This retrospective cohort study characterized the impact of prior antibiotic exposure on distribution and nonsusceptibility profiles of Gram-negative pathogens causing hospital-onset urinary tract infections (UTI).Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and other Enterobacteriaceae ≥3 days after hospital admission were included. Assessment outcomes included the distribution of bacteria in urine cultures, antibiotic susceptibility patterns, and the effect of prior antibiotic exposure, defined as 0, 1, or ≥2 prior antibiotics, on the distribution and antibiotic susceptibility profiles of the Gram-negative organisms.Hospital patients with positive urine culture for E. coli (49.5%), K. pneumoniae (17.1%), and P. aeruginosa (8.2%). P. aeruginosa was significantly more commonly isolated in patients with ≥2 prior antibiotic exposures (12.6%) compared with no exposure  or 1 prior exposure . Two or more prior antibiotic exposures were associated with slightly higher incidences of fluoroquinolone nonsusceptibility, multidrug resistance, and extended-spectrum β-lactamase phenotype compared with 0 or 1 exposure, suggesting an increased risk for resistant Gram-negative pathogens among hospital patients with urinary tract infections occurring ≥3 days after admission.The most commonly isolated pathogens from 5574 unique UTI episodes (2027 with and 3547 without prior antibiotic exposure) were Clinicians should critically assess prior antibiotic exposure when selecting empirical therapy for patients with hospital-onset urinary tract infections caused by Gram-negative pathogens.The online version of this article (doi:10.1186/s12879-017-2270-7) contains supplementary material, which is available to authorized users. Increasing antibiotic resistance among Gram-negative pathogens, particularly in the hospital setting, is well documented and constitutes a major public health concern , 2. GramTwo critical patient characteristics that modify the risk for antibiotic-resistant infection are recent antibiotic exposure and recent admission to, or current residence in, a health care institution , 8, 9. CThis retrospective, observational study used hospital discharge data from the Premier Healthcare Database, which at the time of this study contained data from more than 435 million patient encounters. Laboratory results were available from a subset of approximately 160 facilities in the Premier Healthcare Database. Patients were included in the study if all the following criteria were met: inpatient discharge between January 1, 2012, and March 31, 2013; positive urine culture for any of the prespecified Gram-negative bacteria ≥3 days after hospital admission; and receipt of an antibiotic with activity against Gram-negative pathogens on the index culture date or within the 3-day period thereafter. The first documented urine culture was included in the analysis. Duplicate isolates from subsequent urine cultures within 30 days were excluded. Duplicate isolates recovered from urine cultures >30 days after the index culture were included because we felt these to be representative of either recurrent or new infection. As such, we felt it was important to include these subsequent occurrences as unique episodes within the study.Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and other members of the Enterobacteriaceae family (excluding E. coli and K. pneumoniae). Prior antibiotic exposure was defined as administration of ≥1 prespecified agents with Gram-negative activity during the current hospitalization and before the index urine culture. Antibiotics included for assessment of prior exposure were those available in the working Premier data set: meropenem, doripenem, imipenem, ertapenem, piperacillin/tazobactam, cefepime, ceftazidime, ceftriaxone, cefotaxime, ciprofloxacin, levofloxacin, gentamicin, tobramycin, amikacin, ampicillin/sulbactam, cefazolin, gatifloxacin, cefazolin, tigecycline, and ticarcillin/clavulanic acid (or clavulanate). Data for other groups of antibiotics, including second-generation cephalosporins and trimethoprim/sulfamethoxazole, were not available.The Gram-negative organisms of interest were The first outcome measure was distribution and antibiotic susceptibility patterns among the prespecified Gram-negative organisms for patients meeting inclusion criteria. The second outcome measure was the association of prior antibiotic exposure, defined as 0, 1, or ≥2 prior exposures, with the distribution and antibiotic susceptibility profiles of the Gram-negative organisms of interest. Organisms were assessed for susceptibility to fluoroquinolones , carbapenems , and piperacillin/tazobactam.P. aeruginosa, only meropenem-, imipenem-, or doripenem-nonsusceptibility was used to define carbapenem resistance; P. aeruginosa isolates were considered nonsusceptible to third-generation cephalosporins if they were resistant to ceftazidime, or, if data were unavailable, to cefepime. For Enterobacteriaceae, isolates were considered nonsusceptible to a third-generation cephalosporin if they had documented resistance to at least two agents . Nonsusceptibility to third-generation cephalosporins was considered indicative of an extended-spectrum beta-lacatmase (ESBL) phenotype. For patients with missing carbapenem, piperacillin/tazobactam, or MDR susceptibility data, isolates were considered carbapenem-susceptible, piperacillin/tazobactam-susceptible, or non-MDR if they were susceptible to a third-generation cephalosporin. Otherwise, carbapenem, piperacillin/tazobactam, and MDR susceptibility data were considered missing and were not included in the analyses that evaluated the relationship between prior antibiotic exposures and antibiotic susceptibility profiles.For the purposes of this analysis, antibiotic susceptibility was classified as susceptible versus nonsusceptible , based on the microbiology report. Multidrug resistance was defined as nonsusceptibility to ≥1 agents in ≥3 antibiotic classes . For P. Unadjusted descriptive analysis was used to characterize the distribution and antibiotic susceptibility patterns of the Gram-negative organisms of interest and the effect of prior antibiotic exposure on each of these patterns. Although the study was not powered for prespecified statistical analyses, possible significance between groups (≥2 prior exposures compared with 0 or 1 prior exposure) was calculated using the method of Miettinen and Nurminen.n = 2027 prior antibiotic exposure; n = 3547 no prior exposure), from which 6093 pathogens were isolated . The most commonly isolated pathogens were E. coli , K. pneumoniae , and P. aeruginosa   than in patients with no prior antibiotic exposure  or with 1 prior exposure . For other pathogen groups, the presence or absence of prior antibiotic exposure did not substantially affect distribution trends, and differences were nonsignificant.The distribution of pathogens in relation to prior antibiotic exposure is shown in Fig. p = 0.041), these differences in incidence were not statistically significant. This trend seemed to be driven largely by susceptibility pattern changes observed with K. pneumoniae  was the most commonly isolated pathogen [P. aeruginosa UTIs and with slightly higher incidences of fluoroquinolone-, carbapenem-, and third-generation cephalosporin-nonsusceptibility as well as multidrug resistance. Collectively, our findings suggest that previous exposure to ≥2 antibiotic regimens is associated with an alteration of the distribution and susceptibility profiles of Gram-negative pathogens among patients with hospital-onset UTIs.Our study focusing on the single risk factor of antibiotic exposure produced several notable findings. Consistent with epidemiologic trends, pathogen , 9, 12, Although antibiotic nonsusceptibility was highest among those with ≥2 prior exposures to antibiotics, fluoroquinolone nonsusceptibility rates were >18%, even in the absence of prior antibiotic exposure. This finding is not unexpected given the large-scale use of fluoroquinolones for UTIs in both community and health care settings. Emergence of resistance to these antibiotics is recognized by a number of world health agencies and is considered a major public health concern . BecauseOur findings also highlight the limitations associated with relying solely on antibiograms for patients with hospital-onset UTI, particularly those with ≥2 antibiotic exposures. Although antibiograms are a useful starting point in the empirical drug selection process, they reflect the cumulative susceptibility rates for the first recovered pathogen in a particular patient, regardless of current site, timing of collection , or patient-specific risk factors for resistance  . Recent Some limitations should be considered when interpreting these findings. First, though we acknowledge that several factors may contribute to antibiotic-resistant infections, we purposefully did not assess multiple risk factors because data were not uniformly available for all patients. We also believed that inclusion of other patient factors would complicate our intent to create a simple, straightforward approach to guide empirical antibiotic selection. Because we did not adjust for other factors, our findings cannot substantiate prior use of antibiotics as an independent risk factor for, or as a cause of, resistance, but our findings do show that prior use is an additional easily identifiable variable that can be used to guide empirical therapy. Second, the study was restricted to patients with UTIs occurring ≥3 days after hospital admission. Although we had access to detailed hospitalization data, information on antibiotic and health care exposure before admission was limited. As such, we focused on patients for whom complete, detailed data were available. The 3-day time frame after hospital admission fails to account for recent antibiotic use in outpatient or other institutional settings; therefore, the potential effects of these exposures on our findings are unknown. Third, this study did not specifically quantify the relationship between cumulative duration of exposure to all or any given antibiotic and the presence of resistance. Given that the goal of the study was to provide clinicians with a straightforward, easily adaptable method for empirical antibiotic selection, we did not think it was necessary to specify with increased granularity the relationship between duration of prior antibiotic exposure and resistance. Although we were able to document prior receipt of many commonly used antibiotics with Gram-negative activity, data for other groups of antibiotics, including second-generation cephalosporins, and trimethoprim/sulfamethoxazole, were not available for this analysis. The relatively small number of patients with ≥2 prior antibiotic exposures might also limit the interpretation of findings regarding resistance, though increased rates observed among fewer pathogens is still of concern. In addition, though the statistical analysis suggests some significant differences between patients with ≥2 prior exposures and patients with 0 or 1 prior exposure, it should be noted that the numbers of patients in some of the groups were very small. As such, determination of the true statistical relevance of prior antibiotic exposure will require additional studies adequately powered to determine significance levels. Fourth, given that the presence of the ESBL phenotype among pathogens was not formally tested, the use of select third-generation cephalosporin resistance as a marker for ESBL may underestimate or overestimate the true incidence of ESBL. However, we believe third-generation cephalosporin resistance represented a practical surrogate because it is readily available to clinicians on urine culture susceptibility reports and that routine formal ESBL testing is not recommended by the Clinical and Laboratory Standards Institute (CLSI). Furthermore, given that culture susceptibility reports were from clinical and not reference laboratories, it can be assumed that third-generation cephalosporin resistance was reported using the US Food and Drug Administration–approved minimum inhibitory concentration breakpoint, which may better correlate with the presence of ESBL than CLSI breakpoints . FinallyHaving received ≥2 prior antibiotic regimens was associated with increased risk for a resistant Gram-negative pathogen among patients with hospital-onset UTIs. In addition to considering the hospital antibiogram, which should be stratified for greater usefulness, hospital clinicians should critically assess prior exposure to antibiotics when selecting empirical therapy for patients with UTIs that occur ≥3 days after admission. Awareness of common pathogen distribution and nonsusceptibility trends among hospital patients with UTIs, combined with readily identifiable patient risk factors for infection with resistant pathogens, can provide a useful framework for clinicians during the empirical antibiotic selection process.


