Many inpatients receive little or no rehabilitation on weekends. Our aim was to determine what effect providing additional Saturday rehabilitation during inpatient rehabilitation had on functional independence, quality of life and length of stay compared to 5 days per week of rehabilitation.This was a multicenter, single-blind (assessors) randomized controlled trial with concealed allocation and 12-month follow-up conducted in two publically funded metropolitan inpatient rehabilitation facilities in Melbourne, Australia. Patients were eligible if they were adults (aged ≥18 years) admitted for rehabilitation for any orthopedic, neurological or other disabling conditions excluding those admitted for slow stream rehabilitation/geriatric evaluation and management. Participants were randomly allocated to usual care Monday to Friday rehabilitation (control) or to Monday to Saturday rehabilitation (intervention). The additional Saturday rehabilitation comprised physiotherapy and occupational therapy. The primary outcomes were functional independence ; measured on an 18 to 126 point scale), health-related quality of life , and patient length of stay. Outcome measures were assessed on admission, discharge (primary endpoint), and at 6 and 12 months post discharge.P = 0.01) and health-related quality of life  on discharge and may have had a shorter length of stay by 2 days  when compared to control group participants. Intervention group participants were 17% more likely to have achieved a clinically significant change in functional independence of 22 FIM points or more (risk ratio (RR) 1.17, 95% CI 1.03 to 1.34) and 18% more likely to have achieved a clinically significant change in health-related quality of life  on discharge compared to the control group. There was some maintenance of effect for functional independence and health-related quality of life at 6-month follow-up but not at 12-month follow-up. There was no difference in the number of adverse events between the groups .We randomly assigned 996 adults (mean (SD) age 74 (13) years) to Monday to Saturday rehabilitation (n = 496) or usual care Monday to Friday rehabilitation (n = 500). Relative to admission scores, intervention group participants had higher functional independence (mean difference (MD) 2.3, 95% confidence interval (CI) 0.5 to 4.1, Providing an additional day of rehabilitation improved functional independence and health-related quality of life at discharge and may have reduced length of stay for patients receiving inpatient rehabilitation.ACTRN12609000973213Australian and New Zealand Clinical Trials Registry http://www.biomedcentral.com/10.1186/1741-7015-11-199.Please see related commentary:  Rehabilitation involves specialized, coordinated, multidisciplinary care that aims to restore functional independence in physical and cognitive activities . Allied There has been recent debate in the UK about providing 7-day acute healthcare in the National Health Service ,9. It haDespite the view that most hospitals provide weekend rehabilitation, only 30% of rehabilitation hospitals in Australia offer weekend therapy . AlthougThe primary aim of this study was to determine what effect providing an additional Saturday rehabilitation service in inpatient rehabilitation had on the discharge outcomes of functional independence, quality of life and length of stay. The secondary aim was to investigate if any benefits of providing additional therapy were maintained at 6 and 12 months after discharge from inpatient rehabilitation.This was a multicenter, single-blind, randomized controlled trial. The trial was registered with the Australian and New Zealand Clinical Trials Registry (ACTRN12609000973213) prior to patient recruitment. The trial was conducted according to the published trial protocol . The onlThe trial took place at 2 publically funded metropolitan rehabilitation facilities  with a combined total of 90 rehabilitation beds . Recruitment occurred from 1 July 2010 to 30 June 2011. In Australia, patients admitted for rehabilitation are usually not able to return directly home from acute hospital due to reduced functional independence. Before being accepted for inpatient rehabilitation, patients are typically assessed in an acute hospital as being able to participate actively in rehabilitation with the expectation that they will improve sufficiently to return to community living.Participants were included if they were aged 18 years or older and had been admitted for rehabilitation at either of the two facilities. Participants with any orthopedic , neurological  or other disabling condition  were included. Participants were excluded if they were admitted for slow-stream rehabilitation termed ‘geriatric evaluation and management’ (as this patient group are managed differently to patients admitted for standard rehabilitation) or if they were enrolled in another intervention trial. Participants were not excluded if their primary language was a language other than English (an accredited interpreter assisted with informed consent and outcome measurement) or if they had reduced cognition (the next of kin was approached for informed consent).Participants were randomized to the intervention or the control group using a concealed method, with 1:1 allocation. The block allocation sequence was generated electronically and assignments concealed in sequentially numbered, sealed, opaque envelopes. Only after the participant was enrolled in the trial and had completed baseline testing was assignment made by opening the next envelope in the sequence. A member of the research team who was not involved in recruitment, assessment or treatment of participants prepared the envelopes.Usual care rehabilitation was provided to all participants in both groups daily from Monday to Friday. Rehabilitation therapy focused on task-specific training and discharge planning and was at the discretion of the treating therapist. Patients at the two facilities usually receive about 2 h of physiotherapy and occupational therapy per weekday as well as full nursing, medical and other allied health services.In addition, the intervention group was scheduled to receive a full physiotherapy and occupational therapy service on Saturday . Weekend therapists may or may not have been the patient’s usual therapist but were therapists employed by the hospital network and not research staff. The content of the therapy provided at the weekend was similar to that which was provided during the week as determined by the patient’s Monday to Friday therapists. Instructions were provided by a written handover.Outcome measures were assessed directly at admission and discharge and by telephone at 6 months and 12 months. The primary endpoint was assessment at discharge with follow-up measures of functional independence and health-related quality of life at 6 months and 12 months. Outcome assessors who measured primary and secondary outcomes were blinded to group allocation. The success of blinding was evaluated at the discharge assessment by asking assessors to guess their patient’s group allocation. Treating therapists and other members of the rehabilitation team (who made decisions regarding discharge) were not blinded to group allocation.Functional independence was assessed using the functional independence measure (FIM)  administHealth-related quality of life was assessed using the EuroQoL questionnaire (EQ-5D) and visual analog scale (EQ-VAS) . The EQ-Length of stay was measured as the number of overnight stays in the rehabilitation facility and was included as a primary outcome based on pilot data  that sugSecondary outcome measures included the Personal Care Participation Assessment and Resource Tool (PC-PART) , 10-m waAdverse events, including falls, skin tears and infections were recorded using the health services incident reporting database. Adverse events were classified as severe, moderate, mild, or no harm.Health service utilization and costs for participants in this trial will be reported elsewhere. Subsets of participants enrolled in the current trial had additional measures taken to explore the effects of additional rehabilitation. Physical activity levels were monitored ,30 and iBased on one of the primary outcome measures (length of stay), a sample size of 712 participants was estimated in the trial protocol . To recrAnalysis of covariance (ANCOVA) was used to analyze between-group differences in discharge (primary endpoint), 6-month and 12-month scores with baseline scores as covariate . IntentiOver a 12-month period 1,225 eligible patients were admitted to rehabilitation at the 2 sites. A total of 996 patients provided informed consent to participate and were randomized to receive either Monday to Saturday rehabilitation (intervention) (n = 496) or Monday to Friday rehabilitation (control) (n = 500). Recruitment rates were higher than originally expected and the project steering committee decided to stop recruitment earlier than planned as it appeared that the target sample size would be reached prior to 18 months. Without any interim analyses being performed, it was decided to stop recruitment at 12 months. The primary outcome measure of length of stay was obtained for all participants (100%) on discharge. By the end of the trial (12-month follow-up) 106 participants had died . In all, 86% of participants (852 of 996) were available for follow-up at 6 months and 82% (813 of 996) at 12 months  age of 74 13) years and 637 (64%) were women (Table 3 years aParticipants in the intervention group received a mean of 53 more minutes of rehabilitation therapy (95% CI 31 to 74) per week compared to the control group. A total of 457 (92%) participants in the intervention group and 8 (2%) participants in the control group received at least 1 session of additional Saturday rehabilitation.From available data, assessors correctly guessed group allocation on discharge 55% of the time.P = 0.01), and possibly at 6 months , but not at 12 months  compared to the control group (Table Participants in the intervention group had higher FIM scores on discharge (mean difference (MD) 2.3, 95% CI 0.5 to 4.1, P = 0.009) on discharge and possibly at 6 months  but not at 12 months  when compared to the control group. Participants in both groups scored similarly on the EQ-5D VAS at discharge, 6 months and 12 months  compared to the control group, with length of stay reduced from a mean of 23 (SD 20) days to 21 (SD 16) days. Few participants were discharged on a weekend day; 15 participants in the intervention group and 11 participants in the control group.The intervention group may have had a shorter length of stay by 2 days . In total, 88% of participants who were living independently in the community prior to their admission returned to their previous living accommodation; there were no differences between groups in terms of discharge destination  or need for follow-up outpatient or community allied health services .There were no significant differences between the groups in PC-PART, modified Motor Assessment Scale, or timed up and go test at discharge Table . The intNo serious adverse events occurred during the additional Saturday rehabilitation. There were a total of 240 adverse events reported during inpatient rehabilitation. Adverse events included non-injurious falls  and minor medical issues such as skin tears . No adverse events were classified as causing serious harm and two were classified as causing moderate harm . Participants in the intervention group had an observed adverse event rate of 19% less than participants in the control group , but this did not reach statistical significance.During inpatient rehabilitation, providing additional allied health services helped patients to get better quicker. Patients who received additional Saturday rehabilitation were discharged at a higher level of functional independence and with higher health-related quality of life than those who received Monday to Friday rehabilitation despite being discharged home sooner. The likely reduction in length of stay did not come at the expense of poorer discharge outcomes. Participants who received Monday to Saturday rehabilitation were just as likely to be discharged home  and just as likely to need follow-up outpatient services on discharge compared to those in the control group. These results confirm findings from a systematic review about the benefits of providing additional therapy  and add In this trial, patients who received Monday to Saturday rehabilitation did not receive a great deal more rehabilitation  than patients who received Monday to Friday rehabilitation but this additional rehabilitation did improve outcomes. The amount of additional rehabilitation was somewhat less than the expected, which could be due to missed sessions of therapy as a consequence of feeling unwell, day leave on a Saturday or because patients were admitted late in the week and had not been recruited, assessed and randomized to be scheduled for weekend therapy. However, the additional rehabilitation provided did improve outcomes.Rehabilitation in the form of physiotherapy and occupational therapy typically focused on task specific training and discharge planning. This additional rehabilitation alone may have been enough to improve outcomes if patients made gains during the extra sessions of therapy. However, other factors may have also contributed to improved outcomes. Patients who received Saturday rehabilitation did not have a 2-day break in therapy, which may have reduced time for functional decline due to inactivity. Analysis of the physical activity levels of a subset of participants in the current trial found that those receiving Saturday rehabilitation were more physically active on both days of the weekend compared to those who received Monday to Friday rehabilitation . In addiWe also found that benefits in functional independence and health-related quality of life gained from additional weekend rehabilitation may have been maintained for up to 6 months post discharge suggesting that the more successful outcome achieved during rehabilitation may have had ongoing effects. Most improvement occurred during inpatient rehabilitation when therapy was being provided with only relatively small gains following discharge Table . PreviouThere were no significant differences between groups in terms of most secondary outcomes, including the timed up and go test, PC-PART and the modified Motor Assessment Scale. This may reflect the goals of rehabilitation where interventions were focused on improving overall functional independence for discharge back to living in the community rather than specific activities such as balance, walking speed or upper limb function.Recent debate has highlighted the issue of weekend healthcare provision and the benefits and difficulties in providing weekend healthcare ,9. Our tpost hoc analyses are discouraged [This trial included participants with a variety of health conditions requiring rehabilitation, non-English speaking participants, and participants with cognitive impairment making the results generalizable to many metropolitan inpatient rehabilitation facilities. A limitation is that subgroup analyses were not planned or completed, therefore we do not know if the results are particularly applicable to patients with certain diagnoses. However, our trial was not powered for subgroup analyses and such couraged . In addicouraged ,45. Anotcouraged .Providing additional allied health services  on Saturdays during inpatient rehabilitation helped patients to regain their functional independence faster. Future research could focus on the dose–response relationship of additional weekend rehabilitation services, and explore whether the additional amount of rehabilitation therapy or reducing the consecutive amount of time without rehabilitation therapy improved outcomes.ANCOVA: Analysis of covariance; CI: confidence interval; EQ-5D: EuroQoL five dimensions questionnaire; EQ-VAS: EuroQoL questionnaire visual analog scale; FIM: Functional independence measure; MD: Mean difference; PC-PART: Personal Care Participation Assessment and Resource Tool; RR: Risk ratio.The authors declare that they have no competing interests.NS, NB, JW and NT were responsible for study concept and study design and CP contributed to study design. CP and NB were responsible for data collection. CP, NS and NT were responsible for data analysis and data interpretation. CP wrote the first draft of the manuscript and CP, NS, NB, JW and NT contributed to writing the final manuscript. CP and NT are the guarantors. All authors read and approved the final manuscript.


