Many patients do not eat and drink sufficiently during hospitalisation. The clinical consequences of this under nutrition include lassitude, an increased risk of complications and prolonged convalescence. The aim of the study was 1) to introduce intervention targeting nutritional care for medical inpatients, 2) to investigate the effect of this intervention, and 3) to investigate the occupational groups' attitudes towards nutritional intervention and nutritional care in general.The design was to determinate the extent to which the protein and energy requirements of medical inpatients were met before and after intervention. Dietary protein and energy intakes were assessed by 72-hour weighed food records. A total number of 108 medical patients at four bed sections and occupational groups in the two intervention bed sections, Aarhus University Hospital, Denmark participated. The intervention included introduction and implementation of nursing procedures targeting nutritional care during a five-month investigation period using standard food produced at the hospital. The effect of intervention for independent groups of patients were tested by one-way analysis of variance. After the intervention occupational groups were interviewed in focus groups.Before the intervention hospital food on average met 72% of the patients' protein requirement and 85% of their energy requirement. After intervention hospital food satisfied 85% of the protein and 103% of the energy requirements of 14 patients in one intervention section and 56% of the protein and 76% of the energy requirement of 17 patients in the other intervention section. Hospital food satisfied 61% of the protein and 75% of the energy requirement in a total of 29 controls. From the occupational groups' point of view lack of time, lack of access to food, and lack of knowledge of nutritional care for patients were identified as barriers to better integration of nutritional care into the overall care provision.There was ample room for improving the extent to which standard hospital food satisfies patients' protein and energy requirements, but implementation of procedures addressing nutritional care were difficult, especially at bed sections with a large staff turnover. Many patients do not eat and drink sufficiently during hospitalisation. Thus, 30–50% of the elderly patients are undernourished ,2 and moThe first aim of this research was to examine to which extent standard hospital food met hospitalised medical patients' protein and energy requirements. The second aim was to introduce nursing procedures focusing on the nutritional care based on the Danish nutritional recommendations for inpatients  to invesThe setting was an endocrinology ward with 49 beds and 3481 patients discharged during 2002 (divided into bed sections IA and IB) and a cardiology ward with 53 beds and 4542 patients discharged during 2002 (divided into bed sections IIA and IIB) . All hosMedical patients' pre-intervention dietary protein and energy intakes were assessed by 72-hour weighed food records  at four Both acute and referred medical patients at all ages participated. The inclusion criteria were defined as: 1) the patient was not placed on a prescribed diet, 2) the patient had no contact with, or had not previously received dietary advice from a clinical dietician, 3) the patient did not belong to an ethnic minority, and 4) the patient was hospitalised for at least five days. Patients with dementia and patients who were severely mentally or physically impaired were excluded.Typical patient diagnoses included acute or chronic lung disease , acute or chronic cardiovascular disease , metabolic disorders  or infectious disease . The nursing staff selected the patients meeting all the criteria. The patients received oral and written information about the investigation underlining the voluntary nature of their participation. Three or four patients from a bed section participated at the same time, providing data for the food records.The occupational groups participating in the focus group interviews were: nurses, health care support staff and nurse aides on day or evening duty from one of the two intervention sections IB and IIB (four interviews), two nurses in charge from the two intervention sections (one interview), three maids from the two intervention sections (one interview), two clinical dieticians from the two intervention sections (one interview), and one catering officer from the kitchen . In total 26 informants participated in eight interviews.2) calculated. The patient was asked about ability to chew and swallow and recorded as 'effortless', 'slight difficulty' or 'with difficulty'. On discharge they ascribed to the meals during hospitalisation was recorded as 'very important', 'of some importance', 'almost no importance' and 'no importance'.Data on patient age, date of and diagnosis on hospitalisation, second diagnosis, oedema, dehydration, body weight on hospitalisation (if measured) were collected by the investigators from hospital records. Body temperature (if fever) was collected from the hospitals records during the 72-hour food recording. Patients' body weights were recorded twice: at hospitalisation (or when they were included in the study) and on discharge. This weighing was standardised according to time of the day, the patients dress and the scales. The changes in body weight during hospitalisation were recorded for the patients not having oedema or dehydration. Patient height was measured and body mass index  and energy (kJ) intake by the investigators. The calculations were based on the data from the recipes used in the hospital kitchen and the Danish Database 'Dankost 2000', which contains data from the Danish food tables .Each patient's physical activity was recorded every hour during three days and nights (72 hours) in the period of food recording. It was recorded whether the patient was 'lying asleep', 'lying awake', 'sitting', 'walking' or 'training'. For walking or training the approximate duration of activity was recorded as a fraction of an hour, and a factor of physical activity was estimated for each 24-hour period . The invOfficial Danish food recommendations for institutions propose that patients with chronic diseases have 1.0–1.5 gram of protein per kilogram body weight depending on the degree of stress metabolism . A factoThe estimated energy requirement was calculated as 'basal metabolic rate' (Harris Benedict equation ) x 'the The mean recorded protein (g) and energy (kJ) intake was compared with the estimated protein (g) and energy requirements (kJ), and the degree (in percent) to which the patient's 24-hour requirements were met.The patient's nutritional status is assessed on admission and during hospitalisation.The nurses in charge from the two intervention bed sections IB and IIB received information a) specifying to which degree the patients' protein and energy requirements were being met before intervention and b) detailing the Danish Recommendations for Hospitalised Patients . In ordeAs recommended by the nurses in charge three forms  with different purpose were made to support the staff in relation to the nutritional care.Form A patient data related to the nutritional care were collected upon admission: height, body weight, BMI, usual body weight and changes in body weight for a defined period (if possible), oedema or dehydration, the date and by whom the patient had been informed about the food service, the result of the first assessment of the nutritional status (result from form B), a short description of 1) the patient's ability to eat and drink, and 2) of the action taken by the staff 3) the date of the next assessment of the nutritional status. The form also allowed room for the results of the next five assessments of the patient's nutritional status.In Form B was to assess the patient's nutritional status/risk score and suggesting the action the staff could take. Actions were performed according to detailed English Standards [The purpose of tandards . The assForm C the estimated record of the patient's protein and energy intake could be calculated and compared to data in the nutritional handbook describing what the food items contained of protein and energy. This handbook contained a standardised description of all meals delivered from the kitchen in household measurements  and the estimated protein (g) and energy (kJ) content.In The investigators convened meetings with the nursing staff and the domestic helpers at the two intervention sections IB and IIB. The rationale of the standard was explained detailed and both oral and written instructions about the use of the forms were given. Four meetings were held in bed section IB and six in bed section IIB. At these meetings problems, ideas, etc. related to the standard and the forms were discussed and adjusted according to these discussions. The investigators contacted the staff in bed sections IB and IIB once or twice a week during the five-month investigation period to give support if wanted.The intervention at the two bed sections had no influence on the food production in the hospital. But before the intervention the kitchen produced a 'unrestricted diet' to all patients not placed on a prescribed diet, which contain about 8250 kJ and 70–80 gram of protein with about 15, 41 and 43% of energy from protein, fat and carbohydrates . During The number of patients required was calculated in the following way:The clinically relevant difference between the average extent to which the patient's protein and energy requirement was met before and after the intervention was estimated to 15% . PatientThe dietary protein and energy intake was calculated as a 24-hour mean (SD) for each patient and for each group (SD) of patients at each bed section. The outcome measure was the percentage degree to which the patient's actual protein and energy requirement was covered compared with his/her estimated requirement. Confidence intervals for the outcome measures were estimated.The effect of intervention for independent groups of patients were tested by one-way analysis of variance (ANOVA) using the SPSS version 9.0. The assumptions of independence, normality and identical variances were fulfilled. Analyses of covariance were described for non-comparable variables for the four patient groups after intervention.An interview guide was designed for each of five occupational groups: 1) nurses, health care support staff and nurse aides (four interviews), 2) charge nurses, 3) maids, 4) clinical dieticians and 5) one catering officer from the kitchen . In the The study fulfilled the declaration of Helsinki II and was approved by the Local Scientific Ethics Committee.Food records were completed for 48 patients before and 60 patients after intervention. Table In table The results of the intervention was different at bed section IB and IIB; The intervention significantly improved the degree to which the energy and protein requirements were met among patients in intervention section IB compared with patients in the control sections IA and IIA (protein p = 0.009 and energy p = 0.010). On average, the former had an intake of 85% of their calculated protein requirement and 103% of their energy requirement. In intervention section IIB, the patients only had an intake reaching 56% of their protein and 76% of their energy requirement. These values were on average much lower than for patients in section IB and they were comparable to those obtained in the control sections IA and IIA. Analysis of co-variance for the non-comparable variables age, patient mobility, BMI, type of diet and number of bed-days showed no significant effect on the outcome measure for the degree of meeting the patients' requirement of protein and energy. The patients ability to chew and swallow, and the importance of the meals to the patients