The Catechol-O-methyltransferase (COMT) represents the key enzyme in catecholamine degradation. Recent studies suggest that the COMT rs4680 polymorphism is associated with the response to endogenous and exogenous catecholamines. There are, however, conflicting data regarding the COMT Met/Met phenotype being associated with an increased risk of acute kidney injury (AKI) after cardiac surgery. The aim of the current study is to prospectively investigate the impact of the COMT rs4680 polymorphism on the incidence of AKI in patients undergoing cardiac surgery.In this prospective single center cohort study consecutive patients hospitalized for elective cardiac surgery including cardiopulmonary-bypass (CPB) were screened for participation. Demographic clinical data, blood, urine and tissue samples were collected at predefined time points throughout the clinical stay. AKI was defined according to recent recommendations of the Kidney Disease Improving Global Outcome (KDIGO) group. Genetic analysis was performed after patient enrolment was completed.p = 0.66). There were also no differences in the post-operative period, including ICU or in-hospital stay.Between April and December 2014, 150 patients were recruited. The COMT genotypes were distributed as follows: Val/Met 48.7%, Met/Met 29.3%, Val/Val 21.3%. No significant differences were found for demography, comorbidities, or operative strategy according to the underlying COMT genotype. AKI occurred in 35 patients (23.5%) of the total cohort, and no differences were evident between the COMT genotypes (20.5% Met/Met, 24.7% Val/Met, 25.0% Val/Val, We did not find statistically significant variations in the risk for postoperative AKI, length of ICU or in-hospital stay according to the underlying COMT genotype. The Catechol-O-methyltransferase (COMT) represents one of the key enzymes in catecholamine degradation. The common polymorphism rs4680 in exon four is coding for a Val to Met substitution at position 158 and results in increased thermolability of the COMT enzyme: Val/Val being associated with high enzyme activity, Val/Met with intermediate and Met/Met with low enzyme activity . ReducedRecent studies suggest that this COMT polymorphism is associated with substantial variance in the response to endogenous and exogenous catecholamines, frequently being referred to as “catecholamine resistance” , 4. ThisIn the current prospective cohort study, we investigated the prevalence of the various COMT genotypes in patients undergoing cardiac surgery. We also assessed the risk of AKI dependent on COMT genotype as the study’s primary endpoint, assuming higher risk in particular in the COMT low activity phenotype (Met/Met).CKD-EPI < 30 ml/min, signs of active infection , women during pregnancy and lactation and patients on medication with COMT inhibitors, MAO inhibitors or with immunosuppressive therapy.The study protocol was approved by the Ethics Committee at the University Hospital of Würzburg. All participants provided written informed consent after they were asked at least 24 h prior to surgery (preferably at the day of admission) to participate in the study. Patients (18+ years) were eligible if they were undergoing an elective cardiac surgical procedure involving cardiopulmonary bypass (CPB); such as coronary artery bypass graft (CABG) with or without mammary artery bypass, valve surgery  with or without removal of the atrial auricle, combined CABG and valve surgery, or surgery of the thoracic aorta. Mild hypothermia (32-34 °C) was applied to all patients during surgery and cardiac arrest resulted from blood cardioplegia (Buckberg). Exclusion criteria included chronic kidney disease greater than stage 3, i.e. eGFRCollected data comprised routine clinical data on patient characteristics and comorbidities, surgery procedures, anesthesia and ICU details including laboratory data and medication. Information was collected at various predefined time points during the patient’s hospital stay at admission/pre-operatively, at ICU admission, 24 h and 48 h after ICU admission as well as 6 days post-surgery and/or at hospital discharge. Furthermore, standardized interviews were performed by trained study personnel to collect data on medical history, comorbidities and patient related factors e.g. pain at admission and at discharge. Cardiac dysfunction was defined as LVEF< 50% in a standardized echocardiography prior to surgery.An EDTA-blood sample was drawn preoperatively and COMT genotyping was performed as described previously . In brieThe study’s primary endpoint was defined as the development of AKI within the first 48 h after surgery according to the most recent recommendations by KDIGO  as incre2-test/Fisher’s exact test, respectively. Uni- and multivariate logistic regression analysis was performed to investigate the risk of AKI dependent on the COMT genotype and clinical as well as surgical parameters. Due to limited statistical power, clinical and surgical factors were time on CPB, as the most important indicator of intraoperative stress [p-values of ≤0.05 were considered as statistically significant. Statistical analyses were performed using SPSS Versions 22 and 23.Patient demographics are presented as mean and standard deviations, medians and interquartile ranges and number of observations with proportions (%), as appropriate. Differences across COMT-genotype-groups were assessed by ANOVA, Kruskal-Wallis-H-Test and χe stress , 10 and e stress . The scon = 150 patients, resulting in an assumed distribution of COMT genotype of Val/Val n = 37, Val/Met n = 74, Met/Met n = 37, total n = 148, drop out n = 2. This approach allowed for rejecting the null-hypothesis that the risk for AKI does not vary across phenotypes  with a power of 0.80 and alpha of ≤0.05, if the incidence in the remaining groups are AKI-risk Val/Met = 29% and AKI-risk Met/Met = 53%. Of note, it was planned by protocol, that if signals can be observed that supported our hypothesis that the Met/Met (or any other) genotype was associated with AKI, the sample size shall be increased and to focus on modifiable surgery-related factors as the basis for interventional studies.The current study was a pilot study to investigate the hypothesis that the various COMT genotypes can be observed according to the Hardy-Weinberg equilibrium and that the resulting phenotypes would carry different risk for AKI. The power calculation was based on the following assumptions: the prevalence of the COMT polymorphism would be Val/Val (25%), Val/Met (50%) and Met/Met (25%). The distribution of AKI across COMT phenotype has been described as Met/Met: 31%, Val/Met: 19.5%, and Val/Val: 13.5%, respectively . Since wEligible patients were screened for participation between April and December 2014. During the study, 165 patients (of 915 per se eligible subjects within this time-period undergoing surgery at our department) were included matching our in- and exclusion criteria. There were 15 dropouts due to unplanned off-pump surgery, withdrawal of consent or missing genotypes; thus 150 patients were included in the final analysis.2. Median EUROSCORE 2 was 1.7. Seventy-four (49%) patients had cardiac dysfunction, i.e. left ventricular ejection fraction (LV-EF) < 50%. The prevalence of cardiac dysfunction was lowest in the Val/Met group (31.5%), while in the Val/Val group the prevalence was 71.9%. There were no other significant differences regarding demography, medical history and surgical approach between the three COMT genotypes.Patient characteristics of the entire cohort and stratified according to COMT genotype are displayed in Table p = 0.02), and a statistically non-significant trend was observed for patients with higher EUROSCORE 2 values (p = 0.09). In multivariable analysis, only time on CPB remained statistically significant (p = 0.049) in the model (data not shown).A total of 35 patients 23.5%) experienced AKI within 48 h after surgery (Table 3.5% expep = 0.4), nor in multivariate modelling  controlling for COMT genotype and also including the interaction of COMT genotype and cardiac dysfunction.To further understand the role of cardiac dysfunction, we performed additional analyses but did not find any meaningful and significant association of the COMT genotype, cardiac dysfunction and the occurrence of postoperative AKI. Also in logistic regression analyses, cardiac dysfunction was not associated with the incidence of AKI neither in univariate . These patients per se are at increased risk for AKI [The incidence of AKI was lower than in previous studies at our centre , 15 whic for AKI . On the  for AKI . This ce for AKI . HoweverIt is a major strength of our study that baseline SCr was prospectively measured at a defined time-point (during introduction of anesthesia) followed by subsequent measurements in clinical routine up to 48 h on the ICU. This approach excluded pre-operative AKI and also allowed for classification of AKI following the most recent KDIGO guidelines. We could not observe any variation in the risk of AKI according to COMT genotype, neither in slight elevations of SCr (i.e. AKI stage 1), nor in higher stages of AKI. Only two patients required hemodialysis treatment for AKI and both were in the  intermediate-risk COMT Val/Met group.Evidence is robust that adverse peri- and postoperative events, including vasodilatory or cardiogenic shock and AKI are related to worse subsequent outcomes; such as the development of chronic kidney disease (CKD), and also of systolic and/or diastolic left ventricular dysfunction, congestive heart failure (CHF) and mortality , 7, 19. Data on the risk of AKI being dependent on the COMT genotype is conflicting. In particular, two recent publications report on associations between COMT and AKI or renal damage, respectively. Herein, the Met/Met phenotype of the COMT enzyme was associated with an increased risk of AKI . This waWhile all papers used the RIFLE classification for AKI definition, we used the latest definition by the Kidney Disease Improving Global Outcome (KDIGO) published in 2012 –7. The aThe disparities between the RIFLE and the KDIGO definition of AKI might result in a different incidence of AKI; in contrast to the KDIGO definition, particularly mild alterations in kidney function are not being classified as AKI according to RIFLE. Therefore, any association of the COMT genotype with AKI according to RIFLE may be detected predominantly in more severe stages of AKI. Most of the AKI episodes in our study were of stage 1 and the number of more severe AKI stages was very limited. Therefore the statistical power of our study may be too small to observe significant differences in the AKI incidence according to COMT genotype. Yet, as we did not find any signal for any association of COMT genotype with the risk of AKI, we believe that there may not exist any meaningful association.In our analyses, we found an association between cardiac dysfunction and the COMT polymorphism, which has not been described in the literature. We found a higher prevalence of cardiac dysfunction in the Val/Val genotype group, while the Val/Met genotype was associated with a low prevalence of impaired LV function. Further analyses did not reveal any association between cardiac dysfunction and the incidence of AKI. Our findings of a potentially existing clinical relation of cardiac dysfunction an the underlying COMT genotype, admittedly being based on limited statistical power, need to be confirmed by future studies.We are aware of several limitations of our study. First, reflecting a pilot study, the sample size is limited to detect small differences between the COMT phenotypes and postoperative outcomes and also to control for covariates in multivariable modelling. Overfitting the multivariate models might also be possible and therefore missing or even finding any associations by chance is truly possible. Second, there is no standardized operating procedure at our department considering fluid or catecholamine management of the patients, therefore treatment bias cannot be excluded. However, the treating physicians and nurses as well as the study personnel were unware of the underlying COMT polymorphism, as the COMT genotype were measured after completion of enrolment. Thirds, during 9 months study duration, a total of 915 patients would have been eligible, but due to limited resources on study personnel, only 165 patients were enrolled. Therefore, selection bias is conceivable, but we assume that our study sample overall reflects most of all eligible patients at our center: the study population was distributed by COMT genotype as expected following the Hardy-Weinberg-equilibrium with no differences in pre-operative patient characteristics. It is thus unlikely that a meaningful proportion of Met/Met patients which are theoretically at very high risk for AKI were not enrolled due to whatever clinical reasons, but definitely unconscious to the COMT genotype.The COMT rs4680 genotype was not associated with statistically significant differences in preoperative phenotypes, the surgical strategy, intraoperative and early postoperative complications. We did not find variations of the risk for postoperative AKI according to COMT genotype.