Demographic ageing will lead to increasing pressure on visual rehabilitation services, which need to be efficiently organised in the near future. The Dutch ICF Activity Inventory (D-AI) was developed to assess the rehabilitation needs of visually impaired persons. This pilot study tests the feasibility of the D-AI using a computer-assisted telephone interview.In addition to the regular intake, the first version of the D-AI was assessed in 20 patients. Subsequently, patients and intake assessors were asked to fill in an evaluation form. Based on these evaluations, a new version of the D-AI was developed.Mean administration time of the D-AI was 88.8 (± 41.0) minutes. Overall, patients and assessors were positive about the D-AI assessment. However, professionals and 60% of the patients found the administration time to be too long. All included items were considered relevant and only minor adjustments were recommended.The systematic character of the revised D-AI will prevent topics from being overlooked and indicate which needs have the highest priority from a patient-centred perspective. Moreover, ongoing assessment of the D-AI will enhance evaluation of the rehabilitation process. To decrease administration time, in the revised D-AI only the top priority goals will be fully assessed. Using the D-AI, a rehabilitation plan based on individual needs can be developed for each patient. Moreover, it enables better evaluation of the effects of rehabilitation. A larger validation study is planned. As a result of demographic ageing, the number of people with irreversible visual impairments is expected to rise in the coming decades ,2. This However, MRCs lack an instrument which examines and prioritises specific rehabilitation needs in a systematic way. Such an instrument would also enable more effective evaluation of rehabilitation outcome. Therefore, we developed an adapted and extended Dutch version of the Activity Inventory (AI) by Massof et al. ,24, the MRCs in the Netherlands are planning to make their intake procedure more structured and objective and showed interest in using the D-AI for this purpose. Because implementation of a new intake method needs to be performed accurately and gradually, an important step in this process is a feasibility study; however, this type of study shows considerable variety in both purpose and methods -30.To investigate rehabilitation needs, this limited pilot study (as part of a longer-term investigation): i) tests the feasibility of the D-AI using a computer-assisted telephone interview (CATI), ii) determines whether the most relevant topics are included, and (iii) tests whether all questions and answer categories are clear and satisfactory. For this purpose, patients and assessors were asked about their perceptions of and experiences with the D-AI using a CATI.To assess feasibility of the questionnaire in visually impaired elderly, eligible participants had to be at least 50 years old, speak adequate Dutch, and have sufficient cognitive ability to participate. Persons with low vision from any cause were allowed to participate.Patients were recruited after enrolment at a specific location of an MRC. All patients met the study criteria as defined in the evidence-based guidelines on the referral of visually impaired persons to low-vision services in the Netherlands . FollowiA random sample of 32 patients was taken from patients who had enroled between September and November 2007. Data collection took place before the start of rehabilitation (i.e. between November 2007 and January 2008).The study protocol was approved by the Medical Ethics Committee of the VU University Medical Center Amsterdam and was consistent with the principles of the Declaration of Helsinki. All patients provided written informed consent.The D-AI was administered by four different assessors, including the first author who trained the other three assessors. These three assessors were employees of the MRC where the pilot study took place and were usually involved in the intake process. After the first interview was conducted by the first author, the other three assessors conducted nine, seven and three interviews, respectively.The D-AI consists of many possible rehabilitation goals. Each goal belongs to one of the 'Activity and Participation' chapters of the International Classification of Functioning, Disability and Health (ICF) . AdditioFive hobby-related goals (e.g. creative activities) consist of sub-goals. These sub-goals will only be assessed if the overall goal has a priority score higher than zero. In order to develop the first draft of the D-AI, a literature study was made, and patient records and focus group discussions were employed. This process and the content of the D-AI are described in detail elsewhere by Bruijning et al. .The D-AI and its routing (see above) were programmed using Blaise Enterprise 4.7 , so that it could easily be assessed using a CATI. Depending on the patient's response, the computer automatically displays the following question. Immediately after assessing the D-AI, participants were asked to complete questions about the D-AI and its assessment, with the aim to improve the feasibility of the questionnaire. These questions were: What do you think about the assessment time? How did you feel about completing the questionnaire? What other activities or rehabilitation needs do you suggest should be included? How do you feel about completing the questionnaire by telephone? Do you have any recommendations to improve the questionnaire?The three assessors who were usually involved in the intake process were also requested to fill out an evaluation form about the D-AI. Additionally, the assessors reported each item which they thought was (possibly) misunderstood. Finally, additional recommendations about the content and wording of the D-AI were requested from one expert .Patient characteristics, such as age, visual acuity and eye condition, were collected from the patient's medical files.For all goals, priority scores were calculated, as well as the mean, median and maximum scores of goal importance, goal difficulty and priority scores. Data were analysed using SPSS 15.0. If possible, similar answers on the evaluation forms were combined. Finally, based on the results of this pilot study, each item was discussed and consensus between the first and second authors was reached with the aim to develop an improved version of the D-AI.Of the 32 patients randomly selected to be invited to join this pilot study, 20 (62.5%) agreed to participate. Table The mean time to complete the telephone interview for the D-AI was 88.8  min.The mean number of main goals and sub-goals fully assessed at the task level (with a priority score > 0) was 26.5 (± 15.3). If sub-goals are excluded, this number decreases to 22.7 (± 13.8). For one participant only eight main/sub-goals were fully assessed. The highest number of main goals and sub-goals that were fully assessed was 62.Further analysis of the assessment of the D-AI showed that for 21 main goals and for 38 sub-goals the mean priority score was less than one. There were four main goals  for which none of the participants had a priority score of at least one. To give an impression of the data, Table The three assessors indicated that the D-AI is a more objective way to investigate rehabilitation needs and that it is a practical instrument to systematically assess the most important and difficult goals and tasks for patients. No topics are overlooked and the quality of the intake is less dependent on the characteristics of the intake assessor. Patients and assessors thought there was no need to include or delete any specific item. However, they felt that some questions needed rephrasing to improve the clarity. For example, some assessors reported that in many cases the goal 'social activities and trips' was initially interpreted as 'social events' or as 'going on holiday'. Additionally, 'watching TV or film ' appeared to be too similar to 'watching TV'. Also, the goal 'using specific ICT tools' was reported to be misunderstood. One assessor suggested that is would be useful to establish what percentage of the interview was completed during the CATI.All three assessors agreed that the D-AI interview took too much time, which was 'uncomfortable' and 'unpractical'. Furthermore, they noted that some questions showed up two or more times, which could be annoying for the participants. Moreover, they suggested to include 'not applicable' as an answer category for the question on goal importance. Additionally, one assessor reported that the systematic character and multiple-choice options of the questionnaire made spontaneous conversation difficult and thought that the patients may not like this.A summary of the patient evaluation is presented in Table After analysing the results, some minor changes were made to the order of the items. Furthermore, three main goals, 'social activities and trips', 'watching TV or film ' and 'using specific ICT tools' were deleted. Some tasks that belonged to these goals were moved to other goals. For example, the item 'watching news programs' initially belonging to the goal 'watching TV or film ' was moved to the goal 'watching TV'. Finally, some tasks were added, deleted or rephrased. A summary of the results is given in Tables 'Not applicable' means that you usually do not perform this subject, either because of physical conditions other than your eye condition, or because you are satisfied with the help you get concerning this subject.Furthermore, the answer category 'not applicable' has been added to the question on goal importance. Also, an explanation of this answer category is now included in the introduction of the D-AI, as follows: Another important change concerns the structure of the D-AI. The new version consists of two parts. First, for all goals the 'importance' question and - if the goal is at least 'a little important' (importance score: 1) - the 'difficulty' question will be assessed. Priority scores of all goals are then determined and a priority list (PL) composed, in which all goals are ranked from the highest to the lowest priority score. The second part of the questionnaire consists of assessing all tasks that belong to the goals that have the same or a higher priority score as the fifteenth goal of the list (TPL-15). This means that not all goals with a priority score of at least one will be fully assessed. However, this is possible if the goal with the lowest priority score has the same score as the fifteenth goal. The mean number of goals  per person that would have been administered following the new structure (TPL-15) is 18.75 ± 6.3 (range 8-34). Figure The results of this study support the feasibility of the extended and adapted D-AI for use with visually