formal networks across disciplines and delivery systems. Communication and common understanding supported by use of shared frameworks within and across systems can enhance rehabilitation services. While there is much yet to be learned about the relatively new move to community-based cancer rehabilitation, the experience from Danish municipalities points to key strategies that may improve coordination and coherence of care in other contexts.RM: Rehabilitation Manager; GP: General practice; ICF: International classification system of functioning.Both authors declare no conflict of interest and the authors alone are responsible for the content of the paper.The study was led and conducted by the first author, KlC. KlC performed the main analysis, which was further developed and critically revised in close cooperation with the second author, MC. Both authors approved the final manuscript.MPC, Ph.D. is Professor and Chair, Department of Health Care Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, USA. KlC, Ph.D. is Associate professor and Head of the Research initiative of Activity Studies and Occupational Therapy at the Research unit Health, Man and Society, Institute of Public Health, University of Southern Denmark, DK.This manuscript has not been submitted elsewhere and the authors declare no conflicts of interest with respect to the authorship and/or publication of this article.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/13/339/prepub


This toxin has atypical pharmacological properties, including incomplete inhibition of 3H-prazosin or 125I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the α1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ρ-Da1a for the α1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ρ-Da1a, prazosin and HEAT compete at the α1A-adrenoceptor. ρ-Da1a did not affect the dissociation kinetics of 3H-prazosin or 125I-HEAT, and the IC50 of ρ-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ρ-Da1a remained stable. The effect of ρ-Da1a on agonist-stimulated Ca2+ release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the α1A-adrenoceptor were evaluated for alterations in ρ-Da1a affinity. The D1063.32A and the S1885.42A/S1925.46A receptor mutations reduced toxin affinity moderately , while the F862.64A, F2886.51A and F3127.39A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F862.64 was identified as a key interaction point for 125I-HEAT, as the variant F862.64A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ρ-Da1a interacts with the human α1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist  and agonist (F2886.51 and F3127.39) ligands. Its selectivity for the α1A-adrenoceptor may result, at least partly, from its interaction with the residue F862.64, which appears to be important also for HEAT binding.ρ-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the α Many toxins that interact with voltage- and ligand-gated ion channels display both high affinity and selectivity. For the last 50 years, these properties have been used to identify, purify and classify membrane targets and for structure/function studies. The particular properties of these toxins are now also being exploited pharmacologically, and some toxins are used as drugs and others are currently undergoing preclinical trials Conus tulipa and β-cardiotoxin, from the snake Ophiophagus hannah, are active against α1-ARs Dendroaspis angusticeps: ρ-Da1a, previously called AdTx1, which is highly selective for the α1A-AR 2-ARs 2A-AR Although voltage- and ligand-gated ion channels are the main targets for neurotoxins, other targets, including G Protein-Coupled Receptors (GPCRs), have also been identified. The animal toxins active on GPCRs can be divided into two families 3H-N- methylscopolamine 3H-N- methylscopolamine and 3H-acetylcholine Conus tulipa, and has been classified as a non-competitive α1B-AR antagonist that accelerates 3H-prazosin dissociation kinetics and antagonizes α1B-AR activation by an insurmountable mechanism 1B-AR, consistent with its allosteric properties 1B-AR. ρ-TIA, however, shows only 10 to 25-fold selectivity for the α1B-AR over the other α1-AR subtypes, and has been described as a competitive antagonist at the α1A-AR although it does not fully inhibit 125I-HEAT binding Adrenergic and muscarinic toxins isolated from mamba snake venoms belong to the same three-finger-fold family and display substantial sequence identity (52–97%) 1A-AR, to define the effect of ρ-Da1a on agonist-stimulated Ca2+ release, and to use site-directed mutagenesis to analyze the α1A-AR binding site for this peptide toxin.These observations have led to hypotheses regarding the mode of action of these peptide toxins at receptor targets. The aims of our study were to use equilibrium and kinetic binding experiments to establish the pharmacological behavior of ρ-Da1a at the α125I-HEAT, 3H-prazosin, 3H-rauwolscine and 3H-CGP-12177 were purchased from PerkinElmer . Non radioactive HEAT was obtained from Tocris , and 5-(N-ethyl-N-isopropyl-amiloride (EPA), prazosin, yohimbine, and propranonol were obtained from Sigma-Aldrich .Total protein and membrane protein concentrations were determined using the Bio-Rad protein assay, with bovine serum albumin as standard.1A-AR cDNA inserted in the prK5 vector was kindly provided by Michael Brownstein . Point mutations were introduced into the α1A-AR gene by sense and antisense primers  containing the desired changes, using the QuikChange Site-Directed Mutagenesis kit. The incorporation of each mutation was verified by DNA sequencing. The variants F3087.35A and F3127.39A were generous gifts from Dr. Diane Perez .