Intestinal fatty acid-binding protein (I-FABP) has been shown to be of high diagnostic value in patients with acute mesenteric ischemia. Whether these results can be reproduced in critically ill patients on the ICU was to be investigated.  I-FABP was measured in serum and urine of 43 critically ill patients in ICU when mesenteric ischemia was suspected. Bowel ischemia was confirmed in 21 patients (group 1). 22 patients who survived at least seven days without confirmation of ischemia were assigned to group 2. I-FABP levels were compared between the groups, and interval from the event that has triggered ischemia to I-FABP measurement was recorded.  For the identification of patients with mesenteric ischemia, sensitivity, specificity, and area under the curve (AUC) for serum and urine I-FABP were 33.3%, 95.5%, and 0.565 and 81.3%, 70.0%, and 0.694, respectively. I-FABP measurements performed within 12 to 48 h after the event that triggered ischemia showed a sensitivity, specificity, and AUC for serum and urine of 75%, 100%, and 0.853 and 100%, 73.3%, and 0.856, respectively.  In ICU patients, one single I-FABP measurement at the time of clinical suspicion failed to reliably detect or exclude mesenteric ischemia. A higher diagnostic value of I-FABP was only confirmed in the early stages of mesenteric ischemia. I-FABP may be used most appropriately in perioperative monitoring. Splanchnic hypoperfusion has an important impact on the treatment of critically ill patients in intensive care units, either as transient mesenteric ischemia triggering a gut-derived systemic inflammatory response syndrome (SIRS) or as mesenteric ischemic necrosis , 2. It cIntestinal fatty acid-binding protein (I-FABP) is a valuable marker for epithelial damage of the intestine and, therefore, for mesenteric ischemia. This small cytosolic protein with a molecular weight of approximately 15 kDa is expressed only in mature enterocytes. Due to its low molecular weight and its distribution at the tips of the intestinal villi, it is rapidly released into the circulation and excreted in urine in case of mucosal damage . In humaA number of clinical studies demonstrated the high sensitivity and specificity of I-FABP in detecting mesenteric ischemia in patients presenting with acute abdomen –12 or inWe conducted a prospective observational trial including critically ill patients in the ICU to address the following questions: Are I-FABP values different in patients with and without mesenteric ischemia at the time of initial clinical suspicion? Does the interval between the event which triggered ischemia and the I-FABP test influence the diagnostic value?Clinical signs (prolonged ileus after surgery and increasing need for vasopressors)Hyperlactatemia (>3.5 mmol/l or no drop below 5 mmol/l within 12 h after cardiac surgery)Signs of bowel ischemia found in endoscopy  or computed tomography .In the multidisciplinary ICU of Jena University Hospital, all patients suspected of having bowel ischemia were included immediately when tested positive for at least one of the following inclusion criteria:These criteria were compiled from our own clinical experience and have not been restricted to proof of bowel ischemia by means of CT or endoscopy since clinical suspicion often raises due to unspecific signs like delayed bowel function, distension of the abdomen, or hyperlactatemia.Patients in whom bowel ischemia was confirmed during laparotomy were assigned to group 1 (ischemia). Patients surviving more than 7 days after inclusion into the study without any proof of bowel ischemia were assigned to group 2 (no ischemia). Exclusion criteria were age < 18 years and pregnancy. The study protocol was approved by the local ethics committee of Jena University Hospital (Ref. no. 4028-03/14). Written informed consent was obtained from all patients or their legal representative.A urine specimen was taken at the time of study inclusion (specimen “U0”). Blood was collected from the routine blood samples at the following time points: day of inclusion (“d0”), day before inclusion (“d-1”), and the following three days . No extra blood samples were taken for study purposes. Study samples were aliquoted and stored at −80°C until analysis.If informed consent could not be obtained within 96 h after study inclusion, all aliquots of the respective patient were discarded.n = 161, urine n = 36) were performed after inclusion of the last patient using a commercially available ELISA kit HK406-02 . The specific measurement of human I-FABP is feasible in the range from 47 to 3000 pg/ml using this assay. Samples > 3000 pg/ml were diluted with buffer solution up to 10 times.Laboratory analyses .All continuous variables were expressed as median with interquartile range (IQR), since Kolmogorov–Smirnov test showed non-normal distribution of all I-FABP values. The groups were compared by the Mann–Whitney During a 9-month period, 2072 patients were treated on ICU. 82 patients met the inclusion criteria. Of 32 patients, informed consent could not be obtained within 96 hours after study inclusion. Six patients were excluded because less than three specimens were available since they died before day 1. One patient without confirmation of mesenteric ischemia died on day 5 and, therefore, could not be assigned to any of the groups. Data of 43 patients were analysed, 21 of them being assigned to group 1 and 22 to group 2. Patients' characteristics and the initial reason for admission into the ICU are listed in n = 13); 26 patients met two or three inclusion criteria, as shown in 17 patients met one inclusion criterion . Mesenteric ischemia was confirmed in all cases by histological examination. Histological findings ranged from ischemia-associated mucosal ulcers to complete bowel necrosis. In one patient with embolic occlusion of the superior mesenteric artery (SMA), ischemia of the small bowel was confirmed by the surgeon and resolved by embolectomy without resection.All patients of group 1 underwent laparotomy. In 20 of them, a bowel resection was performed because of ischemia . In the three remaining patients, no potential causative event and no sudden deterioration of the clinical condition within the last 14 days could be identified.In 40 patients, it was possible to identify the event which most probably triggered mesenteric ischemia  showed no statistically significant difference of I-FABP levels in both groups. This may be partially explained by the fact that, according to the study protocol, serum measurements were performed from the daily routine samples which were not always taken at the time of study inclusion.However, the exact point in time when the mesenteric ischemia occurs is often obscure in ICU patients since clinical signs of acute abdomen are frequently masked. I-FABP is abundant only at the tips of the villi of bowel mucosa and rapidly released into the circulation in case of severe mucosal ischemia , 17, 18.The time interval from mesenteric hypoperfusion to I-FABP measurement therefore seems to significantly influence the diagnostic value of the test and may lead to false negative  or false positive  results. Therefore, one single I-FABP measurement in ICU patients at the time of clinical suspicion failed to reliably detect or exclude mesenteric ischemia in our study. It can be assumed that repeated I-FABP measurements in critical situations like major surgery or cardiogenic shock may better reflect severity of intestinal damage and its recovery. For this clinical applicability, availability of a rapid laboratory assay is needed.van der Voort et al. evaluated I-FABP test performance in 44 ICU patients with suspected mesenteric ischemia . MediansSymptoms of mesenteric ischemia in ICU patients are often unspecific. Thus, we applied a wide range of inclusion criteria for this study leading to a balanced recruitment to both groups. The retrospective analysis shows that more than 80% of patients of group 1 had signs of ischemia in endoscopy or CT and a large part of group 2 patients was included because of hyperlactatemia. However, in 12 of 17 patients of group 1 who were positive in endoscopy or CT, clinical signs or hyperlactatemia lead to suspicion of mesenteric ischemia and subsequently to further diagnostic measures. In 5 patients, the diagnosis of mesenteric ischemia was based on findings in CT scans that were performed without prior suspicion of mesenteric hypoperfusion. For this reason, we do not think that the unequal distribution of inclusion criteria to both groups influenced the results.Mesenteric ischemia was confirmed in all patients of group 1 by surgical and histological findings. The definition of the absence of mesenteric ischemia is difficult in general since a comprehensive examination of the entire gut is almost impossible in vivo. Thus, our definition of the negative control group was patient survival of at least 7 days without confirmation of mesenteric ischemia, presuming that a mesenteric ischemia would be fatal within this period. However, some patients of the control group may have had mesenteric hypoperfusion that was resolved and, furthermore, surviving a significant mesenteric ischemia for more than 7 days seems occasionally possible.The diagnostic value of I-FABP was higher using urine samples as compared to using blood samples. However, critically ill patients in ICU frequently suffer from renal insufficiency impairing both serum I-FABP clearance and urine I-FABP levels. Due to the small heterogeneous sample size, we were not able to investigate the effect of renal function or dialysis on the test results.Another factor that might influence I-FABP levels to a more or less relevant degree is surgery or manipulation of the bowel. In our study population, 12 patients in both groups had some form of abdominal surgery prior to inclusion to the study . We did not exclude these patients, because this pilot study aimed to evaluate the performance of the I-FABP test in the real world population of ICU patients. Again, the sample size was too small to perform a subgroup analysis of patients with and without surgical bowel manipulation.In ICU patients, one single I-FABP measurement at the time of clinical suspicion failed to reliably detect or exclude mesenteric ischemia. A high diagnostic value of I-FABP was only confirmed in the time interval between 12 and 48 hours after the onset of mesenteric ischemia. I-FABP may be used most appropriately in perioperative monitoring. Further studies should consider the influence of the interval between mesenteric ischemia and I-FABP measurement on the diagnostic accuracy of the test.


