Objectives. Five different second-generation supraglottic airway devices, ProSeal LMA, Supreme LMA, i-gel, SLIPA, and Laryngeal Tube Suction-D, were studied. Operators were inexperienced users with a military background, combat lifesavers, nurses, and physicians. Methods. This was a prospective, randomized, single-blinded study. Devices were inserted in the operating room in low light conditions after induction of general anesthesia. Primary outcome was successful insertion on the first attempt while secondary aims were insertion time, number of attempts, oropharyngeal seal pressure, ease of insertion, fibre optic position of device, efficacy of ventilation, and intraoperative trauma or regurgitation of gastric contents. Results. In total, 505 patients were studied. First-attempt insertion success rate was higher in the Supreme LMA (96%), i-gel (87.9%), and ProSeal LMA (85.9%) groups than in the Laryngeal Tube Suction-D (80.6%) and SLIPA (69.4%) groups. Insertion time was shortest in the Supreme LMA (70.4 ± 32.5 s) and i-gel (74.4 ± 41.1 s) groups (p < 0.001). Oropharyngeal seal pressures were higher in the Laryngeal Tube Suction-D and ProSeal LMA groups than in other three devices. Conclusions. Most study parameters for the Supreme LMA and i-gel were found to be superior to the other three tested supraglottic airway devices when inserted by novice military operators. Combat lifesavers in the Czech Army have already been trained for several years in easy and effective airway management during practicing medicine in the field. Generally, the algorithms of the Tactical Combat Casualty Care (TCCC) are applied . The rolThis study was designed as randomized, prospective, and single-blinded (patient side). Ethical approval was obtained from the Local Ethical Committee (IRB), number 80-76/39-2012-UVN. The research was performed in full accordance with the Helsinki Declaration and the study was registered with a public database (R&D IS of the Czech Republic).All patients scheduled for elective procedures under general anesthesia during the study period and meeting the inclusion criteria were invited to participate in this study. The study setting was University Military Hospital in Prague, Czech Republic. Approximately 10 000 procedures under general anesthesia are performed in this hospital annually. The study period lasted from August 2012 till December 2013.2), history or prediction of difficult laryngoscopy, pregnancy, and edentulous subjects. The randomization process was performed using a randomization list created with the freeware http://www.psychicscience.org/ and patients were randomized after signing the informed consent with the code provided in sealed envelopes. The following SADs were used in patients, ProSeal laryngeal mask airway , Supreme laryngeal mask airway , i-gel , Streamlined Liner of the Pharyngeal Airway (SLIPA) , and Laryngeal Tube Suction-D   , known a2 was controlled at 1, 5, and 10 minutes after insertion as a marker of efficient ventilation.The primary outcome of the study was to evaluate the first-attempt insertion success rates of the devices and compare these between the groups. Secondary outcomes were evaluated as follows: time needed for successful insertion, number of insertion attempts, oropharyngeal seal pressure (OSP), ease of insertion, fibre optic check of the vocal cords through the devices, and presence of perioperative oropharyngeal trauma or gastric content aspiration/regurgitation. etCO−1) and a continuous infusion of remifentanil (1-2 mcg·kg−1·min−1) until the loss of verbal contact and eyelash reflexes. Anesthesia was maintained using isoflurane in an air : oxygen mixture whereas analgesia was maintained with a continuous infusion of remifentanil at a rate of 0.2–1.0 mcg·kg−1·min−1. No muscle relaxants were given as part of the study protocol.All patients were premedicated using oral midazolam at a dose of 7.5 mg prior to surgery. Induction of general anesthesia was performed without the use of artificial light in gloomy conditions , using p−1 and to eliminate CO2 (etCO2 over 6.5 kPa) at 10 minutes despite repeated insertion attempts. If the operator was not able to achieve successful ventilation on five insertion attempts, tracheal intubation was performed as a rescue procedure by a supervising consultant. Insertion time was defined as time needed for SAD preparation , its insertion, cuff inflation (if applicable), and confirmation of effective ventilation with a visible etCO2 tracking on the monitor and this started immediately after finishing initial bag-mask ventilation. The cuffs of inflatable devices  were inflated according to the manufacturer recommendations. Correct position of PLMA, SLMA, and i-gel was also confirmed using “suprasternal notch” and “bubble” tests. Patients were artificially ventilated using pressure control ventilation (PCV mode) with a target tidal volume of 7 mL·kg−1. Other parameters of ventilation were respiratory rate 12–16 per minute, inspiration/expiration ratio 1 : 2, and PEEP 4 cm H2O. End-tidal CO2 level was maintained between 4.7 and 5.3 kPa. An airway leak test was performed at 5 min after insertion; pressure limit was set to 40 cm H2O, the APL valve was fully closed, and air flow was set to 3 L·min−1. Oropharyngeal seal pressure (leak pressure) was defined as the pressure inside the system when the first sounds were audible above the larynx using a stethoscope [SADs were inserted only by inexperienced operators working or undergoing training in a military hospital. They were defined as users who have not inserted an SAD more than five times in real patients. The operators included combat lifesavers, military paramedics, nurses, surgical scrub nurses, or junior doctors at the beginning of their training. All participants were employed by the Czech Army and completed Basic Airway Skills Course according to their job competencies. The course agenda included bag-mask ventilation, insertion of oral/nasal airways, insertion of supraglottic airway devices, tracheal intubation, and surgical cricothyrotomy. Majority of training is performed on manikins or simulators. In regard of SADs all novice operators observed instructive insertion of these devices, had opportunity to insert them on manikins, and were apprized of each SAD prior to its insertion. A consultant anesthesiologist was always present in order to deal with failures or complications. The consultant performed a short presentation about the SAD to the operator prior to insertion, explaining its preparation, lubrication of the device, insertion technique, fixation, and evaluation of its efficacy. Size of the devices was selected according to the weight of patients and manufacturer recommendations. Level of consciousness was evaluated in all patients after induction of general anesthesia using the AVPU score and Glasgow Coma Scale. The devices were inserted only in patients who did not react to painful stimulus, forced jaw thrust. Failure to effectively insert an SAD was defined as impossibility to achieve tidal volumes of 4 mL·kgFibre optic check of the SAD position was performed through the device using a flexible fibrescope. The following scoring system was used: (1) full view of glottis, (2) vocal cords, arytenoids, and inferior surface of epiglottis visible, (3) only superior surface of epiglottis visible, and (4) no part of epiglottis or larynx visible .This was a subjective evaluation performed by the operator, for which a five-point Likert scale was used ((1) very easy insertion, (2) easy insertion, (3) neither easy nor difficult insertion, (4) difficult insertion, and (5) very difficult insertion).The devices were carefully examined after removal for any signs of blood or gastric fluid. The oral cavity of patients was evaluated for bleeding or signs of regurgitation after removal of the SAD. Postoperative complaints were not evaluated.p values less than 0.05 were considered as significant.The sample size was calculated for an alpha-error of 0.05 and a power of 80% (beta-error of 0.2) to detect a 20% difference in insertion success rate on the first attempt. 65% success rate was considered as a baseline according to the study of Ragazzi et al. . It was In total 520 randomization codes were created and finally 505 patients were included in the study. Fifteen patients dropped out during the study period or their charts were incomplete. p = 0.012), SLIPA (p = 0.0001), or LTS-D (p = 0.0004) groups. Similarly, both PLMA and i-gel showed higher insertion success rate on the first attempt than SLIPA device .Insertion success rate on the first attempt varied between the devices . These sp = 0.005), i-gel and SLIPA (p = 0.01), and PLMA and SLIPA (p = 0.049), respectively.The total insertion success rate was similarly high for the SLMA (99%), PLMA (97%), and i-gel (99%), whereas the LTS-D (93.1%) and SLIPA (90%) showed slightly lower numbers. However, statistically significant difference was achieved only between SLMA and SLIPA (p < 0.001) when compared with the SLMA and i-gel. The highest oropharyngeal seal pressures were achieved with the PLMA and LTS-D devices. The remaining three devices exhibited significantly lower seal pressures: p < 0.001 (Time for successful insertion was shortest in the SLMA (70.4 ± 32.5 s) and i-gel (74.4 ± 41.1 s) groups in comparison with the other three devices . Inserti < 0.001 .p < 0.001. Significant differences in the view of the glottis were also found between the i-gel and SLMA (p < 0.05) or SLIPA (p < 0.05) (p < 0.05).Best view of the glottis, as confirmed by fibre optic evaluation, was achieved with the i-gel airway (88.7% of grade 1 or 2) while this was lowest in the LTS-D group (only 62.2% of grade 1 or 2):  < 0.05) . Fibre oRegarding ease of insertion, the participants reported that the SLMA was the easiest device to insert, 62% very easy and 31% easy, while the LTS-D and SLIPA were most difficult to introduce, less than 50% of very easy or easy insertions .p = 0.005).Perioperative complications such as blood on the device on removal or regurgitation/aspiration of gastric contents occurred only in 18 patients (3.8%). Blood on the device after removal was found in one patient in the PLMA (1%), SLMA (1%), and i-gel (1%) groups, in five patients managed with the LTS-D (4.9%), and in ten patients in the SLIPA group (10%). One patient significantly regurgitated through the gastric lumen of the PLMA but did not aspirate. Insertion of the i-gel was associated with one case of soft palate trauma which presented as minor bleed lasting for four hours. There was no significant difference between the PLMA, SLMA, i-gel, and LTS-D groups in the incidence of blood traces on the device. SLIPA was associated with significantly higher airway morbidity than PLMA, SLMA, and i-gel  in simulated field scenario when all devices were inserted by nonexperienced military personnel. The main findings are that the most suitable devices for use in this scenario are the Supreme LMA and i-gel airway and that the LTS-D and SLIPA have less favorable insertion parameters as well as other features. Important parameters for use in the field are the insertion success rate and speed of insertion. Both the SLMA and i-gel airway not only showed a high first-attempt insertion success rate and faster insertion times than other SADs but also were evaluated by the participants as most “easy to insert.” PLMA has a reasonable insertion profile but it possesses significant disadvantage compared to the other studied devices. The device is not available as a disposable version and the use of reusable device in the field is difficult. The i-gel may offer another potential advantage against the rest of these SADs. It can be used as a conduit for a tracheal tube placement such as with an intubating LMA .The results of our study may be compared with other evidence related to 2nd-generation SADs. Ragazzi et al. compared insertion success rates and other parameters between the SLMA and i-gel when inserted by novice operators . They foERC guidelines  recommenThis study has several limitations related mainly to its emphasis on military medicine. Simulated conditions differ significantly from real situations in the field. Field conditions are very stressful for military paramedics, unconscious victims may struggle, and there is often orofacial or neck trauma with bleeding present, causing airway management to be more difficult. The victims are also often found in various positions although they are subsequently moved to supine position for airway management procedures.Furthermore other factors such as light conditions, neck immobilization, quality of training, or device insertion technique affect success rate significantly in out-of-hospital scenarios. Gastric tubes were also not inserted through the devices to drain gastric contents. We have decided against their insertion because one of the devices (SLIPA) does not allow gastric tube insertion and also because gastric tubes are not part of the emergency equipment carried by combat lifesavers. We also allowed participants to perform more than usual three attempts on the placement of device. Other limitations of the study include that postoperative airway morbidity and other patient complaints were not assessed. However, there has been no study to date published comparing these 2nd-generation supraglottic airway devices, with supposed lower incidence of gastric content aspiration in relation to airway management in the field, and the results of this study could become a starting point for further research projects evaluating the role of these devices in military medicine. Finally, few new devices, such as the 3gLM airway  or CPV GThe Supreme LMA and i-gel supraglottic airway seem to be the most convenient of the 2nd-generation supraglottic airway devices for insertion by relatively inexperienced military healthcare providers in a simulated low light scenario. Other devices tested showed either lower success rates of insertion or a significantly longer insertion time.