α1-ARs were kindly provided by Dr. Hervé Paris  and were grown in a 50∶50 Dulbecco's Modified Eagle's Medium (DMEM)/Ham's F12 medium supplemented with 10% (v/v) foetal bovine serum (FBS), glutamine (2 mM), penicillin (100 units/ml) and streptomycin (100 µg/ml) at 37°C with 5% CO2. COS-7 cells were grown at 37°C under 5% CO2 in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 1% penicillin and 1% glutamine . At 80% confluence, the cells were transfected using a calcium phosphate precipitation method for transient expression of the genetic construct. After 48 h incubation at 37°C, cells were harvested and the membranes were prepared as follow. Cells were washed with ice-cold phosphate buffer and centrifuged at 1700 g for 10 min (4°C). The pellet was suspended in ice-cold buffer  and homogenized using an Potter-Elvehjem homogenizer . The homogenate was centrifuged at 1700 g for 15 min (4°C). The sediment was resuspended in buffer, homogenized, and centrifuged at 1700 g for 15 min (4°C). The combined supernatants were centrifuged at 35,000 g for 30 min (4°C), and the pellet was suspended in the same buffer (0.1 ml/dish). The CHO cells used for Ca2+ release experiments also stably express the human α1A-AR  as selective ligands for α1-ARs, 3H-rauwolscine for α2-ARs and 3H-CCGP-12177 for β-ARs. Non-specific binding to α1, α2 and β-ARs was measured in presence of prazosin (10 µM), yohimbine (10 µM) and propanolol (10 µM), respectively. Binding experiments were performed in a 100 µL reaction mix at room temperature in buffer composed of 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 g/L BSA. Reactions were stopped by filtration through 96 GF/C filter plates pre-incubated with 0.5% polyethylenimine. An aliquot of 25 µL of Microscint 0 was added onto each dry filter and the radioactivity was quantified on a TopCount beta counter with a 33% yield . Saturation binding assays were performed using a fixed amount of receptors and a series of concentrations of 125I-HEAT with an incubation time of 1 h. Competition binding assays were performed by mixing the radioligand  with a range of competitor concentrations before adding membranes , for 16 h of incubation. Dissociation kinetics experiments were performed by pre-equilibrating 125I-HEAT (400 pM) or 3H-prazosin (2 nM) for 3 hours with α1A-AR COS-7 cell membranes . Radiotracer dissociation was then measured following addition of HEAT (5 µM) or prazosin (10 µM) alone or with ρ-Da1a (2.5 µM), 5-(N-ethyl-N-isopropyl)-amiloride  or adrenaline (2 mM).We used 1A-AR were seeded at 2×104 cells per well in 96-well plates overnight. The following morning, the media was removed and cells washed three times in a modified Hanks' buffered saline solution  containing BSA 0.5% (w/v). In light-diminished conditions cells were treated with fluoro-4 . Excess fluoro-4 not taken up by the cells was removed by washing twice in modified HBSS and then cells incubated for a further 30 min in the absence or presence of differing concentrations of ρ-Da1a before the assay plate was transferred to a FlexStation . Real-time fluorescence measurements were recorded every 1.7 seconds over 200 seconds, with agonist  additions occurring after 17 seconds, using an excitation wavelength of 485 nm and reading emission wavelength of 520 nm. All experiments were performed in duplicate. Agonist responses represent the difference between basal fluorescence and peak [Ca2+]i measurements expressed as a percentage of the response to A23187 (1 µM) in each experiment.CHO-K1 cells expressing the αD values and Bmax (number of binding sites) were determined by applying a nonlinear regression to data obtained with saturation binding assays. The nonlinear regression used was BS = (Bmax*A)/KD+A, where BS is the specific binding, Bmax the number of binding sites, A the concentration of radioligand, and KD the dissociation constant of the radioligand. Data resulting from competition binding assays were analyzed using the Hill equation for IC50 and curve slope estimations. The binding affinity (pKi) of ρ-Da1a was determined from the IC50 value of inhibition curves using the Cheng and Prussof equation 50 = Ki+(L/KD)*Ki. Dissociation kinetics were analyzed using a simple equation of exponential decay BS * exp (-Koff*t), where BS is the specific binding at time zero and Koff is the dissociation rate constant. Results are expressed as mean ± s.e. mean from n independent experiments. One-way Anova test was used to compare values. A p<0.05 was accepted for statistical significance.Binding data were analyzed by nonlinear regression using the KaleidaGraph 4.0 software . pK2+ release are expressed as mean ± s.e. mean from n independent experiments. Data were analysed using non-linear curve fitting (Graph Pad PRISM v5.02) to obtain pEC15 values for the [Ca2+]i assays. Antagonists such as ρ-Da1a that have slow dissociation kinetics are prone to display hemi-equilibrium artifacts in functional transient responses such as measurement of intracellular Ca2+ levels. As such, when competing with an agonist, the maximal response achieved by the agonist reduces in the presence of higher antagonist concentrations due to the inaccessibility of a large pool of the receptors in the time taken for the transient response to occur B of ρ-Da1a. In order to account for this, the pKB value for ρ-Da1a was calculated by the modified Lew-Angus method 15 values, based on the extent of reduction in agonist maximal responses in the presence of ρ-Da1a. The pEC15 values were plotted against the concentration of antagonist and non-linear regression applied B values for ρ-Da1a against each of the four different agonists.Values for intracellular Ca1A-AR was generated with MODELLER 1A-AR is the β2-AR subtype, with an overall amino acid sequence identity of 21% 2-AR structures are available  and are very similar (Cα RMSDs<1.5 Å for 253 residues). We used the X-ray structure with the highest resolution (2RH1) as a template A model of the αρ-Da1a, (previously AdTx1) 1-ARs expressed in yeast 1A-ARs, 7.28±0.09 for α1B-ARs, 6.85±0.08 nM for α2C-ARs, and 5.95±0.08 for α1D-ARs. No significant effect was observed with 10 µM of ρ-Da1a at α2A, α2B, β1, or β2-ARs. For α1A- and α1B-ARs, even the highest concentrations of ρ-Da1a did not completely inhibit 3H-prazosin binding, which remained at 18±3% and 17±1%, respectively . However, as observed with 3H-prazosin, ρ-Da1a interacts very efficiently with the α1A-AR .3H-prazosin and 125I-HEAT was studied in comparison with adrenaline and the negative allosteric modulator EPA . In contrast, neither 2 mM adrenaline nor 2.5 µM ρ-Da1a affected the 3H-prazosin dissociation rate  and in the presence of ρ-Da1a (Koff HEAT+ρ-Da1a = 0.058 min−1), prazosin (Koff HEAT+prazosin = 0.06 min−1), and EPA  were dose-dependently inhibited by ρ-Da1a  with Hill slopes between 0.8 and 1.1. Residual binding in the presence of 3H-prazosin fluctuated between 15 to 25% of the total binding, but did not show any trend to concentration-dependence. The curve IC50ρ-Da1a as a function of 3H-prazosin concentration (L) fitted the linear regression IC50ρ-Da1a = 1.067+1.82*L, incompatible with a negative allosteric modulation (50ρ-Da1a = Kiρ-Da1a+(Kiρ-Da1a * Lprazosin/Kdprazosin) ρ-Da1a of 1.067 nM (pKi 8.97) and a Kdprazosin of 0.586 nM (pKd 9.23). An analogous experiment was performed using 125I-HEAT. Ten concentrations  were inhibited by ρ-Da1a  with Hill slopes between 0.9 and 1.4. Residual binding fluctuated between 18 to 28% of the total binding (50ρ-Da1a as a function of 125I-HEAT concentrations fitted the equation