during hospitalisation were comparable in the four groups of patients before and after the intervention.In the control sections the diet met 61% of the patients' protein and 75% of their energy requirements after intervention. These levels were not significantly different from those recorded before the intervention, but 11% and 14% lower than before the kitchen changed the diets.During the intervention period, the nursing staff in bed section IB used the forms for assessing the nutritional care of three patients. In intervention section IIB the forms was used assessing the nutritional care of 17 patients. The patients nutritional status/risk score were not determined otherwise.Analysis of the qualitative data from the eight interviews extracted five templates with questions relevant to an increased risk of insufficient nutritional care:1. Divergent attitudes towards intervention.2. Lack of flexibility during meals.3. Lack of knowledge about nutritional care for patients.4. Nutrition – a subordinate part of the care.5. Lack of recognition of responsibility for nutritional care.Analysis showed that the staff in the intervention sections had not been using the nutritional records systematically. Several nurses thought that the records were too comprehensive and overwhelming. Many mentioned that they had not had the time to learn how to use the records and they were clearly perceived as an extra workload. The nurses in charge mentioned that it was not unproblematic to burden staff with material they did not have the time or resources to read. However, a few staff members, among them two nurse students from bed section IIB, had learned how to use the records. They found that they were utilizable and easy to use.The two nurses in charge had divergent views on the usability of the intervention study. The charge nurse in bed section IIB thought that the intervention had improved their work with the patients' nutrition. The staff had previously accepted that patients would lie without eating for seven to ten days. Intervention caused the staff to use a feeding tube on threatened patients earlier than before the intervention. However, the charge nurse from bed section IB declared that the staff in her section had not shown much commitment to the intervention. The staff had not taken 'ownership' of the intervention study because the decision to participate in the project had not been a staff decision but one taken by the central management. She emphasized that the staff's attitude was rooted in the fact they had to take in new ideas and instructions all the time.Several care providers in bed section IB thought that it was a sizeable extra workload to use the records for recording patients' nutritional statuses and that this had constituted a barrier to their active participation in the process. Other nurses in bed section IB declared that they did not think that it was necessary to continuously register a patient's nutritional status. It sufficed for some nurses to use their 'clinical judgement' and on this basis monitor the patient's weight status. These nurses were not interested in any new initiatives and in tools for nutritional care.The records were not – and are still not – an integral part of the nutritional care in the intervention sections. This impacted on care continuity. The few staff members who had actually been using the records and had been able to identify patients at risk of insufficient nutrition reported that their observations had not been translated into action.Although the food records were only used to a minor extent, the staff generally agreed to the relevance of focusing on the patients' nutrition. Several nurses had not previously paid much attention to the patient's nutrition, but the intervention had made them more conscious of this issue:"I must say that after we have begun to pay attention to the diet, it has become clear to me how important it is. You have always known that it was important, but you do not really expect the patients to be undernourished when they are hospitalised" Nursebefore where they did not pay much attention to the fact that febrile patients constituted a special group at risk of falling into nutritional deficit". The general belief that 'fat is bad' for patients was widespread before the intervention. This belief springs from general dietary recommendations for healthy people. However, the intervention raised consciousness of the fact that public dietary recommendations may be suitable for healthy, but not for ill people.After the intervention the nurses were more conscious of their choice of food rich in energy than "The focus group interviews overall showed that the concept of 'individual nutrition' was not easily introduced in the nutritional practice at the two bed section. The staff was able to offer food five times during 12 hours during a 24-hour period. The duration of the meals was dictated by tight time schedules for maids and the hospital orderly. Lunch and dinner were often served under time pressure. Between the fixed meals, the care providers often lacked the time to offer patients various kinds of between-meals in the form of frozen, heated food. Assuming that nutritional care rests on the efforts of a committed staff, it may be claimed that the very organisation of the food service was counterproductive to individual nutritional care because the staff did not have real opportunities to offer the patients any food outside the fixed meal times. Individual nutritional care was also hampered by the fact that the kitchen ran a 24-hour nutrition schedule. This makes it difficult for the patients themselves to decide which meals they want to eat and hence to involve them in their own nutritional care. This was especially a problem for elderly nibblers.The clinical dieticians disseminated knowledge about nutrition to the staff in the bed sections, e.g. knowledge about a change in diet from 'unrestricted diet' to 'normal diet and 'hospital diet'. However, such knowledge dissemination was obstructed in several ways. A large staff turnover in some sections meant that such knowledge did not stay in the sections. The exchange of permanent nursing staff during a seven month period including the intervention was 9% and 46% in intervention section IB and IIB respectively . It was difficult for the clinical dietician to get through to the entire staff, as those who were willing to listen were those who took interest in the patients' diet:"But those we do see are those among the staff who take active interest. It's the old guard turning up" Clinical dietician"just learning what a 'normal diet' and a 'hospital diet' is takes so much time".Some care providers found that it was time-consuming to acquire knowledge about nutrition. Thus a member of the health care support staff mentioned that Along this line, several staff members, especially nursing assistants and health care support staff mentioned that it would be very useful if they had a resource agent they could ask about nutritional issues. It was possible for the care providers to refer the patients to the clinical dieticians. However, they felt that the dieticians were often so busy giving advice to referred patients that they could hardly assume a role in the daily nutritional care. The clinical dieticians, on their side, indicated that they would like the care providers to involve them more so that they could also give advice to patients who had not been referred. However, it was difficult for the clinical dieticians to be allowed to contribute:"The nurses think that they can manage the patients' nutritional situation. I think that is what they believe today. But if we were there when a question was raised, then they would use us. That's what I think" Clinical dieticianMuch would be gained, according to the clinical dieticians, if the staff knew that the patients' loss of weight during hospitalisation should be avoided and if such knowledge was used in the nutritional care.One nurse put forward the view that recommendations for healthy people also applied to patients. But during the intervention she expressed that she had changed her perception, but she found it difficult to manage nutritional requirements of ill patients and at the same time relate to dietary advice to healthy people, which were also used in the nutritional care of hospitalised patients. The clinical dieticians had also noted that many elderly patients were served the 'normal diet' even if they needed 'hospital diet'. These observations could signal the existence of a gap between the knowledge the nurses had and the knowledge actually needed to asses, among others, which diet suited particular patients best. The introduction of two diets caused some confusion and uncertainty among the occupational groups involved.because it's nutrition and similar things which we must choose not to include when we are busy".The care providers expressed an interest in the patients' nutrition, but also mentioned that they often had to ignore this aspect of care because of their tight work schedule. Some days when they had the time and the resources, they would pay more attention to the patients' nutrition, overseeing for example how much the patients were eating. But on busy days the care providers had to abstain – e.g. from offering the patients an extra portion "Time was a limiting factor in nutritional care. The overall message was that the staff found it difficult to find time for determining the patient's height, calculate BMI, talk with the patient about losing weight, the patient's wishes for diet and his/her possible problems with eating and drinking. Time was hence both a real and an imagined barrier to recording the patients' nutritional status and to including the patients in their own nutritional care.along with laundering and ironing", as mentioned by the charge nurse in section IB. Other care providers in section IB also said that serving food and beverages for the patients was not part of their job:In relation to that several care providers in section IB said that the nutritional care was a secondary priority. Hence, it was not perceived as a part of the care and treatment itself, but rather as a service ""You feel you are in the catering business in some way when you have to wait on the patients" Nursing assistantThis would seem to suggest that for some care providers, nutritional care and the tasks such care demanded was not perceived as a natural part of their care activities. If a part of the staff defined nutritional care and the task of making sure that the patients got enough to eat as a job function outside the normal realm of their occupation this evidently constituted a barrier to an improvement in the patients' nutritional care.The patients' nutrition was hence not a priority area within the overall care work performed by the nurses and it was not an active part of the treatment. Inversely, the nurse in charge in section IIB thought that nutrition should be a first line priority to ensure that the work performed by the other occupational groups could have optimal effect. She pointed, among others, to the restoration of physical strength among stroke patients. She was aware that the work routines and the barriers to knowledge dissemination to other occupational groups was a factor limiting the speed with which changes could be implemented. The nurses in charge' attitudes to intervention and nutritional care were reflected in the attitudes of the rest of the staff.The data paradoxically showed that although nutritional care falls within the nurses' competence area, they only engaged in such care when they had the time to do so. When the nurses were busy, which they often were according to themselves, they gave lower priority to nutritional care. However, continuity of nutritional care was particularly important in nibblers