Clinical benefit of axitinib as a first line agent to treat patients with metastatic renal cell carcinoma (mRCC), or locally advanced renal cell carcinoma (RCC) have not been clearly demonstrated. The aim of this study was to evaluate the efficacy and safety of axitinib as first-line therapy in Japanese patients with locally advanced RCC or mRCC.In this retrospective study, we focused on eighteen patients who underwent first-line therapy with axitinib between May 2012 and May 2014 at Hirosaki University. Axitinib was orally administered at a dose of 10 mg daily. Progression-free survival (PFS) was the primary endpoint, while secondary endpoints included overall response rate (ORR) and adverse events (AEs).All patients had histologically proven clear cell RCC. The median duration of the administration of axitinib was 10.8 months. According to the response evaluation criteria for solid tumors, five patients (27.8%) achieved a partial response and nine (50%) had stable disease. The 1-year PFS rate was 84.4%, and the median PFS was 20.4 months . No serious AEs were reported during the study, and there were no toxicity-related deaths.In the current study, axitinib showed acceptable oncological outcomes and favorable safety profile as first-line therapy for locally advanced RCC or mRCC in treatment-naïve Japanese patients. Thus, first-line therapy with axitinib may provide a feasible option for treatment of advanced RCC or mRCC patients. Although the innate chemoresistance of renal cell carcinoma (RCC) is a limitation in the systemic treatment for metastatic renal cell carcinoma (mRCC) , the cliThere is no doubt that initial treatment of low- or intermediate-risk mRCC patients  with a vAxitinib, an effective and selective second-generation inhibitor of VEGF receptors-1, 2, and 3 , has demThis study, which was carried out at a single institution in Japan, aimed to evaluate the efficacy and safety of axitinib as first-line therapy in patients with advanced or mRCC.In this retrospective study, we reviewed the clinical and pathological records of a total of 39 locally advanced RCC or mRCC patients who were administered VEGFR-TKIs or mTORs between May 2012 and May 2014 at Hirosaki University. We focused on 18 patients who underwent first-line therapy with axitinib. Eligible patients had histologically confirmed clear cell RCC, with local progression or distant metastases. Data on patient demographics and tumor characteristics were obtained from the patients’ medical charts. Memorial Sloan-Kettering Cancer Center (MSKCC) criteria were evaluated based on the five risk factors: low Karnofsky performance status (<80), high LDH , low serum hemoglobin, high corrected serum calcium (>10 mg/dL), and time from initial diagnosis to axitinib treatment of <1 year [The study protocol and informed consent documents were reviewed and approved by the Hirosaki University institutional review board.Axitinib was administered orally at a dose of 10 mg daily. The axitinib dose was reduced in patients with grade 3 AEs based on the Common Terminology Criteria for Adverse Events (version 4) or two readings of systolic blood pressure at 150 mmHg or higher, or diastolic blood pressure at 100 mmHg or higher, while maintaining maximal antihypertensive therapy. In this study, none of the patients received axitinib dose titration.Based on the results of percutaneous ultrasonography-guided biopsy, the diagnosis of RCC was confirmed by a single pathologist at our institution.Baseline evaluations included complete history-taking and physical examinations, assessment of Eastern Cooperative Oncology Group performance status (ECOG PS), abdominal and pelvic computed tomography (CT) or magnetic resonance imaging (MRI), and chest radiography or CT. Tumors were measured at baseline before the administration of axitinib. The response to treatment was assessed using the Response Evaluation Criteria in Solid Tumors, version 1.1 . Bone leAll tumors were staged according to the cancer staging manual (7th edition), published by the American Joint Committee on Cancer .The primary endpoint was the PFS. The secondary endpoints were overall response rate (ORR) and AEs. The PFS was defined as the time between the initiation of axitinib treatment and the date on the CT scan that identified progressive disease (PD), on other records of clear clinical evidence of PD, or death.P values were 2-sided, and the significance level was set at a P value of < 0.05.Data were analyzed using IBM SPSS Statistics 20 . Survival after axitinib administration was estimated using the Kaplan–Meier method. All The pretreatment characteristics of the patients are listed in Table According to the response evaluation criteria in solid tumors (RECIST) criteria, five patients achieved a partial response, nine had stable disease, and four had PD. Median duration of response was 10.8 months . Tumor shrinkage was observed in 15 patients , with a median decrease of 20% in tumor size ; the median estimated blood loss was 225 mL (range: 50–380 mL). No intraoperative or postoperative complications, as defined by the Clavien-Dindo classification , were enP = 0.373). The median PFS was not reached in the locally advanced group, and it was 20.4 months in the metastasis group. According to the MSKCC risk stratification, the PFS did not differ significantly among all risk groups (P = 0.985). Two patients received sunitinib as a second-line treatment, and one patient underwent third-line therapy with pazopanib. One patient received best supportive care. The duration of effectiveness in the patients who were administered sunitinib or pazopanib as second-line treatment were 3 and 6 months, respectively.At the end of the follow-up period, none of the patients had died of cancer or other causes. The 1-year PFS rate was 84.4% Figure . The medThe AEs are shown in Table P < 0.001), respectively, in a randomized phase III trial [Till 2005, the standard of care was limited to cytokine therapy, including interferon-alpha (IFN-α) and/or interleukin-2, and these treatments were frequently associated with limited efficacy and high toxicity . A betteII trial . Sunitinad hoc analyses indicate that several AEs occur at a significantly higher rate in Asian patients relative to Caucasian patients; for example, HFS occurred in 70% of Asian patients compared with 28% of Caucasian patients (P < 0.001) [Several studies have reported widely variable rates and grades of sunitinib-related AEs ,8,9,20. < 0.001) .Common AEs of axitinib include diarrhea, hypertension, fatigue, anorexia and weight loss. The safety profile of axitinib is generally manageable with standard medical intervention . In the P = 0.038) [P = 0.022) [In this study, the treatment-naïve cRCC patients had a relatively longer PFS without axitinib dose titration, compared with other clinical trials. Rini et al. reported that in treatment-naïve mRCC patients who initially tolerated axitinib at a dose of 5 mg twice daily, a significantly higher proportion achieved an objective response with axitinib dose titration than with placebo titration . Further= 0.038) . In addi= 0.022) . In thisThe current study has several limitations. First, it is a retrospective study, with an inherent potential for bias. Second, a relatively small number of patients were enrolled in this study, and the follow-up period was relatively short. In this study, the number of enrolled patients was relatively high age compared with other randomized trials ,27. AEs In the current study, axitinib showed improved oncological outcomes and an acceptable safety profile as the first-line therapy for advanced RCC or mRCC in treatment-naïve patients. Thus, first-line therapy with axitinib may provide a promising treatment option for advanced RCC or mRCC patients. Further trials in the first-line setting are warranted.Written informed consent was obtained from the patient for publication of this case report and the accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


An anaerobic threshold (AT) of <11ml/min/kg can identify patients at high risk of cardiopulmonary complications after major surgery. The aim of this study was to assess the value of cardiopulmonary exercise testing (CPET) in predicting cardiopulmonary complications in high risk patients undergoing oesophagogastric cancer resection.2 peak). Outcome measures were length of high dependency unit stay, length of hospital stay, unplanned intensive care unit (ICU) admission, and postoperative morbidity and mortality.Between March 2008 and October 2010, 108 patients  with a median age of 66 years (range: 38–84 years) underwent CPET before potentially curative resections for oesophagogastric cancers. Measured CPET variables included AT and maximum oxygen uptake at peak exercise  and 15.2ml/min/kg  respectively; 57 patients (55%) had an AT of <11ml/min/ kg and 26 (12%) had an AT of <9ml/min/kg. Postoperative complications occurred in 57 patients (29 cardiopulmonary [28%] and 28 non-cardiopulmonary [27%]). Four patients (4%) died in hospital and 21 (20%) required an unplanned ICU admission. Cardiopulmonary complications occurred in 42% of patients with an AT of <9ml/min/kg compared with 29% of patients with an AT of ≥9ml/min/kg but <11ml/min/kg and 20% of patients with an AT of ≥11ml/min/kg (p=0.04). There was a trend that those with an AT of <11ml/min/kg and a low VO2 peak had a higher rate of unplanned ICU admission.The mean AT and VOThis study has shown a correlation between AT and the development of cardiopulmonary complications although the discriminatory ability was low. Surgical resection remains the best chance of cure for patients presenting with oesophagogastric cancer.Surgery places severe stresses on a patient’s cardiopulmonary reserve, increasing oxygen demand by approximately 40%.Over the past few years, there has been interest in the role of cardiopulmonary exercise testing (CPET).et al have shown that all postoperative cardiopulmonary deaths occur in patients with an anaerobic threshold (AT) of <11ml/min/kg and/or with significant myocardial ischaemia on CPET.Older Between March 2008 and October 2010, 180 consecutive patients underwent CPET as a formal fitness assessment prior to consideration for resection after discussion at the multidisciplinary cancer meeting. In our preoperative staging algorithm, CPET was done before positron emission tomography – computed tomography in patients with oesophageal cancer and before laparoscopy in those with gastric cancer. Patients considered for radical surgical treatment had clinical evaluation, chest x-ray, electrocardiography, pulmonary function tests and echocardiography. Stress echocardiography and thallium myocardial perfusion imaging were performed only in selected patients after review by a cardiologist. CPET was performed along with existing measures of fitness and was not the sole arbiter of fitness.