Directly observed treatment, short course (DOTS) strategy has been a cornerstone for Tuberculosis (TB) control programs in developing countries. However, in Ethiopia satisfaction level of patients’ with TB with the this strategy is not well understood. Therefore, the study aimed to assess the satisfaction level of patients with TB with the DOTS.Explanatory sequential mixed method design was carried out in Addis Ababa, Ethiopia. Interviewer-administered questionnaire with 601 patients with TB who were on follow-up was employed in the quantitative approach. In the qualitative approach telephonic-interview with 25 persons lost to follow-up and focus group discussions with 23 TB experts were conducted.Sixty seven percent of respondent was satisfied with the DOTS. Rural residency , having TB symptoms  and treatment supporter  were associated with satisfaction with DOTS.In qualitative finding, all persons lost to follow-up were dissatisfied while TB experts enlightened lack of evidence to affirm the satisfaction level of patients with DOTS. Explored factors contributing to satisfaction include: on time availability of health care providers, DOTS service delivery process, general condition of health care facilities, nutritional support and transportation.DOTS is limited to satisfy patients with TB and lacks a consistent system that determines the satisfaction level of patients with TB. Therefore, DOTS strategy needs to have a system to captures patients’ satisfaction level to respond on areas that need progress to improve DOTS service quality. In 2015, there were an estimated 10.4 million incident cases of TB and 1.4 million deaths from Tuberculosis (TB) globally [One third of the world’s population, approximately 2 billion persons, is thought to be latently infected with Mycobacterium tuberculosis (M.th position in 2015 [The burden of the TB is higher in 30 high TB burdened countries (HBCs) which account for 84% of all estimated cases worldwide. Among these India leads with the highest number of TB cases, and Ethiopia held the 11In the mid-1990s, WHO developed and recommended Directly Observed Treatment Short-course (DOTS) strategy for TB treatment. It is an international recommended strategy that has been considered as a cornerstone for TB control programs in developing countries including Ethiopia ,4. In EtThe DOTS startegy implementation in Ethiopia evaluated at different times based on the treatment outcome of patients with TB with out due attention to the patients’ satisfaction –12. HoweThe study was used explanatory sequential mixed method study design from September 2015 to November 2015 in Addis Ababa, Ethiopia.2. Administratively, the Addis Ababa is divided into 10 sub-cities, which are in turn divided into 116 wereda, the smallest government administrative units [Addis Ababa has a surface area of 540 Square kilometers. According to projection of Central Statistics Agency (CSA) of Ethiopia, in the year 2014 its population was about 3.2 million . The popve units .Available health institutions in the city are 48 hospitals, of which 34 are private, 11 public and 3 non-governmental (NGO). Among 11 public hospitals, 6 are owned and managed by Addis Ababa City Administration Health Bureau (AACAHB), 3 by Federal Ministry Health of Ethiopia and 2 Police and Defense Ministers . In addiOne hundred twenty one health facilities which were providing DOTS services in Addis Ababa were categorised into three groups. The categories were formed based on the ownership of the health facilities such as government, NGO for not profit and private for profit health facilities. Among listed health facilities in each category, by rule of thumb, approximately 25% of health facilities were randomly selected. Totally 30 health facilities were selected, of which 23 were among government, 6 were among private for profit and 1 was among non-government for not profit categories.The sample size was determined based single population proportion formula with the assumption of 50% expected satisfaction level of patients with TB, 0.05 error allowance, 1.96 two-sided critical value for 95% confidence interval level and 0.05 levels of satisfaction significance. Based on the above information the calculated sample size was 384. The calculated sample size, 384, multiplied by 1.5 for design effect compensation, and 5% contingency for non-response rate added. Hence the final sample size was 605. The determined sample size was proportionally allocated based on the number of patients with TB who were on follow-up at each health facility. Patients with TB who were on follow-up were randomly recruited while they came for follow-up at each selected health facilities until the allotted sample size was reached.The quantitative data were captured using an interviewer-administered structured questionnaire. The questionnaire was prepared through depth review of literatures , 21–26 aThe questionnaire contains 23 measuring items in 5-point Likert scale. Of which 10 items were focused on the structure, 10 items were focused on the process and 3 items were focused on the outcome of the DOTS. In addition, the questionnaire contained one stand-alone overall satisfaction measuring scale item ranged from 0 to 10 scores. The item’s lowest score was zero and the maximum score was 10.Initially the questionnaire was prepared in English and translated into Amharic language  and was translated back to English. The questionnaire was piloted on 27 patients with TB who were on follow-up at a health facility which was not included in actual data collection sites. The questionnaire’s overall reliability test was done in the pilot and actual study. Cronbach’s alpha of 0.88 and 0.85 were obtained from the pilot and actual study, respectively. The structure, process, and outcome measuring items Cronbach alpha values were 0.78, 0.78 and 0.85 respectively.The data were collected from 601 consented patients with TB who were on follow-up of TB treatment. Data were collected by nurses who trained on the questionnaire and ethical aspect of human subject involved study. Data was double entered into Statistical Packages for Social Science version 21.0 for Windows .The satisfaction level of the respondents was determined by two methods. The first method was by 23 measuring items included in Donabedian’s quality assessment in health care model. The mean score of 23 measuring items included in the questionnaire was calculated for each respondent. The calculated mean score equal 2 and less were classed as unsatisfied and the mean score 4 and above were classed as satisfied. For scores between greater than 2 and less than 4, first the median was calculated then the scores less than the median were classed as unsatisfied and the rest classed as satisfied .The second method was by taking the score of stand-alone single overall satisfaction measuring item. A stand-alone single overall satisfaction measuring scale mean was calculated and the mean score was used as cut of value to regard satisfied against dissatisfied . HoweverThe cut of value calculated from Donabedian quality health care proxy model was used to categorize satisfied against not satisfied for bivariate and multi-variable analysis. Predictors for patient satisfaction extracted from the literature related to DOTS strategy were assessed in binary logistic regression. In multi-variable regression variable which had a p value 0.05 in binary logistic regression were considered.In order to keep instrument validation and reliability, a questionnaire was piloted before the actual data collection and Cronbach’s value was calculated. To increase the quality of data, (a) the data collectors were nurses who have proximity with TB treatment strategy, (b) a one day training was given for data collectors before the start of data collection, (c) the overall activities of data collection were monitored by the researcher and there was strict supervision during data collection, (d) all completed questionnaires were examined by the researcher for completeness (e) double data entryentry was done and consistencies of the collected data were checked during analysis.In the qualitative approach, two groups of participants were included: persons lost to follow-up and TB experts. Before data collection, the TB register of each selected health facilities was checked to identify persons lost to follow-up within the six months’ time and their telephone address. A total of 23 TB experts was purposely selected. Two TB experts from each 10 Subcity health offices and 3 TB experts from AACAHB were included.Focus group discussions (FGDs) and telephonic interview guide were developed in English based on literature review for TB experts and persons lost to follow-up, respectively. The FGD and telephonic interview guides, then translated to Amharic. Both the FGDs and telephonic interviews were facilitated in Amharic language. The qualitative data saturation was reached with 25 persons lost to follow-up patients with TB and 23 TB experts' participation during the interview and discussions, respectively .Three FGDs with consented TB experts were conducted. The first and the second FGDs included 8 participants each while the third FGD included 7 participants. The discussions were facilitated based on pre-prepared leading questions. The leading questions were: how TB treatment is being provided in Addis Ababa, what are the challenges with DOTS strategy, how does/did you know either patients with TB are being satisfied or not, what are the factors that may contribute or limit satisfaction of patients with TB and what does/did you suggest to improve satisfaction levels of patient with TB. The FGDs were recorded and notes were taken.Telephonic-interviews with consented persons lost to follow-up from TB treatments were carried out. In the interview probing questions were used. The probing questions were: what extent were you satisfied with the service provided while you were on follow-up of your treatment; what are the factors that made you satisfied/dissatisfied you, and what do you suggest to improve satisfaction of patient with TB.Qualitative data analysis was started simultaneously with the data collection. During data collection, field notes were taken, audio digital recorder (ADR) and non-verbal and gestural cues were observed. All Amharic recorded data were translated and transcribed verbatim by the researcher and research assistant independently. After the verbatim transcription and translation done, the data were compiled, read and re-read of the transcripts and emerging ideas were listed. Then codes, categories and sub-categories for the listed ideas were created. The categories were related by drawing lines between categories. Recoding was done when necessary. Themes were generated from these categories. The transcripts were also given to a public health specialist, who was also an expert in qualitative research methodology to independently create codes and categories, and identify emerging themes. A consensus meeting between the researcher and the public health specialist was held, where the categories and themes identified were compared, revised and then used as research findings for this study .The credibility, reliability and transferability of the study findings were enhanced with a collection of data from multiple sources: patients with TB who were on follow-up, lost to follow-up and TB experts. The study used different methods, quantitative and qualitative approaches using structured interview, telephonic-interview and FGD, and the findings were triangulated during interpretation. Study sites exhibited a varied