according to the clinical dietician:"It is not always big science or intricate calculations; it's almost just a simple matter of remembering to serve the food to the patient" Clinical dieticianThe results suggest that the occupational groups involved in the food service had different guidelines. The assistant catering officer from the kitchen declared that she adopted a 24-hour approach to the planning of menus and distribution of energy percentages. The care providers prioritised that patients ate the meals they chose from the menus. However, this target was compromised by constraints of time and choice. The maids stuck meticulously to the diet previously decided by the nurses for each individual patient, while the care providers did not. Moreover, contrary to the clinical dieticians, some care providers thought that overweight patients should lose weight during their admission. The dietary change introduced to make some of the patients lose weight was, however, criticized by some of care providers in section IIB. They found that the change to 'normal diet' was not clear to the patients and was an expression of abuse of power, because the patients did not have any choice.The data gave no indications that the involved occupational groups shared a common goal as far as nutritional care was concerned. Inversely, the different groups had different priorities and showed neither insight nor any understanding of the professional competences of the other groups. The clinical dieticians also mentioned that a relatively high staff turnover at the bed sections ran counter to continuity of nutritional care and made it difficult to maintain a high, constant level of nutritional knowledge at the bed section derived through instruction and teaching undertaken by the clinical dieticians.Furthermore, the responsibility for the practical aspects of nutritional care could not be precisely located because many different staff groups were involved. This invariably increased the risk that responsibility was diluted, viz. that the individual care provider loses his/her sense of responsibility and overview of the situation. The staff in the bed sections did not see precise definition of responsibility as a central issue as opposed to staff outside the bed sections who would like to see a clear formal distribution of responsibility for nutritional care with a view to improving communication and procedures."Nutritional care, distribution and orders should be given priority from above. It should not be the maids who should work for this. They are often having all the problems because the care providers have other duties they must see to; so the maids are sometimes doing as best they can; and what else can they do? But it should be a priority coming from the very top" Assistant catering officerThe data suggested that dilution of responsibility was accompanied by an element of responsibility evasion. The care providers are, theoretically, responsible for the patients' nutritional care, but the maids assumed the lion's share of this responsibility in practice. The maids were employed in the maintenance section and therefore had no occupational responsibility for the patients' nutrition. However, the maids were very committed and felt responsible for the patients' nutrition. They found it difficult to accept that they had no guarantees that other staff members would take responsibility for the patients' diet when they were not at work. When the maids were having their weekends, holidays etc., the substitutes would often take over their function. The maids declared that they would be happy to take a more active role in the patients' nutritional care. Through their teaching at the bed sections, the clinical dieticians had learned that the maids in general were showing much commitment, attention and responsibility towards the issue of the patients' nutritional status. Inversely, several care providers found it difficult so see themselves take a more active role vis-à-vis nutritional care.As a measure intended to counteract the dilution and evasion of responsibility, the assistant catering officer suggested that central hospital management should issue a clear statement that the patients' nutritional status was a high priority area that deserved serious attention from all occupational groups. Such a message could also give impetus to a process of clarifying responsibilities and tasks related to nutritional care in all bed sections.Prior to intervention food ingested during hospitalisation on average met 72% of the patient' protein table , and thePatients who were severely mentally or physically impaired were not included in the study of ethical reasons, although they as 'nibblers' did not receive a different form of nutritional care. So the sample is not representative for all the medical patients. If this group of patients had been included the quantitative results probably would have been lower, as described in an other Danish study .The average length of the hospital stay for the patients participating in this study was 23 days. The average length for medical patients in the Aarhus County was six days . This siThe introduction of new diets made a difference both to the patients and the staff at the bed sections. Thus the 'normal diet' had a lower fat energy percentage than the other diets. This meant that patients had to consume a very sizeable diet in order to cover their energy requirement, which was rarely manageable for patients with reduced appetite. The clinical dietician mentioned that they had most frequently met patients with a poor or reduced appetite and a simultaneous need for a diet with a high nutrient density. This observation is corroborated by observations made by other Danish clinical dieticians . The resThe common understanding and recognition of the integration of nutritional care as part of the overall care among all occupational groups is a key prerequisite in an effort to see nutritional care as part of the care for the individual patient ,30,31. ANutritional care fell within the competence of the nurses who were therefore able largely to determine to which extent other occupational groups were allowed to contribute with knowledge about nutrition. The clinical dieticians mentioned that they would like a more extensive dialogue with the other care providers about the patients' nutritional status, but these groups did not welcome such cooperation.One of the nurses in charge found that the intervention had made the nurses pay more attention to nutritional issues including, in particular, patients at increased risk of becoming undernourished. However, it was difficult to translate increased attention into specific nutritional care actions such as recording the patients' nutritional status upon admission by using the special food records. In a study of the relationship between nurses' competences and their knowledge about nutrition and diet in a hospital in the South of England, Lin Perry showed that there was no clear association between the nurses' attitudes, knowledge and actions, as neither knowledge nor attitudes were translated into action . The stuHowever, the fact that the staff entertained views on the importance of the food and the food service did not imply that all groups were committed to seeing nutritional care as an element of the overall care effort. And the intervention study was an external project that was not anchored in the bed section's own staff. So that may explain the moderate reluctance to take active part in the study shown by some of the occupational groups . The effThe clinical dieticians, the maids and the assistant catering officer reported poor communication between patients, nursing staff and kitchen. But the care staff did not share this view.The individual bed sections apparently did not have a clear distribution of responsibilities embracing all aspects of nutritional care. On the contrary, the data suggested that dilution of responsibility was accompanied by an element of responsibility evasion.The degree to which patients' energy and protein requirements are covered undoubtedly varies from hospital to hospital depending on the menus served and the commitment to the nutritional care shown by the care staff and management. However, any food service involves a long chain of tasks and work processes reaching from the kitchen to the patient, and the food service is essentially organised in the same way and priorities are generally the same in all Danish hospitals. It is therefore likely that the problems associated with insufficient nutritional care are of a similar nature in Danish hospitals and some European hospitals . The perThe average intake of energy and protein among hospitalised medical patients did not cover their requirements. Prior to intervention, food ingested during hospitalisation on average met 72% of the patients' protein and 85% of their energy requirement. After changing the diets from 'unrestricted diet' to 'normal diet' and 'hospital diet', the diet on average met 61% of the control patients' protein and 75% of their energy requirements. Intervention allowed a significantly better satisfaction of the patients' protein and energy requirements at one of the intervention sections using standard hospital food. However, the implementation of procedures focusing on nutritional care appeared to be difficult, especially at bed sections with a large staff turnover. Consequently, the results of the study call attention to the existence of barriers to efforts aimed at improving the nutritional care of patients.Introduction of nutritional care as part of the overall care met with barriers among the care providers. Focus group interviews identified these barriers as lack of time, lack of knowledge, lack of contact with resource agents concerning nutrition, lack of commitment, resistance towards a additional perceived workload and resistance towards providing service to the patients. Care providers who wished to provide individual nutritional care saw the very organisation of the food service as an obstacle to their freedom of action and flexibility. The effect of this was that it was difficult to accommodate individual patients' requirements.Occupational groups involved in nutritional care worked on the basis of different perceptions, had no shared target and no clear division of responsibility. Improvement of the nutritional care requires that focus be directed towards the final link in the food service chain.This study showed that nutritional care was a subordinate rather than a coordinate element in the overall care effort. The failure of coordination hinged on dimensions of organisation, knowledge and resource utilisation and it significantly affected the degree to which patients' nutritional requirements were met. An increase in the priority given to nutritional care by central hospital management and a concomitant general change in attitude towards nutritional care is needed and is probably a precondition for achieving a level of sufficient nutrition among hospitalised patients.Karin O. Lassen carried out the research design, the fundraising, coordination of organisational communication, food record planning and implementation, performed the data analysis, drafted the manuscript and is the guarantor of the manuscript. Filip Kruse participated in the designing the focus group interviews, in the data analysis, and as author of the manuscript. Merete Bjerrum participated in the data analysis and as author of the manuscript. Lillian Jensen participate in the planning of the food records, performed the calculation of protein and energy intake and participate in the discussion of the manuscript. Kjeld Hermansen contribute to organisational support and discussion of research design and manuscript. All authors read and approved the final manuscript.None declared.