® 600  and the Ergoselect bicycle ergometer  were used for the tests. During the test, patients were exposed to incremental physical exercise on a bicycle ergometer to their maximally tolerated level, which was determined by either exhaustion or, more often, the development of symptoms (pain or breathlessness). Several variables were recorded throughout the procedure including blood pressure, cardiac activity via electrocardiography, and inspiratory and expiratory gases . Two key measurements can be determined from these data, VO2 peak (oxygen uptake at peak exercise) and AT, indicating the point at which anaerobic metabolism is inadequate to maintain high energy phosphate production in exercising muscles, forcing anaerobic metabolism to make up the deficit.CPET was performed in the respiratory function laboratory in the Glasgow Royal Infirmary with a doctor and full resuscitation equipment present. The ZANPatients received the standard care under the responsible consultant surgeon with administration of prophylactic antibiotics and thromboembolic prophylaxis. The operation performed depended on tumour site, tumour size and surgeon preference. Patients were nursed in a high dependency unit (HDU) postoperatively. A diatrizoic acid swallow was performed on day 7 after the oesophagectomy and total gastrectomy to assess anastomotic healing.Common Terminology Criteria for Adverse Events.Length of HDU stay, length of hospital stay, unplanned intensive care unit (ICU) admission, and postoperative morbidity and mortality were recorded. Postoperative morbidity was divided into cardiopulmonary and non-cardiopulmonary complications. Cardiopulmonary complications were defined according to p-value of <0.05 was deemed statistically significant. Analysis was performed using SPSS® version 18.0 .Variables were grouped using standard thresholds. Categorical data were compared using chi-squared tests. Continuous data were compared using Student’s t-test and the Mann–Whitney U test as appropriate. The relationship between length of hospital stay and CPET measurements was assessed by linear regression. A Of the 180 patients, 108  with a mean age of 66 years  were suitable for surgical resection while 72 were not suitable for the reasons shown in 2 peak in the 103 patients undergoing oesophagogastric resection was 15.2ml/min/kg . The mean AT was 10.8ml/min/kg, with 57 patients (55%) having an AT of <11ml/min/kg and 26 patients (25%) an AT of <9ml/min/ kg. The mean AT in patients with significant cardiorespiratory co-morbidity and therefore deemed unfit for surgery after outpatient clinical assessment was 8.6ml/min/kg (SD: 1.9ml/min/kg). This was significantly lower than those patients undergoing resection (p<0.001). There was no significant difference in the mean AT or VO2 peak according to the operative approach, type of operation, tumour location or presence of thoracotomy.The mean VOPostoperative complications occurred in 57 patients (55%) with 29 (28%) developing cardiopulmonary complications and 28 (27%) developing non-cardiopulmonary complications . The meap=0.05) (p=0.04) . No significant increase was found in unplanned ICU admission rates or length of hospital stay in patients with an AT of <11ml/min/kg or a low VO2 peak.The mean AT in those with cardiopulmonary complications was 9.9ml/min/kg compared with a mean of 11.2ml/ min/kg in patients with no cardiopulmonary complications (p=0.05) . The maj(p=0.04) . A low V2 peak seemed to identify patients at risk.2 peak and AT as predictors of cardiopulmonary complications. The area under the ROC curve for VO2 peak and AT was 0.62  and 0.60  respectively. Using Older et al’s recommended AT cut-off value of 11ml/min/kg,p=0.09). We therefore examined a variety of alternative cut-off values, and found that an AT of 9ml/min/kg had a sensitivity of 74% and a specificity of 57% (p=0.04), and this was able to predict the development of postoperative cardiopulmonary complications in our population.This study investigated the relationship between preoperative CPET outcome measures and the development of postoperative complications in 103 patients undergoing resection for oesophagogastric cancer. The complication and mortality rates in our patients were similar to those published for the rest of the UK.2 peak. Other studies report a mean AT of 13.9ml/min/kgThe variables derived from CPET were AT and VOet al.A study from 2012 analysing the effects of a low cardiopulmonary reserve and outcome, particularly anastomotic leak, after a pancreaticoduodenectomy suggested an AT of 10.1ml/ml/kg was an appropriate cut-off level in their cohort using ROC analysis.et al assessed AT initially in relation to major surgery in elderly patients, suggesting a risk grading and treatment protocol.Older In our unit, CPET results are not used primarily to determine which patients may have an operation although they can provide objective evidence to support clinical concern. Studies have shown that preoperative optimisation with beta-blockers and statins can improve the cardiorespiratory function in high risk surgical patients.Our study has some limitations. The study population is heterogeneous with patients undergoing oesophagectomy and gastrectomy, and some requiring thoracotomy. Our study numbers are small, which may account for the lack of statistical significance associated with unplanned ICU admission, and it is difficult to determine primary and secondary complications, for example atrial fibrillation as a secondary response to an anastomotic leak.CPET provides an objective, overall assessment of cardiorespiratory reserve in patients undergoing major surgery such as oesophagogastric cancer resection. We have shown that patients with a low AT have a higher risk of developing cardiopulmonary complications. Further work is required in larger cohorts to corroborate these findings and formulate appropriate management plans for high-risk patients.


To compare quantitative computed tomography (CT) analysis and single-indicator thermodilution to measure pulmonary edema in patients with acute respiratory distress syndrome (ARDS).CT), tissue volume of lung (TVCT), tissue volume index (TVI), mean radiographic attenuation (CTmean) for the whole lung and gas-to-tissue ratio (g/t). Pulmonary thermal volume (PTV) and extravascular lung water index (ELWI) were determined by the PiCCO plus system. CT or single-indicator thermodilution variables were correlated with respiratory system compliance (Crs), PaO2/FiO2, and Acute Physiology And Chronic Health EvaluationII (APACHE II) and Sequential Organ Failure Assessment (SOFA) scores.Ten patients with ARDS were included. All underwent spiral CT of the thorax for estimating gas content of lung  as were TVI and ELWI . The bias between TVCT and PTV as well as TVI and ELWI was -277 ± 217 and 0.62 ± 4.56, respectively. 2) ELWI and CT distribution of lung-tissue compartments were not correlated. 3) CT or single-indicator thermodilution variables were not correlated with Crs, PaO2/FiO2 or APACHE II or SOFA score.1) TVQuantitative CT analysis and single-indicator thermodilution showed good agreement in measuring pulmonary edema. Interstitial lung edema is the most important pathological character of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) ,2. QuantCT) and tissue volume (TVCT) [Quantitative CT analysis provides a measure of lung volume, gas content (GVe (TVCT) . It alsoe (TVCT) . Althouge (TVCT) -11. MoreExtravascular lung water (EVLW) has been widely used to quantify pulmonary edema and guide fluid resuscitation in critically ill patients . Single-The gravimetric method of estimating EVLW is an experimental method and is the gold standard; it can be used to compare the wet and dry weight of the lung in animals or humans on autopsy, for only one measurement. The accuracy of the single-indicator method in measuring lung water has been confirmed by the gravimetric method in animals and human studies -26. In aMeasuring lung edema by the thermal indocyanine green-dye double-dilution method has shown good agreement with that by quantitative CT . Howeverplus system measurement for clinical monitoring. We excluded patients who were < 18 years old, were pregnant, or had chronic obstructive pulmonary disease, recent arrhythmia; unstable angina or myocardial infarction; chest wall trauma; or high intracranial pressure. The Ethics Committee of Qilu Hospital, Shandong University, approved the study protocol and informed consent was obtained from the patient’s next of kin.From March to December 2010, we included 10 consecutive patients with ARDS according to the criteria from the European American Consensus Conference on ARDS [plus system . Ventilation was maintained throughout the protocol.Each patient underwent insertion of a 4 F central venous catheter  in the subclavian vein with an injectate temperature-sensor housing  and a 5 F femoral arterial catheter with a thermistor tip . Both catheters were connected to the PiCCO Spiral CT of the thorax was performed from the apex to diaphragm , with continuous monitoring of heart rate, arterial blood pressure, and oxygen saturation. CT parameters were collimation scan, 20 mm; exposure, 120 kV and 220 mA; table speed, 25 mm/sec; and pitch, 1:1.When patients were transported for CT scan, the ventilatory settings were maintained as in the ICU ward. The CT scan was performed quickly during an end-expiratory pause. Previous ventilatory settings