IC50ρ-Da1a = 0.706+16.22*L which gave a Kiρ-Da1a of 0.706 nM (pKi 9.15) and a KdHEAT of 0.0435 nM (pKd 10.36).The ρ-Da1a ICeriments . Eleven dulation . Using t binding . IC50ρ-D1A-AR, all agonists stimulated Ca2+ release  77.8±1.5, phenylephrine pEC50 7.64±0.16, Emax 64.8±2.4, A61603 pEC50 10.17±0.07, Emax 75.5±1.3, and oxymetazoline pEC50 9.09±0.08, Emax 60.8±1.3. Increasing concentrations of ρ-Da1a reduced the maximal response to each of the four agonists while shifting the curves to the right. The pKB values for ρ-Da1a were calculated by a modified Lew-Angus method We next tested the effect of ρ-Da1a on responses to noradrenaline and phenylephrine (phenethylamine agonists) and A61603 and oxymetazoline (imidazoline agonists). In CHO-K1 cells expressing the α release , with pE1-ARs that are known to interact with agonists and/or antagonists: F862.643.32 (D125 in α1B-AR) 5.415.42 and S1925.466.51 (F310 on α1B-AR) 6.557.35 and F3127.395.47 and F2816.44, predicted to play a role in the stabilization of the active state of α1A-AR 125I-HEAT affinity values for D1063.32A, F1935.47A, F2816.44A, F2886.51A, M2926.55A and F3087.35A were not significantly different from the wild type receptor. One mutated receptor, F1875.41A, had significantly higher affinity for 125I-HEAT with a pKD of 10.70±0.005 compared to wild type pKD 10.05±0.09  and F3087.35A (29 pmol/mg protein) both displayed a similar pKd to each other and to the wild type receptor.We investigated the involvement of residues within the orthosteric pocket of α.05±0.09 , p<0.05.e α1A-AR , however125I-HEAT are shown at wild type, D1063.32A and F862.64A receptors (3.32A variant (pKi 9.74±0.12) and the wild type receptor (pKi 9.57±0.08) but was strongly affected by the F862.64A mutation (pKi 8.21±0.09). ρ-Da1a affinity at the D1063.32A variant was reduced by 6-fold (pKi 8.48±0.11) compared to the wild type receptor while affinity at F862.64A was reduced by 36-fold (pKi 7.70±0.06). The mutation F862.64A affects both HEAT and ρ-Da1a affinities, suggesting that the structural organization of the receptor could have been perturbed by this modification. We used the radioligand 3H-prazosin to examine this point and found that prazosin affinity for the F862.64A mutant (pKd 9.21±0.07) was very close to the one measured for wild type receptor . While this mutation may alter interactions between F862.64 and other aromatic residues within the orthosteric pocket 1A-AR, ρ-Da1a inhibited only 75–80% of 125I-HEAT binding in experiments with these mutant receptors.Curves for competition of HEAT and ρ-Da1a with binding of eceptors . As seen5.41A, M2926.55A and F3087.35A variants, ρ-Da1a inhibited 125I-HEAT binding with affinities similar to the wild type  receptor , the radioligand (3H-prazosin or 125I-HEAT), or the expression system , a finding more consistent with non-competitive interaction. To examine functional antagonism by ρ-Da1a, we measured blockade of intracellular Ca2+ release following 30 min pre-incubation of CHO-α1A-AR cells with the toxin . These data conform to “non-permissive” antagonism, where receptor occupancy by the toxin prevents simultaneous orthosteric agonist interaction B values of the modulator are altered depending on the agonist used B of ρ-Da1a is the same for four agonists belonging to two distinct structural classes, and known to display signaling bias at the α1A-AR 3H-prazosin or 125I-HEAT, and that ρ-Da1a affinity for the α1A-AR is reduced by mutation of residues within the orthosteric pocket.We have characterized the interaction between ρ-Da1a and the αhe toxin . The obs3H-N-methylscopolamine and 3H-acetylcholine in membranes expressing the M1 AChR 3H-prazosin and 125I-HEAT from the α1A-AR  is a major determinant for the interaction with the aromatic ring of catecholamines and with α1-AR antagonists like prazosin and phentolamine 6.51 and F3127.39, and one with phenethylamine-type (F2886.51) and imidazoline-type (F3127.39) agonists. In addition, F862.64 is the only residue unique to the α1A-AR subtype that is important for toxin affinity. In α1B-, α2C- and α1D-ARs, this position is occupied by a leucine, an asparagine and a methionine, respectively. F862.64 was previously identified as a determinant for interaction of the α1A-AR with various antagonists 2.64M receptor mutant did not show any changes in HEAT affinity 2.64A one strongly decreased it while having no effect on prazosin affinity. This residue most likely contributes to the selectivity of ρ-Da1a for the α1A-AR subtype.We found three key mutations that have major importance for ρ-Da1a binding: F861A-AR based on the β2-AR structure 3.32 and the S1885.42/S1925.46 positions are about 16 Å from the surface of the receptor on one side of the orthosteric site, whereas positions F862.64, F2886.51 and F3127.39 that all interact strongly with ρ-Da1a are located on the opposite side and distributed from the surface down to a depth of 12 Å. As seen for toxin binding, mutation of F862.64 had a substantial effect on HEAT affinity, whereas mutation of F2886.51 had no effect, and mutation of F3127.39 to alanine caused a slight increase in the affinity of HEAT. Although we have yet to define additional residues that contribute to HEAT binding, these findings support the idea that ρ-Da1a and the radioligands have overlapping but distinct binding modes.We constructed a homology model of the α1A-AR. This is not the case for MT7, as the experimentally-based model of the MT7-M1 muscarinic receptor complex indicates an extracellular location of the toxin involving mainly the extracellular loop e2, in agreement with its allosteric properties 1 receptor 1A-AR model  decreases ρ-Da1a affinity 18-fold. In TM7, ρ-TIA is sensitive to mutation at position F3307.35 of the α1B-AR  but not at position F3347.39 (corresponding to F3127.39 in α1A-AR). Mutation of the α1A-AR at residue F3127.39, which is one helical turn further from the extracellular face of the α1A-AR than F3087.35, produces a 93-fold reduction in ρ-Da1a affinity, highlighting the difference in binding mode of ρ-Da1a and the α1A-AR compared to ρ-TIA and the α1B-AR. Hence of the three animal toxins for which the mode of action has been described, the two negative allosteric modulators (MT7 and ρ-TIA) interact mostly with the external part of their receptor targets while ρ-Da1a interacts with the orthosteric binding site.A three-finger fold toxin can be represented by a 35 Å isosceles triangle of around 10 Å thickness . It is tAR model  shows a 1A-AR, however the large size of the toxin and/or its slow dissociation rate appear to cause altered pharmacology. In