There are multiple errors throughout the paper. In the second paragraph of the introduction, reference [10] is incorrectly placed after the third sentence. It should be placed after the fourth sentence and read as follows: On the other hand, LL-SSEPs possess latencies greater or equal to 40 ms [10].The first sentence of the Monopolar reference section is missing the phrase “by controlateral MNS,” after the word elicited. The correct sentence is: With regard to SL-SSEPs, a component with mean latency 18.9±1.2 ms and mean amplitude 2.7±1.9 μV was elicited by contralateral MNS in twenty-two out of twenty four STN’s.The first sentence of the third paragraph within the Discussion section incorrectly references the VIM as being independent from the thalamus. The correct sentence is: Focusing on the generators of SL-SSEPs, previous studies directed towards the thalamus [18] and its VIM  came to different conclusions regarding the generators of SL-SSEPs.


For the exposed LWAGC to the elevated temperatures of 100 to 800 °C, the results illustrate that the concretes gain compressive strength after being exposed to elevated temperatures of 100, 200 and 300 °C. Afterward, the strength of the LWAGC started to deteriorate and decrease after being exposed to elevated temperatures of 400 °C, and up to 800 °C. Based on the mechanical strength results of the exposed LWAGCs to elevated temperatures of 100 °C to 800 °C, the relationship between the exposure temperature and the obtained residual compressive strength is statistically analyzed and achieved. In addition, the microstructure investigation of the unexposed LWAGC shows a good bonding between aggregate and mortar at the interface transition zone (ITZ). However, this bonding is subjected to deterioration as the LWAGC is exposed to elevated temperatures of 400, 600 and 800 °C by increasing the microcrack content and swelling of the unreacted silicates.This paper presents the mechanical and microstructural characteristics of a lightweight aggregate geopolymer concrete (LWAGC) synthesized by the alkali-activation of a fly ash source (FA) before and after being exposed to elevated temperatures, ranging from 100 to 800 °C. The results show that the LWAGC unexposed to the elevated temperatures possesses a good strength-to-weight ratio compared with other LWAGCs available in the published literature. The unexposed LWAGC also shows an excellent strength development  Globally, it is well-known that the lightweight concrete (LWC) is more advantageous than the normal weight concrete (NWC) by the reduction of the structure’s dead-weight, better thermal insulation for buildings, and less cost for transportation. Lightweight concretes (LWC) are commonly used in the construction of buildings, bridge deck pavements, and in more limited roles, for entire bridge superstructures ,2,3. The2 emission up to 80%–90% [Recently, geopolymer materials attracted a great deal of consideration as a new environmentally-friendly engineering technology for producing supplementary materials to the ordinary Portland cement (OPC) due to their brilliant mechanical and thermal properties. As compared to the OPC, the geopolymer technology can reduce CO 80%–90% , and it  80%–90% . Further 80%–90% ,11.Nevertheless, only few published studies and researches regarding the utilization of LWA in the production of lightweight aggregate geopolymer concrete (LWAGC) has been reported . To our 3, respectively, at 28 days, which permits its classification as a structural LWAC, according to the American Concrete Institute ACI 213R [et al. [3, respectively, at 28 days. Hence, as they used the same aggregate combinations, the alkali-activation of the FA with a liquid activator appears to be better than the alkali-activated GGBS with a powder activator, as it produces almost the same strength, but with about 11% less density. [et al. . Accordiversus aging times of 3, 7, 28, 56, 91, 180 and 365 days. It can be seen that the LWAGC gains a high rate of strength, increasing at early times of three and seven days, which are equivalent to about 66% and 81% of the 28-day strength, respectively. This trend is attributed to the fact that the geopolymerization process produces excellent early strength at early ages [Moreover, rly ages . Howeverrly ages  reportedSubsequently, the compressive strength of the LWAGC is observed to decrease gradually after being exposed to an elevated temperature of 400 °C, and further up to 800 °C . The strcf is the residual compressive strength (MPa) and t is the exposure temperature (°C), with a fitting error of (1.1048).Furthermore, The observed strength loss of the LWAGC exposed to elevated temperatures, higher than 300 °C, and up to 800 °C, is attributed to the essential thermal shrinkage resulted by the loss of mass due to the evaporation of the structural water at elevated temperatures. The water in the geopolymer structure is transformed to a water vapor when the geopolymer is heated above 100 °C, and its pressure increases continuously with the increasing heating temperature . Thus, aWhereas, the thermal strain data of the LWA presented in 24Moreover, 2/g. The FA was activated with alkaline activator prepared by mixing a technical grade sodium silicate (Na2SiO3) and sodium hydroxide solution (NaOH). The chemical composition of the Na2SiO3 was SiO2 = 30.1%, Na2O = 9.4%, H2O = 60.5% and the modulus ratio (SM) equal to 2 (where SM = SiO2/Na2O). The NaOH of 12 M was prepared by mixing sodium hydroxide pellets of 97%–99% purity with distilled water. The alkaline activator prepared by mixing the Na2SiO3 and 12 M NaOH solution at a constant mass ratio of 1:1.The FA used in this work was provided by the Manjung Power Station, Lumut, Perak, Malaysia. The chemical composition of the FA was determined by X-ray fluorescence (XRF)  as listed in The LWA used for the LWAGC preparation was commercial lightweight expanded clay aggregate (LECA). The LWA granules were spherical in shape with closed surfaces, having a slightly rough and porous texture. The physical properties of the LWA are given in The mix proportioning of the LWAGC was made according to ACI 211.2 standard  and presThe LWAs were prepared in oven-dry condition (OD) by being pre-dried at 105 °C for 24 h in order to remove any residual moisture prior to mixing, due to the highly moisture absorption capability of the LWA. Concurrently, the fine aggregate was prepared in air-dry condition (AD) by firstly washing with a tap water and then drying in shade for 24 h prior to mixing. The absorbed water by the LWAs and fine sand was constrained during mixing by adding extra water, equivalent to the quantity of the water absorption of each aggregate type. In practice, the FA and aggregates (LWA + sand) were mixed in a pan mixer for 3 min. Following, the prepared alkaline activator liquid and the extra water dosages were gradually added to the solid constituents. The wet mixing continued for a further 5 min until the mixture homogenized. Subsequently, the slump of the fresh concrete was measured prior to pouring the mixture into 100 mm × 100 mm × 100 mm cubic plastic molds and compacted in three layers using a steel rod to strike each layer 25 times. The molds were sealed from the ambient to prevent the loss of moisture.All wrapped molds were cured, undisturbed, in an oven at 70 °C for 24 h after casting. Following, the molds were taken out of the furnace and left to cool at room temperature before demolding. The sealed specimens were stored under ambient conditions.Number of LWAGC specimens at 27 days was further exposed to elevated temperatures, ranging from 100 to 800 °C, in increments of 100 °C. The specimens were placed into a furnace and heated at a fixed heating rate of 4.4 °C/min. The geopolymers specimens were kept at each elevated temperatures for 1 h, afterwards, the specimens were left to cool, inside the furnace, to room temperature. Meanwhile, the unexposed specimens were left, undisturbed, at ambient conditions. The compressive strength test for LWAGCs exposed to elevated temperatures of 100 to 800 °C was preformed one day after the heating. Thus, all specimens were left to complete the aging period of 28 days before being tested for their physical and mechanical properties. The density and water absorption of the unexposed geopolymers were preformed according to ASTM C 140-01 , and thel is the length change (mm); and l0 is the initial length of the specimens; ΔT is the temperature difference, respectively.Dilatometry measurements were carried out in order to measure the thermal expansion of the specimens using a Linseis, L75 Laser dilatometer , at temperatures ranging from 20 to 800 °C, with a constant heating rate of 5 °C/min. The test was performed for both geopolymer paste and LWA specimens, as they can present valuable information for the thermal expansion behavior for concrete at high temperatures, as reported in references ,31. The Scanning electron microscopy (SEM) was performed using SEM JSM-6460 LA Jeol., Tokyo, Japan, in order to investigate the microstructure of the LWAGC, unexposed and exposed, to elevated temperatures. The specimen fragments of the compressive strength tested LWAGCs were mounted in epoxy resin and further vacuumed and coated with a thin layer of platinum using a JFC-1600 auto fine coater Jeol. Japan. The test was carried out using secondary, as well as, backscattered electron (BSE) detectors.This paper presents the mechanical and microstructural characteristics of a lightweight aggregate geopolymer concrete (LWAGC) synthesized by the alkali activation of locally sourced FA, before and after exposure to elevated temperatures, ranging from 100 to 800 °C. The mechanical and physical results of the unexposed LWAGC showed a good strength-to-weight ratio when compared with other LWAGCs available in the published literature. In addition, the mechanical strength of the resulting LWAGC showed the possibility of classifying it as a structural LWC that could be used as construction material. Moreover, the continuous gain in the compressive strength of the unexposed LWAGC, with ages of up to 365 days, has added another advantage to these novel materials.For the exposed LWAGC, the results of the current paper showed that the concrete gained a compressive strength, even after exposure to elevated temperatures of 100, 200 and 300 °C, which made it an excellent refractory material for application when high strength and thermal durability, up to 300 °C, is desired. The strength of the LWAGC started to deteriorate after being exposed to elevated temperatures of 400–800 °C, due to vapor effects, as well as the differences in the thermal expansion between the aggregate and the geopolymeric paste. It was also observed that the rate of strength deterioration was continuously increased with the increase in temperature.The microstructural investigation by SEM, for the unexposed LWAGC, showed a good bonding between the aggregate and mortar at the ITZ. This bonding, however, was subjected to a continuous deterioration as the LWAGC was exposed to elevated temperatures of 400, 600 and 800 °C, which explains the strength decrease of the LWAGC exposed to these elevated temperatures.