were promptly reestablished at the end of the scan. CT images were analyzed by use of a home-made dedicated software, ARDS Viewer . The ARDS Viewer allows for drawing the contour of the lung directly on a computer monitor Figure . The medLung volume (gas + tissue) was calculated as the number of voxels present in a given lung region . DespiteCT) and volume of tissue (TVCT) of the overall lung can be obtained. The mean radiographic attenuation value (CTmean) for the whole lung was computed by averaging the attenuation value of all voxels in the region of interest. The gas-to-tissue ratio (g/t) was computed as the ratio between GVCT and TVCT. The tissue volume index (TVI) was obtained by indexing TVCT to predicted body weight of the patient.All voxels in the matrix have the same volume depending on the size of the pixels of the matrix and the thickness of the CT section (0.5 cm). Therefore, the volume of gas  nplus system involves a thermal indicator to determine EVLW, cardiac output (CO), and volumetric measures. A central venous catheter and femoral arterial catheter with a thermistor tip are inserted for measurement. Cold saline (8°C), 15 ml volume, is injected into the central venous catheter. Then the thermistor tip on the femoral arterial catheter measures the downstream temperature change within the abdominal aorta. A transpulmonary thermodilution curve can be drawn. The cardiac output is calculated by the modified Stewart Hamilton equation from the area below the transpulmonary thermodilution curve. From the mean transit time (MTt) and the down slope time (DSt) of the exponential thermodilution curve, preload and lung water values are determined.The PiCCO The intrathoracic thermal volume (ITTV) can be calculated by multiplying the CO by the MTt. The thermal indicator mixes with the largest possible volume of distribution.The ITTV is composed of the pulmonary thermal volume (PTV) and the global end-diastolic volume (GEDV). PTV can be determined as the product of CO and the exponential DSt as follows.Sakka and coworkers  examinedEVLW reflects all fluid, including interstitial and alveolar, that is outside of the pulmonary vasculature during transit of the thermal indicator. EVLW is calculated as the difference between the ITTV and ITBV as follows.plus system, ITBV and GEDV are indexed to the body surface area of each patient to obtain the intrathoracic blood volume index (ITBI) and global end-diastolic volume index (GEDI); EVLW is indexed to predicted body weight for the extravascular lung water index (ELWI). All values were obtained just after the patient returned to the ICU after CT scan.In the PiCCO 2/FiO2 was calculated; and respiratory system compliance (Crs) was recorded. The Acute Physiology And Chronic Health Evaluation II (APACHE II) and Sequential Organ Failure Assessment (SOFA) scores were obtained from the worse record for each physiological variable within the 24 hr before and after CT scan.Immediately after CT scan, arterial blood was drawn for blood gas analysis, PaOCT and ELWI with TVI, we performed a linear regression analysis. Agreement between values was assessed by the Bland and Altman plot method. Correlation was evaluated between the main values  and PaO2/FiO2, Crs, and APACHE II and SOFA scores. To assess whether pulmonary edema was associated with any specific tissue compartment, we correlated the ELWI with the relative tissue volume for each compartment. All correlations were assessed by linear regression analysis with the Pearson correlation coefficient r. Statistical significance was set at P<0.05. Statistical analyses involved use of SAS 9.2 .Data are presented as mean ± SD. To evaluate the association of PTV with TV2/FiO2 < 200 mmHg, with an overall mean 103.87 ± 54.42 mmHg. ARDS was due to pneumonia for 6 patients and multiple trauma for 2, with abdominal infection for 2. Half of the patients presented a large amount of pleural fluid.We included 10 patients ; characteristics are in Table CT was 1221.37 ± 383.68 ml, TVI 18.12 ± 6.79 ml/kg, GVCT 916.58 ± 418.08 ml, CTmean -407.97 ± 118.30 HU, and g/t 0.80 ± 0.40  and PTV values overestimated those of TVCT . The agreement between TVI and ELWI was better than that for TVCT and PTV, although mean TVI was slightly larger than ELWI  and CT or single-indicator thermodilution variables , mechanics , physiologic features , and pharmacologic features (vasoactive drugs) ,31. The CT perhaps because CT cannot differentiate between lung compartments with interstitial fluid, pulmonary tissue, or residual intra-vascular blood. As well, other factors that can limit the reliability of EVLW measured by single-indicator thermodilution include the level of PEEP, the extent of the shunt in the lungs, and recirculation of cold saline. One trail estimated the CT-based ELWI by two different radiologists without analyzing software [The results of this study indicate that in patients with ARDS, EVLW and PTV measured by the single-indicator thermodilution method were highly correlated with lung tissue content measured by CT. However, EVLW and PTV determined with single-indicator thermodilution overestimated TVsoftware . CT-basesoftware . But in software ,35. Howesoftware . Saugel software . Howeversoftware .ELWI and the differences between TVI and ELWI were not correlated with proportions of any lung compartments. This result agreed with Patroniti and coworkers , but we 2/FiO2. This result agreed with some previous research [2/FiO2 was affected by many factors, such as PEEP and inhaled oxygen concentration. Even for the same patient, the position, sedation state, and airway secretions can lead to changes in PaO2/FiO2[We found no correlation between CT or PiCCO variables and Crs or PaOresearch ,37,38, aPaO2/FiO2. DynamicPaO2/FiO2, which sAPACHE II and SOFA scores have been widely used to assess the severity of critically ill patients and were closely related to the prognosis of multiple organ dysfunction syndrome ,42. We fBecause of the small population and the lack of previous data to compare with our findings, further studies are necessary to confirm our results. As well, the time of mechanical ventilation before enrollment and ventilator settings were not the same for each patient, which may have affected our results.Measuring pulmonary edema by quantiative CT analysis shows good agreement with that by single-indicator thermodilution.• Interstitial lung edema is the most important pathological character of acute respiratory distress syndrome. Quantitative measurement of lung edema can provide a useful marker of disease severity and prognosis.• Measurement of pulmonary edema by quantitative CT analysis showed good agreement with that by single-indicator thermodilution, although extravascular lung water and pulmonary thermal volume values were underestimated by the latter method for tissue volume of the lung.• As a noninvasive method, quantitative CT analysis is a gold standard for evaluating lung aeration, which may have clinical relevance in the mechanical ventilation setting and provide a quantitative “in vivo” measurement to estimate pulmonary edema.CT: Computed tomography; ALI/ARDS: Acute lung injury/acute respiratory distress syndrome; APACHE II: Acute Physiology And Chronic Health Evaluation II; CO: Cardiac output; Crs: Compliance of respiratory system; Crs: Compliance of respiratory system; CTmean: Mean radiographic attenuation value for the whole lung; g/t: Gas-to-tissue ratio; DSt: Exponential downslope decay time; EVLW: Extravascular lung water; ELWI: Extravascular lung water index for predicted body weight; ITBI: Intrathoracic blood volume index for patient surface area; ITTV: Intrathoracic thermal volume; ITBV: Intrathoracic blood volume; GEDI: Global end-diastolic volume index for patient surface area; GEDV: Global end-diastolic volume; g/t: Ratio of gas to tissue volume; GVCT: Quantitative CT analysis of gas volume; MODS: Multiple organ dysfunction syndrome; MTt: Mean transit time; PEEP: Positive end-expiratory pressure; PTV: Pulmonary thermal volume; PBV: Pulmonary blood volume; SOFA: Sequential Organ Failure Assessment; TVI: Tissue volume index for predicted body weight; TVCT: Quantitative CT analysis of tissue volume.The authors declare that they have no competing interests.FZ, CL and DW designed the study. FZ, CL and JZ planned and collected the data. FZ, CL and HG wrote the manuscript. All authors read and approved the final manuscript.


Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis (Bt) are spore-forming members of the Bacillus cereus group. Spores of B. cereus group species are encircled by exosporium, which is composed of an external hair-like nap and a paracrystalline basal layer. Despite the extensive studies on the structure of the exosporium-related proteins, little is known about the transcription and regulation of exosporium gene expression in the B. cereus group. Herein, we studied the regulation of several exosporium-related genes in Bt. A SigK consensus sequence is present upstream of genes encoding hair-like nap proteins (bclA and bclB), basal layer proteins , and inosine hydrolase (iunH). Mutation of sigK decreased the transcriptional activities of all these genes, indicating that the transcription of these genes is controlled by SigK. Furthermore, mutation of gerE decreased the transcriptional activities of bclB, bxpB, cotB, and iunH but increased the expression of bxpA, and GerE binds to the promoters of bclB, bxpB, cotB, bxpA, and iunH. These results suggest that GerE directly regulates the transcription of these genes, increasing the expression of bclB, bxpB, cotB, and iunH and decreasing that of bxpA. These findings provide insight into the exosporium assembly process at the transcriptional level. Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis (Bt) are spore-forming members of the Bacillus cereus groupBacillus encompasses species capable of forming highly resistant dormant endospores as a response to harsh environmental conditions. Spores of the B. cereus group are complex, multilayered structures. The nucleoid-containing core is enclosed within a peptidoglycan cortex, which is surrounded by the spore coatB. cereus group species are encircled by an additional loose-fitting layer called the exosporiumBacillus subtilis, for which the coat constitutes the outermost layer of the mature sporeThe genus Many characteristics of the exosporium have been previously elucidated. The exosporium is separated from the spore coat by a region known as the interspace and is the final layer of the spore to be assembled9101113149109161011212628B. cereus group. Herein, we demonstrate that the transcription of bclA, bclB, bxpA, bxpB, cotB, exsY, and iunH are controlled by RNA polymerase sigma factor SigK in Bt HD73. Furthermore, the expression of bclB, bxpA, bxpB, cotB, and iunH is directly regulated by GerE. gerE encodes the terminal transcription factor in the sporulation regulatory cascade in Bacillus subtilis. GerE is a small DNA-binding protein that is both an activator and a repressor in the mother cell that regulates the transcription of many genes involved in spore coat synthesis and assembly in the late stages of sporulation and germination3031Despite the extensive studies on the structure of the exosporium-related proteins, little is known about the transcription and regulation of exosporium gene expression in the B. cereus and B. anthracis in Bt HD73 (HD73_1438 (bclA) and HD73_2664 (bclB) encode BclA and BclB and have 67.8% and 90.0% identity, respecively, to homologous genes in B. anthracis Sterne strain 7702B. cereus ATCC 10876bclA and bclB, 5′-RACE analysis was performed as described in the Methods. The TSSs of bclA and bclB were confirmed to be a single 5′-end nucleotide residue C and G located 120 bp and 150 bp upstream of the start codon according to the sequences of 20 random clones, respectively (bclA and bclB promoter sequences identified sequences CAC(-N16-)CATATGTTA and AGC(-N16-)CATATAATT upstream of the bclA and bclB TSS, respectively, which are similar to the consensus sequences recognized by SigK-containing RNA polymerasebclA and PbclB, PbclA-lacZ and PbclB-lacZ fusions were constructed and transformed into Bt wild-type strain HD73 and mutant strains, HD(ΔsigK) and HD(ΔgerE). The β-galactosidase assay showed that the transcriptional activity of PbclA was sharply decreased from T10 to T23 in HD(ΔsigK) (10 to T18 in HD(ΔgerE), and with no significant difference from T18 to T23 compared with that of wild-type strain HD73 (bclB was sharply decreased from T9 to T23 both in HD(ΔsigK) and HD(ΔgerE) compared with that of HD73  and PbclB (276 bp) promoters was examined by EMSA. FAM-labeled fragments containing the promoter regions of bclB were incubated with different amounts of GerE and assayed for the formation of protein-DNA complexes. Slower-migrating probe-protein complexes were observed upon incubation with increasing amounts of GerE . This may result from the lack of direct binding, from a purified GerE protein partially defective in binding or from unfavorable in vitro binding conditions. These results indicated that transcription of PbclA and PbclB are controlled by SigK in the late stage of sporulation and that PbclB is directly activated by GerE, while PbclA is negatively regulated by GerE.We identified 17 exosporium homologous genes with known functions in  Bt HD73  comprisiectively A and 2A.D(ΔsigK) . It was ain HD73 . However of HD73 . To dete of GerE . It indithe EMSA . A 23-bpr region  was protbxpA (HD73_2410), bxpB (HD73_1452), cotB (HD73_0469), and exsY (HD73_1449). These genes have 75.4%, 97.0%, 76.9%, and 87.0% identity, respectively, to homologous genes in B. anthracis or B. cereus  and decreased in HD(ΔgerE) compared with those of wild-type strain HD73 , whereas it was increased in HD(ΔgerE) compared with HD73  but showed no significant difference in HD(ΔgerE)  . These riunH (HD73_3089) in Bt HD73, which has 93.1% identity to the homologous gene bas2693 in the B. anthracis Ames strainiunH was confirmed to be a single 5′-end nucleotide residue G residue located 10 bp upstream of the start codon (iunH (iunH was abolished from T8 to T22 in HD(ΔsigK) and lower in HD(ΔgerE) than in HD73  controls gene expression in the mother cell during sporulation314B. subtilis sporeB. cereus group species are encircled by the exosporiumB. cereus group. Indeed, only exsB is known to undergo SigK-mediated transcription and is positively regulated by GerE, as shown in our pervious studybclA, bclB, bxpA, bxpB, cotB, exsY, and iunH are controlled by SigK using a β-galactosidase assay. The SigK consensus sequence is located upstream of these and ten other exosporium-related genes in Bt and is predicted to be present in most B. cereus group strains . This finding suggested that the transcription mechanisms of exosporium genes are similar throughout the B. cereus group.In a B. subtilis cascade, the synthesis of each factor depends upon the activity of the prior factor, and there is a feedback loop in which SigK RNAP transcribes gerE, which then negatively regulates transcription of the sigK gene31oxdDgerT cotBcotCyxeEyeeKB. subtilis. We observed similar effects under the current conditions. The transcription of bclA and bxpA is negatively regulated by GerE, which could bind to the promoter of bxpA. Furthermore, the transcription of bclB, bxpB, cotB, and iunH is positively regulated by GerE, and their promoters could bind to GerE.In the 10). BxpA is located below the spore coat associated with the cortex and is synthesized during sporulation and assembled into the spore before mother cell lysis, but it is not found in vegetative cells in B. anthracis AmesbxpAbxpA initiates at T8 and is abolished in the sigK mutant. ExsY is a homologue of B. subtilis cysteine-rich spore coat proteins CotY and CotZexsY begins at T7 under the control of SigK and is similar to the transcriptional mechanism of cotYZ in B. subtilisB. anthracisB. subtilis spore coat protein CotBcotB begins at T10 under the control of SigK, and is regulated by GerE in Bt. The manner of transcription and regulation is similar between Bt and B. subtilis31bclA, bxpB, cotB, bxpA, exsY, and iunH in wild-type HD73 is very similar, increasing from T8 to T17 and decreasing thereafter, suggesting that these proteins are assembled into the basal layer and hair-like nap simultaneously and are nearly complete at T17. However, the transcription of bclB is significantly higher than that of blcA after T17 with continuous transcriptional activity from T8 to T23. These transcriptional data are differ to previous reports, which have suggested that bclB and bclA are transcribed at an identical stage in sporulation, but with bclB transcribed at an approximately two-fold lower level9The collagen-like glycoproteins BclA and BclB require BxpB to assemble the hair-like nap of exosporium, and the assembly timing of the three proteins is similarEscherichia coli strain TG1 was used as the host for cloning experiments. The Dam-/Dcm-E. coli ET12567 strain (laboratory stock) was used to generate unmethylated DNA for the electrotransformation assay. Bt strains were transformed by electroporation, as described previously51E. coli and Bt strains were cultured in Luria-Bertani (LB) medium, with 220 rpm shaking at 37 °C and 30 °C, respectively. The antibiotic concentrations used for bacterial selection were as follows: 100 μg/ml kanamycin and 10 μg/ml erythromycin for Bt, and 100 μg/ml ampicillin for E. coli.The bacterial strains and plasmids used in this study are listed in Taq and KOD DNA polymerase . Amplified fragments were purified using purification kits . Bt chromosomal DNA was extracted with the Puregene kit . Restriction enzymes and T4 DNA ligase  were used according to the manufacturer’s instructions. Oligonucleotide primers .PCR was performed using  primers  were synTM RACE cDNA Amplification Kit  according to the manufacturer’s instructions. Gene-specific primers and the universal primer mix (UPM) ix (UPM)  were useS-transferase (GST) tag was purified from E. coliE. coli BL21(DE3) strain carrying pGEXgerE plasmid was incubated in LB medium. When the optical density at 600 nm (OD600) reached 0.6, IPTG was added to a final concentration of 1 mM. After 4 h of induction at 37 °C, the bacterial cells were harvested by centrifuging the culture at 13,000 × g for 10 min. The pellet was resuspended in phosphate-buffered saline (PBS) and sonicated on ice. All subsequent procedures were carried out at 4 °C. The supernatant was collected by centrifuging the lysate at 13,000 × g for 20 min and loading it onto a glutathione-Sepharose 4B column previously equilibrated with PBS buffer. The column was washed with 50 mM Tris-HCl containing 10 mM reduced glutathione (pH 8.0). The fractions were analyzed by SDS-PAGE. Fractions with the target protein were pooled and dialyzed against PBS buffer. The purified GST-GerE protein was analyzed by SDS-PAGE on a 12% polyacrylamide gel with a protein molecular standard. All the steps described above were performed according to the manufacturer’s instructions .GerE protein with a glutathione cry1Ac promoter  was used as the negative control.The DNA fragment was obtained by PCR of strain HD73 genomic DNA using specific primers  labeled DNase I footprinting assays were performed based on a fluorescence labeling procedurelacZ geneΔsigK and ΔgerE mutant strains. The resultant strains, HD73(Pn), ΔsigK(Pn), and ΔgerE(Pn), were selected by resistance to erythromycin and tested by PCR to confirm the presence of the promoter fragments in the plasmids.The promoters of exosporium genes were amplified from Bt HD73 genomic DNA using specific primers. Promoter restriction fragments were then ligated into the pHT304-18Z vector containing a promoterless lacZ transcriptional fusions were cultured in Schaeffer’s sporulation medium (SSM)8 to T22 , from which cells were harvested by centrifugation for 1 min at 10,000 × g. The supernatant was removed, and the pellet was stored at −20 °C or resuspended in 500 μl Buffer Z  with 1 mM dithiothreitol. The β-galactosidase activity was determined as previously describedBt strains containing How to cite this article: Peng, Q. et al. The Regulation of Exosporium-Related Genes in Bacillus thuringiensis. Sci. Rep.6, 19005; doi: 10.1038/srep19005 (2016).