yeast membranes expressing the α1A-AR, both prazosin and ρ-Da1a cause complete displacement of 125I-ρ-Da1a, whereas like in CHO-K1 and COS-7 cells, ρ-Da1a displaces only 85% of 3H-prazosin binding 3H-rauwolscine binding to α2-ARs despite showing no effect on the 3H-rauwolscine dissociation rate, but their modes of action have still not been fully established 1B-ARs but has been described as a competitive antagonist of the α1A-AR in functional assays 125I-HEAT binding in membranes from HEK-293 cells transfected with the α1A-AR. We initially thought that all α1A-ARs present in membrane preparations may be accessible to small molecule radioligands, but that a sub-population of the receptors might exist in conformations that are inaccessible to the larger toxin. This could reflect steric hindrance or a mixed allosteric/orthosteric mode of action of ρ-Da1a, however if this were the case, and the two populations of receptors were in equilibrium, the residual binding should change over time. We found that this was not the case, as the residual binding showed no time dependence over 2–24 hours. Thus the two receptor pools are not interchangeable, suggesting possible separation between distinct membrane compartments. This question remains to be resolved for ρ-Da1a but also for other toxins that display atypical pharmacological properties.We found one discrepancy in our study, namely that ρ-Da1a shows incomplete competition with radioligands in equilibrium binding studies, a property normally characteristic of allosteric modulators. Our combined data, indicate that ρ-Da1a binds at least in part within the orthosteric pocket of the α1A-AR orthosteric site, and whether additional regions bind to α1A-AR extracellular loops as seen for MT7 and ρ-TIA. Identification of additional receptor binding sites will be of interest because any such extra-orthosteric interaction may contribute to the α1A-AR selectivity of ρ-Da1a, as well as the insurmountable antagonism observed here in cell-based assays, on isolated rat in vivo experiments Key questions arising from our work are to determine which residues of ρ-Da1a bind within the α1A-AR and highlight the crucial role of residues located in the α1A-AR orthosteric site for the toxin interaction. Thus, despite the fact that ρ-Da1a and MT7 belong to the same three-finger fold structural family of toxins, and interact with homologous biogenic amine receptors, the mode of interaction with their respective targets is distinct. Evolution of snake toxins has thus not only generated a wide range of pharmacological activities from a unique peptide scaffold, but also various strategies to interact with similar molecular targets.In conclusion, our findings demonstrate competitive behavior of the ρ-Da1a toxin at the αFile S1Recombinant expression of ρ-Da1a.(DOCX)Click here for additional data file.


The growing demand for physical rehabilitation processes can result in the rising of costs and waiting lists, becoming a threat to healthcare services' sustainability. Telerehabilitation solutions can help in this issue by discharging patients from points of care while improving their adherence to treatment. Sensing devices are used to collect data so that the physiotherapists can monitor and evaluate the patients' activity in the scheduled sessions. This paper presents a software platform that aims to meet the needs of the rehabilitation experts and the patients along a physical rehabilitation plan, allowing its use in outpatient scenarios. It is meant to be low-cost and easy-to-use, improving patients and experts experience. We show the satisfactory results already obtained from its use, in terms of the accuracy evaluating the exercises, and the degree of users' acceptance. We conclude that this platform is suitable and technically feasible to carry out rehabilitation plans outside the point of care. Nowadays there is an ever-growing demand for rehabilitation processes within public and private healthcare services. In 2011 the World Health Organization estimated that one billion people worldwide were in need of rehabilitation treatments [Aging is recognised as one of the main causes of the need for any kind of rehabilitation treatment, and its importance lies in the fact that it is unavoidable. Focusing on physical rehabilitation, besides ageing, there are other prominent causes for these therapies: musculoskeletal injuries, illnesses that affect the locomotor system, and postsurgery scenarios. All these situations cause the loss of physical abilities, having a clear impact on patients' quality of life.Physical rehabilitation plans involve a set of different therapies that aim to return patients to a healthy condition allowing them to recover a normal way of living. The objectives and the design of these plans vary depending on temporary aspects of the disabilities. In case of permanent disabilities, rehabilitation will probably last until the end of the patient's life, having as unique goal the maintenance of his self-sufficiency, or just decelerate the loss of physical abilities. If the disabilities are temporary, the objective of rehabilitation therapies is to help patients to regain as much self-sufficiency and lost skills as possible, in the minimum time, in order to live a normal life.As it is easy to deduce, physical rehabilitation processes are highly cost-consuming, becoming a threat to the sustainability of healthcare services. Rehabilitation plans are required to be as efficient as possible to make them affordable to funding institutions. In these plans, efficiency depends on its logistics, organizational factors, and healthcare policies. In addition to this, patients' attitudes and behaviour have a great impact on this issue due to the concept of patients' treatment adherence. Treatment adherence is related to the degree in which patients follow the experts' guidance and fulfill the scheduled clinical activities prescribed to them . The lacinpatient scenario, in which the patient goes to a point of care in scheduled sessions and performs the exercises partially supervised by experts, since each physiotherapist attends many patients at a time. In  we can soutpatient scenario, in this case after the prescription of a rehabilitation plan, the patient performs, usually at home, the exercises under no supervision. Experts check his evolution through regular meetings at the point of care.Patient's attitude towards rehabilitation treatments can be influenced by several factors, which are related to him, his relation with the rehabilitation experts, and organizational issues of the rehabilitation