The  Estrogens and their receptors are important in human development, physiology and disease. In this study, we utilized an integrated genome-wide molecular and computational approach to characterize the interaction between the activated estrogen receptor (ER) and the regulatory elements of candidate target genes.in vitro and in vivo datasets showed that the expression profiles in T-47D and MCF-7 cells are remarkably similar and overlap with genes differentially expressed between ER-positive and ER-negative tumors. Computational analysis revealed a significant enrichment of putative estrogen response elements (EREs) in the cis-regulatory regions of direct target genes. Chromatin immunoprecipitation confirmed ligand-dependent ER binding at the computationally predicted EREs in our highest ranked ER direct target genes, NRIP1, GREB1 and ABCA3. Wider examination of the cis-regulatory regions flanking the transcriptional start sites showed species conservation in mouse-human comparisons in only 6% of predicted EREs.Of around 19,000 genes surveyed in this study, we observed 137 ER-regulated genes in T-47D cells, of which only 89 were direct target genes. Meta-analysis of heterogeneous cis-regulatory regions of these core ER target genes are poorly conserved suggests that different evolutionary mechanisms are operative at transcriptional control elements than at coding regions. These results predict that certain biological effects of estrogen signaling will differ between mouse and human to a larger extent than previously thought.Only a small core set of human genes, validated across experimental systems and closely associated with ER status in breast tumors, appear to be sufficient to induce ER effects in breast cancer cells. That  Estrogens are involved in a number of vertebrate developmental and physiological processes. Human and animal studies have revealed the roles of estrogen receptor (ER) in female and male sexual development and behavior, reproductive functions, and the regulation of the neuroendocrine and cardiovascular systems and bone metabolism . Moleculcis-regulatory elements of target genes either directly by binding to previously described conserved estrogen response elements  or indirectly by associating with AP-1 and Sp1 transcription factor complexes and their respective binding sites [Two ER subtypes, ERα and ERβ, are known to mediate estrogen signaling; and they function as ligand-dependent transcription factors . After tng sites . Co-acting sites . The cycng sites -13. Tissng sites . At the ng sites -20. Tranin vivo relevance in breast tumors, and cis-regulatory element mapping and molecular characterization and confirmation of the interaction between ER and putative target genes. In this study, we took a combinatorial approach to ERα target gene discovery and characterization by using high-density DNA microarrays to obtain a global gene-expression profile of hormone response in ERα-positive (EPα+) breast tumor cells. This included drug treatments that interrogate ER-mediated and translation-independent regulation, integration of additional in vitro estrogen-response data and human breast tumor sample gene-expression data for candidate gene validation and identification of relevant in vivo targets, computational binding site modeling and promoter analysis to map putative ER-binding sites, and chromatin immunoprecipitation (ChIP) to characterize the interaction between ER and the regulatory elements of candidate target genes. Here we present our findings and discuss the insights they provide into the genome-wide architecture of the ER-mediated transcriptional regulatory network and its conservation in cell lines, breast tumors and through evolution.Microarray experiments have been carried out, particularly in breast tumor cell lines, to study alterations in gene-expression profiles in response to estrogen treatment -27. Many+ breast carcinoma cell lines have yielded a number of novel estrogen-responsive genes and demonstrated the utility of such genome-scale technologies in studying estrogen biology. These earlier studies, however, only included anti-estrogen and cycloheximide (CHX) treatments either at a limited number of time points or only in validation assays for a handful of putative responsive genes. Therefore, to map more comprehensively the transcriptional regulatory network regulated by ER and to generate data in an additional ER+ breast tumor cell context for cross-cell line analysis, we treated the estrogen-dependent T-47D ER+ breast cancer cell line with 17β-estradiol (E2) and with E2 in combination with either the pure anti-estrogen ICI 182,780 (ICI) or the protein synthesis inhibitor CHX and performed high-resolution time-course gene-expression analyses  and filtered for ICI sensitivity (p < 0.057) and CHX insensitivity (p > 0.24) by comparing E2-treated samples with E2+ICI and E2+CHX samples to isolate putative ER downstream targets and direct targets, respectively. Figure Estrogen-responsive genes were determined by statistical analysis of expression ratios in E2-treated samples versus the mock-treated controls using a two-tailed paired MYC proto-oncogene on our list is probably because the probe was not represented in the arrays used in our study.For estrogen-responsive genes Figure , the expTo control for the confounding direct effects of ICI and CHX, independent of E2, we also treated the cells with only ICI for 2, 8, 12 or 24 hours, or only CHX for 2 or 8 hours. CHX treatment partially obscured the responses in 4 of 89 genes (4.5%) that met the selection criteria for putative direct target genes by inducing a detectable E2-like effect following either CHX, E2, or E2+CHX treatments . As this result does not rule out these genes as direct targets, we included them in the direct target list, but noted this caveat in Table in vitro studies of estrogen responses, but most of the data, including those from published microarray studies, have come from experiments using MCF-7 cells. Therefore, we were interested in comparing the expression profiles in response to hormone treatment between MCF-7 cells and the T-47D cells used in our studies. For our analysis, we obtained a publicly available MCF-7 hormone and tamoxifen response dataset [A number of breast cancer cell lines have been used in  dataset  from the dataset .+ cell lines share common estrogen-response pathways.Using Unigene cluster IDs from build 166 as the common identifiers between the two datasets, we extracted expression data from 104 of 137 T-47D ER-regulated genes Figure  that werin vitro observations in cell lines reflected biological significance in vivo. To address this, we explored the association between the ER-regulated genes identified in our in vitro analysis and the ER status-associated expression profiles in breast tumor samples. We hypothesized that putative ER target genes should be differentially expressed in breast tumors in an ER status-dependent manner. For example, pS2/TFF1 and cyclin D1, both upregulated by estrogen treatment in MCF-7 cells, were shown to be expressed at higher levels in ER+ tumors [A key question we wished to address was whether the + tumors ,35.p-value < 0.01 false discovery rate, ER+ vs ER-) in human breast tumor samples with respect to ER status. Of the 137 ER-regulated genes  identified in the T-47D study, 44 genes were differentially expressed in at least one breast cancer study , suggesting that the estrogen-responsive pathways represent only a minor part of the ER-status-associated transcriptome in breast tumors. This is similar to observations made previously by Meltzer and co-workers [p = 0.006 by chi-square analysis).A number of breast cancer microarray studies have shown that ER status remains the most important prognostic marker and tumor classifier. Expression data from six breast cancer microarray studies  were-workers ,39. Howe+/ER-) across all samples from the breast cancer studies . This suggests context-dependent regulation of some downstream pathways, which is likely to be different between primary tumors and experimental cell lines. Taken together, we note that these in vitro validated estrogen-responsive genes are also differentially expressed in ER+ primary tumors, and may therefore have direct biological and clinical significance.To compare the expression profiles of responsive and differentially expressed genes, we plotted the average relative expression ratios of each gene  the transcriptional start site , defined as the most 5' nucleotide of the reference transcript in the NCBI RefSeq database, of candidate genes and queried them for potential ER-binding sites. The size and locations of the sequences flanking the start sites were selected because most of the characterized ER-binding sites have been mapped to these regions in known target genes [y-axis for each graph represents the relative frequency of binding-site prediction as determined by the fraction of genes with predicted binding sites over the total number of genes queried; the x-axis represents the number of most significant genes investigated, ordered by statistical significance, for each of the groups of genes . Since short binding site motifs are ubiquitous in the human genome, we asked whether there was enrichment of such response elements in the 3.5 kilobase (kb) upstream windows of responsive genes as compared to unresponsive genes. Enrichment for each motif is represented by a divergence of the relative frequencies of binding-site predictions for putative target genes . The enrichment of putative Sp1 sites in the target genes was more modest but did not reach statistical significance . This is expected as Sp1 sites are quite common in the human genome and additionally function in general transcriptional regulation. We did not observe any enrichment of AP-1 sites (p = 0.66) or the negative control GATA1 sites (p = 0.51). These findings suggest that the ERE is the major response element mediating the specific regulation of ER target genes on a whole-genome scale. We also surmised that although Sp1 and AP-1 binding sites are known to facilitate ER functions in some target genes they are not used as a common ER-targeted cis-regulatory element within the human genome, at least not sufficiently to distinguish target genes from non-responsive genes.For binding-site predictions we used our previously described ERE model  and AP-1s Figure , and twoTo determine the conservation and potential functionality of the predicted EREs, we also examined the same 3.5 kb window in the 5' upstream regions of mouse orthologs of the 89 putative human ER target genes. Seventy-two human-mouse orthologous gene pairs were extracted from the Mouse Genome Database  and the d = 469 bases) between the human and the mouse sequences. In fact, the relative positions of the conserved EREs only differ by an average of 52 bases if the predicted EREs for GREB1 , which differed by 1.7 kb in their relative position, were excluded from the analysis. For the ERE mouse-human pairs with one or more base deviations in their core sequences, there is little conservation in the flanking sequences and in the relative positions of predicted EREs , also known as receptor-interacting protein 140 (RIP140), first identified as an ER-binding protein and a co-regulator of receptor activity [NRIP1 transcript levels were raised following estrogen treatment, and its expression dynamics in the presence of anti-estrogens and CHX were consistent with other primary response genes [NRIP1 as a putative ER target gene that is upregulated by E2, sensitive to ICI treatment and insensitive to CHX in T-47D cells. Furthermore, we detected a conserved perfect ERE at around 700 bases upstream of the TSS, indicating a potential ER-binding site and direct regulation by the activated receptor. The other direct target gene - gene regulated by estrogen in breast cancer 1 (GREB1) - was identified in a subtractive hybridization screen for estrogen-responsive genes in MCF-7 cells. It has no known function and does not appear to share significant homology with any other gene in the sequence databases [GREB1 and the predicted ERE is also conserved in mouse. Given that both NRIP1 and GREB1 have been conserved during vertebrate evolution, we compared