We derive the Levinson type generalization of the Jensen and the converse Jensen inequality for real Stieltjes measure, not necessarily positive. As a consequence, also the Levinson type generalization of the Hermite-Hadamard inequality is obtained. Similarly, we derive the Levinson type generalization of Giaccardi’s inequality. The obtained results are then applied for establishing new mean-value theorems. The results from this paper represent a generalization of several recent results. The well-known Jensen inequality asserts that for a convex function n-tuple of numbers from the interval I and n-tuple such that Steffensen  showed tBoas  gave theLetbe a continuous convex function, whereIis the range of the continuous monotonic function (either increasing or decreasing) and letbe either continuous or of bounded variation satisfyingThenThe generalization of this result is also given by Boas in . It is tIfis either continuous or of bounded variation satisfyingfor allandand iffis continuous and monotonic (either increasing or decreasing) in each of thenintervalsthen inequality .(i)Bullen  rescalednt}yi≤b,  holds fon}, then  holds.(ii)Pečarić  proved t(iii)i.e. he proved that inequality f is 3-convex in Mercer’s assumptions. Furthermore, Witkowski weakened the assumption  onand convex (concave) on(1)If (2)The function For the class old (see ): (1)If Jakšetić, Pečarić, and Praljak in  gave theLetand letandbe continuous monotonic functions (either increasing or decreasing) with rangesandrespectively. Letandbe continuous or of bounded variation satisfyingfor allandandfor allandIfis continuous and ifholds, thenG defined on s and t. The function G is continuous under s and continuous under t, and it can easily be shown by integrating by parts that any function On the other hand, in  Pečarić,thors in  gave theLetbe continuous function andinterval such that the image ofgis a subset ofLetbe continuous function or the function of bounded variation, such thatandThen the following two statements are equivalent: (1)For every continuous convex functionholds.(2)For allholds, where the functionis defined in  and (2) are also equivalent if we change the sign of inequality in both For every continuous concave functionthe reverse inequality in For allinequality  and (2′) are also equivalent if we change the sign of inequality in both statements (1′) and (2′).The main aim of our paper is to give a Levinson type generalization of the result from Theorem In order to simplify the notation, throughout this paper we use the following notation: Letandbe continuous functions, an interval andsuch thatandLetandbe continuous functions or functions of bounded variation such thatandand such thatandholds.If for alland for allwe havewhere the functionGis defined in If for allandinequalities If for allandthe reverse inequalities in   holds.φ belongs to class φ is convex (resp. concave), the condition  (see ), so if f and g to be monotonic, and the functions λ and μ to satisfy G is continuous and convex in both variables, we can apply the Jensen inequality and see that for all It is easy to see that Theorem ualities  hold, soIn this section we give the results for the discrete case. The proofs are similar to those in the integral case given in the previous section, so we will state these results without the proofs.G defined in If for allandthe inequalities in If for allandthe reversed inequalities in   holds.G is convex in both variables, in the case when all Theorem ualities  hold. NoWe can replace the equality from the condition  by a weaμ on ϕ on f is any μ-measurable function on The Jensen inequality for convex functions implies a whole series of other classical inequalities. One of the most famous ones amongst them is the so-called Edmundson-Lah-Ribarič inequality which states that, for a positive measure It was obtained in 1973. by Lah and Ribarič in their paper . Since tG defined in For every continuous convex functionholds, where(2)For allholds, where the functionis defined in For every continuous concave functionthe reverse inequality in For allinequality  and (2′) are also equivalent if we change the sign of inequality in both statements (1′) and (2′).In the following theorem we give the Levinson type generalization of the upper result, and we use a similar method to Section Letandbe continuous functions, an interval andsuch thatandwhereandLetandbe continuous functions or functions of bounded variation such thatandandIf for allwe haveand for allwe havewhere the functionGis defined in If for allthe inequality in If for allthe reversed inequality in , the condition ], then  becomes (c) (see ), if addIn this section we give the Levinson type generalization for converses of Jensen’s inequality in discrete case. The proofs are similar to those in the integral case given in the previous section, so we give these results with the proofs omitted.G is defined in If for allinequality If for allthe reversed inequality in   holds.We can replace the equality from the condition  by a weaThe classical Hermite-Hadamard inequality states that for a convex function Its weighted form is proved by Fejér in : If \docFink in  discusseRodić Lipanović, Pečarić, and Perić in  obtainedIn this section a Levinson type generalization of the Hermite-Hadamard inequality for signed measures will be given as a consequence of the results given in Sections Here we use the following notation: Letbe an interval andand letandLetandbe continuous functions or functions of bounded variation such thatandand such thatIf for alland for allthe inequalitieshold, where the functionGis defined in If for all ualities  hold, thities in  hold.(ii)If for all ities in  hold, thc  holds.Let the conditions from the previous corollary hold. Note that for the Levinson type generalization of the left-side inequality of the generalized Hermite-Hadamard inequality it is necessary to demand that If in Remark equality  under whequality  holds.x̃ goes outside of the interval ỹ outside of the interval Similarly, from the results given in the fourth section we get the Levinson type generalization of the right-side inequality of the generalized Hermite-Hadamard inequality. Here we allow that the mean value Letbe an interval andand letandLetandbe continuous functions or functions of bounded variation such thatandand such thatIf for allwe haveand for allwe havewhere the functionGis defined in If for all equality  holds anequality  holds, tities in  hold.(ii)If for all ality in  holds anality in  holds, tc  holds.Let the conditions from the previous theorem hold. If in Remark equality  under whequality  holds.It is easy to see that for nditions , 6.7) a a\documenditions  are alwaLetbe an interval andand letand(i)Ifholds, then for every continuous function(ii)Ifholds, then for every continuous functionIfthen the inequalities in (i) and (ii) are reversed.The well-known Petrović inequality  for convThe following generalization of  is givenGiaccardi, Letbe a nonnegativen-tuple andbe a realn-tuple such thatIfis a convex function, thenwheren-tuples p of real numbers which are not necessarily nonnegative. As a simple consequence, we will obtain a Levinson type generalization of the original Giaccardi inequality For every continuous convex functionholds.(2)For allholds, where the functionis defined in  and (2) are also equivalent if we change the sign of the inequality in both inequalities, in For every continuous concave functionthe reverse inequality in For allinequality  and (2′) are also equivalent if we change the sign of inequality in both statements (1′) and (2′).n-tuples p and m-tuples q of arbitrary real numbers instead of nonnegative real numbers.Our first result is a Levinson type generalization of the Giaccardi inequality for Letbe an interval andsuch thatandLetpandxben-tuples of real numbers, and letqandybem-tuples of real numbers such thatandLetwhereIfand for alland the functionGdefined in Ifandand if for allinequality Ifandand if for allinequality  and (ii) also hold if we reverse the signs in all of the inequalities.Let the assumptions from the previous theorem hold. (i)Ifand if for allinequalities Ifand if for allinequalities   holds.(iii)Ifandand if for allinequality Ifandand if for allinequality  and (iv) also hold if we reverse the signs in all of the mentioned inequalities.One needs to notice that if we set φ is convex (resp. concave), the condition  (see ), if addLet Motivated by the results obtained in previous sections, we define the following linear functionals which, respectively, represent the difference between the right and the left side of inequalities  and 4.74.7: 8.1\(i)≥0, when  holds ans2∈  holds;(ii)≥0, when  holds, a}s∈  holds an}s∈  holds.We have: In the following two theorems we give the mean-value theorems of the Lagrange and Cauchy type, respectively.Letandbe continuous functions, an interval andsuch thatandLetandbe continuous functions or functions of bounded variation such thatLetandbe linear functionals defined above, and let(i)If If =0), so  (resp. =0), so ) holds ftisfying  (resp. \tisfying ). □Let the conditions from Theorem hold. LetIfandthen there existsuch thatandLet us define a function ψ to be Note that if in Theorem f, g, λ, and μ from our theorems to fulfill the conditions from Jensen’s integral inequality or Jensen-Steffensen’s, or Jensen-Brunk’s, or Jensen-Boas’ inequality, then - applying that inequality on the function G which is continuous and convex in both variables - we see that in these cases for all Note that if we set the functions ities in  hold, anIf in the definition of the functional Let As before, motivated by the discrete results obtained in previous sections, we define the following linear functionals which, respectively, represent the difference between the right and the left side of inequalities , 5.4), , 5.4), anditions  hold andfined in .(i)≥0, when  holds ans2∈  holds;(ii)≥0, when  holds, a}s∈  holds an}s∈  holds;(iii)Qm>0 and  holds, a}s∈  and If If Ifand  for  and  for  to hold,Letandand let(i)If (holds andthen there existssuch that (holds.If  holds anuch that  holds.(ii)If If (holds andthen there existssuch that (holds.If  holds anuch that  holds.(ii)If (holds andthen there existssuch that (holds.If  holds anuch that  holds.G the inequalities in (Suppose that ities in  hold for


We have proposed a high sensitive photonic crystal fiber (PCF) strain sensor based on the Mach-Zehnder interferometer (MZI). The sensing head is formed by all-fiber in-line single mode-multimode-photonic-crystal-single mode fiber (SMPS) structure, using only the splicing method. Such a strain sensor exhibited a high sensitivity of −2.21 pm/με within a large measurement range of up to 5000 με and a large fringe visibility of up to 24 dB. Moreover, it was found that the strain sensitivity was weekly dependent of the length of PCF or MMF. In addition, the sensor exhibited the advantages of simplicity of fabrication, high sensitivity and larger fringe visibility. Optical fiber sensors have widely attracted many researchers’ attention due to its compact size, simplicity of fabrication, immunity to electromagnetic interference and high resolution ,2. StraiIn this paper, we demonstrated a high-sensitivity strain sensor based on SMPS structure MZI, which was realized by splicing a section of MMF with PCF between two SMFs using direct fusion technology ,30,31. T2 or femtosecond laser manufacturing. When the light was transmitted into the MMF, a part of the power could be coupled into the cladding modes of the PCF at the MMF-PCF collapsed region due to the mode filed mismatch. At the PCF-SMF collapsed region, the part light in the PCF cladding mode would couple back into the core and produce interfere with the PCF core mode. Consequently, they were coupled into the fundamental mode of the right SMF. In order to trace the interference fringes, a C+L ASE source with a wavelength of 1528~1602 nm and an optical spectrum analyzer  with a resolution of 10 pm were connected to the ends of the SMFs during the manufacturing process.Theoretically, we can assume that intensity at the end of PCF stub of length L (which is known as sensing length of the