services. An example of this is the importance of the scenarios in which the rehabilitation processes take place. We can distinguish between two main situations:Inpatient scenarios require higher personnel cost but allow a more regular communication between patients and physiotherapists. On the contrary, in outpatient situations this communication is almost nonexistent. So it is highly likely that the patient does not remember clearly the expert's instructions to perform the exercises in his day-to-day rehabilitation sessions. Even more, as they perform them without supervision, they will not receive any feedback about the correctness of their performance, until the next scheduled visit to the physiotherapist office.In their study, Jack et al. observe the difference between adherence to treatment of patients involved in inpatient and outpatient scenarios, respectively . PatientWithin the field of information and communication technologies (ICT) we can find suitable solutions to face the previously described issues related to physical rehabilitation processes. After meeting with physiotherapists and reviewing related works, we decided to design a system to allow patients to perform rehabilitation exercises in outpatient scenarios but assuring the real-time communication between them and the experts at the point of care. In short, we aim to reduce the workload of healthcare services, making them more efficient, while eliminating the gap between patients and experts improving their experiences by using a low-cost and easy-to-handle platform.To achieve those goals our system should monitor the patients' performance of the prescribed exercises, evaluate its correctness, and securely store these data. Furthermore, patients and physiotherapists should have access at any time (24 × 7 × 365) to all the data related to the rehabilitation plan, to obtain useful feedback. In this work we described the objectives of our project, its final design, and the test and validation processes carried out to show its feasibility to be used for telerehabilitation purposes.This paper is organized as follows. In The objective of improving the quality of health care and the functioning of healthcare services has led to many cases of use of ICT in the medical field. Our work is connected with the concept of telemedicine, which aims to offer ubiquitous healthcare services to patients. Within this concept, we can talk about telerehabilitation as process in which a patient performs tasks related to follow a rehabilitation plan, of any kind, outside a healthcare centre.Telerehabilitation proposals vary in their design since they are focused on different aspects of the rehabilitation process. These variations depend on the targeted user (patient or physiotherapist), their purpose , the disability and part of the body to be treated, and the environment in which the solution will be used.There is a prominent trend of works that use virtual reality and robotics to offer to patients innovative ways to perform prescribed exercises. Some sensing devices are used to offer the virtual reality feeling and to record useful data about patient's state and behaviour , 11. TheIn a ubiquitous scenario patients should be monitored as they perform the rehabilitation exercises. Afterwards, the obtained data should be at physiotherapist disposal to be evaluated or examined. Motion tracking techniques are useful to do this task. A work by Zhou and Hu shows examples of the use of multiple tracking systems for rehabilitation purposes, depending on the sensing devices and information processing algorithms used .Inertial sensors have been extensively used for human motion tracking and recognition because of their low cost, simplicity, and availability. These sensors have been applied to recognize and measure human activities , 19 and Inertial sensors are often combined with other sensors, inertial or not. This is done to avoid some disadvantages of these sensors like drift when estimating 3D coordinates or noise in the gathered data, due to sensor rotations, displacements, or other perturbations –23. The Taking into account the need of a low-cost system and the patient's autonomy and comfort, we tested the possibility of using one single inertial sensor. We choose a gamepad manufactured by Nintendo, the Wii Remote©, that includes a three-axis accelerometer . Wii RemAfter reviewing previous works we had insight of several issues that should be considered to design a low-cost, easy-to-use, and able-to-generalize rehabilitation system. Many of them are focused only on rehabilitating concrete parts of the body , 31, or Our work aims to build a system whose main objective is to improve the efficiency of physical rehabilitation processes due to the benefits of outpatient scenarios. In addition, it is designed to bridge the gap between patients and physiotherapists, trying to simulate the ideal case of having an expert side by side with patients while they perform the prescribed exercises at home. At the same time, the system is intended to be helpful for physiotherapists, offering them accurate data about patients' performance allowing them to gain insight about their evolution.Primary objective of efficiency can be fulfilled through achieving other secondary ones, which can be grouped paying attention to the aspects of the rehabilitation processes they affect: technology, users (patients and physiotherapists), and rehabilitation services' functioning.The system must give to patients and experts suitable mechanisms to interact with each other as if they were one in front of each other. In the case of patients, at the time they perform their exercises they should have at their disposal all information about the schedule of the rehabilitation plan, a full description about how to perform each exercise, and real-time feedback about their performance. For experts the system should gather information about the fulfillment of the plan and the correctness of the patients' performance. This will allow a quick and ubiquitous assessment of the patients' evolution and an update of their rehabilitation plans if needed.System's use will help to increase patients adherence to treatment in two ways. First, since every interaction with the system is recorded, if they do not perform the exercises, if they perform them at a wrong time or in an erroneous way, the physiotherapist will instantaneously know it. Secondly, the system will avoid cases of forgetting the instructions about how to perform the exercises or the schedule of rehabilitation sessions.All the services that the system will