the 5' upstream regions from human, chimpanzee, mouse and rat genome sequences to see whether the predicted regulatory element has been conserved in additional murine and primate species. For all of the regions surveyed, we found that the core ERE has been perfectly conserved , are conserved in ERactivity ,45. It wactivity ,47. Prevse genes ,24,26. Iatabases . A perfed Figure . In addiTFF1/pS2 (positive control) and ATP-binding cassette, subfamily A, member 3 (ABCA3), a gene related to other ABC transporters that are thought to be involved in cellular lipid transport and which is a putative ER direct target gene as determined in this and a previous study [NRIP1 and GREB1 was also recently reported in a study of near-consensus EREs in the human and mouse genomes [To determine the role of the predicted ERE as an ER-binding site, we performed chromatin immunoprecipitations (ChIPs) using anti-ER antibodies. In addition to the two conserved EREs, we also included two non-conserved EREs from us study . Forward genomes .We have conducted a genome-wide analysis of E2-responsive genes. Through a strategy of iterative validation using genomics, informatics and experimental biology we have identified and characterized a core set of 89 ER direct target genes out of the 18,912 genes represented on our microarray (0.5%). This set of direct target genes derived from experiments in T-47D cells show very similar behavior in another cell line, MCF-7, and also overlap with genes that can distinguish ER status in human breast cancers. Taken together, these results suggest common underlying mechanisms for ER transcriptional control and define specific genetic components of the ER transcriptional network that are consistent across model experimental and clinical conditions.p = 0.0027) but that binding sites for AP1, Sp1 or GATA1 (which we used as a negative control response element) are not. The ERE thus appears to be the predominant ER transcriptional control element. Moreover, despite definitive experiments showing the ability of AP-1 and Sp1 sites to mediate ER responses [These results emboldened us to decipher the rules and informational framework underlying ER transcriptional control. The anti-estrogen treatment with the ICI drug and preincubation of the cells with CHX allowed us to identify genes likely to be ER direct targets. We extracted extended promoter regions  and determined potential ER-binding sites by using ERE and AP-1 and Sp1 binding-site models ,42. Becaesponses ,47,50-54Alu repetitive sequence that is frequent in the genome [Alu ERE into our analysis, however, dramatically degrades the enrichment of EREs found in direct ER-responsive genes (p = 0.06). This suggests that though such EREs are experimentally functional, they have little impact on the specific ER transcriptional cassette, functioning as no more than 'noise' in the system. This has been confirmed by negative ChIP data for several Alu-ERE sites (data not shown). These observations highlight the potential confounding factors in genome-wide analysis of functionally relevant response elements.Previous investigations have uncovered a functional ERE embedded within an e genome . InclusiOur use of a 3.5 kb window around the TSS to search for relevant EREs captures the majority of known EREs , indicatin silico identification of EREs does not assure their function in an ER response, we selected three new putative direct ER target genes identified by our stringent criteria for further validation. NRIP1, GREB1, and ABCA3 are all genes found to be ER responsive in at least two cell lines; they have a discernable ERE around the TSS, blocked by ICI and not inhibited by CHX, and their expression can discern ER status in breast cancers. Using ChIP we confirmed that the EREs in all three are directly targeted by ER following estrogen stimulation  of the putative target genes identified in this study have no known biological functions  as rigorously tested direct targets of ER regulation and a well-studied ER direct target, TFF1/pS2, we assessed the evolutionary conservation of the validated upstream EREs between human and mouse homologs. Interestingly, we found highly conserved EREs (including flanking regions) only for NRIP1 and GREB1. ABCA3 and TFF1/pS2 both have upstream functional EREs in the human genes but not in their mouse orthologs  orthologous pairs examined showed conservation of ERE sequences between the human and mouse genes. This is remarkable given the 84.7%  identity+ tumors from a database of six breast cancer microarray studies [+ breast tumors, suggesting that the estrogen-responsive pathways account for only a portion of the receptor-positive molecular signature, an observation also noted by others [+ status of primary breast cancers can be accounted for by concordant effects of an activated ER. Interestingly, a number of these differentially expressed genes (around 30%) were expressed in the opposite direction in the cell lines compared to the tumor consensus. We speculate that this may be due either to consistently different profiles of ER cofactors or to an intense expression signature in tumor-associated stromal cells that is opposite to that of the cancer cells. Nevertheless, those genes that are estrogen-responsive in cell lines and differentially expressed in ER+ tumors represent the most promising candidates for further functional analysis.We suggest the relevance of many of these estrogen-response genes to breast tumor biology by showing significant similarities between estrogen-induced expression profiles in MCF-7 cells and the behavior of these genes in ER studies -40. Not y others . Neverthin vitro and in vivo systems. On examination, this core direct target gene list does not predict a unified biological process controlled by ER. Instead, the gene functions would predict a pleiotropic cellular response. By further in silico analysis of the promoter regions, we observed minimal conservation in the cis-regulatory region of the direct estrogen-response genes between humans and mice. This raises the intriguing possibility that the evolutionary processes governing the configuration of transcriptional regulation will be different from those affecting the functional domains of genes. Moreover, we predict that the estrogen response in the mouse will differ significantly from that in the human, but that a small set of ER direct target genes that are highly conserved in their cis-regulatory regions will act as the key effectors of evolutionarily important core ER functions such as sex differentiation.In summary, we have presented an integrated strategy for discovering and characterizing ER target genes, response elements and the transcriptional regulatory network downstream of ER activation. With this approach, we uncovered a universal set of genes that describe the most direct effects of ER and operate across multiple cis-regulatory regions of putative target genes within this core set revealed the enrichment of the ERE sequence motif but not other known ER-binding sites. Of all the predicted EREs in human direct target genes, only a handful (6%) appear to be conserved in mouse orthologs, although both conserved and non-conserved predicted EREs were shown to bind ER in human cell lines. Taken together, these findings suggest the potential for species-specific mechanisms and effects in response to hormone exposure.Estrogen responses in human breast tumor cells appear to be mediated by a relatively small conserved core set of ER-regulated genes. Examination of the 2. For estrogen treatments, cells were washed with PBS and pre-cultured in phenol-red-free DMEM/F12 medium supplemented with 0.5% charcoal-filtered FCS (Hyclone) for 24 h. For time-course experiments, T-47D cells were treated with 1 nM 17β-estradiol  or 1 nM E2 + 10 nM ICI 182, 780 (Tocris Cookson) for the amount of time specified. To determine the primary response, cells were treated with 5 μg/ml cycloheximide  for 30 min before the start of estrogen treatment. Control treatments with ICI  and CHX (2 and 8 h) alone were also carried out for the times specified and at the same concentrations as above. To extract RNA, cells were washed with PBS, lysed in Trizol (Invitrogen) and samples were harvested by additional phenol-chloroform extraction steps as prescribed by the manufacturer.T-47D and MCF-7 cells were maintained in DMEM/F12 (1:1) medium (Invitrogen) supplemented with 10% fetal calf serum (FCS) (Hyclone) at 37°C and buffered with 5% COt-test between matching treated samples and mock-treated controls at each time point and fold-difference cutoff at multiple time points as described and clustered and visualized using the Eisen Cluster and TreeView programs [Microarrays were generated by spotting the Compugen 19 K human oligo library, made by Sigma-Genosys, on poly-L-lysine-coated glass slides. Twenty-five micrograms of each sample total RNA and human universal reference RNA (Stratagene) were labeled with Cy5-conjugated dUTP and Cy3-conjugated dUTP (PerkinElmer), respectively, and hybridized to the arrays using protocols established by the Patrick O. Brown Laboratory . Array iprograms . Gene on+ and ER- tumors. Each individual dataset was analyzed independently for differentially expressed genes by calculating the false discovery rate for each gene [p-value filter at less than or equal to 0.01. ER-status-associated genes were then cross-referenced with the in vitro estrogen-responsive genes via the UniGene cluster ID (build 166). The log-transformed average mean-centered expression values for each statistically significant study were used for visualization. Raw in vitro MCF-7 estrogen response data [A database containing published and unpublished breast cancer expression data was queried for genes whose expression profiles differentiated ERach gene  and settnse data  were downse data . The datp-value. Most significant direct target genes used in the analysis were ranked by the lowest p-values from analysis of E2-treated and control samples, E2 and E2+ICI samples, and E2 and E2+CHX samples. Non-responsive genes were ranked by highest p-values from the same analysis.The LocusLink and RefSeq  databaseTFF1/pS2 ERE: forward CCATGTTGGCCAGGCTAGTC; reverse ACAACAGTGGCTCACGGGGT. NRIP1 ERE: forward, TGCTCCTGGGTCCTACGTCT; reverse TCCCCTTCACCCCACAACAC. GREB1 ERE: forward AGCAGTGAAAAAAAGTGTGGCAACTGGG; reverse CGACCCACAGAAATGAAAAGGCAGCAAACT. ABCA3 ERE: forward, CACCTTCCATCTGTCCAAAG; reverse, CAACCCTGAGGTTTGGGAAC. Actin exon 3 control: forward, AGACCTTCAACACCCCAGCC; reverse, GTCACGCACGATTTCCCGCT. Amplification products were also assayed for specificity by melting-curves analysis at the end of each run. Relative quantifications were carried out by building standard curves for each primer set and using genomic DNA, similar to the input, as the template. Enrichment of ER binding was determined by comparing the relative quantities of anti-ER and control anti-GST products.MCF7 cells were estrogen deprived for 24 h and treated with 100 nM E2 for 45 min before 1% formaldehyde treatment to cross-link the transcription machinery and the chromatin. Immunoprecipitations were carried out overnight with ERα (HC-20) or GST antibodies (Santa-Cruz Biotechnology) and protein A-sepharose beads (Zymed). Washing and extraction protocols were modified from methods described previously  and PCR The following additional data files are available with the online version of this article: the processed raw data for the time course microarray study (Additional data file The processed raw data for the time course microarray studyClick here for additional data fileReplicate 1 of the control microarray study with the ICI and CHX treatments aloneClick here for additional data fileReplicate 2 of the control microarray study with the ICI and CHX treatments aloneClick here for additional data fileThe complete list of all 387 estrogen-responsive genes described in the article with UniGene cluster numbers from build 166Click here for additional data fileExpression profiles of ICI and CHX responsive genes identified in the control experimentsClick here for additional data fileThe corresponding figure legend to expression profiles of ICI and CHX responsive genes identified in the control experimentsClick here for additional data file