Distinguishing inflammatory bowel disease (IBD) from functional gastrointestinal (GI) disease remains an important issue for gastroenterologists and primary care physicians, and may be difficult on the basis of symptoms alone. Faecal calprotectin (FC) is a surrogate marker for intestinal inflammation but not cancer.This large retrospective study aimed to determine the most effective use of FC in patients aged 16–50 presenting with GI symptoms.FC results were obtained for patients presenting to the GI clinics in Edinburgh between 2005 and 2009 from the Edinburgh Faecal Calprotectin Registry containing FCs from >16,000 patients. Case notes were interrogated to identify demographics, subsequent investigations and diagnoses.895 patients were included in the main analysis, 65% female and with a median age of 33 years. 10.2% were diagnosed with IBD, 7.3% with another GI condition associated with an abnormal GI tract and 63.2% had functional GI disease. Median FC in these three groups were 1251, 50 and 20 μg/g (p < 0.0001). On ROC analysis, the AUC for FC as a predictor of IBD vs. functional disease was 0.97. Using a threshold of ≥ 50 μg/g for IBD vs. functional disease yielded a sensitivity of 0.97, specificity of 0.74, positive predictive value of 0.37 and negative predictive value of 0.99. Combined with alarm symptoms, the sensitivity was 1.00.Implementation of FC in the initial diagnostic workup of young patients with GI symptoms, particularly those without alarm symptoms, is highly accurate in the exclusion of IBD, and can provide reassurance to patients and physicians. The relatively non-specific clinical manifestations of gastrointestinal disease can make it difficult for clinicians to distinguish between functional and organic intestinal disease, especially in patients presenting without rectal bleeding or systemic upset.Faecal calprotectin (FC), a 36.5 kDa calcium-binding cytosolic protein found in neutrophils, is increasingly being used in clinical practice as a surrogate marker for intestinal inflammation. FC correlates with faecal excretion of white cells and a number of studies have demonstrated that FC is significantly elevated in the stool of patients with active inflammatory bowel disease (IBD) compared to control groups.The current recommended upper limit of FC in the faeces of healthy individuals is 50 μg/g. A meta-analysis of adult patients has previously given sensitivity of 95% and a specificity of 91% when using a 50 μg/g cut-off threshold for differentiating IBD from healthy controls.Since 2005 a reliable FC assay has been available in the biochemistry department at the Western General Hospital, Edinburgh. More than 8000 assays had been performed by 2008. Our clinical practice has evolved to utilise FC values in two main areas. First, FC has been used to monitor disease activity in patients with established IBD; second, to exclude IBD in patients presenting to the out-patient clinic with lower GI upset. As confidence in the utility of FC has grown, we have tended in recent years to avoid invasive endoscopic and radiological investigation in young adult patients with a negative FC (< 50 μg/g) and no alarm symptoms.This study aimed to determine the most effective use of FC in the diagnosis of GI disease in patients with no prior known GI disease, at the first presentation to GI services. We assessed how FC can be used as a non-invasive tool to aid referral to GI services, and how this improves cost effectiveness of resource allocation through reduction of unnecessary colonoscopies. Comparison was made against other serum markers to determine the optimal initial diagnostic workup of patients aged 16–50 years.Patient data were analysed from two large teaching hospitals within the same healthcare board (NHS Lothian): Western General Hospital (WGH), Edinburgh, and the Royal Infirmary Edinburgh (RIE). These were identified using the Edinburgh Faecal Calprotectin Register (EFCR), a record kept by the Biochemistry department at WGH. The EFCR contains the name, patient I.D., date of birth, referring hospital/department, and FC concentration for all of the samples analysed.The EFCR contains the data of 22,204 FC samples from 16,267 patients . PatientFor the primary analysis, patients suffering from severe intercurrent illnesses (n = 19) were excluded as were patients receiving NSAID or aspirin therapy (n = 52), aminosalicylates and/or corticosteroids (n = 2), leaving 895 patients in the final cohort.FC was measured in a faecal extract using a standard enzyme-linked immunosorbent assay (ELISA) technique as previously described .Data was collected retrospectively by review of electronic patient records and recorded on a standardised data collection form. The electronic patient record system  logs all patient contacts with secondary care (throughout NHS Lothian), including all endoscopic and radiological investigations, clinic appointments and hospital admissions. This data was then cross-referenced with other hospital electronic databases that store clinic letters and laboratory results in order to ensure the maximum retrieval and accuracy of data. Patients were followed up until at least three years after first presentation using Trak, ensuring all re-presentations and subsequent diagnoses were noted.Parameters recorded were: age, gender, FC level and date of sample, presenting complaints , past medical history, family history (ulcerative colitis (UC), Crohn's disease (CD), IBD unclassified (IBDU), coeliac disease, colon cancer), smoking history , drug history , investigations performed  and blood results , C-reactive protein (CRP), albumin, hematinics , thyroid function tests, glucose, 7 alphahydroxycholestenone and anti-tissue transglutaminase IgA titre). Rectal bleeding, bloody diarrhoea, nocturnal symptoms, weight loss and anaemia were grouped as “alarm symptoms”. Where a laboratory test was reported as greater or less than a threshold, for statistical purposes it was assigned to one more or one less than the threshold respectively.Any investigations performed were recorded as normal, abnormal or incomplete. “Abnormal” endoscopy findings included mucosal abnormalities, such as histologically proven malignancies and inflammation. The normal group includes those where no abnormalities were found as well as non-adenomatous polyps and haemorrhoids.Diagnosis was recorded as had been stated in the clinical notes. The Lennard-Jones criteria were used to diagnose IBD and the Montreal criteria to classify clinical phenotypes.Patients with a definitive organic diagnosis or who had undergone full colonoscopy (n = 467) were censored at the time of initial case note review. Those cases where an organic diagnosis was not made at the time of the FC or where no colonoscopy had been performed (n = 428) were reviewed in the last quarter of 2012 to ensure that no further cases of IBD or other significant GI pathology had been missed. Patients whose symptoms resolved spontaneously, who did not require further investigation and who did not re-present to hospital with GI symptoms were classified as ‘symptoms resolved.’ Those who were lost to follow-up without a definitive diagnosis were classed as ‘lost to follow-up’.The main comparisons have been made in those with functional disease vs. those with IBD or another condition associated with an abnormal tract, since these are the patients in whom endoscopy would be a potentially useful test.Potential cost savings were calculated using 2012 tariff prices quoted by the Department of Health.Statistical analyses of functional vs. organic groups and functional vs. IBD groups were performed. Medians and inter-quartile range are provided. Mann–Whitney U, Kruskal–Wallis, chi-squared and Fisher's exact tests were used to determine statistical significance. Receiver operating characteristic (ROC) curves were used to determine the best cut off point for FC when predicting organic disease and IBD. Comparison of area under the curve (AUC) was performed using the Delong and bootstrap methods. Positive predictive values (PPV) and negative predictive values (NPV) were calculated. Pre-test probabilities were calculated using all individuals regardless of FC concentration. Post-test probabilities were calculated with respect to different thresholds of FC.A two-tailed p-value of less than 0.05 was considered significant. Confidence intervals for sensitivity and specificity were calculated using the method described by Newcombe with continuity correction.64.9% of patients were female, and the median age (interquartile range) at the time of FC was 33.1 years (25.6–40.7) . 566/895FC was not significantly associated with age (p = 0.21), sex (p = 0.18) or current smoking (p = 0.80).FC was significantly higher in patients diagnosed with IBD  than those with other conditions associated with an abnormal gastrointestinal tract  or with a functional diagnosis  .Of the 91 patients ultimately diagnosed with IBD, 40 (44%) had CD, 41 (45%) had UC and 10 (11%) had IBDU. There was no significant difference in FC between the three subtypes of IBD (p = 0.56). Within the group with CD, there were 10 (25%) with L1  disease, including one patient with L1 + 4, 18 (45%) with L2 (colonic) disease and 12 (30%) with L3 (ileocolonic) disease. FC was significantly higher in those with L2 or L3 disease, with a median (IQR) of 1280 (714–2295) μg/g than in those with L1 disease where median (IQR) FC was 495 (288–822) μg/g (p = 0.009) .Within the group with UC, there were 3 (7%) with E1 disease (proctitis), 12 (29%) with E2 disease (left-sided colitis) and 21 (51%) with E3 disease (extensive colitis). In the remaining 5 patients, the disease extended beyond the point of insertion of the sigmoidoscope and complete staging of extent was not achieved during the initial diagnostic period. There was no significant difference in FC by disease extent when those without complete staging were excluded .Across all patients diagnosed with IBD, there was no significant association between time to diagnosis and faecal calprotectin.Receiver operating characteristic (ROC) analysis revealed an area under the curve (AUC) for FC of 0.85 for prediction of conditions with an abnormal GI tract (including IBD) vs. functional disease, and 0.97 for prediction of IBD vs. functional disease . This waAlarm symptoms were present in 25% (140/566) of those ultimately diagnosed with functional disease, 86% (78/91) of those diagnosed with IBD and 54% (35/65) of those diagnosed with another condition associated with an abnormal GI tract (p < 0.0001). The positive predictive value of alarm symptoms for IBD or an abnormal GI tract vs. functional disease was 0.45 (95% CI 0.38–0.51), and the negative predictive value was 0.91 (0.88–0.93) with a sensitivity of 0.72 (0.65–0.79) and specificity of 0.75 (0.71–0.79). For prediction of IBD vs. functional disease, the PPV was 0.36 (0.29–0.43) and NPV was 0.97 (0.95–0.98), with a sensitivity of 0.86 (0.76–0.92) and specificity of 0.75 (0.71–0.79).As can be seen in Fasciola hepatica, 1 giardiasis, 1 presumed infection with response to metronidazole), 2 with gastro-oesophageal reflux disease and 2 non-specific bowel inflammation. One of these patients with non-specific bowel inflammation was initially thought to have CD but had non-specific changes on her index colonoscopic biopsies and subsequently normal colonoscopy and biopsies.Thirteen patients had no alarm symptoms and a FC of < 50 μg/g, but were found to have a disease associated with an abnormal GI tract. These were 1 case of appendicitis, 1 coeliac disease, 3 with colonic adenomatous polyps, 1 with diverticulosis, 3 with GI infections  and were diagnosed with inflammatory bowel disease. All three had alarm symptoms (two had blood in their stool and one had weight loss). Two of these patients were diagnosed with ulcerative proctitis which has not extended further in > 4 years of follow-up. One had mild terminal ileal CD with no subsequent progression.Between 2005 and 2008, our practice evolved with increasing use of FC and reduction in the percentage of these patients subsequently undergoing invasive investigation. In the 2005, 63 patients underwent stool analysis for FC with 84.1% of them undergoing either sigmoidoscopy or colonoscopy. In 2008, 409 patients had stool sent for FC with 56.7% subsequently undergoing invasive investigation (Table S3).Over the study period, 581/895 (64.9%) patients presented without alarm symptoms. 395 of these (68.0%) had a FC of < 50 μg/g. 150 of these patients (38%) had a subsequent colonoscopy and 50 (13%) a flexible sigmoidoscopy, identifying incidental adenomatous polyps in 3 patients and no other significant pathology. If the low FC had been used to triage these patients to a non-invasive approach, this would have saved £88,233 over that time period.This study uses the largest, ‘real-world’ population of undiagnosed patients to determine the best way of using FC at first presentation to the GI clinic to differentiate non-invasively between organic and functional disease. This allows identification of those in need of efficient and effective further investigation. Incorporating FC into the standard work-up of patients presenting with lower GI symptoms may potentially relieve pressure on hospital services by identifying patients who can be managed solely in primary care.Our findings corroborate existing data showing that FC reliably distinguishes between patients with functional disease and IBD. Von Roon et al.'s meta-analysis of adult patients demonstrated a sensitivity of 95% and specificity of 91% when using a 50 μg/g cut-off point for differentiating IBD patients from healthy controls.Both ESR and CRP are markers that are commonly used to identify systemic inflammation in patients with IBD-like symptoms. In accordance with previous research, we show CRP and ESR are raised in patients with organic disease and IBD.One of the most clinically relevant findings from our data is the NPV for IBD of 99.0% when a FC threshold of 50 μg/g is used. When FC less than 50 μg/g is combined with the absence of alarm symptoms, NPV is 100.0% for IBD. This allows the exclusion of IBD from the differential diagnosis of these patients. Furthermore, in patients meeting these criteria, NPV for any GI tract abnormality is 96.1%. Of the 13 patients with no alarm symptoms and FC less than 50 μg/g who had a diagnosis of abnormal GI tract, colonoscopy was helpful in only four patients and these (diverticular disease and colonic polyps) were likely incidental findings. Clinicians can therefore be reassured that referral for colonoscopy will not identify severe organic disease in patients in whom no abnormalities are found in initial investigations. This finding could potentially be applied to a primary care scenario and aid selection of patients for colonoscopy.With the Department of Health pricing a single colonoscopy in adults at £563 there is great potential for FC to aid more cost-effective decision making with regard to further investigation.One of the strengths of this study is that all individuals without a definitive diagnosis or in whom a functional diagnosis had been made without colonoscopy were re-reviewed three years later to identify any possible latent cases of IBD or other GI disease.This study clarifies important, clinically relevant information about FC. Awareness of the high negative predictive value of FC allows clinicians to effectively exclude IBD as a cause for gastrointestinal symptoms in patients with FC levels under 50 μg/g. FC can thus be used as an adjunct to other presenting complaints and investigations, allowing the risk stratification of patients presenting with gastrointestinal symptoms in a cost-effective manner.NAK is funded by a research training fellowship from the Wellcome Trust[grant 097943] and has had financial support to attend meetings from Warner Chillcott, Shire, MSD and Norgine. He has had speaker fees from MSD and Warner Chilcott.JS has had research funding from Abbvie, speaker fees from Ferring and travel support from Shire.IDRA has been on advisory boards for MSD, Hospira and P&G.CWL has been on advisory boards for and had lecture fees from Abbvie, Hospira, MSD, Vifor, Pharmacosmos and P&G.