impaired older adults, assessed by a CATI. In general, participants and assessors were positive about the content of the D-AI and both groups reported that the topics in the D-AI covered all relevant rehabilitation needs. As was confirmed by the assessors, the D-AI had some additional important positive qualities: they felt that the D-AI is a more objective way to assess rehabilitation needs, it makes the intake less dependent on the individual qualities of the intake assessor, and prevents important topics being overlooked by the patient and/or the intake assessor.In this population of visually impaired older adults, a CATI was considered to be an appropriate way to assess the D-AI. Moreover, direct data entry limits the non-response rate to questionnaire items, because of the enforcement of answers. Additionally, it facilitates compiling large databases for the items, which will increase possibilities for future analyses . Most paThe threshold of the TPL-15 was chosen after comparing the assessment time with the number of assessed goals. We expect that this cut-off will decrease the administration time by 15 min. More important, we expect it to lower the outliers with a long assessment time and, simultaneously, to maintain a suitable amount of detailed information. However, because the exact threshold of 15 was arbitrary, more studies are needed to establish whether assessing only those tasks that belong to the goals that have the same or higher priority score as the fifteenth goal of the list, is sufficient to investigate the rehabilitation needs of the visually impaired elderly.All assessors and eleven participants reported that some (similar) task questions were asked two or more times (e.g. reading similar items). Examples are 'reading a TV guide' or 'reading a medicine label'; however, these comparable items belonged to different goals . Not all goals will be assessed and, because the difficulty level of these similar items (e.g. reading) might differ in another context, these double items have not yet been excluded. In an upcoming validation study with a much larger population, we will examine these similar items with the aim to include only the most distinct items.Another important change was adding an extra answer category to the goal-importance question. Patients complained that the answer categories did not always reflect how they felt. For instance, the goal 'household tasks' was often described as 'not applicable' because the participant had a housekeeper. Several participants reported that saying 'not important' did not adequately represent their situation.As can be seen in Table Dutch MRCs are willing to adopt a more structured and objective intake procedure and showed interest in using the D-AI for this purpose. The systematic character of the D-AI prevents topics from being overlooked and indicates which needs have the highest priority from a patient-centred perspective. The results in this study show a wide variety of personal rehabilitation needs. This supports the use of an extensive and systematic instrument that enables each individual to receive their own personal rehabilitation plan. Moreover, ongoing assessment of the D-AI will enhance evaluation of the rehabilitation process. Successful rehabilitation will result in lower priority scores because, after rehabilitation, the goals will be less difficult and/or less important, e.g. due to newly learned alternatives or because of increased acceptance of vision loss. This enables more effective monitoring of the individual patient's progress, as well as evaluation of specific rehabilitation programmes.Concerning implementation of the D-AI, we suggest that MRCs link all possible rehabilitation goals to specific rehabilitation programmes and assistive devices. After assessment of the first part of the D-AI , the patient and a professional from the MRC will discuss which rehabilitation options will be feasible and where to start. From this viewpoint, not only the 'Activity and Participation' domains but also the total ICF scheme will be discussed. After this, a rehabilitation plan can be formulated together. At the end of the rehabilitation trajectory, this rehabilitation plan will be evaluated to establish whether rehabilitation is successful and, if necessary, to reformulate the plan.In conclusion, assessment of the extended and adapted D-AI appears to be a promising way to systematically investigate and evaluate rehabilitation needs immediately after entering an MRC. The results of this feasibility study clearly indicate how to improve the feasibility of the D-AI. The small sample size seemed to be sufficient as the results were relatively homogeneous. The findings in this study support the relevance of further developing the D-AI. The next step in this process is a limited patient file study to compare rehabilitation plans based on the regular intake and on the D-AI. To further improve the D-AI and to be able to report important psychometric properties, a larger validation study will also be conducted.D-AI: Dutch ICF Activity Inventory; MRC: Multidisciplinary Rehabilitation Centre; AI: Activity Inventory; CATI: Computer-Assisted Telephone Interview; ICF: International Classification of Functioning, Disability and Health; PL: Priority List; TPL-15: Top Priority ListThe authors declare that they do not have competing interests. Moreover, the sponsor of the study had no role in the design and conduct of the study, the data collection, data analysis, data interpretation, or writing of the report.JB participated in the design, the acquisition, analysis and interpretation of data, and drafted the manuscript. RvN and GvR participated in the design of the study and were involved in drafting the manuscript or critically revising it for important intellectual content. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/10/318/prepub


Recent studies identifying putative truncated androgen receptor isoforms with ligand-independent activity have shed new light on the acquisition of androgen depletion independent (ADI) growth of prostate cancer. In this study, we present a model system in which a C-terminally truncated variant of androgen receptor (TC-AR) is inducibly expressed in LNCaP, an androgen-dependent cell line, which expresses little truncated receptor. We observed that when TC-AR is overexpressed, the endogenous full length receptor (FL-AR) is transcriptionally downmodulated. This in essence allows us to “replace” FL-AR with TC-AR and compare their individual properties in exactly the same genetic and cellular background, which has not been performed before. We show that the TC-AR translocates to the nucleus, activates transcription of AR target genes in the absence of DHT and is sufficient to confer ADI growth to the normally androgen dependent LNCaP line. We also show that while there is significant overlap in the genes regulated by FL- and TC-AR there are also differences in the respective suites of target genes with each AR form regulating genes that the other does not. Among the genes uniquely activated by TC-AR is RHOB which is shown to be involved in the increased migration and morphological changes observed in LN/TC-AR, suggesting a role of RHOB in the regulation of androgen-independent behavior of prostate cancer cells. Like the AD CaP cell line LNCaP, CWR22Rv1 retains a functional androgen receptor (AR) and, as such, is responsive to the presence or absence of DHT. However, in contrast to LNCaP and more in line with advanced CaP cell lines, CWR22Rv1 is not dependent upon the presence of DHT for growth.Prostate cancer (CaP) initially presents as an androgen dependent (AD) disease, but frequently progresses to an androgen depletion independent (ADI) or castration-resistant state. As the latter escapes therapies which target the androgen receptor signaling axis, considerable efforts have been made to more thoroughly understand both the transition to and biology of ADI disease. The most representative v1 has been studied extensively within the context of acquisition of ADI growth. As expected, considerable research has focused on the CWR22Rv1 androgen receptor (AR) which has been shown to carry the common H874Y mutation v1 and the relapsed CWR22 variant xenograft from which it was derived express an AR with a duplication of exon 3, which is accompanied by a high level of truncated AR. These properties are not present in the original androgen-dependent CWR22 xenograft, and we suggested that the truncated receptor may be responsible for the transition to its androgen-independent state. Using antibodies targeting different regions of AR, we mapped the truncated receptor species to be the N-terminal half of the molecule, consisting of NTD and DBD v1 has been found to carry an intragenically duplicated AR locus Because of the unique niche it occupies within the collection of CaP cell lines, CWR22Rv1. Work by Hu et al v1. Guo et al, went on to develop antibodies against AR3 and conclusively identified the presence of the truncated receptor corresponding to AR3. While the direct demonstration of a cleaved product at the protein level is more challenging than the detection of spliced transcripts, none of the studies have excluded the contributions of truncated receptor generated by proteolytic cleavage. Indeed, Hornberg et al Studies by Libertini et al v567es) from prostate cancer xenografts with exons 5, 6, and 7 deleted, which is constitutively active in prostate cancer cell lines The schematic structure of the prototypic AR truncated variants in our reports as well as in other studies is shown in v1 predominates. The biological and transactivational properties of FL-AR and TC-AR can thus be studied in exactly the same genetic and cell background.Most of the previous studies on truncated receptors have focused on their ability to transactivate androgen-responsive genes and to induce androgen-independent growth RHOB, a gene we show to be upregulated by TC-AR, but not by DHT-bound endogenous FL-AR, to be a possible effector of morphological and migration differences during the transition to ADI disease. As the AR forms in these cell lines represent the minimal common domains shared by all functional ligand-independent AR, the data generated by this study will likely be applicable to similarly truncated receptors.To demonstrate the utilization of this cell line, we report the presence of autoregulation of AR expression levels, acquisition of ADI growth, and changes in cell shape and migration following induction of TC-AR. We also extend upon reporter assays involving C-terminally truncated AR forms to show occupancy at an AR regulated promoter and transcriptional activation of an AR regulated gene. Using microarray and qRT-PCR, we report on the common and unique genes regulated by TC-AR and DHT-bound