MicroRNAs are ~17–24 nt. noncoding RNAs found in all eukaryotes that degrade messenger RNAs via RNA interference (if they bind in a perfect or near-perfect complementarity to the target mRNA), or arrest translation (if the binding is imperfect). Several microRNA targets have been identified in lower organisms, but only one mammalian microRNA target has yet been validated experimentally.We carried out a population-wide statistical analysis of how human microRNAs interact complementarily with human mRNAs, looking for characteristics that differ significantly as compared with scrambled control sequences. These characteristics were used to identify a set of 71 outlier mRNAs unlikely to have been hit by chance.C. elegans and Drosophila, many human microRNAs exhibited long exact matches (10 or more bases in a row), up to and including perfect target complementarity. Human microRNAs hit outlier mRNAs within the protein coding region about 2/3 of the time. And, the stretches of perfect complementarity within microRNA hits onto outlier mRNAs were not biased near the 5'-end of the microRNA. In several cases, an individual microRNA hit multiple mRNAs that belonged to the same functional class.Unlike the case in Drosophila and C. elegans.The analysis supports the notion that sequence complementarity is the basis by which microRNAs recognize their biological targets, but raises the possibility that human microRNA-mRNA target interactions follow different rules than have been previously characterized in  C. elegans and Drosophila [MicroRNAs (miRNAs) are small, ~18–24 nt. noncoding RNAs that are found in all eukaryotes and are cleaved from larger ~70 nt. precursors via the action of Dicer enzyme [reviews: ref. [osophila ,4.Drosophila [C. elegans [Drosophila [Drosophila microRNAs recognize 5–6 nt. common motifs within the target, these motifs are not a general feature of mammalian microRNAs [C. elegans and Drosophila.Five different papers have recently appeared that used computational approaches to predict microRNA targets in osophila -7, and mosophila ,9. Theseosophila ,11. In c elegans  and Drososophila , known microRNAs . Thus, iC. elegans and Drosophila, and identify a short-list of promising candidate microRNA-mRNA target pairs that are unlikely to have arisen by chance.In the present paper, we have performed an unbiased statistical analysis of the manner in which human microRNAs interact complementarily with human mRNAs present in the NCBI human RefSeq database, looking for characteristics that differ significantly as compared with scrambled versions of the same microRNA sequences. The results demonstrate several novel features of human microRNA-mRNA interactions that differ from Population-wide statistical analyses were first carried out by examining the types of complementary interactions that occur between the set of microRNAs listed in Lagos-Quintana et al , and theTo define the types of interactions that can occur by chance, ten independent sets ("replications") of scrambled microRNA counterpart sequences were generated and examined for complementarity with the mRNA population. Our underlying assumption is that scrambled sequences will hit mRNA at random and define the "noise" level in any given situation, whereas microRNA sequences will hit the same number of "noise" interactions plus any true targets. Unless otherwise noted, the scrambled sequences were random permutations of the microRNA sequences, keeping constant the overall nucleotide composition. Because microRNAs have a distinctive nonrandom di-nucleotide composition, we also confirmed that key findings were obtained when using scrambled sequences that had similar di-nucleotide composition to the microRNAs.First, we characterized the length distribution of exact complementarity between the population of mRNAs vs. the set of nonredundant microRNAs (i.e. those that overlapped by 10 or more bases were collected into groups and the longest member of the group was chosen as nonredundant). MicroRNAs produced significantly longer exact "hits" on mRNAs than their scrambled counterparts when G:U matches were excluded (fig. -9) and suggests that about 1/4 of the total hits in this "10+ set" occur upon "true" biological mRNA targets. Our approach is to identify further the mRNAs that represent statistical outliers (i.e. that are unlikely to be hit by chance) within this larger "10+ set" by comparing properties of hits made by the set of microRNA sequences vs. the set of scrambled sequences. At any given parametric value, the number of hits observed in the microRNA set, minus the number of hits in the scrambled set, provides an estimate of the number of true microRNA targets that satisfy that parametric value. We examined three different hit properties – a) exact hit length, b) gapped BLAST score and c) presence of multiple hits – both alone and combined with each other. Starting from the "10+ set" estimated to contain only 26% true targets (see above), we added additional criteria to compile a list of candidates estimated to contain over 80% true targets.As shown in figure The most important single parameter for discriminating hits produced by microRNAs vs. scrambled sequences appears to be exact hit length. At a cut-off of 17 exact hit length, there were 14 mRNAs hit by the microRNA set that satisfied this criterion, vs. an average of 1.9 mRNAs hit by each of the scrambled sequence sets  gave better discrimination power than using any single feature, supporting the idea that they are relevant to identifying biologically relevant mRNA targets. We examined three different combinations of parameter cut-off values: 1) One combination consisted of targets with multiple hits from distinct microRNAs less than 25 bases apart, with at least one exact hit ≥13 bases and with at least one gapped BLAST score ≥185 (not counting G:U). For the next two lists, we scored only exact hits ≥10 bases long and that occurred ≤50 times within the entire mRNA population; this minimized "noise" arising from common or low-complexity target sequences, albeit at the cost of removing some target sequences that are shared within protein families. 2) Criteria required two or more hits from distinct microRNAs ≤100 bases apart, at least one exact hit ≥14 bases and one gapped-BLAST score of ≥190 (not counting G:U). 3) This required hits ≤500 bases apart, at least one exact hit ≥14 bases, and at least one gapped-BLAST score > 89% of the best-possible score including G:U matches (this takes into account the fact that longer microRNAs have greater possible absolute scores than shorter microRNAs). for additional data files including a fully annotated outlier mRNA set, a list of all microRNA hits upon this set (extended with and without including G:U matches), and a list of the nonredundant microRNAs together with their putative mRNA targets.All three approaches produced lists of outlier mRNAs that had overlapping members, shared similar characteristics and exhibited similar discrimination ratios. For simplicity and robustness, these have been combined  into a single list consisting of 71 outlier mRNAs . As well, individual microRNAs hit multiple (up to 17) distinct members of the outlier set, which again happened significantly more often than by chance fig. . These fC. elegans and Drosophila. For example, there was no preference for microRNA hits to be located within 3'-untranslated regions: 5% of hits were located in the 5'-UTR, 1% at the 5'-UTR/coding junction, 67% in the protein coding region, 1% at the coding/3'-UTR junction, and only 26% in the 3'-UTR. This distribution was not significantly different from hits produced by the scrambled sequences. As well, the best microRNA hits upon candidate mRNA targets did not have relatively better target complementarity near their 5'-end: Only 13% of hits had ≥ 7 exact hit length starting at position 1 or 2 relative to the 5'end of the microRNA (vs. 17.5% of hits produced by scrambled sequences).The outlier mRNA set contained very similar types of targets as predicted by previous computational studies -8, inclu), further suggesting that this is likely to be a true biological target for microRNA regulation.MicroRNA 145 is particularly interesting because it hits 17 distinct targets on the candidate list, of which a disproportionate number (6) are in the signal transduction category and three of these are related to GTPase activation (Rho GTPase-activating protein (RICS), G protein gamma 7, and hypothetical protein FLJ32810 – containing RhoGAP and SH3 domains; Table By comparing how the population of microRNAs vs. their scrambled counterparts interact with the population of human RefSeq mRNA sequences, we estimate that the probability of detecting a true microRNA target increases a) as the length of exact complementarity of a "hit" between microRNA and target increases, b) as the overall complementarity of a "hit" increases , and c) as two or more distinct microRNAs hit the same mRNA in closer proximity. Targets in the outlier mRNA set also received more hits per unit length and more multiple hits from distinct microRNAs than expected by chance. Finally, we found cases in which an individual microRNA hit multiple mRNAs that belonged to the same functional class. The analysis suggests that target complementarity is a major factor in identifying biologically relevant mRNA targets: As values of each parameter increase, the difference between the number of hits in the microRNA set vs. the scrambled set increases steadily, and combining all three parameters gives better discrimination power than using any single feature.Drosophila [Drosophila targets: 1) Human microRNAs hit mRNAs with exact hit lengths extending much longer than observed in Drosophila, up to and including perfect complementarity. 2) Human microRNAs hit candidate mRNA targets within the protein coding region about 2/3 of the time.  3) Drosophila and C. elegans was simply obscured by the fact that we searched the large sequence space represented by all human mRNA sequences – the larger the sequence space, the greater the chance that any given target criterion will be satisfied by scrambled sequences, hence the more difficult it can be to detect true targets above the noise level. We agree that this can be a problem using very large sequence databases, such as the human EST database or the entire human genome. As well, using cut-off levels of parameter distributions to define the candidate list probably excludes many true human mRNA targets. However, human RefSeq was demonstrably not too large for our analysis, since very strong trends were observed in a variety of other parameters  Sequences in RefSeq > 20,000 bases long were removed from consideration because they were hit by many, if not all microRNAs, and a few sequences > 15,000 bases long were removed from the final candidate list because they had a relatively high false-positive probability. B) When counting the number of hits over the population of mRNAs, two hits were counted as redundant if the entire region around the hit (plus or minus 25 nucleotides on each side) was identical. C) When counting distinct hits by microRNAs on the same target, two hits were counted as redundant if they shared the same exact hit. This minimized possible artifacts due to overlapping microRNAs, as well as removed cases in which microRNAs hit exactly-repeating sequences within the target. D) In tabulating hits onto mRNA targets, we did not count hits that contained low-complexity sequences as detected by the DUST algorithm encoded by a Perl script provided by Lincoln Stein . E) WhenTo decide whether the number of observed microRNA hits were significantly different from chance, 10 replications of scrambled sequences were used to estimate prediction intervals. The prediction interval allows one to say with 95% confidence that any single new replication of the scrambled set will be below the value of the microRNA set. Prediction intervals were chosen as more conservative and more appropriate than confidence intervals.NS contributed biological expertise, whereas VT contributed statistical and computational expertise. The analyses were carried out together, and both authors read and approved the final manuscript.