Background and Objectives. The intestinal mucosa is extremely sensitive to ischemia. Better intestinal preservation is the first step to improve the results of intestinal transplantation. The aim of the study is to investigate the effect of cold Lifor solution on preservation of swine small bowel. Methods. Swine ileum segments (200 cm) were allotransplanted heterotopically after 9-hour cold storage with UW solution , with Lifor solution , or without storage , respectively. After cold storage, mucosal adenosine triphosphate (ATP) concentrations and histopathologic analysis after preservation were performed. At day 7 after the transplantation, intestinal absorptive function was also observed. Results. After 9 h cold preservation, pathological changes, the content of ATP in the intestinal mucosa, and the intestinal absorptive function after transplantation in group 2 were similar to those of group 1. Conclusion. The effect of cold storage of swine small bowel with Lifor solution is similar to that of UW solution. It may provide additional rationale for further exploration of Lifor as an alternative preservation solution in small bowel transplantation. Intestinal transplantation (ITx) has become an established therapeutic option to patients with irreversible intestinal failure (IF), when complications or failure of parenteral nutrition appears . RecentlCurrently, vascular washout and cold storage (CS) with University of Wisconsin solution (UWS) are considered the gold standard for preservation of abdominal organs . This reLifor is a new type of organ preservation solution comprised of different nutrients, growth factors, and nutrient carriers , 10. RecTherefore, the aim of the study is to examine whether Lifor solution is still much more effective than UW in cold preserving small bowel by a model of swine segmental small bowel allotransplantation.Domestic crossbred pigs of either sex, weighing 20 kg to 25 kg, were used in the study after a 5- to 7-day acclimatization. Food and water were provided ad libitum. Animals were treated humanely by use of protocols that were approved by the Institutional Animal Use and Care Committee of Nanjing University.n = 6) or the Lifor solution . The grafts in the control group  were immediately transplanted without preservation.All animals were randomly divided into three groups. The grafts were preserved for 9 hours at 4°C using UWS . UW  solution contained phosphate, adenosine, lactobionate, raffinose, allopurinol, glutathione, buffers, and pentafraction .Lifor solution  contained sugars, amino acids, salts, buffers, colloids, and lipid nanoparticles  and atropine (0.06 mg/kg) and anesthesia was induced and maintained with intravenous injection of 150 mg/kg/min propofol . In donor animals, a 200 cm segment of the ileum was isolated on a pedicle of the superior mesenteric artery (SMA) and the superior mesenteric vein (SMV) following a midline laparotomy. After cold flushing (0~4°C) via the artery with heparinized preserving liquid, the harvested intestinal segment was preserved in an icebox for 9 hours. At the end of the preservation, the graft was allotransplanted with anastmoses of SMA and SMV to the right external iliac artery and vein of recipient, respectively, in an end-to-end fashion. Both ends of the graft intestine were exteriorized through the bilateral abdominal walls as a Thiry-Vella loop.All animals were allowed to drink only water ad libitum during the period from postoperative day 1 to day 3. FK506  was administered intravenously (0.1 mg/kg per day) for immunosuppression. In addition, they were intravenously administered maintenance infusion therapy of heparin (100 U/kg per day) and antibiotics. Full oral feeding, including solid food, was started on postoperative day 4. At the end of experiment, all animals were euthanized by anesthesia overdose, followed by an intravenous injection of saturated potassium chloride.At the end of the preservation (PRE), 1 h after ischemia-reperfusion (IRI), and day 7 after transplantation (POD7), graft biopsy specimens of terminal harvested ileum were taken and placed in formalin for standard processing (hematoxylin and eosin staining). Histopathology tissue sections, stained with hematoxylin and eosin (H&E), were evaluated by light microscopy. Histological injury of the intestinal samples was quantitatively evaluated according to the scoring system of Regner et al. in a blinded manner . The graAfter 9-hour preservation, graft samples of terminal harvested ileum were snap frozen in liquid nitrogen and preserved below −80°C until later analysis. Frozen samples were weighed and then extracted 1 : 5 w/v in perchloric acid containing 1 mM EDTA. The precipitated protein was removed through centrifugation. Acid extracts were neutralized by the addition of 3 M KOH/0.4 M Tris/0.3 M KCl and then recentrifuged. Aliquots of neutralized extracts were immediately processed via standard enzyme-linked metabolite assays. And then spectrophotometric analysis was performed to measure the absorbance of NADH at 340 nm, providing quantification of ATP. Values are reported as micromoles per gram protein. Protein was measured according to Chiu et al. .Maltose absorption test was performed on postoperative day (POD) 7 to evaluate intestinal absorptive function , 15. Malp value < 0.05 was considered statistically significant.Nonparametric test was used for statistical analysis by using SPSS 16.0  Software. All data are expressed as mean ± SEM and a All animals of the three groups survived without serious complications until day 7 of the graft assessment.p > 0.05). At day 7 after transplantation, no pathologic findings of even mild acute rejection were observed in any grafts that were evaluated and histological studies showed almost normal structure of the small intestinal mucosa in all groups.As shown in p < 0.05)  .p < 0.05), no significant difference was noted between groups 1 and 2 , no significant difference was noted between groups 1 and 2 (p > 0.05, ITx has become the therapy of choice for patients with IF as management strategies evolve in recent years, but unique clinical problems still remain, including the lack of a potent preservation strategy for the small bowel . Graft vLifor, a nanoparticle solution containing amino acids, salts, sugars, and other additives, as a novel organ preservation solution was firstly assessed by Stowe et al. in a model of low-flow perfused swine hearts , 9. Unde+ content of UW is dramatically higher than that of Lifor. Although high K+ solutions can effectively maintain intracellular ion balance during organ preservation, the high K+ concentrations can subsequently impair reperfusion by promoting vasoconstriction [Notably, Lifor has been reported to contain cellular nutrients, buffers, salts, and lipid nanoparticles, but the proprietary nature of Lifor solution precludes a detailed comparison to UW , 9. Nevetriction . Additiotriction , 21. TakIn contrast with solid organ, the intestinal mucosa is much more vulnerable to injury resulting from ischemia , 22. In In this study, we prolonged the preservation period up to 9 h and tested graft viability by assessing the ATP concentration, histologic findings, and intestinal absorptive function after transplantation. At 9 h, the cold Lifor storage succeeded in preserving the graft according to the changes in the parameters obtained in comparison with UWS. With the extension of preservation time, the reduction of ATP concentration is coordinated with the damage degree of intestinal tissue injury, and no significant difference was found between UW and Lifor groups.In summary, the effect of short-term cold storage of canine small bowel with Lifor solution is similar to that of UW solution. In addition, this study provides additional rationale for further exploration of Lifor as an alternative preservation solution in small bowel transplantation.