endogenous AR. Lastly, while its effect is not directly involved in ADI growth, we identify 5′-ggatccaatgtaagtgcagttagggctggg-3′; reverse primer: 5′-ctcgagtcatagtttcagattaccaagtttcttcagcttcc-3′) from cDNA derived from CWR22Rv1, TOPO cloned and verified for sequence accuracy. The insert was then excised from the cloning vector using BamHI and XhoI restriction sites and ligated into a similarly digested modified form of pLenti4/TO/V5-DEST (Invitrogen). Subcloning was done such that TC-AR was placed immediately downstream and in frame with sequence encoding the FLAG epitope to produce the lentiviral expression plasmid pLenti4/TO/FLAG-TC-AR.TC-AR was PCR amplified  according to manufacturer's instructions. Briefly, 293T cells were co-transfected with each of three helper plasmids along with the appropriate expression or knockdown plasmid. Lentiviral particle-containing supernatant was then harvested 48-hours post transfection, filtered and applied to the appropriate parental cells which were then cultured in the presence of blasticidin , zeocin  or puromycin . Drug resistant clonal populations were then isolated, expanded and analyzed for gene expression or gene knockdown by western blot. A summary of cell lines reported here and their derivation  is as follows: LNCaP/TR , LN/TC-AR  and LN/TC-AR/shR-RHOB . Lentiviral plasmids encoding shRNA sequence targeting RHOB were purchased from Open Biosystems. Plasmid name with corresponding mature anti-sense region is pLKO.1/shRHOB; AACTCGTCCTTACTGAACACG.LNCaP and 293T cells were obtained from ATCC and cultured in RPMI or DMEM, respectively, both of which were supplemented with 10% FBS and 1% PSG. All cells were cultured at 37C in the presence of 5% COCells were lysed in RIPA buffer supplemented with Complete Protease Inhibitor Cocktail (Roche). Nuclei and cellular debris were removed by centrifugation at 14,000×g for 15 min. at 4 C. Protein concentration was then assayed using the BCA Protein Assay Reagent (Pierce). 100 µg samples were then separated by denaturing SDS-PAGE and transferred to PVDF membranes (Immobilon). Membranes were blocked for 90 min. in 5% NFDM in TBST. Primary antibodies α-FLAG M2 (Sigma-Aldrich), α-AR (PG-21) (Millipore), α-actin (Sigma-Aldrich) and α-RHOB (Santa Cruz) were diluted in 5% NFDM in TBST according to manufacturer's recommendations and incubated with the membranes overnight at 4 C. Membranes were then washed twice with TBST for 15 min. and incubated with either α-mouse or α-rabbit secondary antibody conjugated to HRP (Santa Cruz) and diluted 1∶5000 in 5% NFDM in TBST for 90 min. at RT. Membranes were washed twice with TBST and developed with Supersignal (Pierce) and exposed to Bio-Max X-Ray film (Kodak).LN/TC-AR cells were grown in RPMI 1640 supplemented with 10% FBS/1% PSG and induced with 4 ng/mL doxyxycline for a period of 48 hours with uninduced cells serving as a negative control. Cells were lysed in Pierce's IP lysis buffer supplemented with Halts protease and phosphatase inhibitors (Pierce Biotechnology) and samples were centrifuged  to clear lysate. Supernatant was then transferred to a fresh 1.5 mL centrifuge tube and concentration was determined by BCA assay. An aliquot of cell lysate was reserved as an input control and 500 µg lysate was mixed with 50 uL of a 50% slurry of α-FLAG M2 agarose beads (Sigma Aldrich) and incubated overnight at 4 C with constant rotation. Beads were washed 3× with 1 mL Pierce's IP buffer and eluted with 30 µL 6× sample buffer. Samples were resolved on a 6% polyacrylamide gel and transferred to a PVDF membrane. Membranes were probed with α-AR(PG-21) followed by HRP-conjugated α-Rabbit developed with SuperSignal and imaged on a Cell Biosciences western blot imager.LN/TC-AR cells were seeded to 24-well plates at a density of 60 K/well and incubated overnight at 37 C. The following day, cells were co-transfected with pPSA6.0-luc (180 ng/well) and pH 48-ren (20 ng/well) using Effectene Transfection Reagent (Qiagen) and treated with either DHT or various concentrations of doxycycline. Forty-eight hours post-transfection/treatment, cells were lysed and assayed for luciferase and renella production using the Dual-luciferase Assay Kit (Promega) as recommended by the manufacturer on an EG&G Berthold LB96V MicroLumatPlus microplate luminometer . All sample groups were completed in triplicate  and have been normalized for transfection efficiency using the renella relative light units (RLU) acquired for each sample.LN/TC-AR cells were plated to 6-well plates with one slide in each well and were hormone-depleted for 3 days then treated with 4.5 ng/mL doxycycline treatment or vehicle only as control for 24 hours. Slides were washed with 1× PBS twice and fixed in 4% paraformaldehyde at 4°C for 30 minutes. After three washes with 1× PBS, slides were permeabilized with 1% Triton X-100 in 1× PBS followed by 1% NP-40 in 1× PBS for 10 minutes. Slides were blocked with 2% BSA in TBST for 30 minutes then washed 3 times with 1× PBS. Anti-FLAG M2 antibody was diluted 1∶50 in 2% BSA in TBST and incubated with slides for one hour at room temperature (RT). Slides were then washed with 1× PBS 3 times and incubated with Alexa Fluor 488 goat-α-mouse secondary antibody (Invitrogen) at RT for one hour. Slides were washed 3 times with 1× PBS and mounted with SlowFade and DAPI (Invitrogen). Images were acquired with an Olympus fluorescent microscope using appropriate filter sets.5 cells/well in RPMI1640 supplemented with 10%CDT-FBS, 1%PSG. After 24 hours, cells were treated with DHT 1 nM, Dox 4.5 ng/mL, Dox 20 ng/mL and vehicle only as control. Medium was refreshed every 72 hours. Individual treatments were in duplicate. At the reported time points, cells were washed gently with PBS and trypsinized 2–3 minutes at RT. Total cells per well were determined via Countess® Automated Cell Counter (Invitrogen) according to the manufacturer's protocol.Cells were plated to 6-well plates at a density of 2×10Cells were plated to 24-well plates at a density of 20 K cells/well in RPMI1640 supplemented with 10%CDT-FBS, 1%PSG. After 24 hours, cells were treated with DHT 1 nM, Dox 4.5 ng/mL, Dox 20 ng/mL and vehicle only as control. At the designated time points, 30 µL MTT labeling reagent was added to each well and incubated 4 hours at RT. Following the four hour incubation, 300 µL of the Solubilization Solution was added to each well and the plates were incubated overnight. The following day, absorbance of the formazan product was measured at A570 on a microplate reader.5 cells were plated into each insert in RPMI1640 with 0.5% BSA and treated with DHT 1 nM, Dox 4.5 ng/mL, Dox 20 ng/mL and vehicle only as control. Individual treatments were completed in duplicate. 500 µls of RPMI1640 supplemented with 10% FBS was added to bottom chambers and cells were incubated for 48 hours. Cells/media from the top side of each insert were removed and inserts were incubated in 225 µls of Cell Detachment Solution for 30 minutes at 37°C. 75 µL of Lysis Buffer/CyQUANT® GR Dye Solution was added to each well containing Cell Detachment Solution and inserts were incubated in it for 15 minutes at room temperature. 200 µL of each mixture was added to one well of a 96-well plate and fluorescence absorbance was measured by a fluorescence plate reader with a 480/520 nm filter set.Cell migration assay kit (ECM 509) was purchased from Millipore (CHEMICON) and the commercial protocol from Millipore was followed. Briefly, cells were incubated in RPMI1640 media supplemented with 1%PSG only for 24 hours. 3×10http://david.abcc.ncifcrf.gov/home.jsp) and Gene Set Analysis Toolkit LN/TC-AR cells were treated with DHT 1 nM, Dox 4.5 ng/mL, Dox 20 ng/mL and vehicle only as control in 10%CDT media for 24 hours after 3 days in 10%CDT media. Total RNA was extracted by using QIAshredder and RNeasy Mini Kit and following the protocols from QIAGEN. Two independent experiments were performed and RNA samples were submitted to the UC Davis Cancer Center Gene Expression Resource facility. Microarray analysis was done using Affymetrix Human Genome U133 Plus 2.0 arrays . Analysis of the expression data from the Dox 4.5 ng/mL-treated cells, Dox 20 ng/mL-treated cells and DHT-treated cells versus the vehicle-treated cells was conducted using DNA-Chip Analyzer software. Filter criteria are p≤0.05, signal log ratio ≥0.6 (1.5fold) and present (P) for detection. Gene Ontology (GO) analysis was performed by DAVID Functional Annotation tools (http://www.gene-quantification.de/download.html#qgene). Sequences for each primer set are in RNA was isolated as described in Microarray section. Complementary DNA synthesis was done using SuperScript® III Reverse Transcriptase Kit (Invitrogen) according to the manufacturer's protocol. 2 ng cDNA was amplified by iQ5 iCycler thermal cycler (Bio-Rad) and monitored by SYBRGreen (Invitrogen) for real time PCR. Threshold cycle values were normalized against actin or GAPDH. Individual samples were performed in triplicate and converted to relative gene expression using QGene96 software (7cells/assay) were performed following the University of California Davis Genome Center ChIP protocol (http://genomics.ucdavis.edu/farnham). The primary antibodies used in the assays were α-FLAG M2 antibody (Sigma) and α-RNA polymerase II 8WG16 monoclonal antibody (Covance). KLK3 promoter primer sequences are: 5′-TCTGCCTTTGTCCCCTAGAT-3′ (forward) and 5′-AACCTTCATTCCCCAGGACT-3′ (reverse) ChIP assays (2×107cells/assay) were performed following the University of California Davis Genome Center ChIP protocol (http://genomics.ucdavis.edu/farnham). The primary antibody used in the assays was α-FLAG M2 antibody (Sigma-Aldrich). LN/TC-AR cells were treated with 10 ng/mL doxycycline for 24 hours after three days in RPMI supplemented with 10% charcoal dextran treated (CDT) fetal bovine serum and 1% PSG. Two independent ChIP experiments were performed and α-FLAG M2 antibody was used for detecting occupancy of FLAG-tagged TC-AR on chromatins. One total input, one IgG control and two LN/TC-AR ChIP samples were collected and sent to the UCD Cancer Center Gene Expression Resource Facility for hybridization ChIP assays (2×10v1 cell line) was achieved when cells were cultured in complete media supplemented with 4.5 ng/mL doxycycline  is expressed following doxycycline induction . Titratiycycline . In subsImmediately apparent in the doxycycline titrations is the inverse relationship between the induced exogenous TC-AR and endogenous FL-AR protein levels . In ordev567es) and FL-AR has been reported As heterodimerization of a similarly truncated AR form  serve as positive and negative controls, respectively. Luciferase production (dependent upon activity of the upstream PSA promoter) was found to be significantly increased in all doxycycline-treated samples