device) is (1)I=I1+IThe m order interference valley can be written as ,26:(2)λmTherefore, the free spectrum range (FSR) of the interference spectrum can be expressed as ,26:(3)FSWhen axial strain is applied along the fiber axis, the length of MZI will be stretched, the resonant wavelength shift can be expressed using Equation (2), which is given as ,14:(4)ΔλIt can be seen that the strain sensitivity is closely related to the change of In order to analyze the number and power distribution of the interference modes, the wavelength spectra in 2 laser, Bragg gratings and most MZIs based on PM-PCF, Nonlinear PCF, TC-PCF and MCF as well as the tapered PCF with up-tapered joint. Meanwhile, the sensitivity we obtained can be comparable to that of modified PCF and partially filled dual-core PCF. More importantly, our sensor is easier to fabricate and has lower cost and larger measurement range. In addition, the strain sensitivity and the measurement range of SMPS structure MZI was slight higher than that of STPS structure MZI, which is we reported. However, the new structure MZI has exhibited higher fringe visibility of 24 dB and smaller size of 60 mm length than those of STPS structure MZI . Since the core diameter of MMF is larger than that of TCF, more light power will be inject into the cladding mode of PCF, leading to excite cladding modes effectively. Therefore, the new MZI has exhibited better spectra performance. The MZIs based on asymmetrical twin core fiber and MMF, S-tapered PCF, twisted MMF and microfiber fabricated by fs laser, have exhibited higher sensitivity than 4 pm/με. However, the twin core fiber is much expensive and tapered or twisted fiber could reduce the mechanical strength of fiber obviously. At the same time, the fabrication of the micro-hole using fs laser is complex and time-consuming. Those types of MZIs have a narrow measurement range of ~1400 με, which limits their application in sensing fields. The MZI with SMPS structure in our paper has many advantages of simple fabrication, larger measurement range of up to 5000 με and a high sensitivity of 2.21 pm/με as well as high fringe visibility.In order to compare the performance of the strain sensor with previously reported ones, a comparison including strain sensitivity and measurement range is listed in Additionally, in order to compare the strain sensitivity with SMF-PCF-SMF (SPS) structure, the strain characteristics of MZI with 40 mm PCF was demonstrated experimentally, as shown in In order to analyze the number and the power distribution of the interference modes, the experimental spectra of two structures are taken fast Fourier transform (FFT) to obtain the spatial frequency spectra, as shown in In order to know the sensitivity of the proposed MZI to temperature and surrounding refractive index, the MZI with 20 mm MMF and 40 mm PCF was chosen to be characterized. From In conclusion, a high-sensitivity strain sensor based on all fiber inline MZI with SMPS structure was proposed and demonstrated experimentally. A high strain sensitivity of −2.21 pm/με is obtained in the range of 0~5000 με. The measurement range of the sensor was larger than most reported MZIs, including the normal PCF and tapered structure MZI as well as PCF interference-based PM-PCF or TC-PCF. Furthermore, the experimental results proved that the strain sensitivity of the sensor did not strongly depend on the length of MMF or PCF. The advantage of the proposed sensor was simplicity of fabrication. In addition, it exhibited large strain measurement range of up to 5000 με and high fringe visibility of ~24 dB and high sensitivity, which means that the structure is attractive for the development of strain sensors.


ET) is a safe and efficacious therapeutic approach for myocardial infarction (MI). Given the numerous benefits of exercise, exercise‐induced mediators may be promising treatment targets for MI. C57BL/6 mice were fed 1‐trifluoromethoxyphenyl‐3‐(1‐propionylpiperidine‐4‐yl) urea (TPPU), a novel soluble epoxide hydrolase inhibitor (sEHI), to increase epoxyeicosatrienoic acid (EET) levels, for 1 week before undergoing MI surgery. After 1‐week recovery, the mice followed a prescribed exercise programme. Bone marrow‐derived endothelial progenitor cells (EPCs) were isolated from the mice after 4 weeks of exercise and cultured for 7 days. Angiogenesis around the ischaemic area, EPC functions, and the expression of microRNA‐126 (miR‐126) and its target gene Spred1 were measured. The results were confirmed in vitro by adding TPPU to EPC culture medium. ET significantly increased serum EET levels and promoted angiogenesis after MI. TPPU enhanced the effects of ET to reduce the infarct area and improve cardiac function after MI. ET increased EPC function and miR‐126 expression, which were further enhanced by TPPU, while Spred1 expression was significantly down‐regulated. Additionally, the protein kinase B/glycogen synthase kinase 3β (AKT/GSK3β) signalling pathway was activated after the administration of TPPU. EETs are a potential mediator of exercise‐induced cardioprotection in mice after MI. TPPU enhances exercise‐induced cardiac recovery in mice after MI by increasing EET levels and promoting angiogenesis around the ischaemic area.Exercise training ( Myocardial infarction (MI) remains a leading cause of mortality and morbidity worldwide despite a growing number of pharmacological interventions. Exercise training (ET)‐based cardiac rehabilitation is a new and emerging therapeutic approach that improves the prognosis of MI Angiogenesis, the formation of new capillaries from pre‐existing vessels, plays an essential role in cardiac repair after MI and is responsible for restoring blood supply to the ischaemic myocardium and for preserving cardiac function When metabolized by P450 enzymes, arachidonic acid (AA) generates four regioisomeric epoxyeicosatrienoic acids (EETs): 5,6‐EET, 8,9‐EET, 11,12‐EET and 14,15‐EET VEGFA, PIK3R2, vascular cell adhesion molecule 1 (VCAM1) and sprouty‐related EVH1 domain containing 1 (SPRED1) SPRED1 is a target of miR‐126 in promoting angiogenesis MicroRNAs (miRNAs) consist of about 22 nucleotides and are a class of small non‐coding RNAs that regulate gene expression by targeting mRNAs for cleavage or translational repression Protein kinase B/glycogen synthase kinase 3β (AKT/GSK3β) is a critical cardioprotective signalling pathway against cardiac injury after myocardial ischaemia and is activated by ischaemia, exercise, and many other factors Based on the above hypothesis, we designed this study in MI mice to demonstrate that exercise promotes EPC functions and cardiac repair after MI by increasing EET concentrations. Moreover, we examined whether TPPU can enhance the exercise‐induced cardioprotection and thereby provide more evidence for the therapeutic function of sEHIs.As with our previous work, a MI model was constructed using male C57BL/6 mice  by ligating the left anterior descending coronary artery. Mice that underwent sham surgery (non‐MI mice), where the coronary artery was not ligated, were used as the control The potent and selective TPPU was synthesized in the laboratory of Prof. Bruce D. Hammock  in vivo study, a MI mouse model was established as described above. After 1‐week recovery, the surviving mice were randomly divided into four groups: non‐myocardial infarction and non‐exercise training (NMI NET), non‐myocardial infarction with exercise training (NMI ET), myocardial infarction and non‐exercise training (MI NET), and myocardial infarction with exercise training (MI ET) . Mice in the ET groups underwent 4 weeks of exercise. Bone marrow‐derived EPCs were isolated and cultured for 7 days to test the relative expression of miR‐126. At the same time, serum 14, 15‐EET was measured.For the n = 18 each in five independent experiments, respectively. The survival rate of MI surgery is more than 80%. The following survival rate was over 90% during exercise processing, and at least 12 mice in each group were studied. The mice were fed TPPU throughout the experiment. Bone marrow‐derived EPCs were isolated from the mice and cultured for 7 days. EPC functions and relative expression of miR‐126 and its target gene Spred1 and the related signalling pathways were tested. At the same time, infarct area and cardiac function were measured.To test the effect of TPPU on exercised mice, mice in the MI ET group were fed 0, 0.2, 1 or 5 mg/l TPPU for 1 week prior to the MI surgery and subsequent 4‐week exercise programme . Bone marrow‐derived EPCs were isolated from the group and cultured for 7 days, and were treated with 0, 0.1, 1 and 10 μM TPPU, or 10 μM TPPU plus 0.5 μM 14,15‐EEZE, a 14,15‐EET–specific antagonist, for 24 hrs. The relative expression of miR‐126 and the related signalling pathways were measured.For the For histological analysis, mouse hearts were sheared at the end of the experiment, rinsed three times with phosphate‐buffered saline (PBS) and fixed with 4% paraformaldehyde. After paraffin‐embedding, Masson's trichrome staining was performed to identify the infarct area, which was expressed as a percentage of the total LV size.Capillary density was determined by immunohistochemical staining with CD31 antibody. Immunohistochemical staining was performed using the two‐step immunohistochemical technique with diaminobenzidine  as described in the manufacturer's instructions. After restaining with hematine, the samples were coverslipped and photographed. The capillaries around the ischaemic area were counted as the mean of five randomly selected fields under ×400 magnification.At the end of the experiment, the LV function variables were assessed by transthoracic echocardiography, which was performed using a 15‐MHz phased array transducer (Siemens ACUSON S3000). Left ventricular (LV) dimensions were examined digitally in M‐mode tracings and averaged from three consecutive cardiac cycles. LV end‐systolic diameter (LVESD) and end‐diastolic diameter (LVEDD), interventricular septal thickness in diastole and LV posterior wall thickness were assessed. The percentage of LV fractional shortening (FS) was calculated as follows: FS% = (LVEDD‐LVESD)/LVEDD x 100 (%). Cardiac function was also measured as the ejection fraction EF (%) = (EDV – ESV)/EDV × 100%, where EDV and ESV are the end‐diastolic and end‐systolic left ventricular internal dimension (LVID), respectively. All measurements were carried out by three experienced technicians who were unaware of the experimental design.14,15‐EET was measured by liquid chromatography coupled with tandem mass spectrometry  in the laboratory of Prof. Yi Zhu at Tianjin Medical University as they previously described After 4 weeks of exercise, bone marrow‐derived EPCs were isolated and cultured by density gradient centrifugation as described in our previous work Migratory ability of the EPCs was measured as we described previously in vitro angiogenesis abilities of the EPCs were assayed as we described previously After 7 days of culture, the proliferative ability and 6 cells was harvested using TRIzol  according to the manufacturer's instructions. Total RNA (800 ng) was reverse‐transcribed with a TaqMan MicroRNA Reverse Transcription Kit. The miRNA qPCR assay was performed using TaqMan microRNA assays and the TaqMan Universal Master Mix II, no UNG, on a 7300 qPCR system . For the mRNA qPCR assay, complementary DNA (cDNA) was generated using a RevertAid First Strand cDNA Synthesis Kit  and was performed with gene‐specific primers and SYBR Green Master Mix (Applied Biosystems). The data were normalized to a single reference gene . The Mir126 and U6 primer sequences are available upon request . The Spred1 and GAPDH primer sequences are as follows:Total RNA from 1 × 10−∆(∆Ct).Relative gene expression was quantitated using the comparative threshold cycle value (∆CT) method with the above primers. The relative gene expression was calculated as fold change = 2As described in our previous work, Western blotting was performed with rabbit antimouse AKT, phosphorylated AKT (p‐AKT), GSK3β and p‐GSK3β antibodies ; mouse antimouse SPRED1  and β‐actin were used as loading controls P < 0.05 was considered statistically significant.All experiments were performed in duplicate three to five times. Results are expressed as the mean ± S.E.M. Comparison among experimental groups of NMI NET, NMI ET, MI NET and MI ET was performed using two‐way analysis of variance (ANOVA). Comparison among TPPU‐treated experimental groups was performed using one‐way ANOVA. Tukey's multiple comparisons test was used to compare the simple effect between groups. P < 0.05)  Fig. A. This iP < 0.05)  . The MI ET mice treated with 5 mg/l TPPU had a significant increase in the two variables when compared with the MI ET mice treated with 0 mg/l TPPU (P < 0.05)  Fig. D and E. 05) Fig. .in vitro angiogenesis and EPC functions P < 0.05)  Fig. A and B.in vitro angiogenesis, were significantly enhanced after TPPU treatment, also in a dose‐dependent manner. Compared with 0 mg/l TPPU‐treated MI ET mice, 5 mg/l TPPU‐treated MI ET mice increased EPC migratory ability by 2.3‐fold and 1.5‐fold measured by scratch wound and transwell assay, respectively, proliferative ability by 1.5‐fold, and in vitro angiogenesis ability by 1.7‐fold   , indicating that TPPU further enhanced the exercise‐induced miR‐126 upregulation in EPCs  Fig. A. FurthePCs Fig. B.in vitro, we isolated and cultured the bone marrow‐derived EPCs from MI ET mice. After 7 days of culture, we treated the EPCs with 0, 0.1, 1, and 10 μM TPPU, or 10 μM TPPU plus 0.5 μM 14,15‐EEZE. We found that 14,15‐EET levels in the culture medium were significantly increased in tandem with TPPU concentration. 