Drosophila, two distinct transporting cell types receive separate neuropeptide signals, whereas in the ancestral exopterygotes, a single, general cell type mediates all signals. Intriguingly, the largest insect Order Coleoptera (beetles) has evolved a unique approach, in which only a small fraction of cells are targets for neuropeptide action. In addition to demonstrating a universal utility of this technology, our results reveal not only a generality of signalling by the evolutionarily ancient neuropeptide families but also a clear functional separation of the types of cells that mediate the signal.Knowledge on neuropeptide receptor systems is integral to understanding animal physiology. Yet, obtaining general insight into neuropeptide signalling in a clade as biodiverse as the insects is problematic. Here we apply fluorescent analogues of three key insect neuropeptides to map renal tissue architecture across systematically chosen representatives of the major insect Orders, to provide an unprecedented overview of insect renal function and control. In endopterygote insects, such as  The evolution of neuropeptide signalling in insects is poorly understood. Here the authors map renal tissue architecture in the major insect Orders, and show that while the ancient neuropeptide families are involved in signalling in nearly all species, there is functional variation in the cell types that mediate the signal. Drosophila melanogaster, the renal  tubules are characterized by two main physiologically distinct secretory cell types—the principal cell (PC) and the secondary (stellate) cell (SC)—that receive multiple separate neuropeptide signals  is confined to SCs91031) receptors have been shown to localize to PCs, where they act through independent cell signalling pathways to increase primary urine production12131431 receptors are markers of PC activity  in strategically selected representatives of every major insect Order—covering nearly 400 million years of evolution and >90% of insect biodiversityIn contrast to their cognate receptors, the functional core of most insect neuropeptide families are evolutionarily highly conserved , with id202122232512272829Drosophila kinin (DK) analogue conjugated to a stable, high quantum-efficiency fluorophore , which we initially tested in Drosophila. This approach allowed us to exploit the availability of existing tissue expression data as well as independent resources for identifying receptors and quantifying function in this powerful genetic model. A bioinformatic analysis (flyatlas.org33Drosophila kinin receptor (DKR) revealed that the receptor is most highly expressed in the central nervous system (CNS) and epithelial tissues such as the hindgut and tubule of both larval and adult Drosophila  compared with basal rates . Fluorophore labelling does therefore not affect the biological activity of the kinin neuropeptide. In addition to the tubule epithelium, we demonstrated a clear colocalization of receptor and DK-F signals in the adult hindgut and the larval CNS of Drosophila, as well as a functional stimulation of hindgut contraction frequency by both the labelled and unlabelled ligands  and validated them using a similar approach. The spatial expression patterns of the corresponding neuropeptide receptors (CapaR and DH31R-1) revealed that they are most highly expressed in the Drosophila adult tubules  in every beetle species studied  and a cricket (Gryllus bimaculatus) species17tiptop expression and kinin signalling, previously considered intrinsically linked, are intertwined yet separable. Beetles lack kinin signalling altogether  further revealed that other signalling systems stereotypically involved in diuresis are greatly expandedUsing a panel of archetypal fluorophore-labelled neuropeptides, we generated an authoritative overview of insect renal function and control  and 6. Sn of iSC . Data miDrosophila kinin was selected as the archetypal peptide for our study because it is amongst the longest kinin sequences known  was synthesized by Cambridge Peptides , and the modified peptide was subsequently coupled to TMR-C5-maleimide Bodipy dye , to make fluorescent TMR-C5-maleimide-CNSVVLGKKQRFHSWGamide (DK-F). The final working concentration was adjusted according to peptide purity . The same rationale was used for selecting Drosophila capa-1 and DH31  and to native  and labelled DH31 . The choice of fluorophores was based on their photophysical properties  as well as their complementarity to our optical set-up .es known . Therefoand DH31  as archeD. melanogaster (Diptera): all fly lines were cultured on standard medium over a 12:12 h photoperiod at 45–55% humidity at 22 or 26 °C. Only ≥7-day-old flies were used for experimentation. The following strains were used: Canton S , c724-Gal4 (ref. Glossina morsitans (Diptera): parasite-free, unfed tsetse flies were a kind gift of Mr Craig Lapsley, University of Glasgow. Three- to five-day-old flies were used. Tipula oleracea (Diptera): adult specimens were wild caught in Glasgow during May–August 2013 and were used immediately for experimentation. A. gambiae (Diptera): mosquito larvae were a kind gift from Dr Lisa Ranford-Cartwright, University of Glasgow. Larvae were reared on fish food at 22 °C, while eclosing adults were maintained on 5% glucose (w/v) at 26 °C. Both larvae and adults were used for mapping kinin receptor localization; however, only tubules of adult females were used in fluid secretion assays. Manduca sexta (Lepidoptera): specimens of M. sexta were purchased as fourth instar larvae from University of Bath and reared as described previouslyBombyx mori (Lepidoptera): silk moth larvae were obtained commercially and reared according to standard procedures (http://www.silkwormstore.co.uk/). Pachnoda marginata (Coleoptera): larvae were acquired commercially (http://livefoods.co.uk) and reared in moist soil and leaf litter at 26 °C. Both larvae and adults were fed on fruit and water ad libitum. Hylobius abietis (Coleoptera): specimens were wild caught in the south of England and were a kind gift of Dr Daegan Inward, Forest Research, UK. T. castaneum (Coleoptera): stock (San Bernadino) of the red flour beetle was a kind gift from Dr Gregor Bucher, University of Göttingen. Both larvae and adults were kept in whole meal organic flour supplemented with 5% dry yeast at 26 °C until experimentation. T. molitor (Coleoptera): larvae were obtained commercially from http://livefoods.co.uk/ and kept on bran ad libitum until experimentation. Potato slices were occasionally introduced to add moisture. Adults were kept under identical conditions. Vespula vulgaris (Hymenoptera): workers of V. vulgaris were wild caught in Glasgow during August 2013 and maintained on 5% glucose (w/v) at 18 °C for a maximum of 2 days before being used for experiments. Philaenus spumarius (Hemiptera): nymphs were collected around the campus of University of Glasgow during July–August 2013 and used immediately for experimentation. M. persicae (Hemiptera): stock of M. persicae was a kind gift of Brian Fenton, The James Hutton Institute, UK. The aphids were cultured on tomato plants, and only winged adults were used. G. assimilis (Orthoptera): adults were acquired commercially (http://livefoods.co.uk/) and were kept on bran, fruit and water ad libitum.al4 ref. , UAS-CD8al4 ref. ) and c72Drosophila and subsequently fixed in 4% paraformaldehyde in PBS for 30 min. Following several washes in PBS, the tissues were incubated in 10% goat serum and 0.2% Triton X-100 (blocking buffer) for 2 h, followed by incubation with rabbit polyclonal anti-DKR primary antibody−1; Invitrogen, CA, USA), Rhodamine/Alexa-633 coupled phalloidin  and/or Alexa Fluor 488 goat-anti-rabbit antibody  in blocking buffer overnight at 4 °C. After extensive washes in PBS, the tissues were mounted on poly-L-lysine  covered 35 mm glass bottom dishes  in Vectashield  and image acquisition was performed on an inverted Zeiss LSM 510 Meta confocal microscope .Adult hindguts and third instar larval CNSs were dissected from ex vivo receptor-binding assay, appropriate tissues were carefully dissected from cold anesthetized animals of each species, under a dedicated insect saline  and DAPI (1 μg ml−1), while the other was incubated in just DAPI; the latter batch was used to adjust baseline filter and exposure settings to minimize autofluorescence during image acquisition. Images were subsequently recorded on an inverted confocal microscope (Zeiss LSM 510 Meta) or an epifluorescent microscope (Ziess Axioskop II) equipped with a high-resolution microscopy camera (Zeiss AxioCam MR) using baseline filter and exposure settings. A concentration of 10−7 M of the peptide analogues was chosen for this and subsequent assays, as this concentration has been shown to be the minimal concentration needed to produce a saturated receptor response9For the t saline , and theAs the formation of ligand–receptor complexes is a reversible process , we performed a ligand competition assay to validate the binding specificity of the labelled neuropeptides. The competitive binding equilibria can be formalized as:−7 M) and unlabelled (10−5 M) ligands, was taken as an indication of binding specificity. Further, to quantify the resulting reduction in fluorescent signal, intensity profiles from optical sections (10 μm) of multiple SCs  from experimental and competitive inhibition treatments were generated. The intensity profiles were produced using the existing Zeiss LSM 510 Meta confocal software, and an average intensity trace±s.e.m. of n=19–22 SCs for both treatments was depicted as a function of distance in x–y position.where R is the receptor, L′ is the labelled peptide and L is the unlabelled ligandDrosophila MTs. Images were collected every 30 s following the application of DK-F (10−7 M) using the time series function of the Zeiss LSM 510 Meta confocal software. Data were exported as individual tiff-files for subsequent analysis.In addition, time-lapse experiments were set-up to investigate the time frame of DK-F binding to kinin receptors on individual SC in adult Drosophila and preincubated in a freshly prepared mixture of Schneider's medium  and Drosophila saline  for 20 min, before being isolated in drops of the corresponding mixture under liquid paraffin. Each end of the alimentary canal was secured by minute metal pins in opposite ends of the droplet, thus applying a small but constant tension to the tissue. Recordings were made on a Sony NEX-C3 HD camera  mounted on a Leica WILD M3C compound microscope , with initial recordings of 2 min under basal conditions followed by 2 min after peptide application for each hindgut. The number of hindgut contractions before and after addition of the peptides was then visually counted, and the contractile response was presented as percent change in contraction frequency, with each hindgut serving as its own control.A hindgut contraction assay was adapted from published techniques525354in vitro preparations by isolating them in drops of a freshly prepared mixture of Schneider's medium and insect saline  under water-saturated liquid paraffin, with one end being wrapped around a metal pin and the other end submerged in the saline drop. Secreted fluid would then accumulate as a discrete droplet from the common ureter or a small hole introduced midway between the drop and the pin. The tubules were left to secrete for ∼30 min, with non-secreting tubules being replaced if necessary, to produce a set of 10–20 working tubules. A drop of secreted fluid was subsequently collected at appropriate intervals and the diameter was measured using an eyepiece graticule. The volume of each droplet was calculated as (4/3)πr3, where r is the radius of the droplet, and secretion rates plotted against time. Secretion was measured under basal conditions in order to establish a steady rate of secretion, before stimulation with the labelled or unlabelled peptides, with an increase in fluid secretion rate being taken as an indication of a diuretic effect. For each species, the above-mentioned protocol was modified to accommodate differences in anatomy, physiology and size of the tubules. For M. sexta, G. morsitans, P. marginata and T. molitor, both cut ends of each tubule were pulled out into the paraffin and wrapped around metal pins, thus creating a pseudo ligature at each end, and a small hole was introduced as described above2839A. gambiae, V. vulgaris and G. assimilis the cut  end of the tubule was wrapped around a metal pin with the intact distal end bathed in the droplet, and a small hole was made5758Fluid secretions were measured according to standard methods−7 M), and the luminescence was measured in the ensuing period. After the experiment, undischarged aequorin was measured by permeabilizing the cells with 300 μl lysis buffer . Real-time calcium concentrations throughout the experiments were then back calculated with an in-house PERL routineCytosolic calcium measurements were performed as previously described352O before being dehydrated through a graded series of ethanol, and critical point dried using an Autosamdri-815 critical point dryer . Next, the tubules were transferred to aluminium stubs and coated with platinum (70 s∼12 nm thickness) in a JEOL JFC-2300HR high-resolution fine coater  before being examined in a JEOL JSM-6335-F scanning electron microscope .Scanning electron microscopy was performed to visualize MT structure6061t-test, while an unpaired, two-sample t-test was used to test differences in fluorescent intensity between experimental and competitive inhibition treatments, as well as differences between DK and DK-F treatments. In all tests, P<0.05 (two tailed) was taken as the critical value.The statistical analyses were performed using the data analysis software OriginPro 8.5 . All data are plotted as mean±s.e.m. Significant difference in hindgut contraction frequency and tubule secretory response following peptide application was tested using a paired-samples J.A.T.D. and K.A.H. conceived and designed the study with input from S.A.D. K.A.H. performed all experiments with assistance from S.T. and P.C. All authors contributed to the interpretation and analyses of data. K.A.H. and J.A.T.D. wrote the paper with inputs from all authors.How to cite this article: Halberg, K. A. et al. Tracing the evolutionary origins of insect renal function. Nat. Commun. 6:6800 doi: 10.1038/ncomms7800 (2015).Supplementary Figures 1-8, Supplementary Tables 1-4 and Supplementary References