number of patients with TB and only few numbers of respondents’ responses were collected per day for three months to reduce single day’s episodic feeling and recruit adequate number of participants.The respondents were informed that they would not face anything that hinders her/his treatment at the time of data collection, during their treatment and beyond due to participating in the interview. So that enables the respondents to describe their real feeling. Moreover, verbatim translation of FGDs was shared with the participants to increase acceptability and dependability of the discussions.This research extracted information employing questionnaires to patients with TB who were on follow-up, telephonic-interview of persons lost-to follow-up and FGD TB experts. After each sort of data set separately analysed, the findings were triangulated at an interpretation level to complement the weakness of one source of data over the other .Ethical clearance from Institutional Research and Higher Degrees committee from the University of South Africa, and permission letter from Addis Ababa City Adminstration Health Buraeu were obtained before the time of data collection. The IRBs at both institutions reviewed and apporved the consent procedures. Data were collected after securing informed written consent from every participant. The confidentiality of information was maintained by excluding personal identifiers, interviewing privately, and the signed consent forms are stored in lockable board at researcher office.Patient satisfaction: patients’ emotions, feelings and their perception of delivered DOTS services.Directly observed treatment (DOT): means a health worker play an active role to ensure that every patient takes the recommended drugs, in the right combinations, on the correct schedule, for the appropriate duration .Our result presented as follows: quantitative study findings: general characteristics of patients with TB, type of TB and the satisfaction level of the patient with TB and logistic regression analysis outcome. Then followed by qualitative study findings.A total of 601 patients with TB were responded to the interviewer-administer a questionnaire, which accounts for 99.3% of the total expected respondents. Of these, 398 (66%) respondents were on intensive phase and 203 (34%) were in the continuation phase of the TB treatment. Three hundred thirty six (56%) of respondents were male .Sixty seven percent of the respondents were satisfied with the DOTS using the Donabedian quality health care model framed measuring items while 53% of the respondents were satisfied using the overall single measuring item . The resAmong the 23 satisfaction measuring items, reduction of TB symptoms mean score was the highest of all measuring items, 4.16  and the lowest mean score of measuring items was friendly-ness of the TB treatment provision environment, 3.13 (SD = 1.3) .Among 601 respondents, 406 (67.7%) respondents were satisfied with DOTS. Of the 406 satisfied respondents, 208 (51.2%) were male respondents. There was a significant association between the satisfaction level and gender (p = 0.001). Among the 406 satisfied respondents, 170 (42%) respondents were with smear positive PTB, 108 (27%) were with smear negative PTB, 105 (26%) were with EPTB and 23 (5%) were with MDR-TB. There was no significant association between satisfaction with DOTS and type of TB (p = 0.28).Among 195 dissatisfied respondents, 155 were new patient with TB, 23 were relapse patients with TB, 9 were treatment after failure patients with TB, 2 were returning after lost to follow-up patients with TB and 5 were transferred-in patients with TB . There wAmong 406 satisfied patients with TB, 93 (15.5%) were diploma and above holders, 129 (21.5%) were secondary school (9–12), 100 (16.6%) were second cycle primary school (5–8), 33 (5.5%) were first cycle primary school (1–4), and 51 (8.5%) had no formal education. There was no significant association between higher education level achieved and satisfaction (p> 0.05).Age category of patient with TB was not significantly associated with satisfaction (p>0.05). Among 406 satisfied patients with TB, 82 (13.6%) were between 18–24 age category, 145 (24.1%) were between 25–34 age category, 106 (17.6%) were between 35–44 age category, 41 (6.8%) were 45–54 age category, 23 (3.8%) were 55–64 age category and 9 (1.5%) were above 65 years old.There was a significant association between income category and satisfaction with DOTS (p = 0.02). Cross tabulation of satisfaction with income category presented in 2 = 5.904, df = 8 and p = 0.658 revealed the model adequacy during the analysis.Among 7 independent variables which showed an association with the satisfaction level in binary logistic regression, gender, presence of TB symptoms and treatment supporter were associated with satisfaction after controlling confounders . The gooAmong 23 TB experts participated in the FGDs, 15 (65%) were male. Thirty five percent of TB experts’ age category was 20–30 year, 13% was 31–40 year, 43.5% was 41–50 year and 8.6% was 51–60 year. The FGDs participants’ TB treatment and control related work experiences were ranged 2–15 years with the median of 5.5 years. Thirteen (55%) of FGDs participants had Master degree and 12 (45%) hold Bachelor degree.Among 25 persons lost to follow-up contacted for the telephonic-interview, 3 (12%) of contacted addresses were wrong, 2 (8%) did not pick-up for repeated telephone calls 1 (4%) did not volunteer to participate in the telephonic-interview and 19 (76%) were fully participated. From 19 fully participated in a telephonic-interview, 11 (58%) were male. The mean age of the telephonic-interview participants was 39.4 years (SD = 9 years).Telephonic-interview participants explained that they were dissatisfied with DOTS service while they were on follow-up. The dissatisfaction of persons lost to follow-up and factors explored with satisfactions is explained in the following categories: DOTS delivery process, general condition of health facilities, aligned services and health care providers (HCPs) related .Most of the telephonic-interview respondents explained that the TB diagnosis and examination was started immediately on arrival in which registration was done, without much time desecrated. Immediate registration and the process start day were explained as:“As I went they registered me and I was seen by doctors. With this perspective, I want to say thank you.”However, the cause of illness was not detected in the same day; lasted for seven days and the time elapsed to have an apparent TB diagnosis was displeasing. One of the telephonic-interview respondents explained the dissatisfaction due to elapsed time as follows:“I started my examination on August 5 2015; I did not receive the definite diagnosis until August 12, 2015.”Another discontented issue described in DOTS service delivery process was an obligation to bring a treatment supporter. This was explained by the telephonic-interview respondent as:“After I diagnosed with TB, I should brought somebody on the behalf of me. Should not I be responsible for me?”Besides, some of the registered as persons lost to follow-up, received controversial diagnosis result; in one health facility received a result which concluded having TB while had not or other disease in another facility. The controversy results exposed the patients to unwanted cost. One of the telephonic-interview participants explained the controversy and its financial consequences as follows:“In one health facility I was told that I had TB but after few days, in another facility they rejected the findings as I had TB. I did not go to third facility due to money shortage.”The existence of the incompatible diagnosis result between health facilities was declared as a cause for dissatisfaction by the telephonic-interview respondents. Two respondents explained the incompatibility of the diagnosis as follows:“I went to health centre where you find my address and had been told that as I had Extra Pulmonary TB and stared TB treatment. After a week I went to private clinic and have been informed as I had no TB.”The telephonic-interview respondents explained that the daily follow-up of TB treatment exposed them for different direct and indirect costs. Furthermore, the difficulty of the daily observation was a cause to lost to follow-up. The telephonic-interview participants explained as follows:“I am factory employee; I should be available at 6 am since the service reach at this time, otherwise I had to pay 12 Ethiopian birr per trip to reach to the factory where I work even I may reach at 10 am or so. However, they obliged me to go health facility daily to take the drug.”“I and my wife were supposed to take him to health centre daily to follow-up. I got few days’ work permission and took him but after that my wife alone could not take him since he was bad-tempered. Now sadly he is mad and on the road.”In addition, the DOTS delivery system was criticized as it is resistant to change treatment facility where to follow once started at a given heath facility due to the provision of little attention to give referral paper. One of the telephonic-interview participants explained the resistance to give a referral paper as:“I told him that my husband should have to will move to another place for work with me and to write me a referral paper. He asked me where?… he responded that I need to give him exact name of the facility where I would follow but I knew only the area but not specific health centre’s name.”The general conditions of the HCOs were not so much appreciated by telephonic-interview respondents. The respondents explained that though there were improvements in availing chairs around the TB room, no water to take the drugs and clean toilet, which enables you to use cosily near to the treatment room. The respondent explained the premise inconvenience as:“I had to go to health facility after I emptied my colon at home since there was no convenient toilet.”The telephonic-interview respondents explained that allied health care services such availing transport, food, spiritual and social support are limited, however, the availability of these services is imperative. As reported in the interview, the existing system has no place alongside the screening of patients with TB for a diagnosis other than TB and HIV. The respondents explained weak integration of DOTS with nutritional support and no transport for patients with TB, even for the seriously ill. The respondent explained the less availability of aligned services as:“I saw the ambulance which stood in the compound and I requested the HCP to tell the driver but the HCP replied as this is only for bleeding mother.”“I had seen that some of patients with TB were receiving nutrition and asked to give me too but the response was as I had no MDR-TB.”Telephonic-interview respondents explained that HCPs who were engaged in the DOTS service delivery were available in working hours though they were not so punctual. With this perspective, the majority of the respondents explained as they had a good feeling about HCPs availability. However, few of telephonic-interview respondents did not agree fully about HCPs availability. One of the telephonic-interview respondents explained the disagreement as follows:“While I was following my treatment HCPs were available usually after 8 am though they appointed us at 7:30 am.”However, when the patients arrived earlier than HCPs at TB treatment room they tend to contempt the patients.