offer must be integrated within the current rehabilitation services workflow. This means that eFisioTrack will not be a standalone application just to monitor outpatient performance of exercises. Besides, in the same environment the expert will be able to carry out all the tasks that involve dealing with patients: administrative work, agenda planning, access to patients' history, examination and prescription of complementary therapies, and so forth.eFisioTrack is not focused on a certain joint or a part of the body. It will be able to cope with the rehabilitation of different disabilities and injuries, offering an upgradable set of exercises to do so.The interaction mechanisms between patients and experts should be presented in a friendly environment, through comprehensive interfaces where the different options are logically disposed following common rehabilitation workflow. This is important to avoid rejection due to complexity of usage.Achieving a low-cost system is a challenge that is taken into account at the time of the system's architecture design. This challenge is behind the election of a sensing device and the technologies used to implement both client-side and server-side software components.To assure its scalability, the system has a modular design and each module interacts with the others through layers that abstract the offered services .Since eFisioTrack is based on communication over the Internet some issues should be born in mind. We are dealing with clinical data which requires to set policies to preserve its privacy and integrity. Moreover, the system will offer 24 × 7 × 365 disponibility but sometimes this is impossible due to network failures. eFisioTrack can be used in these circumstances avoiding loss of information.eFisioTrack will cover all the experts' needs, from the first meeting with a patient until the end of the rehabilitation process, while reducing the visits to a point of care and discharging patients from the rehabilitation services.To achieve that, we had several meetings with physiotherapists who develop their task at a public hospital, one of them being expert on adherence to treatment issues. These meetings helped us to understand the functioning of a physical rehabilitation service and to gain insight of their needs.The use of eFisioTrack introduces little changes in experts' current procedures in order to ease the integration of the system in their workflow. In  we show A case of eFisioTrack use begins when a patient goes to the point of care looking for a physiotherapist's help. The physiotherapist checks his physical condition, evaluating the existence of possible injuries. According to this exploration, the expert introduces data related to patient's condition on the system and proceeds to open a new rehabilitation plan. This plan will contain a schedule of several rehabilitation exercises sessions to be done at home. Then, the expert explains to the patient how to perform each exercise, making him use the sensing device to record a motion example for each gesture. This example will be used as a realistic reference of what the patient is able to do and is useful to check the correctness of the patient's at home performances. Doing it in this way we offer a personalized treatment that fits each patient needs and abilities.Now the patient is expected to perform the scheduled sessions at home, avoiding most of the regular visits to the healthcare centre, reducing them to the mininmum needed. In the meantime, experts can check on the system if the patient is fulfilling the rehabilitation plan and how good are the results that he is obtaining. This information is useful to modify the rehabilitation plan to fit new needs. This can be done remotely using the system or in the next appointed meeting with the patient, after a regular exploration to check his evolution.The telerehabilitation's environment hardware infrastructure consists of the following elements.(i) PC. Computers or other general purpose devices  are suitable since eFisioTrack does not require any high performance computing resources. They are used to install the client side of the application for patients and for physiotherapists. The analysis of hardware and computational scalability made in the design phase showed that, after deploying the system, it will not be necessary to acquire any other equipment in close future. Therefore, the price of the overall system is not expected to increase, making it easier to achieve a low-cost solution.(ii) Sensing Device. This device will be used to monitor the patient's performance of the exercises. It is a Wii Remote gamepad, which is a wireless and wearable low-cost motion capture device. It includes an accelerometer that gathers values of linear acceleration along three perpendicular axes . This sensor is able to measure accelerations over a range of ±3 g with 10% sensitivity and send it to the application using a BCM2042 Broadcom Bluetooth chip.(iii) Server. It is where the server side of the application will be allocated. It will contain too the platform's database.(iv) Communication Elements. Whatever connection to the Internet is valid, all the interactions between the client-side application and the server-side application will take place over the net.Software elements have been designed to fulfill the stated objectives. The set of technologies chosen and the way they interact are not casual and aim to meet those requirements: 24 × 7 × 365 connectivity, the use of low-cost motion capture devices, easy-to-use and intuitive graphical interfaces, integration of different development platforms and operating systems, and experts ubiquitous access to the data stored in the system. A diagram of the software components of eFisioTrack and the places where they are used is shown in .(i) Database. The database is a key part of the platform since it provides the data structures for the main functionalities. Data structures have been refined since the first meeting with physiotherapists in order to offer them a complete experience like medical center consultation. The database contains data related to patients, medical records, assigned treatments, planned sessions, and so forth.(ii) Web Services. The software deployed on the server follows the fourth generation of web server paradigm using XML and WSDL interfaces with SOAP-based messaging. Web services have been deployed into a 2-tier architecture (database connectivity and web server). There are three types of web services , which deal with data types defined by us to represent objects like rehabilitation sessions, exercises, and so forth.