The correlation between treatment satisfaction and demographic characteristics, symptoms, or health-related quality of life (HRQL) in patients with gastroesophageal reflux disease (GERD) is unknown. The objective of this study was to assess correlates of treatment satisfaction in patients with GERD receiving a proton pump inhibitor, esomeprazole.Adult GERD patients (n = 217) completed demography, symptom, HRQL, and treatment satisfaction questionnaires at baseline and/or after treatment with esomeprazole 40 mg once daily for 4 weeks. We used multiple linear regressions with treatment satisfaction as the dependent variable and demographic characteristics, baseline symptoms, baseline HRQL, and change scores in HRQL as independent variables.Among the demographic variables only Caucasian ethnicity was positively associated with treatment satisfaction. Greater vitality assessed by the Quality of Life in Reflux and Dyspepsia (QOLRAD) and worse heartburn assessed by a four-symptom scale at baseline, were associated with greater treatment satisfaction. The greater the improvement on the QOLRAD vitality (change score), the more likely the patient is to be satisfied with the treatment.Ethnicity, baseline vitality, baseline heartburn severity, and change in QOLRAD vitality correlate with treatment satisfaction in patients with GERD. The inclusion of patients' opinions in the assessment of interventions has gained greater prominence over the last decades. Regulator agencies now call for the inclusion of patient-reported outcomes (PRO) in clinical trials evaluating pharmaceuticals interventions -4. PRO oWhereas HRQL measures the patient's physical, psychological, and social level of function, treatment satisfaction assesses the patient's attitude towards the treatment, or the extent to which the patient is satisfied or not with the results of the treatment. Thus, treatment satisfaction focuses on the interaction of expectations and preferences for treatments and is defined as the individual's rating of important attributes of the process and outcomes of the treatment experience . Coyne aEvaluating treatment satisfaction may assist healthcare providers in understanding the issues that influence adherence with therapeutic interventions. In addition, treatment satisfaction can be a useful PRO when treatments show similar efficacy because differences in satisfaction could lead to patient preferences for one treatment over another and greater adherence with various treatment regimens.Demographic variables such as age, ethnicity, and gender may influence satisfaction . Older pThe objectives of this study were to assess correlates of treatment satisfaction, including demographic factors, symptoms, and HRQL, as well as change scores in PRO instruments in patients with moderate to severe GERD receiving a proton pump inhibitor, esomeprazole.No statistical determination of sample size has been done since the study is of exploratory nature. We enrolled 249 patients with GERD in 13 gastroenterology practices and four general practices across Canada between March 2002 and March 2003.Included patients were 18 years of age or older and had a diagnosis of moderate to severe GERD and presence of symptoms for three months or longer [Patients completed PRO instruments at the clinic before and approximately 28 days after treatment. The completed PRO instruments included the Quality of Life in Reflux and Dyspepsia (QOLRAD) , the FeePatients rated their satisfaction with treatment on a seven point scale responding to the question: 'How satisfied are you with the study treatment you received?' with the response options: completely satisfied, very satisfied, quite satisfied, no change, dissatisfied, very dissatisfied, and completely dissatisfied.The QOLRAD is a 25-item disease-specific self-administered instrument asking about the impact of heartburn and acid regurgitation on the patient's HRQL during the previous week. The QOLRAD includes questions related to 5 domains; emotional distress, sleep disturbance, problems with food and drink, limitations in physical and social functioning, and lack of vitality. Patients respond to each question on a seven-point scale on which a higher score indicates better HRQL. The psychometric properties concerning validity, reliability, and responsiveness to change are reported elsewhere ,24. The The FT is a visual analogue scale that resembles a thermometer. It is divided into 100 segments with a mark to represent each segment. Its anchors are dead (0) and full health (100) . PatientThis is a 15 item questionnaire designed to quantify HRQL . Each itThe SF-36 contains 36 items that measure 8 dimensions: physical functioning, role limitations due to physical health problems, bodily pain, general health perceptions, vitality, social functioning, role limitations due to emotional problems, and general mental health. This questionnaire has been extensively tested for validation and reliability . Each doTo assess common symptoms in GERD, patients evaluated their heartburn, acid reflux, stomach pain, and belching for the past week using a seven-point scale ranging from no discomfort to very severe discomfort.The SG involves decision in the face of uncertainty, where in the standard administration the uncertainty involves a risk of death. The SG offers the patients two alternatives from which a choice must be made: Choice A is a hypothetical treatment with two possible outcomes: 1) returning to full health (probability p) for t years, at the end of which they die, or 2) immediate death (probability 1 – p). The alternative (choice B) is a certain outcome that he or she will stay in a health state  for t years until death. t varies depending on the patient's age. The interviewer used a change board with the ping-pong approach varying the probability p in steps of 0.05 to find the value p where the respondent considered choice A = choice B. This value of p is the utility value for the health state in choice A in the interval from dead (0) to full health (1). The greater a patient's willingness to accept the risk of a worse outcome (e.g. dead) to avoid the health state in choice A, then the lower is the utility of the state in choice A to them.Patients documented the severity of overall upper GI symptom on a seven-graded scale  over the past seven days. At baseline, patients who had no, minimal or mild symptoms were not included in this study.We calculated the mean and standard deviation of the basic demographic variables. Our multiple linear regression analysis focused on the outcome variable treatment satisfaction, which we treated as a continuous outcome variable. Evaluation of the data with polynomial regression yielded similar results. Potential correlates were demographic variables and baseline scores, as well as change scores for the PRO instruments described in the previous section. We first modelled these variables univariately as correlates of treatment satisfaction and only those that were significant at p < 0.1 entered into the multiple regression model. After having entered the multiple regression model, only those significant at p < 0.05 remained in the final model.Table Table Table The objective of this study was to assess correlates of treatment satisfaction in patients with moderate to severe GERD receiving esomeprazole. We found that Caucasian ethnicity, greater vitality and more severe heartburn at baseline, correlates with treatment satisfaction. Furthermore, the greater the improvement on vitality change score, the more likely the patient is to be satisfied with the treatment.The strengths of this study include the detailed assessment of a number of demographic characteristics, HRQL and symptoms. However, this study has two important limitations. First, we did not perform a placebo controlled trial limiting our ability to assess satisfaction as a true treatment result versus other reasons for satisfaction. Second, investigators have not conducted a thorough psychometric assessment of the treatment satisfaction instrument we used in this study.Nevertheless, the present study yields four important results. First, in this sample of GERD patients without prior endoscopic evaluation of their symptoms, Caucasian ethnicity was positively associated with treatment satisfaction. Ethnic origin is perhaps one of the most complex demographic characteristics  and it hSecond, higher vitality scores, as assessed by the QOLRAD, were associated with higher treatment satisfaction. A patient's health status prior to receiving treatment may cause the patient to be either more or less satisfied with treatment. Clearly and McNeil  reportedThird, higher scores for heartburn, assessed with the four symptoms scale, were related to higher treatment satisfaction. Thus, in our study population, patients with high discomfort from heartburn at baseline perceived a high satisfaction with treatment.Fourth, the higher the improvement on the QOLRAD vitality (change score), the more likely the person is to be satisfied with the treatment.Patients' age is regarded as the most consistent determinant characteristic of satisfaction -15. The Although some studies have reported that patient gender affects satisfaction values ,34, otheThe current results may be unique to the study sample since no placebo control group was included in the study and, therefore, we were unable to evaluate whether the factors related to treatment satisfaction are related to real treatment effects or patients' need to please and placebo effects. The efficacy, tolerability and safety of esomeprazole versus other proton pump inhibitors has been shown in other studies -40. In tInvestigators often use several PRO instruments, each with many dimensions and single items that are more or less correlated in clinical studies. This can lead to a large number of statistical tests being carried out and an increased risk of statistically significant findings occurring by chance in the absence of adjustment of P-values. In the present report we did not carry out adjustments for multiple