“If I reached earlier than the HCPs I usually sit aside of the TB room to prevent myself from being visible to HCPs when they come in.”The HCPs reception on the first day of the treatment was mentioned by telephonic-interview respondents as it is with due respect, in a polite way and provide required information but the days go on they may throw the drugs through the window. One of the telephonic-interview respondents explained the mood swing with respect and information provision as follows:“I could not tell how the HCP received me, respect and advise… when I was diagnosed as I had TB but it lasts on the same day.”However, majority of telephonic-interview respondents explained that HCPs were not devoted to provide TB care service. The disagreement views of telephonic-interview respondents explained as:“The HCP started writing while I was talking with, even was not willing to see my face and respond.”Whereas another respondent explained that the HCPs’ compassionated care as:“He, the HCP, was so kind while I was tired; even he was the one who hold up me to get into the car at the first week of the treatment.”The telephonic-interview respondents explained that HCPs commitment and psychological support to patients with TB was not consistent among themselves and across health institution. The respondents put the level of commitment at two extremes. The extremes are range from holding up the weak patient to getting into the car to turning the face while the patient's request or raise an issue. These extremes were explained by the respondent as:“I remember that, the HCP gave me my drugs in the car, almost 50 metres far from TB room.”Nonetheless, another respondent explained limited commitment of HCPs as:“The HCP was waiting me until I really combat on to TB room window let alone provision of the drug a place where I was.”The FGDs participants explained that there is no consistent and regular system in the region that specifically capture the satisfaction level of patients with TB except some fragmented systems that is not fully dedicated exclusively for patients with TB. The FGD participants explained their perception in the following categories: satisfaction survey exclusively for patients with TB and integrated patient satisfaction .The FGDs participants explained that there is no regular, similar and standardise satisfaction survey of patients with TB as a strategy to understand the satisfaction level of patients’ with TB particularly with DOTS strategy. The FGDs participants described unavailability in satisfaction survey exclusively for patients with TB as follows:“There is no satisfaction evaluation scheme for the TB treatment individually.”However, at some facilities there are fragmented ways of understanding the satisfaction level of patients with TB. Opinion recording books and suggestion boxes are obtained in the health facilities to capture the perceptions of patients. Random interview of patients with TB during supportive supervision also helps to understand the level of satisfaction of patients’ with TB. The practice so far tried to understand the satisfaction level of patients’ with TB at some facilities was explained in the discussion as:“We conduct supervision quarterly. We interview the patients in person on the treatment they have been given by the professionals in the TB room. We also have the public opinions gathering boxes in every department room. Those who can write drop their opinions there and other uses the chance during the interviews.”The recording books and suggestion boxes were criticised for their lack of effectiveness to measure the level of satisfaction objectively since either, so much dissatisfied or so exited patients usually write their feelings. The weakness of the recording book as a tool for satisfaction survey was explained by FGD participant’s personal experiences as a service user. The FGD participant explained as:“I remember once, I went to a clinic to have my eyes treated and I was very up set. I wrote on paper my temper but forgot to drop it in the box. These are the kinds of opinions we are talking about and they may not be bias free enough to evaluate clients’ satisfaction.”The FGDs participants agreed that there is a need for satisfaction measuring mechanism that apply to patients with TB. The discussant suggests that the indicator of satisfaction survey to patients with TB need to be scrutinised, a way of understanding their satisfaction level and the time interval to be yearly, biannually or quarterly at which the level of satisfaction should be measured.Although the Ethiopian Federal Ministry of Health (FMOH) has given due attention and priority to the quality of health care service since the first health sector development plan in 1997, a health care quality strategy has been developed recently. The Ethiopia health care quality strategy has included patient satisfaction as one of success indicators for the provision of quality service for the patients at HCO . Even duThe study reveals different levels of satisfaction with DOTS among different study participant groups. The majority (67%) of patients with TB that were on follow-up of their TB treatment was satisfied with DOTS similar to elsewhere in Ethiopia  and in oRegular measuring and knowing the satisfaction level of the patients emphasized as indicated in quality service provision , 35. HowTB experts participated in this study could not affirm whether the dissatisfaction of patients with TB with DOTS existed. As a result, the study highlighted the importance of the presence of regular and consistent mechanism to understand satisfaction level of patients with TB across health facilities.Similar with another study , gender Similar to the study conducted in Khyber Pakhtunkhwa, the study shows that attributes of patient satisfaction from the side of HCPs are communication skills, technical skills and responsiveness of HCPs to patient needs . In addiResidence, presence of TB symptoms and treatment supporter revealed a significant association with satisfaction with DOTS. Unlike to another study , in thisThe study included different study groups and described the level of patients with TB satisfaction from a different perspective by using quantitative and qualitative approaches. The outcome of the study can be generalized to different regions and countries at the primary health care level with similar settings. However, Addis Ababa is among the eleven geographic regions in the Ethiopian government structure, and is an autonomous administrative region and the largest and capital city of Ethiopia and the population is totally considered as urban dwellers (17). Hence, the majority of the respondents were urban dwellers; therefore, its generalization might be compromised for rural settings. In addition, satisfaction ratings in this study do not replace TSRs or other treatment outcome indicators in DOTS.The study finding shows that patients with TB who were on follow-up of their treatment were following their treatment with dissatisfaction. All of the lost to follow-up patients with TB were dissatisfied with DOTS. Dissatisfaction is one of the causes to deviate from follow-up. DOTS delivery process, general condition of HCO, allied health care services such as nutritional support and transport services are explored factors with satisfaction. Therefore, the DOTS strategy should include a regular mechanism that informs programmer, policy maker and HCPs about patients with TB satisfaction with DOTS. Moreover, the HCPs, TB-experts, Health programmer need to act on the barriers of patients with TB’ dissatisfaction such as unavailability of nutritional support and transport support, inconvenience of HCOs conditions and long waiting time in health facilities.S1 Satisfaction measurement questionnaire and interview guides(PDF)Click here for additional data file.


On the other hand, litter diversity did not affect the algae dominance irrespective to the rainfall scenario. We suggest that rainfall changes may compromise important self-reinforcing mechanisms responsible for maintaining high levels of algae on tank bromeliads ecosystems. We summarized these results into a theoretical model which suggests that tank bromeliads may show two different regimes, determined by the bromeliad ability in taking up nutrients from the water and by the total amount of light entering the tank. We concluded that predicted climate changes might promote regime shifts in tropical aquatic ecosystems by shaping their structure and the relative importance of other regulating factors.Climate change and biodiversity loss have been reported as major disturbances in the biosphere which can trigger changes in the structure and functioning of natural ecosystems. Nonetheless, empirical studies demonstrating how both factors interact to affect shifts in aquatic ecosystems are still unexplored. Here, we experimentally test how changes in rainfall distribution and litter diversity affect the occurrence of the algae-dominated condition in tank bromeliad ecosystems. Tank bromeliads are miniature aquatic ecosystems shaped by the rainwater and allochthonous detritus accumulated in the bases of their leaves. Here, we demonstrated that changes in the rainfall distribution were able to reduce the chlorophyll- Ecosystems face many concurrent stressors which may cause great changes in their functioning. It has been proposed that climate change and biodiversity loss could promote large shifts in natural ecosystems compromising important ecological services –3. Also,Climate models predict that changes in rainfall distribution will be the prevalent change across different areas of the globe, but especially in tropical and high latitude areas . ExtremeBiodiversity loss is considered one of the major drivers of the current global changes . On the Alternatively, tank bromeliads have been increasingly used as model ecosystems to test many ecological questions ,20–23. TAs these aquatic ecosystems are fully fed by rainwater, changes in rainfall could disrupt important mechanisms that regulate algae growth therein. For example, increases in rainfall can raise the water level in tank bromeliad ecosystems  and it cIn this study, we experimentally test the effects of rainfall changes and biodiversity on the occurrence of algae-dominated conditions in tank bromeliads ecosystems. We controlled biodiversity by manipulating litter diversity in the bromeliads tanks and using all combination of three different species from our field site. Then, we manipulated temporal clustering and amplitude of rainfall by using five different rainfall scenarios predicted to occur in the Southeastern Brazil. We used stable isotopes analysis to verify the potential effect of rainfall changes in regulating the ability of bromeliads in uptaking nutrients from the water in their tank. We hypothesized that (i) changes in rainfall distribution will decrease the occurrence of an algae-dominated condition by suppressing the ability of bromeliads in taking up nutrients from their tanks and (ii) this effect will be minimized by the positive effects of litter diversity on the algae dominance. At last, we discuss whether the occurrence of an algae-dominated condition in tank bromeliads can be regarded as alternative states by providing a conceptual framework based on the actual knowledge about these ecosystems.Neoregelia cruenta(Graham) L. B. Sm.  for our field site during nine years . Ambient rainfall scenario was characterized by a 25 mm rainfall event with a six days interval between rainfall events, distributed over 84 days  with a total 300 mm of rainfall. For the altered rainfall scenarios, we held the total amount of rainfall added constant (300 mm) but increased