14,15‐EET levels were increased about 2.8‐fold after treatment with 10 μM TPPU when compared with treatment with 0 μM TPPU (P < 0.05) . However, the effect of TPPU was significantly decreased by the addition of treatment with 0.5 μM 14,15‐EEZE (P < 0.05)  Fig. C. Consis05) Fig. D.Spred1. To confirm that Spred1 is a target of miR‐126 in EPCs, we used antagomiR‐126 (RIBOBIO) to silence miR‐126 in vitro; the antagomiR negative control (Neg Ctrl) (RIBOBIO) was used as the control. Bone marrow‐derived EPCs from NMI NET mice transfected for 24 hrs with 100 nM antagomiR‐126 had >100‐fold down‐regulated miR‐126 expression (P < 0.05)  , suggesting that Spred1 is a target gene of miR‐126 in EPCs    Fig. A–C. In vway Fig. D–F.via activation of the AKT/GSK3β signalling pathway, we used the AKT‐specific inhibitor MK‐2206 to block the AKT signalling pathway after treating EPCs with 10 μM TPPU in vitro. The relative expression of p‐AKT and p‐GSK3β was significantly reduced after 24‐hr treatment with 3 μM MK‐2206 when compared with TPPU treatment alone, indicating that MK‐2206 blocked the AKT/GSK3β signalling pathway (P < 0.05)   Fig. A–C. More05) Fig. D, suggesSpred1 by elevating EET concentrations, which was associated with activation of the AKT/GSK3β signalling pathway  expression in human dermal microvascular endothelial cell, indicating that EETs stimulated angiogenesis via an EGF receptor‐dependent mechanism. Qu et al. et al. via the AKT signalling pathway.In the present study, we observed that TPPU activates the AKT/GSK3β signalling pathway in EPCs in mice after MI. Using MK‐2206 to block the AKT signalling pathway, we also show that TPPU induces miR‐126 expression at least partially by activating the AKT/GSK3β signalling pathway. Previously, we noted that exercise can activate the AKT/GSK3β signalling pathway, which is consistent with the findings of others VEGFA, PIK3R2 and SPRED1 as above mentioned PIK3R2, another important target gene of miR‐126 that closely related to angiogenesis SPRED1 as the target gene of miR‐126 in MI mice model that underwent exercise owing to our preliminary experiment found it was the most significantly changed one in our studied model. Theoretically, there may exist positive feedback regulation between miR‐126 and AKT signalling pathway, and the expression of miR‐126 may be magnified markedly after AKT pathway activated. However, we did not observe a significantly amplified miR‐126 expression after TPPU treated, indicating the positive feedback in theory did not work, which might result from multiple and compensatory mechanism that induced by exercise and TPPU in organism.In our study, we demonstrate that the AKT/GSK3β signalling pathway is involved in TPPU‐regulated miR‐126 expression, thereby promoting EPC function. The most well‐studied target genes that related to angiogenesis of miR‐126 include Collectively, our findings provide new theoretical evidence for exercise‐based cardiac rehabilitation with the aim of improving cardiac rehabilitation recognition and enrolment. We also found that EETs are a potential exercise‐induced mediator and that TPPU, a novel sEHI that increases EET levels, may be a new agent for MI treatment.in vivo study that discussed the mechanisms of TPPU. We did not use sEH gene knockout mice to observe the effects of blocking sEH in vivo, which would have enabled more direct and authentic observation of the results. Besides, we did not test effects of exercise and TPPU on EPC functions in MI mice after miR‐126 knockout in this study. But exercise and TPPU at least partially exert their cardioprotection though regulating miR‐126 expression. Furthermore, exercise is a comprehensive programme that induces the activation of multiple organs and mechanisms in an organism. In the present study, we only tested EETs as a key exercise‐induced regulator for protecting ischaemic heart after MI. Exercise‐induced EPC function might also involve other factors and mechanisms, and there may be crosstalk between them. Therefore, further studies are needed to investigate this.There are limitations to the The authors reported no relationships that could be construed as a conflict of interest.


Consensus on terminology for multiple diseases is lacking. Because of the clinical relevance and social impact of multiple concurrent diseases, it is important that concepts are clear.To highlight the diversity of terms in the literature referring to the presence of multiple concurrent diseases/conditions and make recommendations.comorbidity, multimorbidity, polymorbidity, polypathology, pluripathology, multipathology, and multicondition, and a review of definitions of multimorbidity found in English-language publications indexed from 1970 to 2012 in the MEDLINE and SCOPUS databases.A bibliometric analysis of English-language publications indexed in the MEDLINE database from 1970 to 2012 for the terms Comorbidity was used in 67,557 publications, multimorbidity in 434, and the other terms in three to 31 publications. At least 144 publications used the term comorbidity without referring to an index disease. Thirteen general definitions of multimorbidity were identified, but only two were frequently used (91% of publications). The most frequently used definition (48% of publications) was “more than one or multiple chronic or long-term diseases/conditions”. Multimorbidity was not defined in 51% of the publications using the term.Comorbidity was overwhelmingly used to describe any clinical entity coexisting with an index disease under study. Multimorbidity was the term most frequently used when no index disease was designated. Several definitions of multimorbidity were found. However, most authors using the term did not define it. The use of clearly defined terms in the literature is recommended until a general consensus on the terminology of multiple coexistent diseases is reached.Journal of Comorbidity 2013;3:4–9 Although patients with more than one diagnosed disease are frequently encountered in modern medical practice, the inter-relationships and effects of multiple diseases have not received suitable taxonomic attention in clinical science”. This is how Alvan R. Feinstein begins his seminal paper, published in 1970, in which he coined the term comorbidity as “any distinct additional clinical entity that has existed or that may occur during the clinical course of a patient who has the index disease under study” [comorbidity, which was well defined by Feinstein, has been used (and sometimes redefined) to describe the coexistence of multiple diseases without considering an index disease. Other definitions of comorbidity found in the literature that do not refer to an index disease are: “the association of two distinct diseases in the same individual at a rate higher than expected by chance” [three or more clinical conditions” [the presence of more than one clinical condition” [the co-existence of multiple chronic conditions in a single individual” [more than one chronic disease” [several chronic conditions simultaneously” [the concurrence of multiple health conditions in the same person” [multiple chronic conditions” [two or more diseases, with distinct aetiopathogenesis , that are present in the same individual in a defined period of time” [“ chance” ; “three ditions” ; “the prndition” ; “the coividual” ; “more tdisease” ; “severa person” ; “multipof time” .multimorbidity in situations where no index disease is considered, and to reserve comorbidity for situations in which one of the diseases is considered as index, as originally defined by Feinstein [comorbidity, multimorbidity, polymorbidity, polypathology, pluripathology, multipathology, multicondition.Inconsistency and ambiguity in the use of the terms and implications for practice and research were pointed out by van den Akker and colleagues in 1996 . They preinstein . Despiteeinstein , 13, ambAll terms intuitively refer to the presence of multiple diseases, but the exact meaning or definition, if and when provided, varies from one author to another. In addition to a lack of general agreement in the definition of terms, it is not uncommon to see them used without any definition, which may imply that many authors presume that the meaning of the term is commonly understood.multiple , the use of indices (definitions based on scores), it may be difficult, on the one hand, to find the information of interest, and to compare the results on the other. A lack of a universal consensus on terminology and definitions is an obstacle to the dissemination of information and the application of new knowledge. Because of the clinical relevance and social impact of the presence of multiple diseases, it is important that the conceptualization of this subject is clear and without ambiguities. Thus we conducted a bibliometric analysis of terms used in the context of multiple diseases and a review of definitions of the term multimorbidity. The objective of this review was to shed light on the diversity of terms and the frequency of their use, to identify the different definitions of multimorbidity, and to provide recommendations for the use of terms.This diversity may have a negative impact on practice and research. Because of the different choices made in the design of studies with regard to terms (see list above), to diseases , the number of diseases considered as http://www.ncbi.nlm.nih.gov/pubmed/). We chose this database because it contains more than 22 million citations for biomedical literature, is freely accessible, and is widely used in bibliographical searches. We used the following search terms: comorbidity (and its variant co-morbidity), multimorbidity (and its variant multi-morbidity), polymorbidity (and its variants poly-morbidity and polymorbidities), polypathology (and its variants poly-pathology and polypathologies), pluripathology, multipathology (and its variants multi-pathology and multipathologies), and multicondition. This list was generated based on our personal experience of reading publications on multiple concurrent diseases for several years. Other terms that we are not aware of may exist. We searched for the terms in all fields of the publications ; the only filters used were: (1) publications written in English, and (2) publications indexed from 1970 to December 2012. In the case of comorbidity, the number of publications indexed under that term was overwhelming. To reduce the number of publications in our list and to target literature in which the subject heading is considered the main idea of the article, the filter “MeSH Major Topic” was additionally used for the term comorbidity. All types of publication  