relative to untreated control . FurtherKLK3 promoter  only. Representative images were taken of each culture 48-hours post-treatment . As has KLK2, KLK3, KLK4, FKBP5 and TMPRSS2, were among the commonly up-regulated genes, which showed that TC-AR shared some common biological influence with endogenous AR  for detection). There were 45 genes commonly up-regulated and 35 genes commonly downregulated by each of the three treatments . Some weenous AR . HoweverPDZD2, SHROOM3, SOCS2, ACVR1B, STYK1, RHOB, FGD4, SSX2IP, CDC25A, and CHPT1 genes showed no significant increase following DHT treatment. qRT-PCR analysis of these genes showed that each was upregulated in the doxycycline treatment groups relative to the DHT treatment group, thus confirming the results obtained via microarray. Among these genes, RHOB seems to have the most robust upregulation by TC-AR, but not by DHT-bound FL-AR. This, together with its role in cell migration, was the basis for further examination.Based on the microarray data, we acquired a list of genes which were upregulated by both Low Dox and High Dox treatment groups . To confRHOB, a small GTPase, is a member of the Ras-homologous (Rho) gene family, which plays a role in cell motility, apoptosis response and actin organization RHOB  and LN/TC-AR/shR-empty in which the shRNA sequence targeting RHOB has been removed. Western blot analysis of these lines revealed efficient knockdown of RHOB expression even following indirect induction with doxycycline via TC-AR-mediated upregulation .Images of LN/TC-AR/shR-RHOB cells were taken following treatment with 1 nM DHT, Low Dox or High Dox and culture in androgen depleted media for 48 hours. The shape of doxycycline-induced LN/TC-AR/shR-RHOB cells remained the same as DHT treated or control cells . We thenIt has been previously reported that simple overexpression of AR is sufficient to circumvent the normal androgen dependency of the LNCaP cell line v1, and, like ARv567es (but not AR3/AR-V7), TC-AR retains the hinge domain and an intact nuclear localization signal. In order to ensure that these features did not confer unique activity relative to other possible forms of truncated AR, we tested a total of six similar forms of AR which varied in location of stop codon  and the presence or absence of the exon 3 duplication and found DHT independent transactivation of each to be highly similar. We have also generated LN/TC-AR* which expresses an AR form with the same translational termination as TC-AR, but only a single exon 3. This receptor corresponds precisely to the aforementioned AR(Q641Stop) CRPC mutation The truncated form of AR expressed in LN/TC-AR amounts to a “hybrid” receptor designed to be representative of naturally occurring C-terminally truncated AR forms. The stop codon is placed at Q641, the site of a non-sense mutation identified in a metastatic tumor taken from a patient whose disease had circumvented androgen deprivation therapy In establishing optimal expression of TC-AR, we observed the downregulation of endogenous AR following TC-AR induction. It has been previously reported that AR mRNA levels can be downregulated or upregulated by androgen depending on cell and tissue types v567es, co-immunoprecipitation assays revealed no detectable heterodimerization between FL-AR and TC-AR either in a transient transfection assay Most recently, AR auto-repression was found to be due to the assembly of a FL-AR repressive complex containing lysine-specific demethylase 1 at the intron 2 enhancer KLK3 promoter occupancy by TC-AR and RNAP2 in the absence of DHT. KLK3 was also found by microarray analysis to be upregulated by TC-AR. Together, these results demonstrate that TC-AR is able to facilitate transcription at an endogenous AR-regulated promoter in the absence of DHT.At a minimalistic level, three criteria must be met for a truncated form of AR to be considered a potential surrogate for DHT-bound FL-AR in the absence of DHT: a) it must retain the ability to localize to the cell nucleus, b) facilitate transcription of AR target genes and, c) “rescue” the effect of growth inhibition. Immunostaining of LN/TC-AR following induction with Low Dox reveals that TC-AR predominantly localizes to the cell nucleus in the absence of DHT. Although a commonly used reporter assay (transient transfection of pPSA-luciferase) served as a first screen of LN/TC-AR, that assay is not representative of gene activation in chromatin. Via ChIP analysis of LN/TC-AR we have shown Lastly, we have shown that TC-AR expression is sufficient to confer ADI growth to the normally AD LNCaP cell line and that this effect is tethered tightly to the physiologically relevant expression of TC-AR. LN/TC-AR induced with 3 ng/mL doxycycline fail to grow in conditions of androgen deprivation while cells induced with 5–10 ng/mL doxycycline show ADI proliferation inversely proportional to doxycycline concentration. This is consistent with recent reports in which the growth-promoting effects of some AR splice variants were mediated through FL-AR Although these basic biochemical properties of TC-AR closely approximate DHT-bound endogenous AR in LNCaP, microarray analysis of regulated genes has shown that, while there is significant overlap between the two as would be expected, there also exist differences in that pool of genes. One explanation for this difference could be the differential assembly of cofactors. Common to both TC-AR and endogenous full-length AR is the N-terminal domain (NTD) which serves as the primary binding region for cofactors such as SRC-1 and GRIP1 v567es was assayed BRP44, DHCR24 & HERC3), seven upregulated by TC-AR only  and five common to the transcriptomes of both FL-AR and TC-AR . Also common to both TC-AR and ARv567es is the hinge region and intact nuclear localization signal. This region is potentially a source of variation in these studies as it is not present in AR3/AR7 which instead terminates with amino acids encoded by a cryptic exon.Comparisons of existing transcriptome studies can be difficult given variances in model systems, growth conditions, sample preparation and the wide selection of platforms for such analyses. This is evident in a comparison of the data acquired here with those of two prior studies in which the transcriptome of a similarly truncated AR form, denoted either AR3 Early in our characterization of LN/TC-AR, we found that cell shape changed rapidly following severe overexpression of TC-AR and at a much slower rate and to a slightly lesser degree following induction of physiologically relevant levels of TC-AR. As deregulated cell adhesion of tumor cells, often first observed as a change in cell shape, can lead to cell detachment and promote cell invasion Rho GTPase, RHOA, RHOB and RHOC are known for regulation of the cytoskeleton, cell motility and cell cycle To summarize, we present here a new cell line to study the effect of truncated AR on the progression of prostate cancer to an androgen depletion independent disease. As part of our initial characterization of one such representative form of truncated AR, we have shown that, similar to DHT-bound endogenous full-length AR, the ligand independent truncated AR retains the ability to translocate to the cell nucleus where it is able to bind AREs within chromatin and facilitate transcription of a host of androgen regulated genes. Beyond this similarity, we have identified differences in the targets of transcriptional upregulation with one such difference being the upregulation of RHOB, a gene whose expression is not influenced by DHT-bound full-length AR. As validation of the usefulness of this model cell line, we have shown the upregulation of RHOB to be involved in the increased motility and morphological changes evident in androgen depletion independent growth conferred by the truncated AR.Figure S1LN/TC-AR cells were grown in the presence of hormone depleted media and treated with 4.5 ng/mL of doxycycline (low dox) or left untreated as control for 6 days. Media and doxycycline were refreshed every 3 days. Bright field images were acquired with an Olympus microscope using 20× magnification.(TIF)Click here for additional data file.Figure S2A Western blot showing TC-AR* levels of LN/TC-AR* lines. Doxycycline concentrations are shown above the membrane image. Membrane were probed with α-AR (PG-21) (primary) followed by α-mouse (secondary). Simultaneous with α-AR (PG-21), each membrane was also probed with α-actin (primary) as a control for loading. B Morphology of LN/TC-AR* cell line following induction of TC-AR*. LN/TC-AR* cells were grown in the presence of hormone depleted media and treated with various concentrations of doxycycline (listed immediately above images). At 48-hours post-treatment representative images of each sample group were acquired. C Androgen independent growth of the LN/TC-AR* cell line. Cell count assay showing the growth of LN/TC-AR*. Cells were cultured in androgen-depleted medium that was supplemented with either 1 nM DHT, low dox, high dox or vehicle only. At the designated time points, total cells per well were determined via Countess® Automated Cell Counter. D Luciferase assay showing androgen-independent activation of TC-AR* Luciferase assay showing that TC-AR*-mediated transactivation is not dependent upon androgen within the context of the LN/TC-AR* cell line. LN/TC-AR* cells were co-transfected with pPSA6.0-luc and pH 48-ren in hormone depleted media and treated with low concentrations of doxycycline (listed below the graph) and either 1 nM DHT or vehicle as control for 24 hours. Fold induction is reported relative to uninduced LN/TC-AR* cells grown in the absence of DHT.(TIF)Click here for additional data file.Figure S3AR mRNA in LN/TC-AR cultured in androgen depleted medium decreases following induction of TC-AR. Graph shows data obtained from two separate microarray probe clusters targeting the LBD and 3′UTR of AR. Neither region is present in TC-AR thus ensuring analysis of only endogenous AR mRNA. Cluster 211110_s_at contains a mix of 11 probes spanning nucleotides 3386–3950 (Exon 5 to 3′UTR) and cluster 211621_at contains a mix of 11 probes spanning nucleotides 4010–4251 (3′UTR). All samples for microarray analysis were prepared as described in the main text. Nucleotide positions are based on NCBI Reference Sequence NM_000044.3.(TIF)Click here for additional data file.Figure S4Quantitative Real Time PCR Analysis of AKT1, CDC20 & UBE2C. qRT-PCR was performed as described in (TIF)Click here for additional data file.Table S1Genes up-regulated by DHT, Low Dox and High Dox treatments(XLSX)Click here for additional data file.Table S2Genes up-regulated by Low Dox and High Dox treatments(XLSX)Click here for additional data file.Table S3Sequences of primers for q-RT-PCR(XLSX)Click here for additional data file.