(iii) Connectivity. eFisioTrack sends and receives data to and from a server, through an Internet connection. Also, an offline connectivity mode has been implemented which can be activated by the physiotherapists in the application. Once it is turned on, rehabilitation programme data are stored in the patient computer so the user does not need to log into the application again. Data remain available in the application instead of retrieving them from the server. In the case of data about the performance of rehabilitation exercises, it can be stored on a USB flash memory or on a hard drive so it can be retrieved later.(iv) Security and Privacy. Since the platform environment is related to healthcare, security controls must be used to protect patient's data. The essential developed security measures are to record user actions so that every step taken in the system is known, for example, incorrect password in a login attempt. Database's structures have been designed so that database instances will be associated with neither concrete patients nor the physiotherapist as required by law. Communications are encrypted using SSL secure protocol. In addition, other precautionary measures have been taken into account such as recording every unexpected application error or filtering IP address and port of the connections made to the application server.(v) Technology Integration. eFisioTrack is the result of an important effort integrating different technologies and platforms, in order to build up a sustainable and powerful system. The client side is a C# based standalone application. On the other side, the server is based on a Unix environment. The motion capture device and the application communicate using an open source library created for this purpose by Brian Peek [ian Peek .During the development of the platform, agile software development methods have been used. Elaboration cycles have been centered in evolving functionalities over the time, as the meetings were held with physiotherapists in order to test parts of the platform. On top of that, collaboration with physiotherapist has been crucial to get the platform finished, keeping the user interface and other features such as usability and window layout design as much precise as the rehabilitation treatment needs. Throughout all the above elements, the priority has always been to use open-source elements because this helps in keeping a final affordable system budget.(vi) User Interfaces. Implementation cycles have been centered in evolving functionalities over the time, as we held several meetings with the physiotherapists staff. This was crucial to design and test certain parts of the platform. An example is the different users' interfaces. Issues such as usability and window layout design were jointly examined with experts. Throughout all the above elements, the priority has always been to use open-source elements because this helps in keeping a final affordable system budget. Once we had a pilot of the system, we collect patient's opinion about the usability and usefulness perception too.The platform has three types of users based on the specific role they play: physiotherapists, patients, and administrators. Each one of them accesses different parts of the client application; for example, the administrator adds new physiotherapist into the platform and a physiotherapist user manages all the data regarding a physiotherapy process such as add new patient and add new rehabilitation exercise. Finally a patient user can access the physiotherapy process information to perform a rehabilitation exercise or look at his scheduled elements of a specific day.When a physiotherapist logs into the application, he can choose between patients data management and physiotherapy process remote monitoring . In the remember to try staying in the same plane. When an administrator logs into the application, a small option panel is shown that allows the administrator to add new users into the platform. It can add a physiotherapist or a new administrator since there could be the need for different administrators in the same medical center.When a patient accesses the application, a menu shows the elements of the active physiotherapy process. Moreover, a rehabilitation exercise can be performed with the motion capture device, and once the exercise is finished, resulting data is sent to the server. After that, the application shows the results to the user and suggests some tips to fix detected mistakes such as (vii) Remote Monitoring. The implementation of remote monitoring features allows the physiotherapist to totally personalize the rehabilitation program of the patient.First, a customized movement is recorded which is considered as the master movement for a specific patient and exercise. Once the raw acceleration data is transmitted to the application, it is filtered using a three-point moving average to avoid noisy values on the signal. Then, the filtered acceleration values are the input to the motion evaluation module. This module uses the dynamic time warping (DTW) algorithm to evaluate the correctness of the patient's performed gesture . DTW triTwo clear benefits rise with this telerehabilitation scenario. First one is that with telerehabilitation we save expenses in the overall budget. The second one is contributing to the fact that the patient can perform rehabilitation exercises at home; in this way we avoid problems when traveling, since rehabilitation patients usually have problems with their mobility. Also, in the moment the physiotherapist adds a new exercise there is the option to record a personalized video which the patient can check afterwards in the moment of the exercise performance, reminding the patient how to do it properly. Furthermore, the remote monitoring has been designed using widely accepted guidelines for exercise prescription.The system has been evaluated at several stages, different issues being tested at each stage. Through the design and implementation stage, each module was twice tested, first separately and once integrated in the system, jointly with the other modules. Notwithstanding this early testing, the main evaluation is made on the complete and deployable version of the system to check if it meets the stated requirements and fulfills their objectives. This process is prior to final deployment with real patients.We designed a twofold evaluation process. On one hand, we wanted to test system's ability to monitor patient's exercises performance. We carried out a quantitative study to test eFisioTracks accuracy on doing that. On the other