the temporal clustering of rainy days (two “clustering” scenarios) and the occurrence of extreme rainfall events (two “amplitude” scenarios). In the clustering scenarios, we decreased the interval between rainfall events; clustering rainfall either as two consecutive rainy days  with a 12-day interval between rain events in a Medium Clustering scenario (MC scenario), or as four consecutive rainy days  with a 24-day interval between rain events in a High Clustering scenario (HC scenario). For the amplitude scenarios, we increased the intensity of each rainfall event and the interval between events. In a Medium Amplitude scenario (MA scenario), a one-day 50 mm rainfall event occurred every 14 days, while in a High Amplitude scenario (HA scenario), a one-day 100 mm rainfall event occurred every 28 days. As we intentionally constrained our rainfall manipulations to have total equivalent amounts of rain, each altered rainfall scenario necessarily altered several rainfall variables simultaneously. For example, increases in the occurrence of extreme events  resulted in more consecutive days without rain, as it is predicted to occur [We established the rainfall scenarios based on precipitation projections for Southeastern Brazil for the second half of this century (2071 to 2100). These projections include an increase in temporal clustering of rainfall and in the occurrence of extreme rainfall events, but no changes in the total amount of rain . We estato occur . For morto occur .3 –Eugenia uniflora, C4 –Cyperus sp. and CAM–Clusia hilariana) that are potential litter source for tank bromeliads at our field site. We collected C4 and CAM litter in the field and then washed and dried at 60°C until its mass stabilized (at least 48 hours). E. uniflora was cultivated in a greenhouse with controlled irrigation for 15N enrichment (see next paragraphs). Each bromeliad received 1.2 g dry weighted of its respective litter combination. We choose this amount of litter because it is comparable to the natural amounts found in the main tank of N. cruenta bromeliads (unpublished data). We accommodate the untied litter in the bromeliad central tanks because they are more susceptible to the other factors that regulate algal biomass in the tank bromeliads . In total, all 1-, 2- and 3-species combinations (seven litter combinations) were applied through a substitutive design. It means that all experimental units received the same amount of litter but in different proportions of species. The 1-specie treatments  received 1.2 g of litter from the same litter species; the 2-species treatments  received 0.6 g of each litter species in the treatment; and in the 3-species treatment (C3-C4-CAM), we added 0.4 g of each litter species.We manipulated litter diversity by choosing plants from different metabolic groups  to allow community assembly. After this period, each bromeliad was covered with an individual transparent shelter to prevent natural inputs of rain but which allowed light to enter the bromeliads tank as well as organisms’ colonization in these systems . Water color was determined spectrophotometrically by light absorption at 430 nm in 1 cm cuvette . The con15N labeled leaves of E. uniflora in all treatments containing C3 litter to determine the indirect effects of rainfall on the ability of bromeliad leaves in removing nutrients from their tank water. However, we only analysed these effects in the C3- litter treatments because i) we were mainly interested in the effects of rainfall in regulating the ability of bromeliads in determining the nutrient loading of their tank and ii) we only have 15N enriched E. uniflora, therefore in the mixed treatments several biases could occur associated with nitrogen competition during bromeliad acquisition of nutrients by unbalanced 14N/15N ratios among litter species. Thus, our experimental design did not allow us to verify the potential effects of litter diversity on the bromeliad ability to uptake nutrients from their tank because this manipulation is restricted to C3 litter treatments.We used a data following the recommendations and procedures are given in literature [a concentration values found in the last sampling to perform this analysis. Bimodality was demonstrated using the Bayesian Information Criterion (BIC) values [We verified the occurrence of different conditions in tank bromeliads by using the bimodality approach . This meterature . We used) values . The low) values .a and the 15N content in the bromeliad leaves, we performed linear models to test the effect of rainfall scenario on the 15N isotope ratio content in the bromeliad leaves.We analyzed the effects of bromeliad maximum volume, rainfall scenario, litter diversity and their interaction on the occurrence of algae dominance by using Generalized Linear Models (GLMs). We also used GLMs to evaluate the effects of water color, turbidity, dissolved nitrogen, dissolved phosphorus and dissolved organic carbon on the occurrence of an algae-dominated condition. For these analyses, we used a binomial distribution and designated the algae-dominated bromeliads by the value “1” and non algae-dominated bromeliads by the value “0”. To check the correlation between chlorophyll-a data in each rainfall scenario using the “flexmix” package.All models were performed using R v. 3.2.2 . We carra  and HC rainfall scenarios . In Ambient rainfall scenario, the number of bromeliads dominated by algae was up to 5 folds greater than in HC rainfall scenario (P>0.36).Rainfall changes affected the algae dominance in tank bromeliads , Fig 3 wscenario . Additio15N content in bromeliad leaves .We found a significant effect of rainfall scenario on the δscenario . In C3 lHA and HC rainfall scenario, but litter diversity was not able to mediate this effect. Here, we suggest that predicted changes in rainfall will affect algal biomass in tank bromeliad ecosystems by regulating the ability of bromeliad in uptaking nutrients from the water in their tanks.The understanding of how ecosystems shift to critical conditions is a challenging question for ecosystem conservation and management in the face of global changes ,7. Our rNeoregelia cruenta. In all rainfall scenarios, we observed a large group of bromeliads where chlorophyll-a concentration was below detection level and a smaller group with a detectable source of algae in the water of their tanks. Particularly, in Ambient rainfall scenario, we observed a bimodal distribution of chlorophyll-a with many observations around zero and a number of observations above 80 μg L-1. Previous studies using the same tank bromeliad, but at natural conditions and containing different amounts of litter, found a much lower maximum chlorophyll-a concentration, around to 27 μg L-1 [We demonstrated that the non algae-dominated condition occurred most in the tank bromeliads 7 μg L-1 ,48. We s7 μg L-1 . Our exp15N in the bromeliad leaves which suggests an enhanced ability in taking up nutrients from the water in these rainfall conditions. This result suggests that, in the Restinga of Jurubatiba, water can be a limiting factor for bromeliads to obtain nutrients. Since bromeliads are able to take up water and nutrients by their foliar trichomes and the availability of water increases the contact of trichomes with nutrients [15N in bromeliad leaves and the chlorophyll-a contents and algal biomass in the water in the tank. Previous studies have reported the interaction between bromeliads nutrition and the behavior of the aquatic ecosystem in their tanks [Our data showed that rainfall changes affected the occurrence of algae-dominated and non-algal conditions in tank bromeliads, which could be indirectly related to the water level in these ecosystems. Interestingly, our isotopic analysis revealed that high changes in rainfall amplitude  increased the content of utrients ,49,50, air tanks ,49,51. Tir tanks . Since tir tanks  or with HA rainfall scenario, tank-bromeliads have received the greatest rainfall input (100 mm) which implies that at in a short term, these ecosystems presented the wetter conditions and as consequence the most suitable habitat for colonization at this moment. As time goes by the absence of rainfall creates a drought condition which may trigger changes in the whole ecosystem and decrease the occurrence of algae-dominated ecosystems. Thus, Ambient rainfall scenario creates a more stable wet condition without great changes in the water level in these ecosystems. In this way, the observed effects of rainfall scenarios reveal the overall balance of all conditions described by them.Otherwise, it is important to highlight that each rainfall scenario is described by changes in frequency, intensity and clustering of rain events. For example, in Ambient rainfall scenario ..Fig 5). This is the first study that empirically demonstrates how changes in the rainfall and litter diversity may lead to shifts in algal conditions in tank bromeliad ecosystems. We propose some self-reinforcing feedback mechanisms  which maS1 Appendix(DOCX)Click here for additional data file.S1 Figa values higher than 80 μg L-1, in each litter diversity level for all rainfall scenarios. Ambient, Medium clustering (MC), Medium amplitude (MA), High clustering (HC) and High amplitude (HA) rainfall scenarios are fully described in the main text. * Non algae-dominated states found in the respective litter diversity and rainfall scenario. For statistical details see We reported the frequency of algae-dominated ecosystems by the percentage of ecosystems that were considered algae-dominated, chlorophyll-(TIF)Click here for additional data file.S2 FigThe lines depict the probability density described by the best fitting model (n = 35).(TIF)Click here for additional data file.


Escherichia coli. However, for several applications, smaller metabolic (core) models are needed. Using a recently introduced reduction algorithm and the latest E. coli genome-scale reconstruction iJO1366, we derived EColiCore2, a model of the central metabolism of E. coli. EColiCore2 is a subnetwork of iJO1366 and preserves predefined phenotypes including optimal growth on different substrates. The network comprises 486 metabolites and 499 reactions, is accessible for elementary-modes analysis and can, if required, be further compressed to a network with 82 reactions and 54 metabolites having an identical solution space as EColiCore2. A systematic comparison of EColiCore2 with its genome-scale parent model iJO1366 reveals that several key properties  of the central metabolism are preserved in EColiCore2 while it neglects redundancies along biosynthetic routes. We also compare calculated metabolic engineering strategies in both models and demonstrate, as a general result, how intervention strategies found in a core model allow the identification of valid strategies in a genome-scale model. Overall, EColiCore2 holds promise to become a reference model of E. coli’s central metabolism.Genome-scale metabolic modeling has become an invaluable tool to analyze properties and capabilities of metabolic networks and has been particularly successful for the model organism  Escherichia coli. The first E. coli genome-scale model, iJE660iJR904iAF1260iJO1366E. coli network reconstruction. Since extensive and reliable data on the biochemistry and genome is available for this model organism, the reconstructed genome-scale models turned out to be exceptionally accurate and proved to be invaluable to gain insights into the metabolism of E. coli or to identify intervention strategies for a targeted modification of the metabolism for biotechnological