Simple sequence repeat (SSR) markers are widely used tools for inferences about genetic diversity, phylogeography and spatial genetic structure. Their applications assume that variation among alleles is essentially caused by an expansion or contraction of the number of repeats and that, accessorily, mutations in the target sequences follow the stepwise mutation model (SMM). Generally speaking, PCR amplicon sizes are used as direct indicators of the number of SSR repeats composing an allele with the data analysis either ignoring the extent of allele size differences or assuming that there is a direct correlation between differences in amplicon size and evolutionary distance. However, without precisely knowing the kind and distribution of polymorphism within an allele (SSR and the associated flanking region (FR) sequences), it is hard to say what kind of evolutionary message is conveyed by such a synthetic descriptor of polymorphism as DNA amplicon size. In this study, we sequenced several SSR alleles in multiple populations of three divergent tree genera and disentangled the types of polymorphisms contained in each portion of the DNA amplicon containing an SSR. The patterns of diversity provided by amplicon size variation, SSR variation itself, insertions/deletions (indels), and single nucleotide polymorphisms (SNPs) observed in the FRs were compared. Amplicon size variation largely reflected SSR repeat number. The amount of variation was as large in FRs as in the SSR itself. The former contributed significantly to the phylogenetic information and sometimes was the main source of differentiation among individuals and populations contained by FR and SSR regions of SSR markers. The presence of mutations occurring at different rates within a marker’s sequence offers the opportunity to analyse evolutionary events occurring on various timescales, but at the same time calls for caution in the interpretation of SSR marker data when the distribution of within-locus polymorphism is not known. Simple sequence repeats  Citrus data represent sequences of individual trees each belonging to a different species, variety or cultivar; the Jacaranda data were collected in four different sampling sites considered as four distinct populations; and the oak data came from a single mixed stand of sessile/pedunculate oaks . The Citrus, Jacaranda, and Quercus data sets were submitted to partially different types of analyses according to the structure of each data set.Three data sets were utilized in this study. The structures of the three data sets analysed here varied in the following ways: the Citrus species being derived by natural hybridization from ancestral species, alleles were chosen to minimize known hybrids from the data set of Barkley et al. Citrus accessions. The individual identity of each accession  is given in the Supplementary Jacaranda data were obtained from three sites in French Guiana  and one in Brazil (Tapajos)  . The QueJacaranda markers were obtained following the method developed for SSR markers in tropical crops by Billotte et al. RsaI, followed by an enrichment step in (GT)n and (GA)n repeats by hybridization of cleaved fragments with biotin-labelled (CA)8 and (CT)8 oligoprobes and capture of the selected sequences with streptavidincoated magnetic beads. Those fragments were ligated into a pGEM-TEasy vector , and plasmids were then used to transform competent XL-1 Blue strain Escherichia coli . Positive colonies were tested using colony polymerase chain reaction (PCR) to check the presence of inserts. Amplified fragments were then transferred from an agarose gel onto a N+ Hybond membrane for SSR presence screening by hybridization with labeled γ−32 P (GT)15 and (GA)15 oligoprobes. One hundred and thirteen bacterial clones containing plasmids with inserts that gave strong hybridization signals, with sizes ranging from 500 to 1000bp, were selected and cultured. Plasmids were extracted and sequenced using the universal T7 primer on an automated ABI 377 capillary sequencer . After discarding duplicates, hybrid clones and clones with the SSR region too close to either end of the sequence, thirteen sequences were suitable for primer design and allowed a successful design using Oligo 3 software . Upon screening for polymorphism and clarity of PCR patterns, three primer pairs were chosen for subsequent analyses. Primer sequences, annealing temperatures and GenBank/EMBL accession numbers are shown in Taq buffer, 0.26 mM of dNTP, 0.03 U/µL Taq DNA polymerase  and 0.54 µM of each primer . For Jc3A10 primers, 0.3% BSA was added. An initial denaturation at 94°C for 5 min was followed by 35 cycles of 94°C for 30 s, annealing temperature for 30 s and 72°C for 30 s, and a final extension at 72°C for 10 min. Genotyping was performed using fluorescently labeled primers  in the previous PCR protocol and fragments were separated on an ABI 3130XL capillary sequencer  using ABI POP4 and Applied Biosystems LIZ-500 as internal standard, following the manufacturer’s instructions. DNA was bidirectionally sequenced directly from PCR amplification products. When necessary, gametic phase was determined in heterozygotes by cloning amplicons and sequencing at least one allele per individual following the TA cloning kit protocol from Invitrogen . PCR products were ligated with the plasmid pCR®2.1 and used to transform competent DH5 cells. After an overnight incubation at 37°C, white colonies were isolated. The presence of an SSR allele was checked by PCR with universal M13 primers. PCRs consisted of 1 µL of cultivated colonies, 1× Taq buffer, 0.125 mM dNTPs, 0.025 U/µL Taq DNA polymerase  and 0.83 µM forward and reverse primers. Cycling conditions consisted of 94°C for 5 min; 35 cycles of: 94°C for 1 min, 50°C for 1 min, 72°C for 2 min; and one cycle of 72°C for 5 min. PCR products were checked on a 4% agarose gel in 0.5× TAE. Plasmids were sequenced with the ABI BigDye® Terminator V3.1 kit  following the manufacturer’s protocol. Cloning products were diluted 1∶40, purified with ExoSap-IT , and separated on an ABI 3130XL capillary sequencer . All sequences were aligned and edited using CodonCode Aligner V1.6.3 .Citrus, Jacaranda, and Quercus), each allele was characterised by the following information: (a) amplicon size variation, (b) SSR variation, (c) FR sequence variation and (d) amplicon sequence variation. Each of these portions of information conveyed by SSR marker data was analysed as a separate source of variation. Results were compared across data sets to assess their evolutionary information content. Indels were coded as SNP mutations to represent them as single mutational events.For each data set (A) or haplotypes (h), Nei’s genetic diversity (eH), number of SNPs and indels) were recorded for each source of variation. Linkage disequilibrium (LD) was computed for FR sequence variation.Levels of polymorphism  for all data sets, at the level of individual alleles; (b) for Citrus loci CCT01 and GT03, which displayed extremely complex repeat sequences; see At the individual-allele level, matrices of pairwise genetic distances were computed between individuals and between alleles based on each source of variation. For amplicon length, distance is represented by difference in length; for SSR length, distance is the difference in number of repeat units; for whole amplicon sequence, distance is the total number of differences, including SNPs, indels and number of repeat units; for flanking region sequences, distance is the total number of SNPs and indels. The correlation between matrices was tested by a Mantel test. LD between all pairs of polymorphic sites in FR sequences was tested with a Markov chain Monte Carlo (MCMC) procedure of 10,000 steps and a burn-in phase of 1,000 steps . Pairwise genetic distances for amplicon and FR sequence variation were computed based on the number of mutational steps between sequences. Pairwise genetic distances for SSR variation and amplicon size variation data were computed as Euclidian distances  to account for their faster mutation rate. Weight choice was based on the observation that SSRs have, in our Jacaranda sample, approximately ten times more alleles than each SNP (see Results section). Considering that rare alleles might be missing from our sample, we estimated a ratio of 1∶20 in allele richness between SNPs and SSRs and used this ratio to establish the relative weight. A hierarchical analysis of molecular variance (AMOVA) was performed to partition genetic variance into within- and among-population components. The significance of variance components was tested by 1,000 permutations ST, RST, and NST matrices were used to construct population-level consensus UPGMA (Unweighted Pair Group Method with Arithmetic Mean) cladograms by averaging genetic distance information of three loci with SplitsTree4 v4.6 At the population level, for Citrus, Jacaranda, and Quercus data sets, respectively. Three, three and one SSRs were genotyped and sequenced for the Citrus, Jacaranda and Quercus data sets, respectively. The lengths of the sequenced fragments varied between 123 and 255 bp , sequence data produced from three divergent tree genera were evaluated. Forty-six, 101, and 47 samples constituted the d 255 bp . WheneveLevels of polymorphism  for each amplicon and each source of variation are displayed in Jacaranda and Quercus data set, An example of the complexity of the polymorphisms observed is shown for markers Jc3H10 and QrZAG30  showed significant LD. For the three SSR loci, LD was very irregular and there was no relationship between LD and sequence distance . For the Jacaranda data set, ten pairs out of 27 (37%) showed significant LD. For marker Jc3F4, the SNP at position 81 was in disequilibrium with two indels and one SNP, all located upstream of the SSR. LD was strongest for Jc3H10, where all three pairs were significant. The relationship between LD and sequence distance was significant . Tests of correlation between LD and distance in bp could not be performed for Jc3A10 and Jc3H10 because only three nucleotide sites were polymorphic . For the Quercus data set (marker QrZAG30), 146 pairs of SNPs and indels out of 325 (44.9%) showed significant LD. In particular, 13 or 14 contiguous SNPs and indels located downstream of the SSR at positions 84–107 formed a disequilibrium block, itself in weaker disequilibrium with upstream polymorphic sites at positions 23, 45 and 46 and another SNP downstream the SSR (position 60).LD was tested for all pairs of SNP and indel sites within the FR sequence variation of each marker . For theWe tested whether different sources of variation conveyed the same information on relatedness among alleles. To do so, genetic distances were computed for each pair of individuals with each source of variation  within each marker, and the correlation between genetic distance matrices was tested by a Mantel test. Among the 36 pairs of matrices, 27 (75%) showed a significant correlation . GeneticJacaranda data set, we tested population genetic differentiation for each source of variation by computing FST, RST and NST as appropriate  and SSR variation the smallest (0.05). RST values were very small (<0.02) and non-significant when computed on both amplicon size variation and SSR variation. On the contrary, NST values were large and significant, with values of 0.50 for FR sequence variation and 0.36 for amplicon sequence variation. In the phylogenetic trees obtained from each combination of source of variation/genetic distance measures, FST and NST provided the same topology for all cases they were applied to, indicating a more or less close link between pairs of populations Tapajos/Saint-Laurent and Counami/Paracou (ST grouped Paracou with Tapajos and Counami with Saint-Laurent (for amplicon size variation) or separated the three Guiana shield populations from Tapajos . A total of seven (dinucleotide and trinucleotide) SSR markers were targeted, including compound and imperfect markers. More genetic diversity was found for amplicon sequence variation than for amplicon size variation, suggesting that much of data polymorphism is neglected when SSRs are described through amplicon size alone. On average, 7.7 SNPs and 3.4 indels were detected in the flanking sequences of each marker. Genetic diversity (eH) ranged between 0.306 and 0.922 for the FRs. LD