were included in our review. No further steps were taken to find publications that the automatic research engine could have missed. We counted the number of publications in which each term was used. In publications using a term for the first time, we also took note of the reference and country of origin of the work as we thought that there could be a link between the mother tongue of the authors and the introduction of new terms used to describe the presence of multiple coexistent conditions.For the bibliometric analysis of terms used when referring to the presence of multiple conditions in one individual, we searched the literature using the MEDLINE database in PubMed (multimorbidity (and its variant multi-morbidity), we used the MEDLINE database in PubMed and the SCOPUS database accessed through the library of the Université de Sherbrooke. In the MEDLINE database, we searched for the terms in all fields of the publications; the only filters used were: (1) publications written in English, and (2) publications indexed from 1970 to December 2012. In the SCOPUS database, we used the same filters, but also considered all subject areas contained in the database: life sciences, health sciences, physical sciences, and social sciences and humanities. All types of publication  were included in our review. For the purpose of this review, we looked for the digital or hard copy of the publications identified. We read all available publications looking for a definition or a description of the term. We extracted all definitions provided.To conduct a review of definitions for the term comorbidity, the bibliographic search initially yielded a total of 67,557 publications. After applying the filter “MeSH Major Topic”, 1,028 publications remained. We read the abstracts to assess how many of these publications using the term comorbidity were papers in which an index disease or medical condition was under study. A total of 162 publications did not have an abstract. In the remaining 866 publications with abstracts, 144 (17%) did not have an index disease or medical condition under study, suggesting that the term comorbidity was not used as originally described by Feinstein.In the case of comorbidity) are shown in multimorbidity (434 publications). The other terms were used in three to 31 publications. The reference and country of origin of publications using each term for the first time are shown in multipathology and multicondition were used for the first time in publications from an English-speaking country.Results of the bibliometric analysis of all other terms  in which the term multimorbidity was used without providing any definition of the term. In the publications providing a definition, we did not consider the exact wording to describe multimorbidity as a unique definition of the term. Instead, we grouped definitions with similar meanings into categories of general definitions. We identified 13 general definitions of multimorbidity, but two of them were predominantly used. multimorbidity used in the literature, as well as the number of publications that used them. The most frequently used definition of multimorbidity was “more than one or multiple chronic or long-term diseases/conditions” , which was closely followed by “more than one disease or condition” without specifying chronic or long-term in the definition.In the review of definitions of comorbidity, only two terms (multipathology and multicondition) were used for the first time in publications from an English-speaking country. This raises the question whether this diversity of terms has originated from the translation of studies originally conducted in other languages into English.The bibliometric analysis of terms referring to the presence of multiple diseases in one individual demonstrates the diversity of terms used in the medical literature to describe this common situation, and differences in the frequency of their use. This diversity of terms is not appropriate for medical language in which unambiguous well-defined terms are needed. Apart from comorbidity was the most often used when referring to the presence of multiple diseases in one individual. However, we determined that 17% of the revised abstracts were papers without an index disease under study, suggesting that the use of the term differed from Feinstein’s original definition [multimorbidity. We found that this term was most often used in the context of multiple coexistent diseases or conditions without designating an index disease. This prompted us to conduct a more detailed review of the literature using the term multimorbidity and the definitions proposed by different authors. As shown in multimorbidity, but the vast majority of publications used only two of them. An important number of publications using the term (51%) did not provide any definition. The authors of these publications may have presumed that the meaning of the term was generally understood.As expected, the term multimorbidity led us to another conceptual situation: the use of the terms disease, condition, or illness to describe the health issues of patients. The term illness is frequently used as a synonym for disease, but in many cases it refers to the patients’ personal experience of their disease. As mentioned previously, the term disease refers to a defined pathologic process with a characteristic set of signs and symptoms. Condition is a broad term that includes disease, but also other health issues that fall outside of the traditional disease model. The terms disease and condition were overwhelmingly used in the definitions of multimorbidity and seem to be more appropriate for describing the coexistence of health issues.Finding different definitions for For the time being, since there is no generally accepted terminology, we should be aware of the coexistence of the many terms, considering that definitions for any given term may vary. A comprehensive search of information on the subject should include all terms. If one is writing an article, our recommendation is that only one term be used to avoid ambiguity and to clearly define the meaning of the term used.comorbidity already exists and is widely accepted and used. Indeed, at the time of writing this text, it is the only term, of those mentioned above, that is accepted as a Medical Subject Heading (MeSH)  by the US National Library of Medicine (http://www.nlm.nih.gov/mesh/MBrowser.html), and is defined as “the presence of co-existing or additional diseases with reference to an initial diagnosis or with reference to the index condition that is the subject of study…”. Redefining or using the term comorbidity for other purposes, particularly in situations where no index disease is considered, should be avoided. Publications in which comorbidity is used with a different meaning from the one defined by Feinstein [comorbidity was redefined to fit other situations may not reach the intended audience.General agreement about terminology and definitions should be reached. We suggest that only two terms be used, one for situations in which multiple diseases/conditions coexist with an index disease under study, and another for situations in which there are multiple coexistent diseases/conditions, but none considered as the index disease/condition. In the first case, the term multimorbidity.With regard to the situation in which there are coexistent diseases, but none considered as an index disease, we recommend using the term that is most widely used, that is, multimorbidity implies that authors may use any definition. However, it should be noted that the high prevalence of multiple diseases/condition these days is generally considered as the result of aging of the population and the growing proportion of patients with coexistent long-term diseases/conditions, although it is clear that the presence of multiple chronic diseases/conditions is not a problem only affecting older patients [multimorbidity that we reviewed were about long-term diseases or conditions. Therefore, the most frequently used general definition of “multiple co-occurring chronic or long-term diseases or conditions, including both physical and mental diseases, and none considered as index disease”, seems a reasonable choice to define multimorbidity.The lack of consensus about a definition of patients , 22. Indmultimorbidity, although we acknowledge that there are authors who might have a different opinion on this. Addressing our words to those sharing our belief in this matter, we think that a first step towards a consensus is to expose the situation and instigate reflection on this, which is one of the intentions of this work. This could be followed by an exchange of ideas including as many people as possible interested in the subject, to end with a consensus that could be followed by a proposal of a MeSH. With regard to a joint definition of multimorbidity, we are aware of the recent effort of a large European research team that undertook a methodic data extraction and proposed a definition that was not included in our list of definitions up to 2012 because it was published in 2013 [multimorbidity was: “any combination of chronic disease with at least one other disease (acute or chronic) or biopsychosocial factor (associated or not) or somatic risk factor”. However, the definition lacks the input from many well-known experts from all over the world, including Europe, who have authored an important number of publications on the subject and whose opinions would have been very valuable in the development of the definition.We believe that a general consensus on the terminology for multiple coexistent diseases should exist, as well as a general definition of multimorbidity, we used only one additional database (SCOPUS) to retrieve publications, and this is a limitation of the study. We chose the SCOPUS database because, in addition to covering MEDLINE 100%, it includes more than 4,300 titles from the life sciences, more than 5,300 titles in social sciences and humanities, and more than 7,200 titles in physical sciences. The use of SCOPUS in addition to MEDLINE increased the number of publications identified as using the term multimorbidity from 434 to 465. Although we tried to be exhaustive with the two databases, it is possible that we were unaware of other relevant publications. Our search strategy was not limited to MeSH or keywords assigned by authors to avoid missing publications that were not indexed under these terms. However, as we were searching for specific terms, our search surely missed publications on the topic of multiple concurrent diseases that did not use any of the terms in our list.A limitation of any bibliographic search is the potential omission of relevant articles. For the bibliometric analysis, we did not try to be exhaustive, but rather highlight the use of multiple terms, when referring to multiple diseases, as well as to provide an estimate of their frequency of use. We considered that one database, such as MEDLINE, was enough for these purposes. In the review of definitions of comorbidity should be reserved for situations in which one or more diseases coexist with an index disease under study. For situations in which there are multiple coexistent diseases, but none considered as index disease, the term multimorbidity was by far the most frequently used. The term multimorbidity was most often defined as “the presence of more than one or multiple chronic or long-term diseases or conditions”. We recommend to clearly define terms until a general consensus on terminology of multiple coexistent diseases is reached.There are many terms currently used in the context of multiple concurrent diseases. This diversity may have a negative impact on practice and research. The term