applications1Constraint-based and stoichiometric modeling has become a standard tool for analyzing key properties and capabilities of metabolic networks and numerous studies have demonstrated the power and wide applicability of this approach12456891011NetworkReducer, a network reduction algorithm that allows the derivation of stoichiometrically consistent submodels from genome-scale modelsNetworkReducer has been demonstrated before, the goal of the current contribution is to derive a widely applicable core model of the central metabolism of E. coli from a reference genome-scale network and to analyze its properties in relation to the genome-scale parent model.However, the increasing size and complexity of genome-scale models have set restrictions on the computational feasibility of some stoichiometric modeling techniques. For example, exhaustive elementary modes analysisE. coli have been used in the literature. However, almost all of them were derived in a bottom-up manner and are therefore not related to and consistent with a corresponding genome-scale model. One exception is the E. coli core model presented by Orth et al.EColiCore1, was manually derived from the genome-scale model iAF1260 for educational purposes. Whereas EColiCore1 perfectly fits the demands it was developed for, the stoichiometric properties are closely related but not fully consistent with its genome-scale parent model iAF1260NetworkReduceriAF1260 having the same scope as EColiCore1. However, even this core model has several drawbacks. First of all, it was not derived from the latest reference genome-scale model iJO1366. Secondly, it lacks some central metabolic pathways such as the Entner-Doudoroff and the methylglyoxal pathway. Furthermore, we wanted to protect a larger set of phenotypes in the core model including feasibility of growth on a selection of standard substrates as well as producibility of standard (fermentation) products by E. coli. One major goal of the present contribution was therefore to derive a stoichiometric model of the central metabolism of E. coli from iJO1366 that fulfills these requirements and therefore holds the potential to become a reference model of the central metabolism of E. coli accessible for all types of constrained-based modeling. Another central goal of this study is to systematically compare key properties of the derived core model, EColiCore2, with those from its genome-scale parent model iJO1366. This analysis, which is the first of this kind, includes a comparison of the solution space with respect to flux ranges, reaction essentialities, and production capabilities. We demonstrate that EColiCore2 is capable to describe several key properties of iJO1366 including those that were not explicitly accounted for. Generally, as intended, the derived core model reflects the degrees of freedom in the central metabolism of E. coli very well while neglecting redundancies along biosynthetic routes. A full enumeration of the complete set of elementary modes or of metabolic intervention strategies becomes possible in EColiCore2. Using ethanol as a relevant product, we also compare metabolic engineering intervention strategies in the core and the genome-scale model and demonstrate how the core model can even be used as a basis for the identification of metabolic engineering strategies with higher cardinalities in the genome-scale model that were computationally not tractable before.Several different small-scale models of the central metabolism of E. coli core model we used the recently introduced network reduction algorithm NetworkReducer which takes a given large-scale metabolic network as input and derives a smaller subnetwork while keeping desired features of the full network intactk is described by a corresponding set of linear inequalities:For deriving the s denoting the number of protected functions and phenotypes. In a first (pruning) step, NetworkReducer iteratively removes non-protected reactions and metabolites while checking that none of the protected functions are violated. Afterwards, an optional network compression step can be applied which, for example, lumps reaction sequences (unbranched pathways) into single reactions. This second operation further reduces the size of the network but does not change the space of feasible steady-state flux distributions. For further details on NetworkReducer we refer to the original workwith iJO1366 was used as the gold standard for a well-curated genome-scale stoichiometric network model for E. coli’s metabolism. We introduced some minor modifications to iJO1366 prior to the application of the network reduction routine:We added external metabolites for all reactions where compounds are secreted into or taken up from the environment. This allows a more convenient consideration of mass balances of those metabolites  but does not change the solution space.Further, we allowed reaction R_nadh16tpp (NADH dehydrogenase) to be reversible as otherwise succinate excretion is obligatory under anaerobic conditions  excretion became essential reactions because they are needed to consume formate and glyoxylate formed as byproducts of some synthesis pathways for biomass precursors. This behavior would not reflect reality. In fact, in the genome-scale model, there are different routes that consume these metabolites , however, these pathways would be lost in the reduction process. To avoid such an undesired behavior of the reduced model, we demanded that growth without formate excretion and malate synthase is possible and replaced metabolites M_glx_c (glyoxylate) and M_for_c (formate) in the reactions R_GLYCTO4, R_GLXCL, R_AMPMS2, R_DB4PS, R_FTHFLi, R_GTPCI and R_GTPCII2 by auxiliary metabolites M_glxBIO_c and B_forBIO_c, respectively. These changes reflect the assumption that consumption of glyoxylate and formate produced during biomass synthesis is realized via these reactions. Importantly, this modification does not produce new steady-state flux distributions, it only excludes those solutions that would render malate synthase and formate (or hydrogen) production essential.When applying the The latest reconstruction iJO1366 model will be denoted by ECGS  throughout the manuscript. Flux bounds were set as in the original model iJO1366, in particular, a maximal substrate uptake rate of 10 mmol/gDW/h was used (if not stated otherwise) and the ATP maintenance demand was set to 3.15 mmol/gDW/h. The flux range of all reactions with unknown capacities were set to  for reversible and  for irreversible reactions.For clarity, the slightly modified NetworkReducer. As the goal is to construct a core model that represents the central metabolism of E. coli in detail while neglecting redundancies in anabolic synthesis pathways we protected the reactions and metabolites involved in glycolysis, pentose phosphate pathway, Entner-Doudoroff pathway, tricarboxylic acid cycle, methylglyoxal pathway and key reactions of the electron transport chain. In addition, six major anaplerotic reactions (including the glyoxylate shunt) and two NAD(P) transhydrogenase reactions were protected. To account for biomass synthesis we demanded that the reaction R_Ec_biomass_iJO1366_core_53p95M of ECGS must be maintained. In total, 65 reactions and their reactants have been protected in the central metabolism as listed in the Next we specified the protected parts and phenotypes of the genome-scale model to be retained by NetworkReducer to protect not only parts but also behaviors or phenotypes. We therefore demand that the reduced network must allow for growth on four representative carbon substrates  with at least 98% of the maximal growth rate achievable with the ECGS model  are not included in this set. Here we exploit the possibility of GS model . This enGS model . For allCellNetAnalyzerEColiCore2) and the compressed pruned (EColiCore2comp) networks are provided in SBML format in CellNetAnalyzer projects on the repository website of CellNetAnalyzer (http://www2.mpi-magdeburg.mpg.de/projects/cna/repository.html).The network reduction and all other calculations were done with NetworkReducer to the iJO1366-based genome-scale model ECGS as described in the Methods section , we obtained EColiCore2 (in the following abbreviated by ECC2), a core model of E. coli’s metabolism where all the specified protected parts and phenotypes have been maintained. ECC2 contains 499 reactions (thereof 198 reversible) and 486 internal as well as 33 external metabolites  preserves major metabolic phenotypes. Therefore, we consider ECC2 as a qualified solution suitable for further in-depth analysis.We emphasize that network ECC2 is not the only possible solution and other subnetworks can solve the reduction problem equally well. However, by definition, all alternative networks share the set of protected parts (metabolites and reactions) and protected phenotypes. Further, by altering the order of removed reactions, we found that differences in alternative reduced networks occur mainly in alternative routes for the synthesis of biomass precursors. Our focus was to obtain a core model that (a) represents possibility of anaerobic production of lactate, succinate and ethanol was demanded, their maximal production rates correspond exactly to those of ECGS . The maximal product for formate, hydrogen, and acetate in ECC2 compare also well with what is known (and expected) from experimental observations, however, they are much higher in ECGS (iJO1366 for aerobic growth on glucose is 2.93 (but only 2.0 in ECC2). We refrained from demanding that ECC2 reproduces these extremely high product yields as they are not expected to be of relevance under physiological conditions .The demanded biomass yields for the different substrates are achieved by ECC2. For all substrates, the maximal biomass yields in ECC2 are even above 99.9% of the genome-scale maximum except for aerobic growth on acetate where 98.8% of the maximal biomass yield of ECGS is achieved . Regardi in ECGS . For exaNetworkReducer to ECC2 generates an even more reduced network, ECC2comp, where coupled reactions  are lumped into overall reactions. The lumped reactions can be classified into two different categories. The vast majority (399) are reactions that are fully coupled (form an enzyme subset) with the biomass synthesis reaction. In addition, 21 reactions of the ECC2 model involved in chains of transport reactions (secreting metabolites from the cytosol via periplasm to the extracellular compartment) have been lumped as well. The compression thus further reduced the number of reactions and metabolites down to 79 reactions and 61 internal and 33 external metabolites. Initially, ECC2comp contained 25 degrees of freedom as ECC2, however, for better handling, we split up the reversible exchange reactions for carbon dioxide (CO2), acetate, and succinate into two irreversible (uptake and excretion) reactions respectively thereby increasing the number of reactions to 82 and the degrees of freedom to 28  and biomass byproducts compared to ECC2 and ECGS. However, the adjusted stoichiometric coefficients and the new precursors reflect the metabolites and their amount needed to produce all the building blocks contained in the original biomass synthesis reaction of ECC2 and ECGS . The growth rate was fixed to its near optimal value (0.98 h−1) and for all remaining reactions the minimal and maximal flux values were calculated for both networks. With ECC2 being a subnetwork of ECGS, it follows that the flux range of a reaction in ECC2 will be contained in the flux range of this reaction in ECGS. For 290 out of the 499 reactions in ECC2 (58.1%) a (close to) perfect