among SNPs and indels in the FR was generally significant. These results demonstrate that SSR alleles are very often riddled with abundant non-SSR indels, plus vast amounts of SNPs both within and near the repeat itself. Similar genetic variation was previously observed at intra- or inter-specific levels SSR polymorphisms are widely used to infer population history and biogeographic patterns. These inferences rely on assumptions about SSR mutational models, and departures from such models are likely to bias divergence and diversity estimates. SSR alleles have been demonstrated to contain other sources of polymorphism in addition to variation in repeat number. In this study we have characterised the contribution of molecular variation occurring outside SSR repeats to SSR marker variability. Whole allele sequencing from SSR markers was analyzed from three tree genera  a strictly geographical clustering (the three Guiana shield populations cluster and are separated from the Amazonian population), and (b) a pattern derived from independent results on chloroplast DNA divergence . Our results support the latter hypothesis, as proven by the convergent topology of trees obtained with FST and NST independently of the source of variation. The purely geographical hypothesis, on the other hand, is only supported by SSR variation in combination with RST . RST estimates have larger variance than FST and NST, particularly when based on small numbers of loci, and RST values obtained here may be unreliable. Nevertheless, the pattern of RST differentiation closely follows geographical distance. This may indicate that repeat number variation has arisen locally from small numbers of founding alleles and reflects recent population divergence, while variation in flanking regions may follow larger phylogeographic patterns and reflect deeper divergence. If RST estimated can be trusted, all sources of variation converge with independent, chloroplast-based results, except SSR variation itself. Therefore, information derived from the “true” nature of SSR data (SSR variation) is actually not representative of the information conveyed by alleles as a whole. This finding strongly hints that other sources of variability, more correctly described by mutation models that apply to DNA sequences, actually provide the bulk of information carried by SSRs. In other words, phylogeographic information carried by amplicon length is not provided by the SSR (which amplicon length is intended to represent) but by variation in flanking regions. Note that this contrasts with analyses carried out at the individual level (see above), where information on genetic distance match when measured as amplicon size and as SSR size. This issue raises the question of the application of the SMM, or other step-based mutational models, to SSRs, as already discussed by Colson and Goldstein et al.Jacaranda SSR markers and by the Citrus SSR marker GT03 The consequences – and usefulness – of hidden sequence variation are shown when we compare how sources of variation perform in detecting population divergence. This analysis was performed in the Jacaranda data set (but larger numbers of markers may reduce estimation variance and mitigate this problem). Taking sequence variation into account actually adds a significant piece of information to phylogenetic or phylogeographic reconstruction. Population structuring emerges more clearly on the basis of sequence data, and the combination of sequences and SSR variation provides higher resolution. Consequently, genetic distance measures assuming the SMM or related models should be restricted to perfect SSRs with invariable FRs. On the other hand, measures such as FST (for amplicon size data) and NST (for amplicon sequences) seem to be the tool of choice for the analysis of the average, not-so-ideal SSR markers that a population geneticist meets in his or her everyday work.Given the complexity of amplicon sequences described here, one may wonder how to properly exploit SSR data. The weight of polymorphisms other than variation in repeat number cannot and should not be overlooked: identity in allele size does not necessarily indicate identity in sequence content or the number of repeats within same sized alleles. When considering individual alleles, amplicon size correlates well with SSR repeat number. Nevertheless, even if repeat number could be obtained directly, it may provide only weak and inconsistent signals of population genetic differentiation, as shown by the Figure S1Complete alignments of DNA fragments for all SSR markers studied. Marker names are shown above each alignment.(PDF)Click here for additional data file.Figure S2Z-statistics andsummary of Mantel tests for the correlations of pairs of genetic distance matrices within species. Colouring (shading) indicates the level of significance of the test: green (light grey*), P>0.05; orange (grey*), 0.05<P>0.001; red (dark grey*), P<0.001. Identity of DNA fragment sections is reported: simple sequence repeat (SSR) variation; amplicon size variation, flanking region (FR) sequence variation, and amplicon sequence variation. Marker names are displayed above each panel and data sets refer to Citrus (C), Jacaranda (J) and Quercus (Q). Sequences with single nucleotide polymorphisms (SNPs) within the repeat were excluded from subsequent analyses because of the complexity to describe the mutation model of SSR repeats bracketing SNPs.(PDF)Click here for additional data file.Table S1List and details of the sampled sites for the Jacaranda (J) data set.(DOC)Click here for additional data file.Table S2Description, amplification conditions and polymorphism of the nuclear simple sequence repeats (SSRs) analysed in Jacaranda copaia.(DOC)Click here for additional data file.Table S3List and details of alleles for the three data sets. Amplicon sizes, frequencies and number of associated haplotypes are indicated for each allele.(DOC)Click here for additional data file.


AZD3514 inhibits and down regulates the androgen receptor (AR) and has undergone clinical trials in prostate cancer. To provide proof-of-mechanism (POM) in patients, an immunohistochemistry (IHC) method for determination of AR in circulating tumour cells (CTC) was developed and validated.After an assessment of specificity validation focused on intra- and inter-operator reproducibility utilising a novel modification of incurred sample reanalysis (ISR). β-Content γ-confidence tolerance intervals (BCTI) and Cohen’s Kappa (κ) were employed in statistical analysis of results.In a first set of IHC reproducibility experiments, almost perfect agreement was recorded (κ=0.94) when two different operators scored CTC as overall positive or negative for AR. However, BCTI analysis identified a specific bias in scoring staining intensity, where one operator favoured moderate over strong assignments, whereas the reverse was the case with the second operator. After a period of additional training involving deployment of a panel of standardised images, a second set of validation experiments were conducted. These showed correction of the inter-operator bias by BCTI with κ for scoring intensity increasing from 0.59 to 0.81, indicative of almost perfect agreement.By application of BCTI to the validation of IHC, operator bias and therefore poor reproducibility can be identified, characterised and corrected to achieve a level of error normally associated with a quantitative biomarker assay, such as an ELISA. The methodological approach described herein can be applied to any generic IHC technique. The androgen receptor (AR) axis is a major effector in the development and progression of prostate cancer and an important target in the rational drug design of new anticancer agents. Prior tAZD3514 [6-(4-{4-[2-(4-acetylpiperazin-1-yl)ethoxy]phenyl}piperidin-1-yl)-3-(trifluoromethyl)-7,8-dihydro[During the phase I trial of AZD3514 , detection and enumeration of circulating tumour cells (CTC) pre- and post-administration by the CellSearch System was the In the present study an immunohistochemistry (IHC) method has been developed for the determination of AR expression in CTC and subject to assay validation for potential application to the clinical evaluation of AZD3514. CTC were harvested from blood samples collected from the Astra Zeneca Sponsored Clinical Study D1330N00013) by an approach termed Isolation by Size of Epithelial Tumour Cells (ISET), which lends itself more readily to IHC than the CellSearch System. After a30N00013 Whole blood samples (minimum of 10 ml) were collected from 8 different patients entered into the Astra Zeneca sponsored clinical study D1330N00013: A Methodology Study to Assess the Variability of and Effect of Hormone Therapy on (i) Putative Androgen Regulated Gene Expression in Hair Samples and (ii) Circulating Tumour Cell Numbers and Androgen Receptor Expression in Patients with Prostate Cancer. The male subjects were from either Group 1 with localised prostate cancer with no hormonal treatment or Group 2 with locally advanced/metastatic prostate cancer on castration treatment and their characteristics are reported in Table Written informed consent was obtained from all subjects and the studies were ethically approved by the North West 6 Research Ethics Committee (REC) - Greater Manchester South  and the Declaration of Helsinki Principles was followed. The REC reference number for the study was 10/H1013/13. Specimens were obtained by venipuncture into EDTA tubes, stored at 4°C and processed within 4 hr by ISET as described below.Isolation of CTC from whole blood by the ISET technique was performed according to the manufacturer’s instructions . Prior tIn order to determine the level of AR expression in CTC, ISET filters were analysed by IHC as briefly detailed below. Each individual ISET filter was attached by a paper clip to a glass slide for further handling. Filters were rehydrated in Tris-buffered saline (TBS). All steps were conducted at room temperature unless otherwise stated. Antigen retrieval of samples was conducted in a water bath at 99°C for 40 min in the presence of 250 ml antigen retrieval solution . Next, the filters were washed in TBS and incubated with permeabilisation buffer (0.2% triton in TBS) for 10 min. Spots were then placed on a clean side and incubated with peroxidase block (3% hydrogen peroxidase in methanol) for 30 min, after which they were washed in water. Spots were again transferred to a clean slide and incubated overnight with anti-androgen receptor antibody  in a humidity chamber at 4°C. After incubation with antibody, filters were washed twice in TBS followed by a wash in water. Filters were then placed on a clean slide, Anti-Mouse EnVision + Dual Link System-HRP (DAKO) was added to each filter, which was then incubated for 1 hr after which they were washed twice in TBS. Filters were stained for 10 min with DAKO Liquid DAB + Substrate Chromogen System for 10 min after which they were washed in water. Filters were counter stained with CytoBlue  and finally mounted using Faramount (DAKO).Filters were scanned on the Bioview Allegro™ Plus Scanner  in brightfield mode, covering the entire area of the 0.6 cm diameter spot (scan area was set at 0.7 cm diameter). This scan area was then presented digitally as multiple image frames, from which CTCs could be selected. The brown staining intensity representing the level of AR expression was graded by the analyst as follows: negative, 1+ − weak, 2++ − moderate, 3+++ − strong.AZD3514 was received as a kind gift from Astra Zeneca  and used as received. LNCaP and PC3 human prostate cancer cell lines were obtained from the American Type Culture Collection  and cultured according to ATCC recommendations in RPMI medium 1640 containing foetal bovine serum. Prior to drug treatment, LNCaP cells were cultured for 24 hr in phenol-red free RPMI  with 10% charcoal stripped foetal bovine serum (Life Technologies). The cells were then incubated for 24 hr with 10 μM AZD3514 in 1% DMSO vehicle or vehicle alone as a control. After drug treatment the cells were harvested. Whole blood for spiking with cell lines was donated by healthy human volunteers according to an ethically approved protocol (North West 6 Research Ethics Committee). Blood was spiked with either LNCaP cells prior to incubation with AZD3514 (positive control), LNCaP cells post drug