hand, we want to know users' opinion about the system's use: its usability, usefulness perception, friendly design, and so forth. Users took part in a qualitative study which consists of a survey of 10 simple and straight questions about their experiences.eFisioTrack is designed to manage any kind of rehabilitation exercises related to injuries that affect any part of the body. It includes a catalog of more than 80 different exercises mainly related to orthopedic injuries like subacromial impingement, rotator cuff tendinopathy, slap lesion, and so forth.To evaluate how well eFisioTracks monitor and evaluate patients at home exercises' performance, we select 4 commonly prescribed exercises: free active shoulder flexion, free active shoulder abduction, leg extension, and free horizontal shoulder adduction . Each ofSetup. The test was performed at the rehabilitation service facilities of a public hospital. Two computers and two Wii Remote gamepads were used to perform the selected exercises. Each user completes each session on different days and at different times, trying to simulate the behaviour of a real patient who will carry out the rehabilitation session whenever he likes within the scheduled day.Participants. 10 volunteers took part in the tests. Since there is no a targeted user, we select people of different gender and from as widest range of age as possible. There were 6 men and 4 women, from 20–35 (5), 36–50 (3), and older than 50 (2).Test Development. A physiotherapist designed a short rehabilitation plan that consisted of 4 sessions on which the testers should perform 10 times each exercise. They perform each session on different days, under the supervision of a physiotherapist. Testers deliberately perform correctly or erroneously each repetition. They even tried to trick the system performing repetitions that were almost correct but were not satisfactory enough to be considered as correct by the supervising expert. This is done to test the accuracy of the motion recognition algorithm, when comparing acceleration sequences that have different degrees of similarity. Finally we counted how many times the system's motion evaluation coincided with the expert's opinion.Results. 1600 cases were considered, 800 performed correctly and 800 performed erroneously. In absolutely not,” while a 5 rate meant “yes, absolutely.”Apart from testing system's functionality, it is crucial to check if its use is appealing to users. We designed a qualitative study on which users are asked to fill a questionnaire of 10 questions. Questions are related to aspects of the system or its use and participants answer rating these aspects using a scale that goes from 0 to 5. A 0 rate meant “In In this paper we present eFisioTrack, which is a platform that allows carrying out physical rehabilitation plans in outpatient scenarios. We developed this platform trying to offer solutions to issues like healthcare services efficiency and patients involvement in the rehabilitation processes.eFisioTrack offers new ways to improve both physiotherapist and patient experience. It aims to reduce healthcare cost allowing performing rehabilitation plans at patient's home, discharging patients from the points of care, allowing experts to simultaneously deal with a higher number of patients, and finally shortening waiting lists. From the patients' point of view, it diminishes the discomfort reducing significantly his visits to the healthcare centre.From the first moment, we met physiotherapists with expertise in physical rehabilitation processes. We analyse their workflow and needs, which was very useful at the time of deciding which services our system will offer. This process allowed us to avoid a later possible rejection to use the system due to not being suitable or easy to integrate in experts' routines. Other considered aspects were the cost of the system, its usability, and the simplicity of its use.In this work we show the results from evaluating 2 key issues of the system. First, we tested how good recognizing and evaluating the patients exercises' performance are, despite of using only one sensing device to gather motion data. This is extremely important since the system should do this as if it were an expert that was near the patient while he performd the exercises. We carried out a qualitative study that consisted of checking the system's behaviour performing 16000 repetitions and counting the number of correct and erroneous repetitions well recognized. Results were quite satisfactory, achieving an accuracy average rate of almost 99% in both cases.Secondly, we made a qualitative study that consisted of a survey about the users' perception of different issues related to the design and use of eFisioTrack. Participants answered 10 simple questions, giving answers that range from 0 to 5, being 5 the higher scoring. The scores were between 4.4 and 5, showing a high opinion of the system and the experience of using it.The results of this evaluation make us conclude that the designed environment is appropriate and technically feasible.Physiotherapists greatly appreciated the fact that our environment includes services that cover their needs throughout the whole rehabilitation process. Thanks to this we avoid the fact that the system was perceived as a disturb in their routine.Another issue that our proposal solves is the gap between patients and experts. Physiotherapists found the interaction mechanisms that the system offers to them useful and suitable. These mechanisms help patients not to forget how to perform the exercises. Video and text descriptions of the rehabilitation sessions, together with a messaging mechanism, try to guide and help patient in the absence of the expert. The strengthening of the communication between physiotherapist and patient will benefit on the long course the results of the treatment, giving to the patient the feeling of a more centered-on-him healthcare process.Regarding adherence to treatment, we consider that eFisioTrack can improve it since the amount of detailed data obtained from patients performance allows experts to be more precise in setting and updating the rehabilitation plans. On the other side, the system has many aspects to increase patients motivation to complete rehabilitation programmes. The use of innovative ways to perform the exercises, usually tedious for them, helps them to do it in a better mood. Besides, they know that the expert will immediately know if they are completing every session of the rehabilitation program, so in the next scheduled meeting they will not be able to lie to the expert.