agreement with the corresponding flux ranges in ECGS can be observed (difference less than 0.1 mmol/gDW/h) and for additional 153 reactions (30.7%) a good agreement can be found   . For onlmax) of the mutant is still above 95% of the wild type μmax. If μmax is reduced by more than 5% but growth still possible with more than 5% of the wild type μmax then the reaction is considered growth-limiting but not essential. Finally, if μmax falls below 5% of the maximal growth rate in the wild type, the reaction is considered to be lethal.Reaction essentialities for growth represent another important functional network property. We focused on reaction essentialities for growth on glucose. Separately for each reaction we fixed the rate to zero and maximized the growth rate. A reaction knockout is considered to be without significant effect if the maximal possible growth rate . For each internal metabolite a corresponding excretion reaction was temporarily introduced and then maximized.2 fixation would be conducted by E. coli which is unlikely as such a behavior has, to our knowledge, not been reported yet for these metabolites.Similar as for reaction essentialities, the maximal yield of any metabolite in ECC2 can never be greater than in ECGS since all feasible flux distributions in ECC2 are also possible in ECGS. For the vast majority of metabolites 77.8%,  we foundProduction envelopes (PEs) are projections of the solution space of feasible steady-state flux distributions on a two-dimensional plane. The growth rate is typically projected on the x-axis and a selected product synthesis (excretion) rate on the y-axis. PEs can be computed by means of sequential FVA where the growth rate is incremented in discrete steps from its minimum to its maximum and the minimal and maximal product synthesis rate is computed for each step. It should be noted again that, since ECC2 is a sub-network of ECGS, any PE of ECC2 will be contained in the corresponding PE of ECGS.2 release under aerobic conditions. It can be seen that the maximal growth and CO2 production rates are well represented by the reduced model while some deviations occur for the lower boundaries of CO2 release. For example, while the genome-scale model ECGS allows for CO2 fixation with acetate and succinate as substrates this behavior is not reflected by ECC2. The second and third row show the PEs for growth versus synthesis rates of typical fermentation products with glucose as substrate under any and under exclusively anaerobic conditions, respectively. Except for acetate, the PEs are either identical or, for the case of ethanol production under anaerobic conditions, show a large overlap confirming that the maximal yields of those products in ECGS are also reachable by ECC2. As was already mentioned above, whether the predicted maximal acetate yield with ECGS of almost 3 mol acetate per mole glucose is a realistic value remains questionable. In particular, homoacetic acid fermentation in E. coli (without incorporation of heterologous pathways) as predicted by ECGS has, to our knowledge, not been observed so far. Therefore, we did not protect this property in ECC2 and consider this deviation rather as correction than as restriction. The last row in A selection of representative PEs is given in As one goal of the whole reduction process, with the reduced degrees of freedom in ECC2 and ECC2comp, these models become accessible for the enumeration and subsequent analysis of elementary modes (EM). In the following we will discuss the EM of ECC2 keeping in mind that, as already mentioned, the EM are equivalent for both networks  and storage of EM from ECC2comp will require less than 20% of the memory needed to store the EM of ECC2 (82 vs. 499 reactions). A comparison of the set of EM from ECC2 and ECGS is not possible. So far, the computation of EM for genome-scale models is limited to approaches that compute the shortest EMIn the following we want to study how metabolic engineering strategies identified in the ECC2 model relate to the corresponding intervention strategies computed in ECGS. As a relevant product we chose ethanol which has previously been used as a “benchmark” case in other studies279293027−1.Here we consider production of ethanol from glucose under anaerobic conditions (oxygen uptake rate set to zero). The maximal glucose uptake rate was increased to 18.5 mmol/gDW/h to reflect the higher glucose conversion rates under anaerobic conditions. We excluded pseudo (transport and excretion) reactions from the list of possible knockout targets leaving only reactions that can be targeted by genetic modifications as knockout candidates. We designed the MCS problem as follows: the engineering goal with respect to product formation was formulated by demanding a minimal product yield of 1.4 mol ethanol per mol glucose consumed while growth should be feasible with a minimal rate of 0.05 hFor these constraints, we calculated the complete set of MCS with up to ten reaction deletions for both models, ECGS and ECC2. In total, we found 2176 MCS in ECC2 and 44066 MCS in the genome-scale network ECGS . Each MCECC2 and a cut set of ECGS with MECGS. We first consider the case that we are given one specific MCS of ECC2 (denoted by M*ECC2) and classify each single MECGS with respect to M*ECC2 , hence, the knockouts of M*ECC2 are also sufficient in ECGS to obtain the desired phenotype. An MCS of ECGS belongs to subclass A.2 if it contains all reactions of M*ECC2 complemented by further targets exclusively contained in the ECGS (and not in the ECC2) network to block additional unfavorable pathways in ECGS. Importantly, if the given M*ECC2 is not a valid MCS in ECGS (no MECGS of type A.1 exists for M*ECC2), then some MECGS of type A.2 must exist: knocking out the targets from M*ECC2 and all reactions from ECGS that are not contained in ECC2 (as they were deleted during the reduction process) will lead to a solution space in ECGS that is identical to the one obtained when deleting only M*ECC2 in ECC2. Clearly, not all the reactions exclusively contained in ECGS need normally be deleted to block flux distributions feasible in ECGS but not in ECC2; a minimal set of reaction deletions will be sufficient which is then combined with M*ECC2. We can thus conclude that any MCS of ECCS, that is not a cut set in ECGS, can always be extended to a valid MCS of type A.2 in ECGS. Hence, for a given M*ECC2, there is always either one MECGS of type A.1 or one or several MECGS of type A.2. The other two possible subclasses A.3 and A.4 arise if M*ECC2 is complemented by deletions of at least one additional reaction from ECC2 either without  or may not (B.1) be combined with deletions of reactions exclusively contained in the ECGS network.Cut sets in ECGS which are not supersets of the given Mgiven cut set M#ECC2 in ECC2 with respect to each cut set in ECGS. Every cut set of ECGS belongs then to exactly one subclass. Notably, for different cut sets from ECC2, a certain cut set M#ECGS can be of class A or B. If it is of type A.1 or A.2 for one particular M*ECC2, then it cannot be of class A for any other MCS of ECC2 . If it is of type A.3 or A.4 for M*ECC2 then it can be of type A.3 or A.4 for other MCS of ECC2 (but it cannot change from A.3 to A.4 or vice versa). Of special interest are MCS of ECGS that are exclusively of type B for all MCS of ECC2 and thus not a superset (extension) of any M*ECC2. Those cut sets of ECGS involve deletion patterns in the ECC2 part that were not allowed in cut sets of ECC2 because they would violate some of the specified desired phenotypes, for example, if some of the reactions knocked-out would be essential for growth or product synthesis in ECC2 (but not in ECGS). For this reason, such MCS in ECGS may, in the extreme case, comprise fewer interventions than the smallest MCS in ECC2 . All relationships between cut sets discussed above hold true not only for ECGS and ECC2 but for any pair of a (full) metabolic network and a subnetwork of it.So far we discussed the relationships between one must exist in ECGS) will have at least 11 reaction deletions. 12 MCS of ECGS are of type A.3 and thus extensions of MCS from ECC2 involving additional reaction deletions only in the ECC2 part. Finally, 1,070 MCS of ECGS establish relationships of type A.4. All found 1,082 MCS in ECGS of class A are supersets of 18 distinct MCS of ECC2. For each of the 4 MCS in ECC2 with cardinality 5 there exist MCS in network ECGS with 9 reaction deletions which include all deletions of the respective MCS of network ECC2 and additional deletions. Further, for 14 MCS in ECC2 with cardinality 6 there exist superset MCS in ECGS with two to four additional reaction deletions. For the remaining 2,154 MCS of ECC2, no supersets have been found among the calculated MCS in network ECGS. Since, as explained above, MCS of type A.1 or A.2 must exist, this indicates that the corresponding MCS in ECGS have cardinalities above ten. Lastly, 44,066 − 1,082 = 42,984 of the MCS of cardinality up to ten in ECGS are not a superset of any MCS in ECC2, hence, are exclusively of type B.1 (221) or B.2 (42763).We analyzed the relationships between the calculated MCS in ECC2 and ECGS for the ethanol case study  with restop-down approach, we could initially combine the knockouts of the seed MCS with knockouts of all reactions from ECGS that are not contained in ECC2. As already discussed, this would result in a valid but, most likely, non-minimal cut set. A constrained minimal cut set for ECGS can afterwards be found by iteratively removing knockout candidates from this cut set and validating a posteriori if the undesired phenotype is still blocked. If the removal of a reaction from the cut set violates the target constraints this reaction is reinserted and the cut set is eventually identified as a valid MCS if no further reaction can be removed.The fact that any MCS found in the reduced network ECC2 will be a subset of one or several corresponding MCS in the full network ECGS can be used to construct MCS for the genome-scale model. The strategy is to augment a “seed” MCS from ECC2 by additional knockouts to obtain valid MCS for ECGS. Although all cut sets of type A.2, A.3, and A.4  could, iseed of reaction knockouts for ECGS and the corresponding reactions are inactivated in the ECGS model. Subsequently, all MCS in this model are computed but allowing knockouts only in reactions contained in the ECGS but not in the ECC2 model. By excluding all reactions from ECC2 as knockout candidates for ECGS, the remaining search space is considerably reduced (here by 499 reactions). The union of the seed MCS together with the MCS found in the ECGS network yields a valid MCS for the full ECGS network. In principle, this procedure can be used to determine all MCS in ECGS of type A.2 (and MCS of type A.1 can be easily identified beforehand).A “bottom-up” or “expansion” approach would tackle the problem of finding smallest MCS in a more directed manner: Again, a MCS of ECC2 is used as a We pursued the expansion approach to identify additional knockout strategies for ECGS based on the MCS from ECC2. Each of the 2176 MCS of ECC2 was used as seed in ECGS and corresponding MCS for ECGS with up to 15 additional reaction deletions were identified. Since the (up to 10) reactions of the respective seed MCS from ECC2 are inactivated prior to the computation of corresponding superset MCS in ECGS, the computed MCS can contain up to 25 interventions. The results of these calculations can be summarized as follows. For 1020 (of the 2176) seed MCS from ECC2 we found at least one MCS in ECGS with maximal 15 