treatment or PC3 cells as a negative control and were then processed by ISET and stained for AR by IHC, as described above.In order to evaluate the degree of inter-operator agreement in the assignment of a staining intensity to each CTC Cohen's kappa coefficient was calculated using GraphPad QuickCalcs  based on formula 1 below.Here, Pr(a) is the relative observed agreement among operators, and Pr(e) is the hypothetical probability of chance agreement, obtained using the observed data to calculate the probabilities of each observer randomly assigning each category.Agreement in the staining intensity assigned by different operators was also calculated as a measure of incurred sample reanalysis (ISR) utilising β-content γ-confidence tolerance intervals (BCTI). This statistic yields an upper and lower interval where a specified (β) proportion of measurements will lie with a specified (γ) level of confidence and was calculated as previously reported. In our Specificity of the ISET/IHC technique for the AR in CTC was investigated employing, as controls, human prostate cancer cell lines of known AR status, together with treatment of cells with AZD3514 in order to modulate the levels of the protein. The positive control AR expressing cell line was LNCaP while the negative control was PC3. AR status was confirmed in these cell lines by Western blot analysis utilising the same antibody (AR441) as that employed in the IHC method through the presence of a band at 110 kDa in LNCaP and the absence of a band in PC3  reports on ISR both in the form of precision/imprecision and trueness/bias. Figure After the first validation experiment a programme of staff training was embarked upon. A gallery of 20 IHC images of CTC harvested by ISET from a number of different patients was constructed in order to facilitate a supervised training workshop. Here analysts were presented with 5 different sets of images where each set included an example of a patient CTC expressing AR at weak, moderate, strong and negative staining levels. One such set of 4 graded images taken from the gallery is illustrated in Figure Since the samples proffered to the analysts in the second validation experiment were identical to those proffered in the first, this allowed for a conventional assessment of ISR see Fig. These dFit-for-purpose biomarker method validation defines 5 categories of assay based on readout ranging from absolute quantitation to a nominal positive/negative result,14,24,25In the present paper, method validation was performed on an IHC method for the determination of AR in CTC. While the focus was obviously reproducibility in addition to an assessment of specificity, the issue of inter-operator variability was addressed in a novel manner, through a modification of ISR,31. ThusAs a categorical assay, IHC has a dynamic range restricted to a limited number  of band widths of staining intensity. Nonetheless, in preclinical studies with AZD3514, and utilising the same scoring structure as the present method , IHC was able to demonstrate a dose dependent reduction on AR in tumour tissue, at doses of drug that produced only a modest inhibition of tumour growth. In the The ISET technique is an example of a tumour-marker-independent technology based on filtration through 8 μm pores, unlike the FDA cleared CellSearch System which relies critically on the presence of epithelial markers (EpCam) and antibody directed capture,33. HoweA novel procedure is presented for the fit-for-purpose evaluation of the reproducibility of an IHC method for the determination of AR receptor expression levels in CTC isolated from patients as a biomarker assay utilising a modification of ISR and BCTI for statistical analysis of results. The procedure was employed to identify and correct inter-operator bias in the assignment of a scoring intensity and poor reproducibility, resulting potentially in a reduction of measurement error to a level normally associated with a quantitative biomarker assay, such as an ELISA. The methodological approach could be applied theoretically to any generic IHC method.AR: Androgen receptor; IHC: Immunohistochemistry; CTC: Circulating tumour cells; BCTI: β-content γ-confidence tolerance intervals; K: Cohen’s Kappa; CRPC: Castration-resistant prostate cancer; POM: Proof-of-mechanism; ISET: Isolation by size of epithelial tumour cells; ISR: Incurred sample reanalysis; REC: Research ethics committee.The authors declare that they have no competing interests.JC, KM, CZ, RS and CD were the main authors of the manuscript. CZ developed and validated all programming code utilised in statistical analysis. JC conducted the statistical analysis and interpretation of data. RS and ML performed all the laboratory analysis of samples. NC and TE collected blood samples and clinical data from patients. All authors have read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/226/prepub


The adverse environmental conditions impose extreme limitation to growth and plant development, restricting the genetic potential and reflecting on plant yield losses. The progress obtained by classic plant breeding methods aiming at increasing abiotic stress tolerances have not been enough to cope with increasing food demands. New target genes need to be identified to reach this goal, which requires extensive studies of the related biological mechanisms. Comparative analyses in ancestral plant groups can help to elucidate yet unclear biological processes.SSRLocator software in non-redundant EST databases made for all eleven species chosen for this study. The dimer motifs are more frequent in lower plant species, such as green algae and mosses, and the trimer motifs are more frequent for the majority of higher plant groups, such as monocots and dicots. With this in silico study we confirm several microsatellite plant survey results made with available bioinformatics tools.In this study, we surveyed the occurrence patterns of expressed sequence tag-derived microsatellite markers for model plants. A total of 13,133 SSR markers were discovered using the The comparative studies of EST-SSR markers among all plant lineages is well suited for plant evolution studies as well as for future studies of transferability of molecular markers. In agriculture, productivity is affected by environmental conditions such as drought, salinity, high radiation and extreme temperatures faced by plants during their life cycle, that impose severe limitations to the growth and propagation, restricting their genetic potential and, ultimately, reflecting yield losses of agricultural crops. Although, advances have been achieved through classical breeding, further progress is needed to increase abiotic stress tolerance in cultivated plants. New gene targets need to be identified in order to reach these goals, requiring extensive studies concerning the biological processes related to abiotic stresses. Comparative analysis between primitive and related groups of cultivated species may shed some light on the understanding of these processes.Microsatellites or SSRs (Simple Sequence Repeats) are sequences in which one or few bases are tandemly repeated, ranging from 1-6 base pair (bp) long units. They are ubiquitous in prokaryotes and eukaryotes, present even in the smallest bacterial genomes . VariatiMany studies indicate UTRs as being more abundant in microsatellites than CDS regions . In a stThe characterization of tandem repeats and their variation within and between different plant families, could facilitate their use as genetic markers and consequently allow plant-breeding strategies that focus on the transfer of markers from model to orphan species to be applied. EST-SSR also have a higher probability of being in linkage disequilibrium with genes/QTLs controlling economic traits, making them more useful in studies involving marker-trait association, QTL mapping and genetic diversity analysis .Arabidopsis thaliana (L.) Heynh, 0.37% of maize (Zea mays L.), 3.21% of tiger puffer (Takifugu rubripes Temminck & Schlegel), 0.21% of the nematode Caenorhabditis elegans Maupas and 0.30% of yeast (Saccharomyces cerevisiae Meyer ex. E.C. Hansen) genomes , two ferns (Selaginella spp. and Adiantum capillus-veneris L.), two gymnosperms (Gnetum gnemon L. and Pinus taeda L.) and two flowering plants, a monocot (Oryza sativa) and a dicot . These species were chosen because the amount of available ESTs data in Genbank (NCBI). As these databases may have redundancy, we used the program CAP3 [An exploratory ram CAP3  for MacOSSRLocator [in silico analyses were assessed for occurrence patterns in chosen taxa databases. The same analysis was performed using MISA script http://pgrc.ipk-gatersleben.de/misa/ software to search for SSR occurrences per contig. Several instructions in the algorithm used in SSRLocator resemble those from MISA [SSRLocator's code. Instead of allowing the overlap of a few nucleotides when two SSRs are adjacent to each other and one of them is shorter than the minimum size for a given class as found in MISA and SSRIT, a module written in Delphi language records the data and eliminates such overlaps. For GC content, Perl scripts were used and the results were stored in text files (.txt) for later comparative analyses.Taxa data were loaded into the software RLocator , to inveRLocator . Data rerom MISA  and SSRIrom MISA . HoweverSSRLocator software. This script determined which amino acids were coded by trimer, hexamer and nonamer motifs found in the EST database analysed [For the predicted amino acid contents in the SSR loci, an additional routine script was written in the analysed .SSRLocator software, the Physcomitrella patens EST database was chosen.To validate the frequencies obtained using the http://www.genome.clemson.edu/resources/online_tools/ssr, SSRIT following the SSR categories defined above [This database was run with other SSR search scripts and softwares, such as MISA  and SPUTed above . The resChlamydomonas reinhardtii, Physcomitrella patens, Oryza sativa and Arabidopsis thaliana) was made comparing with the preferencial codon table for each species available at http://www.kazusa.or.jp/codon/. The sequences containing EST-SSR for Physcomitrella patens was submitted to CodonO server [A codon-bias for the model plants included in this research (O server  to confiO server  was usedO server .Physcomitrella patens SSR results were run through a Gene Ontology (GO) assignment database in order to assess associations between SSR loci and biological processes, cellular components and molecular function of known genes. A fasta file with all EST-SSRs found in P. patens was subjected to Blast2GO software and ran against the GO annotated sequences, and the obtained hits were compiled.The in silico all EST-SSR found for the plant ancestral lineage, and for the derivative plant group, represented here by the green algae Chlamydomonas reinhardtii and Arabidopsis thaliana, across the others species EST database used for the present SSR survey. Electronic PCR [Physcomitrella patens EST-SSR sequences to design primers and Chlamydomonas, rice and Arabidopsis as templates. The virtual amplicons resulted for each primer set tested across species were aligned to verify the homology between the amplicons.To verify the potential transferability of this molecular markers we have tested onic PCR  was usedonic PCR  using thin silico studies, including the SSR survey, the electronic PCR and the sequence alignment for selective sites mining and drafted the manuscript. LCM created the SSR script used and participated in the design of the study. ACO conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript.FCV carried out all Patterns of occurrence for dimer SSR motifs in percentage.Click here for filePatterns of occurrence for trimer SSR motifs in percentage.Click here for filePredominant trinucleotide microsatelites motifs loci occurrences per species.Click here for filePredominant tetramers microsatelites motifs loci occurrences per species.Click here for filePredominant pentamers microsatelites motifs loci occurrences per species.Click here for filePredominant hexamers microsatelites motifs loci occurrences per species.Click here for filedN/dS table for the common most frequent motifs for 11 species tested EST databases.Click here for fileEletronical PCR results table.Click here for file