We used small-angle X-ray scattering (SAXS) and transmission electron microscopy (TEM) to study the transformations of these mesophases, while N2 isotherm sorption curves revealed the porosities of these mesoporous silicate samples. Moreover, 29Si CP/MAS solid state nuclear magnetic resonance spectroscopy allowed us to analyze the compositions of the POSS-containing silicate frameworks. Such functional mesoporous silica samples incorporating microporous POSS building units have potential applications in various systems, including optical and electronic devices.We fabricated a series of mesoporous silicas and mesoporous organosilicates with hierarchical porosity through evaporation-induced self-assembly using Pluronic F127 as a template in this study. We could tailor the mesophase of each mesoporous silica sample by varying the weight ratio of its two silica sources: tetraethyl orthosilicate (TEOS) and triethoxysilane hydrosilylated octavinyl polyhedral oligomeric silsesquioxane (OV-POSS-SILY). The mesophases ranged from an ordered body-centered cubic ( Toluene was distilled over sodium prior to use. TEOS (99%) and hydrochloric acid were obtained from SHOWA. EtOH (95%) and THF (>99%) were purchased form ECHO.OV-POSS was purchased from Hybrid Plastics. Triethoxysilane [HSi(OEt)1H NMR : δ 0.6 , 1.2 , 3.8 , 6.0–5.8 . 13C NMR : δ 1.6, 3.4, 18.2, 58.3. 29Si NMR : δ −45.6, −66.8.Triethoxysilane  and Pt(dvs) complex solution in xylene (10 μL) were added sequentially to a solution of OV-POSS  in toluene (20 mL) and then the mixture was stirred at 80 °C for 12 h. After cooling to ambient temperature, the solvent and the unreacted triethoxysilane were evaporated under vacuum. The resulting hydrosilylated product was a colorless and sticky oil. Syntheses were performed at various ratios of OV-POSS-SILY to TEOS , a constant silica precursor-to-F127 weight ratio (3:1) at a constant HCl(aq) concentration. The silica precursor (a mixture of OV-POSS-SILY and TEOS) was added to a solution of the block copolymer in THF with stirring for 30 min. The sample was poured into a Petri dish and the THF solvent was evaporated over 48 h at room temperature. The transparent film was collected and ground into a powder, which was transferred to a Perfluoroalkoxy alkanes (PFA) bottle containing 1.0 M HCl and treated hydrothermally at 100 °C for one day. The product was washed with water and ethanol, dried at room temperature, and calcined in air at 500 °C for 1 h to produce mesoporous silica material. Calcination processes were performed in a furnace at the heating rate of 1 °C/min.1H, 13C, and 29Si solution NMR spectra were used the Bruker AM 500 (500 MHz) spectrometer  and the deuterated solvent acting as the internal standard. 29Si MAS solid state NMR spectra were used the AVIII 600 WB solid state NMR spectrometer . SAXS experiments were located at the BL17B3 beamline of the NSRRC, Taiwan. The X-ray beam had a diameter of 0.5 mm and a wavelength (λ) of 1.24 Å. TEM samples were suspended in EtOH and supported onto a holey carbon film on a Cu grid by using a JEOL 3010 microscope  operated at 200 kV. Nitrogen adsorption/desorption isotherms were determined at −196 °C using an ASAP 2020 analyzer . The samples were degassed under vacuum at 200 °C for at least 6 h prior to measurements. The specific surface areas and pore volumes; pore size distributions were derived from the adsorption branches of the isotherms by using the Barrett-Joyner-Halenda (BJH) model.29Si CP/MAS solid state NMR spectroscopy. Because of the bifunctional characteristics, the porosity enhancement induced by the cage-like POSS within the silica walls, and the remaining vinyl groups from the OV-POSS-SILY units, these mesoporous organosilicas having ordered structures might have potential applications as low-k interlayer dielectric materials or in catalysis.We present herein the first example of the construction of POSS-based mesoporous silica samples with specific mesophase structures that could be changed upon regulation of the OV-POSS-SILY weight ratio in the silicate precursor when using a simple EISA method. Although destruction occurred during the thermal treatment process, some degree of the POSS structures remained, as evidenced through characterization using


Pulmonary fibrosis represents the terminal stage of a diverse group of lung diseases including scleroderma associated interstitial lung disease. The molecular mechanisms underlying the pathogenesis of lung fibrosis are not well understood and there is a great need for more effective treatment for this lethal disease. We recently discovered a small fragment of hepatocyte growth factor (HGF) receptor MET as a peptide designated “M10,” with strong antifibrotic properties. Furthermore, we showed that aspartic acid at position 1398 of MET is essential for M10 generation. The current study was undertaken to investigate the D1398G variant of MET in which aspartic acid at position 1398 was mutated to glycine resulting in loss of M10. We demonstrate that lung fibroblasts, A549, and primary alveolar epithelial cells (AEC) expressing D1398G MET exhibit reduced auto-phosphorylation on tyrosine residues and reduced activation of Ras and MAPK. HGF treatment of scleroderma lung fibroblasts as well as HGF treatment of TGFβ-treated normal lung fibroblasts transfected with wild type MET is associated with decreased collagen, connective tissue growth factor  and smooth muscle α-actin (SMA). However, HGF has no such effects in cells transfected with MET D1398G. Cisplatin- and FasL-induced apoptosis is significantly reduced in AEC transfected with MET wild type, but not in AEC transfected with MET D1398G. We conclude that the D1398G variant of MET is associated with compromised phosphorylation and impaired HGF signaling in lung fibroblasts and AEC, two cell types implicated in the pathogenesis of pulmonary fibrosis associated with scleroderma. Ongoing studies will explore the frequency of this variant and its relationship to pulmonary outcomes in scleroderma patients. Systemic sclerosis  is a multi-system fibrotic disorder that affects skin and internal organs. Interstitial lung disease (ILD) or pulmonary fibrosis is a major organ complication and a leading cause of mortality and morbidity in SSc –3. In paWe previously reported that a cell-protective and antifibrotic factor, hepatocyte growth factor (HGF), is down-regulated in bronchoalveolar lavage fluid and plasma from African American SSc-ILD patients compared with Caucasian SSc-ILD patients . AdditioMET is a transmembrane protein with structural features of tyrosine kinase receptor , 12. METIn response to HGF binding, MET undergoes autophosphorylation at tyrosine residues in the kinase domain (Y1234 and Y1235 in the 1390 amino acid-isoform) . SubsequD-T, requires aspartic acid at position 1398 , shown above in bold. Mutation of 1398 aspartic acid to glycine prevents this site of MET from being recognized and cleaved by caspase-3, which suggests that the D1398G variant of MET is not able to generate the terminal 10-amino-acid-fragment, TRPASFWETS, designated in our laboratory as “M10” , pY1349, and pY1356 antibodies , Cell Signaling Technology , and Sigma-Aldrich  respectively). Total MET was immunobloted using anti-MET (C12) from Santa Cruz Biotechnology , anti-MET (25H2) was from Cell Signaling Technology , and pro-surfactant protein C antibody was from Seven Hills Bioreagents . Anti-type I collagen antibody , anti-CTGF antibody , and anti-smooth-α-actin antibody  were also used.Cells were collected and analyzed by immunoblotting as previously described , 25. PhoThe phosphorylation of p42/p44 MAPK isoforms and total Erk1/2 levels were analyzed using anti-phospho-Erk1/2 and Erk1/2 antibodies in accordance with the manufacturer’s instructions . Immunoblots were routinely stripped and re-blotted with anti-β-actin antibody  as a loading control.Statistical analyses were performed with KaleidaGraph 4.0 . All data were analyzed using ANOVA with post-hoc testing. The results were considered significant if p<0.05.Transfection of all studied cell lines with adenoviruses carrying MET WT and MET D1398G mutant was adjusted to multiplicity of infection 10 to 12 adenoviruses per one cell . Express1230/1234/1235], pY1349, and pY1356 antibodies. Treatment with HGF induced rapid phosphorylation of MET WT at tyrosine 1230, 1234, and 1235 in both LF and A549 cells. Maximal phosphorylation was observed within 5 minutes of HGF treatment and decreased by 20 minutes following HGF treatment  and AEC (A549 cell line) transfected either with MET WT or with D1398G mutant. MET tyrosine phosphorylation was assessed using anti-phospho-c-Met [pYpYpYreatment . HGF-indScleroderma LF transfected with the D1398G mutant or with the MET WT expressed similar to non-transfected cells basal levels of collagen, SMA, and CCN2. HGF, when added to cell culture medium at a concentration of 50 ng/ml for 48 hours, led to reduced expression of collagen, SMA, and CCN2 in non-transfected LF and LF transfected with MET WT, but not in LF transfected with mutant MET D1398G . To inveWe previously demonstrated that HGF-induced down regulation of collagen and CCN2 in scleroderma LF is mediated by a MAPK-dependent pathway . To inveHGF-induced phosphorylation of Erk1/2 is mediated by Ras . To inveAspartic acid at position 1398 is the terminal amino acid of the caspase-3 cleavage motif, DEVD-T, at the C-terminus of MET . To induTo investigate whether loss of the MET C-terminus occurs in patients with SSc-ILD, we performed immunofluorescent staining of lung tissues with C12 antibody and with anti-MET 4F8.2 antibody generated against the MET extracellular domain. We studied lung tissues isolated from three different patients who died from end-stage SSc-ILD. Hematoxylin and eosin staining of each sample demonstrated severe disarrangement of lung architecture with thickened alveolar septae and residual air spaces . In immuTo confirm that cleavage and loss of the MET C-terminus can occur with the endogenous MET, we used non-transfected scleroderma LF, A549, and ATII cell lines. To induce caspase-3 in cells, we employed the caspase-3-activator cisplatin in concentrations described above. Similarly to the described above immunofluorescent study of scleroderma lung tissue, we used anti-MET 4F8.2 antibody detecting total (cleaved and uncleaved MET) and anti-MET antibody C12 that does not recognize MET after C-terminal cleavage. After challenging cells with cisplatin, we observed a decrease of MET labeled with C12 antibody suggesting a loss of the terminal 10 amino acids of MET, TRPASFWETS. In contrast, amount of MET labeled with 4F8.2 antibody was not affected by cisplatin .A cell-protective effect of HGF and MET have been reported in AEC in numerous studies , 32. To The MET gene is located on chromosome 7q21-q31 and encodes for a protein containing a 50 kDa extracellular α-chain and a transmembrane 140 kDa β-chain, which are linked by disulfide bonds . In cancFollowing binding of HGF, under normal conditions MET undergoes auto-phosphorylation at tyrosine residues in its cytoplasmic domain and initiates a cascade of signal transduction events that lead to specific cellular responses , 36. AspAccumulation of collagen, CCN2 and other extracellular matrix proteins in fibrotic lesions is a hallmark of scleroderma and other fibrotic diseases –41. HGF In a previous study, we showed that HGF down-regulates collagen and CCN2 in lung fibroblasts through MAPK-dependent signaling pathways . MAPK siin vitro and in vivo [We recently described antifibrotic properties of the C-terminal fragment of MET M10  in vivo . Here wein vivo cleavage of MET can occur. Indeed, immunofluorescent studies using antibodies that differentially recognize MET expressing or not expressing the terminal 10 amino acids (TRPASFWETS) support the notion that such cleavage can indeed occur in vivo.Alveolar epithelium is the main source of MET in lung  and is tIn addition to downregulating collagen and other extracellular matrix proteins, antifibrotic properties of HGF and MET are based also on anti-apoptotic effects in AEC , 43, 46.Our study demonstrates that D1398G mutation in MET is associated with compromised HGF signaling in lung fibroblasts and in AEC because of decreased auto-phosphorylation and reduced activation of Ras and MAPK signaling. Generation of the C-terminal fragment of MET requires aspartic acid at position 1398 and can be in part responsible for anti-apoptotic and antifibrotic effects of MET.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.