additional reaction deletions. For 1156 seeds no MCS could be identified in ECGS indicating that the allowed 15 reaction deletions do still not suffice to obtain valid MCS for network ECGS. If the MCS of ECC2 with cardinality 5 are used as seeds, at least 8 additional knockouts (of reactions only contained in ECGS) were necessary to achieve a valid MCS in ECGS . However, the smallest MCS results from seeds of cardinality 6 where it is sufficient to delete 5 additional reactions resulting in valid MCS for network ECGS with cardinality 11. Recall here that we predicted before that the smallest MCS of type A.2 would need at least 11 reaction deletions as no MCS of this type A.2 exist up to cardinality 10. The biggest MCS found in this way for network ECGS had as much as 23 reaction deletions: 8 from the seeds and 15 additional knockouts. Again, all additional knockouts were restricted to ECGS reactions not contained in ECC2.We exemplarily discuss specific knockout strategies that have been identified by the different approaches. A specific MCS identified in the reduced network ECC2 suggests deletion of the six reactions i) acetate kinase (R_ACK), ii) phosphogluconate dehydrogenase (R_GND), iii) hexokinase (R_HEX1), iv) D-lactate dehydrogenase (R_LDH_D), v) pyruvate formate lyase (R_PFL), and vi) glucose-6-phosphate isomerase (R_PGI) (we denote this MCS by M1). This knockout set is a valid MCS in ECC2 but does not yet enforce the desired phenotypic behavior (coupling of growth with ethanol synthesis) in network ECGS. However, by enumerating MCS up to size ten in ECGS we found a valid MCS of ECGS that extends M1 with two further reaction deletions, namely acetaldehyde dehydrogenase (R_ACALD) and either xylose isomerase (R_XYLI2) or hexokinase (HEX7). Reaction R_ACALD is contained in network ECC2 whereas reactions R_XYLI2 and R_HEX7 are only part of ECGS. These two MCS are thus of type A.4 and cannot be found with the described expansion approach starting with M1 as a seed in ECGS. However, if we used the above MCS M1 from ECC2 as a seed in ECGS and allow only reactions from ECGS that are not contained in network ECC2 as knockout candidates, at least six additional reaction knockouts are necessary to obtain a valid MCS. For example, additional deletions of R_XYLI2 or HEX7 together with NAD dependent aldehyde dehydrogenase (R_ALDD2x), NADP dependent aldehyde dehydrogenase (R_ALDD2y), citrate lyase (R_CITL), gluconokinase (R_GNK) and acetyl-CoA:hexanoate-CoA transferase (R_HXCT) result in a valid MCS for ECGS and is found by our procedure. These knockout strategies reveal that the deletion of R_ACALD (in the ECC2 subnetwork) is equivalent to the knockout of R_ALDD2x, R_ALDD2y, R_CITL, R_GNK and R_HXCT in the ECGS network.adhE)) was identified as a knockout candidate in several MCS in ECGS but never in ECC2 (where it is the only pathway to produce ethanol). Some alternative ethanol synthesis pathways must therefore exist in ECGS that, with the suggested knockout strategies, would lead to a high ethanol yield. In fact, in ECGS, acetaldehyde can be derived from acetyl-CoA and glycine that are condensed to L-2-Amino-3-oxobutonate (R_GLYAT) which is then reduced to L-allo-threonine via reaction R_ATHRDHr and finally split to acetaldehyde and glycine by R_THRA2i. However, since the acetaldehyde dehydrogenase (adhE) is part of the main route for ethanol production in E. coli and since adhE knockout mutants are known to show no relevant ethanol excretion at all32Generally, elimination of the R_ACALD reaction  MCS in ECGS require a huge and unrealistic number of knockouts or if a calculation of (large sets of) MCS for a given intervention problem is not feasible at all in the ECGS model, it would thus be practical to start with a MCS calculated in the core network ECC2. If experimental implementation of all knockouts contained in the MCS of ECC2 is not sufficient, one may use our expansion algorithm to expand the seed MCS from the ECC2 model to obtain a valid MCS for the ECGS model (whose existence is guaranteed).To summarize, our case study showed that metabolic intervention strategies can be exhaustively enumerated in the reduced network ECC2. The MCS found in the ECC2 network for enhancing ethanol synthesis show significant overlap with the smallest MCS identifiable in ECGS. Discrepancies mainly arise due to (a) additional pathways in ECGS having a zero or low ethanol yield (which must be deleted in ECGS) and (b) additional high-yield ethanol synthesis pathways (which give rise to new intervention strategies in ECGS). For a product of the central metabolism such as ethanol, we expect that a large number of MCS found in ECC2 are sufficient to reach the desired production phenotype. In fact, successful E. coli that is stoichiometrically consistent to and maintains several key properties of the most recent and well-curated genome-scale model of this organism (iJO1366). In addition, for the first time, we systematically compared key properties of the derived metabolic core model and of its genome-scale parent model.The central goal of this contribution was the development of a reference metabolic core model of the central metabolism of NetworkReducer algorithm together with a list of protected parts and phenotypes. This list was carefully compiled to ensure that the core model reflects the degrees of freedom in the central metabolism of E. coli while it was allowed to neglect redundancy along biosynthetic routes. The derived EColiCore2 network is a subnetwork of iJO1366 still comprising 499 reactions and 486 internal metabolites. However, ECC2 is small enough for detailed studies that would not be possible in the genome-scale model. In particular, all elementary modes for growth on the included substrates can be calculated on a standard personal computer in reasonable time. In addition, by compressing (mainly biosynthetic) linear reaction chains to condensed overall reactions a compressed network version of ECC2, ECC2comp, was derived which contains 82 reactions and 54 internal metabolites. The solution space of ECC2comp is equivalent to ECC2 since every steady state flux distribution in ECC2comp can be uniquely mapped to one of ECC2 and vice versa. ECC2comp might be more practical and efficient for some applications. For example, less working memory is required for sophisticated calculations. Further, the reduced number of reactions and metabolites allows for a clear and concise graphical representation of the whole network . These examples demonstrate the trade-off in analyzing a core vs. a full genome-scale network: unrealistic or overoptimistic behaviors represented in the genome-scale model  can be excluded in a core model, however, unknown and non-intuitive pathways of possibly higher relevance might be missed when focusing only on the well-examined part of the metabolism.We performed a comprehensive analysis and comparison of different properties of ECC2 and its genome-scale parent model ECGS to demonstrate that ECC2 is a reasonable reduction of ECGS. ECC2 preserves key phenotypic behaviors and properties and allows for the analysis of major metabolic fluxes and the elucidation of engineering strategies in the central metabolism of We also studied the value of the core model for calculating relevant metabolic engineering strategies by means of a case study with ethanol as product of interest. As expected, cut sets found in ECC2 for overproduction of ethanol are usually not sufficient to completely block all potentially undesired phenotypes (with low product yield) in ECGS but already indicate key targets (and combinations thereof) which are also relevant for ECGS. Analysis of the smallest cut sets in ECGS showed that non-intuitive intervention strategies may exist in the genome-scale model which either target additional unfavorable routes that were not contained in the ECC2 model or/and redirect carbon flux to hidden (unknown) product synthesis pathways in the full network. While the cut sets of the ECGS model are complete and ensure that the desired phenotypes are really achieved in the full model, the relevance of some of the found alternative targets  and pathways is questionable. Generally, one can expect that intervention strategies calculated in ECC2 for products of the central metabolism  will have a better predictivity (most relevant pathways for their production are contained in the model) than those calculated for products of the anabolism where relevant - favorable and unfavorable - pathways might have been removed in the pruning step.We further clarified the relationships between cuts sets of the full and of a subnetwork. With respect to a given subnetwork (here ECC2) one can distinguish two major classes of cut sets in the full (ECGS) model: a cut set in ECGS is either a superset of one  cut sets found in ECC2 , or it is not a superset of any cut set in ECC2 and therefore involves other deletion patterns in the ECC2 subnetwork possibly combined with further knockouts in reactions exclusively contained in ECGS. Whereas cut sets of the latter type can therefore not be obtained or deduced from ECC2, we demonstrated that cut sets in ECGS that are supersets of cut sets from ECC2 can always be obtained by expanding seed cut sets in ECC2. Thus, ECC2 can initially be used to find intervention strategies leading to desired phenotypes in the core model. If a particular cut set was experimentally not successful (the mutant shows still an unfavorable phenotype in the experiment) or if one is interested to determine (some or more) full intervention strategies in the ECGS model, the respective knockout strategy of ECC2 can be used as a seed and then be extended by further knockouts to a valid MCS for ECGS. We demonstrated that this approach can be used to determine thousands of additional valid MCS for ECGS with higher cardinalities which could not be calculated before.E. coli are well reflected in the reduced model.A major advantage of ECC2 is that it is a strict submodel of its genome-scale parent model ECGS so that results from ECC2 can be directly related to ECGS. In fact, this property was used to determine cut sets in the ECGS model from seed cut sets calculated in ECC2. Furthermore, all flux distributions in ECC2 are valid solutions in ECGS and, likewise, all EM of ECC2 are equally valid in ECGS. Hence, analyzing ECC2 means analyzing a subset of the feasible phenotypes of ECGS and ECC2 was derived in such a way that metabolic fluxes in the central metabolism of As desired, ECC2 allows for the full enumeration of EM and metabolic engineering strategies and it is readily usable for educational purposes due to its appropriate scope, size, and clarity. If certain pathways or/and metabolites contained in ECGS but not in the ECC2 model become of interest in a particular application, they might be added to the core model which should still keep the model size low enough to perform detailed calculations (e.g. EM enumeration). Likewise, if one wants to protect other phenotypes from the genome-scale model, the reduction process can be repeated with adapted constraints.E. coli, similar to its well-curated and evolved genome-scale parent model iJO1366.With the results presented in this study, we believe that ECC2 holds the potential to become a reference metabolic model for the central metabolism of How to cite this article: Hädicke, O. and Klamt, S. EColiCore2: a reference network model of the central metabolism of Escherichia coli and relationships to its genome-scale parent model. Sci. Rep.7, 39647; doi: 10.1038/srep39647 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.