comparisons for two main reasons. Firstly, the analysis of correlations was intended to be exploratory rather than confirmatory. Secondly, there is no consensus on how to adjust in analyses of the nature we conducted in this study. A simple adjustment according to Bonferroni would be too conservative, in part because many of the PRO variables are closely correlated.Different drug therapies may elicit unwanted side-effects, which could compromise the patients' HRQL, and adherence with the treatment. Thus, a challenge in the management of GERD is to achieve as high adherence as possible. In addition, treatment satisfaction can be of use when different drug therapies show similar efficacy since it can lead to a preference for one drug over another and greater adherence.Our study also supports the need for validated treatment satisfaction instruments because the available instruments vary widely in clinical trials  and the We examined correlates of treatment satisfaction, including demographic factors, symptoms, and HRQL, as well as change scores in HRQL, in patients with moderate to severe GERD who were not investigated by endoscopy. We observed that Caucasian ethnicity was positively related to treatment satisfaction. Furthermore, higher vitality and more severe heartburn were associated with treatment satisfaction. Finally, the higher the improvement on the QOLRAD vitality (change score), the more likely the patient is to be satisfied with the treatment.Alessio Degl' Innocenti was the project leader for this manuscript, edited the clinical protocol of the study, interpreted the data, and wrote the final manuscript as well as early versions. Gordon H Guyatt and Holger Schünemann were the principal investigators of the study, wrote the clinical protocol and grant application, are responsible for the study protocol, interpreted data and participated in writing the final as well as early versions of this manuscript. Ingela Wiklund contributed to the study protocol, interpreted the data and edited the manuscript. Diane Heels-Ansdell was responsible for the statistical analysis and edited the final manuscript as well as early versions. David Armstrong was co-principal investigator of the study, revised the clinical protocol, assessed patients, interpreted data and edited the final manuscript as well as early versions. Carlo A Fallone and Sander Veldhuyzen van Zanten revised the clinical protocol, assessed patients, interpreted data and edited the final manuscript as well as early versions. Samer El-Dika, Alan N Barkun, and Peggy Austin revised the clinical protocol, interpreted data and edited the final manuscript as well as early versions. Peggy Austin also co-ordinated the study. Lisa Tanser contributed to co-ordination of the study. All authors read and approved the final manuscript. The AstraZeneca global publications group approved the manuscript.


Fundulus heteroclitus was examined. Only a small subset (31%) of tissue-specific differences was consistent in all three populations, indicating that many tissue-specific differences in gene expression are unique to one population and thus are unlikely to contribute to fundamental differences between tissue types.The expression of a selected suite of 192 metabolic genes in brain, heart and liver in three populations of the teleost fish  Fundulus heteroclitus using a highly replicated experimental design.Variation in gene expression is extensive among tissues, individuals, strains, populations and species. The interactions among these sources of variation are relevant for physiological studies such as disease or toxic stress; for example, it is common for pathologies such as cancer, heart failure and metabolic disease to be associated with changes in tissue-specific gene expression or changes in metabolic gene expression. But how conserved these differences are among outbred individuals and among populations has not been well documented. To address this we examined the expression of a selected suite of 192 metabolic genes in brain, heart and liver in three populations of the teleost fish Half of the genes (48%) were differentially expressed among individuals within a population-tissue group and 76% were differentially expressed among tissues. Differences among tissues reflected well established tissue-specific metabolic requirements, suggesting that these measures of gene expression accurately reflect changes in proteins and their phenotypic effects. Remarkably, only a small subset (31%) of tissue-specific differences was consistent in all three populations.These data indicate that many tissue-specific differences in gene expression are unique to one population and thus are unlikely to contribute to fundamental differences between tissue types. We suggest that those subsets of treatment-specific gene expression patterns that are conserved between taxa are most likely to be functionally related to the physiological state in question. The regulation of gene expression varies extensively among tissues, individuals, strains, populations and species -6 and vaAlthough tissue-specific gene expression patterns are often used as a method to identify functionally relevant genes, how conserved these differences are among outbred individuals and among populations has not been well documented. It is possible that many of these changes represent polymorphism among individuals or populations and are not specifically associated with disease. To address this we used a well established system (tissue-specific gene expression) and genes with well defined function and tissue-specific distributions (metabolic genes).Fundulus heteroclitus. A cDNA microarray was used to measure levels of expression in normal healthy male fish for 192 genes involved in central metabolic pathways. We used this compact array in order to impose a high degree of technical and biological replication . Also, this array was used because metabolic genes are essential, are known to have tissue-specific expression, especially in fish, and are often misused as controls with little characterization of variation in expression among individuals or tissues. Analysis of variance (ANOVA) was used as a statistical test to determine which genes were differentially expressed among tissues and populations. Tissue-specific patterns of gene expression were compared among populations. As expected, we detected extensive variation in gene expression among tissues. Unexpectedly, only a fraction (31%) of tissue-specific differences was conserved between all populations.Given the high variance in gene expression among individuals and populations, our goal was to examine the conservation of tissue-specific gene expression among populations of the same species. Specifically, we assessed the among-population variance of tissue-specific patterns of gene expression  in the teleost fish p < 0.05) among individuals within populations and tissues  differentially expressed among brains, hearts and livers . Selecting the α level at which differences between treatments are considered significant is problematic because of the large number of comparisons performed. As such, we present a volcano plot to illustrate the range of expression differences between tissues and associated p-values , the proportion of significant genes is 67% (129 genes), 50% (96) and 39% (75), respectively. Significant differences in expression ranged from less than 1.2-fold to nearly 16-fold , hepatocyte nuclear factor 4-alpha (a transcription factor) was more highly expressed in liver than in other tissues (p < 0.001), and two genes involved in glycerolipid metabolism -lipoprotein lipase and phopholipase XIII A2 - were more highly expressed in liver than other tissues (p < 0.001 for both genes).Many expected tissue-specific patterns emerged. For example, the brain-specific fatty-acid-binding protein was typically more highly expressed in the brain than in other tissues . However, it should be noted that although the split-plot design is powerful for detecting differences between split-plot factors (tissues), it is considered to have low power for detecting differences between blocks (populations)  α[× SEMSR = Qkv] α[is the studentized range [k = number of groups in the comparison , v = degrees of freedom of MStissue-by-individual within population, and SE is the standard error among tissue-by-individual samples within populations. The T-method following ANOVA was used to identify genes differentially expressed among tissues in each population. These data were then used to contrast tissue-specific and population-specific expression patterns. Robustness of ANOVA data was tested using a permutation test; means for the 27 biological samples were randomly permuted 1,000 times between population and tissue and test statistics were recalculated for differences among populations, tissues and tissue-by-population interaction. Agreement between ANOVA and permutation test results would indicate the robustness of the ANOVA model. Finally, in order to graphically illustrate expression similarity among tissues, expression distance between samples was calculated as the sum of differences of log2 expression values over all genes, and neighbor-joining trees of global similarity of expression patterns among tissues  were constructed [where the critical value Qed range , k = numstructed  for eachp-values associated with statistical tests for differences in expression between populations, tissues, tissue-by-population interaction, and among individuals within populations. Also listed are mean expression for each sample, and columns comparing differences in expression between tissues within each population. Final columns tabulate whether a tissue difference was detected for each comparison, whether this difference was consistent between populations, and whether significant interaction was detected for that gene.The following additional data are available with the online version of this paper. Additional data file The results from statistical analyses for all genesClick here for additional data file